CHROMOSOME-BASED PLATFORMS

Abstract

Artificial chromosomes, including ACes, that have been engineered to contain available sites for site-specific, recombination-directed integration of DNA of interest are provided. These artificial chromosomes provide tractable, efficient and rational engineering of the chromosome for a variety of applications.

Description

RELATED APPLICATIONS

[0001] This application is a continuation of and claims priority under 35 U.S.C. §120 to copending U.S. application Ser. No. 10/161,403, filed May 30, 2002, to EDWARD PERKINS, CARL PEREZ, MICHAEL LINDENBAUM, AMY GREENE, JOSEPHINE LEUNG, ELENA FLEMING, SANDRA STEWART and JOAN SHELLARD, who are the inventors as originally filed, entitled "CHROMOSOME-BASED PLATFORMS," which claims benefit of priority under 35 U.S.C. §119(e) to U.S. provisional application Ser. No. 60/294,758, filed May 30, 2001, to EDWARD PERKINS, CARL PEREZ, MICHAEL LINDENBAUM, AMY GREENE, and JOSEPHINE LEUNG, entitled "CHROMOSOME-BASED PLATFORMS" and benefit of priority to U.S. provisional application Ser. No. 60/366,891, filed Mar. 21, 2002, to EDWARD PERKINS, CARL PEREZ, MICHAEL LINDENBAUM, AMY GREENE, JOSEPHINE LEUNG, ELENA FLEMING, and SANDRA STEWART entitled, "CHROMOSOME-BASED PLATFORMS.".

[0002] This application is related to U.S. application Ser. No. 11/006,076, filed Dec. 6, 2004 by EDWARD PERKINS, CARL PEREZ, MICHAEL LINDENBAUM, AMY GREENE, JOSEPHINE LEUNG, ELENA FLEMING, SANDRA STEWART and JOAN SHELLARD, entitled CHROMOSOME-BASED PLATFORMS.

[0003] This application is related to Provisional Application No. 60/294,687, filed May 30, 2001, by CARL PEREZ AND STEVEN FABIJANSKI entitled PLANT ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING PLANT ARTIFICIAL CHROMOSOMES and to U.S. Provisional Application No. 60/296,329, filed Jun. 4, 2001, by CARL PEREZ AND STEVEN FABIJANSKI entitled PLANT ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING PLANT ARTIFICIAL CHROMOSOMES. This application also is related to U.S. Provisional Application No. 60/294,758, filed May 30, 2001, by EDWARD PERKINS et al. entitled CHROMOSOME-BASED PLATFORMS and to U.S. Provisional Application No. 60/366,891, filed Mar. 21, 2002, by EDWARD PERKINS et al. entitled CHROMOSOME-BASED PLATFORMS. This application is also related to U.S. application Ser. No. 10/161,408 and to International PCT application No. PCT/US02/17451, published as WO2002/096923, each filed on the same day herewith, entitled PLANT ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS OF PREPARING PLANT ARTIFICIAL CHROMOSOMES to Perez et al.

[0004] This application is related to U.S. application Ser. No. 08/695,191, filed Aug. 7, 1996 by GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES, now U.S. Pat. No. 6,025,155. This application is also related to U.S. application Ser. No. 08/682,080, filed Jul. 15, 1996 by GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES, now U.S. Pat. No. 6,077,697. This application is also related U.S. application Ser. No. 08/629,822, filed Apr. 10, 1996 by GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES (now abandoned), and is also related to copending U.S. application Ser. No. 09/096,648, filed Jun. 12, 1998, by GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES and to U.S. application Ser. No. 09/835,682, Apr. 10, 1997 by GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES (now abandoned). This application is also related to copending U.S. application Ser. No. 09/724,726, filed Nov. 28, 2000, U.S. application Ser. No. 09/724,872, filed Nov. 28, 2000, U.S. application Ser. No. 09/724,693, filed Nov. 28, 2000, U.S. application Ser. No. 09/799,462, filed Mar. 5, 2001, U.S. application Ser. No. 09/836,911, filed Apr. 17, 2001, and U.S. application Ser. No. 10/125,767, filed Apr. 17, 2002, each of which is by GYULA HADLACZKY and ALADAR SZALAY, and is entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES. This application is also related to International PCT application No. WO 97/40183.

[0005] The subject matter of all of the above provisional applications, international applications, and applications are herein incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS

[0006] An electronic version on compact disc (CD) ROM of a computer-readable form of the Sequence Listing is filed herewith in duplicate, the contents of which are incorporated by reference in their entirety. The computer-readable file on each of the aforementioned duplicate compact discs created on Jun. 29, 2006, is identical, 396 kilobytes in size, and entitled 420DSEQ.001.txt.

FIELD OF THE INVENTION

[0007] Artificial chromosomes, including ACes, that have been engineered to contain available sites for site-specific, recombination-directed integration of DNA of interest are provided. These artificial chromosomes permit tractable, efficient, rational engineering of the chromosome.

BACKGROUND

[0008] Artificial Chromosomes

[0009] A variety of artificial chromosomes for use in plants and animals, particularly higher plants and animals are available. In particular, U.S. Pat. Nos. 6,025,155 and 6,077,697 provide heterochromatic artificial chromosomes designated therein as satellite artificial chromosomes (SATACs) and now designated artificial chromosome expression systems (ACes). These chromosomes are prepared by introducing heterologous DNA into a selected plant or animal cell under conditions that result in integration into a region of the chromosome that leads to an amplification event resulting in production of a dicentric chromosome. Subsequent treatment and growth of cells with dicentric chromosomes, including further amplifications, ultimately results in the artificial chromosomes provided therein. In order to introduce a desired heterologous gene (or a plurality of heterologous genes) into the artificial chromosome, the process is repeated introducing the desired heterologous genes and nucleic acids in the initial targeting step. This process is time consuming and tedious. Hence, more tractable and efficient methods for introducing heterologous nucleic acid molecules into artificial chromosomes, particularly ACes, are needed.

[0010] Therefore, it is an object herein to provide engineered artificial chromosomes that permit tractable, efficient and rational engineering of artificial chromosomes.

SUMMARY OF THE INVENTION

[0011] Provided herein are artificial chromosomes that permit tractable, efficient and rational engineering thereof. In particular, the artificial chromosomes provided herein contain one or a plurality of loci (sites) for site-specific, recombination-directed integration of DNA. Thus, provided herein are platform artificial chromosome expression systems ("platform ACes") containing single or multiple site-specific, recombination sites. The artificial chromosomes and ACes artificial chromosomes include plant and animal chromosomes. Any recombinase system that effects site-specific recombination is contemplated for use herein.

[0012] In one embodiment, chromosomes, including platform ACes, are provided that contain one or more lambda att sites designed for recombination-directed integration in the presence of lambda integrase, and that are mutated so that they do not require additional factors. Methods for preparing such chromosomes, vectors for use in the methods, and uses of the resulting chromosomes are also provided.

[0013] Platform ACes containing the recombination site(s) and methods for introducing heterologous nucleic acid into such sites and vectors therefor, are provided.

[0014] Also provided herein is a bacteriophage lambda (λ) integrase site-specific recombination system.

[0015] Methods using recombinase mediated recombination target gene expression vectors and/or genes for insertion thereof into platform chromosomes and the resulting chromosomes are provided.

[0016] Combinations and kits containing the combinations of vectors encoding a recombinase and integrase and primers for introduction of the site recognized thereby are also provided. The kits optionally include instructions for performing site-directed integration or preparation of ACes containing such sites.

[0017] Also provided herein are mammalian and plant cells comprising the artificial chromosomes and ACes described herein. The cells can be nuclear donor cells, stem cells, such as a mesenchymal stem cell, a hematopoietic stem cell, an adult stem cell or an embryonic stem cell.

[0018] Also provide is a lamba-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-integrase3. Also provided are transgenic animals and methods for producing a transgenic animal, comprising introducing a ACes into an embryonic cell, such as a stem cell or embryo. The ACes can comprise heterologous nucleic acid that encodes a therapeutic product. The transgenic animal can be a fish, insect, reptile, amphibians, arachnid or mammal. In certain embodiments, the ACes is introduced by cell fusion, lipid-mediated transfection by a carrier system, microinjection, microcell fusion, electroporation, microprojectile bombardment or direct DNA transfer.

[0019] The platform ACes, including plant and animal ACes, such as MACs, provided herein can be introduced into cells, such as, but not limited to, animal cells, including mammalian cells, and into plant cells. Hence plant cells that contain platform MACs, animal cells that contain platform PACs and other combinations of cells and platform ACes are provided.

DESCRIPTION OF THE FIGURES

[0020] FIG. 1 provides a diagram depicting creation of an exemplary ACes artificial chromosome prepared using methods detailed in U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183. In this exemplified embodiment, the nucleic acid is targeted to an acrocentric chromosome in an animal or plant, and the heterologous nucleic acid includes a sequence-specific recombination site and marker genes.

[0021] FIG. 2 provides a map of pWEPuro9K, which is a targeting vector derived from the vector pWE15 (GenBank Accession # X65279; SEQ ID No. 31). Plasmid pWE15 was modified by replacing the SalI (Klenow filled)/SmaI neomycin resistance encoding fragment with the PvuII/BamHI (Klenow filled) puromycin resistance-encoding fragment (isolated from plasmid pPUR, Clontech Laboratories, Inc., Palo Alto, Calif.; GenBank Accession no. U07648; SEQ ID No. 30) resulting in plasmid pWEPuro. Subsequently a 9 Kb NotI fragment from the plasmid pFK161 (see Example 1, see, also Csonka et al. (2000) Journal of Cell Science 113:3207-32161; and SEQ ID NO: 118), containing a portion of the mouse rDNA region, was cloned into the NotI site of pWEPuro resulting in plasmid pWEPuro9K.

[0022] FIG. 3 depicts construction of an ACes platform chromosome with a single recombination site, such as loxP sites or an attP or attB site. This platform ACes chromosome is an exemplary artificial chromosome with a single recombination site.

[0023] FIG. 4 provides a map of plasmid pSV40-193attPsensePur.

[0024] FIG. 5 depicts a method for formation of a chromosome platform with multiple recombination integration sites, such as attP sites.

[0025] FIG. 6 sets forth the sequences of the core region of attP, attB, attL and attR (SEQ ID Nos. 33-36).

[0026] FIG. 7 depicts insertional recombination of a vector encoding a marker gene, DsRed and an attB site with an artificial chromosome containing an attP site.

[0027] FIG. 8 provides a map of plasmid pCXLamIntR (SEQ ID NO: 112), which includes the Lambda integrase (E174R)-encoding nucleic acid.

[0028] FIG. 9 diagrammatically summarizes the platform technology; marker 1 permits selection of the artificial chromosomes containing the integration site; marker 2, which is promoterless in the target gene expression vector, permits selection of recombinants. Upon recombination with the platform marker 2 is expressed under the control of a promoter resident on the platform.

[0029] FIG. 10 provides the vector map for the plasmid p18attBZEO-5'6XHS4eGFP (SEQ ID NO: 116).

[0030] FIG. 11 provides the vector map for the plasmid p18attBZEO-3'6XHS4eGFP (SEQ ID NO: 115).

[0031] FIG. 12 provides the vector map for the plasmid p18attBZEO-(6XHS4)2eGFP (SEQ ID NO: 110).

[0032] FIGS. 13 AND 14 depict the integration of a PCR product by site-specific recombination as set forth in Example 8.

[0033] FIG. 15 provides the vector map for the plasmid pPACrDNA as set forth in Example 9.A.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, Genbank sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. Where reference is made to a URL or other such indentifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0035] As used herein, nucleic acid refers to single-stranded and/or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs or derivatives of either RNA or DNA. Also included in the term "nucleic acid" are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives. When referring to probes or primers, optionally labeled, with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that they are statistically unique and of low copy number (typically less than 5, preferably less than 3) for probing or priming a library. Generally a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleotides long.

[0036] As used herein, DNA is meant to include all types and sizes of DNA molecules including cDNA, plasmids and DNA including modified nucleotides and nucleotide analogs.

[0037] As used herein, nucleotides include nucleoside mono-, di-, and triphosphates. Nucleotides also include modified-nucleotides, such as, but are not limited to, phosphorothioate nucleotides and deazapurine nucleotides and other nucleotide analogs.

[0038] As used herein, heterologous or foreign DNA and RNA are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location or locations and/or in amounts in a genome or cell that differ from that in which it occurs in nature. Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or prepared synthetically. Generally, although not necessarily, such nucleic acid encodes RNA and proteins that are not normally produced by the cell in which it is expressed. Any DNA or RNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by heterologous DNA. Heterologous DNA and RNA may also encode RNA or proteins that mediate or alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes.

[0039] Examples of heterologous DNA include, but are not limited to, DNA that encodes a gene product or gene product(s) of interest, introduced for purposes of modification of the endogenous genes or for production of an encoded protein. For example, a heterologous or foreign gene may be isolated from a different species than that of the host genome, or alternatively, may be isolated from the host genome but operably linked to one or more regulatory regions which differ from those found in the unaltered, native gene. Other examples of heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers traits including, but not limited to, herbicide, insect, or disease resistance; traits, including, but not limited to, oil quality or carbohydrate composition. Antibodies that are encoded by heterologous DNA may be secreted or expressed on the surface of the cell in which the heterologous DNA has been introduced.

[0040] As used herein, operative linkage or operative association, or grammatical variations thereof, of heterologous DNA to regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences refers to the relationship between such DNA and such sequences of nucleotides. For example, operative linkage of heterologous DNA to a promoter refers to the physical relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0041] In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation (i.e., start) codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites (see, e.g., Kozak (1991) J. Biol. Chem. 266:19867-19870) can be inserted immediately 5' of the start codon and may enhance expression.

[0042] As used herein, a sequence complementary to at least a portion of an RNA, with reference to antisense oligonucleotides, means a sequence having sufficient complementarity to be able to hybridize with the RNA, preferably under moderate or high stringency conditions, forming a stable duplex. The ability to hybridize depends on the degree of complementarity and the length of the antisense nucleic acid. The longer the hybridizing nucleic acid, the more base mismatches it can contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

[0043] As used herein, regulatory molecule refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or a polypeptide that is capable of enhancing or inhibiting expression of a gene.

[0044] As used herein, recognition sequences are particular sequences of nucleotides that a protein, DNA, or RNA molecule, or combinations thereof, (such as, but not limited to, a restriction endonuclease, a modification methylase and a recombinase) recognizes and binds. For example, a recognition sequence for Cre recombinase (see, e.g., SEQ ID NO:58) is a 34 base pair sequence containing two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core and designated loxP (see, e.g., Sauer (1994) Current Opinion in Biotechnology 5:521-527). Other examples of recognition sequences, include, but are not limited to, attB and attP, attR and attL and others (see, e.g., SEQ ID Nos. 8, 41-56 and 72), that are recognized by the recombinase enzyme Integrase (see, SEQ ID Nos. 37 and 38 for the nucleotide and encoded amino acid sequences of an exemplary lambda phage integrase).

[0045] The recombination site designated attB is an approximately 33 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region; attP (SEQ ID No. 72) is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins IHF, FIS, and X is (see, e.g., Landy (1993) Current Opinion in Biotechnology 3:699-7071 see, e.g., SEQ ID Nos. 8 and 72).

[0046] As used herein, a recombinase is an enzyme that catalyzes the exchange of DNA segments at specific recombination sites. An integrase herein refers to a recombinase that is a member of the lambda (λ) integrase family.

[0047] As used herein, recombination proteins include excisive proteins, integrative proteins, enzymes, co-factors and associated proteins that are involved in recombination reactions using one or more recombination sites (see, Landy (1993) Current Opinion in Biotechnology 3:699-707). The recombination proteins used herein can be delivered to a cell via an expression cassette on an appropriate vector, such as a plasmid, and the like. In other embodiments, the recombination proteins can be delivered to a cell in protein form in the same reaction mixture used to deliver the desired nucleic acid, such as a platform ACes, donor target vectors, and the like.

[0048] As used herein the expression "lox site" means a sequence of nucleotides at which the gene product of the cre gene, referred to herein as Cre, can catalyze a site-specific recombination event. A LoxP site is a 34 base pair nucleotide sequence from bacteriophage P1 (see, e.g., Hoess et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79:3398-3402). The LoxP site contains two 13 base pair inverted repeats separated by an 8 base pair spacer region as follows: (SEQ ID NO. 57):

TABLE-US-00001 ATAACTTCGTATA ATGTATGC TATACGAAGTTAT

E. coliDH5Δlac and yeast strain BSY23 transformed with plasmid pBS44 carrying two loxP sites connected with a LEU2 gene are available from the American Type Culture Collection (ATCC) under accession numbers ATCC 53254 and ATCC 20773, respectively. The lox sites can be isolated from plasmid pBS44 with restriction enzymes EcoRI and SalI, or XhoI and BamHI. In addition, a preselected DNA segment can be inserted into pBS44 at either the SalI or BamHI restriction enzyme sites. Other lox sites include, but are not limited to, LoxB, LoxL, LoxC2 and LoxR sites, which are nucleotide sequences isolated from E. coli (see, e.g., Hoess et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79:3398). Lox sites can also be produced by a variety of synthetic techniques (see, e.g., Ito et al. (1982) Nuc. Acid Res. 10:1755 and Ogilvie et al. (1981) Science 270:270).

[0049] As used herein, the expression "cre gene" means a sequence of nucleotides that encodes a gene product that effects site-specific recombination of DNA in eukaryotic cells at lox sites. One cre gene can be isolated from bacteriophage P1 (see, e.g., Abremski et al. (1983) Cell 32:1301-1311). E. coliDH1 and yeast strain BSY90 transformed with plasmid pBS39 carrying a cre gene isolated from bacteriophage P1 and a GAL1 regulatory nucleotide sequence are available from the American Type Culture Collection (ATCC) under accession numbers ATCC 53255 and ATCC 20772, respectively. The cre gene can be isolated from plasmid pBS39 with restriction enzymes XhoI and SalI.

[0050] As used herein, site-specific recombination refers to site-specific recombination that is effected between two specific sites on a single nucleic acid molecule or between two different molecules that requires the presence of an exogenous protein, such as an integrase or recombinase.

[0051] For example, Cre-lox site-specific recombination can include the following three events: [0052] a. deletion of a pre-selected DNA segment flanked by lox sites; [0053] b. inversion of the nucleotide sequence of a pre-selected DNA segment flanked by lox sites; and [0054] c. reciprocal exchange of DNA segments proximate to lox sites located on different DNA molecules.

[0055] This reciprocal exchange of DNA segments can result in an integration event if one or both of the DNA molecules are circular. DNA segment refers to a linear fragment of single- or double-stranded deoxyribonucleic acid (DNA), which can be derived from any source. Since the lox site is an asymmetrical nucleotide sequence, two lox sites on the same DNA molecule can have the same or opposite orientations with respect to each other. Recombination between lox sites in the same orientation results in a deletion of the DNA segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule. The deleted DNA segment forms a circular molecule of DNA. The original DNA molecule and the resulting circular molecule each contain a single lox site. Recombination between lox sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two lox sites. In addition, reciprocal exchange of DNA segments proximate to lox sites located on two different DNA molecules can occur. All of these recombination events are catalyzed by the gene product of the cre gene. Thus, the Cre-lox system can be used to specifically delete, invert, or insert DNA. The precise event is controlled by the orientation of lox DNA sequences, in cis the lox sequences direct the Cre recombinase to either delete (lox sequences in direct orientation) or invert (lox sequences in inverted orientation) DNA flanked by the sequences, while in trans the lox sequences can direct a homologous recombination event resulting in the insertion of a recombinant DNA.

[0056] As used herein, a chromosome is a nucleic acid molecule, and associated proteins, that is capable of replication and segregation within a cell upon cell division. Typically, a chromosome contains a centromeric region, replication origins, telomeric regions and a region of nucleic acid between the centromeric and telomeric regions.

[0057] As used herein, a centromere is any nucleic acid sequence that confers an ability to segregate to daughter cells through cell division. A centromere may confer stable segregation of a nucleic acid sequence, including an artificial chromosome containing the centromere, through mitotic or meiotic divisions, including through both mitotic and meiotic divisions. A particular centromere is not necessarily derived from the same species in which it is introduced, but has the ability to promote DNA segregation in cells of that species.

[0058] As used herein, euchromatin and heterochromatin have their recognized meanings. Euchromatin refers to chromatin that stains diffusely and that typically contains genes, and heterochromatin refers to chromatin that remains unusually condensed and that has been thought to be transcriptionally inactive. Highly repetitive DNA sequences (satellite DNA) are usually located in regions of the heterochromatin surrounding the centromere (pericentric or pericentromeric heterochromatin). Constitutive heterochromatin refers to heterochromatin that contains the highly repetitive DNA which is constitutively condensed and genetically inactive.

[0059] As used herein, an acrocentric chromosome refers to a chromosome with arms of unequal length.

[0060] As used herein, endogenous chromosomes refer to genomic chromosomes as found in a cell prior to generation or introduction of an artificial chromosome.

[0061] As used herein, artificial chromosomes are nucleic acid molecules, typically DNA, that stably replicate and segregate alongside endogenous chromosomes in cells and have the capacity to accommodate and express heterologous genes contained therein. It has the capacity to act as a gene delivery vehicle by accommodating and expressing foreign genes contained therein. A mammalian artificial chromosome (MAC) refers to chromosomes that have an active mammalian centromere(s). Plant artificial chromosomes, insect artificial chromosomes and avian artificial chromosomes refer to chromosomes that include centromeres that function in plant, insect and avian cells, respectively. A human artificial chromosome (HAC) refers to chromosomes that include centromeres that function in human cells. For exemplary artificial chromosomes, see, e.g., U.S. Pat. Nos. 6,025,155; 6,077,697; 5,288,625; 5,712,134; 5,695,967; 5,869,294; 5,891,691 and 5,721,118 and published International PCT application Nos, WO 97/40183 and WO 98/08964. Artificial chromosomes include those that are predominantly heterochromatic (formerly referred to as satellite artificial chromosomes (SATACs); see, e.g., U.S. Pat. Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183), minichromosomes that contain a de novo centromere (see, U.S. Pat. Nos. 5,712,134, 5,891,691 and 5,288,625), artificial chromosomes predominantly made up of repeating nucleic acid units and that contain substantially equivalent amounts of euchromatic and heterochromatic DNA and in vitro assembled artificial chromosomes (see, copending U.S. provisional application Ser. No. 60/294,687, filed on May 30, 2001).

[0062] As used herein, the term "satellite DNA-based artificial chromosome (SATAC)" is interchangable with the term "artificial chromosome expression system (ACes)". These artificial chromosomes (ACes) include those that are substantially all neutral non-coding sequences (heterochromatin) except for foreign heterologous, typically gene-encoding nucleic acid, that is interspersed within the heterochromatin for the expression therein (see U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183), or that is in a single locus as provided herein. Also included are ACes that may include euchromatin and that result from the process described in U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183 and outlined herein. The delineating structural feature is the presence of repeating units, that are generally predominantly heterochromatin. The precise structure of the ACes will depend upon the structure of the chromosome in which the initial amplification event occurs; all share the common feature of including a defined pattern of repeating units. Generally ACes have more heterochromatin than euchromatin. Foreign nucleic acid molecules (heterologous genes) contained in these artificial chromosome expression systems can include any nucleic acid whose expression is of interest in a particular host cell. Such foreign nucleic acid molecules, include, but are not limited to, nucleic acid that encodes traceable marker proteins (reporter genes), such as fluorescent proteins, such as green, blue or red fluorescent proteins (GFP, BFP and RFP, respectively), other reporter genes, such as 3-galactosidase and proteins that confer drug resistance, such as a gene encoding hygromycin-resistance. Other examples of heterologous nucleic acid molecules include, but are not limited to, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, DNA that encodes other types of proteins, such as antibodies, and DNA that encodes RNA molecules (such as antisense or siRNA molecules) that are not translated into proteins.

[0063] As used herein, an artificial chromosome platform, also referred to herein as a "platform ACes" or "ACes platform", refers to an artificial chromosome that has been engineered to include one or more sites for site-specific, recombination-directed integration. In particular, ACes that are so-engineered are provided. Any sites, including but not limited to any described herein, that are suitable for such integration are contemplated. Plant and animal platform ACes are provided. Among the ACes contemplated herein are those that are predominantly heterochromatic (formerly referred to as satellite artificial chromosomes (SATACs); see, e.g., U.S. Pat. Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183), artificial chromosomes predominantly made up of repeating nucleic acid units and that contain substantially equivalent amounts of euchromatic and heterochromatic DNA resulting from an amplification event depicted in the referenced patent and herein. Included among the ACes for use in generating platforms, are artificial chromosomes that introduce and express heterologous nucleic acids in plants (see, copending U.S. provisional application Ser. No. 60/294,687, filed on May 30, 2001). These include artificial chromosomes that have a centromere derived from a plant, and, also, artificial chromosomes that have centromeres that may be derived from other organisms but that function in plants.

[0064] As used herein a "reporter ACes" refers to a an ACes that comprises one or a plurality of reporter constructs, where the reporter construct comprises a reporter gene in operative linkage with a regulatory region responsive to test or known compounds.

[0065] As used herein, amplification, with reference to DNA, is a process in which segments of DNA are duplicated to yield two or multiple copies of substantially similar or identical or nearly identical DNA segments that are typically joined as substantially tandem or successive repeats or inverted repeats.

[0066] As used herein, amplification-based artificial chromosomes are artificial chromosomes derived from natural or endogenous chromosomes by virtue of an amplification event, such as one initiated by introduction of heterologous nucleic acid into rDNA in a chromosome. As a result of such an event, chromosomes and fragments thereof exhibiting segmented or repeating patterns arise. Artificial chromosomes can be formed from these chromosomes and fragments. Hence, amplification-based artificial chromosomes refer to engineered chromosomes that exhibit an ordered segmentation that is not observed in naturally occurring chromosomes and that distinguishes them from naturally occurring chromosomes. The segmentation, which can be visualized using a variety of chromosome analysis techniques known to those of skill in the art, correlates with the structure of these artificial chromosomes. In addition to containing one or more centromeres, the amplification-based artificial chromosomes, throughout the region or regions of segmentation are predominantly made up of nucleic acid units also referred to as "amplicons", that is (are) repeated in the region and that have a similar gross structure. Repeats of an amplicon tend to be of similar size and share some common nucleic acid sequences. For example, each repeat of an amplicon may contain a replication site involved in amplification of chromosome segments and/or some heterologous nucleic acid that was utilized in the initial production of the artificial chromosome. Typically, the repeating units are substantially similar in nucleic acid composition and may be nearly identical.

[0067] The amplification-based artificial chromosomes differ depending on the chromosomal region that has undergone amplification in the process of artificial chromosome formation. The structures of the resulting chromosomes can vary depending upon the initiating event and/or the conditions under which the heterologous nucleic acid is introduced, including modification to the endogenous chromosomes. For example, in some of the artificial chromosomes provided herein, the region or regions of segmentation may be made up predominantly of heterochromatic DNA. In other artificial chromosomes provided herein, the region or regions of segmentation may be made up predominantly of euchromatic DNA or may be made up of similar amounts of heterochromatic and euchromatic DNA.

[0068] As used herein an amplicon is a repeated nucleic acid unit. In some of the artificial chromosomes described herein, an amplicon may contain a set of inverted repeats of a megareplicon. A megareplicon represents a higher order replication unit. For example, with reference to some of the predominantly heterochromatic artificial chromosomes, the megareplicon can contain a set of tandem DNA blocks (e.g., ˜7.5 Mb DNA blocks) each containing satellite DNA flanked by non-satellite DNA or may be made up of substantially rDNA. Contained within the megareplicon is a primary replication site, referred to as the megareplicator, which may be involved in organizing and facilitating replication of the pericentric heterochromatin and possibly the centromeres. Within the megareplicon there may be smaller (e.g., 50-300 kb) secondary replicons.

[0069] In artificial chromosomes, such as those provided U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183, the megareplicon is defined by two tandem blocks (˜7.5 Mb DNA blocks in the chromosomes provided therein). Within each artificial chromosome or among a population thereof, each amplicon has the same gross structure but may contain sequence variations. Such variations will arise as a result of movement of mobile genetic elements, deletions or insertions or mutations that arise, particularly in culture. Such variation does not affect the use of the artificial chromosomes or their overall structure as described herein.

[0070] As used herein, amplifiable, when used in reference to a chromosome, particularly the method of generating artificial chromosomes provided herein, refers to a region of a chromosome that is prone to amplification. Amplification typically occurs during replication and other cellular events involving recombination (e.g., DNA repair). Such regions include regions of the chromosome that contain tandem repeats, such as satellite DNA, rDNA, and other such sequences.

[0071] As used herein, a dicentric chromosome is a chromosome that contains two centromeres. A multicentric chromosome contains more than two centromeres.

[0072] As used herein, a formerly dicentric chromosome is a chromosome that is produced when a dicentric chromosome fragments and acquires new telomeres so that two chromosomes, each having one of the centromeres, are produced. Each of the fragments is a replicable chromosome. If one of the chromosomes undergoes amplification of primarily euchromatic DNA to produce a fully functional chromosome that is predominantly (at least more than 50%) euchromatin, it is a minichromosome. The remaining chromosome is a formerly dicentric chromosome. If one of the chromosomes undergoes amplification, whereby heterochromatin (such as, for example, satellite DNA) is amplified and a euchromatic portion (such as, for example, an arm) remains, it is referred to as a sausage chromosome. A chromosome that is substantially all heterochromatin, except for portions of heterologous DNA, is called a predominantly heterochromatic artificial chromosome. Predominantly heterochromatic artificial chromosomes can be produced from other partially heterochromatic artificial chromosomes by culturing the cell containing such chromosomes under conditions such as BrdU treatment that destabilize the chromosome and/or growth under selective conditions so that a predominantly heterochromatic artificial chromosome is produced. For purposes herein, it is understood that the artificial chromosomes may not necessarily be produced in multiple steps, but may appear after the initial introduction of the heterologous DNA. Typically, artificial chromosomes appear after about 5 to about 60, or about 5 to about 55, or about 10 to about 55 or about 25 to about 55 or about 35 to about 55 cell doublings after initiation of artificial chromosome generation, or they may appear after several cycles of growth under selective conditions and BrdU treatment.

[0073] As used herein, an artificial chromosome that is predominantly heterochromatic (i.e., containing more heterochromatin than euchromatin, typically more than about 50%, more than about 70%, or more than about 90% heterochromatin) may be produced by introducing nucleic acid molecules into cells, such as, for example, animal or plant cells, and selecting cells that contain a predominantly heterochromatic artificial chromosome. Any nucleic acid may be introduced into cells in such methods of producing the artificial chromosomes. For example, the nucleic acid may contain a selectable marker and/or optionally a sequence that targets nucleic acid to the pericentric, heterochromatic region of a chromosome, such as in the short arm of acrocentric chromosomes and nucleolar organizing regions. Targeting sequences include, but are not limited to, lambda phage DNA and rDNA for production of predominantly heterochromatic artificial chromosomes in eukaryotic cells.

[0074] After introducing the nucleic acid into cells, a cell containing a predominantly heterochromatic artificial chromosome is selected. Such cells may be identified using a variety of procedures. For example, repeating units of heterochromatic DNA of these chromosomes may be discerned by G-banding and/or fluorescence in situ hybridization (FISH) techniques. Prior to such analyses, the cells to be analyzed may be enriched with artificial chromosome-containing cells by sorting the cells on the basis of the presence of a selectable marker, such as a reporter protein, or by growing (culturing) the cells under selective conditions. It is also possible, after introduction of nucleic acids into cells, to select cells that have a multicentric, typically dicentric, chromosome, a formerly multicentric (typically dicentric) chromosome and/or various heterochromatic structures, such as a megachromosome and a sausage chromosome, that contain a centromere and are predominantly heterochromatic and to treat them such that desired artificial chromosomes are produced. Cells containing a new chromosome are selected. Conditions for generation of a desired structure include, but are not limited to, further growth under selective conditions, introduction of additional nucleic acid molecules and/or growth under selective conditions and treatment with destabilizing agents, and other such methods (see International PCT application No. WO 97/40183 and U.S. Pat. Nos. 6,025,155 and 6,077,697).

[0075] As used herein, a "selectable marker" is a nucleic acid segment, generally DNA, that allows one to select for or against a molecule or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds and compositions. Examples of selectable markers include but are not limited to: (1) nucleic acid segments that encode products that provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) nucleic acid segments that encode products that suppress the activity of a gene product; (4) nucleic acid segments that encode products that can be identified, such as phenotypic markers, including β-galactosidase, red, blue and/or green fluorescent proteins (FPs), and cell surface proteins; (5) nucleic acid segments that bind products that are otherwise detrimental to cell survival and/or function; (6) nucleic acid segments that otherwise inhibit the activity of any of the nucleic acid segments described in Nos. 1-5 above (e.g., antisense oligonucleotides or siRNA molecules for use in RNA interference); (7) nucleic acid segments that bind products that modify a substrate (e.g. restriction endonucleases); (8) nucleic acid segments that can be used to isolate a desired molecule (e.g. specific protein binding sites); (9) nucleic acid segments that encode a specific nucleotide sequence that can be otherwise non-functional, such as for PCR amplification of subpopulations of molecules; and/or (10) nucleic acid segments, which when absent, directly or indirectly confer sensitivity to particular compounds. Thus, for example, selectable markers include nucleic acids encoding fluorescent proteins, such as green fluorescent proteins, β-galactosidase and other readily detectable proteins, such as chromogenic proteins or proteins capable of being bound by an antibody and FACs sorted. Selectable markers such as these, which are not required for cell survival and/or proliferation in the presence of a selection agent, are also referred to herein as reporter molecules. Other selectable markers, e.g., the neomycin phosphotransferase gene, provide for isolation and identification of cells containing them by conferring properties on the cells that make them resistant to an agent, e.g., a drug such as an antibiotic, that inhibits proliferation of cells that do not contain the marker.

[0076] As another example, interference of gene expression by double stranded RNA has been shown in Caenorhabditis elegans, plants, Drosophila, protozoans and mammals. This method is known as RNA interference (RNAi) and utilizes short, double-stranded RNA molecules (siRNAs). The siRNAs are generally composed of a 19-22 bp double-stranded RNA stem, a loop region and a 1-4 bp overhang on the 3' end. The reduction of gene expression has been accomplished by direct introduction of the siRNAs into the cell (Harborth J et al., 2001, J Cell Sci 114(pt 24):4557-65) as well as the introduction of DNA encoding and expressing the siRNA molecule. The encoded siRNA molecules are under the regulation of an RNA polymerase III promoter (see, e.g., Yu et al., 2002, Proc Natl Acad Sci USA 99(9); 6047-52; Brummelkamp et al., 2002, Science 296(5567):550-3; Miyagishi et al., 2002, Nat Biotechnol 20(5):497-500; and the like). In certain embodiments, RNAi in mammalian cells may have advantages over other therapeutic methods. For example, producing siRNA molecules that block viral genetic activities in infected cells may reduce the effects of the virus. Platform ACes provided herein encoding siRNA molecule(s) are an additional utilization of the platform ACes technology. The platform ACes could be engineered to encode one or more siRNA molecules to create gene "knockdowns". In one embodiment, a platform ACes can engineered to encode both the siRNA molecule and a replacement gene. For example, a mouse model or cell culture system could be generated using a platform ACes that has a knockdown of the endogenous mouse gene, by siRNA, and the human gene homolog expressing in place of the mouse gene. The placement of siRNA encoding sequences under the regulation of a regulatable or inducible promoter would allow one to temporally and/or spatially control the knockdown effect of the corresponding gene.

[0077] As used herein, a reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Generally reporter genes are readily detectable. Examples of reporter genes include, but are not limited to nucleic acid encoding a fluorescent protein, CAT (chloramphenicol acetyl transferase) (Alton et al. (1979) Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987) Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984) Proc. Natl. Acad. Sci. U.S.A. 81:4154-4158; Baldwin et al. (1984) Biochemistry 23:3663-3667); and alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182:231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2:101).

[0078] As used herein, growth under selective conditions means growth of a cell under conditions that require expression of a selectable marker for survival.

[0079] As used herein, an agent that destabilizes a chromosome is any agent known by those skilled in the art to enhance amplification events, and/or mutations. Such agents, which include BrdU, are well known to those skilled in the art.

[0080] In order to generate an artificial chromosome containing a particular heterologous nucleic acid of interest, it is possible to include the nucleic acid in the nucleic acid that is being introduced into cells to initiate production of the artificial chromosome. Thus, for example, a nucleic acid can be introduced into a cell along with nucleic acid encoding a selectable marker and/or a nucleic acid that targets to a heterochromatic region of a chromosome. For introducing a heterologous nucleic acid into the cell, it can be included in a fragment that includes a selectable marker or as part of a separate nucleic acid fragment and introduced into the cell with a selectable marker during the process of generating the artificial chromosomes. Alternatively, heterologous nucleic acid can be introduced into an artificial chromosome at a later time after the initial generation of the artificial chromosome.

[0081] As used herein, the minichromosome refers to a chromosome derived from a multicentric, typically dicentric, chromosome that contains more euchromatic than heterochromatic DNA. For purposes herein, the minichromosome contains a de novo centromere (e.g., a neocentromere). In some embodiments, for example, the minichromosome contains a centromere that replicates in animals, e.g., a mammalian centromere or in plants, e.g., a plant centromere.

[0082] As used herein, in vitro assembled artificial chromosomes or synthetic chromosomes can be either more euchromatic than heterochromatic or more heterochromatic than euchromatic and are produced by joining essential components of a chromosome in vitro. These components include at least a centromere, a megareplicator, a telomere and optionally secondary origins of replication.

[0083] As used herein, in vitro assembled plant or animal artificial chromosomes are produced by joining essential components (at least the centromere, telomere(s), megareplicator and optional secondary origins of replication) that function in plants or animals. In particular embodiments, the megareplicator contains sequences of rDNA, particularly plant or animal rDNA.

[0084] As used herein, a plant is a eukaryotic organism that contains, in addition to a nucleus and mitochondria, chloroplasts capable of carrying out photosynthesis. A plant can be unicellular or multicellular and can contain multiple tissues and/or organs. Plants can reproduce sexually or asexually and can be perennial or annual in growth. Plants can also be terrestrial or aquatic. The term "plant" includes a whole plant, plant cell, plant protoplast, plant calli, plant seed, plant organ, plant tissue, and other parts of a whole plant.

[0085] As used herein, stable maintenance of chromosomes occurs when at least about 85%, preferably 90%, more preferably 95%, of the cells retain the chromosome. Stability is measured in the presence of a selective agent. Preferably these chromosomes are also maintained in the absence of a selective agent. Stable chromosomes also retain their structure during cell culturing, suffering no unintended intrachromosomal or interchromosomal rearrangements.

[0086] As used herein, de novo with reference to a centromere, refers to generation of an excess centromere in a chromosome as a result of incorporation of a heterologous nucleic acid fragment using the methods herein.

[0087] As used herein, BrdU refers to 5-bromodeoxyuridine, which during replication is inserted in place of thymidine. BrdU is used as a mutagen; it also inhibits condensation of metaphase chromosomes during cell division.

[0088] As used herein, ribosomal RNA (rRNA) is the specialized RNA that forms part of the structure of a ribosome and participates in the synthesis of proteins. Ribosomal RNA is produced by transcription of genes which, in eukaryotic cells, are present in multiple copies. In human cells, the approximately 250 copies of rRNA genes (i.e., genes which encode rRNA) per haploid genome are spread out in clusters on at least five different chromosomes (chromosomes 13, 14, 15, 21 and 22). In mouse cells, the presence of ribosomal DNA (rDNA, which is DNA containing sequences that encode rRNA) has been verified on at least 11 pairs out of 20 mouse chromosomes (chromosomes 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, and 19) (see e.g., Rowe et al. (1996) Mamm. Genome 7:886-889 and Johnson et al. (1993) Mamm. Genome 4:49-52). In Arabidopsis thaliana the presence of rDNA has been verified on chromosomes 2 and 4 (18S, 5.8S, and 25S rDNA) and on chromosomes 3, 4, and 5 (5S rDNA)(see The Arabidopsis Genome Initiative (2000) Nature 408:796-815). In eukaryotic cells, the multiple copies of the highly conserved rRNA genes are located in a tandemly arranged series of rDNA units, which are generally about 40-45 kb in length and contain a transcribed region and a nontranscribed region known as spacer (i.e., intergenic spacer) DNA which can vary in length and sequence. In the human and mouse, these tandem arrays of rDNA units are located adjacent to the pericentric satellite DNA sequences (heterochromatin). The regions of these chromosomes in which the rDNA is located are referred to as nucleolar organizing regions (NOR) which loop into the nucleolus, the site of ribosome production within the cell nucleus.

[0089] As used herein, a megachromosome refers to a chromosome that, except for introduced heterologous DNA, is substantially composed of heterochromatin. Megachromosomes are made up of an array of repeated amplicons that contain two inverted megareplicons bordered by introduced heterologous DNA (see, e.g., FIG. 3 of U.S. Pat. No. 6,077,697 for a schematic drawing of a megachromosome). For purposes herein, a megachromosome is about 50 to 400 Mb, generally about 250-400 Mb. Shorter variants are also referred to as truncated megachromosomes (about 90 to 120 or 150 Mb), dwarf megachromosomes (˜150-200 Mb), and a micro-megachromosome (˜50-90 Mb, typically 50-60 Mb). For purposes herein, the term megachromosome refers to the overall repeated structure based on an array of repeated chromosomal segments (amplicons) that contain two inverted megareplicons bordered by any inserted heterologous DNA. The size will be specified.

[0090] As used herein, gene therapy involves the transfer or insertion of nucleic acid molecules into certain cells, which are also referred to as target cells, to produce specific products that are involved in preventing, curing, correcting, controlling or modulating diseases, disorders and deleterious conditions. The nucleic acid is introduced into the selected target cells in a manner such that the nucleic acid is expressed and a product encoded thereby is produced. Alternatively, the nucleic acid may in some manner mediate expression of DNA that encodes a therapeutic product. This product may be a therapeutic compound, which is produced in therapeutically effective amounts or at a therapeutically useful time. It may also encode a product, such as a peptide or RNA, that in some manner mediates, directly or indirectly, expression of a therapeutic product. Expression of the nucleic acid by the target cells within an organism afflicted with a disease or disorder thereby provides for modulation of the disease or disorder. The nucleic acid encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.

[0091] For use in gene therapy, cells can be transfected in vitro, followed by introduction of the transfected cells into an organism. This is often referred to as ex vivo gene therapy. Alternatively, the cells can be transfected directly in vivo within an organism.

[0092] As used herein, therapeutic agents include, but are not limited to, growth factors, antibodies, cytokines, such as tumor necrosis factors and interleukins, and cytotoxic agents and other agents disclosed herein and known to those of skill in the art. Such agents include, but are not limited to, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-I (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), pro-coagulants such as tissue factor and tissue factor variants, pro-apoptotic agents such FAS-ligand, fibroblast growth factors (FGF), nerve growth factor and other growth factors.

[0093] As used herein, a therapeutically effective product is a product that is encoded by heterologous DNA that, upon introduction of the DNA into a host, a product is expressed that effectively ameliorates or eliminates the symptoms, manifestations of an inherited or acquired disease or that cures the disease.

[0094] As used herein, transgenic plants and animals refer to plants and animals in which heterologous or foreign nucleic acid is expressed or in which the expression of a gene naturally present in the plant or animal has been altered by virtue of introduction of heterologous or foreign nucleic acid.

[0095] As used herein, IRES (internal ribosome entry site; see, e.g., SEQ ID No. 27 and nucleotides 2736-3308 SEQ ID No. 28) refers to a region of a nucleic acid molecule, such as an mRNA molecule, that allows internal ribosome entry sufficient to initiate translation, which initiation can be detected in an assay for cap-independent translation (see, e.g., U.S. Pat. No. 6,171,821). The presence of an IRES within an mRNA molecule allows cap-independent translation of a linked protein-encoding sequence that otherwise would not be translated.

[0096] Internal ribosome entry site (IRES) elements were first identified in picornaviruses, which elements are considered the paradigm for cap-independent translation. The 5' UTRs of all picornaviruses are long and mediate translational initiation by directly recruiting and binding ribosomes, thereby circumventing the initial cap-binding step. IRES elements are frequently found in viral mRNA, they are rare in non-viral mRNA. Among non-viral mRNA molecules that contain functional IRES elements in their respective 5' UTRs are those encoding immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); Drosophila Antennapedia (Oh et al. (1992) Genes Dev, 6:1643-1653); D. Ultrabithorax (Ye et al. (1997) Mol. Cell. Biol. 17:1714-21); fibroblast growth factor 2 (Vagner et al. (1995) Mol. Cell. Biol. 15:35-44); initiation factor eIF4G (Gan et al. (1998) J. Biol. Chem. 273:5006-5012); proto-oncogene c-myc (Nanbru et al. (1995) J. Biol. Chem. 272:32061-32066; Stoneley (1998) Oncogene 16:423-428); IRES_H; from the 5'UTR of NRF1 gene (Oumard et al. (2000) Mol. and Cell Biol., 20(8):2755-2759); and vascular endothelial growth factor (VEGF) (Stein et al. (1998) Mol. Cell. Biol. 18:3112-9).

[0097] As used herein, a promoter, with respect to a region of DNA, refers to a sequence of DNA that contains a sequence of bases that signals RNA polymerase to associate with the DNA and initiate transcription of RNA (such as pol II for mRNA) from a template strand of the DNA. A promoter thus generally regulates transcription of DNA into mRNA. A particular promoter provided herein is the Ferritin heavy chain promoter (excluding the Iron Response Element, located in the 5'UTR), which was joined to the 37 bp Fer-1 enhancer element. This promoter is set forth as SEQ ID NO:128. The endogenous Fer-1 enhancer element is located upstream of the Fer-1 promoter (e.g., a Fer-1 oligo was cloned proximal to the core promoter).

[0098] As used herein, isolated, substantially pure nucleic acid, such as, for example, DNA, refers to nucleic acid fragments purified according to standard techniques employed by those skilled in the art, such as that found in Sambrook et al. ((2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 3rd edition).

[0099] As used herein, expression refers to the transcription and/or translation of nucleic acid. For example, expression can be the transcription of a gene that may be transcribed into an RNA molecule, such as a messenger RNA (mRNA) molecule. Expression may further include translation of an RNA molecule and translated into peptides, polypeptides, or proteins. If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA. With respect to an antisense construct, expression may refer to the transcription of the antisense DNA. As used herein, vector or plasmid refers to discrete elements that are used to introduce heterologous nucleic acids into cells for either expression of the heterologous nucleic acid or for replication of the heterologous nucleic acid. Selection and use of such vectors and plasmids are well within the level of skill of the art.

[0100] As used herein, transformation/transfection refers to the process by which nucleic acid is introduced into cells. The terms transfection and transformation refer to the taking up of exogenous nucleic acid, e.g., an expression vector, by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, by Agrobacterium-mediated transformation, protoplast transformation (including polyethylene glycol (PEG)-mediated transformation, electroporation, protoplast fusion, and microcell fusion), lipid-mediated delivery, liposomes, electroporation, sonoporation, microinjection, particle bombardment and silicon carbide whisker-mediated transformation and combinations thereof (see, e.g., Paszkowski et al. (1984) EMBO J. 3:2717-2722; Potrykus et al. (1985) Mol. Gen. Genet. 199:169-177; Reich et al. (1986) Biotechnology 4:1001-1004; Klein et al. (1987) Nature 327:70-73; U.S. Pat. No. 6,143,949; Paszkowski et al. (1989) in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J and Vasil, L.K. Academic Publishers, San Diego, Calif., p. 52-68; and Frame et al. (1994) Plant J. 6:941-948), direct uptake using calcium phosphate (CaPO4; see, e.g., Wigler et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:1373-1376), polyethylene glycol (PEG)-mediated DNA uptake, lipofection (see, e.g., Strauss (1996) Meth. Mol. Biol. 54:307-327), microcell fusion (see, EXAMPLES, see, also Lambert (1991) Proc. Natl. Acad. Sci. U.S.A. 88:5907-5911; U.S. Pat. No. 5,396,767, Sawford et al. (1987) Somatic Cell Mol. Genet. 13:279-284; Dhar et al. (1984) Somatic Cell Mol. Genet. 10:547-559; and McNeill-Killary et al. (1995) Meth. Enzymol. 254:133-152), lipid-mediated carrier systems (see, e.g., Teifel et al. (1995) Biotechniques 19:79-80; Albrecht et al. (1996) Ann. Hematol. 72:73-79; Holmen et al. (1995) In Vitro Cell Dev. Biol. Anim. 31:347-351; Remy et al. (1994) Bioconjug. Chem. 5:647-654; Le Bolch et al. (1995) Tetrahedron Lett. 36:6681-6684; Loeffler et al. (1993) Meth. Enzymol. 217:599-618) or other suitable method. Methods for delivery of ACes are described in copending U.S. application Ser. No. 09/815,979. Successful transfection is generally recognized by detection of the presence of the heterologous nucleic acid within the transfected cell, such as, for example, any visualization of the heterologous nucleic acid or any indication of the operation of a vector within the host cell.

[0101] As used herein, "delivery," which is used interchangeably with "transfection," refers to the process by which exogenous nucleic acid molecules are transferred into a cell such that they are located inside the cell. Delivery of nucleic acids is a distinct process from expression of nucleic acids.

[0102] As used herein, injected refers to the microinjection, such as by use of a small syringe, needle, or pipette, for injection of nucleic acid into a cell.

[0103] As used herein, substantially homologous DNA refers to DNA that includes a sequence of nucleotides that is sufficiently similar to another such sequence to form stable hybrids, with each other or a reference sequence, under specified conditions.

[0104] It is well known to those of skill in this art that nucleic acid fragments with different sequences may, under the same conditions, hybridize detectably to the same "target" nucleic acid. Two nucleic acid fragments hybridize detectably, under stringent conditions over a sufficiently long hybridization period, because one fragment contains a segment of at least about 10, 14 or 16 or more nucleotides in a sequence that is complementary (or nearly complementary) to a substantially contiguous sequence of at least one segment in the other nucleic acid fragment. If the time during which hybridization is allowed to occur is held constant, at a value during which, under preselected stringency conditions, two nucleic acid fragments with complementary base-pairing segments hybridize detectably to each other, departures from exact complementarity can be introduced into the base-pairing segments, and base-pairing will nonetheless occur to an extent sufficient to make hybridization detectable. As the departure from complementarity between the base-pairing segments of two nucleic acids becomes larger, and as conditions of the hybridization become more stringent, the probability decreases that the two segments will hybridize detectably to each other.

[0105] Two single-stranded nucleic acid segments have "substantially the same sequence", if (a) both form a base-paired duplex with the same segment, and (b) the melting temperatures of the two duplexes in a solution of 0.5×SSPE differ by less than 10° C. If the segments being compared have the same number of bases, then to have "substantially the same sequence", they will typically differ in their sequences at fewer than 1 base in 10. Methods for determining melting temperatures of nucleic acid duplexes are well known (see, e.g., Meinkoth et al. (1984) Anal. Biochem. 138:267-284 and references cited therein).

[0106] As used herein, a nucleic acid probe is a DNA or RNA fragment that includes a sufficient number of nucleotides to specifically hybridize to DNA or RNA that includes complementary or substantially complementary sequences of nucleotides. A probe may contain any number of nucleotides, from as few as about 10 and as many as hundreds of thousands of nucleotides. The conditions and protocols for such hybridization reactions are well known to those of skill in the art as are the effects of probe size, temperature, degree of mismatch, salt concentration and other parameters on the hybridization reaction. For example, the lower the temperature and higher the salt concentration at which the hybridization reaction is carried out, the greater the degree of mismatch that may be present in the hybrid molecules.

[0107] To be used as a hybridization probe, the nucleic acid is generally rendered detectable by labeling it with a detectable moiety or label, such as ³²P, ³H and ¹⁴C, or by other means, including chemical labeling, such as by nick-translation in the presence of deoxyuridylate biotinylated at the 5'-position of the uracil moiety. The resulting probe includes the biotinylated uridylate in place of thymidylate residues and can be detected (via the biotin moieties) by any of a number of commercially available detection systems based on binding of streptavidin to the biotin. Such commercially available detection systems can be obtained, for example, from Enzo Biochemicals, Inc. (New York, N.Y.). Any other label known to those of skill in the art, including non-radioactive labels, may be used as long as it renders the probes sufficiently detectable, which is a function of the sensitivity of the assay, the time available (for culturing cells, extracting DNA, and hybridization assays), the quantity of DNA or RNA available as a source of the probe, the particular label and the means used to detect the label.

[0108] Once sequences with a sufficiently high degree of homology to the probe are identified, they can readily be isolated by standard techniques (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press).

[0109] As used herein, conditions under which DNA molecules form stable hybrids are considered substantially homologous, and a DNA or nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a polypeptide. By the term "substantially homologous" is meant having at least 75%, preferably 80%, preferably at least 90%, most preferably at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function.

[0110] The terms "homology" and "identity" are often used interchangeably. In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP computer program. The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482 (1981). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0111] By sequence identity, the number of conserved amino acids are determined by standard alignment algorithms programs, and are used with default gap penalties established by each supplier. Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Preferably the two molecules will hybridize under conditions of high stringency. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.

[0112] Whether any two nucleic acid molecules have nucleotide sequences that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" can be determined using known computer algorithms such as the "FAST A" program, using for example, the default parameters as in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988). Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine relative sequence identity.

[0113] In general, sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988). Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)).

[0114] Therefore, as used herein, the term "identity" represents a comparison between a test and a reference polypeptide or polynucleotide. For example, a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide.

[0115] As used herein, the term at least "90% identical to" refers to percent identities from 90 to 99.99 relative to the reference polypeptides. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons may be made between a test and reference polynucleotides. Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, or deletions.

[0116] As used herein: stringency of hybridization in determining percentage mismatch encompass the following conditions or equivalent conditions thereto: [0117] 1) high stringency: 0.1×SSPE or SSC, 0.1% SDS, 65° C. [0118] 2) medium stringency: 0.2×SSPE or SSC, 0.1% SDS, 50° C. [0119] 3) low stringency: 1.0×SSPE or SSC, 0.1% SDS, 50° C. or any combination of salt and temperature and other reagents that result in selection of the same degree of mismatch or matching. Equivalent conditions refer to conditions that select for substantially the same percentage of mismatch in the resulting hybrids. Additions of ingredients, such as formamide, Ficoll, and Denhardt's solution affect parameters such as the temperature under which the hybridization should be conducted and the rate of the reaction. Thus, hybridization in 5×SSC, in 20% formamide at 42° C. is substantially the same as the conditions recited above hybridization under conditions of low stringency. The recipes for SSPE, SSC and Denhardt's and the preparation of deionized formamide are described, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapter 8; see, Sambrook et al., vol. 3, p. B.13, see, also, numerous catalogs that describe commonly used laboratory solutions. It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. As used herein, all assays and procedures, such as hybridization reactions and antibody-antigen reactions, unless otherwise specified, are conducted under conditions recognized by those of skill in the art as standard conditions.

[0120] As used herein, conservative amino acid substitutions, such as those set forth in Table 1, are those that do not eliminate biological activity. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin/Cummings Pub. co., p. 224). Conservative amino acid substitutions are made, for example, in accordance with those set forth in TABLE 1 as follows:

TABLE-US-00002 TABLE 1 Original residue Conservative substitution Ala (A) Gly; Ser, Abu Arg (R) Lys, orn Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val; Met; Nle; Nva Leu (L) Ile; Val; Met; Nle; Nva Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile; NLe Val Ornithine Lys; Arg Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu; Met; Nle; Nva

Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions.

[0121] As used herein, the amino acids, which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations. The nucleotides, which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art.

[0122] As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.

[0123] As used herein, a probe or primer based on a nucleotide sequence includes at least 10, 14, 16, 30 or 100 contiguous nucleotides from the reference nucleic acid molecule.

[0124] As used herein, recombinant production by using recombinant DNA methods refers to the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.

[0125] As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activities may be observed in in vitro systems designed to test or use such activities. Thus, for purposes herein the biological activity of a luciferase is its oxygenase activity whereby, upon oxidation of a substrate, light is produced.

[0126] The terms substantially identical or similar varies with the context as understood by those skilled in the relevant art and generally means at least 40, 60, 80, 90, 95 or 98%.

[0127] As used herein, substantially identical to a product means sufficiently similar so that the property is sufficiently unchanged so that the substantially identical product can be used in place of the product.

[0128] As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers or isomers. In such instances, further purification might increase the specific activity of the compound.

[0129] As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. The vectors typically remain episomal, but may be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art. An expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0130] As used herein, protein-binding-sequence refers to a protein or peptide sequence that is capable of specific binding to other protein or peptide sequences generally, to a set of protein or peptide sequences or to a particular protein or peptide sequence.

[0131] As used herein, a composition refers to any mixture of two or more ingredients. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0132] As used herein, a combination refers to any association between two or more items.

[0133] As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.

[0134] As used herein, a cellular extract refers to a preparation or fraction that is made from a lysed or disrupted cell.

[0135] As used herein, the term "subject" refers to animals, plants, insects, and birds and other phyla, genera and species into which nucleic acid molecules may be introduced. Included are higher organisms, such as mammals, fish, insects and birds, including humans, primates, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, hamsters, cats, dogs, horses, chicken and others.

[0136] As used herein, flow cytometry refers to processes that use a laser based instrument capable of analyzing and sorting out cells and or chromosomes based on size and fluorescence.

[0137] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11:942-944).

B. Recombination Systems

[0138] Site-specific recombination systems typically contain three elements: a pair of DNA sequences (the site-specific recombination sequences) and a specific enzyme (the site-specific recombinase). The site-specific recombinase catalyzes a recombination reaction between two site-specific recombination sequences.

[0139] A number of different site-specific recombinase systems are available and/or known to those of skill in the art, including, but not limited to: the Cre/lox recombination system using CRE recombinase (see, e.g., SEQ ID Nos. 58 and 59) from the Escherichia coli phage P1 (see, e.g., Sauer (1993) Methods in Enzymology 225:890-900; Sauer et al. (1990) The New Biologist 2:441-449), Sauer (1994) Current Opinion in Biotechnology 5:521-527; Odell et al. (1990) Mol Gen Genet. 223:369-378; Lasko et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:6232-6236; U.S. Pat. No. 5,658,772), the FLP/FRT system of yeast using the FLP recombinase (see, SEQ ID Nos. 60 and 61) from the 2μ episome of Saccharomyces cerevisiae (Cox (1983) Proc. Natl. Acad. Sci. U.S.A. 80:4223; Falco et al. (1982) Cell 29:573-584; Golic et al. (1989) Cell 59:499-509; U.S. Pat. No. 5,744,336), the resolvases, including Gin recombinase of phage Mu (Maeser et al. (1991) Mol Gen Genet. 230:170-176; Klippel, A. et al (1993) EMBO J. 12:1047-1057; see, e.g., SEQ ID Nos. 64-67), Cin, Hin, αδ Tn3; the Pin recombinase of E. coli (see, e.g., SEQ ID Nos. 68 and 69; Enomoto et al. (1983) J Bacteria 6:663-668), the R/RS system of the pSR1 plasmid of Zygosaccharomyces rouxii (Araki et al. (1992) J. Mol. Biol. 225:25-37; Matsuzaki et al. (1990)J. Bacteriol. 172: 610-618) and site-specific recombinases from Kluyveromyces drosophilarium (Chen et al. (1986) Nucleic Acids Res. 314:4471-4481) and Kluyveromyces waltii (Chen et al. (1992) J. Gen. Microbiol. 138:337-345). Other systems are known to those of skill in the art (Stark et al. Trends Genet. 8:432-439; Utatsu et al. (1987) J. Bacteriol. 169:5537-5545; see, also, U.S. Pat. No. 6,171,861).

[0140] Members of the highly related family of site-specific recombinases, the resolvase family, such as γδ, Tn3 resolvase, Hin, Gin, and Cin are also available. Members of this family of recombinases are typically constrained to intramolecular reactions (e.g., inversions and excisions) and can require host-encoded factors. Mutants have been isolated that relieve some of the requirements for host factors (Maeser et al. (1991) Mol. Gen. Genet. 230:170-176), as well as some of the constraints of intramolecular recombination (see, U.S. Pat. No. 6,171,861).

[0141] The bacteriophage P1 Cre/lox and the yeast FLP/FRT systems are particularly useful systems for site-specific integration, inversion or excision of heterologous nucleic acid into, and out of, chromosomes, particularly ACes as provided herein. In these systems a recombinase (Cre or FLP) interacts specifically with its respective site-specific recombination sequence (lox or FRT, respectively) to invert or excise the intervening sequences. The sequence for each of these two systems is relatively short (34 bp for lox and 47 bp for FRT).

[0142] The FLP/FRT recombinase system has been demonstrated to function efficiently in plant cells (U.S. Pat. No. 5,744,386), and, thus, can be used for producing plant artificial chromosome platforms. In general, short incomplete FRT sites leads to higher accumulation of excision products than the complete full-length FRT sites. The system catalyzes intra- and intermolecular reactions, and, thus, can be used for DNA excision and integration reactions. The recombination reaction is reversible and this reversibility can compromise the efficiency of the reaction in each direction. Altering the structure of the site-specific recombination sequences is one approach to remedying this situation. The site-specific recombination sequence can be mutated in a manner that the product of the recombination reaction is no longer recognized as a substrate for the reverse reaction, thereby stabilizing the integration or excision event.

[0143] In the Cre-lox system, discovered in bacteriophage P1, recombination between loxP sites occurs in the presence of the Cre recombinase (see, e.g., U.S. Pat. No. 5,658,772). This system can be used to insert, invert or excise nucleic acid located between two lox sites. Cre can be expressed from a vector. Since the lox site is an asymmetrical nucleotide sequence, lox sites on the same DNA molecule can have the same or opposite orientation with respect to each other. Recombination between lox sites in the same orientation results in a deletion of the DNA segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule. The deleted DNA segment forms a circular molecule of DNA. The original DNA molecule and the resulting circular molecule each contain a single lox site. Recombination between lox sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two lox sites. In addition, reciprocal exchange of DNA segments proximate to lox sites located on two different DNA molecules can occur. All of these recombination events are catalyzed by the product of the Cre coding region.

[0144] Any site-specific recombinase system known to those of skill in the art is contemplated for use herein. It is contemplated that one or a plurality of sites that direct the recombination by the recombinase are introduced into an artificial chromosome to produce platform ACes. The resulting platform ACes are introduced into cells with nucleic acid encoding the cognate recombinase, typically on a vector, and nucleic acid encoding heterologous nucleic acid of interest linked to the appropriate recombination site for insertion into the platform ACes. The recombinase-encoding-nucleic acid may be introduced into the cells on the same vector, or a different vector, encoding the heterologous nucleic acid.

[0145] An E. coli phage lambda integrase system for ACes platform engineering and for artificial chromosome engineering is provided (Lorbach et al. (2000) J. Mol. Biol. 296:1175-1181). The phage lambda integrase (Landy, A. (1989) Annu. Rev. Biochem. 58:913-94) is adapted herein and the cognate att sites are provided. Chromosomes, including ACes, engineered to contain one or a plurality of att sites are provided, as are vectors encoding a mutant integrase that functions in the absence other factors. Methods using the modified chromosomes and vectors for introduction of heterologous nucleic acid are also provided.

[0146] For purposes herein, one or more of the sites (e.g., a single site or a pair of sites) required for recombination are introduced into an artificial chromosome, such as an ACes chromosome. The enzyme for catalyzing site-directed recombination is introduced with the DNA of interest, or separately, or is engineered onto the artificial chromosome under the control of a regulatable promoter.

[0147] As described herein, artificial chromosome platforms containing one or multiple recombination sites are provided. The methods and resulting products are exemplified with the lambda phage Att/Int system, but similar methods may be used for production of ACes platforms with other recombination systems.

[0148] The Att/Int system and vectors provided herein are not only intended for engineering ACes platforms, but may be used to engineer an Att/Int system into any chromosome. Introduction of att sites into a chromosome will permit engineering of natural chromosomes, such as by permitting targeted integration genes or regulatory regions, and by controlled excision of selected regions. For example, genes encoding a particular trait may be added to a chromosome, such as plant chromosome engineered to contain one or plurality of att sites. Such chromosomes may be used for screening DNA to identify genes. Large pieces of DNA can be introduced into cells and the cells screened phenotypically to select those having the desired trait.

C. Platforms

[0149] Provided herein are platform artificial chromosomes (platform ACes) containing single or multiple site-specific recombination sites. Chromosome-based platform technology permits efficient and tractable engineering and subsequent expression of multiple gene targets. Methods are provided that use DNA vectors and fragments to create platform artificial chromosomes, including animal, particularly mammalian, artificial chromosomes, and plant artificial chromosomes. The artificial chromosomes contain either single or multiple sequence-specific recombination sites suitable for the placement of target gene expression vectors onto the platform chromosome. The engineered chromosome-based platform ACes technology is applicable for methods, including cellular and transgenic protein production, transgenic plant and animal production and gene therapy. The platform ACes are also useful for producing a library of ACes comprising random portions of a given genome (e.g., a mammalian, plant or prokaryotic genome) for genomic screening; as well as a library of cells comprising different and/or mutually exclusive ACes therein.

[0150] Exemplary of artificial chromosome platforms are those based on ACes. ACes artificial chromosomes are non-viral, self-replicating nucleic acid molecules that function as a natural chromosome, having all the elements required for normal chromosomal replication and maintenance within the cell nucleus. ACes artificial chromosomes do not rely on integration into the genome of the cell to be effective, and they are not limited by DNA carrying capacity and as such the therapeutic gene(s) of interest, including regulatory sequences, can be engineered into the ACes. In addition, ACes are stable in vitro and in vivo and can provide predictable long-term gene expression. Once engineered and delivered to the appropriate cell or embryo, ACes work independently alongside host chromosomes, for ACes that are predominantly heterochromatin producing only the products (proteins) from the genes it carries. As provided herein ACes are modified by introduction of recombination site(s) to provide a platform for ready introduction of heterologous nucleic acid. The ACes platforms can be used for production of transgenic animals and plants; as vectors for genetic therapy; for use as protein production systems; for animal models to identify and target new therapeutics; in cell culture for the development and production of therapeutic proteins; and for a variety of other applications.

[0151] 1. Generation of Artificial Chromosomes

[0152] Artificial chromosomes may be generated by any method known to those of skill in the art. Of particular interest herein are the ACes artificial chromosomes, which contain a repeated unit. Methods for production of ACes are described in detail in U.S. Pat. Nos. 6,025,155 and 6,077,697, which, as with all patents, applications, publications and other disclosure, are incorporated herein in their entirety.

[0153] Generation of de Novo Aces.

[0154] ACes can be generated by cotransfecting exogenous DNA--such as a mammary tissue specific DNA cassette including the gene sequences for a therapeutic protein, with a rDNA fragment and a drug resistance marker gene into the desired eukaryotic cell, such as plant or animal cells, such as murine cells in vitro. DNA with a selectable or detectable marker is introduced, and can be allowed to integrate randomly into pericentric heterochromatin or can be targeted to pericentric heterochromatin, such as that in rDNA gene arrays that reside on acrocentric chromosomes, such as the short arms of acrocentric chromosomes. This integration event activates the "megareplicator" sequence and amplifies the pericentric heterochromatin and the exogenous DNA, and duplicates a centromere. Ensuing breakage of this "dicentric" chromosome can result in the production of daughter cells that contain the substantially-original chromosome and the new artificial chromosome. The resulting ACes contain all the essential elements needed for stability and replication in dividing cells--centromere, origins of replications, and telomeres. ACes have been produced that express marker genes (lacZ, green fluorescent protein, neomycin-resistance, puromycin-resistance, hygromycin-resistance) and genes of interest. Isolated ACes, for example, have been successfully transferred intact to rodent, human, and bovine cells by electroporation, sonoporation, microinjection, and transfection with lipids and dendrimers.

[0155] To render the creation of ACes with desired genes more tractable and efficient, "platform" ACes (platform-ACes) can be produced that contain defined DNA sequences for enzyme-mediated homologous DNA recombination, such as by Cre or FLP recombinases (Bouhassira et al. (1996) Blood 88(supplement 1):190a; Bouhassira et al. (1997) Blood, 90:3332-3344; Siebler et al. (1997) Biochemistry: 36:1740-1747; Siebler et al. (1998) Biochemistry 37: 6229-6234; and Bethke et al. (1997) Nucl. Acids Res. 25:2828-2834), and as exemplified herein the lambda phage integrase. A lox site contains two 13 bp inverted repeats to which Cre-recombinase binds and an intervening 8 bp core region. Only pairs of sites having identity in the central 6 bp of the core region are proficient for recombination; sites having non-identical core sequences (heterospecific lox sites) do not efficiently recombine with each other (Hoess et al. (1986) Nucleic Acids Res. 14:2287-2300).

[0156] Generating Acrocentric Chromosomes for Plant Artificial Chromosome Formation.

[0157] In human and mouse cells de novo formation of a satellite DNA based artificial chromosome (SATAC, also referred to as ACes) can occur in an acrocentric chromosome where the short arm contains only pericentric heterochromatin, the rDNA array, and telomere sequences. Plant species may not have any acrocentric chromosomes with the same physical structure described, but "megareplicator" DNA sequences reside in the plant rDNA arrays, also known as the nucleolar organizing regions (NOR). A structure like those seen in acrocentric mammalian chromosomes can be generated using site-specific recombination between appropriate arms of plant chromosomes.

[0158] Approach

[0159] Qin et al. ((1994) Proc. Natl. Acad. Sci. U.S.A. 91:1706-1710, 1994) describes crossing two Nicotiana tabacum transgenic plants. One plant contains a construct encoding a promoterless hygromycin-resistance gene preceded by a lox site (lox-hpt), the other plant carries a construct containing a cauliflower mosaic virus 35S promoter linked to a lox sequence and the cre DNA recombinase coding region (35S-lox-cre). The constructs were introduced separately by infecting leaf explants with agrobacterium tumefaciens which carries the kanamycin-resistance gene (Kan^R). The resultant Kan^R transgenic plants were crossed. Plants that carried the appropriate DNA recombination event were identified by hygromycin-resistance.

[0160] Modification of the Above for Generation of ACes

[0161] The Kan^R cultivars are initially screened, such as by FISH, to identify two sets of candidate transgenic plants. One set has one construct integrated in regions adjacent to the pericentric heterochromatin on the short arm of any chromosome. The second set of candidate plants has the other construct integrated in the NOR region of appropriate chromosomes. To obtain reciprocal translocation both sites must be in the same orientation. Therefore a series of crosses are required, Kan^R plants generated, and FISH analyses performed to identify the appropriate "acrocentric" plant chromosome for de novo plant ACes formation.

[0162] 2. Bacteriophage Lambda Integrase-Based Site-Specific Recombination System

[0163] An integral part of the platform technology includes a site-specific recombination system that allows the placement of selected gene targets or genomic fragments onto the platform chromosomes. Any such system may be used. In particular, a method is provided for insertion of additional DNA fragments into the platform chromosome residing in the cell via sequence-specific recombination using the recombinase activity of the bacteriophage lambda integrase. The lambda integrase system is exemplary of the recombination systems contemplated for ACes. Any known recombination system, including any described herein, particularly any that operates without the need for additional factors or that, by virtue of mutation, does not require additional factors, is contemplated.

[0164] As noted the lambda integrase system provided herein can be used with natural chromosomes and artificial chromosomes in addition to ACes. Single or a plurality of recombination sites, which may be the same or different, are introduced into artificial chromosomes to produce artificial chromosome platforms.

[0165] 3. Creation of Bacteriophage Lambda Integrase Site-Specific Recombination System

[0166] The lambda phage-encoded integrase (designated Int) is a prototypical member of the integrase family. Int effects integration and excision of the phage in and out of the E. coli genome via recombination between pairs of attachment sites designated attB/attP and attL/attR. Each att site contains two inverted 9 base pair core Int binding sites and a 7 base pair overlap region that is identical in wild-type att sites. Each site, except for attB contains additional Int binding sites. In flanking regions, there are recognition sequences for accessory DNA binding proteins, such as integration host factor (IHF), factor for inversion stimulation (FIS) and the phage encoded excision protein (XIS). Except for attB, Int is a heterobivalent DNA-binding protein and, with assistance from the accessory proteins and negative DNA supercoiling, binds simultaneously to core and arm sites within the same att site.

[0167] Int, like Cre and FLP, executes an ordered sequential pair of strand exchanges during integrative and excisive recombination. The natural pairs of target sequences for Int, attB and attP or attL and attR are located on the same or different DNA molecules resulting in intra or intermolecular recombination, respectively. For example, intramolecular recombination occurs between inversely oriented attB and attP, or between attL and attR sequences, respectively, leading to inversion of the intervening DNA segment.

[0168] Like the recombinase systems, such as Cre and FLP, Int directs site-specific recombination. Unlike the other systems, such Cre and FLP, Int generally requires additional protein factors for integrative and excisive recombination and negative supercoiling for integrative recombination. Hence, the Int system had not been used in eukaryotic targeting systems. Mutant Int proteins, designated Int-h (E174K) and a derivative thereof. Int-h/218(E174K/E218K) do not require accessory proteins to perform intramolecular integrative and excisive recombination in co-transfection assays in human cells (Lorbach et al. (2000) J Mol. Biol. 296:1175-1181); wild-type Int does not catalyze intramolecular recombination in human cells harboring target sites attB and attP. Hence it had been demonstrated that mutant Int can catalyze factor-independent recombination events in human cells.

[0169] There has been no demonstration by others that this system can be used for engineering of eukaryotic genomes or chromosomes. Provided herein are chromosomes, including artificial chromosomes, such as but not limited to ACes that contain att sites (e.g., platform ACes), and the use of such chromosomes for targeted integration of heterologous DNA into such chromosomes in eukaryotic cells, including animal, such as rodent and human, and plant cells. Mutant Int provided herein is shown to effect site-directed recombination between sites in artificial chromosomes and vectors containing cognate sites.

[0170] An additional component of the chromosome-based platform technology is the site-specific integration of target DNA sequences onto the platform. For this the native bacteriophage lambda integrase has been modified to carry out this sequence specific DNA recombination event in eukaryotic cells. The bacteriophage lambda integrase and its cognate DNA substrate att is a member of the site-specific recombinase family that also includes the bacteriophage P1 Cre/lox system as well as the Saccharomyces cerevisiae 2 micron based FLP/FRT system (see, e.g., Landy (1989) Ann. Rev. Biochem 58:913-949; Hoess et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79:3398-3402; Broach et al. (1982) Cell 29:227-234).

[0171] By combining DNA endonuclease and DNA ligase activity these recombinases recognize and catalyze DNA exchanges between sequences flanking the recognition site. During the integration of lambda genome into the E. coli (lambda recombination) genome, the phage integrase (INT) in association with accessory proteins catalyzes the DNA exchange between the attP site of the phage genome and the attB site of the bacterial genome resulting in the formation of attL and attR sites (FIG. 6). The engineered bacteriophage lambda integrase has been produced herein to carry out an intermolecular DNA recombination event between an incoming DNA molecule (primarily on a vector containing the bacterial attB site) and the chromosome-based platform carrying the lambda attP sequence independent of lambda bacteriophage or bacterial accessory proteins.

[0172] In contrast to the bi-directional Cre/lox and FLP/FRT system, the engineered lambda recombination system derived for chromosome-based platform technology is advantageously unidirectional because accessory proteins, which are absent, are required for excision of integrated nucleic acid upon further exposure to the lambda Int recombinase.

[0173] 4. Creation of Platform Chromosome Containing Single or Multiple Sequence-Specific Recombination Sites

[0174] a. Multiple Sites

[0175] For the creation of a platform chromosome containing multiple, sequence-specific recombination sites, artificial chromosomes are produced as depicted in FIG. 5 and Example 3. As discussed above, artificial chromosomes can be produced using any suitable methodology, including those described in U.S. Pat. Nos. 5,288,625; 5,712,134; 5,891,691; 6,025,155. Briefly, to prepare artificial chromosomes containing multiple recombination (e.g., integration) sites, nucleic acid (either in the form a one or more plasmids, such as the plasmid pSV40193attPsensePUR set forth in Example 3) is targeted into an amplifiable region of a chromosome, such as the pericentric region of a chromosome. Among such regions are the rDNA gene loci in acrocentric mammalian chromosomes. Hence, targeting nucleic acid for integration into the rDNA region of mammalian acrocentric chromosomes can include the mouse rDNA fragments (for targeting into rodent cell lines) or large human rDNA regions on BAC/PAC vectors (or subclones thereof in standard vectors) for targeting into human acrocentric chromosomes, such as for human gene therapy applications. The targeting nucleic acid generally includes a detectable or selectable marker, such as antibiotic resistance, such as puromycin and hygromycin, a recombination site (such as attP, attB, attL, attR or the like), and/or human selectable markers as required for gene therapy applications. Cells are grown under conditions that result in amplification and ultimately production of ACes artificial chromosomes having multiple recombination (e.g., integration) sites therein. ACes having the desired size are selected for further engineering.

[0176] b. Creation of Platform Chromosome Containing a Single Sequence-Specific Recombination Site

[0177] In this method a mammalian platform artificial chromosome is generated containing a single sequence-specific recombination site. In the Example below, this approach is demonstrated using a puromycin resistance marker for selection and a mouse rDNA fragment for targeting into the rDNA locus on mouse acrocentric chromosomes. Other selection markers and targeting DNA sequences as desired and known to those of skill in the art can be used. Additional resistance markers include genes conferring resistance to the antibiotics neomycin, blasticidin, hygromycin and zeocin. For applications, such as gene therapy in which potentially immunogenic responses are to be avoided, host, such as human, derived selectable markers or markers detectable with monoclonal antibodies (MAb) followed by fluorescent activated cell sorting (FACS) can be used. Examples in this class include, but are not limited to: human nerve growth factor receptor (detection with MAb); truncated human growth factor receptor (detection with MAb); mutant human dihydrofolate reductase (DHFR; detectable using a fluorescent methotrexate substrate); secreted alkaline phosphatase (SEAP; detectable with fluorescent substrate); thymidylate synthase (TS; confers resistance to fluorodeoxyuridine); human CAD gene (confers resistance to N-phosphonacetyl-L-aspartate (PALA)).

[0178] To construct a platform artificial chromosome with a single site, an ACes artificial chromosome (or other artificial chromosome of interest) can be produced containing a selectable marker. A single sequence specific recombination site is targeted onto ACes via homologous recombination. For this, DNA sequences containing the site-specific recombination sequence are flanked with DNA sequences homologous to a selected sequence in the chromosome. For example, when using a chromosome containing rDNA or satellite DNA, such DNA can be used as homologous sequences to target the site-specific recombination sequence onto the chromosome. A vector is designed to have these homologous sequences flanking the site-specific recombination site and, after the appropriate restriction enzyme digest to generate free ends of homology to the chromosome, the DNA is transfected into cells harboring the chromosome. After transfection and integration of the site-specific cassette, homologous recombination events onto the platform chromosome are subcloned and identified, for example by screening single cell subclones via expression of resistance or a fluorescent marker and PCR analysis. In one embodiment, a platform artificial chromosome, such as a platform ACes, that contains a single copy of the recombination site is selected. Examples 2B and 2D exemplify the process, and FIG. 3 provides a diagram depicting one method for the creation of a platform mammalian chromosome containing a single sequence-specific recombination site.

[0179] 5. Lambda Integrase Mediated Recombination of Target Gene Expression Vector onto Platform Chromosome

[0180] The third component of the chromosome-based platform technology involves the use of target gene expression vectors carrying, for example, genes for gene therapy, genes for transgenic animal or plant production, and those required for cellular protein production of interest. Using lambda integrase mediated site-specific recombination, or any other recombinase-mediated site-specific recombination, the target gene expression vectors are introduced onto the selected chromosome platform. The use of target gene expression vector permits use of the de novo generated chromosome-based platforms for a wide range of gene targets. Furthermore, chromosome platforms containing multiple attP sites provides the opportunity to incorporate multiple gene targets onto a single platform, thereby providing for expression of multiple gene targets, including the expression of cellular and genetic regulatory genes and the expression of all or parts of metabolic pathways. In addition to expressing small target genes, such as cDNA and hybrid cDNA/artificial intron constructs, the chromosome-based platform can be used for engineering and expressing large genomic fragments carrying target genes along with its endogenous genomic promoter sequences. This is of importance, for example, where the therapy requires precise cell specific expression and in instances where expression is best achieved from genomic clones rather than cDNA clones. FIG. 9 provides a diagram summarizing one embodiment of the chromosome-based technology.

[0181] A feature of the target gene expression vector that is of interest to include is a promoterless marker gene, which as exemplified (see, FIG. 9) contains an upstream attB site (marker 2 on FIG. 9). The nucleic acid encoding the marker is not expressed unless it is placed downstream from a promoter sequence. Using the recombinase technology provided herein, such as the lambda integrase technology (λINT.sub.E174R on FIG. 8) provided herein, site-specific recombination between the attB site on the vector and the promoter-attP site (in the "sense" orientation) on the chromosome-based platform results in the expression of marker 2 on the target gene expression vector, thereby providing a positive selection for the lambda INT mediated site-specific recombination event. Site-specific recombination events on the chromosome-based platform versus random integrations next to a promoter in the genome (false positive) can be quickly screened by designing primers to detect the correct event by PCR. Examples of suitable marker 2 genes, include, but are not limited to, genes that confer resistance to toxic compounds or antibiotics, fluorescence activated cell sorting (FACS) sortable cell surface markers and various fluorescent markers. Examples of these genes include, but are not limited to, human L26a^R (human homolog of Saccharomyces cerevisiae CYH⁸ gene), neomycin, puromycin, blasticidin, CD24 (see, e.g., U.S. Pat. Nos. 5,804,177 and 6,074,836), truncated CD4, truncated low affinity nerve growth factor receptor (LNGFR), truncated LDL receptor, truncated human growth hormone receptor, GFP, RFP, BFP.

[0182] The target gene expression vectors contain a gene (target gene) for expression from the chromosome platform. The target gene can be expressed using various constitutive or regulated promoter systems across various mammalian species. For the expression of multiple target genes within the same target gene expression vector, the expression of the multiple targets can be coordinately regulated via viral-based or human internal ribosome entry site (IRES) elements (see, e.g., Jackson et al. (1990) Trends Biochem Sci. 15: 477-83; Oumard et al. (2000) Mol. Cell. Biol. 20: 2755-2759). Furthermore, using IRES type elements linked to a downstream fluorescent marker, e.g., green, red or blue fluorescent proteins (GFP, RFP, BFP) allows for the identification of high expressing clones from the integrated target gene expression vector.

[0183] In certain embodiments described herein, the promoterless marker can be transcriptionally downstream of the heterologous nucleic acid, wherein the heterologous nucleic acid encodes a heterologous protein, and wherein the expression level of the selectable marker is transcriptionally linked to the expression level of the heterologous protein. In addition, the selectable marker and the heterologous nucleic acid can be transcriptionally linked by the presence of a IRES between them. As set forth herein the selectable marker is selected from the group consisting of an antibiotic resistance gene, and a detectable protein, wherein the detectable protein is chromogenic or fluorescent. Expression from the target gene expression vector integrated onto the chromosome-based platform can be further enhanced using genomic insulator/boundary elements. The incorporation of insulator sequences into the target gene expression vector helps define boundaries in chromatin structure and thus minimizes influence of chromatin position effects/gene silencing on the expression of the target gene (Bell et al. (1999) Current Opinion in Genetics and Development 9:191-198; Emery et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:9150-9155). Examples of insulator elements that can be included onto target gene expression vector in order to optimize expression include, but are not limited to: [0184] 1) chicken β-globin HS4 element (Prioleau et al. (1999) EMBO J. 18: 4035-4048); [0185] 2) matrix attachment regions (MAR; see, e.g., Ramakrishnan et al. (2000) Mol. Cell. Biol. 20:868-877); [0186] 3) scaffold attachment regions (SAR; see, e.g., Auten et al. (1999) Human Gene Therapy 10:1389-1399); and [0187] 4) universal chromatin opening elements (UCOE; WO/0005393 and WO/0224930)

[0188] The copy number of the target gene can be controlled by sequentially adding multiple target gene expression vectors containing the target gene onto multiple integration sites on the chromosome platform. Likewise, the copy number of the target gene can be controlled within an individual target gene expression vector by the addition of DNA sequences that promote gene amplification. For example, gene amplification can be induced utilizing the dihydrofolate reductase (DHFR) minigene with subsequent selection with methotrexate (see, e.g., Schimke (1984) Cell 37:705-713) or amplification promoting sequences from the rDNA locus (see, e.g., Wegner et al. (1989) Nucl. Acids Res. 17: 9909-9932).

[0189] 6. Platforms with Other Recombinase System Sites

[0190] A "double lox" targeting strategy mediated by Cre-recombinase (Bethke et al. (1997) Nucl. Acids Res. 25:2828-2834) can be used. This strategy employs a pair of heterospecific lox sites--loxA and loxB, which differ by one nucleotide in the 8 bp spacer region. Both sites are engineered into the artificial chromosome and also onto the targeting DNA vector. This allows for a direct site-specific insertion of a commercially relevant gene or genes by a Cre-catalyzed double crossover event. In essence a platform ACes is engineered with a hygromycin-resistance gene flanked by the double lox sites generating lox-ACes, which is maintained in the thymidine kinase deficient cell, LMtk(-). The gene of interest, for example, for testing purposes, the green fluorescence protein gene, GFP and a HSV thymidine kinase gene (tk) marker, are engineered between the appropriate lox sites of the targeting vector. The vector DNA is cotransfected with plasmid pBS185 (Life Technologies) encoding the Cre recombinase gene into mammalian cells maintaining the dual-lox artificial chromosome. Transient expression of the Cre recombinase catalyzes the site-specific insertion of the gene and the tk-gene onto the artificial chromosome. The transfected cells are grown in HAT medium that selects for only those cells that have integrated and expressed the thymidine kinase gene. The HAT^R colonies are screened by PCR analyses to identify artificial chromosomes with the desired insertion.

[0191] To generate the lox-ACes, Lambda-Hyg^R-lox DNA is transfected into the (-) cell line harboring the precursor ACes. Hygromycin-resistant colonies are analyzed by FISH and Southern blotting for the presence of a single copy insert on the ACes.

[0192] To demonstrate the gene replacement technology, cell lines containing candidate lox ACes are cotransfected with pTK-GFP-lox and pBS185 (encoding the Cre recombinase gene) DNA. After transfection, transient expression of plasmid pBS185 will provide sufficient burst of Cre recombinase activity to catalyze DNA recombination at the lox sites. Thus, a double crossover event between the ACes target and the exogenous targeting plasmid carrying the loxA and loxB permits the simple replacement of the hygromycin-resistance gene on the lox-ACes for the tk-GFP cassette from the targeting plasmid, with no integration of vector DNA. Transfected cells are grown in HAT-media to select for tk-expression. Correct targeting will result in the generation of HAT^R, hygromycin sensitive, and green fluorescent cells. The desired integration event is verified by Southern and PCR analyses. Specific PCR primer sets are used to amplify DNA sequences flanking the individual loxA and loxB sites on the lox-ACes before and after homologous recombination.

D. Exemplary Applications of the Platform Aces

[0193] Platform ACes are applicable and tractable for different/optimized cell lines. Those that include a fluorescent marker, for example, can be purified and isolated using fluorescent activated cell sorting (FACS), and subsequently delivered to a target cell. Those with selectable markers provide for efficient selection and provide a growth advantage. Platform ACes allow multiple payload delivery of donor target vectors via a positive-selection site-specific, recombination system, and they allow for the inclusion of additional genetic factors that improve protein production and protein quality.

[0194] The construction and use of the platform ACes as provided for each application may be similarly applied to other applications. Particular descriptions are for exemplification.

[0195] 1. Cellular Protein Production Platform Aces (CPP ACes)

[0196] As described herein, ACes can be produced from acrocentric chromosomes in rodent (mouse, hamster) cell lines via megareplicator induced amplification of heterochromatin/rDNA sequences. Such ACes are ideal for cellular protein production as well as other applications described herein and known to those of skill in the art. ACes platforms that contain a plurality of recombination sites are particularly suitable for engineering as cellular protein production systems.

[0197] In one embodiment, CPP ACes involve a two-component system: the platform chromosome containing multiple engineering sites and the donor target vector containing a platform-specific recombination site with designed expression cassettes (see FIG. 9).

[0198] The platform ACes can be produced from any artificial chromosome, particularly the amplification-based artificial chromosomes. For exemplification, they are produced from rodent artificial chromosomes produced from acrocentric chromosomes using the technology of U.S. Pat. Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183, in which nucleic acid is targeted to the pericentric heterochromatic, and, particularly into rDNA to initiate the replication event(s). The ACes can be produced directly in the chosen cellular protein production cell lines, such as, but not limited to, CHO cells, hybridomas, plant cells, plant tissues, plant protoplasts, stem cells and plant calli.

[0199] a. Platform Construction

[0200] In the exemplary embodiment, the initial de novo platform construction requires co-transfecting with excess targeting DNA, such as, rDNA or lambda DNA without an attP region, and an engineered selectable marker. The engineered selectable marker should contain promoter, generally a constitutive promoter, such as human, viral, i.e., adenovirus or SV40 promoter, including the human ferritin heavy chain promoter (SEQ ID NO:128), SV40 and EF1α promoters, to control expression of a marker gene that provides a selective growth advantage to the cell. An example of such a marker gene is the E. coli hisD gene (encoding histidinol dehydrogenase) which is homologous and analogous to the S. typhimurium hisD a dominant marker selection system for mammalian cells previously described (see, Hartman et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:8047-8051). Since histidine is an essential amino acid in mammals and a nutritional requirement in cell culture, the E. coli hisD gene can be used to select for histidine prototrophy in defined media. Furthermore more stringent selection can be placed on the cells by including histinol in the medium. Histidinol is itself permeable and toxic to cells. The hisD provides a means of detoxification.

[0201] Placed between the promoter and the marker gene is the bacteriophage lambda attP site to use the bacteriophage lambda integrase dependent site-specific recombination system (described herein). The insertion of an attP site downstream of a promoter element provide forward selection of site-specific recombination events onto the platform ACes.

[0202] b. Donor Target Vector Construction

[0203] A second component of the CPP platform ACes system involves the construction of donor target vectors containing a gene product(s) of interest for the CPP platform ACes. Individual donor target vectors can be designed for each gene product to be expressed thus enabling maximum usage of a de novo constructed platform ACes, so that one or a few CPP platform ACes will be required for many gene targets.

[0204] A key feature of the donor vector target is the promoterless marker gene containing an upstream attB site (marker 2 on FIG. 9). Normally the marker would not be expressed unless it is placed downstream of a promoter sequence. As discussed above, using the lambda integrase technology (λINT.sub.E174R on FIG. 8 and FIG. 9), site-specific recombination between the attB site on the vector and the promoter-attP site on the CPP platform ACes result in the expression of the donor target vector marker providing positive selection for the site-specific event. Site-specific recombination events on the CPP ACes versus random integrations next to a promoter in the genome (false positive) can be quickly screened by designing primers to detect the correct event by PCR. In addition, since the lambda integrase reaction is unidirectional, i.e. excision reaction is not possible, a number of unique targets can be loaded onto the CPP platform ACes limited only by the number of markers available.

[0205] Additional features of the donor target vector include gene target expression cassettes flanked by either chromatin insulator regions, matrix attachment regions (MAR) or scaffold attachment regions (SAR). The use of these regions will provide a more "open" chromatin environment for gene expression and help alleviate silencing. An example of such a cassette for expressing a monoclonal antibody is described. For this purpose, a strong constitutive promoter, e.g. chicken β-actin or RNA PolI, is used to drive the expression of the heavy and light chain open reading frames. The heavy and light chain sequences flank a nonattenuated human IRES (IRES_H; from the 5'UTR of NRF1 gene; see Oumard et al., 2000, Mol. and Cell Biol., 20(8):2755-2759) element thereby coordinating transcription of both heavy and light chain sequence. Distal to the light chain open reading frame resides an additional viral encoded IRES (IRES_V modified ECMV internal ribosomal entry site (IRES)) element attenuating the expression of the fluorescent marker gene hrGFP from Renilla (Stratagene). By linking the hrGFP with an attenuated IRES, the heavy and light chains along with the hrGFP are monocistronic. Thus, the identification of hrGFP fluorescing cells will provide a means to detect protein producing cells. In addition, high producing cell lines can be identified and isolated by FACS thereby decreasing the time frame in finding high expressers. Functional monoclonal antibody will be confirmed by ELISA.

[0206] c. Additional Components in Cellular Protein Production Platform ACes (CPP Aces)

[0207] In addition to the aforementioned CPP ACes system, other genetic factors can be included to enhance the yield and quality of the expressed protein. Again to provide maximum flexibility, these additional factors can be inserted onto the CPP platform ACes by λINTE174R dependent site-specific recombination. Other factors that could be used with a CPP Platform ACes include for example, adenovirus E1a transactivation system which upregulates both cellular and viral promoters (see, e.g., Svensson and Akusjarvi (1984) EMBO 3:789-794; and U.S. Pat. Nos. 5,866,359; 4,775,630 and 4,920,211).

[0208] d. Targets for CHO-ACes Engineering to Enhance Cell Growth, Such as CHO Cell Growth and Protein Production/Quality

[0209] If adding these additional factors onto the CPP ACes is not prudent or desired, the host cell, CHO cells, can be engineered to express these factors (see, below, targets for CHO-ACes engineering to enhance CHO cell growth and protein production/quality). Additional factors to consider including are addition of insulin or IGF-1 to sustain viabililty; human sialyltransferases or related factors to produce more human-like glycoproteins; expression of factors to decrease ammonium accumulation during cell growth; expression of factors to inhibit apoptosis; expression of factors to improve protein secretion and protein folding; and expression of factors to permit serum-free transfection and selection.

[0210] 1) Addition of Insulin or IGF-1 to Sustain Viabililty

[0211] Stimulatory factors and/or their receptors are expressed to set up an autocrine loop, to improve cell growth, such as CHO cell growth. Two exemplary candidates are insulin and IGF-1 (see, Biotechnol Prog 2000 September; 16(5):693-7). Insulin is the most commonly used growth factor for sustaining cell growth and viability in serum-free Chinese hamster ovary (CHO) cell cultures. Insulin and IGF-1 analog (LongR(3) serve as growth and viability factors for CHO cells.

[0212] CHO cells were modified to produce higher levels of essential nutrients and factors. A serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44 cells) was prepared. Chinese hamster ovary cells (DG44 cells), which are normally maintained in 10% serum medium, were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium (see, Kim et al. (199) In Vitro Cell Dev Biol Anim 35(4):178-82). A SF medium (SF-DG44) was formulated by supplementing the basal medium with these components; basal medium was prepared by supplementing Dulbecco's modified Eagle's medium and Ham's nutrient mixture F12 with hypoxanthine (10 mg/l) and thymidine (10 mg/l). Development of a SF medium for DG44 cells was facilitated using a Plackett-Burman design technique and weaning of cells.

[0213] 2) Human Sialyltransferases or Related Factors to Produce More Human-Like Glycoproteins

[0214] CHO cells have been modified by increasing their ability to process protein via addition of complex carbohydrates. This has been achieved by overexpression of relevant processing enzymes, or in some cases, reducing expression of relevant enzymes (see, Bragonzi et al. (2000) Biochim Biophys Acta 1474(3):273-282; see, also Weikert et al. (1999) Nature biotech. 17:1116-11121; Ferrari J et al. (1998) Biotechnol Bioeng 60(5):589-95). A CHO cell line expressing alpha-2,6-sialyltransferase was developed for the production of human-like sialylated recombinant glycoproteins. The sialylation defect of CHO cells can be corrected by transfecting the alpha-2,6-sialyltransferase (alpha-2,6-ST) cDNA into the cells. Glycoproteins produced by such CHO cells display alpha-2,6- and alpha-2,3-linked terminal sialic acid residues, similar to human glycoproteins.

[0215] As another example for improving the production of human-like sialylated recombinant glycoproteins, a CHO cell line has been developed that constitutively expresses sialidase antisense RNA (see, Ferrari J et al. (1998) Biotechnol Bioeng 60(5):589-95). Several antisense expression vectors were prepared using different regions of the sialidase gene. Co-transfection of the antisense constructs with a vector conferring puromycin resistance gave rise to over 40 puromycin resistant clones that were screened for sialidase activity. A 5' 474 bp coding segment of the sialidase cDNA, in the inverted orientation in an SV 40-based expression vector, gave maximal reduction of the sialidase activity to about 40% wild-type values.

[0216] Oligosaccharide biosynthesis pathways in mammalian cells have been engineered for generation of recombinant glycoproteins (see, e.g., Sburlati (1998) Biotechnol Prog 14(2):189-92), which describes a Chinese hamster ovary (CHO) cell line capable of producing bisected oligosaccharides on glycoproteins. This cell line was created by overexpression of a recombinant N-acetylglucosaminyltransferase III (GnT-III) (see, also, Prati et al. (1998) Biotechnol Bioeng 59(4):445-50, which describes antisense strategies for glycosylation engineering of CHO cells).

[0217] 3) Expression of Factors to Decrease Ammonium Accumulation During Cell Growth

[0218] Excess ammonium, which is a by-product of CHO cell metabolism can have detrimental effects on cell growth and protein quality (see, Yang et al. (2000) Biotechnol Bioeng 68(4):370-80). To solve this problem ammonium levels were modified by overexpressing carbamoyl phosphate synthetase I and ornithine transcarbamoylase or glutamine synthetase in CHO cells. Such modification resulted in reduced ammonium levels observed and an increase in the growth rate (see Kim et al. (2000) J Biotechnol 81 (2-3): 129-40; and Enosawa et al. (1997) Cell Transplant 6(5):537-40).

[0219] 4) Expression of Factors to Improve Protein Secretion and Protein Folding

[0220] Overexpression of relevant enzymes can be engineered into the ACes to improve protein secretion and folding.

[0221] 5) Expression of Factors to Permit Serum-Free Transfection and Selection

[0222] It is advantageous to have the ability to convert CHO cells in suspension growing in serum free medium to adherence with out having to resort to serum addition. Laminin or fibronectin addition is sufficient to make cells adherent (see, e.g., Zaworski et al. (1993) Biotechniques 15(5):863-6) so that expressing either of these genes in CHO cells under an inducible promoter should allow for reversible shift to adherence without requiring serum addition.

[0223] 2. Platform Aces and Gene Therapy

[0224] The platform ACes provided herein are contemplated for use in mammalian gene therapy, particularly human gene therapy. Human ACes can be derived from human acrocentric chromosomes from human host cells, in which the amplified sequences are heterochromatic and/or human rDNA. Different platform ACes applicable for different tissue cell types are provided. The ACes for gene therapy can contain a single copy of a therapeutic gene inserted into a defined location on platform ACes. Therapeutic genes include genomic clones, cDNA, hybrid genes and other combinations of sequences. Preferred selectable markers are those from the mammalian host, such as human derived factors so that they are non-immunogenic, non-toxic and allow for efficient selection, such as by FACS and/or drug resistance.

[0225] Platform ACes, useful for gene therapy and other applications, as noted herein, can be generated by megareplicator dependent amplification, such as by the methods in U.S. Pat. Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183. In one embodiment, human ACes are produced using human rDNA constructs that target rDNA arrays on human acrocentric chromosomes and induce the megareplicator in human cells, particularly in primary cell lines (with sufficient number of doublings to form the ACes) or stem cells (such as hematopoietic stem cells, mesenchymal stem cells, adult stem cells or embryonic stem cells) to avoid the introduction of potentially harmful rearranged DNA sequences present in many transformed cell lines. Megareplicator induced ACes formation can result in multiple copies of targeting DNA/selectable markers in each amplification block on both chromosomal arms of the platform ACes.

[0226] In view of the considerations regarding immunogenicity and toxicity, the production of human platform ACes for gene therapy applications employs a two component system analogous to the platform ACes designed for cellular protein production (CPP platform ACes). The system includes a platform chromosome of entirely human DNA origin containing multiple engineering sites and a gene target vector carrying the therapeutic gene of interest.

[0227] a. Platform Construction

[0228] The initial de novo construction of the platform chromosome employs the co-transfection of excess targeting DNA and a selectable marker. In one embodiment, the DNA is targeted to the rDNA arrays on the human acrocentric chromosomes (chromosomes 13, 14, 15, 21 and 22). For example, two large human rDNA containing PAC clones 18714 and 18720 and the human PAC clone 558F8 are used for targeting (Genome Research (ML) now Incyte, BACPAC Resources, 747 52nd Street, Oakland Calif.). The mouse rDNA clone pFK161 (SEQ ID NO: 118), which was used to make the human SATAC from the 94-3 hamster/human hybrid cell line (see, e.g., published International PCT application No. WO 97/40183 and Csonka, et al, Journal of Cell Science 113:3207-32161 and Example 1 for a description of pFK161) can also be used.

[0229] For animal applications, selectable markers should be non-immunogenic in the animal, such as a human, and include, but are not limited to: human nerve growth factor receptor (detected with a MAb, such as described in U.S. Pat. No. 6,365,373); truncated human growth factor receptor (detected with MAb), mutant human dihyrofolate reductase (DHFR; fluorescent MTX substrate available); secreted alkaline phosphatase (SEAP; fluorescent substrate available); human thymidylate synthase (TS; confers resistance to anti-cancer agent fluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1; conjugates glutathione to the stem cell selective alkylator busulfan; chemoprotective selectable marker in CD34+ cells); CD24 cell surface antigen in hematopoietic stem cells; human CAD gene to confer resistance to N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1 (MDR-1; P-glycoprotein surface protein selectable by increased drug resistance or enriched by FACS); human CD25 (IL-2a; detectable by Mab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable by carmustine); and Cytidine deaminase (CD; selectable by Ara-C).

[0230] Since megareplicator induced amplification generates multiple copies of the selectable marker, a second consideration for the selection of the human marker is the resulting dose of the expressed marker after ACes formation. High level of expression of certain markers may be detrimental to the cell and/or result in autoimmunity. One method to decrease the dose of the marker protein is by shortening its half-life, such as via the fusion of the well-conserved human ubiquitin tag (a 76 amino acid sequence) thus leading to increased turnover of the selectable marker. This has been used successfully for a number of reporter systems including DHFR (see, e.g., Stack et al. (2000) Nature Biotechnology 18:1298-1302 and references cited therein).

[0231] Using the ubiquitin tagged protein, a human selectable marker system analogous to the CPP ACes described herein is constructed. Briefly, a tagged selectable marker, such as for example one of those described herein, is cloned downstream of an attP site and expressed from a human promoter. Exemplary promoters contemplated for use herein include, but are not limited to, the human ferritin heavy chain promoter (SEQ ID NO:128); RNA PolI; EF1α; TR; glyceraldehyde-3-phosphate dehydrogenase core promoter (GAP); a GAP core promoter including a proximal insulin inducible element the intervening GAP sequence; phosphofructokinase promoter; and phosphoglycerate kinase promoter. Also contemplated herein is an aldolase A promoter H1 & H2 (representing closely spaced transcriptional start sites) along with the proximal H enhancer. There are 4 promoters (e.g., transcriptional start sites) for this gene, each having different regulatory and tissue activity. The H (most proximal 2) promoters are ubiquitously expressed off the H enhancer. This resulting marker can then be co-transfected along with excess human rDNA targeting sequence into the host cells. An important criteria for the selection of the recipient cells is sufficient number of cell doublings for the formation and detection of ACes. Accordingly, the co-transfections should be attempted in human primary cells that can be cultured for long periods of time, such as for example, stem cells (e.g., hematopoietic, mesenchymal, adult or embryonic stem cells), or the like. Additional cell types, include, but are not limited to: single gene transfected cells exhibiting increased life-span; over-expressing c-myc cells, e.g. MSU1.1 (Morgan et al., 1991, Exp. Cell Res., November; 197(1):125-136); over-expressing telomerase lines, such as TERT cells; SV40 large T-antigen transfected lines; tumor cell lines, such as HT1080; and hybrid human cell lines, such as the 94-3 hamster/human hybrid cell line.

[0232] b. Gene Target Vector

[0233] The second component of the GT platform ACes (GT ACes) system involves the use of engineered target vectors carrying the therapeutic gene of interest. These are introduced onto the GT platform ACes via site-specific recombination. As with the CPP ACes, the use of engineered target vectors maximizes the use of the de novo generated GT platform ACes for most gene targets. Furthermore, using lambda integrase technology, GT platform ACes containing multiple attP sites permits the opportunity to incorporate multiple therapeutic targets onto a single platform. This could be of value in cases where a defined therapy requires multiple gene targets, a single therapeutic target requires an additional gene regulatory factor or a GT ACes requires a "kill" switch.

[0234] Similar to the CPP ACes, a feature of the gene target vector is the promoterless marker gene containing an upstream attB site (marker 2 on FIG. 9). Normally, the marker (in this case, a cell surface antigen that can be sorted by FACS would be ideal) would not be expressed unless it is placed downstream of a promoter sequence. Using the lambda integrase technology (λINT.sub.E174R on FIG. 9), site-specific recombination between the attB site on the vector and the promoter-attP site on the GT platform ACes results in the expression of marker#2 on the gene target vector, i.e. positive selection for the site-specific event. Site-specific recombination events on the GT ACes versus random integrations next to a promoter in the genome (false positive) can be quickly screened by designing primers to detect the correct event by PCR.

[0235] For expression of the therapeutic gene, human specific promoters, such as a ferritin heavy chain promoter (SEQ ID NO:128); EF1α or RNA PolI, are used. These promoters are for high level expression of a cDNA encoded therapeutic protein. In addition to expressing cDNA (or even hybrid cDNA artificial intron constructs), the GT platform ACes are used for engineering and expressing large genomic fragments carrying therapeutic genes of interest expressed from native promoter sequences. This is of importance in situations where the therapy requires precise cell specific expression or in instances where expression is best achieved from genomic clones versus cDNA.

[0236] 3. Selectable Markers for Use, for Example, in Gene Therapy (GT)

[0237] The following are selectable markers that can be incorporated into human ACes and used for selection.

[0238] Dual Resistance to 4-Hydroperoxycyclophosphamide and Methotrexate by Retroviral Transfer of the Human Aldehyde Dehydrogenase Class 1 Gene and a Mutated Dihydrofolate Reductase Gene

[0239] The genetic transfer of drug resistance to hematopoietic cells is one approach to overcoming myelosuppression caused by high-dose chemotherapy. Because cyclophosphamide (CTX) and methotrexate (MTX) are commonly used non-cross-resistant drugs, generation of dual drug resistance in hematopoietic cells that allows dose intensification may increase anti-tumor effects and circumvent the emergence of drug-resistant tumors, a retroviral vector containing a human cytosolic ALDH-1-encoding DNA clone and a human doubly mutated DHFR-encoding clone (Phe22/Ser31; termed F/S in the description of constructs) to generate increased resistance to CTX and MTX were constructed (Takebe et al. (2001) Mol Ther 3(1):88-96). This construct may be useful for protecting patients from high-dose CTX- and MTX-induced myelosuppression. ACes can be similarly constructed.

[0240] Multiple Mechanisms of N-Phosphonacetyl-L-Aspartate Resistance in Human Cell Lines: Carbamyl-P Synthetase/Aspartate Transcarbamylase/Dihydro-Orotase Gene Amplification is Frequent Only when Chromosome 2 is Rearranged

[0241] Rodent cells resistant to N-phosphonacetyl-L-aspartate (PALA) invariably contain amplified carbamyl-P synthetase/aspartate transcarbamylase/dihydro-orotase (CAD) genes, usually in widely spaced tandem arrays present as extensions of the same chromosome arm that carries a single copy of CAD in normal cells (Smith et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:1816-21). In contrast, amplification of CAD is very infrequent in several human tumor cell lines. Cell lines with minimal chromosomal rearrangement and with unrearranged copies of chromosome 2 rarely develop intrachromosomal amplifications of CAD. These cells frequently become resistant to PALA through a mechanism that increases the aspartate transcarbamylase activity with no increase in CAD copy number, or they obtain one extra copy of CAD by forming an isochromosome 2p or by retaining an extra copy of chromosome 2. In cells with multiple chromosomal aberrations and rearranged copies of chromosome 2, amplification of CAD as tandem arrays from rearranged chromosomes is the most frequent mechanism of PALA resistance. All of these different mechanisms of PALA resistance are blocked in normal human fibroblasts. Thus, ACes with multiple copies of the CAD gene would provide PALA resistance.

[0242] Retroviral Coexpression of Thymidylate Synthase and Dihydrofolate Reductase Confers Fluoropyrimidine and Antifolate Resistance

[0243] Retroviral gene transfer of dominant selectable markers into hematopoietic cells can be used to select genetically modified cells in vivo or to attenuate the toxic effects of chemotherapeutic agents. Fantz et al. ((1998) Biochem Biophys Res Comm 243(1):6-12) have shown that retroviral gene transfer of thymidylate synthase (TS) confers resistance to TS directed anticancer agents and that co-expression of TS and dihydrofolate reductase (DHFR) confers resistance to TS and DHFR cytotoxic agents. Retroviral vectors encoding Escherichia coli TS, human TS, and the Tyr-to-His at residue 33 variant of human TS (Y33HhTS) were constructed and fibroblasts transfected with these vectors conferred comparable resistance to the TS-directed agent fluorodeoxyuridine (FdUrd, approximately 4-fold). Retroviral vectors that encode dual expression of Y33HhTS and the human L22Y DHFR (L22YhDHFR) variants conferred resistance to FdUrd (3- to 5-fold) and trimetrexate (30- to 140-fold). A L22YhDHFR-Y33HhTS chimeric retroviral vector was also constructed and transduced cells were resistant to FdUrd (3-fold), AG337 (3-fold), trimetrexate (100-fold) and methotrexate (5-fold). These results show that recombinant retroviruses can be used to transfer the cDNA that encodes TS and DHFR and dual expression in transduced cells is sufficiently high to confer resistance to TS and DHFR directed anticancer agents. ACes can be similarly constructed.

[0244] Human CD34+ Cells do not Express Glutathione S-Transferases Alpha

[0245] The expression of glutathione S-transferases alpha (GST alpha) in human hematopoietic CD34+ cells and bone marrow was studied using RT-PCR and immunoblotting (Czerwinski M, Kiem et al. (1997) Gene Ther 4(3):268-70). The GSTA1 protein conjugates glutathione to the stem cell selective alkylator busulfan. This reaction is the major pathway of elimination of the compound from the human body. Human hematopoietic CD34+ cells and bone marrow do not express GSTA1 message, which was present at a high level in liver, an organ relatively resistant to busulfan toxicity in comparison to bone marrow. Similarly, baboon CD34+ cells and dog bone marrow do not express GSTA1. Thus, human GSTA1 is a chemoprotective selectable marker in human stem cell gene therapy and could be employed in ACes construction.

[0246] Selection of Retrovirally Transduced Hematopoietic Cells Using Cd24 as a Marker of Gene Transfer

[0247] Pawliuk et al. ((1994) Blood 84(9):2868-2877) have investigated the use of a cell surface antigen as a dominant selectable marker to facilitate the detection and selection of retrovirally infected target cells. The small coding region of the human cell surface antigen CD24 (approximately 240 bp) was introduced into a myeloproliferative sarcoma virus (MPSV)-based retroviral vector, which was then used to infect day 45-fluorouracil (5-FU)-treated murine bone marrow cells. Within 48 hours of termination of the infection procedure CD24-expressing cells were selected by fluorescent-activated cell sorting (FACS) with an antibody directed against the CD24 antigen. Functional analysis of these cells showed that they included not only in vitro clonogenic progenitors and day 12 colony-forming unit-spleen but also cells capable of competitive long-term hematopoietic repopulation. Double-antibody labeling studies performed on recipients of retrovirally transduced marrow cells showed that some granulocytes, macrophages, erythrocytes, and, to a lesser extent, B and T lymphocytes still expressed the transduced CD24 gene at high levels 4 months later. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD24-expressing cells. These results show that the use of the CD24 cell surface antigen as a retrovirally encoded marker permits rapid, efficient, and nontoxic selection in vitro of infected primary cells, facilitates tracking and phenotyping of their progeny, and provides a tool to identify elements that regulate the expression of transduced genes in the most primitive hematopoietic cells. ACes could be similarly constructed.

[0248] DeltahGHR, a Biosafe Cell Surface-Labeling Molecule for Analysis and Selection of Genetically Transduced Human Cells

[0249] A selectable marker for retroviral transduction and selection of human and murine cells is known (see, Garcia-Ortiz et al. (2000) Hum Gene Ther 11(2):333-46). The molecule expressed on the cell surface of the transduced population is a truncated version of human growth hormone receptor (deltahGHR), capable of ligand (hGH) binding, but devoid of the domains involved in signal triggering. The engineered molecule is stably expressed in the target cells as an inert protein unable to trigger proliferation or to rescue the cells from apoptosis after ligand binding. This new marker, has a wide application spectrum, since hGHR in the human adult is highly expressed only in liver cells, and lower levels have been reported in certain lymphocyte cell populations. The deltahGHR label has high biosafety potential, as it belongs to a well-characterized hormonal system that is nonessential in adults, and there is extensive clinical experience with hGH administration in humans. The differential binding properties of several monoclonal antibodies (MAbs) are used in a cell rescue method in which the antibody used to select deltahGHR-transduced cells is eluted by competition with hGH or, alternatively biotinylated hGH is used to capture tagged cells. In the latter system, the final purified population is recovered free of attached antibodies in hGH (a substance approved for human use)-containing medium. Such a system could be used to identify ACes containing cells.

[0250] 4. Transgenic Models for Evaluation of Genes and Discovery of New Traits in Plants

[0251] Of interest is the use of plants and plant cells containing artificial chromosomes for the evaluation of new genetic combinations and discovery of new traits. Artificial chromosomes, by virtue of the fact that they can contain significant amounts of DNA can also therefore encode numerous genes and accordingly a multiplicity of traits. It is contemplated here that artificial chromosomes, when formed from one plant species, can be evaluated in a second plant species. The resultant phenotypic changes observed, for example, can indicate the nature of the genes contained within the DNA contained within the artificial chromosome, and hence permit the identification of novel genetic activities. Artificial chromosomes containing euchromatic DNA or partially containing euchromatic DNA can serve as a valuable source of new traits when transferred to an alien plant cell environment. For example, it is contemplated that artificial chromosomes derived from dicot plant species can be introduced into monocot plant species by transferring a dicot artificial chromosome. The dicot artificial chromosome possessing a region of euchromatic DNA containing expressed genes.

[0252] The artificial chromosomes can be designed to allow the artificial chromosome to recombine with the naturally occurring plant DNA in such a fashion that a large region of naturally occurring plant DNA becomes incorporated into the artificial chromosome. This allows the artificial chromosome to contain new genetic activities and hence carry novel traits. For example, an artificial chromosome can be introduced into a wild relative of a crop plant under conditions whereby a portion of the DNA present in the chromosomes of the wild relative is transferred to the artificial chromosome. After isolation of the artificial chromosome, this naturally occurring region of DNA from the wild relative, now located on the artificial chromosome can be introduced into the domesticated crop species and the genes encoded within the transferred DNA expressed and evaluated for utility. New traits and gene systems can be discovered in this fashion. The artificial chromosome can be modified to contain sequences that promote homologous recombination within plant cells, or be modified to contain a genetic system that functions as a site-specific recombination system.

[0253] Artificial chromosomes modified to recombine with plant DNA offer many advantages for the discovery and evaluation of traits in different plant species. When the artificial chromosome containing DNA from one plant species is introduced into a new plant species, new traits and genes can be introduced. This use of an artificial chromosome allows for the ability to overcome the sexual barrier that prevents transfer of genes from one plant species to another species. Using artificial chromosomes in this fashion allows for many potentially valuable traits to be identified including traits that are typically found in wild species. Other valuable applications for artificial chromosomes include the ability to transfer large regions of DNA from one plant species to another, such as DNA encoding potentially valuable traits such as altered oil, carbohydrate or protein composition, multiple genes encoding enzymes capable of producing valuable plant secondary metabolites, genetic systems encoding valuable agronomic traits such as disease and insect resistance, genes encoding functions that allow association with soil bacterium such as growth promoting bacteria or nitrogen fixing bacteria, or genes encoding traits that confer freezing, drought or other stress tolerances. In this fashion, artificial chromosomes can be used to discover regions of plant DNA that encode valuable traits.

[0254] The artificial chromosome can also be designed to allow the transfer and subsequent incorporation of these valuable traits now located on the artificial chromosome into the natural chromosomes of a plant species. In this fashion the artificial chromosomes can be used to transfer large regions of DNA encoding traits normally found in one plant species into another plant species. In this fashion, it is possible to derive a plant cell that no longer needs to carry an artificial chromosome to posses the novel trait. Thus, the artificial chromosome would serve as the transfer mechanism to permit the formation of plants with greater degree of genetic diversity.

[0255] The design of an artificial chromosome to accomplish the afore-mentioned purposes can include within the artificial chromosome the presence of specific DNA sequences capable of acting as sites for homologous recombination to take place. For example, the DNA sequence of Arabidopsis is now known. To construct an artificial chromosome capable of recombining with a specific region of Arabidopsis DNA, a sequence of Arabidopsis DNA, normally located near a chromosomal location encoding genes of potential interest can be introduced into an artificial chromosome by methods provided herein. It may be desirable to include a second region of DNA within the artificial chromosome that provides a second flanking sequence to the region encoding genes of potential interest, to promote a double recombination event which would ensure transfer of the entire chromosomal region, encoding genes of potential interest, to the artificial chromosome. The modified artificial chromosome, containing the DNA sequences capable of homologous recombination region, can then be introduced into Arabidopsis cells and the homologous recombination event selected.

[0256] It is convenient to include a marker gene to allow for the selection of a homologous recombination event. The marker gene is preferably inactive unless activated by an appropriate homologous recombination event. For example, U.S. Pat. No. 5,272,071, describes a method where an inactive plant gene is activated by a recombination event such that desired homologous recombination events can be easily scored. Similarly, U.S. Pat. No. 5,501,967 describes a method for the selection of homologous recombination events by activation of a silent selection gene first introduced into the plant DNA, the gene being activated by an appropriate homologous recombination event. Both of these methods can be applied to enable a selective process to be included to select for recombination between an artificial chromosome and a plant chromosome. Once the homologous recombination event is detected, the artificial chromosome, once selected, is isolated and introduced into a recipient cell, for example, tobacco, corn, wheat or rice, and the expression of the newly introduced DNA sequences evaluated.

[0257] Phenotypic changes in the recipient plant cells containing the artificial chromosome, or in regenerated plants containing the artificial chromosome, allows for the evaluation of the nature of the traits encoded by the Arabidopsis DNA, under conditions naturally found in plant cells, including the naturally occurring arrangement of DNA sequences responsible for the developmental control of the traits in the normal chromosomal environment.

[0258] Traits such as durable fungal or bacterial disease resistance, new oil and carbohydrate compositions, valuable secondary metabolites such as phytosterols, flavonoids, efficient nitrogen fixation or mineral utilization, resistance to extremes of drought, heat or cold are all found within different populations of plant species and are often governed by multiple genes. The use of single gene transformation technologies does not permit the evaluation of the multiplicity of genes controlling many valuable traits. Thus, incorporation of these genes into artificial chromosomes allows the rapid evaluation of the utility of these genetic combinations in heterologous plant species.

[0259] The large scale order and structure of the artificial chromosome provides a number of unique advantages in screening for new utilities or novel phenotypes within heterologous plant species. The size of new DNA that can be carried by an artificial chromosome can be millions of base pairs of DNA, representing potentially numerous genes that may have novel utility in a heterologous plant cell. The artificial chromosome is a "natural" environment for gene expression, the problems of variable gene expression and silencing seen for genes transferred by random insertion into a genome should not be observed. Similarly, there is no need to engineer the genes for expression, and the genes inserted would not need to be recombinant genes. Thus, one expects the expression from the transferred genes to be temporal and spatial, as observed in the species from where the genes were initially isolated. A valuable feature for these utilities is the ability to isolate the artificial chromosomes and to further isolate, manipulate and introduce into other cells artificial chromosomes carrying unique genetic compositions.

[0260] Thus, the use of artificial chromosomes and homologous recombination in plant cells can be used to isolate and identify many valuable crop traits.

[0261] In addition to the use of artificial chromosomes for the isolation and testing of large regions of naturally occurring DNA, methods for the use of artificial chromosomes and cloned DNA are also contemplated. Similar to that described above, artificial chromosomes can be used to carry large regions of cloned DNA, including that derived from other plant species.

[0262] The ability to incorporate novel DNA elements into an artificial chromosome as it is being formed allows for the development of artificial chromosomes specifically engineered as a platform for testing of new genetic combinations, or "genomic" discoveries for model species such as Arabidopsis. It is known that specific "recombinase" systems can be used in plant cells to excise or re-arrange genes. These same systems can be used to derive new gene combinations contained on an artificial chromosome.

[0263] The artificial chromosomes can be engineered as platforms to accept large regions of cloned DNA, such as that contained in Bacterial Artificial Chromosomes (BACs) or Yeast Artificial Chromosomes (YACs). It is further contemplated, that as a result of the typical structure of artificial chromosomes containing tandemly repeated DNA blocks, that sequences other than cloned DNA sequence can be introduced by recombination processes. In particular recombination within a predefined region of the tandemly repeated DNA within the artificial chromosome provides a mechanism to "stack" numerous regions of cloned DNA, including large regions of DNA contained within BACs or YACs clones. Thus, multiple combinations of genes can be introduced onto artificial chromosomes and these combinations tested for functionality. In particular, it is contemplated that multiple YACs or BACs can be stacked onto an artificial chromosomes, the BACs or YACs containing multiple genes of complex pathways or multiple genetic pathways. The BACs or YACs are typically selected based on genetic information available within the public domain, for example from the Arabidopsis Information Management System (http://aims.cps.msu.edu/aims/index.html) or the information related to the plant DNA sequences available from the Institute for Genomic Research (http://www.tigr.org) and other sites known to those skilled in the art. Alternatively, clones can be chosen at random and evaluated for functionality. It is contemplated that combinations providing a desired phenotype can be identified by isolation of the artificial chromosome containing the combination and analyzing the nature of the inserted cloned DNA.

[0264] In this regard, it is contemplated that the use of site-specific recombination sequences can have considerable utility in developing artificial chromosomes containing DNA sequences recognized by recombinase enzymes and capable of accepting DNA sequences containing same. The use of site-specific recombination as a means to target an introduced DNA to a specific locus has been demonstrated in the art and such methods can be employed. The recombinase systems can also be used to transfer the cloned DNA regions contained within the artificial chromosome to the naturally occurring plant or mammalian chromosomes.

[0265] As noted herein, many site-specific recombinases are known and can be identified (Kilby et al. (1993) Trends in Genetics 9:413-418). The three recombinase systems that have been extensively employed include: an activity identified as R encoded by the pSR1 plasmid of Zygosaccharomyes rouxii, FLP encoded for the 2 um circular plasmid from Saccharomyces cerevisiae and Cre-lox from the phage P1.

[0266] The integration function of site-specific recombinases is contemplated as a means to assist in the derivation of genetic combinations on artificial chromosomes. In order to accomplish this, it is contemplated that a first step of introducing site-specific recombinase sites into the genome of a plant cell in an essentially random manner is conducted, such that the plant cell has one or more site-specific recombinase recognition sequences on one or more of the plant chromosomes. An artificial chromosome is then introduced into the plant cell, the artificial chromosome engineered to contain a recombinase recognition site (e.g., integration site) capable of being recognized by a site-specific recombinase. Optionally, a gene encoding a recombinase enzyme is also included, preferably under the control of an inducible promoter. Expression of the site-specific recombinase enzyme in the plant cell, either by induction of a inducible recombinase gene, or transient expression of a recombinase sequence, causes a site-specific recombination event to take place, leading to the insertion of a region of the plant chromosomal DNA (containing the recombinase recognition site) into the recombinase recognition site of the artificial chromosome, and forming an artificial chromosome containing plant chromosomal DNA. The artificial chromosome can be isolated and introduced into a heterologous host, preferably a plant host, and expression of the newly introduced plant chromosomal DNA can be monitored and evaluated for desirable phenotypic changes. Accordingly, carrying out this recombination with a population of plant cells wherein the chromosomally located recombinase recognition site is randomly scattered throughout the chromosomes of the plant, can lead to the formation of a population of artificial chromosomes, each with a different region of plant chromosomal DNA, and each potentially representing a novel genetic combination.

[0267] This method requires the precise site-specific insertion of chromosomal DNA into the artificial chromosome. This precision has been demonstrated in the art. For example, Fukushige and Sauer ((1992) Proc. Natl. Acad. Sci. USA, 89:7905-7909) demonstrated that the Cre-lox homologous recombination system could be successfully employed to introduce DNA into a predefined locus in a chromosome of mammalian cells. In this demonstration a promoter-less antibiotic resistance gene modified to include a lox sequence at the 5' end of the coding region was introduced into CHO cells. Cells were re-transformed by electroporation with a plasmid that contained a promoter with a lox sequence and a transiently expressed Cre recombinase gene. Under the conditions employed, the expression of the Cre enzyme catalyzed the homologous recombination between the lox site in the chromosomally located promoter-less antibiotic resistance gene, and the lox site in the introduced promoter sequence, leading to the formation of a functional antibiotic resistance gene. The authors demonstrated efficient and correct targeting of the introduced sequence, 54 of 56 lines analyzed corresponded to the predicted single copy insertion of the DNA due to Cre catalyzed site-specific homologous recombination between the lox sequences.

[0268] Accordingly a lox sequence may be first added to a genome of a plant species capable of being transformed and regenerated to a whole plant to serve as a recombinase target DNA sequence for recombination with an artificial chromosome. The lox sequence may be optimally modified to further contain a selectable marker which is inactive but can be activated by insertion of the lox recombinase recognition sequence into the artificial chromosome.

[0269] A promoterless marker gene or selectable marker gene linked to the recombinase recognition sequence, which is first inserted into the chromosomes of a plant cell can be used to engineer a platform chromosome. A promoter is linked to a recombinase recognition site, in an orientation that allows the promoter to control the expression of the marker or selectable marker gene upon recombination within the artificial chromosome. Upon a site-specific recombination event between a recombinase recognition site in a plant chromosome and the recombinase recognition site within the introduced artificial chromosome, a cell is derived with a recombined artificial chromosome, the artificial chromosome containing an active marker or selectable marker activity that permits the identification and or selection of the cell.

[0270] The artificial chromosomes can be transferred to other plant or animal species and the functionality of the new combinations tested. The ability to conduct such an inter-chromosomal transfer of sequences has been demonstrated in the art. For example, the use of the Cre-lox recombinase system to cause a chromosome recombination event between two chromatids of different chromosomes has been shown.

[0271] Any number of recombination systems may be employed as described herein, such as, but not limited to, bacterially derived systems such as the att/int system of phage lambda, and the Gin/gix system.

[0272] More than one recombination system may be employed, including, for example, one recombinase system for the introduction of DNA into an artificial chromosome, and a second recombinase system for the subsequent transfer of the newly introduced DNA contained within an artificial chromosome into the naturally occurring chromosome of a second plant species. The choice of the specific recombination system used will be dependent on the nature of the modification contemplated.

[0273] By having the ability to isolate an artificial chromosome, in particular, artificial chromosomes containing plant chromosomal DNA introduced via site-specific recombination, and re-introduce the chromosome into other mammalian or plant cells, particularly plant cells, these new combinations can be evaluated in different crop species without the need to first isolate and modify the genes, or carry out multiple transformations or gene transfers to achieve the same combination isolation and testing combinations of the genes in plants. The use of a site-specific recombinase also allows the convenient recovery of the plant chromosomal region into other recombinant DNA vectors and systems, such as mammalian or insect systems, for manipulation and study.

[0274] Also contemplated herein are ACes, cell lines and methods for use in screening a new chromosomal combinations, deletions, truncations with eucaryotic genome that take advantage of the site-specific recombination systems incorporated onto platform ACes provided herein. For example, provided herein is a cell line useful for making a library of ACes, comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes. Also provided herein is a method of making a library of ACes comprising random portions of a genome, comprising introducing one or more ACes into a cell line comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes, under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites within the cell's chromosomal DNA; and isolating said multiplicity of ACes, thereby producing a library of ACes whereby multiple ACes have different portions of the genome within. Also provided herein is a library of cells useful for genomic screening, said library comprising a multiplicity of cells, wherein each cell comprises an ACes having a mutually exclusive portion of a chromosomal nucleic acid therein. The library of cells can be from a different species and/or cell type than the chromosomal nucleic acid within the ACes. Also provided is a method of making one or more cell lines, comprising [0275] a) integrating into endogenous chromosomal DNA of a selected cell species, a multiplicity of heterologous recombination sites, [0276] b) introducing a multiplicity of ACes under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites integrated within the cell's endogenous chromosomal DNA; [0277] c) isolating said multiplicity of ACes, thereby producing a library of ACes whereby a multiplicity of ACes have mutually exclusive portions of the endogenous chromosomal DNA therein; [0278] d) introducing the isolated multiplicity of ACes of step c) into a multiplicity of cells, thereby creating a library of cells; [0279] e) selecting different cells having mutually exclusive ACes therein and clonally expanding or differentiating said different cells into clonal cell cultures, thereby creating one or more cell lines.

[0280] These ACes, cell lines and methods utilize the site-specific recombination sites on platform ACes analogous YAC manipulation related to: the methods of generating terminal deletions in normal and artificial chromosomes (e.g., ACes; as described in Vollrath et al., 1988, PNAS, USA, 85:6027-66031; and Pavan et al., PNAS, USA, 87:1300-1304); the methods of generating interstitial deletions in normal and artificial chromosomes (as described in Campbell et al., 1991, PNAS, USA, 888:5744-5748); and the methods of detecting homologous recombination between two ACes (as described in Cellini et al., 1991, Nuc. Acid Res., 19(5):997-1000).

[0281] 5. Use of Plateform ACes in Pharmacogenomic/Toxicology Applications (Development of "Reporter ACes")

[0282] In addition to the placement of genes onto ACes chromosomes for therapeutic protein production or gene therapy, the platform can be engineered via the IntR lambda integrase to carry reporter-linked constructs (reporter genes) that monitor changes in cellular physiology as measured by the particular reporter gene (or a series of different reporter genes) readout. The reporter linked constructs are designed to include a gene that can be detected (by for example fluorescence, drug resistance, immunohistochemistry, or transcript production, and the like) with well-known regulatory sequences that would control the expression of the detectable gene.

[0283] Exemplary regulatory promoter sequences are well-known in the art:

[0284] a) Reporter ACes for Drug Pathway Screening

[0285] The ACes can be engineered to carry reporter-linked constructs that indicate a signal is being transduced through one or a number of pathways. For example, transcriptionally regulated promoters from genes at the end (or any other chosen point) of particular signal transduction pathways could be engineered on the ACes to express the appropriate readout (either by fluorescent protein production or drug resistance) when the pathway is activated (or down-regulated as well). In one embodiment, a number of reporters from different can be placed on a ACes chromosome. Cells (and/or whole animals) containing such a Reporter ACes could be exposed to a variety of drugs or compounds and monitored for the effects of the drugs or compounds upon the selected pathway(s) by the reporter gene(s). Thus, drugs or compounds can be classified or identified by particular pathways they excite or down-regulate. Similarly, transcriptional profiles obtained from genomic array experiments can be biologically validated using the reporter ACes provided herein.

[0286] b) Reporter ACes for Toxic Compound Testing

[0287] Environmental or man-made genotoxicants can be tested in cell lines carrying a number of reporter-genes platform ACes linked to promoters that are transcriptionally regulated in response to DNA damage, induced apoptosis or necrosis, and cell-cycle perturbations. Furthermore, new drugs and/or compounds could be tested in a similar manner with the genotoxicant ACes reporter for their cellular/genetic toxicity by such a screen. Likewise, toxic compound testing could be carried out in whole transgenic animals carrying the ACes chromosome that measures genotoxicant exposure ("canary in a coal mine"). Thus, the same or similar type ACes could be used for toxicity testing in either a cell-based or whole animal setting. An example would include ACes that carry reporter-linked genes controlled by various cytochrome P450 profiled promoters and the like.

[0288] c) Reporter ACes for Individualized Pharmacogenomics/Drug Profiling

[0289] A common disease may arise via various mechanisms. In many instances there are multiple treatments available for a given disease. However, the success of a given treatment may depend upon the mechanism by which the disease originated and/or by the genetic background of the patient. In order to establish the most effective treatment for a given patient one could utilize the ACes reporters provided herein. ACes reporters can be used in patient cell samples to determine an individualized drug regimen for the patient. In addition, potential polymorphisms affecting the transcriptional regulation of an individual's particular gene can be assessed by this approach.

[0290] d) Reporter ACes for Classification of Similar Patient Tumors

[0291] As with other diseases as described in 5.C) above, cancer cells arise via different mechanisms. Furthermore, as a cancerous cell propagates it may undergo genomic alterations. An ACes reporter transferred to cells of different patients having the same disease, i.e. similar cancers, could be used to categorize the particular cancer of each patient, thereby facilitating the identification of the most effective therapeutic regimen. Examples would include the validation of array profiling of certain classes of breast cancers. Subsequently, appropriate drug profiling could be carried out as described above.

[0292] e) Reporter ACes as a "Differentiation" Sensor

[0293] Using the ACes reporter as a "differentiation" sensor in stem cells or other progenitor cells in order to enrich by selection (either FACS based screening, drug selection and/or use of suicide gene) for a particular class of differentiated or undifferentiated cells. For example, in one embodiment, this assay could also be used for compound screening for small molecule modifiers of cell differentiation.

[0294] f) Whole Animal Studies with Reporter Aces

[0295] Finally, with whole-body fluorescence imaging technology (Yang et al. (2000) PNAS 97:12278) any of the above Reporter ACes methods could be used in conjunction with whole-body imaging to monitor reporter genes within whole animals without sacrificing the animal. This would allow temporal and spatial analysis of expression patterns under a given set of conditions. The conditions tested may include for example, normal differentiation of a stem cell, response to drug or compound treatment whether targeted to the diseased tissue or presented systemically, response to genotoxicants, and the like.

[0296] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1

[0297] pFK161

[0298] Cosmid pFK161 (SEQ ID NO: 118) was obtained from Dr. Gyula Hadlaczky and contains a 9 kb NotI insert derived from a murine rDNA repeat (see clone 161 described in PCT Application Publication No. WO97/40183 by Hadlaczky et al. for a description of this cosmid). This cosmid, referred to as clone 161 contains sequence corresponding to nucleotides 10,232-15,000 in SEQ ID NO. 26. It was produced by inserting fragments of the megachromosome (see, U.S. Pat. No. 6,077,697 and International PCT application No. WO 97/40183). For example, H1D3, which was deposited at the European Collection of Animal Cell Culture (ECACC) under Accession No. 96040929, is a mouse-hamster hybrid cell line carrying this megachromosome into plasmid pWE15 (Stratagene, La Jolla, Calif.; SEQ ID No. 31) as follows. Half of a 100 μl low melting point agarose block (mega-plug) containing isolated SATACs was digested with Non overnight at 37° C. Plasmid pWE15 was similarly digested with NotI overnight. The mega-plug was then melted and mixed with the digested plasmid, ligation buffer and T4 DNA ligase. Ligation was conducted at 16° C. overnight. Bacterial DH5α cells were transformed with the ligation product and transformed cells were plated onto LB/Amp plates. Fifteen to twenty colonies were grown on each plate for a total of 189 colonies. Plasmid DNA was isolated from colonies that survived growth on LB/Amp medium and analyzed by Southern blot hybridization for the presence of DNA that hybridized to a pUC19 probe. This screening methodology assured that all clones, even clones lacking an insert but yet containing the pWE15 plasmid, would be detected.

[0299] Liquid cultures of all 189 transformants were used to generate cosmid minipreps for analysis of restriction sites within the insert DNA. Six of the original 189 cosmid clones contained an insert. These clones were designated as follows: 28 (˜9-kb insert), 30 (˜9-kb insert), 60 (˜4-kb insert), 113 (˜9-kb insert), 157 (˜9-kb insert) and 161 (˜9-kb insert). Restriction enzyme analysis indicated that three of the clones (113, 157 and 161) contained the same insert.

[0300] For sequence analysis the insert of cosmid clone no. 161 was subcloned as follows. To obtain the end fragments of the insert of clone no. 161, the clone was digested with NotI and BamHI and ligated with NotI/BamHI-digested pBluescript KS (Stratagene, La Jolla, Calif.). Two fragments of the insert of clone no. 161 were obtained: a 0.2-kb and a 0.7-kb insert fragment. To subclone the internal fragment of the insert of clone no. 161, the same digest was ligated with BamHI-digested pUC19. Three fragments of the insert of clone no. 161 were obtained: a 0.6-kb, a 1.8-kb and a 4.8-kb insert fragment.

[0301] The insert corresponds to an internal section of the mouse ribosomal RNA gene (rDNA) repeat unit between positions 7551-15670 as set forth in GENBANK accession no. X82564, which is provided as SEQ ID NO. 18. The sequence data obtained for the insert of clone no. 161 is set forth in SEQ ID NOS. 19-25. Specifically, the individual subclones corresponded to the following positions in GENBANK accession no. X82564 (SEQ ID NO:18) and in SEQ ID NOs. 19-25:

TABLE-US-00003 Start End Subclone in X82564 Site SEQ ID No. 161k1 7579 7755 NotI, BamHI 19 161m5 7756 8494 BamHI 20 161m7 8495 10231 BamHI 21 (shows only sequence corresponding to nt. 8495-8950), 22 (shows only sequence corresponding to nt. 9851-10231) 161m12 10232 15000 BamHI 23 (shows only sequence corresponding to nt. 10232-10600), 24 (shows only sequence corresponding to nt. 14267-15000) 161k2 15001 15676 NotI, BamHI 25

[0302] The sequence set forth in SEQ ID NOs. 19-25 diverges in some positions from the sequence presented in positions 7551-15670 of GENBANK accession no. X82564. Such divergence may be attributable to random mutations between repeat units of rDNA.

[0303] For use herein, the rDNA insert from the clone was prepared by digesting the cosmid with Not1 and Bgl11 and was purified as described above. Growth and maintenance of bacterial stocks and purification of plasmids were performed using standard well known methods (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press), and plasmids were purified from bacterial cultures using Midi- and Maxi-preps Kits (Qiagen, Mississauga, Ontario).

pDsRed 1N1

[0304] This vector is available from Clontech (see SEQ ID No. 29) and encodes the red fluorescent protein (DsRed; Genbank accession no. AF272711; SEQ ID Nos. 39 and 40). DsRed, which has a vivid red fluorescence, was isolated from the IndoPacific sea anemone relative Discosoma species. The plasmid pDsRed1N1 (Clontech; SEQ ID No. 29) constitutively expresses a human codon-optimized variant of the fluorescent protein under control of the CMV promoter. Unmodified, this vector expresses high levels of DsRed1 and includes sites for creating N-terminal fusions by cloning proteins of interest into the multiple cloning site (MCS). It is Kan and Neo resistant for selection in bacterial or eukaryotic cells.

Plasmid pMG

[0305] Plasmid pMG (InvivoGen, San Diego, Calif.; see SEQ. ID. NO. 27 for the nucleotide sequence of pMG) contains the hygromycin phosphotransferase gene under the control of the immediate-early human cytomegalovirus (hCMV) enhancer/promoter with intron A. Vector pMG also contains two transcriptional units allowing for the coexpression of two heterologous genes from a single vector sequence.

[0306] The first transcriptional unit of pMG contains a multiple cloning site for insertion of a gene of interest, the hygromycin phosphotransferase gene (hph) and the immediate-early human cytomegalovirus (hCMV) enhancer/promoter with intron A (see, e.g., Chapman et al. (1991) Nuc. Acids Res. 19:3979-3986) located upstream of hph and the multiple cloning site, which drives the expression of hph and any gene of interest inserted into the multiple cloning site as a polycistronic mRNA. The first transcriptional unit also contains a modified EMCV internal ribosomal entry site (IRES) upstream of the hph gene but downstream of the hCMV promoter and MCS for ribosomal entry in translation of the hph gene (see SEQ ID NO. 27, nucleotides 2736-3308). The IRES is modified by insertion of the constitutive E. coli promoter (EM7) within an intron (IM7) into the end of the IRES. In mammalian cells, the E. coli promoter is treated as an intron and is spliced out of the transcript. A polyadenylation signal from the bovine growth hormone (bGh) gene (see, e.g., Goodwin and Rottman (1992) J. Biol. Chem. 267:16330-16334) and a pause site derived from the 3' flanking region of the human α2 globin gene (see, e.g., Enriquez-Harris et al. (1991) EMBO J. 10:1833-1842) are located at the end of the first transcription unit. Efficient polyadenylation is facilitated by inserting the flanking sequence of the bGh gene 3' to the standard AAUAAA hexanucleotide sequence.

[0307] The second transcriptional unit of pMG contains another multiple cloning site for insertion of a gene of interest and an EF-1α/HTLV hybrid promoter located upstream of this multiple cloning site, which drives the expression of any gene of interest inserted into the multiple cloning site. The hybrid promoter is a modified human elongation factor-1 alpha (EF-1 alpha) gene promoter (see, e.g., Kim et al. (1990) Gene 91:217-223) that includes the R segment and part of the U5 sequence (R-U5') of the human T-cell leukemia virus (HTLV) type I long terminal repeat (see, e.g., Takebe et al. (1988) Mol. Cell. Biol 8:466-472). The Simian Virus 40 (SV40) late polyadenylation signal (see Carswell and Alwine (1989) Mol. Cell. Biol. 9:4248-4258) is located downstream of the multiple cloning site. Vector pMG contains a synthetic polyadenylation site for the first and second transcriptional units at the end of the transcriptional unit based on the rabbit β-globin gene and containing the AATAAA hexanucleotide sequence and a GT/T-rich sequence with 22-23 nucleotides between them (see, e.g., Levitt et al. (1989)Genes Dev. 3:1019-1025). A pause site derived from the C2 complement gene (see, Moreira et al. (1995) EMBO J. 14:3809-3819) is also located at the 3' end of the second transcriptional unit.

[0308] Vector pMG also contains an on sequence (ori pMB1) located between the SV40 polyadenylation signal and the synthetic polyadenylation site.

Example 2

[0309] A. Construction of Targeting Vector and Transfection into LMtk- Cells for the Generation of Platform Chromosomes

[0310] A targeting vector derived from the vector pWE15 (GeneBank Accession # X65279) was modified by replacing the SalI (Klenow filled)/SmaI neomycin resistance containing fragment with the PvuII/BamHI (Klenow filled) puromycin resistance containing fragment (isolated from plasmid pPUR, Clontech Laboratories, Inc. Palo Alto, Calif.; SEQ ID No. 30) resulting in plasmid pWEPuro. Subsequently a 9 Kb NotI fragment from the plasmid pFK161 (SEQ ID NO: 118) containing a portion of the mouse rDNA region was cloned into the NotI site of pWEPuro resulting in plasmid pWEPuro9K (FIG. 2). The vector pWEPuro9K was digested with SpeI to linearize and transfected into LMtk- mouse cells. Puromycin resistant colonies were isolated and subsequently tested for artificial chromosome formation via fluorescent in situ hybridization (FISH) (using mouse major and minor DNA repeat sequences, the puromycin gene and telomeres sequences as probes), and fluorescent activated cell sorting (FACS). From this sort, a subclone was isolated containing an artificial chromosome, designated 5B11.12, which carries 4-8 copies of the puromycin resistance gene contained on the pWEPuro9K vector. FISH analysis of the 5B11.12 subclone demonstrated the presence of telomeres and mouse minor on the ACes. DOT PCR has been done on the 5B 11.12 ACes revealing the absence of uncharacterized euchromatic regions on the ACes. A recombination site, such as an att or loxP engineering site or a plurality thereof, was introduced onto this ACes thereby providing a platform for site-specific introduction of heterologous nucleic acid.

B. Targeting a Single Sequence Specific Recombination Site onto Platform Chromosomes

[0311] After the generation of the 5B11.12 platform, a single sequence-specific recombination site is placed onto the platform chromosome via homologous recombination. For this, DNA sequences containing the site-specific recombination sequence can be flanked with DNA sequences of homology to the platform chromosome. For example, using the platform chromosome made from the pWEPuro9K vector, mouse rDNA sequences or mouse major satellite DNA can be used as homologous sequences to target onto the platform chromosome. A vector is designed to have these homologous sequences flanking the site-specific recombination site and, after the appropriate restriction enzyme digest to generate free ends of homology to the platform chromosome, the DNA is transfected into cells harboring the platform chromosome (FIG. 3). Examples of site-specific cassettes that are targeted to the platform chromosome using either mouse rDNA or mouse major repeat DNA include the SV40-attP-hygro cassette and a red fluorescent protein (RFP) gene flanked by loxP sites (Cre/lox, see, e.g., U.S. Pat. No. 4,959,317 and description herein). After transfection and integration of the site-specific cassette, homologous recombination events onto the platform chromosome are subcloned and identified by FACS (e.g. screen and single cell subclone via expression of resistance or fluorescent marker) and PCR analysis.

[0312] For example, a vector can be constructed containing regions of the mouse rDNA locus flanking a gene cassette containing the SV40 early reporter-bacteriophage lambda attP site-hygromycin selectable marker (see FIG. 4 and described below). The use of the bacteriophage lambda attP site for lambda integrase-mediated site-specific recombination is described below. Homologous recombination event of the SV40-attP-hygro cassette onto the platform chromosome was identified using PCR primers that detect the homologous recombination and further confirmed by FISH analysis. After identifying subcloned colonies containing the platform chromosome with a single site-specific recombination site, cells carrying the platform chromosome with a single site-specific recombination site can now be engineered with site-specific recombinases (e.g. lambda INT, Cre) for integrating a target gene expression vector.

C. Targeting a Red Fluorescent Protein (RFP) Gene Flanked by loxP Sites onto 5B11.12 Platform

[0313] As another example, while loxP recombination sites could have been introduced onto the ACes during de novo biosynthesis, it was thought that this might result in multiple segments of the ACes containing a high number of loxP sites, potentially leading to instability upon Cre-mediated recombination. A gene targeting approach was therefore devised to introduce a more limited number of loxP recombination sites into a locus of the 5B11-12 ACes containing introduced and possibly co-amplified endogenous rDNA sequences. Although there are more than 200 copies of rDNA genes in the haploid mouse genome distributed amongst 5-11 chromosomes (depending on strain), rDNA sequences were chosen as the target on the ACes since they represent a less frequent target than that of the satellite repeat sequences. Moreover, having observed much stronger pWEPuro9K hybridization to the 5B11-12 ACes than to other LMTK.sup.- chromosomes and in light of the observation that the transcribed spacer sequences within the rDNA may be less conserved than the rRNA coding regions, it was contemplated that a targeting vector based on the rDNA gene segment in pWEPuro9K would have a higher probability of targeting to the ACes rather than to other LMTK^chromosomes. Accordingly, a targeting vector, pBSFKLoxDsRedLox, was designed and constructed based on the rDNA sequences contained in pWEPuro9K.

[0314] The plasmid pBSFKLoxDsRedLox was generated in 4 steps. First, the NotI rDNA insert of pWEPuro9K (FIG. 2) was inserted into pBS SK- (Stratagene) giving rise to pBSFK. Second, a loxP polylinker cassette was generated by PCR amplification of pNEB193 (SEQ ID NO:32; New England Biolabs) using primers complementary to the M13 forward and reverse priming sites at their 3' end and a 34 bp 5' extension comprising a LoxP site. This cassette was reinserted into pNEB193 generating p193LoxMCSLox. Third, the DsRed gene from pDsRed1-N1 (SEQ ID NO:29; Clontech) was then cloned into the polylinker between the loxP sites generating p193LoxDsRedLox. Fourth, a fragment consisting of the DsRed gene flanked by loxP sites was cloned into a unique NdeI within the rDNA insert of pBSFK generating pBSFKLoxDsRedLox.

[0315] A gel purified 11 Kb PmlI/EcoRV fragment of pBSFKLoxDsRedLox was used for transfection. To detect targeted integration, PCR primers were designed from rDNA sequences within the 5' NotI-PmlI fragment of pWEPuro9K that is not present on the targeting fragment (5' primer) and sequence within the LoxDsRedLox cassette (3' primer). If the targeting DNA integrated correctly within the rDNA sequences, PCR amplification using these primers would give rise to a 2.3 Kb band. PCR reactions containing 1-4 of genomic DNA were carried out according to the MasterTaq protocol (Eppendorf), using murine rDNA 5' primer (5'-CGGACAATGCGGTTGTGCGT-3'; SEQ ID NO:72) and DsRed 3' primer (5'GGCCCCGTAATGCAGAAGAA-3'; SEQ ID NO:73) and PCR products were analyzed by agarose gel electrophoresis.

[0316] 1.5×10⁶ 5B11-12 LMTK.sup.- cells were transfected with 2 μg of the pBSFKLoxDsRedLox targeting DNA described above using Lipofectamine Plus (Invitrogen). For flow sorting, harvested cells were suspended in medium and applied to the Becton Dickinson Vantage SE cell sorter, equipped with 488 nm lasers for excitation and 585/42 bandpass filter for optimum detection of RFP fluorescence. Cells were sorted using dPBS as sheath buffer. Negative control parental 5B11-12 cells and a positive control LMTK.sup.- cell line stably transfected with DsRed were used to establish the selection gates. The RFP positive gated populations were recovered, diluted in medium supplemented with 1× penicillin-streptomycin (Invitrogen), then plated and cultured as previously described. After 4 rounds of enrichment, the percentage of RFP positive cells reached levels of 50% or higher. DNA from populations was analyzed by PCR for evidence of targeted integration. Ultimately, single cell subclones were established from positive pools and were analyzed by PCR and PCR-positive clones confirmed by FISH as described below. DNA was purified from pools or single cell clones using previously described methods set forth in Lahm et al., Transgenic Res., 1998; 7:131-134, or in some cases using a Wizard Genomic DNA purification kit (Promega). For FISH analysis, a biotinylated DsRed gene probe was generated by PCR using DsRed specific primers and biotin-labeled dUTP (5' RFP primer: 5'-GGTTTAAAGTGCGCTCCTCCAAGAACGTCATC-3', SEQ ID NO:74; and 3' RFP primer: 5'AGATCTAGAGCCGCCGCTACAGGAACAGGTGGTGGCGGCC-3'; SEQ ID NO:75). To maximize the signal intensity of the DsRed probe, Tyramide amplification was carried out according to the manufacturers protocols (NEN).

[0317] The process of testing the feasibility of a more general targeting strategy that would not rely on enrichment via drug selection of stably transfected clones can be summarized as follows. A red fluorescent protein gene (RFP; encoded by the DsRed gene) was inserted between the loxP sites of the targeting vector to form pBSFKLoxDsRedLox. After transfection with PBSFKLoxDsRedLox, sequential rounds of high speed flow sorting and expansion of sorted cells in culture could then be used to enrich for stable transformants expressing RFP. In the event of targeted integration, PCR screening with primers that amplify from a spacer region within the segment of the 45s pre-rRNA gene in pWEPuro9K to a specific anchor sequence within the DsRed gene in the targeting cassette would give rise to a diagnostic 2.3 Kb band. As rDNA clusters are found on several chromosomes, confirmation of targeting to an ACes would require fluorescence in situ hybridization (FISH) analysis. Finally, the flanking of the DsRed gene by loxP sites would allow for its removal and subsequent replacement with other genes of interest.

[0318] After transfection of the targeting sequence into 5B11-12 cells, enrichment for targeted clones was carried out using a combination of flow cytometry to detect red-fluorescing cells and PCR screening. Ultimately 17 single cell subclones were identified as potential targeted clones by PCR and of these 16 were found by FISH to contain the DsRed integration event into the ACes. These subclones are referred to herein as D11-C4, D11-C12, D11-H3, C9-C9, C9-B9, C9-F4, C9-H8, C9-F2, C9-G8, C9-B6, C9-G3, C9-E12, C9-A11, C11-E3, C11-A9 and C11-H4. PCR analysis of genomic DNA isolated from the D11-C4 subclone gave rise to a 2.3 Kb band, indicative of a targeted integration into an rDNA locus. Further analysis of the subclone by FISH analysis with a DsRed gene probe demonstrated integration of the LoxDsRedLox targeting cassette on the ACes co-localizing with one of the regions of rDNA staining seen on the 5B11-12 ACes, consistent with a targeted integration into an rDNA locus of the ACes, while integrations on other chromosomes were not observed. Since transfected cells were maintained as heterogeneous populations through several cycles of sorting and replating it was not possible to estimate the frequency of targeted events. In most mammalian cell lines the frequency of gene targeting via homologous recombination is roughly 10^-5-10^-7 treated cells. Despite the low frequency of these events in mammalian cells, it is clear that an RFP expression based screening paradigm, coupled with PCR analysis, can effectively detect and enrich for such infrequent events in a large population. In instances where drug selection is not possible or not desirable, such a system may provide a useful alternative. It was also verified that the modified ACes in subclone D11-C4 could be purified by flow cytometry. The results indicate that the flow karyogram of the D11-C4 subclone was unaltered from that of the 5B11-12 cell line. Thus, the D11-C4 ACes can be purified in high yield from native chromosomes of the host cell line.

D. Reduction of LoxP on Aces to a Single Site.

[0319] The strong hybridization signal detected by FISH on the ACes using the DsRed gene probe suggests that several copies of the targeting cassette may be present on the ACes in the D11-C4 line. This also suggests that multiple rDNA genes have been correctly targeted.

[0320] Accordingly, in certain embodiments where necessary, the number of loxP sites on the ACes can be reduced to a single site by in situ treatment with Cre recombinase, provided that the sites are co-linear. Such a process is described for multiple loxP-flanked integrations on a native mouse chromosome (Garrick et al., Nature Genet., 1998, January; 18(1):56-59). Reduction to a single loxP site on the D11-C4 ACes would result in the loss of the DsRed gene, forming the basis of a useful screen for this event.

[0321] For this purpose, a Cre expression plasmid pCX-Cre/GFP III has been generated by first deleting the EcoRI fragment of pCX-eGFP (SEQ ID NO:71) containing the eGFP coding sequence and replacing it with that of a PCR amplified Cre recombinase coding sequence (SEQ ID NO:58), generating pCX-Cre. Next, the AseI/SspI fragment of pD2eGFP-N1 (containing the CMV promoter driving the D2EGFP gene with SV40 polyA signal; Clontech; SEQ ID NO:87) was inserted into the filled HindIII site of pCX-Cre, generating pCX-Cre\GFP III. Control plasmid pCX-CreRev\GFP III was generated in similar fashion except that the Cre recombinase coding sequence was inserted in the antisense orientation. LMTK.sup.- cell line D11-C4 (containing first generation platform ACes with multiple loxP-DsRED sites) and 5B11-12 cell line (containing ACes with no loxP-DsRED sites) are maintained in culture as described above. D11C4 cells are transfected with 2 μg of plasmid pCX-Cre\GFP III or 2 μg pCX-CreRev\GFP III using Lipofectamine (Invitrogen) as previously described.

[0322] Forty-eight to seventy-two hours after transfection, transfected D11-C4 cells are harvested and GFP positive cells are sorted by cell cytometry using a FACSta Vantage cell sorter (Beckton-Dickinson) as follows: All D11-C4 cells transfected with pCX-Cre\GFP III or control plasmid pCX-CreRev\GFP III that exhibit GFP fluorescent higher than the gate level established by untransfected cells are collected and placed in culture a further 7-14 days. After 7-14 days the initial D11-C4 cells are harvested and analyzed by cell cytometry as follows: Untransfected D11-C4 cells are used to establish the gate that defines the RFP positive population, while 5B11-12 cells are used to set the RFP negative gate. The GFP positive population of D11-C4 transfected with pCX-Cre\GFP III should show decreased red fluorescence compared to pCX-CreRev\GFP III transfected or untransfected control D11-C4 cells. The cells exhibiting greatly decreased or no RFP expression are collected and single cell clones subsequently established. These clones will be expanded and analyzed by fluorescence in-situ hybridization and Southern blotting to confirm the removal of loxP-DsRed gene copies.

Example 3

[0323] Construction of Targeting Vector and Transfection into LMtk- Cells for the Generation of Platform Chromosomes Containing Multiple Site-Specific Recombination Sites

[0324] An example of a selectable marker system for the creation of a chromosome-based platform is shown in FIG. 4. This system includes a vector containing the SV40 early promoter immediately followed by (1) a 282 base pair (bp) sequence containing the bacteriophage lambda attP site and (2) the puromycin resistance marker. Initially a PvuII/StuI fragment containing the SV40 early promoter from plasmid pPUR (Clontech Laboratories, Inc., Palo Alto, Calif.; Seq ID No. 30) was subcloned into the EcoRI/CRI site of pNEB 193 (a PUC 19 derivative obtained from New England Biolabs, Beverly, Mass.; SEQ ID No. 32) generating the plasmid pSV40193. The only differences between pUC19 and pNEB 193 are in the polylinker region. A unique AscI site (GGCGCGCC) is located between the BamHI site and the SmaI site, a unique PacI site (TTAATTAA) is located between the BamHI site and the XbaI site and a unique PmeI site (GTTTAAAC) is located between the PstI site and the SalI site.

[0325] The attP site was PCR amplified from lambda genome (GenBank Accession #NC 001416) using the following primers:

TABLE-US-00004 attPUP: CCTTGCGCTAATGCTCTGTTACAGG SEQ ID No. 1 attPDWN: CAGAGGCAGGGAGTGGGACAAAATTG SEQ ID No. 2

[0326] After amplification and purification of the resulting fragment, the attP site was cloned into the SmaI site of pSV40193 and the orientation of the attP site was determined by DNA sequence analysis (plasmid pSV40193attP). The gene encoding puromycin resistance (Puro) was isolated by digesting the plasmid pPUR (Clontech Laboratories, Inc. Palo Alto, Calif.) with AgeI/BamHI followed by filling in the overhangs with Klenow and subsequently cloned into the AscI site downstream of the attP site of pSV40193attP generating the plasmid pSV40193attPsensePUR (FIG. 4; SEQ ID NO:113)).

[0327] The plasmid pSV40193attPsensePUR was digested with ScaI and co-transfected with the plasmid pFK161 (SEQ ID NO: 118) into mouse LMtk- Cells and platform artificial chromosomes were identified and isolated as described above. The process for generating this exemplary platform ACes containing multiple site-specific recombination sites is summarized in FIG. 5. One platform ACes resulting from this experiment is designated B19-18. This platform ACes chromosome may subsequently be engineered to contain target gene expression nucleic acids using the lambda integrase mediated site-specific recombination system as described herein in Example 7 and 8.

Example 4

[0328] Lambda Integrase Mediated Site-Specific Recombination of a RFP Expressing Vector onto Artificial Chromosomes

[0329] In this example, a vector expressing the red fluorescent protein (RFP) was produced and recombined into the attP site residing on an artificial chromosome within LMtk- Cells. This recombination is depicted in FIG. 7.

[0330] A. Construction of Expression Vectors Containing Wildtype and Mutant Lambda Integrase

[0331] Mutations at the glutamic acid at position 174 in the lambda integrase protein relaxes the requirement for the accessory protein IHF during recombination and DNA supercoiling in vitro (see, Miller et al. (1980) Cell 20:721-729; Lange-Gustafson et al. (1984) J. Biol. Chem. 259:12724-12732). Mutations at this site promote attP, attB intramolecular recombination in mammalian cells (Lorbach et al. (2000) J. Mol. Biol. 296:1175-1181).

[0332] To construct nucleic acid encoding the mutant, lambda integrase was PCR amplified from bacteriophage lambda DNA (cI857 ind Sam 7; New England Biolabs) using the following primers:

TABLE-US-00005 Lamint1 (SEQ ID No. 3) TTCGAATTCATGGGAAGAAGGCGAAGTCATGAGCG) Lamint2 (SEQ ID No. 4) (TTCGAATTCTTATTTGATTTCAATTTTGTCCCAC).

The resulting PCR product was digested with EcoR I and cloned into the EcoR I site of pUC19. Lambda integrase was mutated at amino acid position 174 using QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the following oligos (generating a glutamic acid to arginine change at position 174):

TABLE-US-00006 LambdaINTE174R (SEQ ID No. 6) (CGCGCAGCAAAATCTAGAGTAAGGAGATCAAGACTTACGGCTGACG), LamintR174rev (SEQ ID No. 7) (CGTCAGCCGTAAGTCTTGATCTCCTTACTCTAGATTTTGCTGCGCG).

The resulting site directed mutant was confirmed by sequence analysis. The wildtype and mutant lambda genes were cloned into the EcoR I site of pCX creating pCX-LamInt (SEQ ID NO: 127) and pCXLamIntR (FIG. 8; SEQ ID NO: 112).

[0333] The plasmid pCX (SEQ ID No. 70) was derived from plasmid pCXeGFP (SEQ ID No. 71). Excision of the EcoRI fragment containing the eGFP marker generated pCX. To generate plasmid pCXLamINTR (SEQ ID NO: 112) an EcoRI fragment containing the lambda integrase E174R (SEQ ID No. 37) mutation was cloned into the EcoRI site of pCX, and to generate plasmid pCX-LamINT, an EcoRI fragment containing the wild-type lambda integrase was cloned into the EcoRI site of pCX.

[0334] B. Construction of Integration Vector Containing attB and DsRED

[0335] The plasmid pDsRedN1 (Clontech Laboratories, Palo Alto, Calif.; SEQ ID No. 29) was digested with Hpa I and ligated to the following annealed oligos:

TABLE-US-00007 attB1 (SEQ ID No. 8) (TGAAGCCTGCTTTTTTATACTAACTTGAGCGAA) attB2 (SEQ ID No. 9) (TTCGCTCAAGTTAGTATAAAAAAGCAGGCTTCA)

The resulting vector (pDsRedN1-attB) was confirmed by PCR and sequence analysis.

[0336] C. Transfection into LMtk- Cells

[0337] LM(tk-) cells containing the Prototype A ACes (L1-18; Chromos Molecular Systems Inc., Burnaby, BC Canada) were co-transfected with pDsRedN1 or pDsRedN1-attB and either pCXLamInt (SEQ ID NO: 127) or pCXLamIntR (SEQ ID NO: 112) using Lipofectamine Plus Reagent (LifeTechnologies, Gaithersburg, Md.). The transfected cells were grown in DMEM (LifeTechnologies, Gaithersburg, Md.) with 10% FBS (CanSera) and G418 (CalBiochem) at a concentration of 1 mg/ml.

[0338] D. Enrichment by Cell Sorting

[0339] The transfected cells were sorted using a FACs Vantage SE cell sorter (Becton Dickenson) to enrich for cells expressing DsRed. The cells were excited with a 488 nm Argon laser at 200 watts and cells fluorescing in the 585/42 detection channel were collected. The sorted cells were returned to growth medium for recovery and expansion. After three successive enrichments for cells expressing DsRed, single cell sorting into 96 well plates was performed using the same parameters. Duplicate plates of the single cell clones were made for PCR analysis.

[0340] E. PCR Analysis of Single Cell Clones

[0341] Pools of cells from each row and column of the 96 well plate were used for DNA isolation. DNA was prepared using a Wizard Genomic DNA purification kit (Promega Inc, Madison, Wis.). Nested PCR analysis on the DNA pools was performed to confirm the site-specific recombination event using the following primer sets:

TABLE-US-00008 attPdwn2 (SEQ ID No. 10) (TCTTCTCGGGCATAAGTCGGACACC) CMVen (SEQ ID No. 11) (CTCACGGGGATTTCCAAGTCTCCAC) followed by: attPdwn (SEQ ID No. 12) (CAGAGGCAGGGAGTGGGACAAAATTG) CMVen2 (SEQ ID No. 13) (CAACTCCGCCCCATTGACGCAAATG).

The resulting PCR reactions were analyzed by gel electrophoresis and the potential individual clones containing the site-specific recombination event were identified by combining the PCR results of all of the pooled rows and columns for each 96 well plate. The individual clones were then further analyzed by PCR using the following primers that flank the recombination junction. L1 for and F1rev flank the attR junction whereas REDfor and L2rev flank the attL junction (see FIG. 7):

TABLE-US-00009 L1for (SEQ ID No. 14) AGTATCGCCGAACGATTAGCTCTTCA F1rev (SEQ ID No. 15) GCCGATTTCGGCCTATTGGTTAAA REDfor (SEQ ID No. 16) CCGCCGACATCCCCGACTACAAGAA L2rev (SEQ ID No. 17) TTCCTTCGAAGGGGATCCGCCTACC.

[0342] F. Sequence Analysis of Recombination Junctions

[0343] PCR products spanning the recombination junction were Topo-cloned into pcDNA3.1DN5H is (Invitrogen Inc., San Diego, Calif.) and then sequenced by cycle-sequencing. The clones were confirmed to have the correct attR and attL junctions by cycle sequencing.

[0344] G. Fluorescent In Situ Hybridization (FISH)

[0345] The cell lines containing the correct recombination junction sequence were further analyzed by fluorescent in situ hybridization (FISH) by probing with the DsRed coding region labeled with biotin and visualizing with the Tyramide Signal Amplification system (TSA; NEN Life Science Products). The results indicate that the RFP sequence is present on the ACes.

[0346] H. Southern Analysis

[0347] Genomic DNA was harvested from the cell lines containing an ACes with the correct recombinant event and digested with EcoR I. The digested DNAs were separated on a 0.7% agarose gel, transferred and fixed to a nylon membrane and probed with RFP coding sequences. The result showed that there is an integrated copy of RFP coding sequence in each clone.

Example 5

[0348] Delivery of a Second Gene Encoding GFP onto the RFP Platform ACes

[0349] A. Construction of Integration Vector Containing attB and GFP (pD2eGFPIresPuroattB).

[0350] The plasmid pIRESpuro2 (Clontech, Palo Alto, Calif.; SEQ ID NO: 88) was digested with EcoRI and NotI then ligated to the D2eGFP EcoRI-NotI fragment from pD2eGFP-N1 (Clontech, Palo Alto, Calif.) to create pD2eGFPIresPuro2. Subsequently, oligos encoding the attB site were annealed and ligated into the NruI site of pD2eGFPIresPuro2 to create pD2eGFPIresPuroattB. The orientation of attB in the NruI site was determined by PCR.

[0351] B. Transfection of LMtk- Cells

[0352] The LMtk- Cells containing the RFP platform ACes produced in Example 4, which has multiple attP sites, were co-transfected with pCXLamIntR and pD2eGFPIresPuroattB using LipofectAMINE PLUS reagent. Five μg of each vector was placed into a tube containing 750 μl of DMEM (Dulbecco's modified Eagles Medium). Twenty μl of the Plus reagent was added to the DNA and incubated at room temperature for 15 minutes. A mixture of 30 μl of lipofectamine and 750 μl DMEM was added to the DNA mixture and incubated an additional 15 minutes at room temperature. The DNA mixture was then added dropwise to approximately 3 million cells attached to a 10 cm dish in 5 mls of DMEM. The cells were incubated 4 hours (37° C., 5% CO₂) with the DNA-lipid mixture, after which DMEM with 20% fetal bovine serum was added to the dishes to bring the culture medium to 10% fetal bovine serum. The dishes were incubated at 37° C. with 5% CO₂.

[0353] Plasmid pD2eGFPIresPuroattB has a puromycin gene transcriptionally linked to the GFP gene via an IRES element. Two days after the transfection the cells were placed in medium containing puromycin at 4 μg/ml to select for cells containing the pD2eGFPIresPuroattB plasmid integrated into the genome. Twenty-three clones were isolated after 17 days of selection with puromycin. These clones were expanded and then analyzed for the presence of the GFP gene on the ACes by 2-color (RFP/biotin & GFP/digoxigenin) TSA-FISH (NEN) according to the manufacturers protocol. Sixteen of the 23 clones produced a positive FISH signal on the ACes with a GFP probe.

Example 6

[0354] Delivery of ACes into Human Mesenchymal Stem Cells (hSMC)

[0355] A. Transfection

[0356] Transfection conditions for the most efficient delivery of the ACes into hMSCs (Cambrex BioWhittaker Product Code PT-2501, lot# F0658, East Rutherford, N.J.) were assayed using LipofectAMINE PLUS and Superfect. One million prototype B ACes, which is a murine derived 60 Mb ACes having primarily murine pericentric heterochromatin, and carrying a "payload" containing a hygromycin B selectable marker gene and a lacZ reporter gene (see, Telenius et al., 1999, Chrom. Res., 7:3-7; and Kereso et al., 1996, Chrom. Res., 4:226-239; each of which is incorporated herein by reference in its entirety), were combined with 1-12 μl of the transfection agent. In the case of LipofectAMINE PLUS, the PLUS reagent was combined with the ACes for 15 minutes followed by LipofectAMINE for a further 15 minutes. Superfect was complexed for 10 minutes at a ratio of 2 μl Superfect per 1 million ACes. The ACes/transfection agent complex was then applied to 0.5 million recipient cells and the transfection was allowed to proceed according to the manufacturer's protocol. Percent transfected cells was determined on a FACS Vantage flow cytometer with argon laser tuned to 488 nm at 200 mW and FITC fluorescence collected through a standard FITC 530/30 nm band pass filter. After 24 hours, IdUrd labeled ACes were delivered to human MSCs in the range of 30-50%, varying with transfection agent and dose. ACes delivery curves were generated from data collected in experiments that varied the dose of the transfection reagents. Dose response curves of Superfect and LipofectAMINE PLUS, showing delivery of ACes into recipient hMSCs cells, were prepared, measured by transfer of IdUrd labeled ACes and detected by flow cytometry. Superfect shows maximum delivery in the range of 30-50% at doses greater than 2 μl per million ACes. LipofectAMINE PLUS has a 42-48% delivery peak around 5-8 μl per million ACes. These dose curves were then correlated with toxicity data to determine the transfection conditions that will allow for highest potential transfection efficiency. Toxicity was determined by a modified plating efficiency assay (de Jong et al., 2001, Chrom. Research, 9:475-485). The population's normalized plating efficiency (at maximum % delivery doses) was in the range of 0.2-0.4 for Superfect and 0.5-0.6 with LipofectAMINE PLUS.

[0357] Due to the transfected population consisting of mixed cell types, flow cytometry allowed for the assessment of ACes delivery into each sub-population and the purification of the target population. Flow profiles showing forward scatter (cell size) and side scatter (internal cell granularity) revealed three distinct hMSC populations that were gated into three regions: R3 (small cell region), R4 (medium cell region), R5 (large cell region). Transfection conditions were further optimized by re-analyzing delivery curves and assessing the differences in delivery to each sub-population. Dose response curves of Superfect and LipofectAMINE were prepared showing % delivery to each sub-population represented by the gating on basis of cell size and granularity properties of the mixed population. Three distinct hMSC populations were gated and % delivery dose curves generated. Using Superfect and LipofectAMINE PLUS the overall % delivery increased with cell size (80-90% delivery in large cells). LipofectAMINE PLUS at high doses (8-12 μl per 1 million ACes) shows an increase in the overall proportion of chromosome transfer to the small population (10-20%). This suggests an advantage to using this transfection agent if the small-undifferentiated cell population is the desired target host cell.

[0358] B. Expression from Genes on ACes in hMSCs Following the delivery screening process conducted in section (A) above, the most promising results were subjected to further analyses to monitor expression and verify the presence of structurally intact ACes. The transfection conditions employed for these experiments were exactly the same as those that had been used during the screening process. Short-term expression was monitored by transfecting hMSCs with ACes containing a RFP gene (red fluorescent protein) set forth in Example 2C as "D11C4". The unselected population was harvested at 72-96 hours post transfection and % positive fluorescent cells measured by flow cytometry. RFP expression was in the range of 1-20%.

[0359] Long term-gene expression was assayed by selecting for hygromycin B resistant cells over a period of 7-10 days. Cytogenetic analysis was done to detect presence of intact ACes by Fluorescent In Situ hybridization (FISH), where metaphase chromosomes were hybridized to a mouse major satellite-DNA probe (targeting murine pericentric heterochromatin) and a lambda probe (hybridizing to the lacZ gene). The human mesenchymal transfected culture could not undergo standard sub-cloning as diffuse colonies form with limited doublings available for expansion. Cytogenetic analysis was performed on the entire population, sampling over a period of 3-10 days post-transfection. The hygromycin resistant population was then blocked in mitosis with colchicine and analyzed for presence of intact ACes by FISH. Preliminary FISH results show approximately 2-8% of the hMSC-transfected population had an intact ACes. This compared to rat skeletal muscle myoblast clones, which were in the range of 60-95%. To increase the % of intact ACes in the hMSC-transfected population an enrichment step can be utilized as described in Example 2C.

[0360] C. Differentiation of the hMSCs

[0361] In initial experiments where transfected hMSCs cells have been induced to differentiate into adipose or osteocytes, the results indicate that the transfected cells appear to be differentiating at a rate comparable to the untransfected controls and the cultures are lineage specific as tested by microscopic examination, FISH, Oil Red O staining (adipocyte assay), and calcium secretion (osteocyte assay).

[0362] Accordingly, these results indicate that the artificial chromosomes (ACes) provided herein can be successfully transferred into hMSC target cells. Targeting MSCs (such as hMSCs) permits gene transfer into cells in an undifferentiated state where the cells are easier to expand and purify. The genetically modified cells can then be differentiated in vitro or injected into a site in vivo where the microenvironment will induce transformation into specific cell lineages.

Example 7

Delivery of a Promoterless Marker Gene to a Platform ACes

[0363] Platform ACes containing pSV40attPsensePURO (FIG. 4) were constructed as set forth in Examples 3 and 4.

A. Construction of Targeting Vectors.

[0364] The base vector p18attBZeo (3166 bp; SEQ ID NO: 114) was constructed by ligating the 1067 bp HindIII-SspI fragment containing attBZeo, obtained from pLITattBZeo (SEQ ID NO:91), into pUC18 (SEQ ID NO: 122) digested with HindIII and SspI.

[0365] 1. p18attBZEO-eGFP (6119 bp; SEQ ID NO: 126) was constructed by inserting the 2977 bp SpeI-HindIII fragment from pCXeGFP (SEQ ID NO:71; Okabe, et al. (1997) FEBS Lett 407:313-319) containing the eGFP gene into p18attBZeo (SEQ ID NO: 114) digested with HindIII and XbaI.

[0366] 2. p18attBZEO-5'6XHS4eGFP (FIG. 10; 7631 bp; SEQ ID NO: 116) was constructed by ligating the 4465 bp HindIII fragment from pCXeGFPattB(6XHS4)2 (SEQ ID NO: 123) which contains the eGFP gene, under the regulation of the chicken beta actin promoter, 6 copies of the HS4 core element located 5' of the chicken beta actin promoter and the polyadenylation signal into the HindIII site of p18attBZeo (SEQ ID NO: 114).

[0367] 3. p18attBZEO-3'6XHS4eGFP (FIG. 11; 7600 bp; SEQ ID NO: 115) was created by removing the 5'6XHS4 element from p18attBZeo-(6XHS4)2eGFP (SEQ ID NO: 110). p18attBZeo-(6XHS4)2eGFP was digested with EcoRV and SpeI, treated with Klenow and religated to form p18attBZeo3'6XHS4eGFP (SEQ ID NO: 115).

[0368] 4. p18attBZEO-(6XHS4)2eGFP (FIG. 12; 9080bp; SEQ ID NO: 110) was created in two steps. First, the EcoRI-SpeI fragment from pCXeGFPattB(6XHS4)2 (SEQ ID NO: 123) which contains 6 copies of the HS4 core element was ligated into p18attBZeo (SEQ ID NO: 114) digested with EcoRI and XbaI to create p18attBZeo6XHS4 (4615 bp; SEQ ID NO: 117). Next, p18attBZeo6XHS4 was digested with HindIII and ligated to the 4465 bp HindIII fragment from pCXeGFPattB(6XHS4)2 which contains the eGFP gene, under the regulation of the chicken beta actin promoter, 6 copies of the HS4 core element located 5' of the chicken beta actin promoter and the polyadenylation signal.

TABLE-US-00010 TABLE 2 No. clones No. zeocin No. clones with with correct resistant expected PCR sequence at Targeting plasmid clones product size recombination junction p18attBZEO-eGFP 12 12 NT* p18attBZEO- 11 11 NT 5'6XHS4eGFP p18attBZEO- 11 11 NT 3'6XHS4eGFP p18attBZEO- 9 9 4/4 (6XHS4)2eGFP *NT = not tested

B. Transfection and Selection with Drug.

[0369] The mouse cell line containing the 2^nd generation platform ACE, B19-38 (constructed as set forth in Example 3), was plated onto four 10 cm dishes at approximately 5 million cells per dish. The cells were incubated overnight in DMEM with 10% fetal calf serum at 37° C. and 5% CO₂. The following day the cells were transfected with 5 μg of each of the 4 vectors listed in Example 7.A. above and 5 μg of pCXLamIntR (SEQ ID NO: 112), for a total of 10βg per 10 cm dish. Lipofectamine Plus reagent was used to transfect the cells according to the manufacturers protocol. Two days post-transfection zeocin was added to the medium at 500 ug/ml. The cells were maintained in selective medium until colonies formed. The colonies were then ring-cloned (see, e.g., McFarland, 2000, Methods Cell Sci, March; 22(1):63-66).

C. Analysis of Clones (PCR, Sequencing).

[0370] Genomic DNA was isolated from each of the candidate clones with the Wizard kit (Promega) and following the manufacturers protocol. The following primer set was used to analyze the genomic DNA isolated from the zeocin resistant clones: 5 PacSV40-CTG'TTAATTAACTGTGGAATGTGTG TCAGTTAGGGTG (SEQ ID NO:76); Antisense Zeo-TGAACAGGGTCACGTCGTCC (SEQ ID NO:77). PCR amplification with the above primers and genomic DNA from the site-specific integration of any of the 4 zeocin vectors would result in a 673 bp PCR product.

[0371] As set forth in Table 2, of the 4 zeocin resistant candidate clones thusfar analyzed by PCR, all 4 exhibit the correct sequence for a site-specific integration event.

Example 8

Integration of a PCR Product by Site-Specific Recombination.

[0372] In this example a gene is integrated onto the platform ACes by site-specific recombination without cloning said gene into a vector.

A. PCR Primer Design.

[0373] PCR primers are designed to contain an attB site at the 5' end of one of the primers in the primer set. The remaining primers, which could be one or more than one primer, do not contain an attB site, but are complementary to sequences flanking the gene or genes of interest and any associated regulatory sequences. In first example, 2 primers (one containing an attB site) are used to amplify a selective gene such as puromycin.

[0374] In a second example as shown in FIG. 13, the primer set includes primers 1 & 2 that amplify the GFP gene without amplification of an upstream promoter. Primer 1 contains the attB site at the 5' end of the oligo. Primers 3 & 4 are designed to amplify the IRES-blasticidin DNA sequences from the vector pIRESblasticidin. The 5' end of primer 3 contains sequences complementary to the 5' end of primer 2 such that annealing can occur between 5' ends of the two primers.

B. PCR Reaction and Subsequent Ligation to Create Circular Molecules from the PCR Product

[0375] In the first example set forth above in Section A, the two PCR primers are combined with a puromycin DNA template such as pPUR (Clontech), a heat stable DNA polymerase and appropriate conditions for DNA amplification. The resulting PCR product (attB-Puromycin) is then then purified and self-ligated to form a circular molecule.

[0376] In the second example set forth above in Section A, amplification of the GFP gene and IRES-blasticidin sequences is accomplished by combining primers 1 & 2 with DNA template pD2eGFP and primers 3 & 4 with template pIRESblasticidin under appropriate conditions to amplify the desired template. After initial amplification of the two products (attB-GFP & IRES-blasticidin) in separate reactions, a second round of amplification using both of the PCR products from the first round of amplification together with primers 1 and 4 amplifies the fusion product attB-GFP-IRES-blasticidin (FIG. 13). This technique of using complementary sequences in primer design to create a fusion product is employed in Saccharomyces cerevisiae for allele replacement (Erdeniz et al (1997) Gen Res 2:1174-1183). The amplified product is then purified from the PCR reaction mixture by standard methods and ligated to form a circular molecule.

C. Introduction of PCR Product onto the Aces Using a Recombinase

[0377] The circular PCR product is then be introduced to the platform ACes using the bacteriphage lambda integrase E174R. The introduction can be performed in vivo by transfecting the pCXLamIntR (SEQ ID NO: 112) vector encoding the lambda integrase mutant E174R together with the circularized PCR product into a cell line containing the platform ACE.

D. Selection for Marker Gene

[0378] The marker gene (in this case either puromycin, blasticidin or GFP) is used to enrich the population for cells containing the proper integration event. A proper integration event in the second example (FIG. 14) juxtaposes a promoter residing on the platform ACes 5' to the attB-GFP-IRES-Blasticidin PCR product, allowing for transcription of both GFP and blasticidin. If enrichment is done by drug selection, blasticidin is added to the medium on the transfected cells 24-48 hours post-transfection. Selection is maintained until colonies are formed on the plates. If enrichment is done by cell sorting, cells are sorted 2-4 days post-transfection to enrich for cells expressing the fluorescent marker (GFP in this case).

E. Analysis of Clones

[0379] Clonal isolates are analyzed by PCR, FISH and sequence analysis to confirm proper integration events.

Example 9

Construction of a Human Platform ACes "ACE 0.1"

[0380] A. Construction of the targeting vector pPACrDNA

[0381] Genome Systems (IncyteGenomics) was supplied with the primers 5'HETS (GGGCCGAAACGATCTCAACCTATT; SEQ ID NO:78), and 3'HETS (CGCAGCGGCCCTCCTACTC; SEQ ID NO:79), which were used to amplify a 538 bp PCR product homologous to nt 9680-10218 of the human rDNA sequences (GenBank Accession No. U13369) and used as a probe to screen a human genomic P1AC(P1 Artificial Chromosome) library constructed in the vector pCYPAC2 (Ioannou et al. (1994) Nat. Genet. 6(1): 84-89). Genome Systems clone #18720 was isolated in this screen and contains three repeats of human rDNA as assessed by restriction analysis. GS clone #18720, was digested with PmeI, a restriction enzyme unique to a single repeat of the human rDNA (45 Kbp), and then religated to form pPACrDNA (FIG. 15). The insert in pPACrDNA was analyzed by restriction digests and sequence analysis of the 5' and 3' termini. The pPACrDNA, rDNA sequences are homologous to Genbank Accession #U13369, containing an insert of about 45 kB comprising a single repeat beginning from the end of one repeat at ˜33980 (relative to the Genbank sequence) through the beginning of the next repeat up to approximately 35120 (the repeat offset from that listed in the GenBank file). Thus, the rDNA sequence is just over 1 copy of the repeat extending from 33980 (+/-10 bp) to the end of the first repeat (43 Kbp) and continuing into the second repeat to by 35120 (+/-10 bp).

B. Transfection and ACes Formation.

[0382] Five hundred thousand MSU1.1 cells (Morgan et al., 1991, Exp. Cell Res., November; 197(1):125-136; provided by Dr. Justin McCormick at Michigan State

[0383] University) were plated per 6 cm plate (3 plates total) and allowed to grow overnight. The cells were 70-80% confluent the following day. One plate was transfected with 15 μg pPACrDNA (linearized with Pme I) and 2 μg pSV40attPsensePuro (linearized with Sca I; see Example 3). The remaining plates were controls and were transfected with either 20 μg pBS (Stratagene) or 20 μg pSV40attBsensePuro (linearized with Sca I). All three plates were transfected using a CaPO₄ protocol.

C. Selection of Puromycin Resistant Colonies

[0384] One day post-transfection the cells were "glycerol shocked" by the addition of PBS medium containing 10% glycerol for 30 seconds. Subsequently, the glycerol was removed and replaced with fresh DMEM. Four days post-transfection selective medium was added. Selective medium contains 1 ug/ml puromycin. The transfection plates were maintained at 37° C. with 5% CO₂ in selective medium for 2 weeks at which point colonies could be seen on the plate transfected with pPACrDNA and pSV40attPsensePuro. The colonies were ring-cloned from the plate on day 17 post-selection and expanded in selective medium for analysis. Only two colonies (M2-2d & M2-2b) were able to proliferate in the selective medium after cloning. No colonies were seen on the control plates after 37 days in selective medium.

D. Analysis of Clones

[0385] FISH analysis was performed on the candidate clones to detect ACes formation. Metaphase spreads from the candidate clones were probed in multiple probe combinations. In one experiment, the probes used were biotin-labeled human alphoid DNA (pPACrDNA) and digoxigenin-labeled mouse major DNA (pFK161) as a negative control. Candidate M2-2d was single cell subcloned by flow sorting and the candidate subclones were reanalyzed by FISH. Subclone 1B1 of M2-2d was determined to be a platform ACes and is also designated human Platform ACE 0.1.

Example 10

[0386] Site-Specific Integration of a Marker Gene onto a Human Platform ACE 0.1

[0387] The promoterless delivery method was used to deliver a promoterless blasticidin marker gene onto the human platform ACes with excellent results. The human ACes platform with a promoterless blasticidin marker gene resulted in 21 of 38 blasticidin resistant clones displaying a PCR product of the expected size from the population co-transfected with pLIT38attBBSRpolyA10 and pCXLamIntR (FIG. 8; SEQ ID NOs. 111 and 112). Whereas, the population transfected with pBlueScript resulted in 0 blasticidin resistant colonies.

A. Construction of pLIT38attB-BSRpolyA10 & pLIT38attB BSRpolyA2.

[0388] The vector pLITMUS 38 (New England Biolabs; U.S. Pat. No. 5,691,140; SEQ ID NO: 119) was digested with EcoRV and ligated to two annealed oligomers, which form an attB site (attB1 5'-TGAAGCCTGCTTTTTTATACTAACTTGAGCGAA-3' (SEQ ID NO:8); attB2 5'-TTCGCTCAAGTTAGTATAAAAAAGCAGGCTTCA-3'; SEQ ID NO:9). This ligation reaction resulted in the vector pLIT38attB (SEQ ID NO: 120). The blasticidin resistance gene and SV40 polyA site was PCR amplified with primers: 5BSD (ACCATGAAAACATTTAACATTTCTCAACA; SEQ ID NO:80) and SV40polyA (TTTATTTGTGAAATTTGTGATGCTATTGC; SEQ ID NO:81) using pPAC4 (Frengen, E., et al. (2000) Genomics 68 (2), 118-126; GenBank Accession No. U75992) as template. The blasticidin-SV40polyA PCR product was then ligated into pLIT38attB at the BamHI site, which was Klenow treated following digestion with BamHI. pLIT38attB-BSDpolyA10 (SEQ ID NO: 111) and pLIT38attB-BSDpolyA2 (SEQ ID NO: 121) are the two resulting orientations of the PCR product ligated into the vector.

B. Transfection of MSU1.1 Cells Containing Human Platform ACE 0.1.

[0389] MSU1.1 cells containing human platform ACE 0.1 (see Example 9) was expanded and plated to five 10 cm dishes with 1.3×10⁶ cells per dish. The cells were incubated overnight in DMEM with 10% fetal bovine serum, at 37° C. and 5% CO₂. The following day the cells were transfected with 5 μg of each plasmid as set forth in Table 3, for a total of 10 μg of DNA per plate of cells transfected (see Table 3) using ExGen 500 in vitro transfection reagent (MBI fermentas, cat. no. R0511). The transfection was performed according to the manufacturers protocol. Cells were incubated at 37° C. with 5% CO₂ in DMEM with 10% fetal bovine serum following the transfection.

TABLE-US-00011 TABLE 3 Plate # Plasmid 1 Plasmid 2 No. Bsd^R Colonies 1 pBS None 0 2 pCXLamInt pLIT38attB- 16 BSRpolyA10 3 pCXLamIntR pLIT38attB- 40 BSRpolyA10 4 pCXLamInt pLIT38attB-BSRpolyA2 28 5 pCXLamIntR pLIT38attB-BSRpolyA2 36

C. Selection of Blasticidin Resistant Clones.

[0390] Three days following the transfection the cells were split from a 10 cm dish to two 15 cm dishes. The cells were maintained in DMEM with 10% fetal bovine serum for 4 days in the 15 cm dishes. Seven days post-transfection blasticidin was introduced into the medium. Stably transfected cells were selected with 1 μg/ml blasticidin. The number of colonies formed on each plate is listed in Table 3. These colonies were ring-cloned and expanded for PCR analysis. Upon expansion in blasticidin containing medium some clones failed to live and therefore do not have corresponding PCR data.

D. PCR Analysis

[0391] Thirty-eight of the 40 clones from plate 3 grew after ring-cloning. Genomic DNA was isolated from these clones with the Promega Wizard Genomic cDNA purification kit, digested with EcoRI and used as template in a PCR reaction with the following primers: 3BSP-TTAATTTCGGG TATATTTGAGTGGA (SEQ ID NO:82); 5 PacSV40-CTGTTAATTAACTGTGGAA TGTGTGTCAGTTAGGGTG (SEQ ID NO:76). The PCR conditions were as follows. 100 ng of genomic DNA was amplified with 0.5ul Herculase polymerase (Stratagene) in a 50ul reaction that contained 12.5 pmole of each primer, 2.5 mM of each dNTP, and 1× Herculase buffer (Stratagene). The reactions were placed in a PerkinElmer thermocycler programmed as follows: Initial denaturation at 95° C. for 10 minutes; 35 cycles of 94° C. for 1 minute, 53° C. for 1 minute, 72° C. for 1 minute, and 72° C. for 1 minute; Final extension for 10 minutes at 72° C.; and 4° C. hold. If pLIT38attB-BSRpolyA10 integrates onto the human platform ACE 0.1 correctly, PCR amplification with the above primers should yield an 804 bp product. Twenty-one of the 38 clones from plate 3 produced a PCR product of the expected 804 bp size.

Example 11

Delivery of a Vector Comprising a Promoterless Marker Gene and a Gene Encoding a Therapeutic Product to a Platform Aces

[0392] Platform ACes containing pSV40attPsensePURO (FIG. 4) were constructed as set forth in Examples 3 and 4.

A. Construction of Delivery Vectors

[0393] 1. Erythropoietin cDNA Vector, p18EPOcDNA.

[0394] The erythropoietin cDNA was PCR amplified from a human cDNA library (E. Perkins et al., 1999, Proc. Natl. Acad. Sci. USA 96(5): 2204-2209) using the following primers: EPO5XBA-TATCTAGAATGGGGGTGC ACGAATGTCCTGCC (SEQ ID NO: 83); EPO3BSI-TACGTACGTCATC TGTCCCCTGTCCTGCAGGC (SEQ ID NO: 84). The cDNA was amplified through two successive rounds of PCR using the following conditions: heat denaturation at 95° C. for 3 minutes; 35 cycles of a 30 second denaturation (95° C.), 30 seconds of annealing (60° C.), and 1 minute extension (72° C.); the last cycle is followed by a 7 minute extension at 72° C. BIO-X-ACT (BIOLINE) was used to amplify the erythropoietin cDNA from 2.5 ng of the human cDNA library in the first round of amplification. Five μl of the first amplification product was used as template for the second round of amplification. Two PCR products were produced from the second amplification with Taq polymerase (Eppendorf), each product was cloned into pCR2.1-Topo (Invitrogen) and sequenced. The larger PCR product contained the expected cDNA sequence for erythropoietin. The erythropoietin cDNA was moved from pTopoEPO into p18attBZeo(6XHS4)2eGFP (SEQ ID NO: 110). pTopoEPO was digested with BsiWI and XbaI to release a 588 bp EPO cDNA. BsrGI and BsiWI create compatable ends. The eGFP gene was removed from p18attBZeo(6XHS4)2eGFP by digestion with BsiWI and XbaI, the 8.3 Kbp vector backbone was gel purified and ligated to the 588 bp EPO cDNA to create p18EPOcDNA (SEQ ID NO: 124).

[0395] 2. Genomic Erythropoietin Vector, P18genEPO.

[0396] The erythropoietin genomic clone was PCR amplified from a human genomic library (Clontech) using the following primers: GENEPO3BSI-CGTACGTCATCTGTCCCCT GTCCTGCA (SEQ ID NO: 85); GENEPO 5XBA-TCTAGAATGGGGGT GCACGGTGAGTACT (SEQ ID NO: 86). The reaction conditions for the amplification were as follows: heat denaturation for 3 minutes (95° C.); 30 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (from 65° C. decreasing 0.5° C. per cycle to 50° C.), and 3 minutes extension (72° C.); 15 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (50° C.), and 3 minute extension (72° C.); the last cycle is followed by a 7 minute extension at 72° C. The erythropoietin genomic PCR product (2147 bp) was gel purified and cloned into pCR2.1Topo to create pTopogenEPO. Sequence analysis revealed 2 bp substitutions and insertions in the intronic sequences of the genomic clone of erythropoietin. A partial digest with XbaI and complete digest with BsiWI excised the erythropoietin genomic insert from pTopogenEPO. The resulting 2158 bp genomic erythropoietin fragment was ligated into the 8.3 Kbp fragment resulting from the digestion of p18attBZeo(6XHS4)2eGFP (SEQ ID NO: 110) with XbaI and BsrGI to create p18genEPO (SEQ ID NO: 125).

B. Transfection and Selection with Drug

[0397] The erythropoietin genomic and cDNA genes were each moved onto the platform ACes B 19-38 (constructed as set forth in Example 3) by co-transfecting with pCXLamIntR. Control transfections were also performed using pCXLamInt (SEQ ID NO: 127) together with either p18EPOcDNA (SEQ ID NO: 124) or p18genEPO (SEQ ID NO: 125). Lipofectamine Plus was used to transfect the DNA's into B19-38 cells according to the manufacturer's protocol. The cells were placed in selective medium (DMEM with 10% FBS and Zeocin @ 500 ug/ml) 48 hours post-transfection and maintained in selective medium for 13 days. Clones were isolated 15 days post-transfection.

C. Analysis of Clones (ELISA, PCR)

[0398] 1. ELISA Assays

[0399] Thirty clones were tested for erythropoietin production by an ELISA assay using a monoclonal anti-human erythropoietin antibody (R&D Systems, Catalogue # MAB287), a polyclonal anti-human erythropoietin antibody (R & D Systems, Catalogue # AB-286-NA) and alkaline phosphotase conjugated goat-anti-rabbit IgG (heavy and light chains) (Jackson ImmunoResearch Laboratories, Inc., Catalogue #111-055-144). The negative control was a Zeocin resistant clone isolated from B19-38 cells transfected with p18attBZeo(6XHS4) (SEQ ID NO: 117; no insert control vector) and pCXLamIntR (SEQ ID NO: 112). The preliminary ELISA assay was executed as follows: 1) Nunc-Immuno Plates (MaxiSorb 96-well, Catalogue #439454) were coated with 75 ul of a 1/200 dilution (in Phosphate buffered Saline, pH 7.4 (PBS), Sigma Catalogue # P-3813) of monoclonal anti-human erythropoietin antibody overnight at 4° C. 2) The following day the plates were washed 3 times with 300ul PBS containing 0.15% Tween 20 (Sigma, Catalogue # P-9416). 3) The plates were then blocked with 300ul of 1% Bovine Serum Albumin (BSA; Sigma Catalogue # A-7030) in PBS for 1 hour at 37° C. 4) Repeat the washes as in step 2. 5) The clonal supernatants (75 ul per clone per well of 96-well plate) were then added to the plate and incubated for 1 hour at 37° C. The clonal supernatant analyzed in the ELISA assay had been maintained on the cells 7 days prior to analysis. 6) Repeat the washes of step 2. 7) Add 75 ul of polyclonal anti-human erythropoietin antibody (1/250 dilution in dilution buffer (0.5% BSA, 0.01% Tween 20, 1×PBS, pH 7.4) and incubate 1 hour at 37° C. 8) Repeat washes of step 2. 9) Add 75 ul of goat anti-rabbit conjugated alkaline phosphatase diluted 1/4000 in dilution buffer and incubate 1 hour at 37° C. 10) Repeat washes of step 2. 11) Add 75 ul substrate, p-nitrophenyl phosphate (Sigma N2640), diluted to 1 mg/ml in substrate buffer (0.1 Ethanolamine-HCl (Sigma, Catalogue # E-6133), 5 mM MgCl2 (Sigma, Catalogue # M-2393), pH 9.8). Incubate the plates in the dark for 1 hour at room temperature (22° C.). 12) Read the absorption at 405 nm (reference wavelength 495 nm) on an Universal Microplate Reader (Bio-Tek Instruments, Inc., model # ELX800 UV). The erythropoietin standard curve was derived from readings of diluted human recombinant Erythropoietin (Roche, catalogue #1-120-166; dilution range 125-7.8mUnits/ml). From this preliminary assay the 21 clones displaying the highest expression of erythropoietin were analyzed a second time in the same manner using medium supernatants that had been on the clones for 24 hours and a 1:3 dilution thereof.

[0400] 2. PCR Analysis

[0401] Genomic DNA was isolated from the 21 clones with the best expression (as assessed by the initial ELISA assay above) as well as the B 19-38 cell line and used for PCR analysis. Genomic DNA was isolated using the Wizard genomic DNA purification kit (Promega) according to the manufacturers protocol. Amplification was performed on 100 ng of genomic DNA as template with MasterTaq DNA Polymerase (Eppendorf) and the primer set 5 PacSV40-CTGTTAATTAACTGTGGAATGTGTG TCAGTTAGGGTG (SEQ ID NO: 76) and Antisense Zeo-TGAACAGGGTCACGTCGTCC (SEQ ID NO: 77). The amplification conditions were as follows: heat denaturation for 3 minutes (95° C.); 30 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (from 65° C. decreasing 0.5° C. per cycle to 50° C.), and 1 minutes extension (72° C.); 15 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (50° C.), and 1 minute extension (72° C.); the last cycle is followed by a 10 minute extension at 72° C. PCR products were size separated by gel electrophoresis. Of the 21 clones analyzed 19 produced a PCR product of 650 bp as expected for a site-specific integration event. All nineteen clones were the result of transformations with p19EPOcDNA (5) or p18genEPO (14) and pCXLamIntR (i.e. mutant integrase). The remaining two clones, both of which were the result of transformation with p18genEPO (SEQ ID NO: 125) and pCXLamInt (i.e. wildtype integrase; SEQ ID NO: 127), produced a 400 bp PCR product.

Example 12

Preparation of a Transformation Vector Useful for the Induction of Plant Artificial Chromosome Formation

[0402] Plant artificial chromosomes (PACs) can be generated by introducing nucleic acid, such as DNA, which can include a targeting DNA, for example rDNA or lambda DNA, into a plant cell, allowing the cell to grow, and then identifying from among the resulting cells those that include a chromosome with a structure that is distinct from that of any chromosome that existed in the cell prior to introduction of the nucleic acid. The structure of a PAC reflects amplification of chromosomal DNA, for example, segmented, repeat region-containing and heterochromatic structures. It is also possible to select cells that contain structures that are precursors to PACs, for example, chromosomes containing more than one centromere and/or fragments thereof, and culture and/or manipulate them to ultimately generate a PAC within the cell.

[0403] In the method of generating PACs, the nucleic acid can be introduced into a variety of plant cells. The nucleic acid can include targeting DNA and/or a plant expressable DNA encoding one or multiple selectable markers (e.g., DNA encoding bialophos (bar) resistance) or scorable markers (e.g., DNA encoding GFP). Examples of targeting DNA include, but are not limited to, N. tabacum rDNA intergenic spacer sequence (IGS) and Arabidopsis rDNA such as the 18S, 5.8S, 26S rDNA and/or the intergenic spacer sequence. The DNA can be introduced using a variety of methods, including, but not limited to Agrobacterium-mediated methods, PEG-mediated DNA uptake and electroporation using, for example, standard procedures according to Hartmann et al [(1998) Plant Molecular Biology 36:741]. The cell into which such DNA is introduced can be grown under selective conditions and can initially be grown under non-selective conditions and then transferred to selective media. The cells or protoplasts can be placed on plates containing a selection agent to grow, for example, individual calli. Resistant calli can be scored for scorable marker expression. Metaphase spreads of resistance cultures can be prepared, and the metaphase chromosomes examined by FISH analysis using specific probes in order to detect amplification of regions of the chromosomes. Cells that have artificial chromosomes with functioning centromeres or artificial chromosomal intermediate structures, including, but not limited to, dicentric chromosomes, formerly dicentric chromosomes, minichromosomes, heterochromatin structures (e.g. sausage chromosomes), and stable self-replicating artificial chromosomal intermediates as described herein, are identified and cultured. In particular, the cells containing self-replicating artificial chromosomes are identified.

[0404] The DNA introduced into a plant cell for the generation of PACs can be in any form, including in the form of a vector. An exemplary vector for use in methods of generating PACs can be prepared as follows.

[0405] For the production of artificial chromosomes, plant transformation vectors, as exemplified by pAgIIa and pAgIIb, containing a selectable marker, a targeting sequence, and a scorable marker were constructed using procedures well known in the art to combine the various fragments. The vectors can be prepared using vector pAg1 as a base vector and inserting the following DNA fragments into pAg1: DNA encoding β-glucoronidase under the control of the nopaline synthase (NOS) promoter fragment and flanked at the 3' end by the NOS terminator fragment, a fragment of mouse satellite DNA and an N. tabacum rDNA intergenic spacer sequence (IGS). In constructing plant transformation vectors, vector pAg2 can also be used as the base vector.

1. Construction of pAG1

[0406] Vector pAg1 (SEQ. ID. NO: 89) is a derivative of the CAMBIA vector named pCambia 3300 (Center for the Application of Molecular Biology to International Agriculture, i.e., CAMBIA, Can berra, Australia; www.cambia.org), which is a modified version of vector pCambia 1300 to which has been added DNA from the bar gene confering resistance to phosphinothricin. The nucleotide sequence of pCambia 3300 is provided in SEQ. ID. NO: 90. pCambia 3300 also contains a lacZ alpha sequence containing a polylinker region.

[0407] pAg1 was constructed by inserting two new functional DNA fragments into the polylinker of pCambia 3300: one sequence containing an attB site and a promoterless zeomycin resistance-encoding DNA flanked at the 3' end by a SV40 polyA signal sequence, and a second sequence containing DNA from the hygromycin resistance gene (hygromycin phosphotransferase) confering resistance to hygromycin for selection in plants. Although the zeomycin-SV40 polyA signal fusion is not expected to function in plant cells, it can be activated in mammalian cells by insertion of a functional promoter element into the attB site by site-specific recombination catalyzed by the Lambda att integrase. Thus, the inclusion of the attB-zeomycin sequences allows for evaluation of functionality of plant artificial chromosomes in mammalian cells by activation of the zeomycin resistance-encoding DNA, and provides an att site for further insertion of new DNA sequences into plant artificial chromosomes formed as a result of using pAg1 for plant transformation. The second functional DNA fragment allows for selection of plant cells with hygromycin. Thus, pAg1 contains DNA from the bar gene confering resisance to phosphinothricin, DNA from the hygromycin resistance gene, both resistance-encoding DNAs under the control of a separate cauliflower mosaic virus (CaMV) 35S promoter, and the attB-promoterless zeomycin resistance-encoding DNA.

[0408] pAg1 is a binary vector containing Agrobacterium right and left T-DNA border sequences for use in Agrobacterium-mediated transformation of plant cells or protoplasts with the DNA located between the border sequences. pAg1 also contains the pBR322 Ori for replication in E. coli. pAg1 was constructed by ligating HindIII/PstI-digested p3300attBZeo with HindIII/PstI-digested pBSCaMV35SHyg as follows.

[0409] a. Generation of p3300attBZeo

[0410] Plasmid pCambia 3300 was digested with PstI/Ecl136 II and ligated with PstI/StuI-digested pLITattBZeo (the nucleotide sequence of pLITattBZeo is provided in SEQ. ID. NO: 91. (containing DNA encoding the zeocin resistance gene and an attB Integrase recognition sequence) to generate p3300attBZeo which contains an attB site, a promoterless zeomycin resistance-encoding DNA flanked at the 3' end by a SV40 polyA signal, and a reconstructed PstI site.

[0411] b. Generation of pBSCaMV35SHyg

[0412] A DNA fragment containing DNA encoding hygromycin phosphotransferase flanked by the CaMV 35S promoter and the CaMV 35S polyA signal sequence was obtained by PCR amplification of plasmid pCambia 1302 (GenBank Accession No. AF234298 and SEQ. ID. NO: 92). The primers used in the amplification reaction were as follows:

TABLE-US-00012 CaMV35SpolyA: SEQ. ID. NO: 93 5'-CTGAATTAACGCCGAATTAATTCGGGGGATCTG-3' CaMV35Spr: SEQ. ID. NO: 94 5'-CTAGAGCAGCTTGCCAACATGGTGGAGCA-3'

The 2100-bp PCR fragment was ligated with EcoRV-digested pBluescript II SK+ (Stratagene, La Jolla, Calif., U.S.A.) to generate pBSCaMV35SHyg.

[0413] c. Generation of pAg1

[0414] To generate pAg1, pBSCaMV35SHyg was digested with HindIII/PstI and ligated with HindIII/PstI-digested p3300attBZeo. Thus, pAg1 contains the pCambia 3300 backbone with DNA conferring resistance to phosphinothricin and hygromycin under the control of separate CaMV 35S promoters, an attB-promoterless zeomycin resistance-encoding DNA recombination cassette and unique sites for adding additional markers, e.g., DNA encoding GFP. The attB site can be used as described herein for the addition of new DNA sequences to plant artificial chromosomes, including PACs formed as a result of using the pAg1 vector, or derivatives thereof, in the production of PACs. The attB site provides a convenient site for recombinase-mediated insertion of DNAs containing a homologous att site.

2. pAG2

[0415] The vector pAg2 (SEQ. ID. NO: 95) is a derivative of vector pAg1 formed by adding DNA encoding a green fluorescent protein (GFP), under the control of a NOS promoter and flanked at the 3' end by a NOS polyA signal, to pAg1. pAg2 was constructed as follows. A DNA fragment containing the NOS promoter was obtained by digestion of pGEM-T-NOS, or pGEMEasyNOS (SEQ. ID. NO: 96), containing the NOS promoter in the cloning vector pGEM-T-Easy (Promega Biotech, Madison, Wis., U.S.A.), with XbaI/NcoI and was ligated to an XbaI/NcoI fragment of pCambia 1302 containing DNA encoding GFP (without the CaMV ³⁵S promoter) to generate p1302NOS (SEQ. ID. NO: 97) containing GFP-encoding DNA in operable association with the NOS promoter. Plasmid p1302NOS was digested with SmaI/BsiWI to yield a fragment containing the NOS promoter and GFP-encoding DNA. The fragment was ligated with PmeI/BsiWI-digested pAg1 to generate pAg2. Thus, pAg2 contains DNA from the bar gene confering resistance to phosphinothricin, DNA conferring resistance to hygromycin, both resistance-encoding DNAs under the control of a cauliflower mosaic virus 35S promoter, DNA encoding kanamycin resistance, a GFP gene under the control of a NOS promoter and the attB-zeomycin resistance-encoding DNA. One of skill in the art will appreciate that other fragments can be used to generate the pAg1 and pAg2 derivatives and that other heterlogous DNA can be incorporated into pAg1 and pAg2 derivatives using methods well known in the art.

3. pAgIIa and pAgIIb Transformation Vectors

[0416] Vectors pAgIIa and pAgIIb were constructed by inserting the following DNA fragments into pAg1: DNA encoding β-glucoronidase, the nopaline synthase terminator fragment, the nopaline synthase (NOS) promoter fragment, a fragment of mouse satellite DNA and an N. tabacum rDNA intergenic spacer sequence (IGS). The construction of pAgIIa and pAgIIb was as follows.

[0417] An N. tabacum rDNA intergenic spacer (IGS) sequence (SEQ. ID. NO: 98; see also GenBank Accession No. Y08422; see also Borysyuk et al. (2000) Nature Biotechnology 18:1303-1306; Borysyuk et al. (1997) Plant Mol. Biol. 35:655-660; U.S. Pat. Nos. 6,100,092 and 6,355,860) was obtained by PCR amplification of tobacco genomic DNA. The IGS can be used as a targeting sequence by virtue of its homology to tobacco rDNA genes; the sequence is also an amplification promoter sequence in plants. This fragment was amplified using standard PCR conditions (e.g., as described by Promega Biotech, Madison, Wis., U.S.A.) from tobacco genomic DNA using the primers shown below:

TABLE-US-00013 NTIGS-F1 (SEQ ID No. 99) 5'-GTG CTA GCC AAT GTT TAA CAA GAT G-3' and NTIGS-R1 (SEQ ID No. 100) 5'-ATG TCT TAA AAA AAA AAA CCC AAG TGA C-3'

Following amplification, the fragment was cloned into pGEM-T Easy to give pIGS-I

[0418] A fragment of mouse satellite DNA (Msat1 fragment; GenBank Accession No. V00846; and SEQ ID No. 101) was amplified via PCR from pSAT-1 using the following primers:

TABLE-US-00014 MSAT-F1 (SEQ ID No. 102) 5'-AAT ACC GCG GAA GCT TGA CCT GGA ATA TCG C-3' and MSAT-Ri (SEQ ID No. 103) 5'-ATA ACC GCG GAG TCC TTC AGT GTG CA T-3'

This amplification added a SacII and a HindIII site at the 5' end and a SacII site at the 3' end of the PCR fragment. This fragment was then cloned into the SacII site in pIGS-1 to give pMIGS-1, providing a eukaryotic centromere-specific DNA and a convenient DNA sequence for detection via FISH.

[0419] A functional marker gene containing a NOS-promoter:GUS:NOS terminator fusion was then constructed containing the NOS promoter (GenBank Accession No. U09365; SEQ ID No. 104), E. coli β-glucuronidase coding sequence (from the GUS gene; GenBank Accession No. S69414; and SEQ ID No. 105), and the nopaline synthase terminator sequence (GenBank Accession No. U09365; SEQ ID No. 107). The NOS promoter in pGEM-T-NOS was added to a promoterless GUS gene in pBlueScript (Stratagene, La Jolla, Calif., U.S.A.) using NotI/SpeI to form pNGN-1, which has the NOS promoter in the opposite orientation relative to the GUS gene.

[0420] pMIGS-1 was digested with NotI/SpeI to yield a fragment containing the mouse major satellite DNA and the tobacco IGS which was then added to NotI-digested pNGN-1 to yield pNGN-2. The NOS promoter was then re-oriented to provide a functional GUS gene, yielding pNGN-3, by digestion and religation with SpeI. Plasmid pNGN-3 was then digested with HindIII, and the HindIII fragment containing the β-glucuronidase coding sequence and the rDNA intergenic spacer, along with the Msat sequence, was added to pAG-1 to form pAgIIa (SEQ ID NO: 108), using the unique HindIII site in pAg1 located near the right T-DNA border of pAg1, within the T-DNA region.

[0421] Another plasmid vector, referred to as pAgIIb, was also recovered, which contained the inserted HindIII fragment (SEQ ID NO: 108) in the opposite orientation relative to that observed in pAgIIa. Thus, pAgIIa and pAgIIb differ only in the orientation of the HindIII fragment containing the mouse major satellite sequence, the GUS DNA sequence and the IGS sequence. The nucleotide sequences of pAgIIa is provided in SEQ. ID. NOS: 109.

[0422] Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

Sequence CWU 1

129125DNAArtificial SequencePrimer attPUP 1ccttgcgcta atgctctgtt acagg 25226DNAArtificial SequencePrimer attPDWN 2cagaggcagg gagtgggaca aaattg 26335DNAArtificial SequencePrimer Lamint 1 3ttcgaattca tgggaagaag gcgaagtcat gagcg 35434DNAArtificial SequencePrimer Lamint 2 4ttcgaattct tatttgattt caattttgtc ccac 34520DNAArtificial SequencePrimer 5cggacaatgc ggttgtgcgt 20646DNAArtificial Sequenceprimer 6cgcgcagcaa aatctagagt aaggagatca agacttacgg ctgacg 46746DNAArtificial SequenceLambdaINTER174rev 7cgtcagccgt aagtcttgat ctccttactc tagattttgc tgcgcg 46833DNAArtificial SequenceattB1 8tgaagcctgc ttttttatac taacttgagc gaa 33933DNAArtificial SequenceattB2 9ttcgctcaag ttagtataaa aaagcaggct tca 331025DNAArtificial SequencePrimer attPdwn2 10tcttctcggg cataagtcgg acacc 251125DNAArtificial SequencePrimerCMVen 11ctcacgggga tttccaagtc tccac 251226DNAArtificial SequencePrimerattPdwn 12cagaggcagg gagtgggaca aaattg 261325DNAArtificial SequencePrimerCMVEN2 13caactccgcc ccattgacgc aaatg 251426DNAArtificial SequencePrimerL1 14agtatcgccg aacgattagc tcttca 261524DNAArtificial SequencePrimerF1 rev 15gccgatttcg gcctattggt taaa 241625DNAArtificial SequencePrimerRED 16ccgccgacat ccccgactac aagaa 251725DNAArtificial SequencePrimerL2rev 17ttccttcgaa ggggatccgc ctacc 251822118DNAMus musculusGenBank X825641996-04-09 18gaattcccct atccctaatc cagattggtg gaataacttg gtatagatgt ttgtgcatta 60aaaaccctgt aggatcttca ctctaggtca ctgttcagca ctggaacctg aattgtggcc 120ctgagtgata ggtcctggga catatgcagt tctgcacaga cagacagaca gacagacaga 180cagacagaca gacagacgtt acaaacaaac acgttgagcc gtgtgccaac acacacacaa 240acaccactct ggccataatt attgaggacg ttgatttatt attctgtgtt tgtgagtctg 300tctgtctgtc tgtctgtctg tctgtctgtc tatcaaacca aaagaaacca aacaattatg 360cctgcctgcc tgcctgcctg cctacacaga gaaatgattt cttcaatcaa tctaaaacga 420cctcctaagt ttgccttttt tctctttctt tatctttttc ttttttcttt tcttcttcct 480tccttccttc cttccttcct tccttccttt ctttctttct ttctttcttt cttactttct 540ttctttcctt cttacattta ttcttttcat acatagtttc ttagtgtaag catccctgac 600tgtcttgaag acactttgta ggcctcaatc ctgtaagagc cttcctctgc ttttcaaatg 660ctggcatgaa tgttgtacct cactatgacc agcttagtct tcaagtctga gttactggaa 720aggagttcca agaagactgg ttatattttt catttattat tgcattttaa ttaaaattta 780atttcaccaa aagaatttag actgaccaat tcagagtctg ccgtttaaaa gcataaggaa 840aaagtaggag aaaaacgtga ggctgtctgt ggatggtcga ggctgcttta gggagcctcg 900tcaccattct gcacttgcaa accgggccac tagaacccgg tgaagggaga aaccaaagcg 960acctggaaac aataggtcac atgaaggcca gccacctcca tcttgttgtg cgggagttca 1020gttagcagac aagatggctg ccatgcacat gttgtctttc agcttggtga ggtcaaagta 1080caaccgagtc acagaacaag gaagtataca cagtgagttc caggtcagcc agagtttaca 1140cagagaaacc acatcttgaa aaaaacaaaa aaataaatta aataaatata atttaaaaat 1200ttaaaaatag ccgggagtga tggcgcatgt ctttaatccc agctctcttc aggcagagat 1260gggaggattt ctgagtttga ggccagcctg gtctgcaaag tgagttccag gacagtcagg 1320gctatacaga gaaaccctgt cttgaaaact aaactaaatt aaactaaact aaactaaaaa 1380aatataaaat aaaaatttta aagaatttta aaaaactaca gaaatcaaac ataagcccac 1440gagatggcaa gtaactgcaa tcatagcaga aatattatac acacacacac acacagactc 1500tgtcataaaa tccaatgtgc cttcatgatg atcaaatttc gatagtcagt aatactagaa 1560gaatcatatg tctgaaaata aaagccagaa ccttttctgc ttttgttttc ttttgcccca 1620agatagggtt tctctcagtg tatccctggc atccctgcct ggaacttcct ttgtaggttt 1680ggtagcctca aactcagaga ggtcctctct gcctgcctgc ctgcctgcct gcctgcctgc 1740ctgcctgcct gcctgcctca cttcttctgc cacccacaca accgagtcga acctaggatc 1800tttatttctt tctctttctc tcttctttct ttctttcttt ctttctttct ttctttcttt 1860ctttctttct ttcttattca attagttttc aatgtaagtg tgtgtttgtg ctctatctgc 1920tgcctatagg cctgcttgcc aggagagggc aacagaacct aggagaaacc accatgcagc 1980tcctgagaat aagtgaaaaa acaacaaaaa aaggaaattc taatcacata gaatgtagat 2040atatgccgag gctgtcagag tgctttttaa ggcttagtgt aagtaatgaa aattgttgtg 2100tgtcttttat ccaaacacag aagagaggtg gctcggcctg catgtctgtt gtctgcatgt 2160agaccaggct ggccttgaac acattaatct gtctgcctct gcttccctaa tgctgcgatt 2220aaaggcatgt gccaccactg cccggactga tttcttcttt tttttttttt tggaaaatac 2280ctttctttct ttttctctct ctctttcttc cttccttcct ttctttctat tctttttttc 2340tttctttttt cttttttttt ttttttttaa aatttgccta aggttaaagg tgtgctccac 2400aattgcctca gctctgctct aattctcttt aaaaaaaaac aaacaaaaaa aaaaccaaaa 2460cagtatgtat gtatgtatat ttagaagaaa tactaatcca ttaataactc ttttttccta 2520aaattcatgt cattcttgtt ccacaaagtg agttccagga cttaccagag aaaccctgtg 2580ttcaaatttc tgtgttcaag gtcaccctgg cttacaaagt gagttccaag tccgataggg 2640ctacacagaa aaaccatatc tcagaaaaaa aaaaagttcc aaacacacac acacacacac 2700acacacacac acacacacac acacacacac acacacacag cgcgccgcgg cgatgagggg 2760aagtcgtgcc taaaataaat atttttctgg ccaaagtgaa agcaaatcac tatgaagagg 2820tactcctaga aaaaataaat acaaacgggc tttttaatca ttccagcact gttttaattt 2880aactctgaat ttagtcttgg aaaagggggc gggtgtgggt gagtgagggc gagcgagcag 2940acgggcgggc gggcgggtga gtggccggcg gcggtggcag cgagcaccag aaaacaacaa 3000accccaagcg gtagagtgtt ttaaaaatga gacctaaatg tggtggaacg gaggtcgccg 3060ccaccctcct cttccactgc ttagatgctc ccttcccctt actgtgctcc cttcccctaa 3120ctgtgcctaa ctgtgcctgt tccctcaccc cgctgattcg ccagcgacgt actttgactt 3180caagaacgat tttgcctgtt ttcaccgctc cctgtcatac tttcgttttt gggtgcccga 3240gtctagcccg ttcgctatgt tcgggcggga cgatggggac cgtttgtgcc actcgggaga 3300agtggtgggt gggtacgctg ctccgtcgtg cgtgcgtgag tgccggaacc tgagctcggg 3360agaccctccg gagagacaga atgagtgagt gaatgtggcg gcgcgtgacg gatctgtatt 3420ggtttgtatg gttgatcgag accattgtcg ggcgacacct agtggtgaca agtttcggga 3480acgctccagg cctctcaggt tggtgacaca ggagagggaa gtgcctgtgg tgaggcgacc 3540agggtgacag gaggccgggc aagcaggcgg gagcgtctcg gagatggtgt cgtgtttaag 3600gacggtctct aacaaggagg tcgtacaggg agatggccaa agcagaccga gttgctgtac 3660gcccttttgg gaaaaatgct agggttggtg gcaacgttac taggtcgacc agaaggctta 3720agtcctaccc ccccccccct tttttttttt tttcctccag aagccctctc ttgtccccgt 3780caccgggggc accgtacatc tgaggccgag aggacgcgat gggcccggct tccaagccgg 3840tgtggctcgg ccagctggcg cttcgggtct tttttttttt tttttttttt ttttcctcca 3900gaagccttgt ctgtcgctgt caccgggggc gctgtacttc tgaggccgag aggacgcgat 3960gggccccggc ttccaagccg gtgtggctcg gccagctgga gcttcgggtc tttttttttt 4020tttttttttt tttttttctc cagaagcctt gtctgtcgct gtcaccgggg gcgctgtact 4080tctgaggccg agaggacgcg atgggtcggc ttccaagccg atgtggcggg gccagctgga 4140gcttcgggtt tttttttttc ctccagaagc cctctcttgt ccccgtcacc gggggcgctg 4200tacttctgag gccgagagga cgtgatgggc ccgggttcca ggcggatgtc gcccggtcag 4260ctggagcttt ggatcttttt tttttttttt cctccagaag ccctctcttg tccccgtcac 4320cgggggcacc ttacatctga gggcgagagg acgtgatggg tccggcttcc aagccgatgt 4380ggcggggcca gctggagctt cgggtttttt ttttttcctc cagaagccct ctcttgtccc 4440cgtcaccggg ggcgctgtac ttctgaggcc gagaggacgt gatgggcccg ggttccaggc 4500ggatgtcgcc cggtcagctg gagctttgga tcattttttt ttttccctcc agaagccctc 4560tcttgtcccc gtcaccgggg gcaccgtaca tctgaggccg agaggacacg atgggcctgt 4620cttccaagcc gatgtggccc ggccagctgg agcttcgggt cttttttttt ttttttcctc 4680cagaagcctt gtctgtcgct gtcacccggg gcgctgtact tctgaggccg agaggacgcg 4740atgggcccgg cttccaagcc ggtgtggctc ggccagctgg agcttcgggt cttttttttt 4800tttttttttt ttcctccaga aaccttgtct gtcgctgtca cccggggcgc ttgtacttct 4860gatgccgaga ggacgcgatg ggcccgtctt ccaggccgat gtggcccggt cagctggagc 4920tttggatctt tttttttttt ttttcctcca gaagccctct cttgtccccg tcaccggggg 4980caccttacat ctgaggccta gaggacacga tgggcccggg ttccaggccg atgtggcccg 5040gtcagctgga gctttggatc tttttttttt ttttcttcca gaagccctct tgtccccgtc 5100accggtggca ctgtacatct gaggcggaga ggacattatg ggcccggctt ccaatccgat 5160gtggcccggt cagctggagc tttggatctt attttttttt taattttttc ttccagaagc 5220cctcttgtcc ctgtcaccgg tggcacggta catctgaggc cgagaggaca ttatgggccc 5280ggcttccagg ccgatgtggc ccggtcagct ggagctttgg atcttttttt ttttttttct 5340tttttcctcc agaagccctc tctgtccctg tcaccggggg ccctgtacgt ctgaggccga 5400gggaaagcta tgggcgcggt tttctttcat tgacctgtcg gtcttatcag ttctccgggt 5460tgtcagggtc gaccagttgt tcctttgagg tccggttctt ttcgttatgg ggtcattttt 5520gggccacctc cccaggtatg acttccaggc gtcgttgctc gcctgtcact ttcctccctg 5580tctcttttat gcttgtgatc ttttctatct gttcctattg gacctggaga taggtactga 5640cacgctgtcc tttccctatt aacactaaag gacactataa agagaccctt tcgatttaag 5700gctgttttgc ttgtccagcc tattcttttt actggcttgg gtctgtcgcg gtgcctgaag 5760ctgtccccga gccacgcttc ctgctttccc gggcttgctg cttgcgtgtg cttgctgtgg 5820gcagcttgtg acaactgggc gctgtgactt tgctgcgtgt cagacgtttt tcccgatttc 5880cccgaggtgt cgttgtcaca cctgtcccgg ttggaatggt ggagccagct gtggttgagg 5940gccaccttat ttcggctcac tttttttttt tttttttctc ttggagtccc gaacctccgc 6000tcttttctct tcccggtctt tcttccacat gcctcccgag tgcatttctt tttgtttttt 6060ttcttttttt tttttttttt ttggggaggt ggagagtccc gagtacttca ctcctgtctg 6120tggtgtccaa gtgttcatgc cacgtgcctc ccgagtgcac ttttttttgt ggcagtcgct 6180cgttgtgttc tcttgttctg tgtctgcccg tatcagtaac tgtcttgccc cgcgtgtaag 6240acattcctat ctcgcttgtt tctcccgatt gcgcgtcgtt gctcactctt agatcgatgt 6300ggtgctccgg agttctcttc gggccagggc caagccgcgc caggcgaggg acggacattc 6360atggcgaatg gcggccgctc ttctcgttct gccagcgggc cctcgtctct ccaccccatc 6420cgtctgccgg tggtgtgtgg aaggcagggg tgcggctctc cggcccgacg ctgccccgcg 6480cgcacttttc tcagtggttc gcgtggtcct tgtggatgtg tgaggcgccc ggttgtgccc 6540tcacgtgttt cactttggtc gtgtctcgct tgaccatgtt cccagagtcg gtggatgtgg 6600ccggtggcgt tgcataccct tcccgtctgg tgtgtgcacg cgctgtttct tgtaagcgtc 6660gaggtgctcc tggagcgttc caggtttgtc tcctaggtgc ctgcttctga gctggtggtg 6720gcgctcccca ttccctggtg tgcctccggt gctccgtctg gctgtgtgcc ttcccgtttg 6780tgtctgagaa gcccgtgaga ggggggtcga ggagagaagg aggggcaaga ccccccttct 6840tcgtcgggtg aggcgcccac cccgcgacta gtacgcctgt gcgtagggct ggtgctgagc 6900ggtcgcggct ggggttggaa agtttctcga gagactcatt gctttcccgt ggggagcttt 6960gagaggcctg gctttcgggg gggaccggtt gcagggtctc ccctgtccgc ggatgctcag 7020aatgcccttg gaagagaacc ttcctgttgc cgcagacccc cccgcgcggt cgcccgcgtg 7080ttggtcttct ggtttccctg tgtgctcgtc gcatgcatcc tctctcggtg gccggggctc 7140gtcggggttt tgggtccgtc ccgccctcag tgagaaagtt tccttctcta gctatcttcc 7200ggaaagggtg cgggcttctt acggtctcga ggggtctctc ccgaatggtc ccctggaggg 7260ctcgccccct gaccgcctcc cgcgcgcgca gcgtttgctc tctcgtctac cgcggcccgc 7320ggcctccccg ctccgagttc ggggagggat cacgcggggc agagcctgtc tgtcgtcctg 7380ccgttgctgc ggagcatgtg gctcggcttg tgtggttggt ggctggggag agggctccgt 7440gcacaccccc gcgtgcgcgt actttcctcc cctcctgagg gccgccgtgc ggacggggtg 7500tgggtaggcg acggtgggct cccgggtccc cacccgtctt cccgtgcctc acccgtgcct 7560tccgtcgcgt gcgtccctct cgctcgcgtc cacgactttg gccgctcccg cgacggcggc 7620ctgcgccgcg cgtggtgcgt gctgtgtgct tctcgggctg tgtggttgtg tcgcctcgcc 7680ccccccttcc cgcggcagcg ttcccacggc tggcgaaatc gcgggagtcc tccttcccct 7740cctcggggtc gagagggtcc gtgtctggcg ttgattgatc tcgctctcgg ggacgggacc 7800gttctgtggg agaacggctg ttggccgcgt ccggcgcgac gtcggacgtg gggacccact 7860gccgctcggg ggtcttcgtc ggtaggcatc ggtgtgtcgg catcggtctc tctctcgtgt 7920cggtgtcgcc tcctcgggct cccggggggc cgtcgtgttt cgggtcggct cggcgctgca 7980ggtgtggtgg gactgctcag gggagtggtg cagtgtgatt cccgccggtt ttgcctcgcg 8040tgccctgacc ggtccgacgc ccgagcggtc tctcggtccc ttgtgaggac ccccttccgg 8100gaggggcccg tttcggccgc ccttgccgtc gtcgccggcc ctcgttctgc tgtgtcgttc 8160ccccctcccc gctcgccgca gccggtcttt tttcctctct ccccccctct cctctgactg 8220acccgtggcc gtgctgtcgg accccccgca tgggggcggc cgggcacgta cgcgtccggg 8280cggtcaccgg ggtcttgggg gggggccgag gggtaagaaa gtcggctcgg cgggcgggag 8340gagctgtggt ttggagggcg tcccggcccc gcggccgtgg cggtgtcttg cgcggtcttg 8400gagagggctg cgtgcgaggg gaaaaggttg ccccgcgagg gcaaagggaa agaggctagc 8460agtggtcatt gtcccgacgg tgtggtggtc tgttggccga ggtgcgtctg gggggctcgt 8520ccggccctgt cgtccgtcgg gaaggcgcgt gttggggcct gccggagtgc cgaggtgggt 8580accctggcgg tgggattaac cccgcgcgcg tgtcccggtg tggcggtggg ggctccggtc 8640gatgtctacc tccctctccc cgaggtctca ggccttctcc gcgcgggctc tcggccctcc 8700cctcgttcct ccctctcgcg gggttcaagt cgctcgtcga cctcccctcc tccgtccttc 8760catctctcgc gcaatggcgc cgcccgagtt cacggtgggt tcgtcctccg cctccgcttc 8820tcgccggggg ctggccgctg tccggtctct cctgcccgac ccccgttggc gtggtcttct 8880ctcgccggct tcgcggactc ctggcttcgc ccggagggtc agggggcttc ccggttcccc 8940gacgttgcgc ctcgctgctg tgtgcttggg gggggcccgc tgcggcctcc gcccgcccgt 9000gagcccctgc cgcacccgcc ggtgtgcggt ttcgcgccgc ggtcagttgg gccctggcgt 9060tgtgtcgcgt cgggagcgtg tccgcctcgc ggcggctaga cgcgggtgtc gccgggctcc 9120gacgggtggc ctatccaggg ctcgcccccg ccgacccccg cctgcccgtc ccggtggtgg 9180tcgttggtgt ggggagtgaa tggtgctacc ggtcattccc tcccgcgtgg tttgactgtc 9240tcgccggtgt cgcgcttctc tttccgccaa cccccacgcc aacccaccac cctgctctcc 9300cggcccggtg cggtcgacgt tccggctctc ccgatgccga ggggttcggg atttgtgccg 9360gggacggagg ggagagcggg taagagaggt gtcggagagc tgtcccgggg cgacgctcgg 9420gttggctttg ccgcgtgcgt gtgctcgcgg acgggttttg tcggaccccg acggggtcgg 9480tccggccgca tgcactctcc cgttccgcgc gagcgcccgc ccggctcacc cccggtttgt 9540cctcccgcga ggctctccgc cgccgccgcc tcctcctcct ctctcgcgct ctctgtcccg 9600cctggtcctg tcccaccccc gacgctccgc tcgcgcttcc ttacctggtt gatcctgcca 9660ggtagcatat gcttgtctca aagattaagc catgcatgtc taagtacgca cggccggtac 9720agtgaaactg cgaatggctc attaaatcag ttatggttcc tttggtcgct cgctcctctc 9780ctacttggat aactgtggta attctagagc taatacatgc cgacgggcgc tgacccccct 9840tcccgggggg ggatgcgtgc atttatcaga tcaaaaccaa cccggtgagc tccctcccgg 9900ctccggccgg gggtcgggcg ccggcggctt ggtgactcta gataacctcg ggccgatcgc 9960acgccccccg tggcggcgac gacccattcg aacgtctgcc ctatcaactt tcgatggtag 10020tcgccgtgcc taccatggtg accacgggtg acggggaatc agggttcgat tccggagagg 10080gagcctgaga aacggctacc acatccaagg aaggcagcag gcgcgcaaat tacccactcc 10140cgacccgggg aggtagtgac gaaaaataac aatacaggac tctttcgagg ccctgtaatt 10200ggaatgagtc cactttaaat cctttaacga ggatccattg gagggcaagt ctggtgccag 10260cagccgcggt aattccagct ccaatagcgt atattaaagt tgctgcagtt aaaaagctcg 10320tagttggatc ttgggagcgg gcgggcggtc cgccgcgagg cgagtcaccg cccgtccccg 10380ccccttgcct ctcggcgccc cctcgatgct cttagctgag tgtcccgcgg ggcccgaagc 10440gtttactttg aaaaaattag agtgttcaaa gcaggcccga gccgcctgga taccgcagct 10500aggaataatg gaataggacc gcggttctat tttgttggtt ttcggaactg aggccatgat 10560taagagggac ggccgggggc attcgtattg cgccgctaga ggtgaaattc ttggaccggc 10620gcaagacgga ccagagcgaa agcatttgcc aagaatgttt tcattaatca agaacgaaag 10680tcggaggttc gaagacgatc agataccgtc gtagttccga ccataaacga tgccgactgg 10740cgatgcggcg gcgttattcc catgacccgc cgggcagctt ccgggaaacc aaagtctttg 10800ggttccgggg ggagtatggt tgcaaagctg aaacttaaag gaattgacgg aagggcacca 10860ccaggagtgg gcctgcggct taatttgact caacacggga aacctcaccc ggcccggaca 10920cggacaggat tgacagattg atagctcttt ctcgattccg tgggtggtgg tgcatggccg 10980ttcttagttg gtggagcgat ttgtctggtt aattccgata acgaacgaga ctctggcatg 11040ctaactagtt acgcgacccc cgagcggtcg gcgtccccca acttcttaga gggacaagtg 11100gcgttcagcc acccgagatt gagcaataac aggtctgtga tgcccttaga tgtccggggc 11160tgcacgcgcg ctacactgac tggctcagcg tgtgcctacc ctgcgccggc aggcgcgggt 11220aacccgttga accccattcg tgatggggat cggggattgc aattattccc catgaacgag 11280gaattcccag taagtgcggg tcataagctt gcgttgatta agtccctgcc ctttgtacac 11340accgcccgtc gctactaccg attggatggt ttagtgaggc cctcggatcg gccccgccgg 11400ggtcggccca cggccctggc ggagcgctga gaagacggtc gaacttgact atctagagga 11460agtaaaagtc gtaacaaggt ttccgtaggt gaacctgcgg aaggatcatt aaacgggaga 11520ctgtggagga gcggcggcgt ggcccgctct ccccgtcttg tgtgtgtcct cgccgggagg 11580cgcgtgcgtc ccgggtcccg tcgcccgcgt gtggagcgag gtgtctggag tgaggtgaga 11640gaaggggtgg gtggggtcgg tctgggtccg tctgggaccg cctccgattt cccctccccc 11700tcccctctcc ctcgtccggc tctgacctcg ccaccctacc gcggcggcgg ctgctcgcgg 11760gcgtcttgcc tctttcccgt ccggctcttc cgtgtctacg aggggcggta cgtcgttacg 11820ggtttttgac ccgtcccggg ggcgttcggt cgtcggggcg cgcgctttgc tctcccggca 11880cccatccccg ccgcggctct ggcttttcta cgttggctgg ggcggttgtc gcgtgtgggg 11940ggatgtgagt gtcgcgtgtg ggctcgcccg tcccgatgcc acgcttttct ggcctcgcgt 12000gtcctccccg ctcctgtccc gggtacctag ctgtcgcgtt ccggcgcgga ggtttaagga 12060ccccgggggg gtcgccctgc cgcccccagg gtcggggggc ggtggggccc gtagggaagt 12120cggtcgttcg ggcggctctc cctcagactc catgaccctc ctccccccgc tgccgccgtt 12180cccgaggcgg cggtcgtgtg ggggggtgga tgtctggagc cccctcgggc gccgtggggg 12240cccgacccgc gccgccggct tgcccgattt ccgcgggtcg gtcctgtcgg tgccggtcgt 12300gggttcccgt gtcgttcccg tgtttttccg ctcccgaccc tttttttttc ctccccccca 12360cacgtgtctc gtttcgttcc tgctggccgg cctgaggcta cccctcggtc catctgttct 12420cctctctctc cggggagagg agggcggtgg tcgttggggg actgtgccgt cgtcagcacc 12480cgtgagttcg ctcacacccg aaataccgat acgactctta gcggtggatc actcggctcg 12540tgcgtcgatg aagaacgcag ctagctgcga gaattaatgt gaattgcagg acacattgat 12600catcgacact tcgaacgcac ttgcggcccc gggttcctcc cggggctacg cctgtctgag 12660cgtcggttga cgatcaatcg cgtcacccgc tgcggtgggt gctgcgcggc tgggagtttg 12720ctcgcagggc caacccccca acccgggtcg ggccctccgt ctcccgaagt tcagacgtgt 12780gggcggttgt cggtgtggcg cgcgcgcccg cgtcgcggag cctggtctcc cccgcgcatc 12840cgcgctcgcg gcttcttccc gctccgccgt tcccgccctc gcccgtgcac cccggtcctg 12900gcctcgcgtc ggcgcctccc ggaccgctgc ctcaccagtc tttctcggtc ccgtgccccg 12960tgggaaccca ccgcgccccc gtggcgcccg ggggtgggcg cgtccgcatc tgctctggtc 13020gaggttggcg gttgagggtg tgcgtgcgcc gaggtggtgg tcggtcccct gcggccgcgg 13080ggttgtcggg gtggcggtcg acgagggccg gtcggtcgcc tgcggtggtt gtctgtgtgt 13140gtttgggtct tgcgctgggg gaggcggggt cgaccgctcg cggggttggc gcggtcgccc 13200ggcgccgcgc accctccggc ttgtgtggag ggagagcgag ggcgagaacg gagagaggtg 13260gtatccccgg tggcgttgcg agggagggtt tggcgtcccg cgtccgtccg tccctccctc 13320cctcggtggg cgccttcgcg ccgcacgcgg ccgctagggg cggtcggggc ccgtggcccc

13380cgtggctctt cttcgtctcc gcttctcctt cacccgggcg gtacccgctc cggcgccggc 13440ccgcgggacg ccgcggcgtc cgtgcgccga tgcgagtcac ccccgggtgt tgcgagttcg 13500gggagggaga gggcctcgct gacccgttgc gtcccggctt ccctgggggg gacccggcgt 13560ctgtgggctg tgcgtcccgg gggttgcgtg tgagtaagat cctccacccc cgccgccctc 13620ccctcccgcc ggcctctcgg ggaccccctg agacggttcg ccggctcgtc ctcccgtgcc 13680gccgggtgcc gtctctttcc cgcccgcctc ctcgctctct tcttcccgcg gctgggcgcg 13740tgtcccccct ttctgaccgc gacctcagat cagacgtggc gacccgctga atttaagcat 13800attagtcagc ggaggaaaag aaactaacca ggattccctc agtaacggcg agtgaacagg 13860gaagagccca gcgccgaatc cccgccgcgc gtcgcggcgt gggaaatgtg gcgtacggaa 13920gacccactcc ccggcgccgc tcgtgggggg cccaagtcct tctgatcgag gcccagcccg 13980tggacggtgt gaggccggta gcggccccgg cgcgccgggc tcgggtcttc ccggagtcgg 14040gttgcttggg aatgcagccc aaagcgggtg gtaaactcca tctaaggcta aataccggca 14100cgagaccgat agtcaacaag taccgtaagg gaaagttgaa aagaactttg aagagagagt 14160tcaagagggc gtgaaaccgt taagaggtaa acgggtgggg tccgcgcagt ccgcccggag 14220gattcaaccc ggcggcgcgc gtccggccgt gcccggtggt cccggcggat ctttcccgct 14280ccccgttcct cccgacccct ccacccgcgc gtcgttcccc tcttcctccc cgcgtccggc 14340gcctccggcg gcgggcgcgg ggggtggtgt ggtggtggcg cgcgggcggg gccgggggtg 14400gggtcggcgg gggaccgccc ccggccggcg accggccgcc gccgggcgca cttccaccgt 14460ggcggtgcgc cgcgaccggc tccgggacgg ccgggaaggc ccggtgggga aggtggctcg 14520gggggggcgg cgcgtctcag ggcgcgccga accacctcac cccgagtgtt acagccctcc 14580ggccgcgctt tcgccgaatc ccggggccga ggaagccaga tacccgtcgc cgcgctctcc 14640ctctcccccc gtccgcctcc cgggcgggcg tgggggtggg ggccgggccg cccctcccac 14700ggcgcgaccg ctctcccacc cccctccgtc gcctctctcg gggcccggtg gggggcgggg 14760cggactgtcc ccagtgcgcc ccgggcgtcg tcgcgccgtc gggtcccggg gggaccgtcg 14820gtcacgcgtc tcccgacgaa gccgagcgca cggggtcggc ggcgatgtcg gctacccacc 14880cgacccgtct tgaaacacgg accaaggagt ctaacgcgtg cgcgagtcag gggctcgtcc 14940gaaagccgcc gtggcgcaat gaaggtgaag ggccccgccc gggggcccga ggtgggatcc 15000cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgccgggg 15060aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ttgggcaggg 15120cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 15180cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 15240tttccctcag gatagctggc gctctcgctc ccgacgtacg cagttttatc cggtaaagcg 15300aatgattaga ggtcttgggg ccgaaacgat ctcaacctat tctcaaactt taaatgggta 15360agaagcccgg ctcgctggcg tggagccggg cgtggaatgc gagtgcctag tgggccactt 15420ttggtaagca gaactggcgc tgcgggatga accgaacgcc gggttaaggc gcccgatgcc 15480gacgctcatc agaccccaga aaaggtgttg gttgatatag acagcaggac ggtggccatg 15540gaagtcggaa tccgctaagg agtgtgtaac aactcacctg ccgaatcaac tagccctgaa 15600aatggatggc gctggagcgt cgggcccata cccggccgtc gccgcagtcg gaacggaacg 15660ggacgggagc ggccgcgggt gcgcgtctct cggggtcggg ggtgcgtggc gggggcccgt 15720cccccgcctc ccctccgcgc gccgggttcg cccccgcggc gtcgggcccc gcggagccta 15780cgccgcgacg agtaggaggg ccgctgcggt gagccttgaa gcctagggcg cgggcccggg 15840tggagccgcc gcaggtgcag atcttggtgg tagtagcaaa tattcaaacg agaactttga 15900aggccgaagt ggagaagggt tccatgtgaa cagcagttga acatgggtca gtcggtcctg 15960agagatgggc gagtgccgtt ccgaagggac gggcgatggc ctccgttgcc ctcggccgat 16020cgaaagggag tcgggttcag atccccgaat ccggagtggc ggagatgggc gccgcgaggc 16080cagtgcggta acgcgaccga tcccggagaa gccggcggga ggcctcgggg agagttctct 16140tttctttgtg aagggcaggg cgccctggaa tgggttcgcc ccgagagagg ggcccgtgcc 16200ttggaaagcg tcgcggttcc ggcggcgtcc ggtgagctct cgctggccct tgaaaatccg 16260ggggagaggg tgtaaatctc gcgccgggcc gtacccatat ccgcagcagg tctccaaggt 16320gaacagcctc tggcatgttg gaacaatgta ggtaagggaa gtcggcaagc cggatccgta 16380acttcgggat aaggattggc tctaagggct gggtcggtcg ggctggggcg cgaagcgggg 16440ctgggcgcgc gccgcggctg gacgaggcgc cgccgccctc tcccacgtcc ggggagaccc 16500cccgtccttt ccgcccgggc ccgccctccc ctcttccccg cggggccccg tcgtcccccg 16560cgtcgtcgcc acctctcttc ccccctcctt cttcccgtcg gggggcgggt cgggggtcgg 16620cgcgcggcgc gggctccggg gcggcgggtc caaccccgcg ggggttccgg agcgggagga 16680accagcggtc cccggtgggg cggggggccc ggacactcgg ggggccggcg gcggcggcga 16740ctctggacgc gagccgggcc cttcccgtgg atcgcctcag ctgcggcggg cgtcgcggcc 16800gctcccgggg agcccggcgg gtgccggcgc gggtcccctc cccgcggggc ctcgctccac 16860ccccccatcg cctctcccga ggtgcgtggc gggggcgggc gggcgtgtcc cgcgcgtgtg 16920gggggaacct ccgcgtcggt gttcccccgc cgggtccgcc ccccgggccg cggttttccg 16980cgcggcgccc ccgcctcggc cggcgcctag cagccgactt agaactggtg cggaccaggg 17040gaatccgact gtttaattaa aacaaagcat cgcgaaggcc cgcggcgggt gttgacgcga 17100tgtgatttct gcccagtgct ctgaatgtca aagtgaagaa attcaatgaa gcgcgggtaa 17160acggcgggag taactatgac tctcttaagg tagccaaatg cctcgtcatc taattagtga 17220cgcgcatgaa tggatgaacg agattcccac tgtccctacc tactatccag cgaaaccaca 17280gccaagggaa cgggcttggc ggaatcagcg gggaaagaag accctgttga gcttgactct 17340agtctggcac ggtgaagaga catgagaggt gtagaataag tgggaggccc ccggcgcccg 17400gccccgtcct cgcgtcgggg tcggggcacg ccggcctcgc gggccgccgg tgaaatacca 17460ctactctcat cgttttttca ctgacccggt gaggcggggg ggcgagcccc gaggggctct 17520cgcttctggc gccaagcgtc cgtcccgcgc gtgcgggcgg gcgcgacccg ctccggggac 17580agtgccaggt ggggagtttg actggggcgg tacacctgtc aaacggtaac gcaggtgtcc 17640taaggcgagc tcagggagga cagaaacctc ccgtggagca gaagggcaaa agctcgcttg 17700atcttgattt tcagtacgaa tacagaccgt gaaagcgggg cctcacgatc cttctgacct 17760tttgggtttt aagcaggagg tgtcagaaaa gttaccacag ggataactgg cttgtggcgg 17820ccaagcgttc atagcgacgt cgctttttga tccttcgatg tcggctcttc ctatcattgt 17880gaagcagaat tcaccaagcg ttggattgtt cacccactaa tagggaacgt gagctgggtt 17940tagaccgtcg tgagacaggt tagttttacc ctactgatga tgtgttgttg ccatggtaat 18000cctgctcagt acgagaggaa ccgcaggttc agacatttgg tgtatgtgct tggctgagga 18060gccaatgggg cgaagctacc atctgtggga ttatgactga acgcctctaa gtcagaatcc 18120gcccaagcgg aacgatacgg cagcgccgaa ggagcctcgg ttggccccgg atagccgggt 18180ccccgtccgt cccgctcggc ggggtccccg cgtcgccccg cggcggcgcg gggtctcccc 18240ccgccgggcg tcgggaccgg ggtccggtgc ggagagccgt tcgtcttggg aaacggggtg 18300cggccggaaa gggggccgcc ctctcgcccg tcacgttgaa cgcacgttcg tgtggaacct 18360ggcgctaaac cattcgtaga cgacctgctt ctgggtcggg gtttcgtacg tagcagagca 18420gctccctcgc tgcgatctat tgaaagtcag ccctcgacac aagggtttgt ctctgcgggc 18480tttcccgtcg cacgcccgct cgctcgcacg cgaccgtgtc gccgcccggg cgtcacgggg 18540gcggtcgcct cggcccccgc gcggttgccc gaacgaccgt gtggtggttg ggggggggat 18600cgtcttctcc tccgtctccc gaggacggtt cgtttctctt tccccttccg tcgctctcct 18660tgggtgtggg agcctcgtgc cgtcgcgacc gcggcctgcc gtcgcctgcc gccgcagccc 18720cttgccctcc ggccttggcc aagccggagg gcggaggagg gggatcggcg gcggcggcga 18780ccgcggcgcg gtgacgcacg gtgggatccc catcctcggc gcgtccgtcg gggacggccg 18840gttggagggg cgggaggggt ttttcccgtg aacgccgcgt tcggcgccag gcctctggcg 18900gccggggggg cgctctctcc gcccgagcat ccccactccc gcccctcctc ttcgcgcgcc 18960gcggcggcga cgtgcgtacg aggggaggat gtcgcggtgt ggaggcggag agggtccggc 19020gcggcgcctc ttccattttt tcccccccaa cttcggaggt cgaccagtac tccgggcgac 19080actttgtttt ttttttttcc cccgatgctg gaggtcgacc agatgtccga aagtgtcccc 19140cccccccccc ccccccggcg cggagcggcg gggccactct ggactctttt tttttttttt 19200tttttttttt ttaaattcct ggaaccttta ggtcgaccag ttgtccgtct tttactcctt 19260catataggtc gaccagtact ccgggtggta ctttgtcttt ttctgaaaat cccagaggtc 19320gaccagatat ccgaaagtcc tctctttccc tttactcttc cccacagcga ttctcttttt 19380tttttttttt tttggtgtgc ctctttttga cttatataca tgtaaatagt gtgtacgttt 19440atatacttat aggaggaggt cgaccagtac tccgggcgac actttgtttt tttttttttt 19500tccaccgatg atggaggtcg accagatgtc cgaaagtgtc ccgtcccccc cctccccccc 19560ccgcgacgcg gcgggctcac tctggactct tttttttttt tttttttttt tttaaatttc 19620tggaacctta aggtcgacca gttgtccgtc tttcactcat tcatataggt cgaccggtgg 19680tactttgtct ttttctgaaa atcgcagagg tcgaccagat gtcagaaagt ctggtggtcg 19740ataaattatc tgatctagat ttgtttttct gtttttcagt tttgtgttgt tttgtgttgt 19800tttgtgttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt 19860tttgtgttgt gttgtgttgt gttgtgttgg gttgggttgg gttgggttgg gttgggttgg 19920gttgggttgg gttgggttgt gttgtttggt tttgtgttgt ttggtgttgt tggttttgtt 19980ttgtttgctg ttgttttgtg ttttgcgggt cgaacagttg tccctaaccg agtttttttg 20040tacacaaaca tgcacttttt ttaaaataaa tttttaaaat aaatgcgaaa atcgaccaat 20100tatccctttc cttctctctc ttttttaaaa attttctttg tgtgtgtgtg tgtgtgtgtg 20160tgtgtgtgtg tgcgtgtgtg tgtgtgtgtg cgtgcagcgt gcgcgcgctc gttttataaa 20220tacttataat aataggtcgc cgggtggtgg tagcttcccg gactccagag gcagaggcag 20280gcagacttct gagttcgagg ccagcctggt ctacagagga accctgtctc gaaaaatgaa 20340aataaataca tacatacata catacataca tacatacata catacataca tacatatgag 20400gttgaccagt tgtcaatcct ttagaatttt gtttttaatt aatgtgatag agagatagat 20460aatagataga tggatagagt gatacaaata taggtttttt tttcagtaaa tatgaggttg 20520attaaccact tttccctttt taggtttttt tttttttccc ctgtccatgt ggttgctggg 20580atttgaactc aggaccctgg caggtcaact ggaaaacgtg ttttctatat atataaatag 20640tggtctgtct gctgtttgtt tgtttgcttg cttgcttgct tgcttgcttg cttgcttgct 20700tgcttttttt tttcttctga gacagtattt ctctgtgtaa cctggtgccc tgaaactcac 20760tctgtagacc agcctggcct caatcgaact cagaaatcct cctgcctctt gtctacctcc 20820caattttgga gtaaaggtgt gctacaccac tgcctggcat tattatcatt atcattatta 20880attttattat tagacagaac gaaatcaact agttggtcct gtttcgttaa ttcatttgaa 20940attagttgga ccaattagtt ggctggtttg ggaggtttct tttgtttccg atttgggtgt 21000ttgtggggct ggggatcagg tatctcaacg gaatgcatga aggttaaggt gagatggctc 21060gatttttgta aagattactt ttcttagtct gaggaaaaaa taaaataata ttgggctacg 21120tttcattgct tcatttctat ttctctttct ttctttcttt ctttcagata aggaggtcgg 21180ccagttcctc ctgccttctg gaagatgtag gcattgcatt gggaaaagca ttgtttgaga 21240gatgtgctag tgaaccagag agtttggatg tcaagccgta taatgtttat tacaatatag 21300aaaagttcta acaaagtgat ctttaacttt tttttttttt tttctccttc tacttctact 21360tgttctcact ctgccaccaa cgcgctttgt acattgaatg tgagctttgt tttgcttaac 21420agacatatat tttttctttt ggttttgctt gacatggttt ccctttctat ccgtgcaggg 21480ttcccagacg gccttttgag aataaaatgg gaggccagaa ccaaagtctt ttgaataaag 21540caccacaact ctaacctgtt tggctgtttt ccttcccaag gcacagatct ttcccagcat 21600ggaaaagcat gtagcagttg taggacacac tagacgagag caccagatct cattgtgggt 21660ggttgtgaac cacccaccat gtggttgcct gggatttgaa ctcaggatct tcagaagacg 21720agtcagggct ctaaaccgat gagccatctc tccagccctc ctacattcct tcttaaggca 21780tgaatgatcc cagcatggga agacagtctg ccctctttgt ggtatatcac catatactca 21840ataaaataat gaaatgaatg aagtctccac gtatttattt cttcgagcta tctaaattct 21900ctcacagcac ctccccctcc cccacactgc ctttctccct atgtttgggt ggggctgggg 21960gaggggtggg gtgggggcag ggatctgcat gtcttcttgc aggtctgtga actatttgcg 22020atggcctggt tctctgaact gttgagcctt gtctatccag aggctgactg gctagttttc 22080tacctgaagt ccctgagtga tgatttccct gtgaattc 2211819175DNAMus musculus 19ctcccgcgcg gcccccgtgt tcgccgttcc cgtggcgcgg acaatgcggt tgtgcgtcca 60cgtgtgcgtg tccgtgcagt gccgttgtgg agtgcctcgc tctcctcctc ctccccggca 120gcgttcccac ggttggggac caccggtgac ctcgccctct tcgggcctgg atccg 17520755DNAMus musculus 20ggtctggtgg gaattgttga cctcgctctc gggtgcggcc tttggggaac ggcggggtcg 60gtcgtgcccg gcgccggacg tgtgtcgggg cccacttccc gctcgagggt ggcggtggcg 120gcggcgttgg tagtctcccg tgttgcgtct tcccgggctc ttgggggggg tgccgtcgtt 180ttcggggccg gcgttgcttg gcttacgcag gcttggtttg ggactgcctc aggagtcgtg 240ggcggtgtga ttcccgccgg ttttgcctcg cgtctgcctg ctttgcctcg ggtttgcttg 300gttcgtgtct cgggagcggt ggtttttttt tttttcgggt cccggggaga ggggtttttc 360cgggggacgt tcccgtcgcc ccctgccgcc ggtgggtttt cgtttcgggc tgtgttcgtt 420tccccttccc cgtttcgccg tcggttctcc ccggtcggtc ggccctctcc ccggtcggtc 480gcccggccgt gctgccggac ccccccttct gggggggatg cccgggcacg cacgcgtccg 540ggcggccact gtggtccggg agctgctcgg caggcgggtg agccagttgg aggggcgtca 600tgcccccgcg ggctcccgtg gccgacgcgg cgtgttcttt gggggggcct gtgcgtgcgg 660gaaggctgcg cacgttgtcg gtccttgcga gggaaagagg cttttttttt ttagggggtc 720gtccttcgtc gtcccgtcgg cggtggatcc ggcct 75521463DNAMus musculus 21ggccgaggtg cgtctgcggg ttggggctcg tccggccccg tcgtcctccg ggaaggcgtt 60tagcgggtac cgtcgccgcg ccgaggtggg cgcacgtcgg tgagataacc ccgagcgtgt 120ttctggttgt tggcggcggg ggctccggtc gatgtcttcc cctccccctc tccccgaggc 180caggtcagcc tccgcctgtg ggcttcgtcg gccgtctccc cccccctcac gtccctcgcg 240agcgagcccg tccgttcgac cttccttccg ccttcccccc atctttccgc gctccgttgg 300ccccggggtt ttcacggcgc cccccacgct cctccgcctc tccgcccgtg gtttggacgc 360ctggttccgg tctccccgcc aaaccccggt tgggttggtc tccggccccg gcttgctctt 420cgggtctccc aacccccggc cggaagggtt cgggggttcc ggg 46322378DNAMus musculus 22ggattcttca ggattgaaac ccaaaccggt tcagtttcct ttccggctcc ggccgggggg 60ggcggccccg ggcggtttgg tgagttagat aacctcgggc cgatcgcacg ccccccgtgg 120cggcgacgac ccattcgaac gtctgcccta tcaactttcg atggtagtcg atgtgcctac 180catggtgacc acgggtgacg gggaatcagg gttcgattcc ggagagggag cctgagaaac 240ggctaccaca tccaaggaag gcagcaggcg cgcaaattac ccactcccga cccggggagg 300tagtgacgaa aaataacaat acaggactct ttcgaggccc tgtaattgga atgagtccac 360tttaaatcct ttaagcag 37823378DNAMus musculus 23gatccattgg agggcaagtc tggtgccagc agccgcggta attccagctc caatagcgta 60tattaaagtt gctgcagtta aaaagctcgt agttggatct tgggagcggg cgggcggtcc 120gccgcgaggc gagtcaccgc ccgtccccgc cccttgcctc tcggcgcccc ctcgatgctc 180ttagctgagt tgtcccgcgg ggcccgaagc gtttactttg aaaaaattag agttgtttca 240aagcaggccc gagccgcctg gataccgcca gctaggaaat aatggaatag gaccgcggtt 300cctattttgt ttggttttcg gaactgagcc catgattaag ggaaacggcc gggggcattc 360ccttattgcg ccccccta 37824719DNAMus musculus 24ggatctttcc cgctccccgt tcctcccggc ccctccaccc gcgcgtctcc ccccttcttt 60tcccctctcc ggaggggggg gaggtggggg cgcgtgggcg gggtcggggg tggggtcggc 120gggggaccgc ccccggccgg caaaaggccg ccgccgggcg cacttcaacc gtagcggtgc 180gccgcgaccg gctacgagac ggctgggaag gcccgacggg gaatgtggct cggggggggc 240ggcgcgtctc agggcgcgcc gaaccacctc accccgagtg ttacagccct ccggccgcgc 300tttcgcggaa tcccggggcc gaggggaagc ccgatacccg tcgccgcgct tttcccctcc 360ccccgtccgc ctcccgggcg ggcgtggggg tgggggccgg gccgcccctc ccacgcccgt 420ggtttctctc tctcccggtc tcggccggtt tggggggggg agcccggttg ggggcggggc 480ggactgtcct cagtgcgccc cgggcgtcgt cgcgccgtcg ggcccggggg gttctctcgg 540tcacgccgcc cccgacgaag ccgagcgcac ggggtcggcg gcgatgtcgg ctacccaccc 600gacccgtctt gaaacacgga ccaaggagtc taacgcgtgc gcgagtcagg ggctcgcacg 660aaagccgccg tggcgcaatg aaggtgaagg gccccgtccg ggggcccgag gtgggatcc 71925685DNAMus musculus 25cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgtcgggg 60aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ctgggcaggg 120cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 180cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 240tttccctcag gatagctggc gctctcgcaa ccttcggaag cagttttatc cgggtaaagg 300cggaatggat taggaggtct tggggccgga aacgatctca aactatttct caaactttaa 360atgggtaagg aagcccggct cgctggcgtg gagccgggcg tggaatgcga gtgcctagtg 420ggccactttt ggtaagcaga actggcgctg cgggatgaac cgaacgccgg gttaaggcgc 480ccgatgccga cgctcatcag accccagaaa aggtgttggt tgatatagac agcaggacgg 540tggccatgga agtcggaatc cgctaaggag tgtgtaacaa ctcacctgcc gaatcaacta 600gccctgaaaa tggatggcgc tggagcgtcg ggcccatacc cggccgtcgc cggcagtcgg 660aacgggacgg gacgggagcg gccgc 685265162DNAArtificial SequenceChimeric bacterial plasmid 26gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900gagctcggat cgatatctgc ggccgcgtcg acggaattca gtggatccac tagtaacggc 960cgccagtgtg ctggaattaa ttcgctgtct gcgagggcca gctgttgggg tgagtactcc 1020ctctcaaaag cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat 1080ttgatattca cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa 1140aagacaatct ttttgttgtc aagcttgagg tgtggcaggc ttgagatctg gccatacact 1200tgagtgacaa tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac 1260tgcaggtcga gcatgcatct agggcggcca attccgcccc tctccctccc ccccccctaa 1320cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgtgattttc 1380caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac 1440gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt 1500gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg 1560caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 1620agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 1680aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt 1740accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc 1800gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 1860acgatgataa gcttgccaca acccgggatc caccggtcgc caccatggtg agcaagggcg 1920aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 1980acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 2040agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 2100cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 2160agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca 2220actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 2280tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 2340acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc

aaggtgaact 2400tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga 2460acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 2520ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 2580ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc cctagagctc 2640gctgatcagc ctcgactgtg cctctagttg ccagccatct gttgtttgcc cctcccccgt 2700gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 2760tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 2820caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 2880ttctgaggcg gaaagaacca gctggggctc gagtgcattc tagttgtggt ttgtccaaac 2940tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat 3000catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 3060gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 3120ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 3180gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 3240tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3300cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 3360gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 3420gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3480gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3540ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3600aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3660tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3720ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3780gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3840ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3900ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3960agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4020ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 4080aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 4140tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 4200cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 4260tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 4320cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 4380ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 4440gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4500gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4560gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 4620gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4680tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4740aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 4800cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4860caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4920cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 4980ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 5040aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 5100tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 5160tc 5162275627DNAArtificial SequencepMG plasmid from InvivoGen; IRES sequence modified EMCV nucleotides 2736-3308 27caccggcgaa ggaggcctag atctatcgat tgtacagcta gctcgacatg ataagataca 60ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa 120tttgtgatgc tattgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 180tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca 240gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtagatccat 300ttaaatgtta attaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 360ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 420acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 480tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 540ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 600ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 660ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 720actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 780gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 840tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 900caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 960atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 1020acgttaaggg attttggtca tggctagtta attaagctgc aataaacaat cattattttc 1080attggatctg tgtgttggtt ttttgtgtgg gcttggggga gggggaggcc agaatgactc 1140caagagctac aggaaggcag gtcagagacc ccactggaca aacagtggct ggactctgca 1200ccataacaca caatcaacag gggagtgagc tggatcgagc tagagtccgt tacataactt 1260acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 1320acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 1380ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 1440attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 1500gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 1560ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 1620caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 1680tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 1740tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt 1800tttgacctcc atagaagaca ccgggaccga tccagcctcc gcggccggga acggtgcatt 1860ggaacgcgga ttccccgtgc caagagtgac gtaagtaccg cctatagagt ctataggccc 1920acccccttgg cttcttatgc atgctatact gtttttggct tggggtctat acacccccgc 1980ttcctcatgt tataggtgat ggtatagctt agcctatagg tgtgggttat tgaccattat 2040tgaccactcc cctattggtg acgatacttt ccattactaa tccataacat ggctctttgc 2100cacaactctc tttattggct atatgccaat acactgtcct tcagagactg acacggactc 2160tgtattttta caggatgggg tctcatttat tatttacaaa ttcacatata caacaccacc 2220gtccccagtg cccgcagttt ttattaaaca taacgtggga tctccacgcg aatctcgggt 2280acgtgttccg gacatgggct cttctccggt agcggcggag cttctacatc cgagccctgc 2340tcccatgcct ccagcgactc atggtcgctc ggcagctcct tgctcctaac agtggaggcc 2400agacttaggc acagcacgat gcccaccacc accagtgtgc cgcacaaggc cgtggcggta 2460gggtatgtgt ctgaaaatga gctcggggag cgggcttgca ccgctgacgc atttggaaga 2520cttaaggcag cggcagaaga agatgcaggc agctgagttg ttgtgttctg ataagagtca 2580gaggtaactc ccgttgcggt gctgttaacg gtggagggca gtgtagtctg agcagtactc 2640gttgctgccg cgcgcgccac cagacataat agctgacaga ctaacagact gttcctttcc 2700atgggtcttt tctgcagtca cccgggggat ccttcgaacg tagctctaga ttgagtcgac 2760gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc 2820accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg 2880agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg 2940aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc 3000aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa 3060gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa 3120agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 3180ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg 3240aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca 3300cgataatacc atgggtaagt gatatctact agttgtgacc ggcgcctagt gttgacaatt 3360aatcatcggc atagtatatc ggcatagtat aatacgactc actataggag ggccaccatg 3420tcgactacta accttcttct ctttcctaca gctgagatca ccggtaggag ggccatcatg 3480aaaaagcctg aactcaccgc gacgtctgtc gcgaagtttc tgatcgaaaa gttcgacagc 3540gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta 3600ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt 3660tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg 3720gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa 3780gacctgcctg aaaccgaact gcccgctgtt ctgcaacccg tcgcggagct catggatgcg 3840atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc 3900ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac 3960tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg 4020atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc 4080aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg 4140ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt 4200atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg 4260ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc 4320aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc 4380gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt 4440gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa 4500tgagtcgaga attcgctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc 4560tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 4620ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 4680gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 4740aagggggagg attgggaaga caatagcagg catgcgcagg gcccaattgc tcgagcggcc 4800gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgtaac 4860taacatacgc tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc 4920ccagtgcaag tgcaggtgcc agaacatttc tctatcgaag gatctgcgat cgctccggtg 4980cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg 5040gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt 5100actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg 5160tgaacgttct ttttcgcaac gggtttgccg ccagaacaca gctgaagctt cgaggggctc 5220gcatctctcc ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc cggttgagtc 5280gcgttctgcc gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc taggtaagtt 5340taaagctcag gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca 5400gccggctctc cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt 5460ctgttctgcg ccgttacaga tccaagctgt gaccggcgcc tacgtaagtg atatctacta 5520gatttatcaa aaagagtgtt gacttgtgag cgctcacaat tgatacttag attcatcgag 5580agggacacgt cgactactaa ccttcttctc tttcctacag ctgagat 562728553DNAArtificial SequencepMG plasmid from InvivoGen EMCV IRES sequence 28aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt 60tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg 120acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc 180gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt 240tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta 300taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg 360gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag 420gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac gtgtgtttag 480tcgaggttaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa 540cacgatgata ata 553294692DNAArtificial SequencepDSred1-N1 plasmid from Clontech 29tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 660gatccaccgg tcgccaccat ggtgcgctcc tccaagaacg tcatcaagga gttcatgcgc 720ttcaaggtgc gcatggaggg caccgtgaac ggccacgagt tcgagatcga gggcgagggc 780gagggccgcc cctacgaggg ccacaacacc gtgaagctga aggtgaccaa gggcggcccc 840ctgcccttcg cctgggacat cctgtccccc cagttccagt acggctccaa ggtgtacgtg 900aagcaccccg ccgacatccc cgactacaag aagctgtcct tccccgaggg cttcaagtgg 960gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg 1020caggacggct gcttcatcta caaggtgaag ttcatcggcg tgaacttccc ctccgacggc 1080cccgtaatgc agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc 1140gacggcgtgc tgaagggcga gatccacaag gccctgaagc tgaaggacgg cggccactac 1200ctggtggagt tcaagtccat ctacatggcc aagaagcccg tgcagctgcc cggctactac 1260tacgtggact ccaagctgga catcacctcc cacaacgagg actacaccat cgtggagcag 1320tacgagcgca ccgagggccg ccaccacctg ttcctgtagc ggccgcgact ctagatcata 1380atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 1440ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 1500aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 1560cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt 1620taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 1680ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 1740tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 1800aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt 1860ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc 1920ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg 1980cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct 2040taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 2100tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 2160ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg 2220aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 2280agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 2340agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 2400cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 2460ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga 2520ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc 2580gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 2640gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 2700gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 2760tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 2820agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 2880ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 2940tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 3000acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 3060ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat 3120gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 3180ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 3240tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 3300ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 3360ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 3420cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 3480ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 3540ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga 3600acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt 3660cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat 3720tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa 3780ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca 3840tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 3900ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 3960gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 4020tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 4080ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 4140ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 4200gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 4260ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 4320tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 4380ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 4440agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 4500agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 4560gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 4620tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 4680accgccatgc at 4692304257DNAArtificial SequencepPur plasmid from Clontech 30ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt 60atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 120gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 180actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 240ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 300tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agcttgcatg cctgcaggtc 360ggccgccacg accggtgccg ccaccatccc ctgacccacg cccctgaccc ctcacaagga 420gacgaccttc catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc 480cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg 540tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg 600tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga 660ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg 720agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc 780ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca 840agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc 900ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca

960ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc 1020ccggtgcctg acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc 1080atggctccga ccgaagccga cccgggcggc cccgccgacc ccgcacccgc ccccgaggcc 1140caccgactct agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta 1200aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 1260aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 1320aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 1380tatcatgtct ggatccccag gaagctcctc tgtgtcctca taaaccctaa cctcctctac 1440ttgagaggac attccaatca taggctgccc atccaccctc tgtgtcctcc tgttaattag 1500gtcacttaac aaaaaggaaa ttgggtaggg gtttttcaca gaccgctttc taagggtaat 1560tttaaaatat ctgggaagtc ccttccactg ctgtgttcca gaagtgttgg taaacagccc 1620acaaatgtca acagcagaaa catacaagct gtcagctttg cacaagggcc caacaccctg 1680ctcatcaaga agcactgtgg ttgctgtgtt agtaatgtgc aaaacaggag gcacattttc 1740cccacctgtg taggttccaa aatatctagt gttttcattt ttacttggat caggaaccca 1800gcactccact ggataagcat tatccttatc caaaacagcc ttgtggtcag tgttcatctg 1860ctgactgtca actgtagcat tttttggggt tacagtttga gcaggatatt tggtcctgta 1920gtttgctaac acaccctgca gctccaaagg ttccccacca acagcaaaaa aatgaaaatt 1980tgacccttga atgggttttc cagcaccatt ttcatgagtt ttttgtgtcc ctgaatgcaa 2040gtttaacata gcagttaccc caataacctc agttttaaca gtaacagctt cccacatcaa 2100aatatttcca caggttaagt cctcatttaa attaggcaaa ggaattcttg aagacgaaag 2160ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 2220tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 2280cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 2340aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 2400ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 2460cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 2520agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 2580gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 2640cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 2700gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 2760ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 2820gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 2880gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta 2940cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 3000ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 3060gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 3120gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 3180gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 3240ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 3300gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 3360gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 3420caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 3480ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 3540tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 3600ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 3660tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 3720cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 3780gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 3840ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 3900gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 3960agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 4020tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 4080tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 4140gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 4200caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccag 4257318136DNAArtificial SequencepWE15 cosmid vector 31ctatagtgag tcgtattatg cggccgcgaa ttcttgaaga cgaaagggcc tcgtgatacg 60cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt 120tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 180tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 240gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gcttcctgtt 300tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 360gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 420gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 480gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 540gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 600agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 660ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 720cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 780gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 840cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 900gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 960ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 1020acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 1080ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 1140aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 1200aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 1260ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 1320ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 1380actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 1440caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 1500gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 1560ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 1620cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 1680ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 1740cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg gtttcgccac 1800ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 1860gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 1920tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 1980accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 2040cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt 2100tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga 2160ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag 2220cacgatcatg cgcacccgtc agatccagac atgataagat acattgatga gtttggacaa 2280accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2340ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2400atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa 2460tgtggtatgg ctgattatga tctctagtca aggcactata catcaaatat tccttattaa 2520cccctttaca aattaaaaag ctaaaggtac acaatttttg agcatagtta ttaatagcag 2580acactctatg cctgtgtgga gtaagaaaaa acagtatgtt atgattataa ctgttatgcc 2640tacttataaa ggttacagaa tatttttcca taattttctt gtatagcagt gcagcttttt 2700cctttgtggt gtaaatagca aagcaagcaa gagttctatt actaaacaca gcatgactca 2760aaaaacttag caattctgaa ggaaagtcct tggggtcttc tacctttctc ttcttttttg 2820gaggagtaga atgttgagag tcagcagtag cctcatcatc actagatggc atttcttctg 2880agcaaaacag gttttcctca ttaaaggcat tccaccactg ctcccattca tcagttccat 2940aggttggaat ctaaaataca caaacaatta gaatcagtag tttaacacat tatacactta 3000aaaattttat atttacctta gagctttaaa tctctgtagg tagtttgtcc aattatgtca 3060caccacagaa gtaaggttcc ttcacaaaga tccggaccaa agcggccatc gtgcctcccc 3120actcctgcag ttcgggggca tggatgcgcg gatagccgct gctggtttcc tggatgccga 3180cggatttgca ctgccggtag aactcgcgag gtcgtccagc ctcaggcagc agctgaacca 3240actcgcgagg ggatcgagcc cggggtgggc gaagaactcc agcatgagat ccccgcgctg 3300gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc 3360ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa 3420ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 3480tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 3540tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 3600ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 3660tcgccatggg tcacgacgag atcctcgccg tcgggatgcg cgccttgagc ctggcgaaca 3720gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg 3780cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg 3840tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg 3900caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt 3960cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca 4020gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct 4080tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc 4140cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac 4200ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag 4260atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc 4320agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc 4380ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct 4440ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc 4500accgtttctg cggactggct ttctacgtgt tccgcttcct ttagcagccc ttgcgccctg 4560agtgcttgcg gcagcgtgaa agctttttgc aaaagcctag gcctccaaaa aagcctcctc 4620actacttctg gaatagctca gaggccgagg cggcctaaat aaaaaaaatt agtcagccat 4680ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg 4740ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc 4800ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc 4860ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac agccggatct 4920gcaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat 4980ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc 5040tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag 5100gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg 5160cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cgcataaatc gccgtgacga 5220tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc 5280cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcggca tcccgatgcc 5340gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag 5400caagacgtag cccagcgcgt cgggccgcca tgccggcgat aatggcctgc ttctcgccga 5460aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata 5520ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga 5580cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg 5640cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca 5700agggcatcgg tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta 5760ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca 5820acagtccccc ggccacgggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc 5880gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc 5940acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatct tggcagtcac 6000agcatgcgca tatccatgct tcgaccatgc gctcacaaag taggtgaatg cgcaatgtag 6060tacccacatc gtcatcgctt tccactgctc tcgcgaataa agatggaaaa tcaatctcat 6120ggtaatagtc catgaaaatc cttgtattca taaatcctcc aggtagctat atgcaaattg 6180aaacaaaaga gatggtgatc tttctaagag atgatggaat ctcccttcag tatcccgatg 6240gtcaatgcgc tggatatggg atagatggga atatgctgat ttttatggga cagagttgcg 6300aactgttccc aactaaaatc attttgcacg atcagcgcac tacgaacttt acccacaaat 6360agtcaggtaa tgaatcctga tataaagaca ggttgataaa tcagtcttct acgcgcatcg 6420cacgcgcaca ccgtagaaag tctttcagtt gtgagcctgg gcaaaccgtt aactttcggc 6480ggctttgctg tgcgacaggc tcacgtctaa aaggaaataa atcatgggtc ataaaattat 6540cacgttgtcc ggcgcggcga cggatgttct gtatgcgctg tttttccgtg gcgcgttgct 6600gtctggtgat ctgccttcta aatctggcac agccgaattg cgcgagcttg gttttgctga 6660aaccagacac acagcaactg aataccagaa agaaaatcac tttacctttc tgacatcaga 6720agggcagaaa tttgccgttg aacacctggt caatacgcgt tttggtgagc agcaatattg 6780cgcttcgatg acgcttggcg ttgagattga tacctctgct gcacaaaagg caatcgacga 6840gctggaccag cgcattcgtg acaccgtctc cttcgaactt attcgcaatg gagtgtcatt 6900catcaaggac gccgctatcg caaatggtgc tatccacgca gcggcaatcg aaacacctca 6960gccggtgacc aatatctaca acatcagcct tggtatccag cgtgatgagc cagcgcagaa 7020caaggtaacc gtcagtgccg ataagttcaa agttaaacct ggtgttgata ccaacattga 7080aacgttgatc gaaaacgcgc tgaaaaacgc tgctgaatgt gcggcgctgg atgtcacaaa 7140gcaaatggca gcagacaaga aagcgatgga tgaactggct tcctatgtcc gcacggccat 7200catgatggaa tgtttccccg gtggtgttat ctggcagcag tgccgtcgat agtatgcaat 7260tgataattat tatcatttgc gggtcctttc cggcgatccg ccttgttacg gggcggcgac 7320ctcgcgggtt ttcgctattt atgaaaattt tccggtttaa ggcgtttccg ttcttcttcg 7380tcataactta atgtttttat ttaaaatacc ctctgaaaag aaaggaaacg acaggtgctg 7440aaagcgagct ttttggcctc tgtcgtttcc tttctctgtt tttgtccgtg gaatgaacaa 7500tggaagtcaa caaaaagcag ctggctgaca ttttcggtgc gagtatccgt accattcaga 7560actggcagga acagggaatg cccgttctgc gaggcggtgg caagggtaat gaggtgcttt 7620atgactctgc cgccgtcata aaatggtatg ccgaaaggga tgctgaaatt gagaacgaaa 7680agctgcgccg ggaggttgaa gaactgcggc aggccagcga ggcagatcca caggacgggt 7740gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag caggactggg 7800cggcggcaaa gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg 7860catatagcgc tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc 7920gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg 7980tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc 8040aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagcggt caaacatgag 8100aattcgcggc cgcaattaac cctcactaaa ggatcc 8136322713DNAArtificial SequencepNEB193 plasmid 32tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccgggggc 420gcgccggatc cttaattaag tctagagtcg actgtttaaa cctgcaggca tgcaagcttg 480gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 540aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 600acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 660cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 720tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 780tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 840gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 900aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 960ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 1020gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 1080ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 1140ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 1200cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 1260attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 1320ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 1380aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 1440gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 1500tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 1560ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 1620taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 1680atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 1740actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 1800cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 1860agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 1920gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 1980gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 2040gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 2100gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 2160cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 2220ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 2280accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 2340aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 2400aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 2460caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 2520ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 2580gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 2640cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 2700aggccctttc gtc 27133321DNAArtificial SequenceattP 33cagctttttt atactaagtt g 213421DNAArtificial SequenceattB 34ctgctttttt atactaactt g 213521DNAArtificial SequenceattL 35ctgctttttt atactaagtt g 213621DNAArtificial SequenceattR 36cagctttttt atactaactt g 21371071DNAArtificial SequenceIntegrase E174R 37atg gga aga agg cga agt cat gag cgc cgg gat tta ccc cct aac ctt 48Met Gly Arg Arg Arg Ser His Glu Arg Arg Asp Leu Pro Pro Asn Leu1 5 10 15tat ata aga aac aat gga tat tac tgc tac agg gac cca agg acg ggt 96Tyr Ile Arg Asn Asn Gly Tyr Tyr Cys Tyr Arg Asp Pro Arg Thr Gly 20 25 30aaa gag ttt gga tta ggc aga

gac agg cga atc gca atc act gaa gct 144Lys Glu Phe Gly Leu Gly Arg Asp Arg Arg Ile Ala Ile Thr Glu Ala 35 40 45ata cag gcc aac att gag tta ttt tca gga cac aaa cac aag cct ctg 192Ile Gln Ala Asn Ile Glu Leu Phe Ser Gly His Lys His Lys Pro Leu 50 55 60aca gcg aga atc aac agt gat aat tcc gtt acg tta cat tca tgg ctt 240Thr Ala Arg Ile Asn Ser Asp Asn Ser Val Thr Leu His Ser Trp Leu65 70 75 80gat cgc tac gaa aaa atc ctg gcc agc aga gga atc aag cag aag aca 288Asp Arg Tyr Glu Lys Ile Leu Ala Ser Arg Gly Ile Lys Gln Lys Thr 85 90 95ctc ata aat tac atg agc aaa att aaa gca ata agg agg ggt ctg cct 336Leu Ile Asn Tyr Met Ser Lys Ile Lys Ala Ile Arg Arg Gly Leu Pro 100 105 110gat gct cca ctt gaa gac atc acc aca aaa gaa att gcg gca atg ctc 384Asp Ala Pro Leu Glu Asp Ile Thr Thr Lys Glu Ile Ala Ala Met Leu 115 120 125aat gga tac ata gac gag ggc aag gcg gcg tca gcc aag tta atc aga 432Asn Gly Tyr Ile Asp Glu Gly Lys Ala Ala Ser Ala Lys Leu Ile Arg 130 135 140tca aca ctg agc gat gca ttc cga gag gca ata gct gaa ggc cat ata 480Ser Thr Leu Ser Asp Ala Phe Arg Glu Ala Ile Ala Glu Gly His Ile145 150 155 160aca aca aac cat gtc gct gcc act cgc gca gca aaa tct aga gta agg 528Thr Thr Asn His Val Ala Ala Thr Arg Ala Ala Lys Ser Arg Val Arg 165 170 175aga tca aga ctt acg gct gac gaa tac ctg aaa att tat caa gca gca 576Arg Ser Arg Leu Thr Ala Asp Glu Tyr Leu Lys Ile Tyr Gln Ala Ala 180 185 190gaa tca tca cca tgt tgg ctc aga ctt gca atg gaa ctg gct gtt gtt 624Glu Ser Ser Pro Cys Trp Leu Arg Leu Ala Met Glu Leu Ala Val Val 195 200 205acc ggg caa cga gtt ggt gat tta tgc gaa atg aag tgg tct gat atc 672Thr Gly Gln Arg Val Gly Asp Leu Cys Glu Met Lys Trp Ser Asp Ile 210 215 220gta gat gga tat ctt tat gtc gag caa agc aaa aca ggc gta aaa att 720Val Asp Gly Tyr Leu Tyr Val Glu Gln Ser Lys Thr Gly Val Lys Ile225 230 235 240gcc atc cca aca gca ttg cat att gat gct ctc gga ata tca atg aag 768Ala Ile Pro Thr Ala Leu His Ile Asp Ala Leu Gly Ile Ser Met Lys 245 250 255gaa aca ctt gat aaa tgc aaa gag att ctt ggc gga gaa acc ata att 816Glu Thr Leu Asp Lys Cys Lys Glu Ile Leu Gly Gly Glu Thr Ile Ile 260 265 270gca tct act cgt cgc gaa ccg ctt tca tcc ggc aca gta tca agg tat 864Ala Ser Thr Arg Arg Glu Pro Leu Ser Ser Gly Thr Val Ser Arg Tyr 275 280 285ttt atg cgc gca cga aaa gca tca ggt ctt tcc ttc gaa ggg gat ccg 912Phe Met Arg Ala Arg Lys Ala Ser Gly Leu Ser Phe Glu Gly Asp Pro 290 295 300cct acc ttt cac gag ttg cgc agt ttg tct gca aga ctc tat gag aag 960Pro Thr Phe His Glu Leu Arg Ser Leu Ser Ala Arg Leu Tyr Glu Lys305 310 315 320cag ata agc gat aag ttt gct caa cat ctt ctc ggg cat aag tcg gac 1008Gln Ile Ser Asp Lys Phe Ala Gln His Leu Leu Gly His Lys Ser Asp 325 330 335acc atg gca tca cag tat cgt gat gac aga ggc agg gag tgg gac aaa 1056Thr Met Ala Ser Gln Tyr Arg Asp Asp Arg Gly Arg Glu Trp Asp Lys 340 345 350att gaa atc aaa taa 1071Ile Glu Ile Lys * 35538356PRTArtificial SequenceIntegrase E147R 38Met Gly Arg Arg Arg Ser His Glu Arg Arg Asp Leu Pro Pro Asn Leu1 5 10 15Tyr Ile Arg Asn Asn Gly Tyr Tyr Cys Tyr Arg Asp Pro Arg Thr Gly 20 25 30Lys Glu Phe Gly Leu Gly Arg Asp Arg Arg Ile Ala Ile Thr Glu Ala 35 40 45Ile Gln Ala Asn Ile Glu Leu Phe Ser Gly His Lys His Lys Pro Leu 50 55 60Thr Ala Arg Ile Asn Ser Asp Asn Ser Val Thr Leu His Ser Trp Leu65 70 75 80Asp Arg Tyr Glu Lys Ile Leu Ala Ser Arg Gly Ile Lys Gln Lys Thr 85 90 95Leu Ile Asn Tyr Met Ser Lys Ile Lys Ala Ile Arg Arg Gly Leu Pro 100 105 110Asp Ala Pro Leu Glu Asp Ile Thr Thr Lys Glu Ile Ala Ala Met Leu 115 120 125Asn Gly Tyr Ile Asp Glu Gly Lys Ala Ala Ser Ala Lys Leu Ile Arg 130 135 140Ser Thr Leu Ser Asp Ala Phe Arg Glu Ala Ile Ala Glu Gly His Ile145 150 155 160Thr Thr Asn His Val Ala Ala Thr Arg Ala Ala Lys Ser Arg Val Arg 165 170 175Arg Ser Arg Leu Thr Ala Asp Glu Tyr Leu Lys Ile Tyr Gln Ala Ala 180 185 190Glu Ser Ser Pro Cys Trp Leu Arg Leu Ala Met Glu Leu Ala Val Val 195 200 205Thr Gly Gln Arg Val Gly Asp Leu Cys Glu Met Lys Trp Ser Asp Ile 210 215 220Val Asp Gly Tyr Leu Tyr Val Glu Gln Ser Lys Thr Gly Val Lys Ile225 230 235 240Ala Ile Pro Thr Ala Leu His Ile Asp Ala Leu Gly Ile Ser Met Lys 245 250 255Glu Thr Leu Asp Lys Cys Lys Glu Ile Leu Gly Gly Glu Thr Ile Ile 260 265 270Ala Ser Thr Arg Arg Glu Pro Leu Ser Ser Gly Thr Val Ser Arg Tyr 275 280 285Phe Met Arg Ala Arg Lys Ala Ser Gly Leu Ser Phe Glu Gly Asp Pro 290 295 300Pro Thr Phe His Glu Leu Arg Ser Leu Ser Ala Arg Leu Tyr Glu Lys305 310 315 320Gln Ile Ser Asp Lys Phe Ala Gln His Leu Leu Gly His Lys Ser Asp 325 330 335Thr Met Ala Ser Gln Tyr Arg Asp Asp Arg Gly Arg Glu Trp Asp Lys 340 345 350Ile Glu Ile Lys 35539876DNADiscosoma speciesCDS(45)...(737)Nucleotide sequence encoding red flourescent protein (FP593) 39agtttcagcc agtgacaggg tgagctgcca ggtattctaa caag atg agt tgt tcc 56 Met Ser Cys Ser 1aag aat gtg atc aag gag ttc atg agg ttc aag gtt cgt atg gaa gga 104Lys Asn Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg Met Glu Gly5 10 15 20acg gtc aat ggg cac gag ttt gaa ata aaa ggc gaa ggt gaa ggg agg 152Thr Val Asn Gly His Glu Phe Glu Ile Lys Gly Glu Gly Glu Gly Arg 25 30 35cct tac gaa ggt cac tgt tcc gta aag ctt atg gta acc aag ggt gga 200Pro Tyr Glu Gly His Cys Ser Val Lys Leu Met Val Thr Lys Gly Gly 40 45 50cct ttg cca ttt gct ttt gat att ttg tca cca caa ttt cag tat gga 248Pro Leu Pro Phe Ala Phe Asp Ile Leu Ser Pro Gln Phe Gln Tyr Gly 55 60 65agc aag gta tat gtc aaa cac cct gcc gac ata cca gac tat aaa aag 296Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Lys Lys 70 75 80ctg tca ttt cct gag gga ttt aaa tgg gaa agg gtc atg aac ttt gaa 344Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu85 90 95 100gac ggt ggc gtg gtt act gta tcc caa gat tcc agt ttg aaa gac ggc 392Asp Gly Gly Val Val Thr Val Ser Gln Asp Ser Ser Leu Lys Asp Gly 105 110 115tgt ttc atc tac gag gtc aag ttc att ggg gtg aac ttt cct tct gat 440Cys Phe Ile Tyr Glu Val Lys Phe Ile Gly Val Asn Phe Pro Ser Asp 120 125 130gga cct gtt atg cag agg agg aca cgg ggc tgg gaa gcc agc tct gag 488Gly Pro Val Met Gln Arg Arg Thr Arg Gly Trp Glu Ala Ser Ser Glu 135 140 145cgt ttg tat cct cgt gat ggg gtg ctg aaa gga gac atc cat atg gct 536Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Asp Ile His Met Ala 150 155 160ctg agg ctg gaa gga ggc ggc cat tac ctc gtt gaa ttc aaa agt att 584Leu Arg Leu Glu Gly Gly Gly His Tyr Leu Val Glu Phe Lys Ser Ile165 170 175 180tac atg gta aag aag cct tca gtg cag ttg cca ggc tac tat tat gtt 632Tyr Met Val Lys Lys Pro Ser Val Gln Leu Pro Gly Tyr Tyr Tyr Val 185 190 195gac tcc aaa ctg gat atg acg agc cac aac gaa gat tac aca gtc gtt 680Asp Ser Lys Leu Asp Met Thr Ser His Asn Glu Asp Tyr Thr Val Val 200 205 210gag cag tat gaa aaa acc cag gga cgc cac cat ccg ttc att aag cct 728Glu Gln Tyr Glu Lys Thr Gln Gly Arg His His Pro Phe Ile Lys Pro 215 220 225ctg cag tga actcggctca gtcatggatt agcggtaatg gccacaaaag 777Leu Gln * 230gcacgatgat cgttttttag gaatgcagcc aaaaattgaa ggttatgaca gtagaaatac 837aagcaacagg ctttgcttat taaacatgta attgaaaac 87640230PRTDiscosoma species 40Met Ser Cys Ser Lys Asn Val Ile Lys Glu Phe Met Arg Phe Lys Val1 5 10 15Arg Met Glu Gly Thr Val Asn Gly His Glu Phe Glu Ile Lys Gly Glu 20 25 30Gly Glu Gly Arg Pro Tyr Glu Gly His Cys Ser Val Lys Leu Met Val 35 40 45Thr Lys Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ser Pro Gln 50 55 60Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro65 70 75 80Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val 85 90 95Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Ser Gln Asp Ser Ser 100 105 110Leu Lys Asp Gly Cys Phe Ile Tyr Glu Val Lys Phe Ile Gly Val Asn 115 120 125Phe Pro Ser Asp Gly Pro Val Met Gln Arg Arg Thr Arg Gly Trp Glu 130 135 140Ala Ser Ser Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Asp145 150 155 160Ile His Met Ala Leu Arg Leu Glu Gly Gly Gly His Tyr Leu Val Glu 165 170 175Phe Lys Ser Ile Tyr Met Val Lys Lys Pro Ser Val Gln Leu Pro Gly 180 185 190Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Met Thr Ser His Asn Glu Asp 195 200 205Tyr Thr Val Val Glu Gln Tyr Glu Lys Thr Gln Gly Arg His His Pro 210 215 220Phe Ile Lys Pro Leu Gln225 2304125DNAArtificial Sequencem-att; 41rkycwgcttt yktrtacnaa stsgb 254225DNAArtificial Sequencem-attB; 42agccwgcttt yktrtacnaa ctsgb 254325DNAArtificial Sequencem-attR 43gttcagcttt cktrtacnaa ctsgb 254425DNAArtificial Sequencem-attL 44agccwgcttt cktrtacnaa gtsgb 254525DNAArtificial Sequencem-attP1 45gttcagcttt yktrtacnaa gtsgb 254625DNAArtificial SequenceattB1 46agcctgcttt tttgtacaaa cttgt 254725DNAArtificial SequenceattB2 47agcctgcttt cttgtacaaa cttgt 254825DNAArtificial SequenceattB3 48acccagcttt cttgtacaaa cttgt 254925DNAArtificial SequenceattR1 49gttcagcttt tttgtacaaa cttgt 255025DNAArtificial SequenceattR2 50gttcagcttt cttgtacaaa cttgt 255125DNAArtificial SequenceattR3 51gttcagcttt cttgtacaaa gttgg 255225DNAArtificial SequenceattL1 52agcctgcttt tttgtacaaa gttgg 255325DNAArtificial SequenceattL2 53agcctgcttt cttgtacaaa gttgg 255425DNAArtificial SequenceattL3 54acccagcttt cttgtacaaa gttgg 255525DNAArtificial SequenceattP1 55gttcagcttt tttgtacaaa gttgg 255625DNAArtificial SequenceattP2,P3 56gttcagcttt cttgtacaaa gttgg 255734DNAArtificial SequenceLox P site 57ataacttcgt ataatgtatg ctatacgaag ttat 34581032DNAEscherichia coliCDS(1)...(1032)nucleotide sequence encoding Cre recombinase 58atg tcc aat tta ctg acc gta cac caa aat ttg cct gca tta ccg gtc 48Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val1 5 10 15gat gca acg agt gat gag gtt cgc aag aac ctg atg gac atg ttc agg 96Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg 20 25 30gat cgc cag gcg ttt tct gag cat acc tgg aaa atg ctt ctg tcc gtt 144Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val 35 40 45tgc cgg tcg tgg gcg gca tgg tgc aag ttg aat aac cgg aaa tgg ttt 192Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe 50 55 60ccc gca gaa cct gaa gat gtt cgc gat tat ctt cta tat ctt cag gcg 240Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala65 70 75 80cgc ggt ctg gca gta aaa act atc cag caa cat ttg ggc cag cta aac 288Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn 85 90 95atg ctt cat cgt cgg tcc ggg ctg cca cga cca agt gac agc aat gct 336Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala 100 105 110gtt tca ctg gtt atg cgg cgg atc cga aaa gaa aac gtt gat gcc ggt 384Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly 115 120 125gaa cgt gca aaa cag gct cta gcg ttc gaa cgc act gat ttc gac cag 432Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln 130 135 140gtt cgt tca ctc atg gaa aat agc gat cgc tgc cag gat ata cgt aat 480Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn145 150 155 160ctg gca ttt ctg ggg att gct tat aac acc ctg tta cgt ata gcc gaa 528Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu 165 170 175att gcc agg atc agg gtt aaa gat atc tca cgt act gac ggt ggg aga 576Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg 180 185 190atg tta atc cat att ggc aga acg aaa acg ctg gtt agc acc gca ggt 624Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly 195 200 205gta gag aag gca ctt agc ctg ggg gta act aaa ctg gtc gag cga tgg 672Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp 210 215 220att tcc gtc tct ggt gta gct gat gat ccg aat aac tac ctg ttt tgc 720Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys225 230 235 240cgg gtc aga aaa aat ggt gtt gcc gcg cca tct gcc acc agc cag cta 768Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu 245 250 255tca act cgc gcc ctg gaa ggg att ttt gaa gca act cat cga ttg att 816Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile 260 265 270tac ggc gct aag gat gac tct ggt cag aga tac ctg gcc tgg tct gga 864Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly 275 280 285cac agt gcc cgt gtc gga gcc gcg cga gat atg gcc cgc gct gga gtt 912His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val 290 295 300tca ata ccg gag atc atg caa gct ggt ggc tgg acc aat gta aat att 960Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile305 310 315 320gtc atg aac tat atc cgt aac ctg gat agt gaa aca ggg gca atg gtg 1008Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val 325 330 335cgc ctg ctg gaa gat ggc gat tag 1032Arg Leu Leu Glu Asp Gly Asp * 34059343PRTEscherichia coli 59Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val1 5 10 15Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg 20 25 30Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val 35 40 45Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe 50 55 60Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala65 70 75 80Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn 85 90

95Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala 100 105 110Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly 115 120 125Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln 130 135 140Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn145 150 155 160Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu 165 170 175Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg 180 185 190Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly 195 200 205Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp 210 215 220Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys225 230 235 240Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu 245 250 255Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile 260 265 270Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly 275 280 285His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val 290 295 300Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile305 310 315 320Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val 325 330 335Arg Leu Leu Glu Asp Gly Asp 340601272DNASaccharomyces cerevisiaeCDS(1)...(1272)nucleotide sequence encoding Flip recombinase 60atg cca caa ttt ggt ata tta tgt aaa aca cca cct aag gtg ctt gtt 48Met Pro Gln Phe Gly Ile Leu Cys Lys Thr Pro Pro Lys Val Leu Val1 5 10 15cgt cag ttt gtg gaa agg ttt gaa aga cct tca ggt gag aaa ata gca 96Arg Gln Phe Val Glu Arg Phe Glu Arg Pro Ser Gly Glu Lys Ile Ala 20 25 30tta tgt gct gct gaa cta acc tat tta tgt tgg atg att aca cat aac 144Leu Cys Ala Ala Glu Leu Thr Tyr Leu Cys Trp Met Ile Thr His Asn 35 40 45gga aca gca atc aag aga gcc aca ttc atg agc tat aat act atc ata 192Gly Thr Ala Ile Lys Arg Ala Thr Phe Met Ser Tyr Asn Thr Ile Ile 50 55 60agc aat tcg ctg agt ttc gat att gtc aat aaa tca ctc cag ttt aaa 240Ser Asn Ser Leu Ser Phe Asp Ile Val Asn Lys Ser Leu Gln Phe Lys65 70 75 80tac aag acg caa aaa gca aca att ctg gaa gcc tca tta aag aaa ttg 288Tyr Lys Thr Gln Lys Ala Thr Ile Leu Glu Ala Ser Leu Lys Lys Leu 85 90 95att cct gct tgg gaa ttt aca att att cct tac tat gga caa aaa cat 336Ile Pro Ala Trp Glu Phe Thr Ile Ile Pro Tyr Tyr Gly Gln Lys His 100 105 110caa tct gat atc act gat att gta agt agt ttg caa tta cag ttc gaa 384Gln Ser Asp Ile Thr Asp Ile Val Ser Ser Leu Gln Leu Gln Phe Glu 115 120 125tca tcg gaa gaa gca gat aag gga aat agc cac agt aaa aaa atg ctt 432Ser Ser Glu Glu Ala Asp Lys Gly Asn Ser His Ser Lys Lys Met Leu 130 135 140aaa gca ctt cta agt gag ggt gaa agc atc tgg gag atc act gag aaa 480Lys Ala Leu Leu Ser Glu Gly Glu Ser Ile Trp Glu Ile Thr Glu Lys145 150 155 160ata cta aat tcg ttt gag tat act tcg aga ttt aca aaa aca aaa act 528Ile Leu Asn Ser Phe Glu Tyr Thr Ser Arg Phe Thr Lys Thr Lys Thr 165 170 175tta tac caa ttc ctc ttc cta gct act ttc atc aat tgt gga aga ttc 576Leu Tyr Gln Phe Leu Phe Leu Ala Thr Phe Ile Asn Cys Gly Arg Phe 180 185 190agc gat att aag aac gtt gat ccg aaa tca ttt aaa tta gtc caa aat 624Ser Asp Ile Lys Asn Val Asp Pro Lys Ser Phe Lys Leu Val Gln Asn 195 200 205aag tat ctg gga gta ata atc cag tgt tta gtg aca gag aca aag aca 672Lys Tyr Leu Gly Val Ile Ile Gln Cys Leu Val Thr Glu Thr Lys Thr 210 215 220agc gtt agt agg cac ata tac ttc ttt agc gca agg ggt agg atc gat 720Ser Val Ser Arg His Ile Tyr Phe Phe Ser Ala Arg Gly Arg Ile Asp225 230 235 240cca ctt gta tat ttg gat gaa ttt ttg agg aat tct gaa cca gtc cta 768Pro Leu Val Tyr Leu Asp Glu Phe Leu Arg Asn Ser Glu Pro Val Leu 245 250 255aaa cga gta aat agg acc ggc aat tct tca agc aat aaa cag gaa tac 816Lys Arg Val Asn Arg Thr Gly Asn Ser Ser Ser Asn Lys Gln Glu Tyr 260 265 270caa tta tta aaa gat aac tta gtc aga tcg tac aat aaa gct ttg aag 864Gln Leu Leu Lys Asp Asn Leu Val Arg Ser Tyr Asn Lys Ala Leu Lys 275 280 285aaa aat gcg cct tat tca atc ttt gct ata aaa aat ggc cca aaa tct 912Lys Asn Ala Pro Tyr Ser Ile Phe Ala Ile Lys Asn Gly Pro Lys Ser 290 295 300cac att gga aga cat ttg atg acc tca ttt ctt tca atg aag ggc cta 960His Ile Gly Arg His Leu Met Thr Ser Phe Leu Ser Met Lys Gly Leu305 310 315 320acg gag ttg act aat gtt gtg gga aat tgg agc gat aag cgt gct tct 1008Thr Glu Leu Thr Asn Val Val Gly Asn Trp Ser Asp Lys Arg Ala Ser 325 330 335gcc gtg gcc agg aca acg tat act cat cag ata aca gca ata cct gat 1056Ala Val Ala Arg Thr Thr Tyr Thr His Gln Ile Thr Ala Ile Pro Asp 340 345 350cac tac ttc gca cta gtt tct cgg tac tat gca tat gat cca ata tca 1104His Tyr Phe Ala Leu Val Ser Arg Tyr Tyr Ala Tyr Asp Pro Ile Ser 355 360 365aag gaa atg ata gca ttg aag gat gag act aat cca att gag gag tgg 1152Lys Glu Met Ile Ala Leu Lys Asp Glu Thr Asn Pro Ile Glu Glu Trp 370 375 380cag cat ata gaa cag cta aag ggt agt gct gaa gga agc ata cga tac 1200Gln His Ile Glu Gln Leu Lys Gly Ser Ala Glu Gly Ser Ile Arg Tyr385 390 395 400ccc gca tgg aat ggg ata ata tca cag gag gta cta gac tac ctt tca 1248Pro Ala Trp Asn Gly Ile Ile Ser Gln Glu Val Leu Asp Tyr Leu Ser 405 410 415tcc tac ata aat aga cgc ata taa 1272Ser Tyr Ile Asn Arg Arg Ile * 42061422PRTSaccharomyces cerevisiae 61Pro Gln Phe Gly Ile Leu Cys Lys Thr Pro Pro Lys Val Leu Val Arg1 5 10 15Gln Phe Val Glu Arg Phe Glu Arg Pro Ser Gly Glu Lys Ile Ala Leu 20 25 30Cys Ala Ala Glu Leu Thr Tyr Leu Cys Trp Met Ile Thr His Asn Gly 35 40 45Thr Ala Ile Lys Arg Ala Thr Phe Met Ser Tyr Asn Thr Ile Ile Ser 50 55 60Asn Ser Leu Ser Phe Asp Ile Val Asn Lys Ser Leu Gln Phe Lys Tyr65 70 75 80Lys Thr Gln Lys Ala Thr Ile Leu Glu Ala Ser Leu Lys Lys Leu Ile 85 90 95Pro Ala Trp Glu Phe Thr Ile Ile Pro Tyr Tyr Gly Gln Lys His Gln 100 105 110Ser Asp Ile Thr Asp Ile Val Ser Ser Leu Gln Leu Gln Phe Glu Ser 115 120 125Ser Glu Glu Ala Asp Lys Gly Asn Ser His Ser Lys Lys Met Leu Lys 130 135 140Ala Leu Leu Ser Glu Gly Glu Ser Ile Trp Glu Ile Thr Glu Lys Ile145 150 155 160Leu Asn Ser Phe Glu Tyr Thr Ser Arg Phe Thr Lys Thr Lys Thr Leu 165 170 175Tyr Gln Phe Leu Phe Leu Ala Thr Phe Ile Asn Cys Gly Arg Phe Ser 180 185 190Asp Ile Lys Asn Val Asp Pro Lys Ser Phe Lys Leu Val Gln Asn Lys 195 200 205Tyr Leu Gly Val Ile Ile Gln Cys Leu Val Thr Glu Thr Lys Thr Ser 210 215 220Val Ser Arg His Ile Tyr Phe Phe Ser Ala Arg Gly Arg Ile Asp Pro225 230 235 240Leu Val Tyr Leu Asp Glu Phe Leu Arg Asn Ser Glu Pro Val Leu Lys 245 250 255Arg Val Asn Arg Thr Gly Asn Ser Ser Ser Asn Lys Gln Glu Tyr Gln 260 265 270Leu Leu Lys Asp Asn Leu Val Arg Ser Tyr Asn Lys Ala Leu Lys Lys 275 280 285Asn Ala Pro Tyr Ser Ile Phe Ala Ile Lys Asn Gly Pro Lys Ser His 290 295 300Ile Gly Arg His Leu Met Thr Ser Phe Leu Ser Met Lys Gly Leu Thr305 310 315 320Glu Leu Thr Asn Val Val Gly Asn Trp Ser Asp Lys Arg Ala Ser Ala 325 330 335Val Ala Arg Thr Thr Tyr Thr His Gln Ile Thr Ala Ile Pro Asp His 340 345 350Tyr Phe Ala Leu Val Ser Arg Tyr Tyr Ala Tyr Asp Pro Ile Ser Lys 355 360 365Glu Met Ile Ala Leu Lys Asp Glu Thr Asn Pro Ile Glu Glu Trp Gln 370 375 380His Ile Glu Gln Leu Lys Gly Ser Ala Glu Gly Ser Ile Arg Tyr Pro385 390 395 400Ala Trp Asn Gly Ile Ile Ser Gln Glu Val Leu Asp Tyr Leu Ser Ser 405 410 415Tyr Ile Asn Arg Arg Ile 4206248DNAArtificial SequenceIR2 62gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttc 486348DNAArtificial SequenceIR1 63gaagttccta tactttctag agaataggaa cttcggaata ggaacttc 486466DNABacteriophage muCDS(1)...(66)nucleotide sequence encoding GIN recombinase 64tca act ctg tat aaa aaa cac ccc gcg aaa cga gcg cat ata gaa aac 48Ser Thr Leu Tyr Lys Lys His Pro Ala Lys Arg Ala His Ile Glu Asn1 5 10 15gac gat cga atc aat taa 66Asp Asp Arg Ile Asn * 206521PRTbacteriophage mu 65Ser Thr Leu Tyr Lys Lys His Pro Ala Lys Arg Ala His Ile Glu Asn1 5 10 15Asp Asp Arg Ile Asn 206669DNABacteriophage muCDS(1)...(69)nucleotide sequence encoding Gin recombinase 66tat aaa aaa cat ccc gcg aaa cga acg cat ata gaa aac gac gat cga 48Tyr Lys Lys His Pro Ala Lys Arg Thr His Ile Glu Asn Asp Asp Arg1 5 10 15atc aat caa atc gat cgg taa 69Ile Asn Gln Ile Asp Arg * 206722PRTbacteriophage muGin recombinase of bacteriophage mu 67Tyr Lys Lys His Pro Ala Lys Arg Thr His Ile Glu Asn Asp Asp Arg1 5 10 15Ile Asn Gln Ile Asp Arg 2068555DNAEscherichia coliCDS(1)...(555)nucleotide sequence encoding PIN recombinase 68atg ctt att ggc tat gta cgc gta tca aca aat gac cag aac aca gat 48Met Leu Ile Gly Tyr Val Arg Val Ser Thr Asn Asp Gln Asn Thr Asp1 5 10 15cta caa cgt aat gcg ctg aac tgt gca gga tgc gag ctg att ttt gaa 96Leu Gln Arg Asn Ala Leu Asn Cys Ala Gly Cys Glu Leu Ile Phe Glu 20 25 30gac aag ata agc ggc aca aag tcc gaa agg ccg gga ctg aaa aaa ctg 144Asp Lys Ile Ser Gly Thr Lys Ser Glu Arg Pro Gly Leu Lys Lys Leu 35 40 45ctc agg aca tta tcg gca ggt gac act ctg gtt gtc tgg aag ctg gat 192Leu Arg Thr Leu Ser Ala Gly Asp Thr Leu Val Val Trp Lys Leu Asp 50 55 60cgg ctg ggg cgt agt atg cgg cat ctt gtc gtg ctg gtg gag gag ttg 240Arg Leu Gly Arg Ser Met Arg His Leu Val Val Leu Val Glu Glu Leu65 70 75 80cgc gaa cga ggc atc aac ttt cgt agt ctg acg gat tca att gat acc 288Arg Glu Arg Gly Ile Asn Phe Arg Ser Leu Thr Asp Ser Ile Asp Thr 85 90 95agc aca cca atg gga cgc ttt ttc ttt cat gtg atg ggt gcc ctg gct 336Ser Thr Pro Met Gly Arg Phe Phe Phe His Val Met Gly Ala Leu Ala 100 105 110gaa atg gag cgt gaa ctg att gtt gaa cga aca aaa gct gga ctg gaa 384Glu Met Glu Arg Glu Leu Ile Val Glu Arg Thr Lys Ala Gly Leu Glu 115 120 125act gct cgt gca cag gga cga att ggt gga cgt cgt ccc aaa ctt aca 432Thr Ala Arg Ala Gln Gly Arg Ile Gly Gly Arg Arg Pro Lys Leu Thr 130 135 140cca gaa caa tgg gca caa gct gga cga tta att gca gca gga act cct 480Pro Glu Gln Trp Ala Gln Ala Gly Arg Leu Ile Ala Ala Gly Thr Pro145 150 155 160cgc cag aag gtg gcg att atc tat gat gtt ggt gtg tca act ttg tat 528Arg Gln Lys Val Ala Ile Ile Tyr Asp Val Gly Val Ser Thr Leu Tyr 165 170 175aag agg ttt cct gca ggg gat aaa taa 555Lys Arg Phe Pro Ala Gly Asp Lys * 18069184PRTEscherichia coli 69Met Leu Ile Gly Tyr Val Arg Val Ser Thr Asn Asp Gln Asn Thr Asp1 5 10 15Leu Gln Arg Asn Ala Leu Asn Cys Ala Gly Cys Glu Leu Ile Phe Glu 20 25 30Asp Lys Ile Ser Gly Thr Lys Ser Glu Arg Pro Gly Leu Lys Lys Leu 35 40 45Leu Arg Thr Leu Ser Ala Gly Asp Thr Leu Val Val Trp Lys Leu Asp 50 55 60Arg Leu Gly Arg Ser Met Arg His Leu Val Val Leu Val Glu Glu Leu65 70 75 80Arg Glu Arg Gly Ile Asn Phe Arg Ser Leu Thr Asp Ser Ile Asp Thr 85 90 95Ser Thr Pro Met Gly Arg Phe Phe Phe His Val Met Gly Ala Leu Ala 100 105 110Glu Met Glu Arg Glu Leu Ile Val Glu Arg Thr Lys Ala Gly Leu Glu 115 120 125Thr Ala Arg Ala Gln Gly Arg Ile Gly Gly Arg Arg Pro Lys Leu Thr 130 135 140Pro Glu Gln Trp Ala Gln Ala Gly Arg Leu Ile Ala Ala Gly Thr Pro145 150 155 160Arg Gln Lys Val Ala Ile Ile Tyr Asp Val Gly Val Ser Thr Leu Tyr 165 170 175Lys Arg Phe Pro Ala Gly Asp Lys 180704778DNAArtificial Sequencepcx plasmid 70gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcactcct caggtgcagg 1740ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag 1800atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt 1860ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct 1920cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt 1980agagtttggc aacatatgcc atatgctggc tgccatgaac aaaggtggct ataaagaggt 2040catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact 2100tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa 2160ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata 2220gctgtccctc ttctcttatg aagatccctc gacctgcagc ccaagcttgg cgtaatcatg 2280gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 2340cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 2400gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tccgcatctc 2460aattagtcag

caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc 2520agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 2580gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc 2640ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 2700tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 2760tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg 2820gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 2880ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 2940agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 3000ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 3060caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 3120gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 3180cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 3240tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 3300gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 3360cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 3420tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 3480tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 3540caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 3600aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 3660cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 3720ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 3780tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 3840atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 3900tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 3960aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 4020catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 4080gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 4140ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 4200aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 4260atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 4320cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 4380gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 4440agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 4500gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 4560caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 4620ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 4680tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 4740aggggttccg cgcacatttc cccgaaaagt gccacctg 4778715510DNAArtificial SequencepCXeGFP plasmid 71gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcgccacc atggtgagca 1740agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 1800acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 1860ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 1920ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 1980tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 2040acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 2100tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 2160acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 2220tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 2280agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 2340cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 2400tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcactc 2460ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca 2520aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 2580agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 2640ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg 2700agtatttggt ttagagtttg gcaacatatg ccatatgctg gctgccatga acaaaggtgg 2760ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga 2820aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta 2880acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta 2940ctcccagtca tagctgtccc tcttctctta tgaagatccc tcgacctgca gcccaagctt 3000ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 3060caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 3120cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg 3180gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3240ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 3300gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt 3360ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat 3420aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 3480gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg 3540ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 3600ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 3660acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 3720aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 3780tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 3840aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 3900gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 3960acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4020accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4080ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4140gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4200gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4260ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4320gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4380cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4440cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4500gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4560tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4620gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4680agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4740tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4800agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 4860gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 4920catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 4980ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5040atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5100tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 5160cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5220cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5280atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5340aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5400ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5460aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 551072282DNAArtificial Sequenceattp 72ccttgcgcta atgctctgtt acaggtcact aataccatct aagtagttga ttcatagtga 60ctgcatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa 120tatattgata tttatatcat tttacgtttc tcgttcagct tttttatact aagttggcat 180tataaaaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa 240aatcattatt tgatttcaat tttgtcccac tccctgcctc tg 2827320DNAArtificial SequencePrimer 73ggccccgtaa tgcagaagaa 207432DNAArtificial SequencePrimer 74ggtttaaagt gcgctcctcc aagaacgtca tc 327540DNAArtificial SequencePrimer 75agatctagag ccgccgctac aggaacaggt ggtggcggcc 407637DNAArtificial SequencePrimer 5PacSV40 76ctgttaatta actgtggaat gtgtgtcagt tagggtg 377720DNAArtificial SequencePrimer Antisense Zeo 77tgaacagggt cacgtcgtcc 207824DNAArtificial SequencePrimer 5' HETS 78gggccgaaac gatctcaacc tatt 247919DNAArtificial SequencePrimer 3' HETS 79cgcagcggcc ctcctactc 198029DNAArtificial SequencePrimer 5BSD 80accatgaaaa catttaacat ttctcaaca 298129DNAArtificial SequencePrimer SV40polyA 81tttatttgtg aaatttgtga tgctattgc 298225DNAArtificial SequencePrimer 3BSP 82ttaatttcgg gtatatttga gtgga 258332DNAArtificial SequencePrimer EPO5XBA 83tatctagaat gggggtgcac gaatgtcctg cc 328432DNAArtificial SequencePrimer EPO3SBI 84tacgtacgtc atctgtcccc tgtcctgcag gc 328527DNAArtificial SequencePrimer GENEPO3BSI 85cgtacgtcat ctgtcccctg tcctgca 278628DNAArtificial SequencePrimer GENEPO5XBA 86tctagaatgg gggtgcacgg tgagtact 28874862DNAArtificial SequencepD2eGFP-1N plasmid from Clontech 87tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 660gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 720atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 780gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 840cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 900taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 960caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 1020ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 1080ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 1140gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 1200ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 1260ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 1320aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 1380gacgagctgt acaagaagct tagccatggc ttcccgccgg aggtggagga gcaggatgat 1440ggcacgctgc ccatgtcttg tgcccaggag agcgggatgg accgtcaccc tgcagcctgt 1500gcttctgcta ggatcaatgt gtagatgcgc ggccgcgact ctagatcata atcagccata 1560ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 1620aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 1680aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 1740gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg 1800ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 1860ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 1920tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 1980tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 2040tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 2100aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 2160ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 2220ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 2280tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 2340caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc 2400agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 2460tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 2520aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 2580ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 2640atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt 2700ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg 2760attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 2820tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 2880caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa 2940gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 3000gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 3060ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 3120cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 3180gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 3240catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc 3300gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 3360cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 3420gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 3480gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 3540gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 3600catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 3660tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 3720accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta 3780tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg 3840gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat 3900acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc 3960tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt 4020agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 4080atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 4140aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 4200caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 4260ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 4320cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 4380tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 4440gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 4500ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 4560gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 4620caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 4680ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 4740tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 4800ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc 4860at 4862885192DNAArtificial SequencepIRESpuro2 plasmid from Clontech 88gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc

420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtcgtacgt 960ctccggattc gaattcggat ccgcggccgc atagataact gatccagtgt gctggaatta 1020attcgctgtc tgcgagggcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga 1080cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg 1140cggtgatgcc tttgagggtg gccgcgtcca tctggtcaga aaagacaatc tttttgttgt 1200caagcttgag gtgtggcagg cttgagatct ggccatacac ttgagtgaca atgacatcca 1260ctttgccttt ctctccacag gtgtccactc ccaggtccaa ctgcaggtcg agcatgcatc 1320tagggcggcc aattccgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc 1380ttggaataag gccggtgtgc gtttgtctat atgtgatttt ccaccatatt gccgtctttt 1440ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt 1500tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg 1560gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca 1620cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg 1680gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc 1740tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct 1800gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta 1860ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata agcttgccac 1920aacccacaag gagacgacct tccatgaccg agtacaagcc cacggtgcgc ctcgccaccc 1980gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca 2040cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg caagaactct 2100tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg 2160tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc 2220cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc 2280tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg 2340accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc 2400gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc 2460ggctcggctt caccgtcacc gccgacgtcg agtgcccgaa ggaccgcgcg acctggtgca 2520tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga 2580gcgcacgacc ccatggctcc gaccgaagcc gacccgggcg gccccgccga ccccgcaccc 2640gcccccgagg cccaccgact ctagagctcg ctgatcagcc tcgactgtgc cttctagttg 2700ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 2760cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 2820tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 2880gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 2940gagtgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac 3000cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 3060gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 3120gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 3180cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 3240tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 3300tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 3360ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 3420ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 3480gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 3540gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 3600ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg 3660tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 3720gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 3780tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 3840tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 3900tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3960ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 4020ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 4080gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 4140aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 4200aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 4260cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 4320ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 4380cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 4440ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 4500ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 4560ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 4620gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 4680ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 4740ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 4800gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 4860ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 4920cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 4980ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 5040aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 5100gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5160gcacatttcc ccgaaaagtg ccacctgacg tc 51928911182DNAArtificial SequencepAg1 Plasmid 89catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2040ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 2580tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 2700gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3300catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 6480ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 6540ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 6600atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 6660gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 6720cggggggaga cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 6780gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 6840cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 6900aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 6960gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 7020gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 7080ttccttatat agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 7140agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 7200cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 7260aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 7320tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 7380taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 7440cttggagtag acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga 7500agacgtggtt ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt 7560gggaccactg tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca 7620tttgtaggtg ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca 7680atggaatccg aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg 7740gtcttctgag actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat 7800gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7860taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7920aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7980atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 8040tacgaattcg agccttgact agagggtcga cggtatacag acatgataag atacattgat 8100gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 8160gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa 8220gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat 8280tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgta gcacgtgtca 8340gtcctgctcc tcggccacga agtgcacgca gttgccggcc gggtcgcgca gggcgaactc 8400ccgcccccac ggctgctcgc cgatctcggt catggccggc ccggaggcgt cccggaagtt 8460cgtggacacg acctccgacc actcggcgta cagctcgtcc aggccgcgca cccacaccca 8520ggccagggtg ttgtccggca ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc 8580gtcccggacc acaccggcga agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc 8640ggtccagaac tcgaccgctc cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt 8700caacttggcc atggatccag atttcgctca agttagtata aaaaagcagg cttcaatcct 8760gcaggaattc gatcgacact ctcgtctact ccaagaatat caaagataca gtctcagaag 8820accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc ctcggattcc 8880attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca 8940aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc 9000ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt 9060cttcaaagca agtggattga tgtgataaca tggtggagca cgacactctc gtctactcca 9120agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg 9180taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcaaaagga 9240cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga aaggctatcg 9300ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 9360tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca 9420ctgacgtaag ggatgacgca caatcccact atccttcgca agaccttcct ctatataagg 9480aagttcattt catttggaga ggacacgctg aaatcaccag tctctctcta caaatctatc 9540tctctcgagc tttcgcagat ccgggggggc aatgagatat gaaaaagcct gaactcaccg 9600cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc 9660tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc 9720tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg 9780catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggagtttagc gagagcctga 9840cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac 9900tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc gatcgctgcg gccgatctta 9960gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc 10020gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg 10080acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact 10140gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 10200atggccgcat aacagcggtc

attgactgga gcgaggcgat gttcggggat tcccaatacg 10260aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct 10320acttcgagcg gaggcatccg gagcttgcag gatcgccacg actccgggcg tatatgctcc 10380gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt 10440gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 10500aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata 10560gtggaaaccg acgccccagc actcgtccga gggcaaagaa atagagtaga tgccgaccgg 10620atctgtcgat cgacaagctc gagtttctcc ataataatgt gtgagtagtt cccagataag 10680ggaattaggg ttcctatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat 10740gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc 10800agtactaaaa tccagatccc ccgaattaat tcggcgttaa ttcagatcaa gcttggcact 10860ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 10920tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 10980ttcccaacag ttgcgcagcc tgaatggcga atgctagagc agcttgagct tggatcagat 11040tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 11100cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta 11160tccgttcgtc catttgtatg tg 11182908428DNAArtificial SequencepCambia3300 Plasmid 90catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2040ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 2580tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 2700gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3300catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 6480ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 6540ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 6600atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 6660gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 6720cggggggaga cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 6780gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 6840cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 6900aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 6960gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 7020gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 7080ttccttatat agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 7140agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 7200cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 7260aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 7320tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 7380taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 7440cttggagtag acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga 7500agacgtggtt ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt 7560gggaccactg tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca 7620tttgtaggtg ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca 7680atggaatccg aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg 7740gtcttctgag actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat 7800gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7860taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7920aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7980atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 8040tacgaattcg agctcggtac ccggggatcc tctagagtcg acctgcaggc atgcaagctt 8100ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 8160tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 8220tcgcccttcc caacagttgc gcagcctgaa tggcgaatgc tagagcagct tgagcttgga 8280tcagattgtc gtttcccgcc ttcagtttaa actatcagtg tttgacagga tatattggcg 8340ggtaaaccta agagaaaaga gcgtttatta gaataacgga tatttaaaag ggcgtgaaaa 8400ggtttatccg ttcgtccatt tgtatgtg 8428913438DNAArtificial SequencepLIT38attBZeo Plasmid 91tcgaccctct agtcaaggcc ttaagtgagt cgtattacgg actggccgtc gttttacaac 60gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 120tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 180gcctgaatgg cgaatggcgc ttcgcttggt aataaagccc gcttcggcgg gctttttttt 240gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 300tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 360ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 420ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 480tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 540gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 600gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 660acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 720tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 780caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 840gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 900cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 960tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 1020agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 1080tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 1140ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 1200acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1260ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1320aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1380aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1440aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1500ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1560ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1620aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1680gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1740cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1800atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1860ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1920ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1980ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 2040ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 2100ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 2160tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 2220tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2280tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2340tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2400gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2460tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2520ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2580accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2640acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2700ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gattgaagcc 2760tgctttttta tactaacttg agcgaaatct ggatccatgg ccaagttgac cagtgccgtt 2820ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg 2880ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg 2940ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg 3000cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac 3060gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg 3120cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta 3180cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg 3240gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc 3300aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 3360aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 3420tatcatgtct gtataccg 34389210549DNAArtificial SequencepCambia1302 Plasmid 92catggtagat ctgactagta aaggagaaga acttttcact ggagttgtcc caattcttgt 60tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 120tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc 180gtggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttcaa gatacccaga 240tcatatgaag cggcacgact tcttcaagag cgccatgcct gagggatacg tgcaggagag 300gaccatcttc ttcaaggacg acgggaacta caagacacgt gctgaagtca agtttgaggg 360agacaccctc gtcaacagga tcgagcttaa gggaatcgat ttcaaggagg acggaaacat 420cctcggccac aagttggaat acaactacaa ctcccacaac gtatacatca tggccgacaa 480gcaaaagaac ggcatcaaag ccaacttcaa gacccgccac aacatcgaag acggcggcgt 540gcaactcgct gatcattatc aacaaaatac tccaattggc gatggccctg tccttttacc 600agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga 660ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact 720atacaaagct agccaccacc accaccacca cgtgtgaatt ggtgaccagc tcgaatttcc 780ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 840cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 900gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 960acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 1020ctatgttact agatcgggaa ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 1080cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta 1140tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga tcaaagtact 1200ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt gcagccgtct 1260tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc cctgcccttt 1320tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg cgactagaac 1380cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc 1440agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc 1500aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct ggccaggatg 1560cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg cctggcccgc 1620agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc gggcctgcgt 1680agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt gttgaccgtg 1740ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc 1800gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac cccggcacag 1860atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca 1920ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg

1980cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc 2040ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc 2100aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg 2160tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt 2220ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc 2280gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata 2340tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact 2400taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc gcgccctgca 2460actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcgattg 2520ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga 2580ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag cgccccaggc 2640ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc 2700aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga 2760ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg 2820catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc ttgagtcccg 2880tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc 2940agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa 3000actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc 3060ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca 3120gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac 3180gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca 3240gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat 3300ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg 3360cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc 3420caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggtacaa atcggcgcgg 3480cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca 3540tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag 3600aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc aagggcgacg 3660agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca 3720tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc 3780gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc atggccagtg 3840tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc atgaaccgat 3900accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac 3960tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca 4020ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc 4080tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa 4140ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag 4200aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca 4260tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt 4320tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca 4380ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag gaggaggcgg 4440ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg 4500ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc 4560gaaaaggtct ctttcctgtg gatagcacgt acattgggaa cccaaagccg tacattggga 4620accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag 4680tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta 4740aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc 4800tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc 4860ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc 4920gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc accctgcctc 4980gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 5040gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 5100ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc 5160ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac 5220cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg 5280actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5340tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 5400aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5460ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5520aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5580cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5640cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5700aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5760cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 5820ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 5880ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 5940gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6000agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 6060acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcattct aggtactaaa 6120acaattcatc cagtaaaata taatatttta ttttctccca atcaggcttg atccccagta 6180agtcaaaaaa tagctcgaca tactgttctt ccccgatatc ctccctgatc gaccggacgc 6240agaaggcaat gtcataccac ttgtccgccc tgccgcttct cccaagatca ataaagccac 6300ttactttgcc atctttcaca aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa 6360gttcctcttc gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg 6420gagtgtcttc ttcccagttt tcgcaatcca catcggccag atcgttattc agtaagtaat 6480ccaattcggc taagcggctg tctaagctat tcgtataggg acaatccgat atgtcgatgg 6540agtgaaagag cctgatgcac tccgcataca gctcgataat cttttcaggg ctttgttcat 6600cttcatactc ttccgagcaa aggacgccat cggcctcact catgagcaga ttgctccagc 6660catcatgccg ttcaaagtgc aggacctttg gaacaggcag ctttccttcc agccatagca 6720tcatgtcctt ttcccgttcc acatcatagg tggtcccttt ataccggctg tccgtcattt 6780ttaaatatag gttttcattt tctcccacca gcttatatac cttagcagga gacattcctt 6840ccgtatcttt tacgcagcgg tatttttcga tcagtttttt caattccggt gatattctca 6900ttttagccat ttattatttc cttcctcttt tctacagtat ttaaagatac cccaagaagc 6960taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc ttaaatacca 7020gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat cgacggagcc 7080gattttgaaa ccgcggtgat cacaggcagc aacgctctgt catcgttaca atcaacatgc 7140taccctccgc gagatcatcc gtgtttcaaa cccggcagct tagttgccgt tcttccgaat 7200agcatcggta acatgagcaa agtctgccgc cttacaacgg ctctcccgct gacgccgtcc 7260cggactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg gtcggggagc 7320tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct tagacaactt 7380aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat tcgggggatc 7440tggattttag tactggattt tggttttagg aattagaaat tttattgata gaagtatttt 7500acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg aaaccctata 7560ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa actcgagctt 7620gtcgatcgac agatccggtc ggcatctact ctatttcttt gccctcggac gagtgctggg 7680gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct 7740tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca 7800tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg 7860gtcaagacca atgcggagca tatacgcccg gagtcgtggc gatcctgcaa gctccggatg 7920cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa 7980gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc 8040tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg 8100ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga 8160cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc 8220gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa 8280cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atagcctccg cgaccggttg 8340tagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg 8400ggagatgcaa taggtcaggc tctcgctaaa ctccccaatg tcaagcactt ccggaatcgg 8460gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca 8520gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc 8580ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt 8640ctcgacagac gtcgcggtga gttcaggctt tttcatatct cattgccccc cgggatctgc 8700gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt gtcctctcca 8760aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg attgtgcgtc 8820atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac 8880gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc 8940agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc 9000ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag 9060gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt 9120atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat cacatcaatc 9180cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg 9240gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc 9300gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca 9360gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc 9420aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc 9480gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct ccccgcgcgt 9540tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 9600cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 9660cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 9720tatgaccatg attacgaatt cgagctcggt acccggggat cctctagagt cgacctgcag 9780gcatgcaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 9840tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 9900ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gctagagcag 9960cttgagcttg gatcagattg tcgtttcccg ccttcagttt agcttcatgg agtcaaagat 10020tcaaatagag gacctaacag aactcgccgt aaagactggc gaacagttca tacagagtct 10080cttacgactc aatgacaaga agaaaatctt cgtcaacatg gtggagcacg acacacttgt 10140ctactccaaa aatatcaaag atacagtctc agaagaccaa agggcaattg agacttttca 10200acaaagggta atatccggaa acctcctcgg attccattgc ccagctatct gtcactttat 10260tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa 10320ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag 10380gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga 10440tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc 10500tatataagga agttcatttc atttggagag aacacggggg actcttgac 105499333DNAArtificial SequenceCaMV35SpolyA Primer 93ctgaattaac gccgaattaa ttcgggggat ctg 339429DNAArtificial SequenceCaMV35Spr Primer 94ctagagcagc ttgccaacat ggtggagca 299512592DNAArtificial SequencepAg2 Plasmid 95gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa gccttgatta 60gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag atcgagctag 120ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg acggttcacc 180ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg gcacgccgcg 240ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc agtggcagcg 300ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca aatgacctgc 360cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc atgcgctacc 420gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag atgctagggc 480aaattgccct agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat agcacgtaca 540ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac ccaaagccgt 600acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa ggcgattttt 660ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc tgtgcataac 720tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg tcgctgcgct 780ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca aaaatggctg 840gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca ctcgaccgcc 900ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga aaacctctga 960cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 1020gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca 1080cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga 1140gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 1200ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 1260cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 1320gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 1380tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 1440agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 1500tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 1560cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 1620ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 1680ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1740ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1800ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1860cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1920gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1980atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 2040ttttggtcat gcattctagg tactaaaaca attcatccag taaaatataa tattttattt 2100tctcccaatc aggcttgatc cccagtaagt caaaaaatag ctcgacatac tgttcttccc 2160cgatatcctc cctgatcgac cggacgcaga aggcaatgtc ataccacttg tccgccctgc 2220cgcttctccc aagatcaata aagccactta ctttgccatc tttcacaaag atgttgctgt 2280ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg cttttccgtc tttaaaaaat 2340catacagctc gcgcggatct ttaaatggag tgtcttcttc ccagttttcg caatccacat 2400cggccagatc gttattcagt aagtaatcca attcggctaa gcggctgtct aagctattcg 2460tatagggaca atccgatatg tcgatggagt gaaagagcct gatgcactcc gcatacagct 2520cgataatctt ttcagggctt tgttcatctt catactcttc cgagcaaagg acgccatcgg 2580cctcactcat gagcagattg ctccagccat catgccgttc aaagtgcagg acctttggaa 2640caggcagctt tccttccagc catagcatca tgtccttttc ccgttccaca tcataggtgg 2700tccctttata ccggctgtcc gtcattttta aatataggtt ttcattttct cccaccagct 2760tatatacctt agcaggagac attccttccg tatcttttac gcagcggtat ttttcgatca 2820gttttttcaa ttccggtgat attctcattt tagccattta ttatttcctt cctcttttct 2880acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca attcactgtt 2940ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt tttcaaagtt 3000ggcgtataac atagtatcga cggagccgat tttgaaaccg cggtgatcac aggcagcaac 3060gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc 3120ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt 3180acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat 3240tttgtgccga gctgccggtc ggggagctgt tggctggctg gtggcaggat atattgtggt 3300gtaaacaaat tgacgcttag acaacttaat aacacattgc ggacgttttt aatgtactga 3360attaacgccg aattaattcg ggggatctgg attttagtac tggattttgg ttttaggaat 3420tagaaatttt attgatagaa gtattttaca aatacaaata catactaagg gtttcttata 3480tgctcaacac atgagcgaaa ccctatagga accctaattc ccttatctgg gaactactca 3540cacattatta tggagaaact cgagtcaaat ctcggtgacg ggcaggaccg gacggggcgg 3600taccggcagg ctgaagtcca gctgccagaa acccacgtca tgccagttcc cgtgcttgaa 3660gccggccgcc cgcagcatgc cgcggggggc atatccgagc gcctcgtgca tgcgcacgct 3720cgggtcgttg ggcagcccga tgacagcgac cacgctcttg aagccctgtg cctccaggga 3780cttcagcagg tgggtgtaga gcgtggagcc cagtcccgtc cgctggtggc ggggggagac 3840gtacacggtc gactcggccg tccagtcgta ggcgttgcgt gccttccagg ggcccgcgta 3900ggcgatgccg gcgacctcgc cgtccacctc ggcgacgagc cagggatagc gctcccgcag 3960acggacgagg tcgtccgtcc actcctgcgg ttcctgcggc tcggtacgga agttgaccgt 4020gcttgtctcg atgtagtggt tgacgatggt gcagaccgcc ggcatgtccg cctcggtggc 4080acggcggatg tcggccgggc gtcgttctgg gctcatggta gactcgagag agatagattt 4140gtagagagag actggtgatt tcagcgtgtc ctctccaaat gaaatgaact tccttatata 4200gaggaaggtc ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca gtggagatat 4260cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 4320tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct 4380ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga 4440tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt 4500gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga 4560cgagagtgtc gtgctccacc atgttatcac atcaatccac ttgctttgaa gacgtggttg 4620gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt 4680cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc 4740caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga 4800ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga 4860ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttggcaagct 4920gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 4980gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 5040gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 5100aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgaattcga 5160gccttgacta gagggtcgac ggtatacaga catgataaga tacattgatg agtttggaca 5220aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc 5280tttatttgta accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca 5340tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca 5400acctttcata gaaggcggcg gtggaatcga aatctcgtag cacgtgtcag tcctgctcct 5460cggccacgaa gtgcacgcag ttgccggccg ggtcgcgcag ggcgaactcc cgcccccacg 5520gctgctcgcc gatctcggtc atggccggcc cggaggcgtc ccggaagttc gtggacacga 5580cctccgacca ctcggcgtac agctcgtcca ggccgcgcac ccacacccag gccagggtgt 5640tgtccggcac cacctggtcc tggaccgcgc tgatgaacag ggtcacgtcg tcccggacca 5700caccggcgaa gtcgtcctcc acgaagtccc gggagaaccc gagccggtcg gtccagaact 5760cgaccgctcc ggcgacgtcg cgcgcggtga gcaccggaac ggcactggtc aacttggcca 5820tggatccaga tttcgctcaa gttagtataa aaaagcaggc ttcaatcctg caggaattcg 5880atcgacactc tcgtctactc caagaatatc aaagatacag tctcagaaga ccaaagggct 5940attgagactt ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct 6000atctgtcact tcatcaaaag gacagtagaa aaggaaggtg gcacctacaa atgccatcat 6060tgcgataaag gaaaggctat cgttcaagat gcctctgccg acagtggtcc caaagatgga 6120cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa 6180gtggattgat gtgataacat ggtggagcac gacactctcg

tctactccaa gaatatcaaa 6240gatacagtct cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga 6300aacctcctcg gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag 6360gaaggtggca cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc 6420tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa 6480gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg atatctccac tgacgtaagg 6540gatgacgcac aatcccacta tccttcgcaa gaccttcctc tatataagga agttcatttc 6600atttggagag gacacgctga aatcaccagt ctctctctac aaatctatct ctctcgagct 6660ttcgcagatc cgggggggca atgagatatg aaaaagcctg aactcaccgc gacgtctgtc 6720gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 6780gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 6840agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 6900ctcccgattc cggaagtgct tgacattggg gagtttagcg agagcctgac ctattgcatc 6960tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 7020ctacaaccgg tcgcggaggc tatggatgcg atcgctgcgg ccgatcttag ccagacgagc 7080gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 7140tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 7200gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 7260cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 7320acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 7380atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 7440aggcatccgg agcttgcagg atcgccacga ctccgggcgt atatgctccg cattggtctt 7500gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 7560cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 7620agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 7680cgccccagca ctcgtccgag ggcaaagaaa tagagtagat gccgaccgga tctgtcgatc 7740gacaagctcg agtttctcca taataatgtg tgagtagttc ccagataagg gaattagggt 7800tcctataggg tttcgctcat gtgttgagca tataagaaac ccttagtatg tatttgtatt 7860tgtaaaatac ttctatcaat aaaatttcta attcctaaaa ccaaaatcca gtactaaaat 7920ccagatcccc cgaattaatt cggcgttaat tcagatcaag cttggcactg gccgtcgttt 7980tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 8040cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 8100tgcgcagcct gaatggcgaa tgctagagca gcttgagctt ggatcagatt gtcgtttccc 8160gccttcagtt tggggatcct ctagactgaa ggcgggaaac gacaatctga tcatgagcgg 8220agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc gttttacgtt 8280tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc tggagtttaa 8340tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc taaggtcact 8400atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa attcccctcg 8460gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg gatctcgaga 8520atcgaattcc cgcggccgcc atggtagatc tgactagtaa aggagaagaa cttttcactg 8580gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca 8640gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta 8700ctggaaaact acctgttccg tggccaacac ttgtcactac tttctcttat ggtgttcaat 8760gcttttcaag atacccagat catatgaagc ggcacgactt cttcaagagc gccatgcctg 8820agggatacgt gcaggagagg accatcttct tcaaggacga cgggaactac aagacacgtg 8880ctgaagtcaa gtttgaggga gacaccctcg tcaacaggat cgagcttaag ggaatcgatt 8940tcaaggagga cggaaacatc ctcggccaca agttggaata caactacaac tcccacaacg 9000tatacatcat ggccgacaag caaaagaacg gcatcaaagc caacttcaag acccgccaca 9060acatcgaaga cggcggcgtg caactcgctg atcattatca acaaaatact ccaattggcg 9120atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc ctttcgaaag 9180atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta 9240cacatggcat ggatgaacta tacaaagcta gccaccacca ccaccaccac gtgtgaattg 9300gtgaccagct cgaatttccc cgatcgttca aacatttggc aataaagttt cttaagattg 9360aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta cgttaagcat 9420gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc 9480ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa 9540ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat taaactatca gtgtttgaca 9600ggatatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataac ggatatttaa 9660aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt gcatgccaac cacagggttc 9720ccctcgggat caaagtactt tgatccaacc cctccgctgc tatagtgcag tcggcttctg 9780acgttcagtg cagccgtctt ctgaaaacga catgtcgcac aagtcctaag ttacgcgaca 9840ggctgccgcc ctgccctttt cctggcgttt tcttgtcgcg tgttttagtc gcataaagta 9900gaatacttgc gactagaacc ggagacatta cgccatgaac aagagcgccg ccgctggcct 9960gctgggctat gcccgcgtca gcaccgacga ccaggacttg accaaccaac gggccgaact 10020gcacgcggcc ggctgcacca agctgttttc cgagaagatc accggcacca ggcgcgaccg 10080cccggagctg gccaggatgc ttgaccacct acgccctggc gacgttgtga cagtgaccag 10140gctagaccgc ctggcccgca gcacccgcga cctactggac attgccgagc gcatccagga 10200ggccggcgcg ggcctgcgta gcctggcaga gccgtgggcc gacaccacca cgccggccgg 10260ccgcatggtg ttgaccgtgt tcgccggcat tgccgagttc gagcgttccc taatcatcga 10320ccgcacccgg agcgggcgcg aggccgccaa ggcccgaggc gtgaagtttg gcccccgccc 10380taccctcacc ccggcacaga tcgcgcacgc ccgcgagctg atcgaccagg aaggccgcac 10440cgtgaaagag gcggctgcac tgcttggcgt gcatcgctcg accctgtacc gcgcacttga 10500gcgcagcgag gaagtgacgc ccaccgaggc caggcggcgc ggtgccttcc gtgaggacgc 10560attgaccgag gccgacgccc tggcggccgc cgagaatgaa cgccaagagg aacaagcatg 10620aaaccgcacc aggacggcca ggacgaaccg tttttcatta ccgaagagat cgaggcggag 10680atgatcgcgg ccgggtacgt gttcgagccg cccgcgcacg tctcaaccgt gcggctgcat 10740gaaatcctgg ccggtttgtc tgatgccaag ctggcggcct ggccggccag cttggccgct 10800gaagaaaccg agcgccgccg tctaaaaagg tgatgtgtat ttgagtaaaa cagcttgcgt 10860catgcggtcg ctgcgtatat gatgcgatga gtaaataaac aaatacgcaa ggggaacgca 10920tgaaggttat cgctgtactt aaccagaaag gcgggtcagg caagacgacc atcgcaaccc 10980atctagcccg cgccctgcaa ctcgccgggg ccgatgttct gttagtcgat tccgatcccc 11040agggcagtgc ccgcgattgg gcggccgtgc gggaagatca accgctaacc gttgtcggca 11100tcgaccgccc gacgattgac cgcgacgtga aggccatcgg ccggcgcgac ttcgtagtga 11160tcgacggagc gccccaggcg gcggacttgg ctgtgtccgc gatcaaggca gccgacttcg 11220tgctgattcc ggtgcagcca agcccttacg acatatgggc caccgccgac ctggtggagc 11280tggttaagca gcgcattgag gtcacggatg gaaggctaca agcggccttt gtcgtgtcgc 11340gggcgatcaa aggcacgcgc atcggcggtg aggttgccga ggcgctggcc gggtacgagc 11400tgcccattct tgagtcccgt atcacgcagc gcgtgagcta cccaggcact gccgccgccg 11460gcacaaccgt tcttgaatca gaacccgagg gcgacgctgc ccgcgaggtc caggcgctgg 11520ccgctgaaat taaatcaaaa ctcatttgag ttaatgaggt aaagagaaaa tgagcaaaag 11580cacaaacacg ctaagtgccg gccgtccgag cgcacgcagc agcaaggctg caacgttggc 11640cagcctggca gacacgccag ccatgaagcg ggtcaacttt cagttgccgg cggaggatca 11700caccaagctg aagatgtacg cggtacgcca aggcaagacc attaccgagc tgctatctga 11760atacatcgcg cagctaccag agtaaatgag caaatgaata aatgagtaga tgaattttag 11820cggctaaagg aggcggcatg gaaaatcaag aacaaccagg caccgacgcc gtggaatgcc 11880ccatgtgtgg aggaacgggc ggttggccag gcgtaagcgg ctgggttgtc tgccggccct 11940gcaatggcac tggaaccccc aagcccgagg aatcggcgtg acggtcgcaa accatccggc 12000ccggtacaaa tcggcgcggc gctgggtgat gacctggtgg agaagttgaa ggccgcgcag 12060gccgcccagc ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg gcaagcggcc 12120gctgatcgaa tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc gtcgattagg 12180aagccgccca agggcgacga gcaaccagat tttttcgttc cgatgctcta tgacgtgggc 12240acccgcgata gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa gcgtgaccga 12300cgagctggcg aggtgatccg ctacgagctt ccagacgggc acgtagaggt ttccgcaggg 12360ccggccggca tggccagtgt gtgggattac gacctggtac tgatggcggt ttcccatcta 12420accgaatcca tgaaccgata ccgggaaggg aagggagaca agcccggccg cgtgttccgt 12480ccacacgttg cggacgtact caagttctgc cggcgagccg atggcggaaa gcagaaagac 12540gacctggtag aaacctgcat tcggttaaac accacgcacg ttgccatgca gc 12592963357DNAArtificial SequencepGEMEasyNOS Plasmid 96tatcactagt gaattcgcgg ccgcctgcag gtcgaccata tgggagagct cccaacgcgt 60tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta atcatggtca 120tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 180agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 240cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 300caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 360tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 420cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 480aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 540gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 600agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 660cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 720cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 780ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 840gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 900tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 960acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 1020tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 1080attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 1140gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 1200ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 1260taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 1320ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 1380ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 1440gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 1500ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 1560gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 1620tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 1680atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 1740gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 1800tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 1860atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 1920agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 1980ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 2040tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 2100aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 2160tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 2220aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga 2280aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt 2340ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 2400atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca 2460gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 2520gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg 2580aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 2640ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 2700gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg 2760ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 2820tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 2880gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt 2940aatacgactc actatagggc gaattgggcc cgacgtcgca tgctcccggc cgccatggcg 3000gccgcgggaa ttcgattctc gagatccggt gcagattatt tggattgaga gtgaatatga 3060gactctaatt ggataccgag gggaatttat ggaacgtcag tggagcattt ttgacaagaa 3120atatttgcta gctgatagtg accttaggcg acttttgaac gcgcaataat ggtttctgac 3180gtatgtgctt agctcattaa actccagaaa cccgcggctg agtggctcct tcaacgttgc 3240ggttctgtca gttccaaacg taaaacggct tgtcccgcgt catcggcggg ggtcataacg 3300tgactccctt aattctccgc tcatgatcag attgtcgttt cccgccttca gtctaga 33579710122DNAArtificial Sequencep1302NOS Plasmid 97catggtagat ctgactagta aaggagaaga acttttcact ggagttgtcc caattcttgt 60tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 120tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc 180gtggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttcaa gatacccaga 240tcatatgaag cggcacgact tcttcaagag cgccatgcct gagggatacg tgcaggagag 300gaccatcttc ttcaaggacg acgggaacta caagacacgt gctgaagtca agtttgaggg 360agacaccctc gtcaacagga tcgagcttaa gggaatcgat ttcaaggagg acggaaacat 420cctcggccac aagttggaat acaactacaa ctcccacaac gtatacatca tggccgacaa 480gcaaaagaac ggcatcaaag ccaacttcaa gacccgccac aacatcgaag acggcggcgt 540gcaactcgct gatcattatc aacaaaatac tccaattggc gatggccctg tccttttacc 600agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga 660ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact 720atacaaagct agccaccacc accaccacca cgtgtgaatt ggtgaccagc tcgaatttcc 780ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 840cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 900gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 960acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 1020ctatgttact agatcgggaa ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 1080cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta 1140tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga tcaaagtact 1200ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt gcagccgtct 1260tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc cctgcccttt 1320tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg cgactagaac 1380cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc 1440agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc 1500aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct ggccaggatg 1560cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg cctggcccgc 1620agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc gggcctgcgt 1680agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt gttgaccgtg 1740ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc 1800gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac cccggcacag 1860atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca 1920ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg 1980cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc 2040ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc 2100aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg 2160tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt 2220ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc 2280gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata 2340tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact 2400taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc gcgccctgca 2460actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcgattg 2520ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga 2580ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag cgccccaggc 2640ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc 2700aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga 2760ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg 2820catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc ttgagtcccg 2880tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc 2940agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa 3000actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc 3060ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca 3120gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac 3180gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca 3240gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat 3300ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg 3360cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc 3420caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggtacaa atcggcgcgg 3480cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca 3540tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag 3600aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc aagggcgacg 3660agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca 3720tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc 3780gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc atggccagtg 3840tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc atgaaccgat 3900accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac 3960tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca 4020ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc 4080tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa 4140ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag 4200aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca 4260tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt 4320tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca 4380ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag gaggaggcgg 4440ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg 4500ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc 4560gaaaaggtct ctttcctgtg gatagcacgt acattgggaa cccaaagccg tacattggga 4620accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag 4680tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta 4740aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc 4800tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc 4860ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc 4920gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc accctgcctc 4980gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 5040gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 5100ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc 5160ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac

5220cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg 5280actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5340tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 5400aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5460ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5520aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5580cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5640cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5700aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5760cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 5820ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 5880ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 5940gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6000agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 6060acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcattct aggtactaaa 6120acaattcatc cagtaaaata taatatttta ttttctccca atcaggcttg atccccagta 6180agtcaaaaaa tagctcgaca tactgttctt ccccgatatc ctccctgatc gaccggacgc 6240agaaggcaat gtcataccac ttgtccgccc tgccgcttct cccaagatca ataaagccac 6300ttactttgcc atctttcaca aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa 6360gttcctcttc gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg 6420gagtgtcttc ttcccagttt tcgcaatcca catcggccag atcgttattc agtaagtaat 6480ccaattcggc taagcggctg tctaagctat tcgtataggg acaatccgat atgtcgatgg 6540agtgaaagag cctgatgcac tccgcataca gctcgataat cttttcaggg ctttgttcat 6600cttcatactc ttccgagcaa aggacgccat cggcctcact catgagcaga ttgctccagc 6660catcatgccg ttcaaagtgc aggacctttg gaacaggcag ctttccttcc agccatagca 6720tcatgtcctt ttcccgttcc acatcatagg tggtcccttt ataccggctg tccgtcattt 6780ttaaatatag gttttcattt tctcccacca gcttatatac cttagcagga gacattcctt 6840ccgtatcttt tacgcagcgg tatttttcga tcagtttttt caattccggt gatattctca 6900ttttagccat ttattatttc cttcctcttt tctacagtat ttaaagatac cccaagaagc 6960taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc ttaaatacca 7020gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat cgacggagcc 7080gattttgaaa ccgcggtgat cacaggcagc aacgctctgt catcgttaca atcaacatgc 7140taccctccgc gagatcatcc gtgtttcaaa cccggcagct tagttgccgt tcttccgaat 7200agcatcggta acatgagcaa agtctgccgc cttacaacgg ctctcccgct gacgccgtcc 7260cggactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg gtcggggagc 7320tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct tagacaactt 7380aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat tcgggggatc 7440tggattttag tactggattt tggttttagg aattagaaat tttattgata gaagtatttt 7500acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg aaaccctata 7560ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa actcgagctt 7620gtcgatcgac agatccggtc ggcatctact ctatttcttt gccctcggac gagtgctggg 7680gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct 7740tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca 7800tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg 7860gtcaagacca atgcggagca tatacgcccg gagtcgtggc gatcctgcaa gctccggatg 7920cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa 7980gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc 8040tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg 8100ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga 8160cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc 8220gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa 8280cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atagcctccg cgaccggttg 8340tagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg 8400ggagatgcaa taggtcaggc tctcgctaaa ctccccaatg tcaagcactt ccggaatcgg 8460gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca 8520gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc 8580ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt 8640ctcgacagac gtcgcggtga gttcaggctt tttcatatct cattgccccc ccggatctgc 8700gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt gtcctctcca 8760aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg attgtgcgtc 8820atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac 8880gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc 8940agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc 9000ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag 9060gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt 9120atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat cacatcaatc 9180cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg 9240gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc 9300gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca 9360gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc 9420aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc 9480gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct ccccgcgcgt 9540tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 9600cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 9660cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 9720tatgaccatg attacgaatt cgagctcggt acccggggat cctctagact gaaggcggga 9780aacgacaatc tgatcatgag cggagaatta agggagtcac gttatgaccc ccgccgatga 9840cgcgggacaa gccgttttac gtttggaact gacagaaccg caacgttgaa ggagccactc 9900agccgcgggt ttctggagtt taatgagcta agcacatacg tcagaaacca ttattgcgcg 9960ttcaaaagtc gcctaaggtc actatcagct agcaaatatt tcttgtcaaa aatgctccac 10020tgacgttcca taaattcccc tcggtatcca attagagtct catattcact ctcaatccaa 10080ataatctgca ccggatctcg agaatcgaat tcccgcggcc gc 1012298621DNAArtificial SequenceN. tabacum rDNA intergnic spacer (IGS) sequence 98gtgctagcca atgtttaaca agatgtcaag cacaatgaat gttggtggtt ggtggtcgtg 60gctggcggtg gtggaaaatt gcggtggttc gagcggtagt gatcggcgat ggttggtgtt 120tgcagcggtg tttgatatcg gaatcactta tggtggttgt cacaatggag gtgcgtcatg 180gttattggtg gttggtcatc tatatatttt tataataata ttaagtattt tacctatttt 240ttacatattt tttattaaat ttatgcattg tttgtatttt taaatagttt ttatcgtact 300tgttttataa aatattttat tattttatgt gttatattat tacttgatgt attggaaatt 360ttctccattg ttttttctat atttataata attttcttat ttttttttgt tttattatgt 420attttttcgt tttataataa atatttatta aaaaaaatat tatttttgta aaatatatca 480tttacaatgt ttaaaagtca tttgtgaata tattagctaa gttgtacttc tttttgtgca 540tttggtgttg tacatgtcta ttatgattct ctggccaaaa catgtctact cctgtcactt 600gggttttttt ttttaagaca t 6219925DNAArtificial SequenceNTIGS-F1 Primer 99gtgctagcca atgtttaaca agatg 2510028DNAArtificial SequenceNTIGS-R1 Primer 100atgtcttaaa aaaaaaaacc caagtgac 28101233DNAMus MusculusGenbank #V008461989-07-06 101gacctggaat atggcgagaa aactgaaaat cacggaaaat gagaaataca cactttagga 60cgtgaaatat ggcgaggaaa actgaaaaag gtggaaaatt tagaaatgtc cactgtagga 120cgtggaatat ggcaagaaaa ctgaaaatca tggaaaatga gaaacatcca cttgacgact 180tgaaaaatga cgaaatcact aaaaaacgtg aaaaatgaga aatgcacact gaa 23310231DNAArtificial SequenceMSAT-F1 Primer 102aataccgcgg aagcttgacc tggaatatcg c 3110327DNAArtificial SequenceMSAT-Ri Primer 103ataaccgcgg agtccttcag tgtgcat 27104277DNAArtificial SequenceNopaline Synthase Promoter Sequence 104gagctcgaat ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc 60tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat 120aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca 180attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc 240gcgcgcggtg tcatctatgt tactagatcg ggaattc 2771051812DNAEscherichia coliCDS(1)...(1812)Beta-Glucuronidase 105atg tta cgt cct gta gaa acc cca acc cgt gaa atc aaa aaa ctc gac 48Met Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Asp1 5 10 15ggc ctg tgg gca ttc agt ctg gat cgc gaa aac tgt gga att gat cag 96Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile Asp Gln 20 25 30cgt tgg tgg gaa agc gcg tta caa gaa agc cgg gca att gct gtg cca 144Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg Ala Ile Ala Val Pro 35 40 45ggc agt ttt aac gat cag ttc gcc gat gca gat att cgt aat tat gcg 192Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp Ile Arg Asn Tyr Ala 50 55 60ggc aac gtc tgg tat cag cgc gaa gtc ttt ata ccg aaa ggt tgg gca 240Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile Pro Lys Gly Trp Ala65 70 75 80ggc cag cgt atc gtg ctg cgt ttc gat gcg gtc act cat tac ggc aaa 288Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr Gly Lys 85 90 95gtg tgg gtc aat aat cag gaa gtg atg gag cat cag ggc ggc tat acg 336Val Trp Val Asn Asn Gln Glu Val Met Glu His Gln Gly Gly Tyr Thr 100 105 110cca ttt gaa gcc gat gtc acg ccg tat gtt att gcc ggg aaa agt gta 384Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val 115 120 125cgt atc acc gtt tgt gtg aac aac gaa ctg aac tgg cag act atc ccg 432Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr Ile Pro 130 135 140ccg gga atg gtg att acc gac gaa aac ggc aag aaa aag cag tct tac 480Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln Ser Tyr145 150 155 160ttc cat gat ttc ttt aac tat gcc gga atc cat cgc agc gta atg ctc 528Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val Met Leu 165 170 175tac acc acg ccg aac acc tgg gtg gac gat atc acc gtg gtg acg cat 576Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val Thr His 180 185 190gtc gcg caa gac tgt aac cac gcg tct gtt gac tgg cag gtg gtg gcc 624Val Ala Gln Asp Cys Asn His Ala Ser Val Asp Trp Gln Val Val Ala 195 200 205aat ggt gat gtc agc gtt gaa ctg cgt gat gcg gat caa cag gtg gtt 672Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln Val Val 210 215 220gca act gga caa ggc act agc ggg act ttg caa gtg gtg aat ccg cac 720Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn Pro His225 230 235 240ctc tgg caa ccg ggt gaa ggt tat ctc tat gaa ctg tgc gtc aca gcc 768Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val Thr Ala 245 250 255aaa agc cag aca gag tgt gat atc tac ccg ctt cgc gtc ggc atc cgg 816Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly Ile Arg 260 265 270tca gtg gca gtg aag ggc gaa cag ttc ctg att aac cac aaa ccg ttc 864Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile Asn His Lys Pro Phe 275 280 285tac ttt act ggc ttt ggt cgt cat gaa gat gcg gac ttg cgt ggc aaa 912Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg Gly Lys 290 295 300gga ttc gat aac gtg ctg atg gtg cac gac cac gca tta atg gac tgg 960Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met Asp Trp305 310 315 320att ggg gcc aac tcc tac cgt acc tcg cat tac cct tac gct gaa gag 1008Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu 325 330 335atg ctc gac tgg gca gat gaa cat ggc atc gtg gtg att gat gaa act 1056Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp Glu Thr 340 345 350gct gct gtc ggc ttt aac ctc tct tta ggc att ggt ttc gaa gcg ggc 1104Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu Ala Gly 355 360 365aac aag ccg aaa gaa ctg tac agc gaa gag gca gtc aac ggg gaa act 1152Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly Glu Thr 370 375 380cag caa gcg cac tta cag gcg att aaa gag ctg ata gcg cgt gac aaa 1200Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg Asp Lys385 390 395 400aac cac cca agc gtg gtg atg tgg agt att gcc aac gaa ccg gat acc 1248Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro Asp Thr 405 410 415cgt ccg caa ggt gca cgg gaa tat ttc gcg cca ctg gcg gaa gca acg 1296Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu Ala Thr 420 425 430cgt aaa ctc gac ccg acg cgt ccg atc acc tgc gtc aat gta atg ttc 1344Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe 435 440 445tgc gac gct cac acc gat acc atc agc gat ctc ttt gat gtg ctg tgc 1392Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val Leu Cys 450 455 460ctg aac cgt tat tac gga tgg tat gtc caa agc ggc gat ttg gaa acg 1440Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu Glu Thr465 470 475 480gca gag aag gta ctg gaa aaa gaa ctt ctg gcc tgg cag gag aaa ctg 1488Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gln Glu Lys Leu 485 490 495cat cag ccg att atc atc acc gaa tac ggc gtg gat acg tta gcc ggg 1536His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu Ala Gly 500 505 510ctg cac tca atg tac acc gac atg tgg agt gaa gag tat cag tgt gca 1584Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln Cys Ala 515 520 525tgg ctg gat atg tat cac cgc gtc ttt gat cgc gtc agc gcc gtc gtc 1632Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val 530 535 540ggt gaa cag gta tgg aat ttc gcc gat ttt gcg acc tcg caa ggc ata 1680Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Ile545 550 555 560ttg cgc gtt ggc ggt aac aag aaa ggg atc ttc act cgc gac cgc aaa 1728Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp Arg Lys 565 570 575ccg aag tcg gcg gct ttt ctg ctg caa aaa cgc tgg act ggc atg aac 1776Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg Trp Thr Gly Met Asn 580 585 590ttc ggt gaa aaa ccg cag cag gga ggc aaa caa tga 1812Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln * 595 600106603PRTEscherichia coliGenbank #S694141994-09-23 106Met Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Asp1 5 10 15Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile Asp Gln 20 25 30Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg Ala Ile Ala Val Pro 35 40 45Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp Ile Arg Asn Tyr Ala 50 55 60Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile Pro Lys Gly Trp Ala65 70 75 80Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr Gly Lys 85 90 95Val Trp Val Asn Asn Gln Glu Val Met Glu His Gln Gly Gly Tyr Thr 100 105 110Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val 115 120 125Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr Ile Pro 130 135 140Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln Ser Tyr145 150 155 160Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val Met Leu 165 170 175Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val Thr His 180 185 190Val Ala Gln Asp Cys Asn His Ala Ser Val Asp Trp Gln Val Val Ala 195 200 205Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln Val Val 210 215 220Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn Pro His225 230 235 240Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val Thr Ala 245 250 255Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly Ile Arg 260 265 270Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile Asn His Lys Pro Phe 275 280 285Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg Gly Lys 290 295 300Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met Asp Trp305 310 315 320Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu 325 330 335Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp Glu Thr 340 345 350Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu Ala Gly 355 360 365Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly Glu Thr 370 375

380Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg Asp Lys385 390 395 400Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro Asp Thr 405 410 415Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu Ala Thr 420 425 430Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe 435 440 445Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val Leu Cys 450 455 460Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu Glu Thr465 470 475 480Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gln Glu Lys Leu 485 490 495His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu Ala Gly 500 505 510Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln Cys Ala 515 520 525Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val 530 535 540Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Ile545 550 555 560Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp Arg Lys 565 570 575Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg Trp Thr Gly Met Asn 580 585 590Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln 595 600107277DNAArtificial SequenceNopaline Synthase Terminator Sequence 107gagctcgaat ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc 60tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat 120aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca 180attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc 240gcgcgcggtg tcatctatgt tactagatcg ggaattc 2771083451DNAArtificial SequenceHindIII Fragment containing the beta-glucuronidase coding sequence, the rDNA intergenic spacer, and the Mast1 sequence 108aagcttgacc tggaatatcg cgagtaaact gaaaatcacg gaaaatgaga aatacacact 60ttaggacgtg aaatatggcg aggaaaactg aaaaaggtgg aaaatttaga aatgtccact 120gtaggacgtg gaatatggca agaaaactga aaatcatgga aaatgagaaa catccacttg 180acgacttgaa aaatgacgaa atcactaaaa aacgtgaaaa atgagaaatg cacactgaag 240gactccgcgg gaattcgatt gtgctagcca atgtttaaca agatgtcaag cacaatgaat 300gttggtggtt ggtggtcgtg gctggcggtg gtggaaaatt gcggtggttc gagcggtagt 360gatcggcgat ggttggtgtt tgcagcggtg tttgatatcg gaatcactta tggtggttgt 420cacaatggag gtgcgtcatg gttattggtg gttggtcatc tatatatttt tataataata 480ttaagtattt tacctatttt ttacatattt tttattaaat ttatgcattg tttgtatttt 540taaatagttt ttatcgtact tgttttataa aatattttat tattttatgt gttatattat 600tacttgatgt attggaaatt ttctccattg ttttttctat atttataata attttcttat 660ttttttttgt tttattatgt attttttcgt tttataataa atatttatta aaaaaaatat 720tatttttgta aaatatatca tttacaatgt ttaaaagtca tttgtgaata tattagctaa 780gttgtacttc tttttgtgca tttggtgttg tacatgtcta ttatgattct ctggccaaaa 840catgtctact cctgtcactt gggttttttt ttttaagaca taatcactag tgattatatc 900tagactgaag gcgggaaacg acaatctgat catgagcgga gaattaaggg agtcacgtta 960tgacccccgc cgatgacgcg ggacaagccg ttttacgttt ggaactgaca gaaccgcaac 1020gttgaaggag ccactcagcc gcgggtttct ggagtttaat gagctaagca catacgtcag 1080aaaccattat tgcgcgttca aaagtcgcct aaggtcacta tcagctagca aatatttctt 1140gtcaaaaatg ctccactgac gttccataaa ttcccctcgg tatccaatta gagtctcata 1200ttcactctca atccaaataa tctgcaccgg atctcgagat cgaattcccg cggccgcgaa 1260ttcactagtg gatccccggg tacggtcagt cccttatgtt acgtcctgta gaaaccccaa 1320cccgtgaaat caaaaaactc gacggcctgt gggcattcag tctggatcgc gaaaactgtg 1380gaattgagca gcgttggtgg gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag 1440gcagttttaa cgatcagttc gccgatgcag atattcgtaa ttatgtgggc aacgtctggt 1500atcagcgcga agtctttata ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg 1560atgcggtcac tcattacggc aaagtgtggg tcaataatca ggaagtgatg gagcatcagg 1620gcggctatac gccatttgaa gccgatgtca cgccgtatgt tattgccggg aaaagtgtac 1680gtatcacagt ttgtgtgaac aacgaactga actggcagac tatcccgccg ggaatggtga 1740ttaccgacga aaacggcaag aaaaagcagt cttacttcca tgatttcttt aactacgccg 1800ggatccatcg cagcgtaatg ctctacacca cgccgaacac ctgggtggac gatatcaccg 1860tggtgacgca tgtcgcgcaa gactgtaacc acgcgtctgt tgactggcag gtggtggcca 1920atggtgatgt cagcgttgaa ctgcgtgatg cggatcaaca ggtggttgca actggacaag 1980gcaccagcgg gactttgcaa gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc 2040tctatgaact gtacgtcaca gccaaaagcc agacagagtg tgatatctac ccgctgcgcg 2100tcggcatccg gtcagtggca gtgaagggcg aacagttcct gatcaaccac aaaccgttct 2160actttactgg ctttggccgt catgaagatg cggatttgcg cggcaaagga ttcgataacg 2220tgctgatggt gcacgatcac gcattaatgg actggattgg ggccaactcc taccgtacct 2280cgcattaccc ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggtga 2340ttgatgaaac tgcagctgtc ggctttaacc tctctttagg cattggtttc gaagcgggca 2400acaagccgaa agaactgtac agcgaagagg cagtcaacgg ggaaactcag caggcgcact 2460tacaggcgat taaagagctg atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga 2520gtattgccaa cgaaccggat acccgtccgc aaggtgcacg ggaatatttc gcgccactgg 2580cggaagcaac gcgtaaactc gatccgacgc gtccgatcac ctgcgtcaat gtaatgttct 2640gcgacgctca caccgatacc atcagcgatc tctttgatgt gctgtgcctg aaccgttatt 2700acggttggta tgtccaaagc ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac 2760ttctggcctg gcaggagaaa ctgcatcagc cgattatcat caccgaatac ggcgtggata 2820cgttagccgg gctgcactca atgtacaccg acatgtggag tgaagagtat cagtgtgcat 2880ggctggatat gtatcaccgc gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat 2940ggaatttcgc cgattttgcg acctcgcaag gcatattgcg cgttggcggt aacaagaagg 3000ggatcttcac ccgcgaccgc aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga 3060ctggcatgaa cttcggtgaa aaaccgcagc agggaggcaa acaatgaatc aacaactctc 3120ctggcgcacc atcgtcggct acagcctcgg gaattgcgta ccgagctcga atttccccga 3180tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat 3240gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat 3300gacgttattt atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc 3360gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat 3420gttactagat cgggaattcg atatcaagct t 345110914627DNAArtificial SequencepAg11a Plasmid 109catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2040ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 2580tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 2700gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3300catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 6480ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 6540ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 6600atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 6660gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 6720cggggggaga cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 6780gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 6840cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 6900aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 6960gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 7020gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 7080ttccttatat agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 7140agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 7200cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 7260aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 7320tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 7380taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 7440cttggagtag acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga 7500agacgtggtt ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt 7560gggaccactg tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca 7620tttgtaggtg ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca 7680atggaatccg aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg 7740gtcttctgag actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat 7800gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7860taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7920aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7980atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 8040tacgaattcg agccttgact agagggtcga cggtatacag acatgataag atacattgat 8100gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 8160gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa 8220gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat 8280tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgta gcacgtgtca 8340gtcctgctcc tcggccacga agtgcacgca gttgccggcc gggtcgcgca gggcgaactc 8400ccgcccccac ggctgctcgc cgatctcggt catggccggc ccggaggcgt cccggaagtt 8460cgtggacacg acctccgacc actcggcgta cagctcgtcc aggccgcgca cccacaccca 8520ggccagggtg ttgtccggca ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc 8580gtcccggacc acaccggcga agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc 8640ggtccagaac tcgaccgctc cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt 8700caacttggcc atggatccag atttcgctca agttagtata aaaaagcagg cttcaatcct 8760gcaggaattc gatcgacact ctcgtctact ccaagaatat caaagataca gtctcagaag 8820accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc ctcggattcc 8880attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca 8940aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc 9000ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt 9060cttcaaagca agtggattga tgtgataaca tggtggagca cgacactctc gtctactcca 9120agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg 9180taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcaaaagga 9240cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga aaggctatcg 9300ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 9360tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca 9420ctgacgtaag ggatgacgca caatcccact atccttcgca agaccttcct ctatataagg 9480aagttcattt catttggaga ggacacgctg aaatcaccag tctctctcta caaatctatc 9540tctctcgagc tttcgcagat ccgggggggc aatgagatat gaaaaagcct gaactcaccg 9600cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc 9660tctcggaggg cgaagaatct cgtgctttca gcttcgatgt

aggagggcgt ggatatgtcc 9720tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg 9780catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggagtttagc gagagcctga 9840cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac 9900tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc gatcgctgcg gccgatctta 9960gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc 10020gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg 10080acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact 10140gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 10200atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg 10260aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct 10320acttcgagcg gaggcatccg gagcttgcag gatcgccacg actccgggcg tatatgctcc 10380gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt 10440gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 10500aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata 10560gtggaaaccg acgccccagc actcgtccga gggcaaagaa atagagtaga tgccgaccgg 10620atctgtcgat cgacaagctc gagtttctcc ataataatgt gtgagtagtt cccagataag 10680ggaattaggg ttcctatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat 10740gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc 10800agtactaaaa tccagatccc ccgaattaat tcggcgttaa ttcagatcaa gcttgacctg 10860gaatatcgcg agtaaactga aaatcacgga aaatgagaaa tacacacttt aggacgtgaa 10920atatggcgag gaaaactgaa aaaggtggaa aatttagaaa tgtccactgt aggacgtgga 10980atatggcaag aaaactgaaa atcatggaaa atgagaaaca tccacttgac gacttgaaaa 11040atgacgaaat cactaaaaaa cgtgaaaaat gagaaatgca cactgaagga ctccgcggga 11100attcgattgt gctagccaat gtttaacaag atgtcaagca caatgaatgt tggtggttgg 11160tggtcgtggc tggcggtggt ggaaaattgc ggtggttcga gcggtagtga tcggcgatgg 11220ttggtgtttg cagcggtgtt tgatatcgga atcacttatg gtggttgtca caatggaggt 11280gcgtcatggt tattggtggt tggtcatcta tatattttta taataatatt aagtatttta 11340cctatttttt acatattttt tattaaattt atgcattgtt tgtattttta aatagttttt 11400atcgtacttg ttttataaaa tattttatta ttttatgtgt tatattatta cttgatgtat 11460tggaaatttt ctccattgtt ttttctatat ttataataat tttcttattt ttttttgttt 11520tattatgtat tttttcgttt tataataaat atttattaaa aaaaatatta tttttgtaaa 11580atatatcatt tacaatgttt aaaagtcatt tgtgaatata ttagctaagt tgtacttctt 11640tttgtgcatt tggtgttgta catgtctatt atgattctct ggccaaaaca tgtctactcc 11700tgtcacttgg gttttttttt ttaagacata atcactagtg attatatcta gactgaaggc 11760gggaaacgac aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg 11820atgacgcggg acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc 11880actcagccgc gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg 11940cgcgttcaaa agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct 12000ccactgacgt tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat 12060ccaaataatc tgcaccggat ctcgagatcg aattcccgcg gccgcgaatt cactagtgga 12120tccccgggta cggtcagtcc cttatgttac gtcctgtaga aaccccaacc cgtgaaatca 12180aaaaactcga cggcctgtgg gcattcagtc tggatcgcga aaactgtgga attgagcagc 12240gttggtggga aagcgcgtta caagaaagcc gggcaattgc tgtgccaggc agttttaacg 12300atcagttcgc cgatgcagat attcgtaatt atgtgggcaa cgtctggtat cagcgcgaag 12360tctttatacc gaaaggttgg gcaggccagc gtatcgtgct gcgtttcgat gcggtcactc 12420attacggcaa agtgtgggtc aataatcagg aagtgatgga gcatcagggc ggctatacgc 12480catttgaagc cgatgtcacg ccgtatgtta ttgccgggaa aagtgtacgt atcacagttt 12540gtgtgaacaa cgaactgaac tggcagacta tcccgccggg aatggtgatt accgacgaaa 12600acggcaagaa aaagcagtct tacttccatg atttctttaa ctacgccggg atccatcgca 12660gcgtaatgct ctacaccacg ccgaacacct gggtggacga tatcaccgtg gtgacgcatg 12720tcgcgcaaga ctgtaaccac gcgtctgttg actggcaggt ggtggccaat ggtgatgtca 12780gcgttgaact gcgtgatgcg gatcaacagg tggttgcaac tggacaaggc accagcggga 12840ctttgcaagt ggtgaatccg cacctctggc aaccgggtga aggttatctc tatgaactgt 12900acgtcacagc caaaagccag acagagtgtg atatctaccc gctgcgcgtc ggcatccggt 12960cagtggcagt gaagggcgaa cagttcctga tcaaccacaa accgttctac tttactggct 13020ttggccgtca tgaagatgcg gatttgcgcg gcaaaggatt cgataacgtg ctgatggtgc 13080acgatcacgc attaatggac tggattgggg ccaactccta ccgtacctcg cattaccctt 13140acgctgaaga gatgctcgac tgggcagatg aacatggcat cgtggtgatt gatgaaactg 13200cagctgtcgg ctttaacctc tctttaggca ttggtttcga agcgggcaac aagccgaaag 13260aactgtacag cgaagaggca gtcaacgggg aaactcagca ggcgcactta caggcgatta 13320aagagctgat agcgcgtgac aaaaaccacc caagcgtggt gatgtggagt attgccaacg 13380aaccggatac ccgtccgcaa ggtgcacggg aatatttcgc gccactggcg gaagcaacgc 13440gtaaactcga tccgacgcgt ccgatcacct gcgtcaatgt aatgttctgc gacgctcaca 13500ccgataccat cagcgatctc tttgatgtgc tgtgcctgaa ccgttattac ggttggtatg 13560tccaaagcgg cgatttggaa acggcagaga aggtactgga aaaagaactt ctggcctggc 13620aggagaaact gcatcagccg attatcatca ccgaatacgg cgtggatacg ttagccgggc 13680tgcactcaat gtacaccgac atgtggagtg aagagtatca gtgtgcatgg ctggatatgt 13740atcaccgcgt ctttgatcgc gtcagcgccg tcgtcggtga acaggtatgg aatttcgccg 13800attttgcgac ctcgcaaggc atattgcgcg ttggcggtaa caagaagggg atcttcaccc 13860gcgaccgcaa accgaagtcg gcggcttttc tgctgcaaaa acgctggact ggcatgaact 13920tcggtgaaaa accgcagcag ggaggcaaac aatgaatcaa caactctcct ggcgcaccat 13980cgtcggctac agcctcggga attgcgtacc gagctcgaat ttccccgatc gttcaaacat 14040ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata 14100atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat 14160gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa 14220aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg 14280ggaattcgat atcaagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc 14340ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 14400gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgct 14460agagcagctt gagcttggat cagattgtcg tttcccgcct tcagtttaaa ctatcagtgt 14520ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag aataacggat 14580atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtg 146271109080DNAArtificial Sequencep18attBZeo(6XHS4)2eGFP Plasmid 110cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt 1500ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg 1680atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1980gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 2400cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 2460ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 2580ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 2760gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg 2820tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc 3120ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg 3180ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 3240gggcgcaggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc 3540tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt tggcaaagaa ttcgccacca 3600tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 3660gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 3720gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 3780tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 3840agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 3900tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 3960tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 4020agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 4080gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 4140accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 4200acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 4260tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag 4320aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc 4380tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga 4440agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 4500gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac 4560atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa 4620caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta 4680ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt 4740ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc 4800tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag 4860cccaagcttg catgcctgca ggtcgactct agtggatccc ccgccccgta tcccccaggt 4920gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc 4980cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc 5040ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc 5100cctgggggct ttgggggggg gctgtccccg tgagcggatc cgcggccccg tatcccccag 5160gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct 5220tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga 5280ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca 5340tccctggggg ctttgggggg gggctgtccc cgtgagcgga tccgcggccc cgtatccccc 5400aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat cccgtgccac 5460cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg gggagcgccg 5520gaccggagcg gagccccggg cggctcgctg ctgcccccta gcgggggagg gacgtaatta 5580catccctggg ggctttgggg gggggctgtc cccgtgagcg gatccgcggc cccgtatccc 5640ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg atcccgtgcc 5700accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg gggggagcgc 5760cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga gggacgtaat 5820tacatccctg ggggctttgg gggggggctg tccccgtgag cggatccgcg gccccgtatc 5880ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg 5940ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc 6000gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta 6060attacatccc tgggggcttt gggggggggc tgtccccgtg agcggatccg cggccccgta 6120tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg 6180tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga 6240gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg 6300taattacatc cctgggggct ttgggggggg gctgtccccg tgagcggatc cgcggggctg 6360caggaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 6420tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 6480ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 6540ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 6600ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 6660agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 6720catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 6780tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 6840gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 6900ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 6960cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 7020caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 7080ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 7140taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 7200taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 7260cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 7320tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 7380gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 7440catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 7500atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 7560ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 7620gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 7680agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 7740gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 7800agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 7860catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 7920aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 7980gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 8040taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 8100caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 8160ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 8220ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 8280tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 8340aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 8400actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 8460catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 8520agtgccacct gacgtagtta acaaaaaaaa gcccgccgaa gcgggcttta ttaccaagcg 8580aagcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 8640tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 8700ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtccgtaata cgactcactt 8760aaggccttga ctagagggtc gacggtatac agacatgata agatacattg atgagtttgg 8820acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 8880tgctttattt gtaaccatta taagctgcaa taaacaagtt ggggtgggcg aagaactcca 8940gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg attccgaagc 9000ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tagcacgtgt cagtcctgct 9060cctcggccac gaagtgcacg 90801114223DNAArtificial SequencepLIT38attBBSRpolyA10 Plasmid 111gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840tggagccggt gagcgtgggt ctcgcggtat

cattgcagca ctggggccag atggtaagcc 900ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gattgaagcc 2520tgctttttta tactaacttg agcgaaatct ggatcaccat gaaaacattt aacatttctc 2580aacaagatct agaattagta gaagtagcga cagagaagat tacaatgctt tatgaggata 2640ataaacatca tgtgggagcg gcaattcgta cgaaaacagg agaaatcatt tcggcagtac 2700atattgaagc gtatatagga cgagtaactg tttgtgcaga agccattgcg attggtagtg 2760cagtttcgaa tggacaaaag gattttgaca cgattgtagc tgttagacac ccttattctg 2820acgaagtaga tagaagtatt cgagtggtaa gtccttgtgg tatgtgtagg gagttgattt 2880cagactatgc accagattgt tttgtgttaa tagaaatgaa tggcaagtta gtcaaaacta 2940cgattgaaga actcattcca ctcaaatata cccgaaatta aaagttttac cataccaagc 3000ttggctgctg cctgaggctg gacgacctcg cggagttcta ccggcagtgc aaatccgtcg 3060gcatccagga aaccagcagc ggctatccgc gcatccatgc ccccgaactg caggagtggg 3120gaggcacgat ggccgctttg gtccggatct ttgtgaagga accttacttc tgtggtgtga 3180cataattgga caaactacct acagagattt aaagctctaa ggtaaatata aaatttttaa 3240gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt ccaacctatg 3300gaactgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg ttttgctcag 3360aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct actcctccaa 3420aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta agttttttga 3480gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc acaaaggaaa 3540aagctgcact gctatacaag aaaattatgg aaaaatattc tgtaaccttt ataagtaggc 3600ataacagtta taatcataac atactgtttt ttcttactcc acacaggcat agagtgtctg 3660ctattaataa ctatgctcaa aaattgtgta cctttagctt tttaatttgt aaaggggtta 3720ataaggaata tttgatgtat agtgccttga ctagagatca taatcagcca taccacattt 3780gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa 3840atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc 3900aatagcatca caaatttcac aaataaagat ccacgaattc gctagcttcg gccgtgacgc 3960gtctccggat gtacaggcat gcgtcgaccc tctagtcaag gccttaagtg agtcgtatta 4020cggactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 4080tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 4140tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcttcgctt ggtaataaag 4200cccgcttcgg cgggcttttt ttt 42231125855DNAArtificial SequencepCX-LamIntR Plasmid 112gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcatggga agaaggcgaa 1740gtcatgagcg ccgggattta ccccctaacc tttatataag aaacaatgga tattactgct 1800acagggaccc aaggacgggt aaagagtttg gattaggcag agacaggcga atcgcaatca 1860ctgaagctat acaggccaac attgagttat tttcaggaca caaacacaag cctctgacag 1920cgagaatcaa cagtgataat tccgttacgt tacattcatg gcttgatcgc tacgaaaaaa 1980tcctggccag cagaggaatc aagcagaaga cactcataaa ttacatgagc aaaattaaag 2040caataaggag gggtctgcct gatgctccac ttgaagacat caccacaaaa gaaattgcgg 2100caatgctcaa tggatacata gacgagggca aggcggcgtc agccaagtta atcagatcaa 2160cactgagcga tgcattccga gaggcaatag ctgaaggcca tataacaaca aaccatgtcg 2220ctgccactcg cgcagcaaaa tctagagtaa ggagatcaag acttacggct gacgaatacc 2280tgaaaattta tcaagcagca gaatcatcac catgttggct cagacttgca atggaactgg 2340ctgttgttac cgggcaacga gttggtgatt tatgcgaaat gaagtggtct gatatcgtag 2400atggatatct ttatgtcgag caaagcaaaa caggcgtaaa aattgccatc ccaacagcat 2460tgcatattga tgctctcgga atatcaatga aggaaacact tgataaatgc aaagagattc 2520ttggcggaga aaccataatt gcatctactc gtcgcgaacc gctttcatcc ggcacagtat 2580caaggtattt tatgcgcgca cgaaaagcat caggtctttc cttcgaaggg gatccgccta 2640cctttcacga gttgcgcagt ttgtctgcaa gactctatga gaagcagata agcgataagt 2700ttgctcaaca tcttctcggg cataagtcgg acaccatggc atcacagtat cgtgatgaca 2760gaggcaggga gtgggacaaa attgaaatca aataagaatt cactcctcag gtgcaggctg 2820cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc 2880tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg 2940gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac 3000tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga 3060gtttggcaac atatgccata tgctggctgc catgaacaaa ggtggctata aagaggtcat 3120cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga 3180ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt 3240tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct 3300gtccctcttc tcttatgaag atccctcgac ctgcagccca agcttggcgt aatcatggtc 3360atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 3420aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 3480gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat 3540tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 3600tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 3660gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 3720tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3780caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3840tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag 3900aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 3960cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4020atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4080taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 4140aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 4200tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 4260gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct 4320cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 4380cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 4440atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 4500tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 4560ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 4620acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 4680aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 4740aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 4800tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 4860cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 4920catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 4980ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5040aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5100ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 5160caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 5220attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 5280agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 5340actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 5400ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 5460ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 5520gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 5580atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 5640cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 5700gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 5760gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 5820ggttccgcgc acatttcccc gaaaagtgcc acctg 58551134346DNAArtificial SequencepSV40-193AttpsensePur Plasmid 113ccggtgccgc caccatcccc tgacccacgc ccctgacccc tcacaaggag acgaccttcc 60atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta 120cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac 180cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 240atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 300agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 360tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 420cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc 480agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 540gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 600gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 660cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tggctccgac 720cgaagccgac ccgggcggcc ccgccgaccc cgcacccgcc cccgaggccc accgactcta 780gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc 840acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat 900tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 960tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 1020gatccgcgcc ggatccttaa ttaagtctag agtcgactgt ttaaacctgc aggcatgcaa 1080gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 1140cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 1200aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 1260agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 1320ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 1380ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 1440tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 1500tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 1560gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 1620ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 1680tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 1740agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 1800atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 1860acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 1920actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 1980tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 2040tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 2100tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 2160tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 2220caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 2280cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 2340agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 2400acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 2460gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 2520ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 2580tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 2640ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 2700tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 2760attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 2820agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 2880ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 2940ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 3000cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 3060gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 3120tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 3180tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 3240tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta 3300tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc 3360agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc 3420agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc 3480agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa 3540aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 3600tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 3660gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattcg 3720agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 3780gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 3840cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 3900taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 3960gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 4020agtagtgagg aggctttttt ggaggctcgg tacccccttg cgctaatgct ctgttacagg 4080tcactaatac catctaagta gttgattcat agtgactgca tatgttgtgt tttacagtat 4140tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac 4200gtttctcgtt cagctttttt atactaagtt ggcattataa aaaagcattg cttatcaatt 4260tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgatt tcaattttgt 4320cccactccct gcctctgggg ggcgcg 43461143166DNAArtificial Sequencep18attBZeo Plasmid 114cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgcatgc ctgcaggtcg 420actctagagg atccccgggt accgagctcg aattcgtaat catggtcata gctgtttcct 480gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 780gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200ttatcgccac tggcagcagc cactggtaac aggattagca

gagcgaggta tgtaggcggt 1260gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1320atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tagttaacaa aaaaaagccc 2640gccgaagcgg gctttattac caagcgaagc gccattcgcc attcaggctg cgcaactgtt 2700gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg 2760ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga 2820cggccagtcc gtaatacgac tcacttaagg ccttgactag agggtcgacg gtatacagac 2880atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 2940tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 3000caagttgggg tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc 3060ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa 3120atctcgtagc acgtgtcagt cctgctcctc ggccacgaag tgcacg 31661157600DNAArtificial Sequencep18attBZeo3'6XHS4eGFP Plasmid 115cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatcta gttattaata 420gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact 480tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat 540gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggacta 600tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc 660tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg 720ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgggtcgagg 780tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 840atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg 900ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca 960gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg 1020ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc 1080ccgctccgcg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag 1140gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg 1200ctcgtttctt ttctgtggct gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc 1260gggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg 1320cccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc 1380gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg 1440gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc 1500ggtcgggctg taaccccccc ctgcaccccc ctccccgagt tgctgagcac ggcccggctt 1560cgggtgcggg gctccgtgcg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc 1620ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc tcgggggagg 1680ggcgcggcgg ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct 1740tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct ggcggagccg 1800aaatctggga ggcgccgccg caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg 1860gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc 1920atctccagcc tcggggctgc cgcaggggga cggctgcctt cgggggggac ggggcagggc 1980ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 2040cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt 2100tggcaaagaa ttcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc 2160catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg 2220cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct 2280gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg 2340ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt 2400ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa 2460gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga 2520cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat 2580ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga 2640cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt 2700gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga 2760gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat 2820ggacgagctg tacaagtaag aattcactcc tcaggtgcag gctgcctatc agaaggtggt 2880ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa 2940aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt 3000tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg 3060gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc 3120catatgctgg ctgccatgaa caaaggtggc tataaagagg tcatcagtat atgaaacagc 3180cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta 3240tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac 3300tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat 3360gaagatccct cgacctgcag cccaagcttg catgcctgca ggtcgactct agtggatccc 3420ccgccccgta tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa 3480agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga 3540tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg 3600gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg tgagcggatc 3660cgcggccccg tatcccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg 3720aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg 3780gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc 3840gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc cgtgagcgga 3900tccgcggccc cgtatccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga 3960ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg 4020gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta 4080gcgggggagg gacgtaatta catccctggg ggctttgggg gggggctgtc cccgtgagcg 4140gatccgcggc cccgtatccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca 4200gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct 4260cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc 4320tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg tccccgtgag 4380cggatccgcg gccccgtatc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt 4440cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg 4500ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc 4560cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc tgtccccgtg 4620agcggatccg cggccccgta tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg 4680ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc 4740ggctcgggga tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc 4800cccctagcgg gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg 4860tgagcggatc cgcggggctg caggaattcg taatcatggt catagctgtt tcctgtgtga 4920aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 4980tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 5040cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 5100ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 5160cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 5220ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 5280aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 5340cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 5400cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 5460gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 5520tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 5580cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 5640ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 5700gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 5760gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 5820accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 5880ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 5940tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 6000aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 6060taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 6120gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 6180agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 6240cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 6300tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 6360gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 6420agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 6480gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 6540atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 6600gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 6660tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 6720atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 6780agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 6840gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 6900cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 6960tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 7020ccgcgcacat ttccccgaaa agtgccacct gacgtagtta acaaaaaaaa gcccgccgaa 7080gcgggcttta ttaccaagcg aagcgccatt cgccattcag gctgcgcaac tgttgggaag 7140ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 7200ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 7260gtccgtaata cgactcactt aaggccttga ctagagggtc gacggtatac agacatgata 7320agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt 7380tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt 7440ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc 7500ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg 7560tagcacgtgt cagtcctgct cctcggccac gaagtgcacg 76001167631DNAArtificial Sequencep18attBZeo5'6XHS4eGFP Plasmid 116cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt 1500ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg 1680atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1980gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 2400cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 2460ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 2580ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 2760gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg 2820tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc 3120ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg 3180ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 3240gggcgcaggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc 3540tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt tggcaaagaa ttcgccacca 3600tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 3660gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 3720gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 3780tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 3840agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 3900tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 3960tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 4020agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 4080gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 4140accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 4200acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 4260tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag 4320aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc 4380tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga 4440agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 4500gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac 4560atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa 4620caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta 4680ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt 4740ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc 4800tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag 4860cccaagcttg catgcctgca ggtcgactct agaggatccc cgggtaccga gctcgaattc 4920gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 4980catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 5040attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 5100ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 5160ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 5220aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 5280aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 5340gctccgcccc cctgacgagc atcacaaaaa

tcgacgctca agtcagaggt ggcgaaaccc 5400gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 5460tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 5520ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 5580ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 5640tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 5700tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 5760ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 5820aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 5880ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 5940tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 6000atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 6060aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 6120ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 6180tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 6240ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 6300tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 6360aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 6420gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 6480tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 6540cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 6600tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 6660ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 6720cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 6780actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 6840ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 6900aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 6960ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 7020atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 7080tgacgtagtt aacaaaaaaa agcccgccga agcgggcttt attaccaagc gaagcgccat 7140tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 7200cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt 7260tcccagtcac gacgttgtaa aacgacggcc agtccgtaat acgactcact taaggccttg 7320actagagggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac 7380aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 7440tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat 7500ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt 7560catagaaggc ggcggtggaa tcgaaatctc gtagcacgtg tcagtcctgc tcctcggcca 7620cgaagtgcac g 76311174615DNAArtificial Sequencep18attBZeo6XHS4 Plasmid 117cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgcatgc ctgcaggtcg 420actctagtgg atcccccgcc ccgtatcccc caggtgtctg caggctcaaa gagcagcgag 480aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg 540ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct 600gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt 660ccccgtgagc ggatccgcgg ccccgtatcc cccaggtgtc tgcaggctca aagagcagcg 720agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca 780cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg 840ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg ggggggggct 900gtccccgtga gcggatccgc ggccccgtat cccccaggtg tctgcaggct caaagagcag 960cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg 1020cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcggct 1080cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt tggggggggg 1140ctgtccccgt gagcggatcc gcggccccgt atcccccagg tgtctgcagg ctcaaagagc 1200agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc 1260cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg 1320ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg 1380ggctgtcccc gtgagcggat ccgcggcccc gtatccccca ggtgtctgca ggctcaaaga 1440gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc 1500cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc 1560ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg 1620ggggctgtcc ccgtgagcgg atccgcggcc ccgtatcccc caggtgtctg caggctcaaa 1680gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg 1740tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg 1800gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg 1860ggggggctgt ccccgtgagc ggatccgcgg ggctgcagga attcgtaatc atggtcatag 1920ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 1980ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 2040tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 2100cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 2160ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 2220ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 2280gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 2340gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 2400taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 2460accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 2520tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 2580cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 2640agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 2700gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 2760gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 2820tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 2880acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2940cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 3000acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 3060acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 3120tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 3180ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 3240ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 3300tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 3360aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 3420ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 3480ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 3540gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 3600gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 3660cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 3720actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 3780ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 3840tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 3900ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 3960agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 4020aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt agttaacaaa 4080aaaaagcccg ccgaagcggg ctttattacc aagcgaagcg ccattcgcca ttcaggctgc 4140gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag 4200ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt 4260gtaaaacgac ggccagtccg taatacgact cacttaaggc cttgactaga gggtcgacgg 4320tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga 4380aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc 4440tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat 4500catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt 4560ggaatcgaaa tctcgtagca cgtgtcagtc ctgctcctcg gccacgaagt gcacg 461511817384DNAArtificial SequencepFK161 Plasmid 118gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcggggtttc 60gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 120aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 180tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 240ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 300aagagcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat 360gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc 420ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac 480aggagcacga tcatgcgcac ccgtcagatc cagacatgat aagatacatt gatgagtttg 540gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 600ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc 660attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct 720acaaatgtgg tatggctgat tatgatctct agtcaaggca ctatacatca aatattcctt 780attaacccct ttacaaatta aaaagctaaa ggtacacaat ttttgagcat agttattaat 840agcagacact ctatgcctgt gtggagtaag aaaaaacagt atgttatgat tataactgtt 900atgcctactt ataaaggtta cagaatattt ttccataatt ttcttgtata gcagtgcagc 960tttttccttt gtggtgtaaa tagcaaagca agcaagagtt ctattactaa acacagcatg 1020actcaaaaaa cttagcaatt ctgaaggaaa gtccttgggg tcttctacct ttctcttctt 1080ttttggagga gtagaatgtt gagagtcagc agtagcctca tcatcactag atggcatttc 1140ttctgagcaa aacaggtttt cctcattaaa ggcattccac cactgctccc attcatcagt 1200tccataggtt ggaatctaaa atacacaaac aattagaatc agtagtttaa cacattatac 1260acttaaaaat tttatattta ccttagagct ttaaatctct gtaggtagtt tgtccaatta 1320tgtcacacca cagaagtaag gttccttcac aaagatccgg accaaagcgg ccatcgtgcc 1380tccccactcc tgcagttcgg gggcatggat gcgcggatag ccgctgctgg tttcctggat 1440gccgacggat ttgcactgcc ggtagaactc gcgaggtcgt ccagcctcag gcagcagctg 1500aaccaactcg cgaggggatc gagcccgggg tgggcgaaga actccagcat gagatccccg 1560cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag 1620aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt 1680tcgaacccca gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct 1740gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa 1800gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca 1860gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc 1920aggcatcgcc atgggtcacg acgagatcct cgccgtcggg atgcgcgcct tgagcctggc 1980gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag 2040accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg 2100gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt 2160ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag 2220ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt 2280ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc 2340ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga 2400gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg 2460agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg 2520atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac 2580tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc 2640tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt 2700tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg 2760tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc agcccttgcg 2820ccctgagtgc ttgcggcagc gtgaaagctt tttgcaaaag cctaggcctc caaaaaagcc 2880tcctcactac ttctggaata gctcagaggc cgaggcggcc taaataaaaa aaattagtca 2940gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag 3000gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg 3060ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact 3120tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccacagccg 3180gatctgcagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac 3240gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga 3300ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg 3360tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg 3420cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcgcat aaatcgccgt 3480gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag 3540ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cggcatcccg 3600atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac 3660gccagcaaga cgtagcccag cgcgtcgggc cgccatgccg gcgataatgg cctgcttctc 3720gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc 3780gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa 3840aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat 3900aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc 3960tctcaagggc atcggtcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4020tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4080gcccaacagt cccccggcca cgggcctgcc accataccca cgccgaaaca agcgctcatg 4140agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca 4200accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcttggca 4260gtcacagcat gcgcatatcc atgcttcgac catgcgctca caaagtaggt gaatgcgcaa 4320tgtagtaccc acatcgtcat cgctttccac tgctctcgcg aataaagatg gaaaatcaat 4380ctcatggtaa tagtccatga aaatccttgt attcataaat cctccaggta gctatatgca 4440aattgaaaca aaagagatgg tgatctttct aagagatgat ggaatctccc ttcagtatcc 4500cgatggtcaa tgcgctggat atgggataga tgggaatatg ctgattttta tgggacagag 4560ttgcgaactg ttcccaacta aaatcatttt gcacgatcag cgcactacga actttaccca 4620caaatagtca ggtaatgaat cctgatataa agacaggttg ataaatcagt cttctacgcg 4680catcgcacgc gcacaccgta gaaagtcttt cagttgtgag cctgggcaaa ccgttaactt 4740tcggcggctt tgctgtgcga caggctcacg tctaaaagga aataaatcat gggtcataaa 4800attatcacgt tgtccggcgc ggcgacggat gttctgtatg cgctgttttt ccgtggcgcg 4860ttgctgtctg gtgatctgcc ttctaaatct ggcacagccg aattgcgcga gcttggtttt 4920gctgaaacca gacacacagc aactgaatac cagaaagaaa atcactttac ctttctgaca 4980tcagaagggc agaaatttgc cgttgaacac ctggtcaata cgcgttttgg tgagcagcaa 5040tattgcgctt cgatgacgct tggcgttgag attgatacct ctgctgcaca aaaggcaatc 5100gacgagctgg accagcgcat tcgtgacacc gtctccttcg aacttattcg caatggagtg 5160tcattcatca aggacgccgc tatcgcaaat ggtgctatcc acgcagcggc aatcgaaaca 5220cctcagccgg tgaccaatat ctacaacatc agccttggta tccagcgtga tgagccagcg 5280cagaacaagg taaccgtcag tgccgataag ttcaaagtta aacctggtgt tgataccaac 5340attgaaacgt tgatcgaaaa cgcgctgaaa aacgctgctg aatgtgcggc gctggatgtc 5400acaaagcaaa tggcagcaga caagaaagcg atggatgaac tggcttccta tgtccgcacg 5460gccatcatga tggaatgttt ccccggtggt gttatctggc agcagtgccg tcgatagtat 5520gcaattgata attattatca tttgcgggtc ctttccggcg atccgccttg ttacggggcg 5580gcgacctcgc gggttttcgc tatttatgaa aattttccgg tttaaggcgt ttccgttctt 5640cttcgtcata acttaatgtt tttatttaaa ataccctctg aaaagaaagg aaacgacagg 5700tgctgaaagc gagctttttg gcctctgtcg tttcctttct ctgtttttgt ccgtggaatg 5760aacaatggaa gtcaacaaaa agcagctggc tgacattttc ggtgcgagta tccgtaccat 5820tcagaactgg caggaacagg gaatgcccgt tctgcgaggc ggtggcaagg gtaatgaggt 5880gctttatgac tctgccgccg tcataaaatg gtatgccgaa agggatgctg aaattgagaa 5940cgaaaagctg cgccgggagg ttgaagaact gcggcaggcc agcgaggcag atccacagga 6000cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga 6060ctgggcggcg gcaaagcggt cggacagtgc tccgagaacg ggtgcgcata gaaattgcat 6120caacgcatat agcgctagca gcacgccata gtgactggcg atgctgtcgg aatggacgat 6180atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt 6240gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg 6300ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa gcggtcaaac 6360atgagaattc gcggccgctc ttctcgttct gccagcgggc cctcgtctct ccaccccatc 6420cgtctgccgg tggtgtgtgg aaggcagggg tgcggctctc cggcccgacg ctgccccgcg 6480cgcacttttc tcagtggttc gcgtggtcct tgtggatgtg tgaggcgccc ggttgtgccc 6540tcacgtgttt cactttggtc gtgtctcgct tgaccatgtt cccagagtcg gtggatgtgg 6600ccggtggcgt tgcataccct tcccgtctgg tgtgtgcacg cgctgtttct tgtaagcgtc 6660gaggtgctcc tggagcgttc caggtttgtc tcctaggtgc ctgcttctga gctggtggtg 6720gcgctcccca ttccctggtg tgcctccggt gctccgtctg gctgtgtgcc ttcccgtttg 6780tgtctgagaa gcccgtgaga ggggggtcga ggagagaagg aggggcaaga ccccccttct 6840tcgtcgggtg aggcgcccac cccgcgacta gtacgcctgt gcgtagggct ggtgctgagc 6900ggtcgcggct ggggttggaa agtttctcga gagactcatt gctttcccgt ggggagcttt 6960gagaggcctg gctttcgggg gggaccggtt gcagggtctc ccctgtccgc ggatgctcag 7020aatgcccttg gaagagaacc ttcctgttgc cgcagacccc cccgcgcggt cgcccgcgtg 7080ttggtcttct ggtttccctg tgtgctcgtc gcatgcatcc tctctcggtg gccggggctc 7140gtcggggttt tgggtccgtc ccgccctcag tgagaaagtt tccttctcta gctatcttcc 7200ggaaagggtg cgggcttctt acggtctcga ggggtctctc ccgaatggtc ccctggaggg 7260ctcgccccct gaccgcctcc cgcgcgcgca gcgtttgctc tctcgtctac cgcggcccgc 7320ggcctccccg ctccgagttc ggggagggat cacgcggggc agagcctgtc tgtcgtcctg 7380ccgttgctgc ggagcatgtg gctcggcttg tgtggttggt ggctggggag agggctccgt 7440gcacaccccc gcgtgcgcgt actttcctcc cctcctgagg gccgccgtgc ggacggggtg 7500tgggtaggcg acggtgggct cccgggtccc cacccgtctt cccgtgcctc acccgtgcct 7560tccgtcgcgt gcgtccctct cgctcgcgtc cacgactttg gccgctcccg cgacggcggc 7620ctgcgccgcg cgtggtgcgt gctgtgtgct tctcgggctg tgtggttgtg tcgcctcgcc 7680ccccccttcc cgcggcagcg ttcccacggc tggcgaaatc gcgggagtcc tccttcccct 7740cctcggggtc gagagggtcc gtgtctggcg ttgattgatc tcgctctcgg ggacgggacc 7800gttctgtggg agaacggctg ttggccgcgt ccggcgcgac gtcggacgtg gggacccact 7860gccgctcggg ggtcttcgtc ggtaggcatc ggtgtgtcgg catcggtctc tctctcgtgt 7920cggtgtcgcc tcctcgggct cccggggggc cgtcgtgttt cgggtcggct cggcgctgca 7980ggtgtggtgg gactgctcag gggagtggtg cagtgtgatt

cccgccggtt ttgcctcgcg 8040tgccctgacc ggtccgacgc ccgagcggtc tctcggtccc ttgtgaggac ccccttccgg 8100gaggggcccg tttcggccgc ccttgccgtc gtcgccggcc ctcgttctgc tgtgtcgttc 8160ccccctcccc gctcgccgca gccggtcttt tttcctctct ccccccctct cctctgactg 8220acccgtggcc gtgctgtcgg accccccgca tgggggcggc cgggcacgta cgcgtccggg 8280cggtcaccgg ggtcttgggg gggggccgag gggtaagaaa gtcggctcgg cgggcgggag 8340gagctgtggt ttggagggcg tcccggcccc gcggccgtgg cggtgtcttg cgcggtcttg 8400gagagggctg cgtgcgaggg gaaaaggttg ccccgcgagg gcaaagggaa agaggctagc 8460agtggtcatt gtcccgacgg tgtggtggtc tgttggccga ggtgcgtctg gggggctcgt 8520ccggccctgt cgtccgtcgg gaaggcgcgt gttggggcct gccggagtgc cgaggtgggt 8580accctggcgg tgggattaac cccgcgcgcg tgtcccggtg tggcggtggg ggctccggtc 8640gatgtctacc tccctctccc cgaggtctca ggccttctcc gcgcgggctc tcggccctcc 8700cctcgttcct ccctctcgcg gggttcaagt cgctcgtcga cctcccctcc tccgtccttc 8760catctctcgc gcaatggcgc cgcccgagtt cacggtgggt tcgtcctccg cctccgcttc 8820tcgccggggg ctggccgctg tccggtctct cctgcccgac ccccgttggc gtggtcttct 8880ctcgccggct tcgcggactc ctggcttcgc ccggagggtc agggggcttc ccggttcccc 8940gacgttgcgc ctcgctgctg tgtgcttggg gggggcccgc tgcggcctcc gcccgcccgt 9000gagcccctgc cgcacccgcc ggtgtgcggt ttcgcgccgc ggtcagttgg gccctggcgt 9060tgtgtcgcgt cgggagcgtg tccgcctcgc ggcggctaga cgcgggtgtc gccgggctcc 9120gacgggtggc ctatccaggg ctcgcccccg ccgacccccg cctgcccgtc ccggtggtgg 9180tcgttggtgt ggggagtgaa tggtgctacc ggtcattccc tcccgcgtgg tttgactgtc 9240tcgccggtgt cgcgcttctc tttccgccaa cccccacgcc aacccaccac cctgctctcc 9300cggcccggtg cggtcgacgt tccggctctc ccgatgccga ggggttcggg atttgtgccg 9360gggacggagg ggagagcggg taagagaggt gtcggagagc tgtcccgggg cgacgctcgg 9420gttggctttg ccgcgtgcgt gtgctcgcgg acgggttttg tcggaccccg acggggtcgg 9480tccggccgca tgcactctcc cgttccgcgc gagcgcccgc ccggctcacc cccggtttgt 9540cctcccgcga ggctctccgc cgccgccgcc tcctcctcct ctctcgcgct ctctgtcccg 9600cctggtcctg tcccaccccc gacgctccgc tcgcgcttcc ttacctggtt gatcctgcca 9660ggtagcatat gcttgtctca aagattaagc catgcatgtc taagtacgca cggccggtac 9720agtgaaactg cgaatggctc attaaatcag ttatggttcc tttggtcgct cgctcctctc 9780ctacttggat aactgtggta attctagagc taatacatgc cgacgggcgc tgacccccct 9840tcccgggggg ggatgcgtgc atttatcaga tcaaaaccaa cccggtgagc tccctcccgg 9900ctccggccgg gggtcgggcg ccggcggctt ggtgactcta gataacctcg ggccgatcgc 9960acgccccccg tggcggcgac gacccattcg aacgtctgcc ctatcaactt tcgatggtag 10020tcgccgtgcc taccatggtg accacgggtg acggggaatc agggttcgat tccggagagg 10080gagcctgaga aacggctacc acatccaagg aaggcagcag gcgcgcaaat tacccactcc 10140cgacccgggg aggtagtgac gaaaaataac aatacaggac tctttcgagg ccctgtaatt 10200ggaatgagtc cactttaaat cctttaacga ggatccattg gagggcaagt ctggtgccag 10260cagccgcggt aattccagct ccaatagcgt atattaaagt tgctgcagtt aaaaagctcg 10320tagttggatc ttgggagcgg gcgggcggtc cgccgcgagg cgagtcaccg cccgtccccg 10380ccccttgcct ctcggcgccc cctcgatgct cttagctgag tgtcccgcgg ggcccgaagc 10440gtttactttg aaaaaattag agtgttcaaa gcaggcccga gccgcctgga taccgcagct 10500aggaataatg gaataggacc gcggttctat tttgttggtt ttcggaactg aggccatgat 10560taagagggac ggccgggggc attcgtattg cgccgctaga ggtgaaattc ttggaccggc 10620gcaagacgga ccagagcgaa agcatttgcc aagaatgttt tcattaatca agaacgaaag 10680tcggaggttc gaagacgatc agataccgtc gtagttccga ccataaacga tgccgactgg 10740cgatgcggcg gcgttattcc catgacccgc cgggcagctt ccgggaaacc aaagtctttg 10800ggttccgggg ggagtatggt tgcaaagctg aaacttaaag gaattgacgg aagggcacca 10860ccaggagtgg gcctgcggct taatttgact caacacggga aacctcaccc ggcccggaca 10920cggacaggat tgacagattg atagctcttt ctcgattccg tgggtggtgg tgcatggccg 10980ttcttagttg gtggagcgat ttgtctggtt aattccgata acgaacgaga ctctggcatg 11040ctaactagtt acgcgacccc cgagcggtcg gcgtccccca acttcttaga gggacaagtg 11100gcgttcagcc acccgagatt gagcaataac aggtctgtga tgcccttaga tgtccggggc 11160tgcacgcgcg ctacactgac tggctcagcg tgtgcctacc ctgcgccggc aggcgcgggt 11220aacccgttga accccattcg tgatggggat cggggattgc aattattccc catgaacgag 11280gaattcccag taagtgcggg tcataagctt gcgttgatta agtccctgcc ctttgtacac 11340accgcccgtc gctactaccg attggatggt ttagtgaggc cctcggatcg gccccgccgg 11400ggtcggccca cggccctggc ggagcgctga gaagacggtc gaacttgact atctagagga 11460agtaaaagtc gtaacaaggt ttccgtaggt gaacctgcgg aaggatcatt aaacgggaga 11520ctgtggagga gcggcggcgt ggcccgctct ccccgtcttg tgtgtgtcct cgccgggagg 11580cgcgtgcgtc ccgggtcccg tcgcccgcgt gtggagcgag gtgtctggag tgaggtgaga 11640gaaggggtgg gtggggtcgg tctgggtccg tctgggaccg cctccgattt cccctccccc 11700tcccctctcc ctcgtccggc tctgacctcg ccaccctacc gcggcggcgg ctgctcgcgg 11760gcgtcttgcc tctttcccgt ccggctcttc cgtgtctacg aggggcggta cgtcgttacg 11820ggtttttgac ccgtcccggg ggcgttcggt cgtcggggcg cgcgctttgc tctcccggca 11880cccatccccg ccgcggctct ggcttttcta cgttggctgg ggcggttgtc gcgtgtgggg 11940ggatgtgagt gtcgcgtgtg ggctcgcccg tcccgatgcc acgcttttct ggcctcgcgt 12000gtcctccccg ctcctgtccc gggtacctag ctgtcgcgtt ccggcgcgga ggtttaagga 12060ccccgggggg gtcgccctgc cgcccccagg gtcggggggc ggtggggccc gtagggaagt 12120cggtcgttcg ggcggctctc cctcagactc catgaccctc ctccccccgc tgccgccgtt 12180cccgaggcgg cggtcgtgtg ggggggtgga tgtctggagc cccctcgggc gccgtggggg 12240cccgacccgc gccgccggct tgcccgattt ccgcgggtcg gtcctgtcgg tgccggtcgt 12300gggttcccgt gtcgttcccg tgtttttccg ctcccgaccc tttttttttc ctccccccca 12360cacgtgtctc gtttcgttcc tgctggccgg cctgaggcta cccctcggtc catctgttct 12420cctctctctc cggggagagg agggcggtgg tcgttggggg actgtgccgt cgtcagcacc 12480cgtgagttcg ctcacacccg aaataccgat acgactctta gcggtggatc actcggctcg 12540tgcgtcgatg aagaacgcag ctagctgcga gaattaatgt gaattgcagg acacattgat 12600catcgacact tcgaacgcac ttgcggcccc gggttcctcc cggggctacg cctgtctgag 12660cgtcggttga cgatcaatcg cgtcacccgc tgcggtgggt gctgcgcggc tgggagtttg 12720ctcgcagggc caacccccca acccgggtcg ggccctccgt ctcccgaagt tcagacgtgt 12780gggcggttgt cggtgtggcg cgcgcgcccg cgtcgcggag cctggtctcc cccgcgcatc 12840cgcgctcgcg gcttcttccc gctccgccgt tcccgccctc gcccgtgcac cccggtcctg 12900gcctcgcgtc ggcgcctccc ggaccgctgc ctcaccagtc tttctcggtc ccgtgccccg 12960tgggaaccca ccgcgccccc gtggcgcccg ggggtgggcg cgtccgcatc tgctctggtc 13020gaggttggcg gttgagggtg tgcgtgcgcc gaggtggtgg tcggtcccct gcggccgcgg 13080ggttgtcggg gtggcggtcg acgagggccg gtcggtcgcc tgcggtggtt gtctgtgtgt 13140gtttgggtct tgcgctgggg gaggcggggt cgaccgctcg cggggttggc gcggtcgccc 13200ggcgccgcgc accctccggc ttgtgtggag ggagagcgag ggcgagaacg gagagaggtg 13260gtatccccgg tggcgttgcg agggagggtt tggcgtcccg cgtccgtccg tccctccctc 13320cctcggtggg cgccttcgcg ccgcacgcgg ccgctagggg cggtcggggc ccgtggcccc 13380cgtggctctt cttcgtctcc gcttctcctt cacccgggcg gtacccgctc cggcgccggc 13440ccgcgggacg ccgcggcgtc cgtgcgccga tgcgagtcac ccccgggtgt tgcgagttcg 13500gggagggaga gggcctcgct gacccgttgc gtcccggctt ccctgggggg gacccggcgt 13560ctgtgggctg tgcgtcccgg gggttgcgtg tgagtaagat cctccacccc cgccgccctc 13620ccctcccgcc ggcctctcgg ggaccccctg agacggttcg ccggctcgtc ctcccgtgcc 13680gccgggtgcc gtctctttcc cgcccgcctc ctcgctctct tcttcccgcg gctgggcgcg 13740tgtcccccct ttctgaccgc gacctcagat cagacgtggc gacccgctga atttaagcat 13800attagtcagc ggaggaaaag aaactaacca ggattccctc agtaacggcg agtgaacagg 13860gaagagccca gcgccgaatc cccgccgcgc gtcgcggcgt gggaaatgtg gcgtacggaa 13920gacccactcc ccggcgccgc tcgtgggggg cccaagtcct tctgatcgag gcccagcccg 13980tggacggtgt gaggccggta gcggccccgg cgcgccgggc tcgggtcttc ccggagtcgg 14040gttgcttggg aatgcagccc aaagcgggtg gtaaactcca tctaaggcta aataccggca 14100cgagaccgat agtcaacaag taccgtaagg gaaagttgaa aagaactttg aagagagagt 14160tcaagagggc gtgaaaccgt taagaggtaa acgggtgggg tccgcgcagt ccgcccggag 14220gattcaaccc ggcggcgcgc gtccggccgt gcccggtggt cccggcggat ctttcccgct 14280ccccgttcct cccgacccct ccacccgcgc gtcgttcccc tcttcctccc cgcgtccggc 14340gcctccggcg gcgggcgcgg ggggtggtgt ggtggtggcg cgcgggcggg gccgggggtg 14400gggtcggcgg gggaccgccc ccggccggcg accggccgcc gccgggcgca cttccaccgt 14460ggcggtgcgc cgcgaccggc tccgggacgg ccgggaaggc ccggtgggga aggtggctcg 14520gggggggcgg cgcgtctcag ggcgcgccga accacctcac cccgagtgtt acagccctcc 14580ggccgcgctt tcgccgaatc ccggggccga ggaagccaga tacccgtcgc cgcgctctcc 14640ctctcccccc gtccgcctcc cgggcgggcg tgggggtggg ggccgggccg cccctcccac 14700ggcgcgaccg ctctcccacc cccctccgtc gcctctctcg gggcccggtg gggggcgggg 14760cggactgtcc ccagtgcgcc ccgggcgtcg tcgcgccgtc gggtcccggg gggaccgtcg 14820gtcacgcgtc tcccgacgaa gccgagcgca cggggtcggc ggcgatgtcg gctacccacc 14880cgacccgtct tgaaacacgg accaaggagt ctaacgcgtg cgcgagtcag gggctcgtcc 14940gaaagccgcc gtggcgcaat gaaggtgaag ggccccgccc gggggcccga ggtgggatcc 15000cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgccgggg 15060aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ttgggcaggg 15120cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 15180cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 15240tttccctcag gatagctggc gctctcgctc ccgacgtacg cagttttatc cggtaaagcg 15300aatgattaga ggtcttgggg ccgaaacgat ctcaacctat tctcaaactt taaatgggta 15360agaagcccgg ctcgctggcg tggagccggg cgtggaatgc gagtgcctag tgggccactt 15420ttggtaagca gaactggcgc tgcgggatga accgaacgcc gggttaaggc gcccgatgcc 15480gacgctcatc agaccccaga aaaggtgttg gttgatatag acagcaggac ggtggccatg 15540gaagtcggaa tccgctaagg agtgtgtaac aactcacctg ccgaatcaac tagccctgaa 15600aatggatggc gctggagcgt cgggcccata cccggccgtc gccgcagtcg gaacggaacg 15660ggacgggagc ggccgcgaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat 15720aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 15780tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 15840gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 15900atttccgtgt cgcccttatt cccttttttg cggcattttg cttcctgttt ttgctcaccc 15960agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 16020cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 16080aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg 16140gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 16200agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 16260aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 16320gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 16380ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc 16440aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 16500aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 16560tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 16620agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 16680ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 16740ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 16800ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 16860acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 16920agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 16980ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 17040cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 17100gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 17160cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 17220gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 17280caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc cgaagggaga 17340aaggcggaca ggtatccggt aagcggcagg gtcggaacag gaga 173841192814DNAArtificial SequencepLITMUS38 Plasmid 119gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gatatctgga 2520tccacgaatt cgctagcttc ggccgtgacg cgtctccgga tgtacaggca tgcgtcgacc 2580ctctagtcaa ggccttaagt gagtcgtatt acggactggc cgtcgtttta caacgtcgtg 2640actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 2700gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 2760atggcgaatg gcgcttcgct tggtaataaa gcccgcttcg gcgggctttt tttt 28141202847DNAArtificial SequencepLIT38attB Plasmid 120gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gattgaagcc 2520tgctttttta tactaacttg agcgaaatct ggatccacga attcgctagc ttcggccgtg 2580acgcgtctcc ggatgtacag gcatgcgtcg accctctagt caaggcctta agtgagtcgt 2640attacggact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 2700ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca

2760ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttc gcttggtaat 2820aaagcccgct tcggcgggct ttttttt 28471214223DNAArtificial SequencepLIT38attBBSRpolyA2 Plasmid 121accatgaaaa catttaacat ttctcaacaa gatctagaat tagtagaagt agcgacagag 60aagattacaa tgctttatga ggataataaa catcatgtgg gagcggcaat tcgtacgaaa 120acaggagaaa tcatttcggc agtacatatt gaagcgtata taggacgagt aactgtttgt 180gcagaagcca ttgcgattgg tagtgcagtt tcgaatggac aaaaggattt tgacacgatt 240gtagctgtta gacaccctta ttctgacgaa gtagatagaa gtattcgagt ggtaagtcct 300tgtggtatgt gtagggagtt gatttcagac tatgcaccag attgttttgt gttaatagaa 360atgaatggca agttagtcaa aactacgatt gaagaactca ttccactcaa atatacccga 420aattaaaagt tttaccatac caagcttggc tgctgcctga ggctggacga cctcgcggag 480ttctaccggc agtgcaaatc cgtcggcatc caggaaacca gcagcggcta tccgcgcatc 540catgcccccg aactgcagga gtggggaggc acgatggccg ctttggtccg gatctttgtg 600aaggaacctt acttctgtgg tgtgacataa ttggacaaac tacctacaga gatttaaagc 660tctaaggtaa atataaaatt tttaagtgta taatgtgtta aactactgat tctaattgtt 720tgtgtatttt agattccaac ctatggaact gatgaatggg agcagtggtg gaatgccttt 780aatgaggaaa acctgttttg ctcagaagaa atgccatcta gtgatgatga ggctactgct 840gactctcaac attctactcc tccaaaaaag aagagaaagg tagaagaccc caaggacttt 900ccttcagaat tgctaagttt tttgagtcat gctgtgttta gtaatagaac tcttgcttgc 960tttgctattt acaccacaaa ggaaaaagct gcactgctat acaagaaaat tatggaaaaa 1020tattctgtaa cctttataag taggcataac agttataatc ataacatact gttttttctt 1080actccacaca ggcatagagt gtctgctatt aataactatg ctcaaaaatt gtgtaccttt 1140agctttttaa tttgtaaagg ggttaataag gaatatttga tgtatagtgc cttgactaga 1200gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca 1260cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc 1320agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagatccaga 1380tttcgctcaa gttagtataa aaaagcaggc ttcaatcctg cagagaagct tggcgccagc 1440cggcttcaat tgcacgggcc ccactagtga gtcgtattac gtagcttggc gtaatcatgg 1500tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 1560ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attacatgtg 1620agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 1680taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 1740cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 1800tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 1860gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 1920gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 1980tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2040gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2100cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2160aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2220tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 2280ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 2340attatcaaaa aggatcttca cctagatcct tttacgcgcc ctgtagcggc gcattaagcg 2400cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 2460ctcctttcgc tttcttccct tcctttctcg ccacgttcgc tttccccgtc aagctctaaa 2520tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 2580tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 2640gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 2700ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 2760aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 2820aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 2880ggtaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 2940agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 3000gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 3060ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 3120gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 3180cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 3240acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 3300cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 3360cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 3420ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 3480tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 3540acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggagaacgt 3600tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 3660actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 3720aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 3780ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 3840ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 3900cgaaaagtgc cacctgacgt agttaacaaa aaaaagcccg ccgaagcggg ctttattacc 3960aagcgaagcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 4020cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 4080taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtccg taatacgact 4140cacttaaggc cttgactaga gggtcgacgc atgcctgtac atccggagac gcgtcacggc 4200cgaagctagc gaattcgtgg atc 42231222686DNAArtificial SequencepUC18 Plasmid 122tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg 420actctagagg atccccgggt accgagctcg aattcgtaat catggtcata gctgtttcct 480gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 780gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1260gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1320atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 2640atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 26861238521DNAArtificial SequencepCXeGFPattB(6xHS4)2 Plasmid 123tacggggcgg gggatccact agttattaat agtaatcaat tacggggtca ttagttcata 60gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcgccacc atggtgagca 1740agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 1800acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 1860ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 1920ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 1980tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 2040acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 2100tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 2160acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 2220tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 2280agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 2340cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 2400tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcactc 2460ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca 2520aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 2580agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 2640ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg 2700agtatttggt ttagagtttg gcaacatatg ccatatgctg gctgccatga acaaaggtgg 2760ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga 2820aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta 2880acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta 2940ctcccagtca tagctgtccc tcttctctta tgaagatccc tcgacctgca gcccaagctt 3000ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 3060caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 3120cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg 3180gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3240ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 3300gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt 3360ggaggctagt ggatcccccg ccccgtatcc cccaggtgtc tgcaggctca aagagcagcg 3420agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca 3480cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg 3540ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg ggggggggct 3600gtccccgtga gcggatccgc ggccccgtat cccccaggtg tctgcaggct caaagagcag 3660cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg 3720cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcggct 3780cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt tggggggggg 3840ctgtccccgt gagcggatcc gcggccccgt atcccccagg tgtctgcagg ctcaaagagc 3900agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc 3960cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg 4020ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg 4080ggctgtcccc gtgagcggat ccgcggcccc gtatccccca ggtgtctgca ggctcaaaga 4140gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc 4200cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc 4260ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg 4320ggggctgtcc ccgtgagcgg atccgcggcc ccgtatcccc caggtgtctg caggctcaaa 4380gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg 4440tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg 4500gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg 4560ggggggctgt ccccgtgagc ggatccgcgg ccccgtatcc cccaggtgtc tgcaggctca 4620aagagcagcg agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc 4680tgtccccgca cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc 4740gggcggctcg ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg 4800ggggggggct gtccccgtga gcggatccgc ggggctgcag gaattcgatt gaagcctgct 4860tttttatact aacttgagcg aaatcaagct cctaggcttt tgcaaaaagc taacttgttt 4920attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 4980tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 5040tggatccgct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5100gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5160tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5220agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5280cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5340ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5400tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5460gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc 5520gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5580gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5640ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5700ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5760ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5820gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5880ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 5940tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6000ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 6060gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 6120tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 6180cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 6240ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 6300gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 6360caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 6420gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 6480ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 6540tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 6600caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 6660tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 6720cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 6780ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 6840aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 6900tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 6960gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 7020gaaaagtgcc acctggtcga cggtatcgat aagcttgata tcgaattcct gcagccccgc 7080ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc 7140cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc 7200atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc 7260tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg atacggggcc 7320gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa ttacgtccct 7380cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg cgctcccccc 7440gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg gcacgggatc 7500gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg ggatacgggg 7560ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt aattacgtcc 7620ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc 7680ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga 7740tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg ggggatacgg 7800ggccgcggat ccgctcacgg ggacagcccc

cccccaaagc ccccagggat gtaattacgt 7860ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc 7920ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg 7980gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc tgggggatac 8040ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 8100gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 8160ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 8220gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 8280acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 8340acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 8400ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 8460acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 8520a 85211248851DNAArtificial Sequencep18EPOcDNA Plasmid 124cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt 1500ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg 1680atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1980gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 2400cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 2460ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 2580ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 2760gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg 2820tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc 3120ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg 3180ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 3240gggcgcaggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480tgaccggcgg ctctagaatg ggggtgcacg aatgtcctgc ctggctgtgg cttctcctgt 3540ccctgctgtc gctccctctg ggcctcccag tcctgggcgc cccaccacgc ctcatctgtg 3600acagccgagt cctggagagg tacctcttgg aggccaagga ggccgagaat atcacgacgg 3660gctgtgctga acactgcagc ttgaatgaga atatcactgt cccagacacc aaagttaatt 3720tctatgcctg gaagaggatg gaggtcgggc agcaggccgt agaagtctgg cagggcctgg 3780ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt ggtcaactct tcccagccgt 3840gggagcccct gcagctgcat gtggataaag ccgtcagtgg ccttcgcagc ctcaccactc 3900tgcttcgggc tctgggagcc cagaaggaag ccatctcccc tccagatgcg gcctcagctg 3960ctccactccg aacaatcact gctgacactt tccgcaaact cttccgagtc tactccaatt 4020tcctccgggg aaagctgaag ctgtacacag gggaggcctg caggacaggg gacagatgac 4080gtacaagtaa gaattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt 4140ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg 4200ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat 4260tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa 4320tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg ccatatgctg 4380gctgccatga acaaaggtgg ctataaagag gtcatcagta tatgaaacag ccccctgctg 4440tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt 4500ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat 4560ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tgaagatccc 4620tcgacctgca gcccaagctt gcatgcctgc aggtcgactc tagtggatcc cccgccccgt 4680atcccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga aagcgatccc 4740gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg atgcgggggg 4800agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg ggggagggac 4860gtaattacat ccctgggggc tttggggggg ggctgtcccc gtgagcggat ccgcggcccc 4920gtatccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc 4980ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg 5040ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg 5100acgtaattac atccctgggg gctttggggg ggggctgtcc ccgtgagcgg atccgcggcc 5160ccgtatcccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga 5220tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg 5280ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag 5340ggacgtaatt acatccctgg gggctttggg ggggggctgt ccccgtgagc ggatccgcgg 5400ccccgtatcc cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc 5460gatcccgtgc caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc 5520ggggggagcg ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg 5580agggacgtaa ttacatccct gggggctttg ggggggggct gtccccgtga gcggatccgc 5640ggccccgtat cccccaggtg tctgcaggct caaagagcag cgagaagcgt tcagaggaaa 5700gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg gctcggggat 5760gcggggggag cgccggaccg gagcggagcc ccgggcggct cgctgctgcc ccctagcggg 5820ggagggacgt aattacatcc ctgggggctt tggggggggg ctgtccccgt gagcggatcc 5880gcggccccgt atcccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga 5940aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg 6000atgcgggggg agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg 6060ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtcccc gtgagcggat 6120ccgcggggct gcaggaattc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 6180ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 6240taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 6300aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 6360attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 6420cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 6480gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 6540ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 6600agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 6660tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 6720ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 6780gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 6840ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 6900gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 6960aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 7020aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 7080ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 7140gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 7200gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 7260tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 7320ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 7380ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 7440atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 7500ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 7560tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 7620attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 7680tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 7740ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 7800gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 7860gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 7920gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 7980aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 8040taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 8100tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 8160tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 8220atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 8280tttccccgaa aagtgccacc tgacgtagtt aacaaaaaaa agcccgccga agcgggcttt 8340attaccaagc gaagcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 8400tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 8460gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtccgtaat 8520acgactcact taaggccttg actagagggt cgacggtata cagacatgat aagatacatt 8580gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt 8640tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc 8700gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac 8760gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtagcacgtg 8820tcagtcctgc tcctcggcca cgaagtgcac g 885112510474DNAArtificial Sequencep18genEPO Plasmid 125cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt 1500ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg 1680atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1980gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 2400cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 2460ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 2580ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 2760gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg 2820tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc 3120ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg 3180ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 3240gggcgcaggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480tgaccggcgg ctctagatgc atgctcgagc ggccgccagt gtgatggata tctgcagaat 3540tcgccctttc tagaatgggg gtgcacggtg agtactcgcg ggctgggcgc tcccgcccgc 3600ccgggtccct gtttgagcgg ggatttagcg ccccggctat tggccaggag gtggctgggt 3660tcaaggaccg gcgacttgtc aaggaccccg gaagggggag gggggtgggg tgcctccacg 3720tgccagcggg gacttggggg agtccttggg gatggcaaaa acctgacctg tgaaggggac 3780acagtttggg ggttgagggg aagaaggttt gggggttctg ctgtgccagt ggagaggaag 3840ctgataagct gataacctgg gcgctggagc caccacttat ctgccagagg ggaagcctct 3900gtcacaccag gattgaagtt tggccggaga agtggatgct ggtagctggg ggtggggtgt 3960gcacacggca gcaggattga atgaaggcca gggaggcagc acctgagtgc ttgcatggtt 4020ggggacagga aggacgagct ggggcagaga cgtggggatg aaggaagctg tccttccaca 4080gccacccttc tccctccccg cctgactctc agcctggcta tctgttctag aatgtcctgc 4140ctggctgtgg cttctcctgt ccctgctgtc gctccctctg ggcctcccag tcctgggcgc 4200cccaccacgc ctcatctgtg acagccgagt cctggagagg tacctcttgg aggccaagga 4260ggccgagaat atcacggtga gaccccttcc ccagcacatt ccacagaact cacgctcagg 4320gcttcaggga actcctccca gatccaggaa cctggcactt ggtttggggt ggagttggga 4380agctagacac tgccccccta cataagaata agtctggtgg ccccaaacca tacctggaaa 4440ctaggcaagg agcaaagcca gcagatccta cggcctgtgg gccagggcca gagccttcag 4500ggacccttga ctccccgggc tgtgtgcatt tcagacgggc tgtgctgaac actgcagctt 4560gaatgagaat atcactgtcc cagacaccaa agttaatttc tatgcctgga agaggatgga 4620ggtgagttcc tttttttttt tttttccttt cttttggaga atctcatttg cgagcctgat 4680tttggatgaa agggagaatg atcgagggaa aggtaaaatg gagcagcaga gatgaggctg 4740cctgggcgca gaggctcacg tctataatcc caggctgaga tggccgagat gggagaattg 4800cttgagccct ggagtttcag accaacctag gcagcatagt gagatccccc atctctacaa 4860acatttaaaa aaattagtca ggtgaagtgg tgcatggtgg tagtcccaga tatttggaag 4920gctgaggcgg gaggatcgct tgagcccagg aatttgaggc tgcagtgagc tgtgatcaca 4980ccactgcact ccagcctcag tgacagagtg aggccctgtc tcaaaaaaga aaagaaaaaa 5040gaaaaataat gagggctgta tggaatacat tcattattca ttcactcact cactcactca 5100ttcattcatt cattcattca acaagtctta ttgcatacct tctgtttgct cagcttggtg 5160cttggggctg ctgaggggca ggagggagag ggtgacatgg gtcagctgac tcccagagtc 5220cactccctgt aggtcgggca gcaggccgta gaagtctggc agggcctggc cctgctgtcg 5280gaagctgtcc tgcggggcca ggccctgttg gtcaactctt cccagccgtg ggagcccctg

5340cagctgcatg tggataaagc cgtcagtggc cttcgcagcc tcaccactct gcttcgggct 5400ctgggagccc aggtgagtag gagcggacac ttctgcttgc cctttctgta agaaggggag 5460aagggtcttg ctaaggagta caggaactgt ccgtattcct tccctttctg tggcactgca 5520gcgacctcct gttttctcct tggcagaagg aagccatctc ccctccagat gcggcctcag 5580ctgctccact ccgaacaatc actgctgaca ctttccgcaa actcttccga gtctactcca 5640atttcctccg gggaaagctg aagctgtaca caggggaggc ctgcaggaca ggggacagat 5700gacgtacaag taagaattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg 5760tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta 5820tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt 5880cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc 5940aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgccatatg 6000ctggctgcca tgaacaaagg tggctataaa gaggtcatca gtatatgaaa cagccccctg 6060ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt 6120gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca 6180gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatgaagat 6240ccctcgacct gcagcccaag cttgcatgcc tgcaggtcga ctctagtgga tcccccgccc 6300cgtatccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat 6360cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg 6420gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta gcgggggagg 6480gacgtaatta catccctggg ggctttgggg gggggctgtc cccgtgagcg gatccgcggc 6540cccgtatccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg 6600atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg 6660gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga 6720gggacgtaat tacatccctg ggggctttgg gggggggctg tccccgtgag cggatccgcg 6780gccccgtatc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag 6840cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg 6900cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg 6960gagggacgta attacatccc tgggggcttt gggggggggc tgtccccgtg agcggatccg 7020cggccccgta tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa 7080agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga 7140tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg 7200gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg tgagcggatc 7260cgcggccccg tatcccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg 7320aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg 7380gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc 7440gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc cgtgagcgga 7500tccgcggccc cgtatccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga 7560ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg 7620gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta 7680gcgggggagg gacgtaatta catccctggg ggctttgggg gggggctgtc cccgtgagcg 7740gatccgcggg gctgcaggaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 7800tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 7860gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 7920ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 7980cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 8040cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 8100aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 8160gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 8220tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 8280agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 8340ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 8400taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 8460gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 8520gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 8580ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 8640ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 8700gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 8760caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 8820taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 8880aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 8940tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 9000tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 9060gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 9120gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 9180aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 9240gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 9300ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 9360tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 9420atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 9480ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 9540ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 9600ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 9660atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 9720gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 9780tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 9840ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 9900acatttcccc gaaaagtgcc acctgacgta gttaacaaaa aaaagcccgc cgaagcgggc 9960tttattacca agcgaagcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat 10020cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat 10080taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtccgt 10140aatacgactc acttaaggcc ttgactagag ggtcgacggt atacagacat gataagatac 10200attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa 10260atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg 10320ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa 10380aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtagcac 10440gtgtcagtcc tgctcctcgg ccacgaagtg cacg 104741266119DNAArtificial Sequencep18attBZeoeGFP Plasmid 126cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgggctg caggtcgagg 420gatcttcata agagaagagg gacagctatg actgggagta gtcaggagag gaggaaaaat 480ctggctagta aaacatgtaa ggaaaatttt agggatgtta aagaaaaaaa taacacaaaa 540caaaatataa aaaaaatcta acctcaagtc aaggcttttc tatggaataa ggaatggaca 600gcagggggct gtttcatata ctgatgacct ctttatagcc acctttgttc atggcagcca 660gcatatggca tatgttgcca aactctaaac caaatactca ttctgatgtt ttaaatgatt 720tgccctccca tatgtccttc cgagtgagag acacaaaaaa ttccaacaca ctattgcaat 780gaaaataaat ttcctttatt agccagaagt cagatgctca aggggcttca tgatgtcccc 840ataatttttg gcagagggaa aaagatctca gtggtatttg tgagccaggg cattggccac 900accagccacc accttctgat aggcagcctg cacctgagga gtgaattctt acttgtacag 960ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc 1020gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg 1080cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac 1140gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt 1200gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat 1260gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc 1320ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg 1380ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc 1440ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg 1500cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc 1560gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac 1620caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgg tggcgaattc 1680tttgccaaaa tgatgagaca gcacaacaac cagcacgttg cccaggagct gtaggaaaaa 1740gaagaaggca tgaacatggt tagcagaggc tctagagccg ccggtcacac gccagaagcc 1800gaaccccgcc ctgccccgtc ccccccgaag gcagccgtcc ccctgcggca gccccgaggc 1860tggagatgga gaaggggacg gcggcgcggc gacgcacgaa ggccctcccc gcccatttcc 1920ttcctgccgg cgccgcaccg cttcgcccgc gcccgctaga gggggtgcgg cggcgcctcc 1980cagatttcgg ctccgccaga tttgggacaa aggaagtccc tgcgccctct cgcacgatta 2040ccataaaagg caatggctgc ggctcgccgc gcctcgacag ccgccggcgc tccggggccg 2100ccgcgcccct cccccgagcc ctccccggcc cgaggcggcc ccgccccgcc cggcaccccc 2160acctgccgcc accccccgcc cggcacggcg agccccgcgc cacgccccgc acggagcccc 2220gcacccgaag ccgggccgtg ctcagcaact cggggagggg ggtgcagggg ggggttacag 2280cccgaccgcc gcgcccacac cccctgctca cccccccacg cacacacccc gcacgcagcc 2340tttgttcccc tcgcagcccc cccgcaccgc ggggcaccgc ccccggccgc gctcccctcg 2400cgcacacgcg gagcgcacaa agccccgcgc cgcgcccgca gcgctcacag ccgccgggca 2460gcgcgggccg cacgcggcgc tccccacgca cacacacacg cacgcacccc ccgagccgct 2520cccccccgca caaagggccc tcccggagcc ctttaaggct ttcacgcagc cacagaaaag 2580aaacgagccg tcattaaacc aagcgctaat tacagcccgg aggagaaggg ccgtcccgcc 2640cgctcacctg tgggagtaac gcggtcagtc agagccgggg cgggcggcgc gaggcggcgc 2700ggagcggggc acggggcgaa ggcaacgcag cgactcccgc ccgccgcgcg cttcgctttt 2760tatagggccg ccgccgccgc cgcctcgcca taaaaggaaa ctttcggagc gcgccgctct 2820gattggctgc cgccgcacct ctccgcctcg ccccgccccg cccctcgccc cgccccgccc 2880cgcctggcgc gcgccccccc cccccccgcc cccatcgctg cacaaaataa ttaaaaaata 2940aataaataca aaattggggg tggggagggg ggggagatgg ggagagtgaa gcagaacgtg 3000gggctcacct cgacccatgg taatagcgat gactaatacg tagatgtact gccaagtagg 3060aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac 3120gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta 3180ccgtaaatag tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac 3240atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt 3300taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat 3360tgattactat taataactag aggatccccg ggtaccgagc tcgaattcgt aatcatggtc 3420atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 3480aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 3540gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 3600ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 3660ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 3720acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 3780aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 3840tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 3900aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 3960gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 4020acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4080accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4140ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4200gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4260gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4320ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4380gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4440cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4500cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4560gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4620tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4680gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4740agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4800tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4860agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 4920gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 4980catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 5040ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5100atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5160tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 5220cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5280cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5340atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5400aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5460ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5520aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtagttaa 5580caaaaaaaag cccgccgaag cgggctttat taccaagcga agcgccattc gccattcagg 5640ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg 5700aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga 5760cgttgtaaaa cgacggccag tccgtaatac gactcactta aggccttgac tagagggtcg 5820acggtataca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 5880gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 5940aagctgcaat aaacaagttg gggtgggcga agaactccag catgagatcc ccgcgctgga 6000ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg 6060cggtggaatc gaaatctcgt agcacgtgtc agtcctgctc ctcggccacg aagtgcacg 61191275855DNAArtificial SequencepCXLamInt Plasmid (Wildtype Integrase) 127gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcatggga agaaggcgaa 1740gtcatgagcg ccgggattta ccccctaacc tttatataag aaacaatgga tattactgct 1800acagggaccc aaggacgggt aaagagtttg gattaggcag agacaggcga atcgcaatca 1860ctgaagctat acaggccaac attgagttat tttcaggaca caaacacaag cctctgacag 1920cgagaatcaa cagtgataat tccgttacgt tacattcatg gcttgatcgc tacgaaaaaa 1980tcctggccag cagaggaatc aagcagaaga cactcataaa ttacatgagc aaaattaaag 2040caataaggag gggtctgcct gatgctccac ttgaagacat caccacaaaa gaaattgcgg 2100caatgctcaa tggatacata gacgagggca aggcggcgtc agccaagtta atcagatcaa 2160cactgagcga tgcattccga gaggcaatag ctgaaggcca tataacaaca aaccatgtcg 2220ctgccactcg cgcagcaaaa tcagaggtaa ggagatcaag acttacggct gacgaatacc 2280tgaaaattta tcaagcagca gaatcatcac catgttggct cagacttgca atggaactgg 2340ctgttgttac cgggcaacga gttggtgatt tatgcgaaat gaagtggtct gatatcgtag 2400atggatatct ttatgtcgag caaagcaaaa caggcgtaaa aattgccatc ccaacagcat 2460tgcatattga tgctctcgga atatcaatga aggaaacact tgataaatgc aaagagattc 2520ttggcggaga aaccataatt gcatctactc gtcgcgaacc gctttcatcc ggcacagtat 2580caaggtattt tatgcgcgca cgaaaagcat caggtctttc cttcgaaggg gatccgccta 2640cctttcacga gttgcgcagt ttgtctgcaa gactctatga gaagcagata agcgataagt 2700ttgctcaaca tcttctcggg cataagtcgg acaccatggc atcacagtat cgtgatgaca 2760gaggcaggga gtgggacaaa attgaaatca aataagaatt cactcctcag gtgcaggctg 2820cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc 2880tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg 2940gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac 3000tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga 3060gtttggcaac atatgccata tgctggctgc catgaacaaa ggtggctata aagaggtcat 3120cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga 3180ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt 3240tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct 3300gtccctcttc tcttatgaag atccctcgac ctgcagccca agcttggcgt aatcatggtc 3360atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 3420aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 3480gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat 3540tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 3600tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc

3660gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 3720tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3780caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3840tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag 3900aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 3960cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4020atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4080taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 4140aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 4200tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 4260gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct 4320cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 4380cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 4440atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 4500tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 4560ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 4620acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 4680aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 4740aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 4800tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 4860cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 4920catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 4980ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5040aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5100ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 5160caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 5220attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 5280agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 5340actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 5400ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 5460ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 5520gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 5580atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 5640cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 5700gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 5760gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 5820ggttccgcgc acatttcccc gaaaagtgcc acctg 5855128303DNAArtificial SequenceHuman FER-1 Promoter 128tccatgacaa agcacttttt gagcccaagc ccagcctagc tcgagctaaa cgggcacaga 60gacgccaccg ctgtcccaga ggcagtcggc taccggtccc cgctcccgag ctccgccaga 120gcgcgcgagg gcctccagcg gccgcccctc ccccacagca ggggcggggt cccgcgccca 180ccggaaggag cgggctcggg gcgggcggcg ctgattggcc ggggcgggcc tgacgccgac 240gcggctataa gagaccacaa gcgacccgca gggccagacg ttcttcgccg agagtcgggt 300acc 3031296521DNAArtificial SequencepIRES-BSR Plasmid 129tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg 660cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 720agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac 780agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt 840gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa 900ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact 960cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac 1020aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact 1080ataggctagc ctcgagaatt cacgcgtcga gcatgcatct agggcggcca attccgcccc 1140tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg 1200tttgtctata tgtgattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa 1260cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg 1320caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca 1380acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc 1440ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt 1500gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg 1560ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca 1620tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac 1680gtggttttcc tttgaaaaac acgatgataa gcttgccaca acccaccatg aaaacattta 1740acatttctca acaagatcta gaattagtag aagtagcgac agagaagatt acaatgcttt 1800atgaggataa taaacatcat gtgggagcgg caattcgtac gaaaacagga gaaatcattt 1860cggcagtaca tattgaagcg tatataggac gagtaactgt ttgtgcagaa gccattgcga 1920ttggtagtgc agtttcgaat ggacaaaagg attttgacac gattgtagct gttagacacc 1980cttattctga cgaagtagat agaagtattc gagtggtaag tccttgtggt atgtgtaggg 2040agttgatttc agactatgca ccagattgtt ttgtgttaat agaaatgaat ggcaagttag 2100tcaaaactac gattgaagaa ctcattccac tcaaatatac ccgaaattaa aagttttacc 2160ataccaagct tggcgggcgg ccgcttccct ttagtgaggg ttaatgcttc gagcagacat 2220gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 2280tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 2340agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt 2400tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc cgataaggat cgatccgggc 2460tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 2520ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2580gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2640ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2700tccgatttag agctttacgg cacctcgacc gcaaaaaact tgatttgggt gatggttcac 2760gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 2820ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 2880ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 2940aaatatttaa cgcgaatttt aacaaaatat taacgtttac aatttcgcct gatgcggtat 3000tttctcctta cgcatctgtg cggtatttca caccgcatac gcggatctgc gcagcaccat 3060ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc 3120agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 3180gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 3240cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 3300taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 3360gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 3420agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagcttgat tcttctgaca 3480caacagtctc gaacttaagg ctagagccac catgattgaa caagatggat tgcacgcagg 3540ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 3600ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 3660gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct 3720ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 3780ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 3840cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 3900ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 3960cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 4020gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 4080tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 4140ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 4200agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 4260ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg 4320ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gatggccgca ataaaatatc 4380tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga taaggatccg 4440cgtatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 4500cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 4560acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 4620acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 4680aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 4740tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 4800gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 4860tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 4920aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 4980cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 5040agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 5100ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 5160tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 5220tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 5280caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 5340accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 5400attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 5460ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 5520taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 5580taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 5640aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 5700agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 5760ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 5820ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 5880cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 5940tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 6000tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 6060tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 6120tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 6180ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 6240acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 6300ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 6360gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 6420ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 6480ggccttttgc tggccttttg ctcacatggc tcgacagatc t 6521

Claims

1. A method of introducing one or more heterologous nucleic acid(s) into an artificial chromosome, wherein the artificial chromosome is a mammalian artificial chromosome and is predominantly heterochromatin, the method comprising: (a) introducing one or a plurality of recombination site(s) comprising a heterologous att site into the artificial chromosome (b) mixing the artificial chromosome containing the one or a plurality of recombination site(s) comprising a heterologous att site with a first vector comprising the heterologous nucleic acid and one or a plurality of cognate recombination site(s), wherein the cognate recombination site(s) is a site that participates in recombinase catalyzed recombination with att site(s) in the artificial chromosome; (c) incubating the resulting mixture in the presence of at least one lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR under conditions whereby recombination between the att site and cognate recombination site is effected, thereby introducing the heterologous nucleic acid into the artificial chromosome.

2. The method of claim 1, wherein the artificial chromosome contains a plurality of att sites.

3.-13. (canceled)

14. The method of claim 1, wherein the att sites are selected from the group consisting of attP with attB and attL with attR.

15.-19. (canceled)

20. The method of claim 1, wherein the lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR is encoded by a nucleic acid molecule, wherein the nucleic acid molecule is provided on a second vector or on the first vector, or on the artificial chromosome and its expression is under the control of an inducible promoter.

21. The method of claim 20, wherein the second vector is the plasmid pCXLamIntR.

22. The method of claim 20, wherein the first vector is the plasmid pDsRedN1-attB.

23.-27. (canceled)

28. The method of claim 1 wherein the one or a plurality of recombination site(s) is introduced into the artificial chromosome together with a selectable marker.

29. The method of claim 28 wherein the selectable marker is selected from the group consisting of a gene that provides a selective growth advantage, an antibiotic resistance gene, and a gene encoding a detectable protein, wherein the detectable protein is chromogenic, fluorescent, or capable of being bound by an antibody and FACs sorted.

30. The method of claim 29 wherein the selectable marker is a puromycin-resistance gene.

31. The method of claim 28 wherein the one or a plurality of recombination site(s) and a selectable marker are comprised in pSV40-193attPsensePur (FIG. 4, SEQ ID NO:113).

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