PROMOTER AND PLASMID SYSTEM FOR GENETIC ENGINEERING

Abstract

series of low-copy number plasmids comprising restriction endonuclease recognition sites useful for cloning at least three different genes or operons, each flanked by a terminator sequence, the plasmids containing variants of glucose isomerase promoters for varying levels of protein expression. The materials and methods are useful for genetic engineering in microorganisms, especially where multiple genetic insertions are sought.

Description

[0001]This application claims the benefit of U.S. Provisional Application No. 60/374,931, filed Apr. 22, 2002.

FIELD OF THE INVENTION

[0002]This invention is in the field of molecular biology. More specifically, this invention pertains to a series of low-copy-number plasmids comprising restriction endonuclease recognition sites useful for cloning at least three different genes or operons, each site flanked by a terminator sequence and a set of promoters for varying levels of protein expression. The invention is useful for genetic engineering in microorganisms, especially where multiple genetic insertions are sought.

BACKGROUND OF THE INVENTION

[0003]Molecular biotechnology is a discipline that is based on the ability of researchers to transfer specific units of genetic information from one organism to another. This process, known as cloning, relies on the techniques of recombinant DNA technology to produce a useful product or a commercial process (Glick, B. R.; Pasternak, J. J., Molecular Biotechnoloy Principles and Applications of Recombinant DNA, 2^nd ed. American Society for Microbiology, Washington, D.C. (1998)).

[0004]Commercial processes often require that proteins encoded by the cloned gene are produced at high rates of expression. There is no single strategy for achieving maximal expression of every cloned gene. Most cloned genes have distinctive molecular properties that require the investment of considerable time and effort before a specific set of conditions that result in an acceptable level of expression is found.

[0005]Merely inserting a gene into a cloning vector does not ensure that it will be successfully expressed at the level needed. In response to the need for a high expression rate, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation, and secretion from the host cell. More specifically, the molecular features that have been manipulated to control gene expression include: (1) the nature of the relevant transcriptional promoter and terminator sequences, (2) the strength of the ribosome binding site, (3) the number of copies of the cloned gene and whether the gene is plasmid-borne or integrated into the genome of the host cell, (4) the final cellular location of the synthesized foreign protein, (5) the efficiency of translation in the host organism, and (6) the intrinsic stability of the cloned gene protein within the host cell.

[0006]Additionally, the introduction and expression of foreign DNA in a host organism often changes the metabolism of the organism in ways that may impair normal cellular functioning. This phenomenon is due to a metabolic load or burden imposed upon the host by the foreign DNA. The metabolic load may result from a variety of conditions including 1) increasing plasmid copy number, 2) overproduction of proteins, 3) saturation of export sites, and/or 4) interference of cellular function by the foreign protein itself.

[0007]Techniques to address some of the obstacles presented above are known. Several groups have used multiple promoters in tandem to express genes at different phases of cell growth (CN 1186856), from different RNA polymerases or in different phage species (U.S. Pat. No. 5,547,862; J. Biotechnol. 2(5):303-316 (1985); Biotechniques, 18(1):152-154, 156-157 (1995)). Another group has used tandem repeated multiple cloning sites (MCS) (Gene, 139 (1):83-86 (1994)) to facilitate moving DNA in and out of the plasmid vector. One group has reported the use of a high-copy-number vector with three multiple cloning sites each behind a different promoter for expression of different genes in mammalian cells (Biotech. Bioeng., 57(1):1-10 (1998)).

[0008]Despite these techniques, the problem to be solved remains how to easily and quickly clone multiple genes or operons while minimizing the impact of metabolic load, controlling the yield of the recombinant protein to meet production needs, and enhancing the stability of the transformed host cell.

SUMMARY OF THE INVENTION

[0009]Applicants have created novel glucose isomerase promoter sequences that allow varying levels of gene expression in production organisms. Applicants incorporated expression cassettes containing the variant GI promoters into a low-copy-number plasmid derived from pCL1920 to construct a series of plasmids for genetic engineering. Transcription terminators isolate the associated promoter from transcription from other promoters located outside this construct.

[0010]Applicants also have constructed a unique nucleotide sequence containing cloning sites for multiple rare restriction enzymes, further facilitating cloning in this construct or transfer of this construct to alternate plasmid or vector backbones. The unique cloning sites allow introduction of genes or operons to be expressed under the control of suitable promoters of varying strengths.

[0011]The invention encompasses:

[0012]1. an isolated or recombinant nucleic acid molecule encoding a Streptomyces lividins glucose isomerase variant, the nucleic acid molecule selected from the group consisting of SEQ ID NOS:9-28;

[0013]2. an isolated or recombinant nucleic acid molecule encoding a Streptomyces lividins glucose isomerase variant, the nucleic acid molecule comprising a nucleotide sequence of any of SEQ ID NOs:9-28;

[0014]3. a library of isolated or recombinant nucleic acid molecules encoding a Streptomyces lividins glucose isomerase variant, the library comprising the nucleotide sequences of SEQ ID NOS:9-28;

[0015]4. an expression cassette comprising the nucleic acid molecule of the various GI variants set out above, and;

[0016]5. a kit comprising the nucleic acid molecules encoding the various Streptomyes lividins glucose isomerase variants set out above.

[0017]A further embodiment of the invention is a DNA construct comprising at least three transcriptional terminators and at least one cloning site situated between any two transcriptional terminators. A preferred embodiment of this DNA construct comprises the transcriptional terminators tonB, thrA, or aspA, and the cloning sites are selected from the group consisting of AvrII, NheI, BfaI, Cac8I, BsaJI, and StyI. Preferred cloning sites are NheI or AvrII. A library of these constructs is also encompassed in the invention.

[0018]The invention includes the following DNA constructs: [0019]the pSYCO109mcs plasmid consisting of SEQ ID NO:30, [0020]the short 1.5 GI promoter consisting of SEQ ID NO:31, [0021]the short 1.20 GI promoter consisting of SEQ ID NO:32, [0022]the pAH105 plasmid consisting of SEQ ID NO:70, [0023]the pSYCO101 plasmid consisting of SEQ ID NO:71, [0024]the pSYCO103 plasmid consisting of SEQ ID NO:72, [0025]the pSYCO106 plasmid consisting of SEQ ID NO:73, [0026]the pSYCO109 plasmid consisting of SEQ ID NO:74, [0027]the pSCYO106mcs plasmid consisting of SEQ ID NO:78, and [0028]the pRJ50 plasmid consisting of SEQ ID NO:79.

[0029]A further embodiment of the invention is a vector having a multiple cloning site containing restriction recognition site sequences specific for the restriction endonucleases AscI, NheI, PacI, RsrII, NsiI, SacI, MluI, AgeI, SapI, and SnaBI. A particular embodiment of this vector is the nucleotide sequence of SEQ ID NO:77.

[0030]The genetic materials of this invention include transformed host cells containing the nucleic acid molecules described above and the polypeptides encoded by the polynucleotides.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND BIOLOGICAL DEPOSIT

[0031]Applicants have provided 83 sequences in conformity with Rules for the Standard Representation of Nucleotide and Amino Acid Sequences in patent applications (Annexes I and II to the Decision of the President of the EPO, published in Supplement No. 2 to OJ EPO, 12/1992), with 37 C.F.R. 1.821-1.825 and Appendices A and B (Requirements for Application Disclosures Containing Nucleotides and/or Amino Acid Sequences) with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IYUB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):345-373 (1984) which are herein incorporated by reference.

[0032]SEQ ID NO:1 is the nucleotide sequence for the wildtype Streptomyces lividans glucose isomerase (GI) promoter.

[0033]SEQ ID NOs:2-8 are oligonucleotide primers used for saturation mutagenesis of the GI promoter. In SEQ ID NOs:3-8, "N" represents either A, T, C, or G.

[0034]SEQ ID NOs:9-28 are nucleotide sequences for the GI promoter variants.

[0035]SEQ ID NO:29 is the nucleotide sequence for the yqhD gene from E. coli.

[0036]SEQ ID NO:30 is the nucleotide sequence for the pSYCO109mcs plasmid.

[0037]SEQ ID NO:31 is the nucleotide sequence for the short 1.5 GI promoter.

[0038]SEQ ID NO:32 is the nucleotide sequence for the short 1.20 GI promoter.

[0039]SEQ ID NO:33 is the nucleotide sequence for the short wild-type GI promoter.

[0040]SEQ ID NOs:34-37 are the oligonucleotide primers used for amplification of yqhD with incorporation of the short GI promoters.

[0041]SEQ ID NOs:38-39 are oligonucleotide primers used to construct the yqhD disruption.

[0042]SEQ ID NOs:40-43 are oligonucleotide primers used to confirm disruption of yqhD.

[0043]SEQ ID NOs:44-46 are oligonucleotide primers used for replacement of the chromosomal ppc promoter with the short wild-type GI promoter.

[0044]SEQ ID NO:47 is the nucleotide sequence for a multiple cloning site and terminator.

[0045]SEQ ID NO:48 is the nucleotide sequence for the pHK28-26 plasmid.

[0046]SEQ ID NOs:49-50 are oligonucleotide primers used to amplify dhaB3.

[0047]SEQ ID NOs:51-52 are oligonucleotide primers used to amplify dhaB1.

[0048]SEQ ID NOs:53-54 are oligonucleotide primers used to create the dhaT deletion.

[0049]SEQ ID NOs:55-56 are oligonucleotides used to create a linker.

[0050]SEQ ID NO:57 is a nucleotide sequence encoding three transcriptional terminators separated by restriction sites.

[0051]SEQ ID NOs:58-59 are oligonucleotides used to create SEQ ID NO:60.

[0052]SEQ ID NO:60 is the nucleotide sequence encoding three transcriptional terminators flanked by EcoRI and KpnI sites.

[0053]SEQ ID NOs:61-62 are oligonucleotide primers used to amplify SEQ ID NO:60.

[0054]SEQ ID NOs:63-66 are oligonucleotide primers used to amplify an expression cassette.

[0055]SEQ ID NO:67 is the nucleotide sequence of a double-stranded linker used to generate pCR-pCL1920.

[0056]SEQ ID NOs:68-69 are oligonucleotide primers used to amplify the rrn BT1T2 terminator from pTrc99A.

[0057]SEQ ID NO:70 is the nucleotide sequence for the pAH105 plasmid.

[0058]SEQ ID NO:71 is the nucleotide sequence for the pSYCO101 plasmid.

[0059]SEQ ID NO:72 is the nucleotide sequence for the pSYCO103 plasmid.

[0060]SEQ ID NO:73 is the nucleotide sequence for the pSYCO106 plasmid.

[0061]SEQ ID NO:74 is the nucleotide sequence for the pSYCO109 plasmid.

[0062]SEQ ID NOs:75-76 are oligonucleotide primers used to form SEQ ID NO:77.

[0063]SEQ ID NO:77 is the nucleotide sequence of a multiple cloning fragment containing restriction recognition sites for the following enzymes: NheI, RsrIl, SacI, AgeI, SnaBI, AscI, PacI, NsiI, MluI, and SapI.

[0064]SEQ ID NO:78 is the nucleotide sequence for the pSCYO106mcs plasmid.

[0065]SEQ ID NO:79 is the nucleotide sequence for the pRJ50 plasmid.

[0066]SEQ ID NOs:80-81 are oligonucleotide primers used to amplify the orf operon.

[0067]SEQ ID NOs:82-83 are oligonucleotide primers to check transformants in Example 4.

[0068]Applicants have made the following biological deposit under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure:

TABLE-US-00001 Depositor Identification Int'l. Depository Reference Designation Date of Deposit Escherichia coli RJ8n ATCC PTA-4216 9 Apr. 2002

[0069]As used herein, "ATCC" refers to the American Type Culture Collection International Depository located 10801 University Blvd., Manassas, Va. 20110-1109, U.S.A. The "ATCC No." is the accession number to cultures on deposit with the ATCC.

[0070]The listed deposits will be maintained in the indicated international depository for at least thirty (30) years and will be made available to the public upon grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.

DETAILED DESCRIPTION OF THE INVENTION

[0071]The Applicants have solved the stated problem by creating a series of constructs containing at least three unique cloning sites, each cloning site operably separated from each other by transcription terminators and promoters of different strengths. The promoters of different strengths are variants of the Streptomyces lividans glucose isomerase (GI) promoter (SEQ ID NO:1). Combining the variant GI promoters in expression cassettes with a SYCO plasmid as the production platform provides a system useful for biocatalyst development in a wide variety of bioprocess projects.

[0072]The invention allows the facile and stable incorporation of endogenous or exogenous genes or operons in a vector controlling the levels of gene expression. The use of the single plasmid to express multiple genes or operons reduces the number of antibiotic markers needed to maintain the multiple plamids in the E. coli host that previous methods required to produce a gene product. Use of the invention can minimize the impact of metabolic load, optimize the yield of the recombinant protein, and enhance the stability of the transformed host cell. The invention is especially useful for genetic engineering in bioprocesses where expressing two or more genes or operons may be required for product formation.

[0073]Applicants have created novel GI promoter sequences that allow varying levels of gene expression. Applicants incorporated expression cassettes containing the variant GI promoters into a low-copy-number plasmid derived from pCL1920 to construct a series of plasmids for genetic engineering. The transcription terminators isolate the associated promoter from transcription from other promoters located outside this construct.

[0074]Applicants also have constructed a unique nucleotide sequence containing cloning sites for at least ten rare restriction enzymes, further facilitating cloning in this construct or transfer of this construct to alternate plasmid or vector backbones. The unique cloning sites allow introduction of genes or operons to be expressed under the control of suitable promoters of varying strengths. Further, a given construct may be flanked by unique cloning sites for facile integration into any number of plasmid backbones including pUC, pBR322, PACYC, pSC101, or others known and contemplated by those skilled in the art.

[0075]Applicants have demonstrated a specific utility of the invention in the biosynthesis of 1,3-propanediol (3G) from glucose in E. coli transformed with the claimed materials. Expression cassettes were constructed in a low-copy-number plasmid as described herein and genes for production of 1,3-propanediol were cloned into this vector. The invention may be used to vary gene expression in other expression systems.

DEFINITIONS

[0076]The following definitions and abbreviations are to be used to interpret the claims and specification.

[0077]"Open reading frame" is abbreviated ORF.

[0078]"Polymerase chain reaction" is abbreviated PCR.

[0079]The terms "host cell" or "host organism" refer to a microorganism capable of receiving foreign or heterologous genes or multiple copies of endogenous genes and of expressing those genes to produce an active gene product.

[0080]The terms "DNA construct" or "construct" refer to an artificially constructed fragment of DNA "Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign", "exogenous", or "heterologous" gene refers to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. A "gene construct" refers to a nucleic acid fragment that encodes for expression of one or more specific proteins. In the gene construct the gene may be native, chimeric, or foreign in nature.

[0081]The term "isolated nucleic acid" refers to a nucleic acid (e.g., an RNA, DNA, or a mixed polymer) which is substantially separated from other components that naturally accompany a native sequence (e.g., ribosomes, polymerases, and/or flanking genomic sequences from the originating species). The term includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0082]The terms "encoding" and "coding" refer to the process by which a gene, through the mechanisms of transcription and translation, produces an amino acid sequence. The process of encoding a specific amino acid sequence includes DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated. For example, alterations in the gene sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue (such as glycine), or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes that result in substitution of one negatively charged residue for another (such as aspartic acid for glutamic acid), or one positively charged residue for another (such as lysine for arginine) can also be expected to produce a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity in the encoded products. Moreover, the skilled artisan recognizes that sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C.), with the sequences exemplified herein.

[0083]The term "expression" refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product.

[0084]The term "promoter" refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene.

[0085]The terms "transcription terminator" or "terminator" refer to the genetic element that ends protein synthesis.

[0086]The term "operon" refers to a cluster of genes that are coordinately regulated.

[0087]The terms "polypeptide" and "protein" are used interchangeably to refer to the gene product.

[0088]The terms "plasmid", "vector", and "cassette" refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences (linear or circular) of a single- or double-stranded DNA or RNA, derived from any source. Such elements contain a number of nucleotide sequences that have been joined or recombined into a unique construction capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in its host.

[0089]The term "restriction endonucleases" refers to a class of enzymes that cut a given length of DNA at a specific and unique internal location. By creating the cut in the DNA, restriction endonucleases enable the subsequent splicing or insertion of segments of DNA into the internal location. The terms "restriction site" or "restriction recognition site" refer to a nucleotide sequence (of base pairs) in a DNA molecule that is "recognized" and cut by a given restriction enzyme.

[0090]The term "rare", as applied to restriction enzyme sites, refers to the low frequency of occurrence of a given sequence in a gene. A preferred group of rare restriction enzymes sites for purposes of this specification are AscI, NheI, PacI, RsrII, NsiI, SacI, MluI, AgeI, SapI, and SnaBI.

[0091]The term "cloning site" refers to a location on a vector into which DNA can be inserted. The term "multiple cloning site" or "mcs" refers to a synthetic DNA sequence that contains any one or a number of different restriction enzyme sites to permit insertion at a defined locus (the restriction site) on a vector. The term "unique cloning site" refers to a cloning site that appears one time with a given DNA sequence.

[0092]In describing the relative locations of the elements of a vector, a given site or locus of interest is "between" two others if it is situated in the intermediate length of DNA that separates the two others. In the case of a circular vector, the given site or locus of interest is "between" two others if it is situated within the shortest length of DNA that separates the two other sites on the vector. The given site or locus is said to be "flanked" by another situated either preceding or following the site or locus of interest.

[0093]The term "genetically altered" refers to the process of changing hereditary material by transformation or mutation. The terms "transformation" and "transfection" refer to the acquisition of new genes in a cell after the incorporation of nucleic acid. The acquired genes may be integrated into chromosomal DNA or introduced as extrachromosomal replicating sequences. The term "transformant" refers to the product of a transformation.

[0094]The terms "glycerol dehydratase" or "dehydratase enzyme" refer to the polypeptide(s) responsible for a coenzyme B₁₂-dependent enzyme activity that is capable of isomerizing or converting a glycerol molecule to the product 3-hydroxypropionaldehyde. For the purposes of the present invention, the dehydratase enzymes include a glycerol dehydratase (GenBank U09771, U30903) and a diol dehydratase (GenBank D45071) having preferred substrates of glycerol and 1,2-propanediol, respectively. Glycerol dehydratase of K. pneumoniae ATCC 25955 is encoded by the genes dhaB1, dhaB2, and dhaB3 (GenBank U30903). The dhaB1, dhaB2 and dhaB3 genes code for the α, β, and γ subunits of the glycerol dehydratase enzyme, respectively. Glycerol dehydratase and diol dehydratase are complexes (with an α₂β₂γ₂ subunit composition) that utilize coenzyme B₁₂.

[0095]Glycerol and diol hydratases are subject to mechanism-based suicide inactivation by glycerol and some other substrates (Daniel et al., FEMS Microbiol. Rev. 22:553 (1999)). The term "dehydratase reactivation factor" refers to those proteins responsible for reactivating the dehydratase activity. The terms "dehydratase reactivating activity", "reactivating the dehydratase activity", or "regenerating the dehydratase activity" refer to the phenomenon of converting a dehydratase not capable of catalysis of a substrate to one capable of catalysis of a substrate or to the phenomenon of inhibiting the inactivation of a dehydratase or the phenomenon of extending the useful half-life of the dehydratase enzyme in vivo. Two proteins have been identified as being involved as the dehydratase reactiviation factor (see WO 9821341 (U.S. Pat. No. 6,013,494 herein incorporated by reference) and references therein; Daniel et al., supra; Toraya and Mori, J. Biol. Chem. 274:3372 (1999); and Tobimatsu et al., J. Bacteriol. 181:4110 (1999)).

[0096]The terms "oxidoreductase" or "1,3-propanediol oxidoreductase" refer to the polypeptide(s) responsible for an enzyme activity that is capable of catalyszing the reduction of 3-hydroxypropionaldehyde to 1,3-propanediol. 1,3-Propanediol oxidoreductase includes, for example, the polypeptide encoded by the dhat gene (GeneBank U09771, U30903). Alternatively, yqhD, an E. coli open reading frame with 40% identity to the gene adhB in Clostridium (a probable NADH-dependent butanol dehydratase 2), encodes a polypeptide that functions as a 1,3-propanediol oxidoreductase (WO 0112833).

[0097]The enzymes expressed by the pSYCO plasmids (pSYCO101, pSYCO103, pSYCO106, pSYCO109, pSYCO106mcs, and pSYCO109mcs) can all be said to comprise genes required to express glycerol dehydratase, dehydratase reactiviation factor, glycerol-3-phosphate dehydrogenase, and glycerol-3-phosphatase.

[0098]The terms "fermentable carbon substrate" and "fermentable carbon source" refer to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, glycerol, dihydroxyacetone and one-carbon substrates or mixtures thereof.

Gene Expression System with GI Promoter Variants of Differing Strengths

[0099]The minimal requirement for an effective gene expression system is the presence of a promoter (a site on DNA where the RNA polymerase binds and begins transcription) upstream from a cloned gene. Often a strong promoter, one that has a high affinity for RNA polymerase, is used with the result that the adjacent downstream region is highly or frequently transcribed.

[0100]In the promoter, the main sequence determinant of promoter strength (the level at which the downstream gene is transcribed) is the most highly conserved base pairs. Promoters that have deviations from the conserved sequences have decreased transcription initiation frequency (Hawley, D. K.; McClure, W. R., Nucleic Acids Res., 11:2237-2255 (1983)).

[0101]Promoters for E. coli RNA polymerase have been shown to contain two regions of conserved DNA sequences, located about 10 and 35 base pairs upstream of the transcription start site. Twelve base pairs were determined to be the most highly conserved among promoters. These bases are TTGACA around 35 base pairs upstream, the so called -35 region, and TATAAT around 10 base pairs upstream, the so called -10 region. Optimum spacing between the -10 and -35 regions is 17 base pairs. The promoter is stronger if the spacing is closer to 17 base pairs; however, promoters with interspacings of 15 and 20 base pairs retain partial function.

[0102]Applicants have created a series of constructs incorporating variants of the Streptomyces lividans glucose isomerase (GI) promoter. The constructs form a library or kit of promoter variants with a range of different strengths conferring the ability to tailor varying levels of gene expression as needed. The Streptomyces glucose isomerase (EC 5.3.1.9) catalyzes the conversion of glucose-6-phosphate to fructose-6-phosphate. Transcription of the gene encoding phosphoglucose isomerase (pgi) is controlled by a promoter that contains a characteristic -10 signature sequence (AATAAT) and a characteristic -35 signature sequence (TTGACA). Although saturation mutagenesis was carried out in the -35 region of the promoter, changes to a SpeI restriction site approximately 122 bp upstream from the -35 region also had effects on expressed gene activity. Further, a 25 bp deletion between the -10 and the end of this promoter allowed retention of 86% of the enzyme's activity even with the changes to the SpeI restriction site. These particular results had not been previously reported.

[0103]Transcription termination of RNA synthesis occurs at specific base sequences on the DNA and regulates termination of transcription. A common termination sequence on the DNA is one containing an inverted repeat with a central non-repeating segment. When such a DNA sequence is transcribed, the RNA can form a stem-loop structure by intrastrand base pairing. When such stem-loop structures in the RNA are followed by runs of uridines, they are effective transcription terminators. Other termination sites are regions where a GC-rich sequence is followed by an AT-rich sequence. Such kinds of structures lead to termination of transcription without adding any extra factors and are sometimes termed intrinsic terminators or rho-independent terminators.

[0104]Other types of terminator sequences have been discovered that require protein factors like Rho from E. coli in addition to RNA polymerase to function. Rho does not bind to RNA polymerase or to DNA but binds tightly to RNA and moves down the chain towards the RNA polymerase-DNA complex. Once RNA polymerase has paused at a Rho-dependent termination site, Rho can then cause the RNA and polymerase to leave the DNA, thus terminating transcription. Other proteins involved in transcription termination are, like Rho, RNA-binding proteins. In all cases the sequences involved in termination operate at the level of RNA. However, RNA is transcribed from DNA, and so transcription termination is ultimately determined by specific nucleotide sequences on the DNA. (Madigan, M. T.; Martinko, J. M.; Parker, J.; Brock Biology of Microorganisms, 8^th ed., Prentice Hall; Upper Saddle River, N.J. (1997)).

[0105]Applicants have constructed a termination region in which three different terminator sequences have been placed in tandem. These three terminators are flanked by unique restriction enzyme sites useful for the cloning of genes or operons. The tonB terminator is a bidirectional rho-independent transcriptional terminator found between the E. coli tonB gene and an opposing gene (Postle, K.; Good, R. F., Cell, 41, 577-585 (1985)). The thr attenuator, similar in structure to other rho-independent terminators facilitates transcriptional termination of the E. coli threonine operon (Yanget et al., J. Biol. Chem., 270:23330-23336 (1995)). The aspA terminator with a structure characteristic of rho-independent terminators, facilitates transcriptional termination of the E. coli aspartase operon (Takagi et al., Nucleic Acid Res., 13:2063-2074 (1985)).

[0106]As autonomous, self-replicating genetic elements, plasmids have the basic attributes to make them potential vectors for carrying cloned DNA. Naturally-occurring plasmids often lack several important features required in a high-quality cloning vector. These features include (1) a small size (necessary for efficient transfer of exogenous DNA into a host), (2) unique restriction endonuclease recognition sites into which the insert DNA can be cloned, and (3) one or more selectable genetic markers for identifying recipient cells that carry the cloning vector-insert DNA construct. Consequently, plasmid cloning vectors have to be genetically engineered (Glick, B. R., Pasternak, J. J., Molecular BiotechnoloqV Principles and Applications of Recombinant DNA, 2^nd ed., American Society for Microbiology, Washington, D.C. (1998)).

[0107]pCL1920/21 vectors are a pair of low-copy-number plasmids that contain a 580 bp BstUI fragment carrying the lac promoter/operator, a multiple cloning site and lacZ fragment of pUC19 cloned in place of the polylinker region in pGB2, a pSC101-derived plasmid which confers spectinomycin and streptomycin resistance in E. coli. pCL1920/21 vectors (five copies per cell) have a 40-fold difference in plasmid copy number between pCL1920/21 vectors and pUC vectors (200 copies per cell). Thus, the pCL1920/21 vectors allow regulated low-level expression of genes inserted downstream of the promoter-operator when transformed into strains. They should also be useful for cloning genes that may be deleterious at high copy number. Since the pCL1920/21 vectors are compatible with ColE1-derived plasmids they can be used to form stable co-transformants together with pBR322 or pUC derived plasmids (Lerner et al., Nucleic Acids Res., 18:4631 (1990)).

[0108]The plamids of the inventions may be used in a variety of hosts for the controlled bioproduction of materials.

Replacing Chromosomally-Located Native Promoters of any Endogenous Gene or Operon to Alter Transcriptional Level.

[0109]Claimed promoter variants (constructs comprising SEQ ID NOs:31 and 32) may be used in a method to replace chromosomally-located native promoters associated with any endogenous gene or operon in order to alter the transcription level of the gene or operon. The result is changed protein production levels. The promoter to be replaced can be any gene in any microorganism where the Llambda red method of Datsenko and Wanner [(2000) PNAS 97:6640-6645] or an equivalent method is operable.

[0110]In the method, a chimeric DNA molecule comprising a selectable marker operably linked to a divergently-arranged non-native promoter operably linked to the 5' coding region of a target gene is synthesized by polymerase chain reaction (PCR). The synthesis is accomplished using: (1) a pair of chemically synthesized primers, (a) the first primer comprising: (i) a DNA region distal to the target natural promoter to be replaced, (ii) a non-native promoter, and (iii) a DNA region from either the 3' or 5' end of the selectable marker; and (b) the second primer comprising: (i) a DNA region proximal to the targeted insertion site, and (ii) a DNA region from the opposite end of the selectable marker than was used in the first primer; and (2) a DNA template encoding a selectable marker. This product is integrated in the DNA product synthesized above at the chromosomal target site of any host cell using the method of Datsenko and Wanner (supra). The result of this protocol is that the target native promoter(s) are replaced with the PCR-synthesized chimeric molecule that carries the non-native promoter.

[0111]An extension of the method may be used to assess the effect of the varying gene expression level on biocatalyst performance.

Biosynthesis of 1,3-propanediol (3G) from Glucose in E. coli

[0112]The plasmids of the invention may be used in E. coli for the biosynthesis of 1,3-propanediol (3G) from glucose. The examples herein include the construction of a production organism that incorporates the claimed invention and the genetic machinery necessary to convert a fermentable carbon substrate to 1,3-propanediol.

[0113]The genes involved in 1,3-propanediol production include a dehydratase gene (typically a glycerol or diol dehydratase) and an oxidoreductase as well as other proteins expected to aid in the assembly or in maintaining the stability of the dehydratase enzyme. These genes may be transgenes introduced into the host cell, or may be endogenous. At least one of these genes will be a transgene and introduced into the production cell. Recombinant organisms containing the necessary genes that encode the enzymatic pathway to convert a carbon substrate to 1,3-propanediol may be constructed using techniques well known in the art. The transformed production cell is then grown under appropriate conditions for the production of 1,3-propanediol.

[0114]Production of 1,3-propanediol in E. coli has been previously described (U.S. Pat. No. 5,633,362; U.S. Pat. No. 5,821,092; U.S. Pat. No. 5,686,276; U.S. Pat. No. 6,025,184; U.S. Pat. No. 6,013,494; U.S. Pat. No. 5,599,689; U.S. Pat. No. 6,136,576). Expression of many different genes are involved in the production from glucose of 1,3-propanediol by a recombinant E. coli. Genes encoding glycerol dehydratase (dhaB) and 1,3-propanediol oxidoreductase (dhaT) were isolated from a native host such as Klebsiella and used to transform host strains such as E. coli strain DH5α or FM5; K. pneumoniae strain ATCC 25955; K. oxytoca strain ATCC 8724 or M5a1, S. cerevisiae strain YPH499, P. pastoris strain GTS115, and A. niger strain FS1.

[0115]In Klebsiella pneumonia, Citrobacter freundii, and Clostridium pasteurianum, the genes encoding the three structural subunits of glycerol dehydratase (dhaB1-3 or dhaB, C, and E) are located adjacent to a gene encoding a specific 1,3-propanediol oxidoreductase (dhaT). Although the genetic organization differs somewhat among these microorganisms, these genes are clustered in a group that also includes orfX and orfZ (genes encoding a dehydratase reactivation factor for glycerol dehydratase), as well as orfY and orfW (genes of unknown function). The specific 1,3-propanediol oxidoreductases (dhaT's) of these microorganisms are known to belong to the family of type III alcohol dehydrogenases; each exhibits a conserved iron-binding motif and has a preference for the NAD.sup.+/NADH linked interconversion of 1,3-propanediol and 3-HPA. However, the NAD.sup.+/NADH linked interconversion of 1,3-propanediol and 3-HPA is also catalyzed by alcohol dehydrogenases which are not specifically linked to dehydratase enzymes (for example, horse liver and baker's yeast alcohol dehydrogenases (E.C. 1.1.1.1)), albeit with less efficient kinetic parameters. Glycerol dehydratase (E.C. 4.2.1.30) and diol [1,2-propanediol] dehydratase (E.C. 4.2.1.28) are related but distinct enzymes that are encoded by distinct genes. Diol dehydratase genes from Klebsiella oxytoca and Salmonella typhimurium are similar to glycerol dehydratase genes and are clustered in a group which comprises genes analogous to orfX (and orfZ (Daniel et al., FEMS Microbiol. Rev. 22:553 (1999); Toraya and Mori, J. Biol. Chem. 274:3372 (1999); GenBank AF026270).

[0116]The gene encoding glycerol-3-phosphate dehydrogenase (DAR1, GPD1) has been cloned and sequenced from S. diastaticus (Wang et al., J. Bact. 176:7091-7095 (1994)). The DAR1 gene was cloned into a shuttle vector and used to transform E. coli where expression produced active enzyme. Wang et al. (supra) recognize that DAR1 is regulated by the cellular osmotic environment but do not suggest how the gene might be used to enhance 1,3-propanediol production in a recombinant microorganism.

[0117]Other glycerol-3-phosphate dehydrogenase enzymes have been isolated. For example, sn-glycerol-3-phosphate dehydrogenase has been cloned and sequenced from Saccharomyces cerevisiae (Larason et al., Mol. Microbiol. 10: 1101 (1993)). Albertyn et al. (Mol. Cell. Biol. 14:4135 (1994)) teach the cloning of GPD1 encoding a glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae. Like Wang et al. (supra), both Albertyn et al. and Larason et al. recognize the osmo-sensitivity of the regulation of this gene but do not suggest how the gene might be used in the production of 1,3-propanediol in a recombinant microorganism.

[0118]As with G3PDH, glycerol-3-phosphatase has been isolated from Saccharomyces cerevisiae and the protein identified as being encoded by the GPP1 and GPP2 genes (Norbeck et al., J. Biol. Chem. 271:13875 (1996)). Like the genes encoding G3PDH, it appears that GPP2 is osmosensitive.

EXAMPLES

[0119]The present invention is further defined in the following Examples that indicate preferred embodiments of the invention. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

General Methods

[0120]Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).

[0121]Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg, and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), New England Biolabs (Beverly, Mass.) or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified.

[0122]The meaning of abbreviations is as follows: "h" means hour(s), "min" means minute(s), "sec" means second(s), "d" means day(s), "mL" means milliliters, "L" means liters, "mm" means millimeters, "nm" means nanometers, "mM" means millimolar, "M" means molar, "mmol" means millimole(s), "μmole" mean micromole", "g" means gram, "μg" means microgram.

Example 1

Construction of Glucose Isomerase Promoter Variants

[0123]The Streptomyces lividans glucose isomerase (GI) promoter (SEQ ID NO:1) contains a characteristic -10 signature sequence (AATAAT) and a characteristic -35 signature sequence (-35 T, -34 T, -33 G, -32 A, -31 C, -30 A). Using mixed base oligonucleotides, saturation mutagenesis of the -35 region of the GI promoter in pMP38 (as described in Example 6 following) was performed by standard PCR. In six individual PCR reactions, an upstream primer (SEQ ID NO:2) was paired with one of six downstream primers (SEQ ID NOs:3-8), each of which contains an equal mixture of all four possible bases at a single position in the -35 region, designated as N. The upstream primer also incorporates two single base pair changes that change a SpeI restriction site (ACTAGT) immediately following the EcoRI site to an AvrII restriction site (CCTAGG). The six PCR products were digested with EcoRI and HindIII, and individually ligated to EcoRI/HindIII digested pMP38. Ligations were transformed into E. coli, and recombinant plasmids were identified through restriction analysis by the SpeI to AvrII conversion, and subjected to nucleotide sequencing. Only recombinant plasmids would be expected to harbor possible -35 region changes. Of the twenty-four possible recombinant outcomes (4 bases in 6 positions), 18 were obtained, of which 13 represent changes in the -35 region (Table 1).

TABLE-US-00002 TABLE 1 GI promoter variants obtained by saturation mutagenesis PCR SEQ ID Position Base Name Comments NO. -30A A P1.6 No change in -35 region 9 T P1.5 10 G P1.20 11 C P1.10 12 -31C C P2.8 No change in -35 region 9 A P2.39 13 -32A A P3.4 No change in -35 region; 14 25 bp deletion between -10 and -HindIII site C P3.5 15 -33G G P4.49 No change in -35 region 9 A P4.15 16 C P4.1 17 -34T T P5.10 No change in -35 region 9 C P5.12 18 A P5.17 19 G P5.19 20 -35T T NA No change in -35 region 9 G P6.5 21 A P6.14 22 C P6.20 23 *NA = Not Applicable

[0124]Although five of the possible 18 changes in the -35 region were not isolated, these may also be useful for varying expression levels of cloned or chromosomally-encoded native and non-native genes or operons. These five additional GI promoter variants are described in Table 2.

TABLE-US-00003 TABLE 2 Other Potential GI Promoter Variants Position Base SEQ ID NO. -31C G 24 T 25 -32A G 26 T 27 -33G T 28

Example 2

Analysis of Glucose Isomerase Promoter Variants By Measuring Glycerol Dehydratase Activity

[0125]Glycerol dehydratase (GDH; encoded by dhaB1-3) activity was used as a reporter to measure the effect of the GI promoter mutations (Table 3). It was observed that even in the absence of a change in the -35 region, GDH activity dropped significantly due to the two base pair changes which converted SpeI to AvrII (for example, P1.6). It was also determined that P3.4 did not have a -35 mutation, but did have a 25 base pair deletion immediately following the -10 region, and had nearly wild-type (86%) promoter strength.

[0126]Dehydratase activity in cell-free extracts was determined using either glycerol or 1,2-propanediol as substrate. Cell-free extracts were prepared by cell disruption using a French press followed by centrifugation of the cellular debris. The assay, based on the reaction of aldehydes with methylbenzo-2-thiazolone hydrazone, has been described by Forage and Foster (Biochim. Biophys. Acta 569:249 (1979)).

TABLE-US-00004 TABLE 3 Measure of Relative GDH Activity In GI Promoter Variants relative GDH Plasmid activity pMP38 100 pMP38/1.6 13 pMP38/1.5 3 pMP38/1.20 1 pMP38/1.10 1 pMP38/2.39 0 pMP38/3.4 86 pMP38/3.5 1 pMP38/4.1 0 pMP38/4.15 0 pMP38/5.12 0 pMP38/5.17 0 pMP38/5.19 0 pMP38/6.5 0 pMP38/6.14 1 pMP38/6.20 2

Example 3

Analysis of GI Promoter Variants Using LUX Assays

[0127]A second type of reporter was used to measure levels of expression driven from the GI promoter variants. Bacterial bioluminescence is a phenomenon in which the products of 5 structural genes (luxA, luxB, luxC, IuxD, and luxE) work in concert to produce light. The IuxD product generates a C14 fatty acid from a precursor. The C14 fatty acid is activated in an ATP dependent reaction to an acyl-enzyme conjugate through the action of the luxE product, which couples bacterial bioluminescence to the cellular energetic state. The acyl-enzyme (luxE product) serves as a transfer agent, donating the acyl group to the luxC product. The acyl-LuxC binary complex is then reduced in a reaction in which NADPH serves as an electron pair and proton donor reducing the acyl conjugate to the C14 aldehyde. This reaction couples the reducing power of the cell to bacterial light emission. The light production reaction, catalyzed by luciferase (the product of luxA and luxB), generates light. The energy for light emission is provided by the aldehyde to fatty acid conversion and FMNH₂ oxidation, providing another couple between light production and the cellular energy state.

[0128]The Photorabdus luminenscens luxAB genes were used as reporters for GI promoter variant strength (Van Dyk et al., Appl. Environ. Microbiol., 180:785-792 (1995)). A PCR fragment carrying the P. luminenscens luxAB genes and containing SpeI sites at the 3' and 5' ends and an NcoI site engineered at the initiation codon of luxA was subcloned into the SpeI site in pMCS5 (MobiTec, Gottingen, Germany) yielding pJT13. Then a gene SOEing PCR-based kanamycin cassette with SwaI/NcoI ends was cloned into SwaI/NcoI-digested pJT13 to make pJT14.HIGHCOPY, the high copy luxAB promoter probe. pJT14.HIGHCOPY was then digested with SpeI producing the luxAB::kanamycin cassette, which was subcloned into the unique NheI site (compatible with SpeI), in pRJ50 (SEQ ID NO:79) to make pJT14.LOWCOPY.1, the low copy luxAB promoter probe. The GI promoters 1.6, 1.5, 1.20, and native were cloned into pJT14.HIGHCOPY and pJT14.LOWCOPY as NotI/NcoI fragments to make the high-copy constructs pJT18, pJT19, pJT20, and pJT25, respectively, and low-copy constructs pJT21.1, pJT22.1, pJT23.1, and pJT26.1, respectively. The plasmids were then transformed into select E. coli strains for in vivo bioluminescence measurements.

[0129]Promoter strengths were measured by luminometry using broth cultures of E. coli reporter strains, n-decanal as the aldehyde substrate, and a luminometer, as described by Van Dyk and Rosson (Methods in Molecular Biology, Vol. 102: Bioluminescence Methods and Protocols, 85 (1998)). E. coli clones were inoculated from a fresh agar plate into test tubes containing standard Luria-Bertani liquid growth medium with the appropriate antibiotic and grown aerobically (with shaking) at 37° C. for approximately 16 h. Cells were then subcultured into 100-mL flasks containing 25 mL of fresh medium and grown under the same conditions for approximately 8-10 h. Aliquots (200 μL) were then taken from each culture and placed into 96-well clear and white plates for optical density measurements at 600 nm (SpectraMax 190 Plater Reader, Molecular Devices Corporation, Sunnyvale, Calif.) and luminometer measurements (Luminoscan Ascent TAype 392, LabSystems, Helsinki, Finland), respectively. For the luminometry readings, 2 μL of exogenous aldehyde (n-decanal) was added to each well and measurements made. Results from these assays are listed in Table 4. These luminometry measurements indicated a level of promoter strength similar to that indicated by glycerol dehydratase assays.

TABLE-US-00005 TABLE 4 Bioluminescence Measurements GI Promoter Plasmid construct Relative Bioluminescence GI wildtype pJT26.1 100% GI 1.6 pJT21.1 12.9% GI 1.5 pJT22.1 3.0% GI 1.20 pJT23.1 1.3%

Example 4

Use of Shortened GI Promoter Sequences to Achieve Different Levels of Gene Expression

[0130]A subset of the GI promoter sequences described and used in Examples 1-3 were used to vary levels of expression of E. coli yqhD (SEQ ID NO:29) from the pSYCO109mcs plasmid (as described in Example 8 and SEQ ID NO:30) in strain RJ8n in which the yqhD gene was disrupted on the chromosome to create strain RJ8n (yqhD-).

[0131]Three expression cassettes for yqhD were constructed. These cassettes contain (i) one of the shortened GI promoters designated short 1.5 GI (SEQ ID NO:31), short 1.20 GI (SEQ ID NO:32), or short wild-type GI (SEQ ID NO:33); (ii) yqhD from E. coli KLP23 (WO9928480); and (iii) the threonine terminator (Lynn et al., J. Mol. Biol., 183:529-541 (1985)). The yqhD gene was isolated by PCR amplification from genomic KLP23 DNA using forward synthetic primers for short 1.5 GI (SEQ ID NO:34), short 1.20 GI (SEQ ID NO:35), or short wild-type GI (SEQ ID NO:36) which contain one of the shortened GI promoters and also incorporate a RsrII restriction site, and the reverse primer for yqhD (SEQ ID NO:37) that contained the threonine terminator and included a SacI site. Plasmid pSYCO109mcs was digested with RsrII/SacI and the RsrII/SacI digested PCR products were ligated into the plasmid. The ligation mixture was transformed into the RJ8n (yqhD-) strain by electroporation and the enzyme activity levels in each of the strains were compared (Table 5.)

[0132]The enzyme activity expressed by yqhD will reduce the aldehydes 3-hydroxypropionaldehyde (3-HPA) and butanal with similar rates using NADPH as the source of reducing equivalents. Since 3-HPA is not commercially available, butanal is generally used. The assay mixture contained in 1 mL total volume: 200 mM potassium phosphate buffer (pH 7.5), 10 mM butanal, 0.2 mM NADPH, and approximately 0.01 mg protein from cell-free extracts to be assayed. The initial rate of oxidation of NADPH after addition of protein sample was followed by measuring the change in absorbance at 340 nm (Δε=6.22 mM-1). A unit of activity is defined as that required to oxidize 1 micromole of NADPH in 1 minute in the presence of 10 mM butanal at 35° C. The activities of various strains are given in Table 5 below and were consistent with levels of expression allowed by the longer GI promoter variants.

TABLE-US-00006 TABLE 5 YqhD Activity Activity Strain and construct (U/mg) % Activity RJ8n(yqhD-) 0.015 0.8 RJ8n(yqhD-)/pSYCO109mcs 0.010 0.5 RJ8n(yqhD-)/pSYCO109mcs-short 1.20 GI yqhD 0.14 7.3 RJ8n(yqhD-)/pSYCO109mcs-short 1.5 GI yqhD 0.29 15 RJ8n(yqhD-)/pSYCO109mcs-short wild-type 1.92 100 GI yqhD

[0133]To create RJ8n (yqhD-) the yqhD gene was disrupted in E. coli MG1655 using the procedure as described by Wanner and Datsenko (PNAS, 97(12):6640-6645 (2000)) for Red-mediated homologous recombination. The forward PCR primer H1::6574 (SEQ ID NO:38) (containing 42 bp of sequence homologous to yqhD and the primer binding site P1 to pKD13) and the reverse PCR primer H2::6706 (SEQ ID NO:39) (containing 47 bp of homologous yqhD sequence and the primer binding site P4 to pKD13) were prepared. PCR amplification with pKD13 as the template produced a PCR product that had yqhD sequence on each end followed by FRT (FLP recognition target) sites that flank a kanamycin resistance (kanR) marker. The PCR product was electrotransformed into E. coli MG1655 cells and kanamycin-resistant transformants were selected. Correct insertion in the transformants was confirmed by PCR using primers yqhDUP (SEQ ID NO:82) and yqhDDN (SEQ ID NO:83) flanking the yqhD gene. The temperature-sensitive plasmid containing the Lambda Red system was cured by growth of the strains at 42° C.

[0134]The yqhD::kan disruption was moved into RJ8n by P1 transduction and confirmed by PCR using the yqhDUP2 (SEQ ID NO:40) and yqhDDN2 (SEQ ID NO:41) primers paired with primers internal to the kanR gene (Vec 61; SEQ ID NO:42 and Vec 60; SEQ ID NO:43). To remove the kanamycin marker, integrants were transformed with the temperature-sensitive replicon, pCP20, which contains the gene for the FLP recombinase. FLP recombinase excises the kanamycin marker at the flanking FRT (FLP recognition target) sites. Kanamycin-sensitive cells were then grown at 42° C. to cure pCP20. The resultant strain was RJ8n (yqhD-).

Example 5

Replacement of the E. Coli Phosphoenolpyruvate Carboxylase Chromosomal Promoter with a GI Promoter

[0135]Example 5 describes the replacement in the Escherichia coli genome of the natural ppc (encoding the phosphoenolpyruvate carboxylase or PEP carboxylase) promoter by the short wild-type GI promoter (SEQ ID NO:33).

Design of the Oligonucleotides for the ppc Promoter Replacement

[0136]Two oligonucleotides (ppcF, SEQ ID NO:44 and ppcR, SEQ ID NO:45) were designed to amplify by PCR a cassette containing an 80-bp sequence homologous to the upstream region of the natural ppc promoter, a chloramphenicol-resistance encoding gene (cat) flanked by baker yeast FRT sites, the short wild-type GI promoter sequence (SEQ ID NO:33), and a 40-bp sequence homologous to the downstream region of the +1 transcription start site of the natural ppc promoter.

[0137]The ppcR primer (SEQ ID NO:45) is 100 nucleotides long and includes: the entire sequence from the +1 of P1 (natural ppc promoter) transcription start to 41 bp upstream the ATG of ppc, the short wild-type GI promoter sequence (SEQ ID NO. 33) from 4 bp upstream of the -35 to 9 bp downstream of the -10, and the priming site for pKD3 (Wanner and Datsenko, supra), an R6K plasmid containing the cat gene flanked by two FRT sites. The ppcF primer (SEQ ID NO:44) is 100 nucleotides long and includes 80 bp of sequence upstream of the natural ppc promoter and the priming site for pKD3.

[0138]Primers ppcF and ppcR (SEQ ID NOs:44 and 45) were used to amplify the promoter replacement cassette using plasmid pKD3 as a template. The 1.15-kb PCR product was purified by agarose gel electrophoresis followed by QIAquick gel extraction Kit (Qiagen, Inc., Valencia, Calif.).

Replacement of the Natural Ppc Promoter into Escherichia coli Genome by Homologous Recombination Using Linear DNA

[0139]Competent Escherichia coli MG1655 cells containing pKD46 (Datsenko and Wanner, supra), a Red-recombinase plasmid expressing γ, β, and exo under the control of the arabinose promoter, were electrotransformed with 0.5 μg of the above 1.15-kb linear DNA and the resulting transformants were screened for chloramphenicol resistance (15 μg/mL). The recombinant strains were checked by PCR using primers ppcF and seqppcR (SEQ ID NO:46). Non-specific integration of the cassette gives no PCR products while true recombinants give a 1.25-kb PCR product. The sequence of the short wild-type GI promoter was confirmed by sequencing the 1.25-kb PCR product with the seqppcR primer (SEQ ID NO:46).

Measurement of Enzymatic Activity

[0140]The PEP carboxylase activities in MG1655 and in MG1655 (short wild-type GI-ppc) were measured on ultra-centrifuged cell-free extract using the following assay and are indicated in Table 6. The activity of PPC under control of the short wild-type GI promoter was over three times higher than under control of the natural promoter.

[0141]The decrease at 340 nm (due to consumption of NADH) was measured in a mixture containing: 0.11 M Tris buffer (pH 8.5), NADH (0.22 mM), Magnesium sulfate (11.1 mM), Sodium bicarbonate (11.1 mM), Acetyl-CoA (0.25 mM), MalateDH (Sigma), 50 μL of 6 U cell extracts and 0.03 Phosphoenolpyruvate (1.11 mM). The following formula was used to determine activity:

Units / mg protein = Δ A 340 / min ( test ) - Δ A 340 / min ( blank ) 6.22 × mg protein / mL reaction mixture ##EQU00001##

TABLE-US-00007 TABLE 6 Activity of PPC from natural and GI1.6 promoters Strain Activity (U/mg) MG1655 0.05 MG1655 (1.6GI ppc) 0.164

Example 6

Construction of an Expression Plasmid for Use in Transformation of Escherichia Coli with Genes from the Klebsiella pneumoniae dha Regulon

[0142]Construction of the Expression Vector or pTacIQ:

[0143]The E. coli expression vector pTacIQ was prepared by inserting the lacI^Q gene (Farabaugh, Nature, 274(5673):765-769 (1978)) and tac promoter (Amann et al., Gene 25:167-178 (1983)) into the EcoRI site of pBR322 (Sutcliffe, Cold Spring Harb. Symp. Quant. Biol. 43:77-90 (1979)). A multiple cloning site and terminator sequence (SEQ ID NO:47) replaced the pBR322 sequence from EcoRI to SphI.

Subcloning the Glycerol Dehydratase Genes (dhaB1, 2, 3×):

[0144]The open reading frame for the dhaB3 gene was amplified from pHK28-26 (SEQ ID NO:48) by PCR using primers (SEQ ID NOs:49-50) incorporating an EcoRI site at the 5' end and a XbaI site at the 3' end. The product was subcloned into pLitmus29 (New England Biolabs) to generate the plasmid pDHAB3 containing dhaB3.

[0145]The region (containing the entire coding region for dhaB1, dhaB2, dhaB3, and dhaBX of the dhaB operon from pHK28-26) was cloned into pBluescriptilKS+(Stratagene, La Jolla, Calif.) using the restriction enzymes KpnI and EcoRI to create the plasmid pM7.

[0146]The dhaBX gene was removed by digesting plasmid pM7 with ApaI and XbaI, purifying the 5.9-kb fragment and ligating it with the 325-bp ApaI-XbaI fragment from plasmid pDHAB3 to create pM11 (containing dhaB1, dhaB2, and dhaB3).

[0147]The open reading frame for the dhaB1 gene was amplified from pHK28-26 by PCR using primers (SEQ ID NOs:51-52) incorporating a HindIII site and a consensus ribosome-binding site (RBS) at the 5' end and a XbaI site at the 3' end. The product was subcloned into pLitmus28 (New England Biolabs) to generate the plasmid pDT1 containing dhaB1.

[0148]A NotI-XbaI fragment from pM11 (containing part of the dhaB1 gene, the dhaB2 gene, and the dhaB3 gene) was inserted into pDT1 to create the dhaB expression plasmid, pDT2. The HindIII-XbaI fragment (containing the dhaB(1,2,3) genes from pDT2) was inserted into pTacIQ to create pDT3.

Subcloning the 1,3-propanediol Dehydrogenase Gene (dhaT):

[0149]The KpnI-SacI fragment of pHK28-26 (containing the 1,3-propanediol dehydrogenase (dhaT) gene) was subcloned into pBluescriptil KS+ creating plasmid pAH1. The dhat gene was amplified by PCR using pAH1 as template DNA and the synthetic primers (SEQ ID NOs:53-54) which incorporated an XbaI site at the 5' end and a BamHI site at the 3' end. The product was subcloned into pCR-Script (Stratagene) at the SrfI site to generate the plasmids pAH4 and pAH5 containing dhaT. The plasmid pAH4 contains the dhat gene in the correct orientation for expression from the lac promoter in pCR-Script and pAH5 contains dhat gene in the opposite orientation. The XbaI-BamHI fragment from pAH4 (containing the dhat gene) was inserted into pTacIQ to generate plasmid pAH8. The HindIII-BamHI fragment from pAH8 (containing the RBS and dhat gene) was inserted into pBluescriptilKS+ to create pAH1.

Construction of an Expression Cassette for dhaT and dhaB(1,2,3):

[0150]An expression cassette for dhaT and dhaB(1,2,3) was assembled from the individual dhaB(1,2,3) and dhaT subclones described previously using standard molecular biology methods. A SpeI-SacI fragment (containing the dhaB(1,2,3) genes from pDT3) was inserted into pAH11 at the SpeI-SacI sites to create pAH24. A SalI-XbaI linker created from SEQ ID NOs:55-56 was inserted into pAH5 that was digested with the restriction enzymes SalI-XbaI to create pDT16. The linker destroys the XbaI site. The 1-kb SalI-MluI fragment from pDT16 was then inserted into pAH24 replacing the existing SalI-MluI fragment to create pDT18. pDT21 was constructed by inserting the SalI-NotI fragment from pDT18 and the NotI-XbaI fragment from pM7 into pCL1920 (GenBank AX085428). The glucose isomerase promoter sequence from Streptomyces lividans (SEQ ID NO:1) was cloned by PCR and inserted into EcoRI-HindIII sites of pLitmus28 to construct pDT5. pCL1925 was constructed by inserting the EcoRI-PvuII fragment of pDT5 (containing the GI promoter) into the EcoRI-PvuII site of pCL1920 (GenBank AX085428).

Construction of Expression Vector for Glycerol Dehydratase Under the Control of the Streptomyces Glucose Isomerase Promoter:

[0151]The HindIII restriction fragment (containing dhaT) was deleted from pDT24 to generate pRN105. The pDT24 plasmid was constructed by cloning the HindIII-MluI fragment of pDT21 and the MluI-XbaI fragment of pDT21 into the HindIII-XbaI sites of pCL1925. A PCR product (comprising the 3' region of dhaX, from a unique HpaI restriction site to the end of dhaX, and incorporating the HpaI restriction site at the 5' end and an XbaI restriction site at the 3' end) was generated from pRN105 template and used to replace the existing HpaI/XbaI restriction fragment in pRN105, generating pMP37. A PCR product (comprising the 5' region of dhaB1, from a unique HindIII restriction site just upstream of the start codon to a unique NotI restriction site within dhaB1, and incorporating the HindIII restriction site at the 5' end and the NotI restriction site at the 3' end) was generated from pDT29 template and used to replace the small HindIII/NotI restriction fragment in pRN105, generating pRJ25. The pDT29 had been constructed by inserting the SacI-EcoRI fragment of pHK28-26 into SacI-EcoRI sites of pCL1925. The small HpaI/XbaI restriction fragment (containing the 5' region of dhaX from pMP37) was ligated to the large XbaI/HpaI restriction fragment from pRJ25 to generate pMP38, in which the Streptomyces lividans glucose isomerase promoter (SEQ ID NO:1) drives expression of the K. pneumoniae dhaB1-3,X operon using the native ribosome-binding site.

Example 7

Construction of SYCO Plasmids for Production of 1,3-propanediol

[0152]To produce 1,3-propanediol from glucose in an E. coli host, several operons from different sources can be expressed. These include genes coding for a glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate phosphatase, and glycerol dehydratase activity. These genes may come from sources such as the dha operon from Klebsiella pnuemoniae (containing dhaR, dhaT, dhaX, and dhaB1-3), and the orf operon also from Klebsiella pnuemoniae (containing orf YXW), and an operon containing DAR1 and GPP2 from Saccharomyces. In order to maintain strain stability in fermentation it is preferable to maintain as few plasmids as possible in the E. coli host. To this end a series of plasmids were constructed to enable cloning of at least three different operons on a single plasmid. Three transcriptional terminators were used to flank unique cloning sites in order to prevent read-through of the RNA polymerase. These transcriptional terminators included the tonB terminator, thr attenuator and aspA terminator. The tonB terminator is a bi-directional rho-independent terminator located between the E. Coli tonB gene and an opposing gene (Postle, K. and Good, R. F., Cell, 41:577-585 (1985)). The thr attenuator facilitates transcriptional termination of the E. Coli threonine operon (Lynn et al., J. Mol. Biol., 183:529-541 (1985)). The aspA terminator facilitates transcriptional termination of the E. Coli aspartase operon (Takagi et al., Nucleic Acid Research. 13(6):2063-2072 (1985)).

Construction of pRJ50 Comprising Three Transcriptional Terminators Flanked by Unique Cloning Sites:

[0153]A synthetic DNA fragment (comprising the tonB, thr, and aspA transcriptional terminators (SEQ ID NO:57) and several restriction sites) was assembled using PCR-mediated overlap extension (Horton et al., BioTechniques, 8:528-535, (1990)). Two 100 base oligonucleotides (SEQ ID NOs:58-59) that complement each other for a span of 25 base pairs at the 3' ends were annealed to generate a 175-base DNA fragment (SEQ ID NO:60). Two additional oligonucleotide primers (SEQ ID NOs:61-62) were used to further amplify the 175-base fragment, which is flanked by EcoRI and KpnI restriction sites. The 175-base pair PCR product was digested with EcoRI and KpnI and subcloned into EcoRI/KKpnI digested plasmid pCL1925 to generate pRJ50 (SEQ ID NO:79).

Construction of an Expression Cassette for dhaR, orfY, orfX, orfW and dhaB(1, 2,3,X):

[0154]A derivative of plasmid pDT29 was constructed in which all except the first five and the last five codons (plus stop codon) of the gene dhaT were deleted by a technique known as PCR-mediated overlap extension. Using pDT29 as the template, two primary PCR products were generated using the following primers:

TABLE-US-00008 SEQ ID NO:63 = 5'GAC GCA ACA GTA TTC CGT CGC3'; SEQ ID NO:64 = 5'ATG AGC TAT CGT ATG TTC CGC CAG GCA TTC TGA GTG TTA ACG3'; SEQ ID NO:65 = 5'GCC TGG CGG AAC ATA CGA TAG CTC ATA ATA TAC3'; SEQ ID NO:66 = 5'CGG GGC GCT GGG CCA GTA CTG3'.

[0155]SEQ ID NO:65 was paired with SEQ ID NO:66 to generate a product of 931 bps and encompassing nucleic acid including 5' dhaB1 (to unique ScaI site), all of orfY, and the first five codons of dhaT. SEQ ID NO:63 was paired with SEQ ID NO:64 to generate a product of 1348 bps and encompassing nucleic acid including the last five codons (plus stop codon) of dhaT, all of orfX, all of orfW, and 5' dhaR (to unique SapI site). The 15 bases at the 5' end of SEQ ID NO:64 constitute a tail that is the inverse complement of a 15-base portion of SEQ ID NO:65. Similarly, the 11 bases at the 5' end of SEQ ID NO:65 constitute a tail that is the inverse complement of an 11-base portion of SEQ ID NO:64. Thus, the two primary PCR products were joined together after annealing (via 26-bp tail overlap) and extending by PCR, to generate a third nucleic acid product of 2253 bps. This third PCR product was digested with SapI and ScaI and ligated into pDT29 which was also digested with SapI and ScaI, to generate the plasmid pKP32, which is identical to pDT29, except for the large, in-frame deletion within dhaT.

Construction of Plasmids for Expression of orfWXY and dhaB1-3 Containing Different GI Promoter Variants:

[0156]The orf operon from pKP32 was PCR-amplified (SEQ ID NOs:80-81) with HindIII at the 5' end and AvrII at the 3' end, and subcloned between HindIII and AvrII in pLitmus28 (New England Biolabs) to generate pKP38. The EcoRI/HindIII restriction fragment (containing the GI mutant promoter P1.6 (SEQ ID NO:9) from pMP38/1.6) was subcloned between EcoRI and HindIII in pKP38 to generate pKP39. The AvrII/XbaI restriction fragment (containing the dhaB expression cassette from pMP38/1.6) was subcloned between AvrII and XbaI in pLitmus28 (New England Biolabs) to generate pMP39. The AvrII/XbaI restriction fragment (containing the dhaB expression cassette from pMP39) was subcloned into the AvrII site of pRJ50 to generate pSYCO11. The AvrII restriction fragment (containing the orf expression cassette from pKP39) was subcloned into the NheI site of pSYCO11 to generate pSYCO12. The plasmids pSYCO11 and pSYCO12 are identical except that pSYCO11 does not contain the orf operon.

[0157]The EcoRI/HindIII restriction fragment (containing the GI mutant promoter P1.5 (SEQ ID NO:10) from pMP38/1.5) was subcloned between EcoRI and HindIII in pKP38 to generate pKP40. The AvrII restriction fragment (containing the orf operon driven by P1.5 from pKP40) was subcloned into the NheI site of pSYCO11 to generate pSYCO13. The AvrII/NotI restriction fragment (containing the P1.6 and 5' end of dhaB1 in pSYCO13) was replaced with the corresponding AvrII/NotI restriction fragment from pMP38/1.5 to generate pSYCO19.

Construction of pSYCO101.1pSYCO103. pSYCO106 and pSYCO109 Vectors with Three Operons Each Isolated by Transcriptional Terminators:

[0158]A double-stranded nucleic acid linker (SEQ ID NO:67) was subcloned between the XbaI and SmaI restriction sites in pCL1920 (GenBank AX085428) to generate pCR-pCL1920. The glycerol pathway expression cassette in pAH48 comprising the trc promoter which was derived from pTrc99A (Amersham Pharmacia Biotech, Piscataway, N.J.), the coding sequences for DAR1 and GPP2, of S. cerevisiae, and the terminator rrnBT1T2 (from pTrc99A) was PCR-amplified (SEQ ID NOs:68-69) and subcloned into the SrfI restriction site of pCR-pCL1920 to generate pAH105 (SEQ ID NO:70).

[0159]The PvuII(2)/PvuII(4) restriction fragment (containing the DAR1/GPP2 expression cassette from pAH105) was subcloned into the Bst1107I site of pSYCO12 to generate pSYCO101 (SEQ ID NO:71). The DAR1/GPP2 operon is in the opposite orientation relative to the orf operon and the dhaB operon. The NheI restriction fragment (containing the DAR1/GPP2 expression cassette from pAH105) was subcloned into the XbaI site of pSYCO19 to generate pSYCO103 (SEQ ID NO:72).

[0160]The plasmid pSYCO103 comprises (a) a set of two exogenous genes obtained from Saccharomyces cerevisiae (DAR1 (a gene encoding glycerol-3-phosphate dehydrogenase) and GPP2 (a gene encoding glycerol-3-phosphatase)); (b) a set of three exogenous genes obtained from Klebsiella pneumoniae (dhaB1 (a gene encoding the "α" subunit of glycerol dehydratase), dhaB2 (a gene encoding the "β" subunit of glycerol dehydratase), and dhaB3 (a gene encoding the "γ" subunit of glycerol dehydratase)); and (c) a set of two exogenous genes obtained from Klebsiella pneumoniae (dhaBX (a gene encoding the "α" subunit of dehydratase reactivation factor) and orfX (a gene encoding the "β" subunit of dehydratase reactivation factor)). In pSYCO103 the DAR1/GPP2 operon is in the same orientation relative to the orf operon and the dhaB operon.

[0161]The NheI restriction fragment (containing the DAR1/GPP2 expression cassette from pAH105) was subcloned into the XbaI site of pSYCO12 to generate pSYCO106 (SEQ ID NO:73). The DAR1/GPP2 operon is in the same orientation relative to the orf operon and the dhaB operon. The Pm/I/NotI restriction fragment in pSYCO106 was removed and replaced with the overlapping StuI/NotI restriction fragment from pSYCO106, resulting in a 141 base pairs deletion near the 3' end of orfW to generate pSYCO109 (SEQ ID NO:74).

Example 8

A Novel Nucleotide Sequence with Ten Rare Restriction Enzyme Sites Useful for Cloning

[0162]A novel nucleotide sequence was designed to encode ten rare restriction endonuclease sites useful for cloning of additional genes, operons, or cassettes and as sites for transferring cassettes from this plasmid to another. The plasmid pSCYCO106deltaS was constructed by restricting pSYCO106 with SpeI, filling in the ends with Klenow and religating. The pSYCO106deltaS was digested with EcoRI to isolate the vector backbone and then recircularized by ligation to form pSpREPds. Oligonucleotides (SEQ ID NOs:75-76) were annealed at 60° C. and digested with KpnI/StuI. The multiple cloning fragment (SEQ ID NO:77) contains recognition sites for the following enzymes: NheI, RsrII, SacI, Agel, SnaBI, AscI, PacI, NsiI, MluI, and SapI. The fragments were gel-purified and cloned to pSpREPds to form pSpREPmcs. The pSpREPmcs was linearized with EcoRI and the EcoRI fragments (containing the pathway genes from pSYCO106deltaS and pSYCO109) were ligated to pSpREPmcs to form pSYCO106mcs (SEQ ID NO:78) and pSYCO109mcs (SEQ ID NO: 30), respectively.

Example 9

Production of 1,3-Propanediol Using E. coli Strain RJ8N/pSYCO101

[0163]The plasmid pSYCO101 (SEQ ID NO:71) was used to transform electrocompetent E. coli RJ8n cells, resulting in the E. coli strain, RJ8n/pSYCO101.

[0164]RJ8n/pSYCO101 was pre-cultured for seeding a fermenter in 2YT medium (10 g/L yeast extract, 16 g/L tryptone, and 10 g/L NaCl) containing 50 mg/L spectinomycin. Cultures were started from frozen stocks (10% glycerol as cryoprotectant) in 500 mL of medium in a 2-L Erlenmeyer flask, grown at 35° C. in a shaker at 250 rpm until an OD₅₅₀ of approximately 1.0 was reached and used to seed the fermenter.

[0165]The following components were sterilized together in the fermenter vessel: 45 g KH₂PO₄, 12 g citric acid monohydrate, 12 g MgSO₄.7H₂O, 30 g yeast extract, 1.8 g ferric ammonium citrate, 5 mL Mazu DF204 as antifoam, 1.2 g CaCl₂.2H₂O, 7.2 mL sulfuric acid and 60 mL of a trace element solution. After sterilization, the pH was raised to 6.8 with 20-28% NH₄OH and the following components were added: 0.30 g spectinomycin, and glucose (from a 67 weight % feed). The solution of trace elements contained (g/L): citric acid. H₂O (4.0), MnSO₄.H₂O (3.0), NaCl (1.0), FeSO₄. 7H₂O (0.10), CoCl₂. 6H₂O (0.10), ZnSO₄ 7H₂O (0.10), CuSO₄. 5H₂O (0.010), H₃BO₃ (0.010), and Na₂MoO₄. 2H₂O (0.010). After inoculation, the volume was 6.0 L and the glucose concentration was 10 g/L.

[0166]A 15-L stirred tank fermenter was prepared with the medium described above. The temperature was controlled at 34° C. and aqueous ammonia (20-28 weight %) was used to control pH at 6.8. Dissolved oxygen (DO) control was set at 10% and back pressure was controlled at 0.5 bar. Except for minor excursions, glucose was maintained at between 10 g/L and 25 g/L with a 67% (wt) feed. An addition of 10 mg vitamin B₁₂ was made at 10 h elapsed fermentation time and a co-feed (2.64 mg/h of a 0.0167 mg/mL solution) begun one hour later. A titer of 99 g/L 1,3-propanediol was obtained after 64 h.

Sequence CWU 1

831199DNAartificial sequencepromoter 1gaattcacta gtcgatctgt gctgtttgcc acggtatgca gcaccagcgc gagattatgg 60gctcgcacgc tcgactgtcg gacgggggca ctggaacgag aagtcaggcg agccgtcacg 120cccttgacaa tgccacatcc tgagcaaata attcaaccac taaacaaatc aaccgcgttt 180cccggaggta accaagctt 199236DNAartificial sequenceprimer 2cgggaattcc ctaggcgatc tgtgctgttt gccacg 36384DNAartificial sequenceprimer 3cttaagcttg gttacctccg ggaaacgcgg ttgatttgtt tagtggttga attatttgct 60caggatgtgg catngtcaag ggcg 84484DNAartificial sequenceprimer 4cttaagcttg gttacctccg ggaaacgcgg ttgatttgtt tagtggttga attatttgct 60caggatgtgg cattntcaag ggcg 84584DNAartificial sequenceprimer 5cttaagcttg gttacctccg ggaaacgcgg ttgatttgtt tagtggttga attatttgct 60caggatgtgg cattgncaag ggcg 84684DNAartificial sequenceprimer 6cttaagcttg gttacctccg ggaaacgcgg ttgatttgtt tagtggttga attatttgct 60caggatgtgg cattgtnaag ggcg 84784DNAartificial sequenceprimer 7cttaagcttg gttacctccg ggaaacgcgg ttgatttgtt tagtggttga attatttgct 60caggatgtgg cattgtcnag ggcg 84884DNAartificial sequenceprimer 8cttaagcttg gttacctccg ggaaacgcgg ttgatttgtt tagtggttga attatttgct 60caggatgtgg cattgtcang ggcg 849187DNAartificial sequencepromoter 9cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttg 120acaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18710187DNAartificial sequencepromoter 10cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttg 120actatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18711187DNAartificial sequencepromoter 11cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttg 120acgatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18712187DNAartificial sequencepromoter 12cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttg 120accatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18713186DNAartificial sequencepromoter 13ctaggcgatc tgtgctgttt gccacggtat gcagcaccag cgcgagatta tgggctcgca 60cgctcgactg tcggacgggg gcactggaac gagaagtcag gcgagccgtc acgcccttga 120aaatgccaca tcctgagcaa ataattcaac cactaaacaa atcaaccgcg tttcccggag 180gtaacc 18614162DNAartificial sequencepromoter 14cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttg 120acaatgccac atcctgagca aataattttc ccggaggtaa cc 16215187DNAartificial sequencepromoter 15cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttg 120ccaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18716187DNAartificial sequencepromoter 16cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgccctta 120acaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18717187DNAartificial sequencepromoter 17cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttc 120acaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18718187DNAartificial sequencepromoter 18cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgccctcg 120acaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18719187DNAartificial sequencepromoter 19cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgccctag 120acaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18720187DNAartificial sequencepromoter 20cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgccctgg 120acaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18721187DNAartificial sequencepromoter 21cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccgtg 120acaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18722187DNAartificial sequencepromoter 22cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccatg 120acaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18723187DNAartificial sequencepromoter 23cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccctg 120acaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18724187DNAartificial sequencepromoter 24cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttg 120agaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18725187DNAartificial sequencepromoter 25cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttg 120ataatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18726187DNAartificial sequencepromoter 26cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttg 120gcaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18727187DNAartificial sequencepromoter 27cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttg 120tcaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 18728187DNAartificial sequencepromoter 28cctaggcgat ctgtgctgtt tgccacggta tgcagcacca gcgcgagatt atgggctcgc 60acgctcgact gtcggacggg ggcactggaa cgagaagtca ggcgagccgt cacgcccttt 120acaatgccac atcctgagca aataattcaa ccactaaaca aatcaaccgc gtttcccgga 180ggtaacc 187291164DNAEscherichia coli K-12 29atgaacaact ttaatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140cgtatatacg aagccgcccg ctaa 11643013470DNAartificial sequenceplasmid 30ccttaagtga gtcgtattac ggactggccg tcgttttaca acgtcgtgac tgggaaaacc 60ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 120gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 180gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 240ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 300ccgctgacga gcttagtaaa gccctcgcta gattttaatg cggatgttgc gattacttcg 360ccaactattg cgataacaag aaaaagccag cctttcatga tatatctccc aatttgtgta 420gggcttatta tgcacgctta aaaataataa aagcagactt gacctgatag tttggctgtg 480agcaattatg tgcttagtgc atctaacgct tgagttaagc cgcgccgcga agcggcgtcg 540gcttgaacga attgttagac attatttgcc gactaccttg gtgatctcgc ctttcacgta 600gtggacaaat tcttccaact gatctgcgcg cgaggccaag cgatcttctt cttgtccaag 660ataagcctgt ctagcttcaa gtatgacggg ctgatactgg gccggcaggc gctccattgc 720ccagtcggca gcgacatcct tcggcgcgat tttgccggtt actgcgctgt accaaatgcg 780ggacaacgta agcactacat ttcgctcatc gccagcccag tcgggcggcg agttccatag 840cgttaaggtt tcatttagcg cctcaaatag atcctgttca ggaaccggat caaagagttc 900ctccgccgct ggacctacca aggcaacgct atgttctctt gcttttgtca gcaagatagc 960cagatcaatg tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat tgcgctgcca 1020ttctccaaat tgcagttcgc gcttagctgg ataacgccac ggaatgatgt cgtcgtgcac 1080aacaatggtg acttctacag cgcggagaat ctcgctctct ccaggggaag ccgaagtttc 1140caaaaggtcg ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg tcaccgtaac 1200cagcaaatca atatcactgt gtggcttcag gccgccatcc actgcggagc cgtacaaatg 1260tacggccagc aacgtcggtt cgagatggcg ctcgatgacg ccaactacct ctgatagttg 1320agtcgatact tcggcgatca ccgcttccct catgatgttt aactttgttt tagggcgact 1380gccctgctgc gtaacatcgt tgctgctcca taacatcaaa catcgaccca cggcgtaacg 1440cgcttgctgc ttggatgccc gaggcataga ctgtacccca aaaaaacagt cataacaagc 1500catgaaaacc gccactgcgc cgttaccacc gctgcgttcg gtcaaggttc tggaccagtt 1560gcgtgagcgc atacgctact tgcattacag cttacgaacc gaacaggctt atgtccactg 1620ggttcgtgcc ttcatccgtt tccacggtgt gcgtcacccg gcaaccttgg gcagcagcga 1680agtcgaggca tttctgtcct ggctggcgaa cgagcgcaag gtttcggtct ccacgcatcg 1740tcaggcattg gcggccttgc tgttcttcta cggcaaggtg ctgtgcacgg atctgccctg 1800gcttcaggag atcggaagac ctcggccgtc gcggcgcttg ccggtggtgc tgaccccgga 1860tgaagtggtt cgcatcctcg gttttctgga aggcgagcat cgtttgttcg cccagcttct 1920gtatggaacg ggcatgcgga tcagtgaggg tttgcaactg cgggtcaagg atctggattt 1980cgatcacggc acgatcatcg tgcgggaggg caagggctcc aaggatcggg ccttgatgtt 2040acccgagagc ttggcaccca gcctgcgcga gcaggggaat taattcccac gggttttgct 2100gcccgcaaac gggctgttct ggtgttgcta gtttgttatc agaatcgcag atccggcttc 2160agccggtttg ccggctgaaa gcgctatttc ttccagaatt gccatgattt tttccccacg 2220ggaggcgtca ctggctcccg tgttgtcggc agctttgatt cgataagcag catcgcctgt 2280ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg ttcaatttca 2340tgttctagtt gctttgtttt actggtttca cctgttctat taggtgttac atgctgttca 2400tctgttacat tgtcgatctg ttcatggtga acagctttga atgcaccaaa aactcgtaaa 2460agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatatgga cagttttccc 2520tttgatatgt aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg 2580atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta tgttctctag 2640tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat acttactttg 2700catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgcag ttaaagcatc 2760gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct gatgtaatgg ttgttggtat 2820tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat ccatttgtct 2880atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat caaccaccaa 2940tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc attggttaag 3000ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt catcaaggct 3060aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata accactcata 3120aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat attttatgaa 3180tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt ctaatttttc 3240gcttgagaac ttggcatagt ttgtccactg gaaaatctca aagcctttaa ccaaaggatt 3300cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata caccataagc 3360attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg ataccgtccg 3420ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc ataaaattag 3480cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg ctttgaaaac 3540aactaattca gacatacatc tcaattggtc taggtgattt taatcactat accaattgag 3600atgggctagt caatgataat tactagctag tccttttcct ttgagttgtg ggtatctgta 3660aattctgcta gacctttgct ggaaaacttg taaattctgc tagaccctct gtaaattccg 3720ctagaccttt gtgtgttttt tttgtttata ttcaagtggt tataatttat agaataaaga 3780aagaataaaa aaagataaaa agaatagatc ccagccctgt gtataactca ctactttagt 3840cagttccgca gtattacaaa aggatgtcgc aaacgctgtt tgctcctcta caaaacagac 3900cttaaaaccc taaaggctta agtagcaccc tcgcaagctc gggcaaatcg ctgaatattc 3960cttttgtctc cgaccatcag gcacctgagt cgctgtcttt ttcgtgacat tcagttcgct 4020gcgctcacgg ctctggcagt gaatgggggt aaatggcact acaggcgcct tttatggatt 4080catgcaagga aactacccat aatacaagaa aagcccgtca cgggcttctc agggcgtttt 4140atggcgggtc tgctatgtgg tgctatctga ctttttgctg ttcagcagtt cctgccctct 4200gattttccag tctgaccact tcggattatc ccgtgacagg tcattcagac tggctaatgc 4260acccagtaag gcagcggtat catcaacagg cttacccgtc ttactgtcgg gaattcattt 4320aaatagtcaa aagcctccga ccggaggctt ttgactgcta ggcgatctgt gctgtttgcc 4380acggtatgca gcaccagcgc gagattatgg gctcgcacgc tcgactgtcg gacgggggca 4440ctggaacgag aagtcaggcg agccgtcacg cccttgacaa tgccacatcc tgagcaaata 4500attcaaccac taaacaaatc aaccgcgttt cccggaggta accaagcttg cgggagagaa 4560tgatgaacaa gagccaacaa gttcagacaa tcaccctggc cgccgcccag caaatggcgg 4620cggcggtgga aaaaaaagcc actgagatca acgtggcggt ggtgttttcc gtagttgacc 4680gcggaggcaa cacgctgctt atccagcgga tggacgaggc cttcgtctcc agctgcgata 4740tttccctgaa taaagcctgg agcgcctgca gcctgaagca aggtacccat gaaattacgt 4800cagcggtcca gccaggacaa tctctgtacg gtctgcagct aaccaaccaa cagcgaatta 4860ttatttttgg cggcggcctg ccagttattt ttaatgagca ggtaattggc gccgtcggcg 4920ttagcggcgg tacggtcgag caggatcaat tattagccca gtgcgccctg gattgttttt 4980ccgcattata acctgaagcg agaaggtata ttatgagcta tcgtatgttc cgccaggcat 5040tctgagtgtt aacgagggga ccgtcatgtc gctttcaccg ccaggcgtac gcctgtttta 5100cgatccgcgc gggcaccatg ccggcgccat caatgagctg tgctgggggc tggaggagca 5160gggggtcccc tgccagacca taacctatga cggaggcggt gacgccgctg cgctgggcgc 5220cctggcggcc agaagctcgc ccctgcgggt gggtatcggg ctcagcgcgt ccggcgagat 5280agccctcact catgcccagc tgccggcgga cgcgccgctg gctaccggac acgtcaccga 5340tagcgacgat caactgcgta cgctcggcgc caacgccggg cagctggtta aagtcctgcc 5400gttaagtgag agaaactgaa tgtatcgtat ctatacccgc accggggata aaggcaccac 5460cgccctgtac ggcggcagcc gcatcgagaa agaccatatt cgcgtcgagg cctacggcac 5520cgtcgatgaa ctgatatccc agctgggcgt ctgctacgcc acgacccgcg acgccgggct 5580gcgggaaagc ctgcaccata ttcagcagac gctgttcgtg ctgggggctg aactggccag 5640cgatgcgcgg ggcctgaccc gcctgagcca gacgatcggc gaagaggaga tcaccgccct 5700ggagcggctt atcgaccgca atatggccga gagcggcccg ttaaaacagt tcgtgatccc 5760ggggaggaat ctcgcctctg cccagctgca ccctgatgct tgcgcttgaa ctggcctagc 5820aaacacagaa aaaagcccgc acctgacagt gcgggctttt tttttcctag gcgatctgtg 5880ctgtttgcca cggtatgcag caccagcgcg agattatggg ctcgcacgct cgactgtcgg 5940acgggggcac tggaacgaga agtcaggcga gccgtcacgc ccttgacaat gccacatcct 6000gagcaaataa ttcaaccact aaacaaatca accgcgtttc ccggaggtaa ccaagcttca 6060ccttttgagc cgatgaacaa tgaaaagatc aaaacgattt gcagtactgg cccagcgccc 6120cgtcaatcag gacgggctga ttggcgagtg gcctgaagag gggctgatcg ccatggacag 6180cccctttgac ccggtctctt cagtaaaagt ggacaacggt ctgatcgtcg aactggacgg 6240caaacgccgg gaccagtttg acatgatcga ccgatttatc gccgattacg cgatcaacgt 6300tgagcgcaca gagcaggcaa tgcgcctgga ggcggtggaa atagcccgta tgctggtgga 6360tattcacgtc agccgggagg agatcattgc catcactacc gccatcacgc cggccaaagc 6420ggtcgaggtg atggcgcaga tgaacgtggt ggagatgatg atggcgctgc agaagatgcg 6480tgcccgccgg accccctcca accagtgcca cgtcaccaat ctcaaagata atccggtgca 6540gattgccgct gacgccgccg aggccgggat ccgcggcttc tcagaacagg agaccacggt 6600cggtatcgcg cgctacgcgc cgtttaacgc cctggcgctg ttggtcggtt cgcagtgcgg 6660ccgccccggc gtgttgacgc agtgctcggt ggaagaggcc accgagctgg agctgggcat 6720gcgtggctta accagctacg ccgagacggt gtcggtctac ggcaccgaag cggtatttac 6780cgacggcgat gatacgccgt ggtcaaaggc gttcctcgcc tcggcctacg cctcccgcgg 6840gttgaaaatg cgctacacct ccggcaccgg atccgaagcg ctgatgggct attcggagag 6900caagtcgatg ctctacctcg aatcgcgctg catcttcatt actaaaggcg ccggggttca 6960gggactgcaa aacggcgcgg tgagctgtat cggcatgacc ggcgctgtgc cgtcgggcat 7020tcgggcggtg ctggcggaaa acctgatcgc ctctatgctc gacctcgaag tggcgtccgc 7080caacgaccag actttctccc actcggatat tcgccgcacc gcgcgcaccc tgatgcagat 7140gctgccgggc accgacttta ttttctccgg ctacagcgcg gtgccgaact acgacaacat

7200gttcgccggc tcgaacttcg atgcggaaga ttttgatgat tacaacatcc tgcagcgtga 7260cctgatggtt gacggcggcc tgcgtccggt gaccgaggcg gaaaccattg ccattcgcca 7320gaaagcggcg cgggcgatcc aggcggtttt ccgcgagctg gggctgccgc caatcgccga 7380cgaggaggtg gaggccgcca cctacgcgca cggcagcaac gagatgccgc cgcgtaacgt 7440ggtggaggat ctgagtgcgg tggaagagat gatgaagcgc aacatcaccg gcctcgatat 7500tgtcggcgcg ctgagccgca gcggctttga ggatatcgcc agcaatattc tcaatatgct 7560gcgccagcgg gtcaccggcg attacctgca gacctcggcc attctcgatc ggcagttcga 7620ggtggtgagt gcggtcaacg acatcaatga ctatcagggg ccgggcaccg gctatcgcat 7680ctctgccgaa cgctgggcgg agatcaaaaa tattccgggc gtggttcagc ccgacaccat 7740tgaataaggc ggtattcctg tgcaacagac aacccaaatt cagccctctt ttaccctgaa 7800aacccgcgag ggcggggtag cttctgccga tgaacgcgcc gatgaagtgg tgatcggcgt 7860cggccctgcc ttcgataaac accagcatca cactctgatc gatatgcccc atggcgcgat 7920cctcaaagag ctgattgccg gggtggaaga agaggggctt cacgcccggg tggtgcgcat 7980tctgcgcacg tccgacgtct cctttatggc ctgggatgcg gccaacctga gcggctcggg 8040gatcggcatc ggtatccagt cgaaggggac cacggtcatc catcagcgcg atctgctgcc 8100gctcagcaac ctggagctgt tctcccaggc gccgctgctg acgctggaga cctaccggca 8160gattggcaaa aacgctgcgc gctatgcgcg caaagagtca ccttcgccgg tgccggtggt 8220gaacgatcag atggtgcggc cgaaatttat ggccaaagcc gcgctatttc atatcaaaga 8280gaccaaacat gtggtgcagg acgccgagcc cgtcaccctg cacatcgact tagtaaggga 8340gtgaccatga gcgagaaaac catgcgcgtg caggattatc cgttagccac ccgctgcccg 8400gagcatatcc tgacgcctac cggcaaacca ttgaccgata ttaccctcga gaaggtgctc 8460tctggcgagg tgggcccgca ggatgtgcgg atctcccgcc agacccttga gtaccaggcg 8520cagattgccg agcagatgca gcgccatgcg gtggcgcgca atttccgccg cgcggcggag 8580cttatcgcca ttcctgacga gcgcattctg gctatctata acgcgctgcg cccgttccgc 8640tcctcgcagg cggagctgct ggcgatcgcc gacgagctgg agcacacctg gcatgcgaca 8700gtgaatgccg cctttgtccg ggagtcggcg gaagtgtatc agcagcggca taagctgcgt 8760aaaggaagct aagcggaggt cagcatgccg ttaatagccg ggattgatat cggcaacgcc 8820accaccgagg tggcgctggc gtccgactac ccgcaggcga gggcgtttgt tgccagcggg 8880atcgtcgcga cgacgggcat gaaagggacg cgggacaata tcgccgggac cctcgccgcg 8940ctggagcagg ccctggcgaa aacaccgtgg tcgatgagcg atgtctctcg catctatctt 9000aacgaagccg cgccggtgat tggcgatgtg gcgatggaga ccatcaccga gaccattatc 9060accgaatcga ccatgatcgg tcataacccg cagacgccgg gcggggtggg cgttggcgtg 9120gggacgacta tcgccctcgg gcggctggcg acgctgccgg cggcgcagta tgccgagggg 9180tggatcgtac tgattgacga cgccgtcgat ttccttgacg ccgtgtggtg gctcaatgag 9240gcgctcgacc gggggatcaa cgtggtggcg gcgatcctca aaaaggacga cggcgtgctg 9300gtgaacaacc gcctgcgtaa aaccctgccg gtggtggatg aagtgacgct gctggagcag 9360gtccccgagg gggtaatggc ggcggtggaa gtggccgcgc cgggccaggt ggtgcggatc 9420ctgtcgaatc cctacgggat cgccaccttc ttcgggctaa gcccggaaga gacccaggcc 9480atcgtcccca tcgcccgcgc cctgattggc aaccgttccg cggtggtgct caagaccccg 9540cagggggatg tgcagtcgcg ggtgatcccg gcgggcaacc tctacattag cggcgaaaag 9600cgccgcggag aggccgatgt cgccgagggc gcggaagcca tcatgcaggc gatgagcgcc 9660tgcgctccgg tacgcgacat ccgcggcgaa ccgggcaccc acgccggcgg catgcttgag 9720cgggtgcgca aggtaatggc gtccctgacc ggccatgaga tgagcgcgat atacatccag 9780gatctgctgg cggtggatac gtttattccg cgcaaggtgc agggcgggat ggccggcgag 9840tgcgccatgg agaatgccgt cgggatggcg gcgatggtga aagcggatcg tctgcaaatg 9900caggttatcg cccgcgaact gagcgcccga ctgcagaccg aggtggtggt gggcggcgtg 9960gaggccaaca tggccatcgc cggggcgtta accactcccg gctgtgcggc gccgctggcg 10020atcctcgacc tcggcgccgg ctcgacggat gcggcgatcg tcaacgcgga ggggcagata 10080acggcggtcc atctcgccgg ggcggggaat atggtcagcc tgttgattaa aaccgagctg 10140ggcctcgagg atctttcgct ggcggaagcg ataaaaaaat acccgctggc caaagtggaa 10200agcctgttca gtattcgtca cgagaatggc gcggtggagt tctttcggga agccctcagc 10260ccggcggtgt tcgccaaagt ggtgtacatc aaggagggcg aactggtgcc gatcgataac 10320gccagcccgc tggaaaaaat tcgtctcgtg cgccggcagg cgaaagagaa agtgtttgtc 10380accaactgcc tgcgcgcgct gcgccaggtc tcacccggcg gttccattcg cgatatcgcc 10440tttgtggtgc tggtgggcgg ctcatcgctg gactttgaga tcccgcagct tatcacggaa 10500gccttgtcgc actatggcgt ggtcgccggg cagggcaata ttcggggaac agaagggccg 10560cgcaatgcgg tcgccaccgg gctgctactg gccggtcagg cgaattaaac gggcgctcgc 10620gccagcctct aggtacaaat aaaaaaggca cgtcagatga cgtgcctttt ttcttgtcta 10680gcgtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg 10740tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt 10800ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg acaattaatc 10860atccggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca ggaaacagac 10920catgactagt aaggaggaca attccatggc tgctgctgct gatagattaa acttaacttc 10980cggccacttg aatgctggta gaaagagaag ttcctcttct gtttctttga aggctgccga 11040aaagcctttc aaggttactg tgattggatc tggtaactgg ggtactacta ttgccaaggt 11100ggttgccgaa aattgtaagg gatacccaga agttttcgct ccaatagtac aaatgtgggt 11160gttcgaagaa gagatcaatg gtgaaaaatt gactgaaatc ataaatacta gacatcaaaa 11220cgtgaaatac ttgcctggca tcactctacc cgacaatttg gttgctaatc cagacttgat 11280tgattcagtc aaggatgtcg acatcatcgt tttcaacatt ccacatcaat ttttgccccg 11340tatctgtagc caattgaaag gtcatgttga ttcacacgtc agagctatct cctgtctaaa 11400gggttttgaa gttggtgcta aaggtgtcca attgctatcc tcttacatca ctgaggaact 11460aggtattcaa tgtggtgctc tatctggtgc taacattgcc accgaagtcg ctcaagaaca 11520ctggtctgaa acaacagttg cttaccacat tccaaaggat ttcagaggcg agggcaagga 11580cgtcgaccat aaggttctaa aggccttgtt ccacagacct tacttccacg ttagtgtcat 11640cgaagatgtt gctggtatct ccatctgtgg tgctttgaag aacgttgttg ccttaggttg 11700tggtttcgtc gaaggtctag gctggggtaa caacgcttct gctgccatcc aaagagtcgg 11760tttgggtgag atcatcagat tcggtcaaat gtttttccca gaatctagag aagaaacata 11820ctaccaagag tctgctggtg ttgctgattt gatcaccacc tgcgctggtg gtagaaacgt 11880caaggttgct aggctaatgg ctacttctgg taaggacgcc tgggaatgtg aaaaggagtt 11940gttgaatggc caatccgctc aaggtttaat tacctgcaaa gaagttcacg aatggttgga 12000aacatgtggc tctgtcgaag acttcccatt atttgaagcc gtataccaaa tcgtttacaa 12060caactaccca atgaagaacc tgccggacat gattgaagaa ttagatctac atgaagatta 12120gatttattgg atccaggaaa cagactagaa ttatgggatt gactactaaa cctctatctt 12180tgaaagttaa cgccgctttg ttcgacgtcg acggtaccat tatcatctct caaccagcca 12240ttgctgcatt ctggagggat ttcggtaagg acaaacctta tttcgatgct gaacacgtta 12300tccaagtctc gcatggttgg agaacgtttg atgccattgc taagttcgct ccagactttg 12360ccaatgaaga gtatgttaac aaattagaag ctgaaattcc ggtcaagtac ggtgaaaaat 12420ccattgaagt cccaggtgca gttaagctgt gcaacgcttt gaacgctcta ccaaaagaga 12480aatgggctgt ggcaacttcc ggtacccgtg atatggcaca aaaatggttc gagcatctgg 12540gaatcaggag accaaagtac ttcattaccg ctaatgatgt caaacagggt aagcctcatc 12600cagaaccata tctgaagggc aggaatggct taggatatcc gatcaatgag caagaccctt 12660ccaaatctaa ggtagtagta tttgaagacg ctccagcagg tattgccgcc ggaaaagccg 12720ccggttgtaa gatcattggt attgccacta ctttcgactt ggacttccta aaggaaaaag 12780gctgtgacat cattgtcaaa aaccacgaat ccatcagagt tggcggctac aatgccgaaa 12840cagacgaagt tgaattcatt tttgacgact acttatatgc taaggacgat ctgttgaaat 12900ggtaacccgg gctgcaggca tgcaagcttg gctgttttgg cggatgagag aagattttca 12960gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg 13020gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg 13080ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa 13140cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct 13200ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga 13260gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc 13320ctgacggatg gcctttttgc gtttctacaa actccagctg gatcgggcgc tagagtatac 13380atttaaatgg taccggcgcg ccgctagctt aattaacgga ccgatgcatg agctcacgcg 13440taccggtgct cttcgatcta cgtaagaagg 134703142DNAartificial sequencepromoter 31gcccttgact atgccacatc ctgagcaaat aattcaacca ct 423242DNAartificial sequencepromoter 32gcccttgacg atgccacatc ctgagcaaat aattcaacca ct 423342DNAartificial sequencepromoter 33gcccttgaca atgccacatc ctgagcaaat aattcaacca ct 423481DNAartificial sequenceprimer 34cattcggacc ggcccttgac tatgccacat cctgagcaaa taattcaacc actacagcaa 60agggagcaag taatgaacaa c 813581DNAartificial sequenceprimer 35cattcggacc ggcccttgac gatgccacat cctgagcaaa taattcaacc actacagcaa 60agggagcaag taatgaacaa c 813681DNAartificial sequenceprimer 36cattcggacc ggcccttgac aatgccacat cctgagcaaa taattcaacc actacagcaa 60agggagcaag taatgaacaa c 813782DNAartificial sequenceprimer 37cattgagctc aaaaaaaaag cccgcactgt caggtgcggg cttttttctg tgtttaagct 60tagcgggcgg cttcgtatat ac 823862DNAartificial sequenceprimer 38atgtgcgcgc caacgtcatg tgggcggcga ctcaggcgct gagtgtaggc tggagctgct 60tc 623967DNAartificial sequenceprimer 39cagacgcgtt cagcatattg cagcagctta gcgcgcttgg tatcgcgatt ccggggatcc 60gtcgacc 674025DNAartificial sequenceprimer 40gccagcaagc ggcaaatctc ttcac 254125DNAartificial sequenceprimer 41gaggcgtaaa aagcttagcg ggcgg 254224DNAartificial sequenceprimer 42gcttcctcgt gctttacggt atcg 244323DNAartificial sequenceprimer 43cctgcgtgca atccatcttg ttc 2344100DNAartificial sequenceprimer 44cgatttttta acatttccat aagttacgct tatttaaagc gtcgtgaatt taatgacgta 60aattcctgct atttattcgt gtgtaggctg gagctgcttc 10045100DNAartificial sequenceprimer 45tcgcattggc gcgaatatgc tcgggctttg cttttcgtca gtggttgaat tatttgctca 60ggatgtggca ttgtcaaggg catatgaata tcctccttag 1004630DNAartificial sequenceprimer 46gcggaatatt gttcgttcat attaccccag 304794DNAartificial sequenceterminator 47agcttaggag tctagaatat tgagctcgaa ttcccgggca tgcggtaccg gatccagaaa 60aaagcccgca cctgacagtg cgggcttttt tttt 944812145DNAartificial sequenceplasmid 48gtcgaccacc acggtggtga ctttaatgcc gctctcatgc agcagctcgg tggcggtctc 60aaaattcagg atgtcgccgg tatagttttt gataatcagc aagacgcctt cgccgccgtc 120aatttgcatc gcgcattcaa acattttgtc cggcgtcggc gaggtgaata tttcccccgg 180acaggcgccg gagagcatgc cctggccgat atagccgcag tgcatcggtt catgtccgct 240gccgccgccg gagagcaggg ccaccttgcc agccaccggc gcgtcggtgc gggtcacata 300cagcgggtcc tgatgcaggg tcagctgcgg atgggcttta gccagcccct gtaattgttc 360attcagtaca tcttcaacac ggttaatcag ctttttcatt attcagtgct ccgttggaga 420aggttcgatg ccgcctctct gctggcggag gcggtcatcg cgtaggggta tcgtctgacg 480gtggagcgtg cctggcgata tgatgattct ggctgagcgg acgaaaaaaa gaatgccccg 540acgatcgggt ttcattacga aacattgctt cctgattttg tttctttatg gaacgttttt 600gctgaggata tggtgaaaat gcgagctggc gcgctttttt tcttctgcca taagcggcgg 660tcaggatagc cggcgaagcg ggtgggaaaa aattttttgc tgattttctg ccgactgcgg 720gagaaaaggc ggtcaaacac ggaggattgt aagggcatta tgcggcaaag gagcggatcg 780ggatcgcaat cctgacagag actagggttt tttgttccaa tatggaacgt aaaaaattaa 840cctgtgtttc atatcagaac aaaaaggcga aagatttttt tgttccctgc cggccctaca 900gtgatcgcac tgctccggta cgctccgttc aggccgcgct tcactggccg gcgcggataa 960cgccagggct catcatgtct acatgcgcac ttatttgagg gtgaaaggaa tgctaaaagt 1020tattcaatct ccagccaaat atcttcaggg tcctgatgct gctgttctgt tcggtcaata 1080tgccaaaaac ctggcggaga gcttcttcgt catcgctgac gatttcgtaa tgaagctggc 1140gggagagaaa gtggtgaatg gcctgcagag ccacgatatt cgctgccatg cggaacggtt 1200taacggcgaa tgcagccatg cggaaatcaa ccgtctgatg gcgattttgc aaaaacaggg 1260ctgccgcggc gtggtcggga tcggcggtgg taaaaccctc gataccgcga aggcgatcgg 1320ttactaccag aagctgccgg tggtggtgat cccgaccatc gcctcgaccg atgcgccaac 1380cagcgcgctg tcggtgatct acaccgaagc gggcgagttt gaagagtatc tgatctatcc 1440gaaaaacccg gatatggtgg tgatggacac ggcgattatc gccaaagcgc cggtacgcct 1500gctggtctcc ggcatgggcg atgcgctctc cacctggttc gaggccaaag cttgctacga 1560tgcgcgcgcc accagcatgg ccggaggaca gtccaccgag gcggcgctga gcctcgcccg 1620cctgtgctat gatacgctgc tggcggaggg cgaaaaggcc cgtctggcgg cgcaggccgg 1680ggtagtgacc gaagcgctgg agcgcatcat cgaggcgaac acttacctca gcggcattgg 1740ctttgaaagc agtggcctgg ccgctgccca tgcaatccac aacggtttca ccattcttga 1800agagtgccat cacctgtatc acggtgagaa agtggccttc ggtaccctgg cgcagctggt 1860gctgcagaac agcccgatgg acgagattga aacggtgcag ggcttctgcc agcgcgtcgg 1920cctgccggtg acgctcgcgc agatgggcgt caaagagggg atcgacgaga aaatcgccgc 1980ggtggcgaaa gctacctgcg cggaagggga aaccatccat aatatgccgt ttgcggtgac 2040cccggagagc gtccatgccg ctatcctcac cgccgatctg ttaggccagc agtggctggc 2100gcgttaattc gcggtggcta aaccgctggc ccaggtcagc ggtttttctt tctcccctcc 2160ggcagtcgct gccggagggg ttctctatgg tacaacgcgg aaaaggatat gactgttcag 2220actcaggata ccgggaaggc ggtctcttcc gtcattgccc agtcatggca ccgctgcagc 2280aagtttatgc agcgcgaaac ctggcaaacg ccgcaccagg cccagggcct gaccttcgac 2340tccatctgtc ggcgtaaaac cgcgctgctc accatcggcc aggcggcgct ggaagacgcc 2400tgggagttta tggacggccg cccctgcgcg ctgtttattc ttgatgagtc cgcctgcatc 2460ctgagccgtt gcggcgagcc gcaaaccctg gcccagctgg ctgccctggg atttcgcgac 2520ggcagctatt gtgcggagag cattatcggc acctgcgcgc tgtcgctggc cgcgatgcag 2580ggccagccga tcaacaccgc cggcgatcgg cattttaagc aggcgctaca gccatggagt 2640ttttgctcga cgccggtgtt tgataaccac gggcggctgt tcggctctat ctcgctttgc 2700tgtctggtcg agcaccagtc cagcgccgac ctctccctga cgctggccat cgcccgcgag 2760gtgggtaact ccctgcttac cgacagcctg ctggcggaat ccaaccgtca cctcaatcag 2820atgtacggcc tgctggagag catggacgat ggggtgatgg cgtggaacga acagggcgtg 2880ctgcagtttc tcaatgttca ggcggcgaga ctgctgcatc ttgatgctca ggccagccag 2940gggaaaaata tcgccgatct ggtgaccctc ccggcgctgc tgcgccgcgc catcaaacac 3000gcccgcggcc tgaatcacgt cgaagtcacc tttgaaagtc agcatcagtt tgtcgatgcg 3060gtgatcacct taaaaccgat tgtcgaggcg caaggcaaca gttttattct gctgctgcat 3120ccggtggagc agatgcggca gctgatgacc agccagctcg gtaaagtcag ccacaccttt 3180gagcagatgt ctgccgacga tccggaaacc cgacgcctga tccactttgg ccgccaggcg 3240gcgcgcggcg gcttcccggt gctactgtgc ggcgaagagg gggtcgggaa agagctgctg 3300agccaggcta ttcacaatga aagcgaacgg gcgggcggcc cctacatctc cgtcaactgc 3360cagctatatg ccgacagcgt gctgggccag gactttatgg gcagcgcccc taccgacgat 3420gaaaatggtc gcctgagccg ccttgagctg gccaacggcg gcaccctgtt tctggaaaag 3480atcgagtatc tggcgccgga gctgcagtcg gctctgctgc aggtgattaa gcagggcgtg 3540ctcacccgcc tcgacgcccg gcgcctgatc ccggtggatg tgaaggtgat tgccaccacc 3600accgtcgatc tggccaatct ggtggaacag aaccgcttta gccgccagct gtactatgcg 3660ctgcactcct ttgagatcgt catcccgccg ctgcgcgccc gacgcaacag tattccgtcg 3720ctggtgcata accggttgaa gagcctggag aagcgtttct cttcgcgact gaaagtggac 3780gatgacgcgc tggcacagct ggtggcctac tcgtggccgg ggaatgattt tgagctcaac 3840agcgtcattg agaatatcgc catcagcagc gacaacggcc acattcgcct gagtaatctg 3900ccggaatatc tcttttccga gcggccgggc ggggatagcg cgtcatcgct gctgccggcc 3960agcctgactt ttagcgccat cgaaaaggaa gctattattc acgccgcccg ggtgaccagc 4020gggcgggtgc aggagatgtc gcagctgctc aatatcggcc gcaccaccct gtggcgcaaa 4080atgaagcagt acgatattga cgccagccag ttcaagcgca agcatcaggc ctagtctctt 4140cgattcgcgc catggagaac agggcatccg acaggcgatt gctgtagcgt ttgagcgcgt 4200cgcgcagcgg atgcgcgcgg tccatggccg tcagcaggcg ttcgagccga cgggactggg 4260tgcgcgccac gtgcagctgg gcagaggcga gattcctccc cgggatcacg aactgtttta 4320acgggccgct ctcggccata ttgcggtcga taagccgctc cagggcggtg atctcctctt 4380cgccgatcgt ctggctcagg cgggtcaggc cccgcgcatc gctggccagt tcagccccca 4440gcacgaacag cgtctgctga atatggtgca ggctttcccg cagcccggcg tcgcgggtcg 4500tggcgtagca gacgcccagc tgggatatca gttcatcgac ggtgccgtag gcctcgacgc 4560gaatatggtc tttctcgatg cggctgccgc cgtacagggc ggtggtgcct ttatccccgg 4620tgcgggtata gatacgatac attcagtttc tctcacttaa cggcaggact ttaaccagct 4680gcccggcgtt ggcgccgagc gtacgcagtt gatcgtcgct atcggtgacg tgtccggtag 4740ccagcggcgc gtccgccggc agctgggcat gagtgagggc tatctcgccg gacgcgctga 4800gcccgatacc cacccgcagg ggcgagcttc tggccgccag ggcgcccagc gcagcggcgt 4860caccgcctcc gtcataggtt atggtctggc aggggacccc ctgctcctcc agcccccagc 4920acagctcatt gatggcgccg gcatggtgcc cgcgcggatc gtaaaacagg cgtacgcctg 4980gcggtgaaag cgacatgacg gtcccctcgt taacactcag aatgcctggc ggaaaatcgc 5040ggcaatctcc tgctcgttgc ctttacgcgg gttcgagaac gcattgccgt cttttagagc 5100catctccgcc atgtagggga agtcggcctc ttttaccccc agatcgcgca gatgctgcgg 5160aataccgata tccatcgaca gacgcgtgat agcggcgatg gctttttccg ccgcgtcgag 5220agtggacagt ccggtgatat tttcgcccat cagttcagcg atatcggcga atttctccgg 5280gttggcgatc aggttgtagc gcgccacatg cggcagcagg acagcgttgg ccacgccgtg 5340cggcatgtcg tacaggccgc ccagctggtg cgccatggcg tgcacgtagc cgaggttggc 5400gttattgaaa gccatcccgg ccagcagaga agcataggcc atgttttccc gcgcctgcag 5460attgctgccg agggccacgg cctggcgcag gttgcgggcg atgaggcgga tcgcctgcat 5520ggcggcggcg tccgtcaccg ggttagcgtc tttggagata taggcctcta cggcgtgggt 5580cagggcatcc atcccggtcg ccgcggtcag ggcggccggt ttaccgatca tcagcagtgg 5640atcgttgata gagaccgacg gcagtttgcg ccagctgacg atcacaaact tcactttggt 5700ttcggtgttg gtcaggacgc agtggcgggt gacctcgctg gcggtgccgg cggtggtatt 5760gaccgcgacg ataggcggca gcgggttggt cagggtctcg attccggcat actggtacag 5820atcgccctca tgggtggcgg cgatgccgat gcctttgccg caatcgtgcg ggctgccgcc 5880gcccacggtg acgatgatgt cgcactgttc gcggcgaaac acggcgaggc cgtcgcgcac 5940gttggtgtct ttcgggttcg gctcgacgcc gtcaaagatc gccacctcga tcccggcctc 6000ccgcagataa tgcagggttt tgtccaccgc gccatcttta attgcccgca ggcctttgtc 6060ggtgaccagc agggcttttt tcccccccag cagctggcag cgttcgccga ctacggaaat 6120ggcgttgggg ccaaaaaagt taacgtttgg caccagataa tcaaacatac gatagctcat 6180aatatacctt ctcgcttcag gttataatgc ggaaaaacaa tccagggcgc actgggctaa 6240taattgatcc tgctcgaccg taccgccgct aacgccgacg gcgccaatta cctgctcatt 6300aaaaataact ggcaggccgc cgccaaaaat aataattcgc tgttggttgg ttagctgcag 6360accgtacaga gattgtcctg gctggaccgc tgacgtaatt tcatgggtac cttgcttcag 6420gctgcaggcg ctccaggctt tattcaggga aatatcgcag ctggagacga aggcctcgtc 6480catccgctgg ataagcagcg tgttgcctcc gcggtcaact acggaaaaca ccaccgccac 6540gttgatctca gtggcttttt tttccaccgc cgccgccatt tgctgggcgg cggccagggt 6600gattgtctga acttgttggc tcttgttcat

cattctctcc cgcaccagga taacgctggc 6660gcgaatagtc agtagggggc gatagtaaaa aactattacc attcggttgg cttgctttat 6720ttttgtcagc gttattttgt cgcccgccat gatttagtca atagggttaa aatagcgtcg 6780gaaaaacgta attaagggcg ttttttatta attgatttat atcattgcgg gcgatcacat 6840tttttatttt tgccgccgga gtaaagtttc atagtgaaac tgtcggtaga tttcgtgtgc 6900caaattgaaa cgaaattaaa tttatttttt tcaccactgg ctcatttaaa gttccgctat 6960tgccggtaat ggccgggcgg caacgacgct ggcccggcgt attcgctacc gtctgcggat 7020ttcacctttt gagccgatga acaatgaaaa gatcaaaacg atttgcagta ctggcccagc 7080gccccgtcaa tcaggacggg ctgattggcg agtggcctga agaggggctg atcgccatgg 7140acagcccctt tgacccggtc tcttcagtaa aagtggacaa cggtctgatc gtcgaactgg 7200acggcaaacg ccgggaccag tttgacatga tcgaccgatt tatcgccgat tacgcgatca 7260acgttgagcg cacagagcag gcaatgcgcc tggaggcggt ggaaatagcc cgtatgctgg 7320tggatattca cgtcagccgg gaggagatca ttgccatcac taccgccatc acgccggcca 7380aagcggtcga ggtgatggcg cagatgaacg tggtggagat gatgatggcg ctgcagaaga 7440tgcgtgcccg ccggaccccc tccaaccagt gccacgtcac caatctcaaa gataatccgg 7500tgcagattgc cgctgacgcc gccgaggccg ggatccgcgg cttctcagaa caggagacca 7560cggtcggtat cgcgcgctac gcgccgttta acgccctggc gctgttggtc ggttcgcagt 7620gcggccgccc cggcgtgttg acgcagtgct cggtggaaga ggccaccgag ctggagctgg 7680gcatgcgtgg cttaaccagc tacgccgaga cggtgtcggt ctacggcacc gaagcggtat 7740ttaccgacgg cgatgatacg ccgtggtcaa aggcgttcct cgcctcggcc tacgcctccc 7800gcgggttgaa aatgcgctac acctccggca ccggatccga agcgctgatg ggctattcgg 7860agagcaagtc gatgctctac ctcgaatcgc gctgcatctt cattactaaa ggcgccgggg 7920ttcagggact gcaaaacggc gcggtgagct gtatcggcat gaccggcgct gtgccgtcgg 7980gcattcgggc ggtgctggcg gaaaacctga tcgcctctat gctcgacctc gaagtggcgt 8040ccgccaacga ccagactttc tcccactcgg atattcgccg caccgcgcgc accctgatgc 8100agatgctgcc gggcaccgac tttattttct ccggctacag cgcggtgccg aactacgaca 8160acatgttcgc cggctcgaac ttcgatgcgg aagattttga tgattacaac atcctgcagc 8220gtgacctgat ggttgacggc ggcctgcgtc cggtgaccga ggcggaaacc attgccattc 8280gccagaaagc ggcgcgggcg atccaggcgg ttttccgcga gctggggctg ccgccaatcg 8340ccgacgagga ggtggaggcc gccacctacg cgcacggcag caacgagatg ccgccgcgta 8400acgtggtgga ggatctgagt gcggtggaag agatgatgaa gcgcaacatc accggcctcg 8460atattgtcgg cgcgctgagc cgcagcggct ttgaggatat cgccagcaat attctcaata 8520tgctgcgcca gcgggtcacc ggcgattacc tgcagacctc ggccattctc gatcggcagt 8580tcgaggtggt gagtgcggtc aacgacatca atgactatca ggggccgggc accggctatc 8640gcatctctgc cgaacgctgg gcggagatca aaaatattcc gggcgtggtt cagcccgaca 8700ccattgaata aggcggtatt cctgtgcaac agacaaccca aattcagccc tcttttaccc 8760tgaaaacccg cgagggcggg gtagcttctg ccgatgaacg cgccgatgaa gtggtgatcg 8820gcgtcggccc tgccttcgat aaacaccagc atcacactct gatcgatatg ccccatggcg 8880cgatcctcaa agagctgatt gccggggtgg aagaagaggg gcttcacgcc cgggtggtgc 8940gcattctgcg cacgtccgac gtctccttta tggcctggga tgcggccaac ctgagcggct 9000cggggatcgg catcggtatc cagtcgaagg ggaccacggt catccatcag cgcgatctgc 9060tgccgctcag caacctggag ctgttctccc aggcgccgct gctgacgctg gagacctacc 9120ggcagattgg caaaaacgct gcgcgctatg cgcgcaaaga gtcaccttcg ccggtgccgg 9180tggtgaacga tcagatggtg cggccgaaat ttatggccaa agccgcgcta tttcatatca 9240aagagaccaa acatgtggtg caggacgccg agcccgtcac cctgcacatc gacttagtaa 9300gggagtgacc atgagcgaga aaaccatgcg cgtgcaggat tatccgttag ccacccgctg 9360cccggagcat atcctgacgc ctaccggcaa accattgacc gatattaccc tcgagaaggt 9420gctctctggc gaggtgggcc cgcaggatgt gcggatctcc cgccagaccc ttgagtacca 9480ggcgcagatt gccgagcaga tgcagcgcca tgcggtggcg cgcaatttcc gccgcgcggc 9540ggagcttatc gccattcctg acgagcgcat tctggctatc tataacgcgc tgcgcccgtt 9600ccgctcctcg caggcggagc tgctggcgat cgccgacgag ctggagcaca cctggcatgc 9660gacagtgaat gccgcctttg tccgggagtc ggcggaagtg tatcagcagc ggcataagct 9720gcgtaaagga agctaagcgg aggtcagcat gccgttaata gccgggattg atatcggcaa 9780cgccaccacc gaggtggcgc tggcgtccga ctacccgcag gcgagggcgt ttgttgccag 9840cgggatcgtc gcgacgacgg gcatgaaagg gacgcgggac aatatcgccg ggaccctcgc 9900cgcgctggag caggccctgg cgaaaacacc gtggtcgatg agcgatgtct ctcgcatcta 9960tcttaacgaa gccgcgccgg tgattggcga tgtggcgatg gagaccatca ccgagaccat 10020tatcaccgaa tcgaccatga tcggtcataa cccgcagacg ccgggcgggg tgggcgttgg 10080cgtggggacg actatcgccc tcgggcggct ggcgacgctg ccggcggcgc agtatgccga 10140ggggtggatc gtactgattg acgacgccgt cgatttcctt gacgccgtgt ggtggctcaa 10200tgaggcgctc gaccggggga tcaacgtggt ggcggcgatc ctcaaaaagg acgacggcgt 10260gctggtgaac aaccgcctgc gtaaaaccct gccggtggtg gatgaagtga cgctgctgga 10320gcaggtcccc gagggggtaa tggcggcggt ggaagtggcc gcgccgggcc aggtggtgcg 10380gatcctgtcg aatccctacg ggatcgccac cttcttcggg ctaagcccgg aagagaccca 10440ggccatcgtc cccatcgccc gcgccctgat tggcaaccgt tccgcggtgg tgctcaagac 10500cccgcagggg gatgtgcagt cgcgggtgat cccggcgggc aacctctaca ttagcggcga 10560aaagcgccgc ggagaggccg atgtcgccga gggcgcggaa gccatcatgc aggcgatgag 10620cgcctgcgct ccggtacgcg acatccgcgg cgaaccgggc acccacgccg gcggcatgct 10680tgagcgggtg cgcaaggtaa tggcgtccct gaccggccat gagatgagcg cgatatacat 10740ccaggatctg ctggcggtgg atacgtttat tccgcgcaag gtgcagggcg ggatggccgg 10800cgagtgcgcc atggagaatg ccgtcgggat ggcggcgatg gtgaaagcgg atcgtctgca 10860aatgcaggtt atcgcccgcg aactgagcgc ccgactgcag accgaggtgg tggtgggcgg 10920cgtggaggcc aacatggcca tcgccggggc gttaaccact cccggctgtg cggcgccgct 10980ggcgatcctc gacctcggcg ccggctcgac ggatgcggcg atcgtcaacg cggaggggca 11040gataacggcg gtccatctcg ccggggcggg gaatatggtc agcctgttga ttaaaaccga 11100gctgggcctc gaggatcttt cgctggcgga agcgataaaa aaatacccgc tggccaaagt 11160ggaaagcctg ttcagtattc gtcacgagaa tggcgcggtg gagttctttc gggaagccct 11220cagcccggcg gtgttcgcca aagtggtgta catcaaggag ggcgaactgg tgccgatcga 11280taacgccagc ccgctggaaa aaattcgtct cgtgcgccgg caggcgaaag agaaagtgtt 11340tgtcaccaac tgcctgcgcg cgctgcgcca ggtctcaccc ggcggttcca ttcgcgatat 11400cgcctttgtg gtgctggtgg gcggctcatc gctggacttt gagatcccgc agcttatcac 11460ggaagccttg tcgcactatg gcgtggtcgc cgggcagggc aatattcggg gaacagaagg 11520gccgcgcaat gcggtcgcca ccgggctgct actggccggt caggcgaatt aaacgggcgc 11580tcgcgccagc ctctctcttt aacgtgctat ttcaggatgc cgataatgaa ccagacttct 11640accttaaccg ggcagtgcgt ggccgagttt cttggcaccg gattgctcat tttcttcggc 11700gcgggctgcg tcgctgcgct gcgggtcgcc ggggccagct ttggtcagtg ggagatcagt 11760attatctggg gccttggcgt cgccatggcc atctacctga cggccggtgt ctccggcgcg 11820cacctaaatc cggcggtgac cattgccctg tggctgttcg cctgttttga acgccgcaag 11880gtgctgccgt ttattgttgc ccagacggcc ggggccttct gcgccgccgc gctggtgtat 11940gggctctatc gccagctgtt tctcgatctt gaacagagtc agcatatcgt gcgcggcact 12000gccgccagtc ttaacctggc cggggtcttt tccacgtacc cgcatccaca tatcactttt 12060atacaagcgt ttgccgtgga gaccaccatc acggcaatcc tgatggcgat gatcatggcc 12120ctgaccgacg acggcaacgg aattc 121454937DNAartificial sequenceprimer 49ggaattcaga tctcagcaat gagcgagaaa accatgc 375027DNAartificial sequenceprimer 50gctctagatt agcttccttt acgcagc 275133DNAartificial sequenceprimer 51ggccaagctt aaggaggtta attaaatgaa aag 335226DNAartificial sequenceprimer 52gctctagatt attcaatggt gtcggg 265326DNAartificial sequenceprimer 53gctctagatt attcaatggt gtcggg 265442DNAartificial sequenceprimer 54gcgccgtcta gaattatgag ctatcgtatg tttgattatc tg 425518DNAartificial sequenceprimer 55tcgacgaatt caggagga 185618DNAartificial sequenceprimer 56ctagtcctcc tgaattcg 1857133DNAartificial sequencefragment 57agtcaaaagc ctccgaccgg aggcttttga ctgctagcaa acacagaaaa aagcccgcac 60ctgacagtgc gggctttttt tttcctaggt acaaataaaa aaggcacgtc agatgacgtg 120ccttttttct tgt 13358100DNAartificial sequenceprimer 58ggaattcatt taaatagtca aaagcctccg accggaggct tttgactgct agcaaacaca 60gaaaaaagcc cgcacctgac agtgcgggct ttttttttcc 10059100DNAartificial sequenceprimer 59ggggtaccat ttaaatgtat actctagaca agaaaaaagg cacgtcatct gacgtgcctt 60ttttatttgt acctaggaaa aaaaaagccc gcactgtcag 10060175DNAartificial sequencefragment 60ggaattcatt taaatagtca aaagcctccg accggaggct tttgactgct agcaaacaca 60gaaaaaagcc cgcacctgac agtgcgggct ttttttttcc taggtacaaa taaaaaaggc 120acgtcagatg acgtgccttt tttcttgtct agagtataca tttaaatggt acccc 1756120DNAartificial sequenceprimer 61ggaattcatt taaatagtca 206220DNAartificial sequenceprimer 62ggggtaccat ttaaatgtat 206321DNAartificial sequenceprimer 63gacgcaacag tattccgtcg c 216442DNAartificial sequenceprimer 64atgagctatc gtatgttccg ccaggcattc tgagtgttaa cg 426533DNAartificial sequenceprimer 65gcctggcgga acatacgata gctcataata tac 336621DNAartificial sequenceprimer 66cggggcgctg ggccagtact g 216753DNAartificial sequencelinker 67tctagaggat ccgctagcac tagtagcccg ggcgctagcg cggccgcccc ggg 536837DNAartificial sequenceprimer 68tattcagctg gctagcgtgc accaatgctt ctggcgt 376935DNAartificial sequenceprimer 69gatccagctg gagtttgtag aaacgcaaaa aggcc 35707283DNAartificial sequenceplasmid 70tagtaaagcc ctcgctagat tttaatgcgg atgttgcgat tacttcgcca actattgcga 60taacaagaaa aagccagcct ttcatgatat atctcccaat ttgtgtaggg cttattatgc 120acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc 180ttagtgcatc taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt 240gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 300tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 360gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 420acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 480actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 540tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 600cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 660atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 720agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 780tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 840atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 900tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 960gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 1020gcgatcaccg cttccctcat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 1080acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 1140gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 1200actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata 1260cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc 1320atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt cgaggcattt 1380ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg 1440gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgccctggct tcaggagatc 1500ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc 1560atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc agcttctgta tggaacgggc 1620atgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 1680atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 1740gcacccagcc tgcgcgagca ggggaattaa ttcccacggg ttttgctgcc cgcaaacggg 1800ctgttctggt gttgctagtt tgttatcaga atcgcagatc cggcttcagc cggtttgccg 1860gctgaaagcg ctatttcttc cagaattgcc atgatttttt ccccacggga ggcgtcactg 1920gctcccgtgt tgtcggcagc tttgattcga taagcagcat cgcctgtttc aggctgtcta 1980tgtgtgactg ttgagctgta acaagttgtc tcaggtgttc aatttcatgt tctagttgct 2040ttgttttact ggtttcacct gttctattag gtgttacatg ctgttcatct gttacattgt 2100cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat 2160ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac 2220ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag 2280ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 2340ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2400aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2460cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2520atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2580tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2640taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca 2700tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2760gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2820tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2880aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2940gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 3000gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 3060atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 3120gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 3180tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 3240atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 3300tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3360gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3420ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3480aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac 3540aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc 3600ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat 3660caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc 3720agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc 3780cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg 3840tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc 3900acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg 3960tatcatcaac aggcttaccc gtcttactgt cgggaattcg cgttggccga ttcattaatg 4020cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt 4080gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt 4140gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc 4200caagcttgca tgcctgcagg tcgactctag aggatccgct agcactagta gccctattca 4260gctggctagc gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc 4320tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat 4380aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctgttgac 4440aattaatcat ccggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg 4500aaacagacca tgactagtaa ggaggacaat tccatggctg ctgctgctga tagattaaac 4560ttaacttccg gccacttgaa tgctggtaga aagagaagtt cctcttctgt ttctttgaag 4620gctgccgaaa agcctttcaa ggttactgtg attggatctg gtaactgggg tactactatt 4680gccaaggtgg ttgccgaaaa ttgtaaggga tacccagaag ttttcgctcc aatagtacaa 4740atgtgggtgt tcgaagaaga gatcaatggt gaaaaattga ctgaaatcat aaatactaga 4800catcaaaacg tgaaatactt gcctggcatc actctacccg acaatttggt tgctaatcca 4860gacttgattg attcagtcaa ggatgtcgac atcatcgttt tcaacattcc acatcaattt 4920ttgccccgta tctgtagcca attgaaaggt catgttgatt cacacgtcag agctatctcc 4980tgtctaaagg gttttgaagt tggtgctaaa ggtgtccaat tgctatcctc ttacatcact 5040gaggaactag gtattcaatg tggtgctcta tctggtgcta acattgccac cgaagtcgct 5100caagaacact ggtctgaaac aacagttgct taccacattc caaaggattt cagaggcgag 5160ggcaaggacg tcgaccataa ggttctaaag gccttgttcc acagacctta cttccacgtt 5220agtgtcatcg aagatgttgc tggtatctcc atctgtggtg ctttgaagaa cgttgttgcc 5280ttaggttgtg gtttcgtcga aggtctaggc tggggtaaca acgcttctgc tgccatccaa 5340agagtcggtt tgggtgagat catcagattc ggtcaaatgt ttttcccaga atctagagaa 5400gaaacatact accaagagtc tgctggtgtt gctgatttga tcaccacctg cgctggtggt 5460agaaacgtca aggttgctag gctaatggct acttctggta aggacgcctg ggaatgtgaa 5520aaggagttgt tgaatggcca atccgctcaa ggtttaatta cctgcaaaga agttcacgaa 5580tggttggaaa catgtggctc tgtcgaagac ttcccattat ttgaagccgt ataccaaatc 5640gtttacaaca actacccaat gaagaacctg ccggacatga ttgaagaatt agatctacat 5700gaagattaga tttattggat ccaggaaaca gactagaatt atgggattga ctactaaacc 5760tctatctttg aaagttaacg ccgctttgtt cgacgtcgac ggtaccatta tcatctctca 5820accagccatt gctgcattct ggagggattt cggtaaggac aaaccttatt tcgatgctga 5880acacgttatc caagtctcgc atggttggag aacgtttgat gccattgcta agttcgctcc 5940agactttgcc aatgaagagt atgttaacaa attagaagct gaaattccgg tcaagtacgg 6000tgaaaaatcc attgaagtcc caggtgcagt taagctgtgc aacgctttga acgctctacc 6060aaaagagaaa tgggctgtgg caacttccgg tacccgtgat atggcacaaa aatggttcga 6120gcatctggga atcaggagac caaagtactt cattaccgct aatgatgtca aacagggtaa 6180gcctcatcca gaaccatatc tgaagggcag gaatggctta ggatatccga tcaatgagca 6240agacccttcc aaatctaagg tagtagtatt tgaagacgct ccagcaggta ttgccgccgg 6300aaaagccgcc ggttgtaaga tcattggtat tgccactact ttcgacttgg acttcctaaa 6360ggaaaaaggc tgtgacatca ttgtcaaaaa ccacgaatcc atcagagttg gcggctacaa 6420tgccgaaaca gacgaagttg aattcatttt tgacgactac ttatatgcta aggacgatct 6480gttgaaatgg taacccgggc tgcaggcatg caagcttggc tgttttggcg gatgagagaa 6540gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt 6600gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg 6660ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc 6720aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg 6780tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac 6840ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga 6900aggccatcct gacggatggc ctttttgcgt ttctacaaac tccagctgga tcgggcgcta 6960gcgcggccgc cccgggtacc gagctcgaat tcactggccg tcgttttaca acgtcgtgac 7020tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 7080tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 7140ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 7200atatggtgca ctctcagtac aatctgctct

gatgccgcat agttaagcca gccccgacac 7260ccgccaacac ccgctgacga gct 72837113669DNAartificial sequenceplasmid 71tagtaaagcc ctcgctagat tttaatgcgg atgttgcgat tacttcgcca actattgcga 60taacaagaaa aagccagcct ttcatgatat atctcccaat ttgtgtaggg cttattatgc 120acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc 180ttagtgcatc taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt 240gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 300tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 360gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 420acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 480actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 540tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 600cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 660atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 720agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 780tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 840atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 900tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 960gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 1020gcgatcaccg cttccctcat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 1080acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 1140gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 1200actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata 1260cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc 1320atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt cgaggcattt 1380ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg 1440gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgccctggct tcaggagatc 1500ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc 1560atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc agcttctgta tggaacgggc 1620atgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 1680atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 1740gcacccagcc tgcgcgagca ggggaattaa ttcccacggg ttttgctgcc cgcaaacggg 1800ctgttctggt gttgctagtt tgttatcaga atcgcagatc cggcttcagc cggtttgccg 1860gctgaaagcg ctatttcttc cagaattgcc atgatttttt ccccacggga ggcgtcactg 1920gctcccgtgt tgtcggcagc tttgattcga taagcagcat cgcctgtttc aggctgtcta 1980tgtgtgactg ttgagctgta acaagttgtc tcaggtgttc aatttcatgt tctagttgct 2040ttgttttact ggtttcacct gttctattag gtgttacatg ctgttcatct gttacattgt 2100cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat 2160ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac 2220ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag 2280ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 2340ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2400aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2460cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2520atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2580tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2640taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca 2700tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2760gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2820tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2880aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2940gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 3000gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 3060atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 3120gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 3180tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 3240atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 3300tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3360gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3420ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3480aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac 3540aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc 3600ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat 3660caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc 3720agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc 3780cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg 3840tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc 3900acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg 3960tatcatcaac aggcttaccc gtcttactgt cgggaattca tttaaatagt caaaagcctc 4020cgaccggagg cttttgactg ctaggcgatc tgtgctgttt gccacggtat gcagcaccag 4080cgcgagatta tgggctcgca cgctcgactg tcggacgggg gcactggaac gagaagtcag 4140gcgagccgtc acgcccttga caatgccaca tcctgagcaa ataattcaac cactaaacaa 4200atcaaccgcg tttcccggag gtaaccaagc ttgcgggaga gaatgatgaa caagagccaa 4260caagttcaga caatcaccct ggccgccgcc cagcaaatgg cggcggcggt ggaaaaaaaa 4320gccactgaga tcaacgtggc ggtggtgttt tccgtagttg accgcggagg caacacgctg 4380cttatccagc ggatggacga ggccttcgtc tccagctgcg atatttccct gaataaagcc 4440tggagcgcct gcagcctgaa gcaaggtacc catgaaatta cgtcagcggt ccagccagga 4500caatctctgt acggtctgca gctaaccaac caacagcgaa ttattatttt tggcggcggc 4560ctgccagtta tttttaatga gcaggtaatt ggcgccgtcg gcgttagcgg cggtacggtc 4620gagcaggatc aattattagc ccagtgcgcc ctggattgtt tttccgcatt ataacctgaa 4680gcgagaaggt atattatgag ctatcgtatg ttccgccagg cattctgagt gttaacgagg 4740ggaccgtcat gtcgctttca ccgccaggcg tacgcctgtt ttacgatccg cgcgggcacc 4800atgccggcgc catcaatgag ctgtgctggg ggctggagga gcagggggtc ccctgccaga 4860ccataaccta tgacggaggc ggtgacgccg ctgcgctggg cgccctggcg gccagaagct 4920cgcccctgcg ggtgggtatc gggctcagcg cgtccggcga gatagccctc actcatgccc 4980agctgccggc ggacgcgccg ctggctaccg gacacgtcac cgatagcgac gatcaactgc 5040gtacgctcgg cgccaacgcc gggcagctgg ttaaagtcct gccgttaagt gagagaaact 5100gaatgtatcg tatctatacc cgcaccgggg ataaaggcac caccgccctg tacggcggca 5160gccgcatcga gaaagaccat attcgcgtcg aggcctacgg caccgtcgat gaactgatat 5220cccagctggg cgtctgctac gccacgaccc gcgacgccgg gctgcgggaa agcctgcacc 5280atattcagca gacgctgttc gtgctggggg ctgaactggc cagcgatgcg cggggcctga 5340cccgcctgag ccagacgatc ggcgaagagg agatcaccgc cctggagcgg cttatcgacc 5400gcaatatggc cgagagcggc ccgttaaaac agttcgtgat cccggggagg aatctcgcct 5460ctgcccagct gcacgtggcg cgcacccagt cccgtcggct cgaacgcctg ctgacggcca 5520tggaccgcgc gcatccgctg cgcgacgcgc tcaaacgcta cagcaatcgc ctgtcggatg 5580ccctgttctc catggcgcga atcgaagaga ctaggcctga tgcttgcgct tgaactggcc 5640tagcaaacac agaaaaaagc ccgcacctga cagtgcgggc tttttttttc ctaggcgatc 5700tgtgctgttt gccacggtat gcagcaccag cgcgagatta tgggctcgca cgctcgactg 5760tcggacgggg gcactggaac gagaagtcag gcgagccgtc acgcccttga caatgccaca 5820tcctgagcaa ataattcaac cactaaacaa atcaaccgcg tttcccggag gtaaccaagc 5880ttcacctttt gagccgatga acaatgaaaa gatcaaaacg atttgcagta ctggcccagc 5940gccccgtcaa tcaggacggg ctgattggcg agtggcctga agaggggctg atcgccatgg 6000acagcccctt tgacccggtc tcttcagtaa aagtggacaa cggtctgatc gtcgaactgg 6060acggcaaacg ccgggaccag tttgacatga tcgaccgatt tatcgccgat tacgcgatca 6120acgttgagcg cacagagcag gcaatgcgcc tggaggcggt ggaaatagcc cgtatgctgg 6180tggatattca cgtcagccgg gaggagatca ttgccatcac taccgccatc acgccggcca 6240aagcggtcga ggtgatggcg cagatgaacg tggtggagat gatgatggcg ctgcagaaga 6300tgcgtgcccg ccggaccccc tccaaccagt gccacgtcac caatctcaaa gataatccgg 6360tgcagattgc cgctgacgcc gccgaggccg ggatccgcgg cttctcagaa caggagacca 6420cggtcggtat cgcgcgctac gcgccgttta acgccctggc gctgttggtc ggttcgcagt 6480gcggccgccc cggcgtgttg acgcagtgct cggtggaaga ggccaccgag ctggagctgg 6540gcatgcgtgg cttaaccagc tacgccgaga cggtgtcggt ctacggcacc gaagcggtat 6600ttaccgacgg cgatgatacg ccgtggtcaa aggcgttcct cgcctcggcc tacgcctccc 6660gcgggttgaa aatgcgctac acctccggca ccggatccga agcgctgatg ggctattcgg 6720agagcaagtc gatgctctac ctcgaatcgc gctgcatctt cattactaaa ggcgccgggg 6780ttcagggact gcaaaacggc gcggtgagct gtatcggcat gaccggcgct gtgccgtcgg 6840gcattcgggc ggtgctggcg gaaaacctga tcgcctctat gctcgacctc gaagtggcgt 6900ccgccaacga ccagactttc tcccactcgg atattcgccg caccgcgcgc accctgatgc 6960agatgctgcc gggcaccgac tttattttct ccggctacag cgcggtgccg aactacgaca 7020acatgttcgc cggctcgaac ttcgatgcgg aagattttga tgattacaac atcctgcagc 7080gtgacctgat ggttgacggc ggcctgcgtc cggtgaccga ggcggaaacc attgccattc 7140gccagaaagc ggcgcgggcg atccaggcgg ttttccgcga gctggggctg ccgccaatcg 7200ccgacgagga ggtggaggcc gccacctacg cgcacggcag caacgagatg ccgccgcgta 7260acgtggtgga ggatctgagt gcggtggaag agatgatgaa gcgcaacatc accggcctcg 7320atattgtcgg cgcgctgagc cgcagcggct ttgaggatat cgccagcaat attctcaata 7380tgctgcgcca gcgggtcacc ggcgattacc tgcagacctc ggccattctc gatcggcagt 7440tcgaggtggt gagtgcggtc aacgacatca atgactatca ggggccgggc accggctatc 7500gcatctctgc cgaacgctgg gcggagatca aaaatattcc gggcgtggtt cagcccgaca 7560ccattgaata aggcggtatt cctgtgcaac agacaaccca aattcagccc tcttttaccc 7620tgaaaacccg cgagggcggg gtagcttctg ccgatgaacg cgccgatgaa gtggtgatcg 7680gcgtcggccc tgccttcgat aaacaccagc atcacactct gatcgatatg ccccatggcg 7740cgatcctcaa agagctgatt gccggggtgg aagaagaggg gcttcacgcc cgggtggtgc 7800gcattctgcg cacgtccgac gtctccttta tggcctggga tgcggccaac ctgagcggct 7860cggggatcgg catcggtatc cagtcgaagg ggaccacggt catccatcag cgcgatctgc 7920tgccgctcag caacctggag ctgttctccc aggcgccgct gctgacgctg gagacctacc 7980ggcagattgg caaaaacgct gcgcgctatg cgcgcaaaga gtcaccttcg ccggtgccgg 8040tggtgaacga tcagatggtg cggccgaaat ttatggccaa agccgcgcta tttcatatca 8100aagagaccaa acatgtggtg caggacgccg agcccgtcac cctgcacatc gacttagtaa 8160gggagtgacc atgagcgaga aaaccatgcg cgtgcaggat tatccgttag ccacccgctg 8220cccggagcat atcctgacgc ctaccggcaa accattgacc gatattaccc tcgagaaggt 8280gctctctggc gaggtgggcc cgcaggatgt gcggatctcc cgccagaccc ttgagtacca 8340ggcgcagatt gccgagcaga tgcagcgcca tgcggtggcg cgcaatttcc gccgcgcggc 8400ggagcttatc gccattcctg acgagcgcat tctggctatc tataacgcgc tgcgcccgtt 8460ccgctcctcg caggcggagc tgctggcgat cgccgacgag ctggagcaca cctggcatgc 8520gacagtgaat gccgcctttg tccgggagtc ggcggaagtg tatcagcagc ggcataagct 8580gcgtaaagga agctaagcgg aggtcagcat gccgttaata gccgggattg atatcggcaa 8640cgccaccacc gaggtggcgc tggcgtccga ctacccgcag gcgagggcgt ttgttgccag 8700cgggatcgtc gcgacgacgg gcatgaaagg gacgcgggac aatatcgccg ggaccctcgc 8760cgcgctggag caggccctgg cgaaaacacc gtggtcgatg agcgatgtct ctcgcatcta 8820tcttaacgaa gccgcgccgg tgattggcga tgtggcgatg gagaccatca ccgagaccat 8880tatcaccgaa tcgaccatga tcggtcataa cccgcagacg ccgggcgggg tgggcgttgg 8940cgtggggacg actatcgccc tcgggcggct ggcgacgctg ccggcggcgc agtatgccga 9000ggggtggatc gtactgattg acgacgccgt cgatttcctt gacgccgtgt ggtggctcaa 9060tgaggcgctc gaccggggga tcaacgtggt ggcggcgatc ctcaaaaagg acgacggcgt 9120gctggtgaac aaccgcctgc gtaaaaccct gccggtggtg gatgaagtga cgctgctgga 9180gcaggtcccc gagggggtaa tggcggcggt ggaagtggcc gcgccgggcc aggtggtgcg 9240gatcctgtcg aatccctacg ggatcgccac cttcttcggg ctaagcccgg aagagaccca 9300ggccatcgtc cccatcgccc gcgccctgat tggcaaccgt tccgcggtgg tgctcaagac 9360cccgcagggg gatgtgcagt cgcgggtgat cccggcgggc aacctctaca ttagcggcga 9420aaagcgccgc ggagaggccg atgtcgccga gggcgcggaa gccatcatgc aggcgatgag 9480cgcctgcgct ccggtacgcg acatccgcgg cgaaccgggc acccacgccg gcggcatgct 9540tgagcgggtg cgcaaggtaa tggcgtccct gaccggccat gagatgagcg cgatatacat 9600ccaggatctg ctggcggtgg atacgtttat tccgcgcaag gtgcagggcg ggatggccgg 9660cgagtgcgcc atggagaatg ccgtcgggat ggcggcgatg gtgaaagcgg atcgtctgca 9720aatgcaggtt atcgcccgcg aactgagcgc ccgactgcag accgaggtgg tggtgggcgg 9780cgtggaggcc aacatggcca tcgccggggc gttaaccact cccggctgtg cggcgccgct 9840ggcgatcctc gacctcggcg ccggctcgac ggatgcggcg atcgtcaacg cggaggggca 9900gataacggcg gtccatctcg ccggggcggg gaatatggtc agcctgttga ttaaaaccga 9960gctgggcctc gaggatcttt cgctggcgga agcgataaaa aaatacccgc tggccaaagt 10020ggaaagcctg ttcagtattc gtcacgagaa tggcgcggtg gagttctttc gggaagccct 10080cagcccggcg gtgttcgcca aagtggtgta catcaaggag ggcgaactgg tgccgatcga 10140taacgccagc ccgctggaaa aaattcgtct cgtgcgccgg caggcgaaag agaaagtgtt 10200tgtcaccaac tgcctgcgcg cgctgcgcca ggtctcaccc ggcggttcca ttcgcgatat 10260cgcctttgtg gtgctggtgg gcggctcatc gctggacttt gagatcccgc agcttatcac 10320ggaagccttg tcgcactatg gcgtggtcgc cgggcagggc aatattcggg gaacagaagg 10380gccgcgcaat gcggtcgcca ccgggctgct actggccggt caggcgaatt aaacgggcgc 10440tcgcgccagc ctctaggtac aaataaaaaa ggcacgtcag atgacgtgcc ttttttcttg 10500tctagagtac tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg 10560ttttcccagt cacgacgttg taaaacgacg gccagtgaat tcgagctcgg tacccggggc 10620ggccgcgcta gcgcccgatc cagctggagt ttgtagaaac gcaaaaaggc catccgtcag 10680gatggccttc tgcttaattt gatgcctggc agtttatggc gggcgtcctg cccgccaccc 10740tccgggccgt tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag 10800agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg 10860ttttatttga tgcctggcag ttccctactc tcgcatgggg agaccccaca ctaccatcgg 10920cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc gcgctactgc 10980cgccaggcaa attctgtttt atcagaccgc ttctgcgttc tgatttaatc tgtatcaggc 11040tgaaaatctt ctctcatccg ccaaaacagc caagcttgca tgcctgcagc ccgggttacc 11100atttcaacag atcgtcctta gcatataagt agtcgtcaaa aatgaattca acttcgtctg 11160tttcggcatt gtagccgcca actctgatgg attcgtggtt tttgacaatg atgtcacagc 11220ctttttcctt taggaagtcc aagtcgaaag tagtggcaat accaatgatc ttacaaccgg 11280cggcttttcc ggcggcaata cctgctggag cgtcttcaaa tactactacc ttagatttgg 11340aagggtcttg ctcattgatc ggatatccta agccattcct gcccttcaga tatggttctg 11400gatgaggctt accctgtttg acatcattag cggtaatgaa gtactttggt ctcctgattc 11460ccagatgctc gaaccatttt tgtgccatat cacgggtacc ggaagttgcc acagcccatt 11520tctcttttgg tagagcgttc aaagcgttgc acagcttaac tgcacctggg acttcaatgg 11580atttttcacc gtacttgacc ggaatttcag cttctaattt gttaacatac tcttcattgg 11640caaagtctgg agcgaactta gcaatggcat caaacgttct ccaaccatgc gagacttgga 11700taacgtgttc agcatcgaaa taaggtttgt ccttaccgaa atccctccag aatgcagcaa 11760tggctggttg agagatgata atggtaccgt cgacgtcgaa caaagcggcg ttaactttca 11820aagatagagg tttagtagtc aatcccataa ttctagtctg tttcctggat ccaataaatc 11880taatcttcat gtagatctaa ttcttcaatc atgtccggca ggttcttcat tgggtagttg 11940ttgtaaacga tttggtatac ggcttcaaat aatgggaagt cttcgacaga gccacatgtt 12000tccaaccatt cgtgaacttc tttgcaggta attaaacctt gagcggattg gccattcaac 12060aactcctttt cacattccca ggcgtcctta ccagaagtag ccattagcct agcaaccttg 12120acgtttctac caccagcgca ggtggtgatc aaatcagcaa caccagcaga ctcttggtag 12180tatgtttctt ctctagattc tgggaaaaac atttgaccga atctgatgat ctcacccaaa 12240ccgactcttt ggatggcagc agaagcgttg ttaccccagc ctagaccttc gacgaaacca 12300caacctaagg caacaacgtt cttcaaagca ccacagatgg agataccagc aacatcttcg 12360atgacactaa cgtggaagta aggtctgtgg aacaaggcct ttagaacctt atggtcgacg 12420tccttgccct cgcctctgaa atcctttgga atgtggtaag caactgttgt ttcagaccag 12480tgttcttgag cgacttcggt ggcaatgtta gcaccagata gagcaccaca ttgaatacct 12540agttcctcag tgatgtaaga ggatagcaat tggacacctt tagcaccaac ttcaaaaccc 12600tttagacagg agatagctct gacgtgtgaa tcaacatgac ctttcaattg gctacagata 12660cggggcaaaa attgatgtgg aatgttgaaa acgatgatgt cgacatcctt gactgaatca 12720atcaagtctg gattagcaac caaattgtcg ggtagagtga tgccaggcaa gtatttcacg 12780ttttgatgtc tagtatttat gatttcagtc aatttttcac cattgatctc ttcttcgaac 12840acccacattt gtactattgg agcgaaaact tctgggtatc ccttacaatt ttcggcaacc 12900accttggcaa tagtagtacc ccagttacca gatccaatca cagtaacctt gaaaggcttt 12960tcggcagcct tcaaagaaac agaagaggaa cttctctttc taccagcatt caagtggccg 13020gaagttaagt ttaatctatc agcagcagca gccatggaat tgtcctcctt actagtcatg 13080gtctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacattata cgagccggat 13140gattaattgt caacagctca tttcagaata tttgccagaa ccgttatgat gtcggcgcaa 13200aaaacattat ccagaacggg agtgcgcctt gagcgacacg aattatgcag tgatttacga 13260cctgcacagc cataccacag cttccgatgg ctgcctgacg ccagaagcat tggtgcacgc 13320tagccagtac atttaaatgg taccctctag tcaaggcctt aagtgagtcg tattacggac 13380tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc 13440ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc 13500cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta 13560cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg 13620ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgagct 136697213543DNAartificial sequenceplasmid 72tagtaaagcc ctcgctagat tttaatgcgg atgttgcgat tacttcgcca actattgcga 60taacaagaaa aagccagcct ttcatgatat atctcccaat ttgtgtaggg cttattatgc 120acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc 180ttagtgcatc taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt 240gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 300tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 360gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 420acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 480actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 540tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 600cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 660atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 720agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 780tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 840atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 900tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 960gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 1020gcgatcaccg cttccctcat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 1080acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 1140gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 1200actgcgccgt taccaccgct

gcgttcggtc aaggttctgg accagttgcg tgagcgcata 1260cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc 1320atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt cgaggcattt 1380ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg 1440gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgccctggct tcaggagatc 1500ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc 1560atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc agcttctgta tggaacgggc 1620atgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 1680atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 1740gcacccagcc tgcgcgagca ggggaattaa ttcccacggg ttttgctgcc cgcaaacggg 1800ctgttctggt gttgctagtt tgttatcaga atcgcagatc cggcttcagc cggtttgccg 1860gctgaaagcg ctatttcttc cagaattgcc atgatttttt ccccacggga ggcgtcactg 1920gctcccgtgt tgtcggcagc tttgattcga taagcagcat cgcctgtttc aggctgtcta 1980tgtgtgactg ttgagctgta acaagttgtc tcaggtgttc aatttcatgt tctagttgct 2040ttgttttact ggtttcacct gttctattag gtgttacatg ctgttcatct gttacattgt 2100cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat 2160ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac 2220ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag 2280ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 2340ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2400aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2460cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2520atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2580tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2640taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca 2700tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2760gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2820tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2880aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2940gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 3000gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 3060atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 3120gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 3180tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 3240atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 3300tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3360gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3420ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3480aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac 3540aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc 3600ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat 3660caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc 3720agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc 3780cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg 3840tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc 3900acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg 3960tatcatcaac aggcttaccc gtcttactgt cgggaattca tttaaatagt caaaagcctc 4020cgaccggagg cttttgactg ctaggcgatc tgtgctgttt gccacggtat gcagcaccag 4080cgcgagatta tgggctcgca cgctcgactg tcggacgggg gcactggaac gagaagtcag 4140gcgagccgtc acgcccttga ctatgccaca tcctgagcaa ataattcaac cactaaacaa 4200atcaaccgcg tttcccggag gtaaccaagc ttgcgggaga gaatgatgaa caagagccaa 4260caagttcaga caatcaccct ggccgccgcc cagcaaatgg cggcggcggt ggaaaaaaaa 4320gccactgaga tcaacgtggc ggtggtgttt tccgtagttg accgcggagg caacacgctg 4380cttatccagc ggatggacga ggccttcgtc tccagctgcg atatttccct gaataaagcc 4440tggagcgcct gcagcctgaa gcaaggtacc catgaaatta cgtcagcggt ccagccagga 4500caatctctgt acggtctgca gctaaccaac caacagcgaa ttattatttt tggcggcggc 4560ctgccagtta tttttaatga gcaggtaatt ggcgccgtcg gcgttagcgg cggtacggtc 4620gagcaggatc aattattagc ccagtgcgcc ctggattgtt tttccgcatt ataacctgaa 4680gcgagaaggt atattatgag ctatcgtatg ttccgccagg cattctgagt gttaacgagg 4740ggaccgtcat gtcgctttca ccgccaggcg tacgcctgtt ttacgatccg cgcgggcacc 4800atgccggcgc catcaatgag ctgtgctggg ggctggagga gcagggggtc ccctgccaga 4860ccataaccta tgacggaggc ggtgacgccg ctgcgctggg cgccctggcg gccagaagct 4920cgcccctgcg ggtgggtatc gggctcagcg cgtccggcga gatagccctc actcatgccc 4980agctgccggc ggacgcgccg ctggctaccg gacacgtcac cgatagcgac gatcaactgc 5040gtacgctcgg cgccaacgcc gggcagctgg ttaaagtcct gccgttaagt gagagaaact 5100gaatgtatcg tatctatacc cgcaccgggg ataaaggcac caccgccctg tacggcggca 5160gccgcatcga gaaagaccat attcgcgtcg aggcctacgg caccgtcgat gaactgatat 5220cccagctggg cgtctgctac gccacgaccc gcgacgccgg gctgcgggaa agcctgcacc 5280atattcagca gacgctgttc gtgctggggg ctgaactggc cagcgatgcg cggggcctga 5340cccgcctgag ccagacgatc ggcgaagagg agatcaccgc cctggagcgg cttatcgacc 5400gcaatatggc cgagagcggc ccgttaaaac agttcgtgat cccggggagg aatctcgcct 5460ctgcccagct gcacgtggcg cgcacccagt cccgtcggct cgaacgcctg ctgacggcca 5520tggaccgcgc gcatccgctg cgcgacgcgc tcaaacgcta cagcaatcgc ctgtcggatg 5580ccctgttctc catggcgcga atcgaagaga ctaggcctga tgcttgcgct tgaactggcc 5640tagcaaacac agaaaaaagc ccgcacctga cagtgcgggc tttttttttc ctaggcgatc 5700tgtgctgttt gccacggtat gcagcaccag cgcgagatta tgggctcgca cgctcgactg 5760tcggacgggg gcactggaac gagaagtcag gcgagccgtc acgcccttga ctatgccaca 5820tcctgagcaa ataattcaac cactaaacaa atcaaccgcg tttcccggag gtaaccaagc 5880ttcacctttt gagccgatga acaatgaaaa gatcaaaacg atttgcagta ctggcccagc 5940gccccgtcaa tcaggacggg ctgattggcg agtggcctga agaggggctg atcgccatgg 6000acagcccctt tgacccggtc tcttcagtaa aagtggacaa cggtctgatc gtcgaactgg 6060acggcaaacg ccgggaccag tttgacatga tcgaccgatt tatcgccgat tacgcgatca 6120acgttgagcg cacagagcag gcaatgcgcc tggaggcggt ggaaatagcc cgtatgctgg 6180tggatattca cgtcagccgg gaggagatca ttgccatcac taccgccatc acgccggcca 6240aagcggtcga ggtgatggcg cagatgaacg tggtggagat gatgatggcg ctgcagaaga 6300tgcgtgcccg ccggaccccc tccaaccagt gccacgtcac caatctcaaa gataatccgg 6360tgcagattgc cgctgacgcc gccgaggccg ggatccgcgg cttctcagaa caggagacca 6420cggtcggtat cgcgcgctac gcgccgttta acgccctggc gctgttggtc ggttcgcagt 6480gcggccgccc cggcgtgttg acgcagtgct cggtggaaga ggccaccgag ctggagctgg 6540gcatgcgtgg cttaaccagc tacgccgaga cggtgtcggt ctacggcacc gaagcggtat 6600ttaccgacgg cgatgatacg ccgtggtcaa aggcgttcct cgcctcggcc tacgcctccc 6660gcgggttgaa aatgcgctac acctccggca ccggatccga agcgctgatg ggctattcgg 6720agagcaagtc gatgctctac ctcgaatcgc gctgcatctt cattactaaa ggcgccgggg 6780ttcagggact gcaaaacggc gcggtgagct gtatcggcat gaccggcgct gtgccgtcgg 6840gcattcgggc ggtgctggcg gaaaacctga tcgcctctat gctcgacctc gaagtggcgt 6900ccgccaacga ccagactttc tcccactcgg atattcgccg caccgcgcgc accctgatgc 6960agatgctgcc gggcaccgac tttattttct ccggctacag cgcggtgccg aactacgaca 7020acatgttcgc cggctcgaac ttcgatgcgg aagattttga tgattacaac atcctgcagc 7080gtgacctgat ggttgacggc ggcctgcgtc cggtgaccga ggcggaaacc attgccattc 7140gccagaaagc ggcgcgggcg atccaggcgg ttttccgcga gctggggctg ccgccaatcg 7200ccgacgagga ggtggaggcc gccacctacg cgcacggcag caacgagatg ccgccgcgta 7260acgtggtgga ggatctgagt gcggtggaag agatgatgaa gcgcaacatc accggcctcg 7320atattgtcgg cgcgctgagc cgcagcggct ttgaggatat cgccagcaat attctcaata 7380tgctgcgcca gcgggtcacc ggcgattacc tgcagacctc ggccattctc gatcggcagt 7440tcgaggtggt gagtgcggtc aacgacatca atgactatca ggggccgggc accggctatc 7500gcatctctgc cgaacgctgg gcggagatca aaaatattcc gggcgtggtt cagcccgaca 7560ccattgaata aggcggtatt cctgtgcaac agacaaccca aattcagccc tcttttaccc 7620tgaaaacccg cgagggcggg gtagcttctg ccgatgaacg cgccgatgaa gtggtgatcg 7680gcgtcggccc tgccttcgat aaacaccagc atcacactct gatcgatatg ccccatggcg 7740cgatcctcaa agagctgatt gccggggtgg aagaagaggg gcttcacgcc cgggtggtgc 7800gcattctgcg cacgtccgac gtctccttta tggcctggga tgcggccaac ctgagcggct 7860cggggatcgg catcggtatc cagtcgaagg ggaccacggt catccatcag cgcgatctgc 7920tgccgctcag caacctggag ctgttctccc aggcgccgct gctgacgctg gagacctacc 7980ggcagattgg caaaaacgct gcgcgctatg cgcgcaaaga gtcaccttcg ccggtgccgg 8040tggtgaacga tcagatggtg cggccgaaat ttatggccaa agccgcgcta tttcatatca 8100aagagaccaa acatgtggtg caggacgccg agcccgtcac cctgcacatc gacttagtaa 8160gggagtgacc atgagcgaga aaaccatgcg cgtgcaggat tatccgttag ccacccgctg 8220cccggagcat atcctgacgc ctaccggcaa accattgacc gatattaccc tcgagaaggt 8280gctctctggc gaggtgggcc cgcaggatgt gcggatctcc cgccagaccc ttgagtacca 8340ggcgcagatt gccgagcaga tgcagcgcca tgcggtggcg cgcaatttcc gccgcgcggc 8400ggagcttatc gccattcctg acgagcgcat tctggctatc tataacgcgc tgcgcccgtt 8460ccgctcctcg caggcggagc tgctggcgat cgccgacgag ctggagcaca cctggcatgc 8520gacagtgaat gccgcctttg tccgggagtc ggcggaagtg tatcagcagc ggcataagct 8580gcgtaaagga agctaagcgg aggtcagcat gccgttaata gccgggattg atatcggcaa 8640cgccaccacc gaggtggcgc tggcgtccga ctacccgcag gcgagggcgt ttgttgccag 8700cgggatcgtc gcgacgacgg gcatgaaagg gacgcgggac aatatcgccg ggaccctcgc 8760cgcgctggag caggccctgg cgaaaacacc gtggtcgatg agcgatgtct ctcgcatcta 8820tcttaacgaa gccgcgccgg tgattggcga tgtggcgatg gagaccatca ccgagaccat 8880tatcaccgaa tcgaccatga tcggtcataa cccgcagacg ccgggcgggg tgggcgttgg 8940cgtggggacg actatcgccc tcgggcggct ggcgacgctg ccggcggcgc agtatgccga 9000ggggtggatc gtactgattg acgacgccgt cgatttcctt gacgccgtgt ggtggctcaa 9060tgaggcgctc gaccggggga tcaacgtggt ggcggcgatc ctcaaaaagg acgacggcgt 9120gctggtgaac aaccgcctgc gtaaaaccct gccggtggtg gatgaagtga cgctgctgga 9180gcaggtcccc gagggggtaa tggcggcggt ggaagtggcc gcgccgggcc aggtggtgcg 9240gatcctgtcg aatccctacg ggatcgccac cttcttcggg ctaagcccgg aagagaccca 9300ggccatcgtc cccatcgccc gcgccctgat tggcaaccgt tccgcggtgg tgctcaagac 9360cccgcagggg gatgtgcagt cgcgggtgat cccggcgggc aacctctaca ttagcggcga 9420aaagcgccgc ggagaggccg atgtcgccga gggcgcggaa gccatcatgc aggcgatgag 9480cgcctgcgct ccggtacgcg acatccgcgg cgaaccgggc acccacgccg gcggcatgct 9540tgagcgggtg cgcaaggtaa tggcgtccct gaccggccat gagatgagcg cgatatacat 9600ccaggatctg ctggcggtgg atacgtttat tccgcgcaag gtgcagggcg ggatggccgg 9660cgagtgcgcc atggagaatg ccgtcgggat ggcggcgatg gtgaaagcgg atcgtctgca 9720aatgcaggtt atcgcccgcg aactgagcgc ccgactgcag accgaggtgg tggtgggcgg 9780cgtggaggcc aacatggcca tcgccggggc gttaaccact cccggctgtg cggcgccgct 9840ggcgatcctc gacctcggcg ccggctcgac ggatgcggcg atcgtcaacg cggaggggca 9900gataacggcg gtccatctcg ccggggcggg gaatatggtc agcctgttga ttaaaaccga 9960gctgggcctc gaggatcttt cgctggcgga agcgataaaa aaatacccgc tggccaaagt 10020ggaaagcctg ttcagtattc gtcacgagaa tggcgcggtg gagttctttc gggaagccct 10080cagcccggcg gtgttcgcca aagtggtgta catcaaggag ggcgaactgg tgccgatcga 10140taacgccagc ccgctggaaa aaattcgtct cgtgcgccgg caggcgaaag agaaagtgtt 10200tgtcaccaac tgcctgcgcg cgctgcgcca ggtctcaccc ggcggttcca ttcgcgatat 10260cgcctttgtg gtgctggtgg gcggctcatc gctggacttt gagatcccgc agcttatcac 10320ggaagccttg tcgcactatg gcgtggtcgc cgggcagggc aatattcggg gaacagaagg 10380gccgcgcaat gcggtcgcca ccgggctgct actggccggt caggcgaatt aaacgggcgc 10440tcgcgccagc ctctaggtac aaataaaaaa ggcacgtcag atgacgtgcc ttttttcttg 10500tctagcgtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg 10560caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg 10620ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt 10680aatcatccgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac 10740agaccatgac tagtaaggag gacaattcca tggctgctgc tgctgataga ttaaacttaa 10800cttccggcca cttgaatgct ggtagaaaga gaagttcctc ttctgtttct ttgaaggctg 10860ccgaaaagcc tttcaaggtt actgtgattg gatctggtaa ctggggtact actattgcca 10920aggtggttgc cgaaaattgt aagggatacc cagaagtttt cgctccaata gtacaaatgt 10980gggtgttcga agaagagatc aatggtgaaa aattgactga aatcataaat actagacatc 11040aaaacgtgaa atacttgcct ggcatcactc tacccgacaa tttggttgct aatccagact 11100tgattgattc agtcaaggat gtcgacatca tcgttttcaa cattccacat caatttttgc 11160cccgtatctg tagccaattg aaaggtcatg ttgattcaca cgtcagagct atctcctgtc 11220taaagggttt tgaagttggt gctaaaggtg tccaattgct atcctcttac atcactgagg 11280aactaggtat tcaatgtggt gctctatctg gtgctaacat tgccaccgaa gtcgctcaag 11340aacactggtc tgaaacaaca gttgcttacc acattccaaa ggatttcaga ggcgagggca 11400aggacgtcga ccataaggtt ctaaaggcct tgttccacag accttacttc cacgttagtg 11460tcatcgaaga tgttgctggt atctccatct gtggtgcttt gaagaacgtt gttgccttag 11520gttgtggttt cgtcgaaggt ctaggctggg gtaacaacgc ttctgctgcc atccaaagag 11580tcggtttggg tgagatcatc agattcggtc aaatgttttt cccagaatct agagaagaaa 11640catactacca agagtctgct ggtgttgctg atttgatcac cacctgcgct ggtggtagaa 11700acgtcaaggt tgctaggcta atggctactt ctggtaagga cgcctgggaa tgtgaaaagg 11760agttgttgaa tggccaatcc gctcaaggtt taattacctg caaagaagtt cacgaatggt 11820tggaaacatg tggctctgtc gaagacttcc cattatttga agccgtatac caaatcgttt 11880acaacaacta cccaatgaag aacctgccgg acatgattga agaattagat ctacatgaag 11940attagattta ttggatccag gaaacagact agaattatgg gattgactac taaacctcta 12000tctttgaaag ttaacgccgc tttgttcgac gtcgacggta ccattatcat ctctcaacca 12060gccattgctg cattctggag ggatttcggt aaggacaaac cttatttcga tgctgaacac 12120gttatccaag tctcgcatgg ttggagaacg tttgatgcca ttgctaagtt cgctccagac 12180tttgccaatg aagagtatgt taacaaatta gaagctgaaa ttccggtcaa gtacggtgaa 12240aaatccattg aagtcccagg tgcagttaag ctgtgcaacg ctttgaacgc tctaccaaaa 12300gagaaatggg ctgtggcaac ttccggtacc cgtgatatgg cacaaaaatg gttcgagcat 12360ctgggaatca ggagaccaaa gtacttcatt accgctaatg atgtcaaaca gggtaagcct 12420catccagaac catatctgaa gggcaggaat ggcttaggat atccgatcaa tgagcaagac 12480ccttccaaat ctaaggtagt agtatttgaa gacgctccag caggtattgc cgccggaaaa 12540gccgccggtt gtaagatcat tggtattgcc actactttcg acttggactt cctaaaggaa 12600aaaggctgtg acatcattgt caaaaaccac gaatccatca gagttggcgg ctacaatgcc 12660gaaacagacg aagttgaatt catttttgac gactacttat atgctaagga cgatctgttg 12720aaatggtaac ccgggctgca ggcatgcaag cttggctgtt ttggcggatg agagaagatt 12780ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct 12840ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt 12900agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat 12960aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa 13020cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc 13080cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc 13140catcctgacg gatggccttt ttgcgtttct acaaactcca gctggatcgg gcgctagagt 13200atacatttaa atggtaccct ctagtcaagg ccttaagtga gtcgtattac ggactggccg 13260tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 13320cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 13380aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc 13440tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 13500agttaagcca gccccgacac ccgccaacac ccgctgacga gct 135437313543DNAartificial sequenceplasmid 73tagtaaagcc ctcgctagat tttaatgcgg atgttgcgat tacttcgcca actattgcga 60taacaagaaa aagccagcct ttcatgatat atctcccaat ttgtgtaggg cttattatgc 120acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc 180ttagtgcatc taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt 240gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 300tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 360gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 420acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 480actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 540tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 600cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 660atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 720agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 780tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 840atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 900tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 960gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 1020gcgatcaccg cttccctcat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 1080acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 1140gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 1200actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata 1260cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc 1320atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt cgaggcattt 1380ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg 1440gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgccctggct tcaggagatc 1500ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc 1560atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc agcttctgta tggaacgggc 1620atgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 1680atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 1740gcacccagcc tgcgcgagca ggggaattaa ttcccacggg ttttgctgcc cgcaaacggg 1800ctgttctggt gttgctagtt tgttatcaga atcgcagatc cggcttcagc cggtttgccg 1860gctgaaagcg ctatttcttc cagaattgcc atgatttttt ccccacggga ggcgtcactg 1920gctcccgtgt tgtcggcagc tttgattcga taagcagcat cgcctgtttc aggctgtcta 1980tgtgtgactg ttgagctgta acaagttgtc tcaggtgttc aatttcatgt tctagttgct 2040ttgttttact ggtttcacct gttctattag gtgttacatg ctgttcatct gttacattgt 2100cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat 2160ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac 2220ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag 2280ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 2340ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2400aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2460cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2520atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2580tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2640taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct

tttaaactca 2700tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2760gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2820tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2880aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2940gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 3000gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 3060atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 3120gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 3180tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 3240atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 3300tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3360gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3420ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3480aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac 3540aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc 3600ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat 3660caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc 3720agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc 3780cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg 3840tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc 3900acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg 3960tatcatcaac aggcttaccc gtcttactgt cgggaattca tttaaatagt caaaagcctc 4020cgaccggagg cttttgactg ctaggcgatc tgtgctgttt gccacggtat gcagcaccag 4080cgcgagatta tgggctcgca cgctcgactg tcggacgggg gcactggaac gagaagtcag 4140gcgagccgtc acgcccttga caatgccaca tcctgagcaa ataattcaac cactaaacaa 4200atcaaccgcg tttcccggag gtaaccaagc ttgcgggaga gaatgatgaa caagagccaa 4260caagttcaga caatcaccct ggccgccgcc cagcaaatgg cggcggcggt ggaaaaaaaa 4320gccactgaga tcaacgtggc ggtggtgttt tccgtagttg accgcggagg caacacgctg 4380cttatccagc ggatggacga ggccttcgtc tccagctgcg atatttccct gaataaagcc 4440tggagcgcct gcagcctgaa gcaaggtacc catgaaatta cgtcagcggt ccagccagga 4500caatctctgt acggtctgca gctaaccaac caacagcgaa ttattatttt tggcggcggc 4560ctgccagtta tttttaatga gcaggtaatt ggcgccgtcg gcgttagcgg cggtacggtc 4620gagcaggatc aattattagc ccagtgcgcc ctggattgtt tttccgcatt ataacctgaa 4680gcgagaaggt atattatgag ctatcgtatg ttccgccagg cattctgagt gttaacgagg 4740ggaccgtcat gtcgctttca ccgccaggcg tacgcctgtt ttacgatccg cgcgggcacc 4800atgccggcgc catcaatgag ctgtgctggg ggctggagga gcagggggtc ccctgccaga 4860ccataaccta tgacggaggc ggtgacgccg ctgcgctggg cgccctggcg gccagaagct 4920cgcccctgcg ggtgggtatc gggctcagcg cgtccggcga gatagccctc actcatgccc 4980agctgccggc ggacgcgccg ctggctaccg gacacgtcac cgatagcgac gatcaactgc 5040gtacgctcgg cgccaacgcc gggcagctgg ttaaagtcct gccgttaagt gagagaaact 5100gaatgtatcg tatctatacc cgcaccgggg ataaaggcac caccgccctg tacggcggca 5160gccgcatcga gaaagaccat attcgcgtcg aggcctacgg caccgtcgat gaactgatat 5220cccagctggg cgtctgctac gccacgaccc gcgacgccgg gctgcgggaa agcctgcacc 5280atattcagca gacgctgttc gtgctggggg ctgaactggc cagcgatgcg cggggcctga 5340cccgcctgag ccagacgatc ggcgaagagg agatcaccgc cctggagcgg cttatcgacc 5400gcaatatggc cgagagcggc ccgttaaaac agttcgtgat cccggggagg aatctcgcct 5460ctgcccagct gcacgtggcg cgcacccagt cccgtcggct cgaacgcctg ctgacggcca 5520tggaccgcgc gcatccgctg cgcgacgcgc tcaaacgcta cagcaatcgc ctgtcggatg 5580ccctgttctc catggcgcga atcgaagaga ctaggcctga tgcttgcgct tgaactggcc 5640tagcaaacac agaaaaaagc ccgcacctga cagtgcgggc tttttttttc ctaggcgatc 5700tgtgctgttt gccacggtat gcagcaccag cgcgagatta tgggctcgca cgctcgactg 5760tcggacgggg gcactggaac gagaagtcag gcgagccgtc acgcccttga caatgccaca 5820tcctgagcaa ataattcaac cactaaacaa atcaaccgcg tttcccggag gtaaccaagc 5880ttcacctttt gagccgatga acaatgaaaa gatcaaaacg atttgcagta ctggcccagc 5940gccccgtcaa tcaggacggg ctgattggcg agtggcctga agaggggctg atcgccatgg 6000acagcccctt tgacccggtc tcttcagtaa aagtggacaa cggtctgatc gtcgaactgg 6060acggcaaacg ccgggaccag tttgacatga tcgaccgatt tatcgccgat tacgcgatca 6120acgttgagcg cacagagcag gcaatgcgcc tggaggcggt ggaaatagcc cgtatgctgg 6180tggatattca cgtcagccgg gaggagatca ttgccatcac taccgccatc acgccggcca 6240aagcggtcga ggtgatggcg cagatgaacg tggtggagat gatgatggcg ctgcagaaga 6300tgcgtgcccg ccggaccccc tccaaccagt gccacgtcac caatctcaaa gataatccgg 6360tgcagattgc cgctgacgcc gccgaggccg ggatccgcgg cttctcagaa caggagacca 6420cggtcggtat cgcgcgctac gcgccgttta acgccctggc gctgttggtc ggttcgcagt 6480gcggccgccc cggcgtgttg acgcagtgct cggtggaaga ggccaccgag ctggagctgg 6540gcatgcgtgg cttaaccagc tacgccgaga cggtgtcggt ctacggcacc gaagcggtat 6600ttaccgacgg cgatgatacg ccgtggtcaa aggcgttcct cgcctcggcc tacgcctccc 6660gcgggttgaa aatgcgctac acctccggca ccggatccga agcgctgatg ggctattcgg 6720agagcaagtc gatgctctac ctcgaatcgc gctgcatctt cattactaaa ggcgccgggg 6780ttcagggact gcaaaacggc gcggtgagct gtatcggcat gaccggcgct gtgccgtcgg 6840gcattcgggc ggtgctggcg gaaaacctga tcgcctctat gctcgacctc gaagtggcgt 6900ccgccaacga ccagactttc tcccactcgg atattcgccg caccgcgcgc accctgatgc 6960agatgctgcc gggcaccgac tttattttct ccggctacag cgcggtgccg aactacgaca 7020acatgttcgc cggctcgaac ttcgatgcgg aagattttga tgattacaac atcctgcagc 7080gtgacctgat ggttgacggc ggcctgcgtc cggtgaccga ggcggaaacc attgccattc 7140gccagaaagc ggcgcgggcg atccaggcgg ttttccgcga gctggggctg ccgccaatcg 7200ccgacgagga ggtggaggcc gccacctacg cgcacggcag caacgagatg ccgccgcgta 7260acgtggtgga ggatctgagt gcggtggaag agatgatgaa gcgcaacatc accggcctcg 7320atattgtcgg cgcgctgagc cgcagcggct ttgaggatat cgccagcaat attctcaata 7380tgctgcgcca gcgggtcacc ggcgattacc tgcagacctc ggccattctc gatcggcagt 7440tcgaggtggt gagtgcggtc aacgacatca atgactatca ggggccgggc accggctatc 7500gcatctctgc cgaacgctgg gcggagatca aaaatattcc gggcgtggtt cagcccgaca 7560ccattgaata aggcggtatt cctgtgcaac agacaaccca aattcagccc tcttttaccc 7620tgaaaacccg cgagggcggg gtagcttctg ccgatgaacg cgccgatgaa gtggtgatcg 7680gcgtcggccc tgccttcgat aaacaccagc atcacactct gatcgatatg ccccatggcg 7740cgatcctcaa agagctgatt gccggggtgg aagaagaggg gcttcacgcc cgggtggtgc 7800gcattctgcg cacgtccgac gtctccttta tggcctggga tgcggccaac ctgagcggct 7860cggggatcgg catcggtatc cagtcgaagg ggaccacggt catccatcag cgcgatctgc 7920tgccgctcag caacctggag ctgttctccc aggcgccgct gctgacgctg gagacctacc 7980ggcagattgg caaaaacgct gcgcgctatg cgcgcaaaga gtcaccttcg ccggtgccgg 8040tggtgaacga tcagatggtg cggccgaaat ttatggccaa agccgcgcta tttcatatca 8100aagagaccaa acatgtggtg caggacgccg agcccgtcac cctgcacatc gacttagtaa 8160gggagtgacc atgagcgaga aaaccatgcg cgtgcaggat tatccgttag ccacccgctg 8220cccggagcat atcctgacgc ctaccggcaa accattgacc gatattaccc tcgagaaggt 8280gctctctggc gaggtgggcc cgcaggatgt gcggatctcc cgccagaccc ttgagtacca 8340ggcgcagatt gccgagcaga tgcagcgcca tgcggtggcg cgcaatttcc gccgcgcggc 8400ggagcttatc gccattcctg acgagcgcat tctggctatc tataacgcgc tgcgcccgtt 8460ccgctcctcg caggcggagc tgctggcgat cgccgacgag ctggagcaca cctggcatgc 8520gacagtgaat gccgcctttg tccgggagtc ggcggaagtg tatcagcagc ggcataagct 8580gcgtaaagga agctaagcgg aggtcagcat gccgttaata gccgggattg atatcggcaa 8640cgccaccacc gaggtggcgc tggcgtccga ctacccgcag gcgagggcgt ttgttgccag 8700cgggatcgtc gcgacgacgg gcatgaaagg gacgcgggac aatatcgccg ggaccctcgc 8760cgcgctggag caggccctgg cgaaaacacc gtggtcgatg agcgatgtct ctcgcatcta 8820tcttaacgaa gccgcgccgg tgattggcga tgtggcgatg gagaccatca ccgagaccat 8880tatcaccgaa tcgaccatga tcggtcataa cccgcagacg ccgggcgggg tgggcgttgg 8940cgtggggacg actatcgccc tcgggcggct ggcgacgctg ccggcggcgc agtatgccga 9000ggggtggatc gtactgattg acgacgccgt cgatttcctt gacgccgtgt ggtggctcaa 9060tgaggcgctc gaccggggga tcaacgtggt ggcggcgatc ctcaaaaagg acgacggcgt 9120gctggtgaac aaccgcctgc gtaaaaccct gccggtggtg gatgaagtga cgctgctgga 9180gcaggtcccc gagggggtaa tggcggcggt ggaagtggcc gcgccgggcc aggtggtgcg 9240gatcctgtcg aatccctacg ggatcgccac cttcttcggg ctaagcccgg aagagaccca 9300ggccatcgtc cccatcgccc gcgccctgat tggcaaccgt tccgcggtgg tgctcaagac 9360cccgcagggg gatgtgcagt cgcgggtgat cccggcgggc aacctctaca ttagcggcga 9420aaagcgccgc ggagaggccg atgtcgccga gggcgcggaa gccatcatgc aggcgatgag 9480cgcctgcgct ccggtacgcg acatccgcgg cgaaccgggc acccacgccg gcggcatgct 9540tgagcgggtg cgcaaggtaa tggcgtccct gaccggccat gagatgagcg cgatatacat 9600ccaggatctg ctggcggtgg atacgtttat tccgcgcaag gtgcagggcg ggatggccgg 9660cgagtgcgcc atggagaatg ccgtcgggat ggcggcgatg gtgaaagcgg atcgtctgca 9720aatgcaggtt atcgcccgcg aactgagcgc ccgactgcag accgaggtgg tggtgggcgg 9780cgtggaggcc aacatggcca tcgccggggc gttaaccact cccggctgtg cggcgccgct 9840ggcgatcctc gacctcggcg ccggctcgac ggatgcggcg atcgtcaacg cggaggggca 9900gataacggcg gtccatctcg ccggggcggg gaatatggtc agcctgttga ttaaaaccga 9960gctgggcctc gaggatcttt cgctggcgga agcgataaaa aaatacccgc tggccaaagt 10020ggaaagcctg ttcagtattc gtcacgagaa tggcgcggtg gagttctttc gggaagccct 10080cagcccggcg gtgttcgcca aagtggtgta catcaaggag ggcgaactgg tgccgatcga 10140taacgccagc ccgctggaaa aaattcgtct cgtgcgccgg caggcgaaag agaaagtgtt 10200tgtcaccaac tgcctgcgcg cgctgcgcca ggtctcaccc ggcggttcca ttcgcgatat 10260cgcctttgtg gtgctggtgg gcggctcatc gctggacttt gagatcccgc agcttatcac 10320ggaagccttg tcgcactatg gcgtggtcgc cgggcagggc aatattcggg gaacagaagg 10380gccgcgcaat gcggtcgcca ccgggctgct actggccggt caggcgaatt aaacgggcgc 10440tcgcgccagc ctctaggtac aaataaaaaa ggcacgtcag atgacgtgcc ttttttcttg 10500tctagcgtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg 10560caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg 10620ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt 10680aatcatccgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac 10740agaccatgac tagtaaggag gacaattcca tggctgctgc tgctgataga ttaaacttaa 10800cttccggcca cttgaatgct ggtagaaaga gaagttcctc ttctgtttct ttgaaggctg 10860ccgaaaagcc tttcaaggtt actgtgattg gatctggtaa ctggggtact actattgcca 10920aggtggttgc cgaaaattgt aagggatacc cagaagtttt cgctccaata gtacaaatgt 10980gggtgttcga agaagagatc aatggtgaaa aattgactga aatcataaat actagacatc 11040aaaacgtgaa atacttgcct ggcatcactc tacccgacaa tttggttgct aatccagact 11100tgattgattc agtcaaggat gtcgacatca tcgttttcaa cattccacat caatttttgc 11160cccgtatctg tagccaattg aaaggtcatg ttgattcaca cgtcagagct atctcctgtc 11220taaagggttt tgaagttggt gctaaaggtg tccaattgct atcctcttac atcactgagg 11280aactaggtat tcaatgtggt gctctatctg gtgctaacat tgccaccgaa gtcgctcaag 11340aacactggtc tgaaacaaca gttgcttacc acattccaaa ggatttcaga ggcgagggca 11400aggacgtcga ccataaggtt ctaaaggcct tgttccacag accttacttc cacgttagtg 11460tcatcgaaga tgttgctggt atctccatct gtggtgcttt gaagaacgtt gttgccttag 11520gttgtggttt cgtcgaaggt ctaggctggg gtaacaacgc ttctgctgcc atccaaagag 11580tcggtttggg tgagatcatc agattcggtc aaatgttttt cccagaatct agagaagaaa 11640catactacca agagtctgct ggtgttgctg atttgatcac cacctgcgct ggtggtagaa 11700acgtcaaggt tgctaggcta atggctactt ctggtaagga cgcctgggaa tgtgaaaagg 11760agttgttgaa tggccaatcc gctcaaggtt taattacctg caaagaagtt cacgaatggt 11820tggaaacatg tggctctgtc gaagacttcc cattatttga agccgtatac caaatcgttt 11880acaacaacta cccaatgaag aacctgccgg acatgattga agaattagat ctacatgaag 11940attagattta ttggatccag gaaacagact agaattatgg gattgactac taaacctcta 12000tctttgaaag ttaacgccgc tttgttcgac gtcgacggta ccattatcat ctctcaacca 12060gccattgctg cattctggag ggatttcggt aaggacaaac cttatttcga tgctgaacac 12120gttatccaag tctcgcatgg ttggagaacg tttgatgcca ttgctaagtt cgctccagac 12180tttgccaatg aagagtatgt taacaaatta gaagctgaaa ttccggtcaa gtacggtgaa 12240aaatccattg aagtcccagg tgcagttaag ctgtgcaacg ctttgaacgc tctaccaaaa 12300gagaaatggg ctgtggcaac ttccggtacc cgtgatatgg cacaaaaatg gttcgagcat 12360ctgggaatca ggagaccaaa gtacttcatt accgctaatg atgtcaaaca gggtaagcct 12420catccagaac catatctgaa gggcaggaat ggcttaggat atccgatcaa tgagcaagac 12480ccttccaaat ctaaggtagt agtatttgaa gacgctccag caggtattgc cgccggaaaa 12540gccgccggtt gtaagatcat tggtattgcc actactttcg acttggactt cctaaaggaa 12600aaaggctgtg acatcattgt caaaaaccac gaatccatca gagttggcgg ctacaatgcc 12660gaaacagacg aagttgaatt catttttgac gactacttat atgctaagga cgatctgttg 12720aaatggtaac ccgggctgca ggcatgcaag cttggctgtt ttggcggatg agagaagatt 12780ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct 12840ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt 12900agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat 12960aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa 13020cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc 13080cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc 13140catcctgacg gatggccttt ttgcgtttct acaaactcca gctggatcgg gcgctagagt 13200atacatttaa atggtaccct ctagtcaagg ccttaagtga gtcgtattac ggactggccg 13260tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 13320cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 13380aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc 13440tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 13500agttaagcca gccccgacac ccgccaacac ccgctgacga gct 135437413402DNAartificial sequenceplasmid 74tagtaaagcc ctcgctagat tttaatgcgg atgttgcgat tacttcgcca actattgcga 60taacaagaaa aagccagcct ttcatgatat atctcccaat ttgtgtaggg cttattatgc 120acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc 180ttagtgcatc taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt 240gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 300tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 360gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 420acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 480actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 540tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 600cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 660atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 720agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 780tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 840atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 900tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 960gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 1020gcgatcaccg cttccctcat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 1080acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 1140gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 1200actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata 1260cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc 1320atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt cgaggcattt 1380ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg 1440gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgccctggct tcaggagatc 1500ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc 1560atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc agcttctgta tggaacgggc 1620atgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 1680atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 1740gcacccagcc tgcgcgagca ggggaattaa ttcccacggg ttttgctgcc cgcaaacggg 1800ctgttctggt gttgctagtt tgttatcaga atcgcagatc cggcttcagc cggtttgccg 1860gctgaaagcg ctatttcttc cagaattgcc atgatttttt ccccacggga ggcgtcactg 1920gctcccgtgt tgtcggcagc tttgattcga taagcagcat cgcctgtttc aggctgtcta 1980tgtgtgactg ttgagctgta acaagttgtc tcaggtgttc aatttcatgt tctagttgct 2040ttgttttact ggtttcacct gttctattag gtgttacatg ctgttcatct gttacattgt 2100cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat 2160ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac 2220ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag 2280ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 2340ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2400aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2460cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2520atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2580tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2640taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca 2700tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2760gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2820tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2880aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2940gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 3000gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 3060atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 3120gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 3180tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 3240atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 3300tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3360gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3420ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3480aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac 3540aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc 3600ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat 3660caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc 3720agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc 3780cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg 3840tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc 3900acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg 3960tatcatcaac aggcttaccc gtcttactgt cgggaattca tttaaatagt caaaagcctc 4020cgaccggagg cttttgactg ctaggcgatc tgtgctgttt gccacggtat gcagcaccag 4080cgcgagatta tgggctcgca cgctcgactg tcggacgggg gcactggaac gagaagtcag 4140gcgagccgtc

acgcccttga caatgccaca tcctgagcaa ataattcaac cactaaacaa 4200atcaaccgcg tttcccggag gtaaccaagc ttgcgggaga gaatgatgaa caagagccaa 4260caagttcaga caatcaccct ggccgccgcc cagcaaatgg cggcggcggt ggaaaaaaaa 4320gccactgaga tcaacgtggc ggtggtgttt tccgtagttg accgcggagg caacacgctg 4380cttatccagc ggatggacga ggccttcgtc tccagctgcg atatttccct gaataaagcc 4440tggagcgcct gcagcctgaa gcaaggtacc catgaaatta cgtcagcggt ccagccagga 4500caatctctgt acggtctgca gctaaccaac caacagcgaa ttattatttt tggcggcggc 4560ctgccagtta tttttaatga gcaggtaatt ggcgccgtcg gcgttagcgg cggtacggtc 4620gagcaggatc aattattagc ccagtgcgcc ctggattgtt tttccgcatt ataacctgaa 4680gcgagaaggt atattatgag ctatcgtatg ttccgccagg cattctgagt gttaacgagg 4740ggaccgtcat gtcgctttca ccgccaggcg tacgcctgtt ttacgatccg cgcgggcacc 4800atgccggcgc catcaatgag ctgtgctggg ggctggagga gcagggggtc ccctgccaga 4860ccataaccta tgacggaggc ggtgacgccg ctgcgctggg cgccctggcg gccagaagct 4920cgcccctgcg ggtgggtatc gggctcagcg cgtccggcga gatagccctc actcatgccc 4980agctgccggc ggacgcgccg ctggctaccg gacacgtcac cgatagcgac gatcaactgc 5040gtacgctcgg cgccaacgcc gggcagctgg ttaaagtcct gccgttaagt gagagaaact 5100gaatgtatcg tatctatacc cgcaccgggg ataaaggcac caccgccctg tacggcggca 5160gccgcatcga gaaagaccat attcgcgtcg aggcctacgg caccgtcgat gaactgatat 5220cccagctggg cgtctgctac gccacgaccc gcgacgccgg gctgcgggaa agcctgcacc 5280atattcagca gacgctgttc gtgctggggg ctgaactggc cagcgatgcg cggggcctga 5340cccgcctgag ccagacgatc ggcgaagagg agatcaccgc cctggagcgg cttatcgacc 5400gcaatatggc cgagagcggc ccgttaaaac agttcgtgat cccggggagg aatctcgcct 5460ctgcccagct gcaccctgat gcttgcgctt gaactggcct agcaaacaca gaaaaaagcc 5520cgcacctgac agtgcgggct ttttttttcc taggcgatct gtgctgtttg ccacggtatg 5580cagcaccagc gcgagattat gggctcgcac gctcgactgt cggacggggg cactggaacg 5640agaagtcagg cgagccgtca cgcccttgac aatgccacat cctgagcaaa taattcaacc 5700actaaacaaa tcaaccgcgt ttcccggagg taaccaagct tcaccttttg agccgatgaa 5760caatgaaaag atcaaaacga tttgcagtac tggcccagcg ccccgtcaat caggacgggc 5820tgattggcga gtggcctgaa gaggggctga tcgccatgga cagccccttt gacccggtct 5880cttcagtaaa agtggacaac ggtctgatcg tcgaactgga cggcaaacgc cgggaccagt 5940ttgacatgat cgaccgattt atcgccgatt acgcgatcaa cgttgagcgc acagagcagg 6000caatgcgcct ggaggcggtg gaaatagccc gtatgctggt ggatattcac gtcagccggg 6060aggagatcat tgccatcact accgccatca cgccggccaa agcggtcgag gtgatggcgc 6120agatgaacgt ggtggagatg atgatggcgc tgcagaagat gcgtgcccgc cggaccccct 6180ccaaccagtg ccacgtcacc aatctcaaag ataatccggt gcagattgcc gctgacgccg 6240ccgaggccgg gatccgcggc ttctcagaac aggagaccac ggtcggtatc gcgcgctacg 6300cgccgtttaa cgccctggcg ctgttggtcg gttcgcagtg cggccgcccc ggcgtgttga 6360cgcagtgctc ggtggaagag gccaccgagc tggagctggg catgcgtggc ttaaccagct 6420acgccgagac ggtgtcggtc tacggcaccg aagcggtatt taccgacggc gatgatacgc 6480cgtggtcaaa ggcgttcctc gcctcggcct acgcctcccg cgggttgaaa atgcgctaca 6540cctccggcac cggatccgaa gcgctgatgg gctattcgga gagcaagtcg atgctctacc 6600tcgaatcgcg ctgcatcttc attactaaag gcgccggggt tcagggactg caaaacggcg 6660cggtgagctg tatcggcatg accggcgctg tgccgtcggg cattcgggcg gtgctggcgg 6720aaaacctgat cgcctctatg ctcgacctcg aagtggcgtc cgccaacgac cagactttct 6780cccactcgga tattcgccgc accgcgcgca ccctgatgca gatgctgccg ggcaccgact 6840ttattttctc cggctacagc gcggtgccga actacgacaa catgttcgcc ggctcgaact 6900tcgatgcgga agattttgat gattacaaca tcctgcagcg tgacctgatg gttgacggcg 6960gcctgcgtcc ggtgaccgag gcggaaacca ttgccattcg ccagaaagcg gcgcgggcga 7020tccaggcggt tttccgcgag ctggggctgc cgccaatcgc cgacgaggag gtggaggccg 7080ccacctacgc gcacggcagc aacgagatgc cgccgcgtaa cgtggtggag gatctgagtg 7140cggtggaaga gatgatgaag cgcaacatca ccggcctcga tattgtcggc gcgctgagcc 7200gcagcggctt tgaggatatc gccagcaata ttctcaatat gctgcgccag cgggtcaccg 7260gcgattacct gcagacctcg gccattctcg atcggcagtt cgaggtggtg agtgcggtca 7320acgacatcaa tgactatcag gggccgggca ccggctatcg catctctgcc gaacgctggg 7380cggagatcaa aaatattccg ggcgtggttc agcccgacac cattgaataa ggcggtattc 7440ctgtgcaaca gacaacccaa attcagccct cttttaccct gaaaacccgc gagggcgggg 7500tagcttctgc cgatgaacgc gccgatgaag tggtgatcgg cgtcggccct gccttcgata 7560aacaccagca tcacactctg atcgatatgc cccatggcgc gatcctcaaa gagctgattg 7620ccggggtgga agaagagggg cttcacgccc gggtggtgcg cattctgcgc acgtccgacg 7680tctcctttat ggcctgggat gcggccaacc tgagcggctc ggggatcggc atcggtatcc 7740agtcgaaggg gaccacggtc atccatcagc gcgatctgct gccgctcagc aacctggagc 7800tgttctccca ggcgccgctg ctgacgctgg agacctaccg gcagattggc aaaaacgctg 7860cgcgctatgc gcgcaaagag tcaccttcgc cggtgccggt ggtgaacgat cagatggtgc 7920ggccgaaatt tatggccaaa gccgcgctat ttcatatcaa agagaccaaa catgtggtgc 7980aggacgccga gcccgtcacc ctgcacatcg acttagtaag ggagtgacca tgagcgagaa 8040aaccatgcgc gtgcaggatt atccgttagc cacccgctgc ccggagcata tcctgacgcc 8100taccggcaaa ccattgaccg atattaccct cgagaaggtg ctctctggcg aggtgggccc 8160gcaggatgtg cggatctccc gccagaccct tgagtaccag gcgcagattg ccgagcagat 8220gcagcgccat gcggtggcgc gcaatttccg ccgcgcggcg gagcttatcg ccattcctga 8280cgagcgcatt ctggctatct ataacgcgct gcgcccgttc cgctcctcgc aggcggagct 8340gctggcgatc gccgacgagc tggagcacac ctggcatgcg acagtgaatg ccgcctttgt 8400ccgggagtcg gcggaagtgt atcagcagcg gcataagctg cgtaaaggaa gctaagcgga 8460ggtcagcatg ccgttaatag ccgggattga tatcggcaac gccaccaccg aggtggcgct 8520ggcgtccgac tacccgcagg cgagggcgtt tgttgccagc gggatcgtcg cgacgacggg 8580catgaaaggg acgcgggaca atatcgccgg gaccctcgcc gcgctggagc aggccctggc 8640gaaaacaccg tggtcgatga gcgatgtctc tcgcatctat cttaacgaag ccgcgccggt 8700gattggcgat gtggcgatgg agaccatcac cgagaccatt atcaccgaat cgaccatgat 8760cggtcataac ccgcagacgc cgggcggggt gggcgttggc gtggggacga ctatcgccct 8820cgggcggctg gcgacgctgc cggcggcgca gtatgccgag gggtggatcg tactgattga 8880cgacgccgtc gatttccttg acgccgtgtg gtggctcaat gaggcgctcg accgggggat 8940caacgtggtg gcggcgatcc tcaaaaagga cgacggcgtg ctggtgaaca accgcctgcg 9000taaaaccctg ccggtggtgg atgaagtgac gctgctggag caggtccccg agggggtaat 9060ggcggcggtg gaagtggccg cgccgggcca ggtggtgcgg atcctgtcga atccctacgg 9120gatcgccacc ttcttcgggc taagcccgga agagacccag gccatcgtcc ccatcgcccg 9180cgccctgatt ggcaaccgtt ccgcggtggt gctcaagacc ccgcaggggg atgtgcagtc 9240gcgggtgatc ccggcgggca acctctacat tagcggcgaa aagcgccgcg gagaggccga 9300tgtcgccgag ggcgcggaag ccatcatgca ggcgatgagc gcctgcgctc cggtacgcga 9360catccgcggc gaaccgggca cccacgccgg cggcatgctt gagcgggtgc gcaaggtaat 9420ggcgtccctg accggccatg agatgagcgc gatatacatc caggatctgc tggcggtgga 9480tacgtttatt ccgcgcaagg tgcagggcgg gatggccggc gagtgcgcca tggagaatgc 9540cgtcgggatg gcggcgatgg tgaaagcgga tcgtctgcaa atgcaggtta tcgcccgcga 9600actgagcgcc cgactgcaga ccgaggtggt ggtgggcggc gtggaggcca acatggccat 9660cgccggggcg ttaaccactc ccggctgtgc ggcgccgctg gcgatcctcg acctcggcgc 9720cggctcgacg gatgcggcga tcgtcaacgc ggaggggcag ataacggcgg tccatctcgc 9780cggggcgggg aatatggtca gcctgttgat taaaaccgag ctgggcctcg aggatctttc 9840gctggcggaa gcgataaaaa aatacccgct ggccaaagtg gaaagcctgt tcagtattcg 9900tcacgagaat ggcgcggtgg agttctttcg ggaagccctc agcccggcgg tgttcgccaa 9960agtggtgtac atcaaggagg gcgaactggt gccgatcgat aacgccagcc cgctggaaaa 10020aattcgtctc gtgcgccggc aggcgaaaga gaaagtgttt gtcaccaact gcctgcgcgc 10080gctgcgccag gtctcacccg gcggttccat tcgcgatatc gcctttgtgg tgctggtggg 10140cggctcatcg ctggactttg agatcccgca gcttatcacg gaagccttgt cgcactatgg 10200cgtggtcgcc gggcagggca atattcgggg aacagaaggg ccgcgcaatg cggtcgccac 10260cgggctgcta ctggccggtc aggcgaatta aacgggcgct cgcgccagcc tctaggtaca 10320aataaaaaag gcacgtcaga tgacgtgcct tttttcttgt ctagcgtgca ccaatgcttc 10380tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata 10440attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa 10500cggttctggc aaatattctg aaatgagctg ttgacaatta atcatccggc tcgtataatg 10560tgtggaattg tgagcggata acaatttcac acaggaaaca gaccatgact agtaaggagg 10620acaattccat ggctgctgct gctgatagat taaacttaac ttccggccac ttgaatgctg 10680gtagaaagag aagttcctct tctgtttctt tgaaggctgc cgaaaagcct ttcaaggtta 10740ctgtgattgg atctggtaac tggggtacta ctattgccaa ggtggttgcc gaaaattgta 10800agggataccc agaagttttc gctccaatag tacaaatgtg ggtgttcgaa gaagagatca 10860atggtgaaaa attgactgaa atcataaata ctagacatca aaacgtgaaa tacttgcctg 10920gcatcactct acccgacaat ttggttgcta atccagactt gattgattca gtcaaggatg 10980tcgacatcat cgttttcaac attccacatc aatttttgcc ccgtatctgt agccaattga 11040aaggtcatgt tgattcacac gtcagagcta tctcctgtct aaagggtttt gaagttggtg 11100ctaaaggtgt ccaattgcta tcctcttaca tcactgagga actaggtatt caatgtggtg 11160ctctatctgg tgctaacatt gccaccgaag tcgctcaaga acactggtct gaaacaacag 11220ttgcttacca cattccaaag gatttcagag gcgagggcaa ggacgtcgac cataaggttc 11280taaaggcctt gttccacaga ccttacttcc acgttagtgt catcgaagat gttgctggta 11340tctccatctg tggtgctttg aagaacgttg ttgccttagg ttgtggtttc gtcgaaggtc 11400taggctgggg taacaacgct tctgctgcca tccaaagagt cggtttgggt gagatcatca 11460gattcggtca aatgtttttc ccagaatcta gagaagaaac atactaccaa gagtctgctg 11520gtgttgctga tttgatcacc acctgcgctg gtggtagaaa cgtcaaggtt gctaggctaa 11580tggctacttc tggtaaggac gcctgggaat gtgaaaagga gttgttgaat ggccaatccg 11640ctcaaggttt aattacctgc aaagaagttc acgaatggtt ggaaacatgt ggctctgtcg 11700aagacttccc attatttgaa gccgtatacc aaatcgttta caacaactac ccaatgaaga 11760acctgccgga catgattgaa gaattagatc tacatgaaga ttagatttat tggatccagg 11820aaacagacta gaattatggg attgactact aaacctctat ctttgaaagt taacgccgct 11880ttgttcgacg tcgacggtac cattatcatc tctcaaccag ccattgctgc attctggagg 11940gatttcggta aggacaaacc ttatttcgat gctgaacacg ttatccaagt ctcgcatggt 12000tggagaacgt ttgatgccat tgctaagttc gctccagact ttgccaatga agagtatgtt 12060aacaaattag aagctgaaat tccggtcaag tacggtgaaa aatccattga agtcccaggt 12120gcagttaagc tgtgcaacgc tttgaacgct ctaccaaaag agaaatgggc tgtggcaact 12180tccggtaccc gtgatatggc acaaaaatgg ttcgagcatc tgggaatcag gagaccaaag 12240tacttcatta ccgctaatga tgtcaaacag ggtaagcctc atccagaacc atatctgaag 12300ggcaggaatg gcttaggata tccgatcaat gagcaagacc cttccaaatc taaggtagta 12360gtatttgaag acgctccagc aggtattgcc gccggaaaag ccgccggttg taagatcatt 12420ggtattgcca ctactttcga cttggacttc ctaaaggaaa aaggctgtga catcattgtc 12480aaaaaccacg aatccatcag agttggcggc tacaatgccg aaacagacga agttgaattc 12540atttttgacg actacttata tgctaaggac gatctgttga aatggtaacc cgggctgcag 12600gcatgcaagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa 12660tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc 12720ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg 12780tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa 12840agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa 12900tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg 12960cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt 13020tgcgtttcta caaactccag ctggatcggg cgctagagta tacatttaaa tggtaccctc 13080tagtcaaggc cttaagtgag tcgtattacg gactggccgt cgttttacaa cgtcgtgact 13140gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 13200ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 13260gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 13320tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 13380cgccaacacc cgctgacgag ct 1340275110DNAartificial sequenceprimer 75gcagtacaaa tgttacgggg taccggcgcg ccgctagctt aattaacgga ccgatgcatg 60agctcacgcg taccggtgct cttcgatcta cgtaagaagg ccttcctatc 11076110DNAartificial sequenceprimer 76gataggaagg ccttcttacg tagatcgaag agcaccggta cgcgtgagct catgcatcgg 60tccgttaatt aagctagcgg cgcgccggta ccccgtaaca tttgtactgc 1107777DNAartificial sequencefragment 77cggcgcgccg ctagcttaat taacggaccg atgcatgagc tcacgcgtac cggtgctctt 60cgatctacgt aagaagg 777813611DNAartificial sequenceplasmid 78aagtgagtcg tattacggac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 60cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 120agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct 180gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 240cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 300tgacgagctt agtaaagccc tcgctagatt ttaatgcgga tgttgcgatt acttcgccaa 360ctattgcgat aacaagaaaa agccagcctt tcatgatata tctcccaatt tgtgtagggc 420ttattatgca cgcttaaaaa taataaaagc agacttgacc tgatagtttg gctgtgagca 480attatgtgct tagtgcatct aacgcttgag ttaagccgcg ccgcgaagcg gcgtcggctt 540gaacgaattg ttagacatta tttgccgact accttggtga tctcgccttt cacgtagtgg 600acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg tccaagataa 660gcctgtctag cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag 720tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac 780aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt 840aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc 900gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga 960tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct 1020ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca 1080atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa 1140aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc 1200aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta caaatgtacg 1260gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc 1320gatacttcgg cgatcaccgc ttccctcatg atgtttaact ttgttttagg gcgactgccc 1380tgctgcgtaa catcgttgct gctccataac atcaaacatc gacccacggc gtaacgcgct 1440tgctgcttgg atgcccgagg catagactgt accccaaaaa aacagtcata acaagccatg 1500aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt 1560gagcgcatac gctacttgca ttacagctta cgaaccgaac aggcttatgt ccactgggtt 1620cgtgccttca tccgtttcca cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc 1680gaggcatttc tgtcctggct ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag 1740gcattggcgg ccttgctgtt cttctacggc aaggtgctgt gcacggatct gccctggctt 1800caggagatcg gaagacctcg gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa 1860gtggttcgca tcctcggttt tctggaaggc gagcatcgtt tgttcgccca gcttctgtat 1920ggaacgggca tgcggatcag tgagggtttg caactgcggg tcaaggatct ggatttcgat 1980cacggcacga tcatcgtgcg ggagggcaag ggctccaagg atcgggcctt gatgttaccc 2040gagagcttgg cacccagcct gcgcgagcag gggaattaat tcccacgggt tttgctgccc 2100gcaaacgggc tgttctggtg ttgctagttt gttatcagaa tcgcagatcc ggcttcagcc 2160ggtttgccgg ctgaaagcgc tatttcttcc agaattgcca tgattttttc cccacgggag 2220gcgtcactgg ctcccgtgtt gtcggcagct ttgattcgat aagcagcatc gcctgtttca 2280ggctgtctat gtgtgactgt tgagctgtaa caagttgtct caggtgttca atttcatgtt 2340ctagttgctt tgttttactg gtttcacctg ttctattagg tgttacatgc tgttcatctg 2400ttacattgtc gatctgttca tggtgaacag ctttgaatgc accaaaaact cgtaaaagct 2460ctgatgtatc tatctttttt acaccgtttt catctgtgca tatggacagt tttccctttg 2520atatgtaacg gtgaacagtt gttctacttt tgtttgttag tcttgatgct tcactgatag 2580atacaagagc cataagaacc tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg 2640gttcgttgtt tttgcgtgag ccatgagaac gaaccattga gatcatactt actttgcatg 2700tcactcaaaa attttgcctc aaaactggtg agctgaattt ttgcagttaa agcatcgtgt 2760agtgtttttc ttagtccgtt atgtaggtag gaatctgatg taatggttgt tggtattttg 2820tcaccattca tttttatctg gttgttctca agttcggtta cgagatccat ttgtctatct 2880agttcaactt ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac caccaatttc 2940atattgctgt aagtgtttaa atctttactt attggtttca aaacccattg gttaagcctt 3000ttaaactcat ggtagttatt ttcaagcatt aacatgaact taaattcatc aaggctaatc 3060tctatatttg ccttgtgagt tttcttttgt gttagttctt ttaataacca ctcataaatc 3120ctcatagagt atttgttttc aaaagactta acatgttcca gattatattt tatgaatttt 3180tttaactgga aaagataagg caatatctct tcactaaaaa ctaattctaa tttttcgctt 3240gagaacttgg catagtttgt ccactggaaa atctcaaagc ctttaaccaa aggattcctg 3300atttccacag ttctcgtcat cagctctctg gttgctttag ctaatacacc ataagcattt 3360tccctactga tgttcatcat ctgagcgtat tggttataag tgaacgatac cgtccgttct 3420ttccttgtag ggttttcaat cgtggggttg agtagtgcca cacagcataa aattagcttg 3480gtttcatgct ccgttaagtc atagcgacta atcgctagtt catttgcttt gaaaacaact 3540aattcagaca tacatctcaa ttggtctagg tgattttaat cactatacca attgagatgg 3600gctagtcaat gataattact agctagtcct tttcctttga gttgtgggta tctgtaaatt 3660ctgctagacc tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa attccgctag 3720acctttgtgt gttttttttg tttatattca agtggttata atttatagaa taaagaaaga 3780ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat aactcactac tttagtcagt 3840tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa acagacctta 3900aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc aaatcgctga atattccttt 3960tgtctccgac catcaggcac ctgagtcgct gtctttttcg tgacattcag ttcgctgcgc 4020tcacggctct ggcagtgaat gggggtaaat ggcactacag gcgcctttta tggattcatg 4080caaggaaact acccataata caagaaaagc ccgtcacggg cttctcaggg cgttttatgg 4140cgggtctgct atgtggtgct atctgacttt ttgctgttca gcagttcctg ccctctgatt 4200ttccagtctg accacttcgg attatcccgt gacaggtcat tcagactggc taatgcaccc 4260agtaaggcag cggtatcatc aacaggctta cccgtcttac tgtcgggaat tcatttaaat 4320agtcaaaagc ctccgaccgg aggcttttga ctgctaggcg atctgtgctg tttgccacgg 4380tatgcagcac cagcgcgaga ttatgggctc gcacgctcga ctgtcggacg ggggcactgg 4440aacgagaagt caggcgagcc gtcacgccct tgacaatgcc acatcctgag caaataattc 4500aaccactaaa caaatcaacc gcgtttcccg gaggtaacca agcttgcggg agagaatgat 4560gaacaagagc caacaagttc agacaatcac cctggccgcc gcccagcaaa tggcggcggc 4620ggtggaaaaa aaagccactg agatcaacgt ggcggtggtg ttttccgtag ttgaccgcgg 4680aggcaacacg ctgcttatcc agcggatgga cgaggccttc gtctccagct gcgatatttc 4740cctgaataaa gcctggagcg cctgcagcct gaagcaaggt acccatgaaa ttacgtcagc 4800ggtccagcca ggacaatctc tgtacggtct gcagctaacc aaccaacagc gaattattat 4860ttttggcggc ggcctgccag ttatttttaa tgagcaggta attggcgccg tcggcgttag 4920cggcggtacg gtcgagcagg atcaattatt agcccagtgc gccctggatt gtttttccgc 4980attataacct gaagcgagaa ggtatattat gagctatcgt atgttccgcc aggcattctg 5040agtgttaacg aggggaccgt catgtcgctt tcaccgccag gcgtacgcct gttttacgat 5100ccgcgcgggc accatgccgg cgccatcaat gagctgtgct gggggctgga ggagcagggg 5160gtcccctgcc agaccataac ctatgacgga ggcggtgacg ccgctgcgct gggcgccctg 5220gcggccagaa gctcgcccct gcgggtgggt atcgggctca gcgcgtccgg cgagatagcc

5280ctcactcatg cccagctgcc ggcggacgcg ccgctggcta ccggacacgt caccgatagc 5340gacgatcaac tgcgtacgct cggcgccaac gccgggcagc tggttaaagt cctgccgtta 5400agtgagagaa actgaatgta tcgtatctat acccgcaccg gggataaagg caccaccgcc 5460ctgtacggcg gcagccgcat cgagaaagac catattcgcg tcgaggccta cggcaccgtc 5520gatgaactga tatcccagct gggcgtctgc tacgccacga cccgcgacgc cgggctgcgg 5580gaaagcctgc accatattca gcagacgctg ttcgtgctgg gggctgaact ggccagcgat 5640gcgcggggcc tgacccgcct gagccagacg atcggcgaag aggagatcac cgccctggag 5700cggcttatcg accgcaatat ggccgagagc ggcccgttaa aacagttcgt gatcccgggg 5760aggaatctcg cctctgccca gctgcacgtg gcgcgcaccc agtcccgtcg gctcgaacgc 5820ctgctgacgg ccatggaccg cgcgcatccg ctgcgcgacg cgctcaaacg ctacagcaat 5880cgcctgtcgg atgccctgtt ctccatggcg cgaatcgaag agactaggcc tgatgcttgc 5940gcttgaactg gcctagcaaa cacagaaaaa agcccgcacc tgacagtgcg ggcttttttt 6000ttcctaggcg atctgtgctg tttgccacgg tatgcagcac cagcgcgaga ttatgggctc 6060gcacgctcga ctgtcggacg ggggcactgg aacgagaagt caggcgagcc gtcacgccct 6120tgacaatgcc acatcctgag caaataattc aaccactaaa caaatcaacc gcgtttcccg 6180gaggtaacca agcttcacct tttgagccga tgaacaatga aaagatcaaa acgatttgca 6240gtactggccc agcgccccgt caatcaggac gggctgattg gcgagtggcc tgaagagggg 6300ctgatcgcca tggacagccc ctttgacccg gtctcttcag taaaagtgga caacggtctg 6360atcgtcgaac tggacggcaa acgccgggac cagtttgaca tgatcgaccg atttatcgcc 6420gattacgcga tcaacgttga gcgcacagag caggcaatgc gcctggaggc ggtggaaata 6480gcccgtatgc tggtggatat tcacgtcagc cgggaggaga tcattgccat cactaccgcc 6540atcacgccgg ccaaagcggt cgaggtgatg gcgcagatga acgtggtgga gatgatgatg 6600gcgctgcaga agatgcgtgc ccgccggacc ccctccaacc agtgccacgt caccaatctc 6660aaagataatc cggtgcagat tgccgctgac gccgccgagg ccgggatccg cggcttctca 6720gaacaggaga ccacggtcgg tatcgcgcgc tacgcgccgt ttaacgccct ggcgctgttg 6780gtcggttcgc agtgcggccg ccccggcgtg ttgacgcagt gctcggtgga agaggccacc 6840gagctggagc tgggcatgcg tggcttaacc agctacgccg agacggtgtc ggtctacggc 6900accgaagcgg tatttaccga cggcgatgat acgccgtggt caaaggcgtt cctcgcctcg 6960gcctacgcct cccgcgggtt gaaaatgcgc tacacctccg gcaccggatc cgaagcgctg 7020atgggctatt cggagagcaa gtcgatgctc tacctcgaat cgcgctgcat cttcattact 7080aaaggcgccg gggttcaggg actgcaaaac ggcgcggtga gctgtatcgg catgaccggc 7140gctgtgccgt cgggcattcg ggcggtgctg gcggaaaacc tgatcgcctc tatgctcgac 7200ctcgaagtgg cgtccgccaa cgaccagact ttctcccact cggatattcg ccgcaccgcg 7260cgcaccctga tgcagatgct gccgggcacc gactttattt tctccggcta cagcgcggtg 7320ccgaactacg acaacatgtt cgccggctcg aacttcgatg cggaagattt tgatgattac 7380aacatcctgc agcgtgacct gatggttgac ggcggcctgc gtccggtgac cgaggcggaa 7440accattgcca ttcgccagaa agcggcgcgg gcgatccagg cggttttccg cgagctgggg 7500ctgccgccaa tcgccgacga ggaggtggag gccgccacct acgcgcacgg cagcaacgag 7560atgccgccgc gtaacgtggt ggaggatctg agtgcggtgg aagagatgat gaagcgcaac 7620atcaccggcc tcgatattgt cggcgcgctg agccgcagcg gctttgagga tatcgccagc 7680aatattctca atatgctgcg ccagcgggtc accggcgatt acctgcagac ctcggccatt 7740ctcgatcggc agttcgaggt ggtgagtgcg gtcaacgaca tcaatgacta tcaggggccg 7800ggcaccggct atcgcatctc tgccgaacgc tgggcggaga tcaaaaatat tccgggcgtg 7860gttcagcccg acaccattga ataaggcggt attcctgtgc aacagacaac ccaaattcag 7920ccctctttta ccctgaaaac ccgcgagggc ggggtagctt ctgccgatga acgcgccgat 7980gaagtggtga tcggcgtcgg ccctgccttc gataaacacc agcatcacac tctgatcgat 8040atgccccatg gcgcgatcct caaagagctg attgccgggg tggaagaaga ggggcttcac 8100gcccgggtgg tgcgcattct gcgcacgtcc gacgtctcct ttatggcctg ggatgcggcc 8160aacctgagcg gctcggggat cggcatcggt atccagtcga aggggaccac ggtcatccat 8220cagcgcgatc tgctgccgct cagcaacctg gagctgttct cccaggcgcc gctgctgacg 8280ctggagacct accggcagat tggcaaaaac gctgcgcgct atgcgcgcaa agagtcacct 8340tcgccggtgc cggtggtgaa cgatcagatg gtgcggccga aatttatggc caaagccgcg 8400ctatttcata tcaaagagac caaacatgtg gtgcaggacg ccgagcccgt caccctgcac 8460atcgacttag taagggagtg accatgagcg agaaaaccat gcgcgtgcag gattatccgt 8520tagccacccg ctgcccggag catatcctga cgcctaccgg caaaccattg accgatatta 8580ccctcgagaa ggtgctctct ggcgaggtgg gcccgcagga tgtgcggatc tcccgccaga 8640cccttgagta ccaggcgcag attgccgagc agatgcagcg ccatgcggtg gcgcgcaatt 8700tccgccgcgc ggcggagctt atcgccattc ctgacgagcg cattctggct atctataacg 8760cgctgcgccc gttccgctcc tcgcaggcgg agctgctggc gatcgccgac gagctggagc 8820acacctggca tgcgacagtg aatgccgcct ttgtccggga gtcggcggaa gtgtatcagc 8880agcggcataa gctgcgtaaa ggaagctaag cggaggtcag catgccgtta atagccggga 8940ttgatatcgg caacgccacc accgaggtgg cgctggcgtc cgactacccg caggcgaggg 9000cgtttgttgc cagcgggatc gtcgcgacga cgggcatgaa agggacgcgg gacaatatcg 9060ccgggaccct cgccgcgctg gagcaggccc tggcgaaaac accgtggtcg atgagcgatg 9120tctctcgcat ctatcttaac gaagccgcgc cggtgattgg cgatgtggcg atggagacca 9180tcaccgagac cattatcacc gaatcgacca tgatcggtca taacccgcag acgccgggcg 9240gggtgggcgt tggcgtgggg acgactatcg ccctcgggcg gctggcgacg ctgccggcgg 9300cgcagtatgc cgaggggtgg atcgtactga ttgacgacgc cgtcgatttc cttgacgccg 9360tgtggtggct caatgaggcg ctcgaccggg ggatcaacgt ggtggcggcg atcctcaaaa 9420aggacgacgg cgtgctggtg aacaaccgcc tgcgtaaaac cctgccggtg gtggatgaag 9480tgacgctgct ggagcaggtc cccgaggggg taatggcggc ggtggaagtg gccgcgccgg 9540gccaggtggt gcggatcctg tcgaatccct acgggatcgc caccttcttc gggctaagcc 9600cggaagagac ccaggccatc gtccccatcg cccgcgccct gattggcaac cgttccgcgg 9660tggtgctcaa gaccccgcag ggggatgtgc agtcgcgggt gatcccggcg ggcaacctct 9720acattagcgg cgaaaagcgc cgcggagagg ccgatgtcgc cgagggcgcg gaagccatca 9780tgcaggcgat gagcgcctgc gctccggtac gcgacatccg cggcgaaccg ggcacccacg 9840ccggcggcat gcttgagcgg gtgcgcaagg taatggcgtc cctgaccggc catgagatga 9900gcgcgatata catccaggat ctgctggcgg tggatacgtt tattccgcgc aaggtgcagg 9960gcgggatggc cggcgagtgc gccatggaga atgccgtcgg gatggcggcg atggtgaaag 10020cggatcgtct gcaaatgcag gttatcgccc gcgaactgag cgcccgactg cagaccgagg 10080tggtggtggg cggcgtggag gccaacatgg ccatcgccgg ggcgttaacc actcccggct 10140gtgcggcgcc gctggcgatc ctcgacctcg gcgccggctc gacggatgcg gcgatcgtca 10200acgcggaggg gcagataacg gcggtccatc tcgccggggc ggggaatatg gtcagcctgt 10260tgattaaaac cgagctgggc ctcgaggatc tttcgctggc ggaagcgata aaaaaatacc 10320cgctggccaa agtggaaagc ctgttcagta ttcgtcacga gaatggcgcg gtggagttct 10380ttcgggaagc cctcagcccg gcggtgttcg ccaaagtggt gtacatcaag gagggcgaac 10440tggtgccgat cgataacgcc agcccgctgg aaaaaattcg tctcgtgcgc cggcaggcga 10500aagagaaagt gtttgtcacc aactgcctgc gcgcgctgcg ccaggtctca cccggcggtt 10560ccattcgcga tatcgccttt gtggtgctgg tgggcggctc atcgctggac tttgagatcc 10620cgcagcttat cacggaagcc ttgtcgcact atggcgtggt cgccgggcag ggcaatattc 10680ggggaacaga agggccgcgc aatgcggtcg ccaccgggct gctactggcc ggtcaggcga 10740attaaacggg cgctcgcgcc agcctctagg tacaaataaa aaaggcacgt cagatgacgt 10800gccttttttc ttgtctagcg tgcaccaatg cttctggcgt caggcagcca tcggaagctg 10860tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc 10920gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga 10980gctgttgaca attaatcatc cggctcgtat aatgtgtgga attgtgagcg gataacaatt 11040tcacacagga aacagaccat gactagtaag gaggacaatt ccatggctgc tgctgctgat 11100agattaaact taacttccgg ccacttgaat gctggtagaa agagaagttc ctcttctgtt 11160tctttgaagg ctgccgaaaa gcctttcaag gttactgtga ttggatctgg taactggggt 11220actactattg ccaaggtggt tgccgaaaat tgtaagggat acccagaagt tttcgctcca 11280atagtacaaa tgtgggtgtt cgaagaagag atcaatggtg aaaaattgac tgaaatcata 11340aatactagac atcaaaacgt gaaatacttg cctggcatca ctctacccga caatttggtt 11400gctaatccag acttgattga ttcagtcaag gatgtcgaca tcatcgtttt caacattcca 11460catcaatttt tgccccgtat ctgtagccaa ttgaaaggtc atgttgattc acacgtcaga 11520gctatctcct gtctaaaggg ttttgaagtt ggtgctaaag gtgtccaatt gctatcctct 11580tacatcactg aggaactagg tattcaatgt ggtgctctat ctggtgctaa cattgccacc 11640gaagtcgctc aagaacactg gtctgaaaca acagttgctt accacattcc aaaggatttc 11700agaggcgagg gcaaggacgt cgaccataag gttctaaagg ccttgttcca cagaccttac 11760ttccacgtta gtgtcatcga agatgttgct ggtatctcca tctgtggtgc tttgaagaac 11820gttgttgcct taggttgtgg tttcgtcgaa ggtctaggct ggggtaacaa cgcttctgct 11880gccatccaaa gagtcggttt gggtgagatc atcagattcg gtcaaatgtt tttcccagaa 11940tctagagaag aaacatacta ccaagagtct gctggtgttg ctgatttgat caccacctgc 12000gctggtggta gaaacgtcaa ggttgctagg ctaatggcta cttctggtaa ggacgcctgg 12060gaatgtgaaa aggagttgtt gaatggccaa tccgctcaag gtttaattac ctgcaaagaa 12120gttcacgaat ggttggaaac atgtggctct gtcgaagact tcccattatt tgaagccgta 12180taccaaatcg tttacaacaa ctacccaatg aagaacctgc cggacatgat tgaagaatta 12240gatctacatg aagattagat ttattggatc caggaaacag actagaatta tgggattgac 12300tactaaacct ctatctttga aagttaacgc cgctttgttc gacgtcgacg gtaccattat 12360catctctcaa ccagccattg ctgcattctg gagggatttc ggtaaggaca aaccttattt 12420cgatgctgaa cacgttatcc aagtctcgca tggttggaga acgtttgatg ccattgctaa 12480gttcgctcca gactttgcca atgaagagta tgttaacaaa ttagaagctg aaattccggt 12540caagtacggt gaaaaatcca ttgaagtccc aggtgcagtt aagctgtgca acgctttgaa 12600cgctctacca aaagagaaat gggctgtggc aacttccggt acccgtgata tggcacaaaa 12660atggttcgag catctgggaa tcaggagacc aaagtacttc attaccgcta atgatgtcaa 12720acagggtaag cctcatccag aaccatatct gaagggcagg aatggcttag gatatccgat 12780caatgagcaa gacccttcca aatctaaggt agtagtattt gaagacgctc cagcaggtat 12840tgccgccgga aaagccgccg gttgtaagat cattggtatt gccactactt tcgacttgga 12900cttcctaaag gaaaaaggct gtgacatcat tgtcaaaaac cacgaatcca tcagagttgg 12960cggctacaat gccgaaacag acgaagttga attcattttt gacgactact tatatgctaa 13020ggacgatctg ttgaaatggt aacccgggct gcaggcatgc aagcttggct gttttggcgg 13080atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa 13140acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga 13200agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg 13260ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt 13320gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg 13380cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa 13440ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact ccagctggat 13500cgggcgctag agtatacatt taaatggtac cggcgcgccg ctagcttaat taacggaccg 13560atgcatgagc tcacgcgtac cggtgctctt cgatctacgt aagaaggcct t 13611794490DNAartificial sequenceplasmid 79tagtaaagcc ctcgctagat tttaatgcgg atgttgcgat tacttcgcca actattgcga 60taacaagaaa aagccagcct ttcatgatat atctcccaat ttgtgtaggg cttattatgc 120acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc 180ttagtgcatc taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt 240gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 300tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 360gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 420acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 480actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 540tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 600cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 660atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 720agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 780tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 840atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 900tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 960gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 1020gcgatcaccg cttccctcat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 1080acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 1140gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 1200actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata 1260cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc 1320atccgtttcc acggtgtgcg tcacccggca accttgggca gcagcgaagt cgaggcattt 1380ctgtcctggc tggcgaacga gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg 1440gccttgctgt tcttctacgg caaggtgctg tgcacggatc tgccctggct tcaggagatc 1500ggaagacctc ggccgtcgcg gcgcttgccg gtggtgctga ccccggatga agtggttcgc 1560atcctcggtt ttctggaagg cgagcatcgt ttgttcgccc agcttctgta tggaacgggc 1620atgcggatca gtgagggttt gcaactgcgg gtcaaggatc tggatttcga tcacggcacg 1680atcatcgtgc gggagggcaa gggctccaag gatcgggcct tgatgttacc cgagagcttg 1740gcacccagcc tgcgcgagca ggggaattaa ttcccacggg ttttgctgcc cgcaaacggg 1800ctgttctggt gttgctagtt tgttatcaga atcgcagatc cggcttcagc cggtttgccg 1860gctgaaagcg ctatttcttc cagaattgcc atgatttttt ccccacggga ggcgtcactg 1920gctcccgtgt tgtcggcagc tttgattcga taagcagcat cgcctgtttc aggctgtcta 1980tgtgtgactg ttgagctgta acaagttgtc tcaggtgttc aatttcatgt tctagttgct 2040ttgttttact ggtttcacct gttctattag gtgttacatg ctgttcatct gttacattgt 2100cgatctgttc atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat 2160ctatcttttt tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac 2220ggtgaacagt tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag 2280ccataagaac ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 2340ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2400aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2460cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2520atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2580tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2640taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca 2700tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2760gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2820tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2880aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2940gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 3000gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 3060atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 3120gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 3180tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 3240atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 3300tgataattac tagtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3360gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3420ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3480aaaagaatag atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac 3540aaaaggatgt cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc 3600ttaagtagca ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat 3660caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc 3720agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc 3780cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg 3840tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc 3900acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg 3960tatcatcaac aggcttaccc gtcttactgt cgggaattca tttaaatagt caaaagcctc 4020cgaccggagg cttttgactg ctagcaaaca cagaaaaaag cccgcacctg acagtgcggg 4080cttttttttt cctaggtaca aataaaaaag gcacgtcaga tgacgtgcct tttttcttgt 4140ctagagtata catttaaatg gtaccctcta gtcaaggcct taagtgagtc gtattacgga 4200ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 4260cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 4320ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt 4380acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat 4440gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgagct 44908033DNAartificial sequenceprimer 80cataagcttg cgggagagaa tgatgaacaa gag 338131DNAartificial sequenceprimer 81acgcctaggc cagttcaagc gcaagcatca g 318225DNAartificial sequenceprimer 82ctttaatctg cacaccccaa cccgc 258325DNAartificial sequenceprimer 83ggcggtatca atcgagcgat aaccc 25

Claims

1-5. (canceled)

6. A DNA construct comprising at least three transcriptional terminators and at least one cloning site situated between any two transcriptional terminators.

7. The DNA construct of claim 6 wherein the transcriptional terminators are tonB, thrA, or aspA, and the cloning sites are selected from the group consisting of AvrII, NheI, BfaI, Cac81, BsaJI, and StyI.

8. A library of DNA constructs, each DNA construct comprising at least three transcriptional terminators and at least one cloning site situated between any two transcriptional terminators.

9. A DNA construct selected from the group consisting ofa) A pSYCO109mcs plasmid consisting of SEQ ID NO:30,b) A DNA construct for the short 1.5 GI promoter consisting of SEQ ID NO:31,c) A DNA construct for the short 1.20 GI promoter consisting of SEQ ID NO:32,d) A DNA construct for the pAH105 plasmid consisting of SEQ ID NO:70,e) A DNA construct for the pSYCO101 plasmid consisting of SEQ ID NO:71,f) A DNA construct for the pSYCO103 plasmid consisting of SEQ ID NO:72,g) A DNA construct for the pSYCO106 plasmid consisting of SEQ ID NO:73,h) A DNA construct for the pSYCO109 plasmid consisting of SEQ ID NO:74,i) A DNA construct for the pSCYO106mcs plasmid consisting of SEQ ID NO:78, andj) A DNA construct for the pRJ50 plasmid consisting of SEQ ID NO:79.

10. A vector having a multiple cloning site containing restriction recognition site sequences specific for the restriction endonucleases AscI, NheI, PacI, RsrII, NsiI, SacI, MluI, AgeI, SapI, and SnaBI.

11. The vector of claim 10 wherein the multiple cloning site has the nucleotide sequence of SEQ ID NO:77.

12-13. (canceled)