Novel method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same

Abstract

te specific integration of a desired DNA at a target site in a mammalian cell via homologous recombination is described. This method provides for the reproducible selection of cell lines wherein a desired DNA is integrated at a predetermined transcriptionally active site previously marked with a marker plasmid. The method is particularly suitable for the production of mammalian cell lines which secrete mammalian proteins at high levels, in particular immunoglobulins. Novel vectors and vector combinations for use in the subject cloning method are also provided.

Description

RELATED APPLICATIONS

[0001]This application is a continuation-in-part of U.S. Ser. No. 08/819,866, filed on Mar. 14, 1997.

FIELD OF THE INVENTION

[0002]The present invention relates to a process of targeting the integration of a desired exogenous DNA to a specific location within the genome or a mammalian cell. More specifically, the invention describes a novel method for identifying a transcriptionally active target site ("hot spot") in the mammalian genome, and inserting a desired DNA at this site via homologous recombination. The invention also optionally provides the ability for gene amplification of the desired DNA at this location by co-integrating an amplifiable selectable marker, e.g., DHFR, in combination with the exogenous DNA. The invention additionally describes the construction of novel vectors suitable for accomplishing the above, and further provides mammalian cell lines produced by such methods which contain a desired exogenous DNA integrated at a target hot spot.

BACKGROUND

[0003]Technology for expressing recombinant proteins in both prokaryotic and eukaryotic organisms is well established. Mammalian cells offer significant advantages over bacteria or yeast for protein production, resulting from their ability to correctly assemble, glycosylate and post-translationally modify recombinantly expressed proteins. After transfection into the host cells, recombinant expression constructs can be maintained as extrachromosomal elements, or may be integrated into the host cell genome. Generation of stably transfected mammalian cell lines usually involves the latter; a DNA construct encoding a gene of interest along with a drug resistance gene (dominant selectable marker) is introduced into the host cell, and subsequent growth in the presence of the drug allows for the selection of cells that have successfully integrated the exogenous DNA. In many instances, the gene of interest is linked to a drug resistant selectable marker which can later be subjected to gene amplification. The gene encoding dihydrofolate reductase (DHFR) is most commonly used for this purpose. Growth of cells in the presence of methotrexate, a competitive inhibitor of DHFR, leads to increased DHFR production by means of amplification of the DHFR gene. As flanking regions of DNA will also become amplified, the resultant coamplification of a DHFR linked gene in the transfected cell line can lead to increased protein production, thereby resulting in high level expression of the gene of interest.

[0004]While this approach has proven successful, there are a number of problems with the system because of the random nature of the integration event. These problems exist because expression levels are greatly influenced by the effects of the local genetic environment at the gene locus, a phenomena well documented in the literature and generally referred to as "position effects" (for example, see Al-Shawi et al, Mol. Cell. Biol., 10:1192-1198 (1990); Yoshimura et al, Mol. Cell. Biol., 7:1296-1299 (1987)). As the vast majority of mammalian DNA is in a transcriptionally inactive state, random integration methods offer no control over the transcriptional fate of the integrated DNA. Consequently, wide variations in the expression level of integrated genes can occur, depending on the site of integration. For example, integration of exogenous DNA into inactive, or transcriptionally "silent" regions of the genome will result in little or no expression. By contrast integration into a transcriptionally active site may result in high expression.

[0005]Therefore, when the goal of the work is to obtain a high level of gene expression, as is typically the desired outcome of genetic engineering methods, it is generally necessary to screen large numbers of transfectants to find such a high producing clone. Additionally, random integration of exogenous DNA into the genome can in some instances disrupt important cellular genes, resulting in an altered phenotype. These factors can make the generation of high expressing stable mammalian cell lines a complicated and laborious process.

[0006]Recently, our laboratory has described the use of DNA vectors containing translationally impaired dominant selectable markers in mammalian gene expression. (This is disclosed in U.S. Ser. No. 08/147,696 filed Nov. 3, 1993, recently allowed).

[0007]These vectors contain a translationally impaired neomycin phosphotransferase (neo) gene as the dominant selectable marker, artificially engineered to contain an intron into which a DHFR gene along with a gene or genes of interest is inserted. Use of these vectors as expression constructs has been found to significantly reduce the total number of drug resistant colonies produced, thereby facilitating the screening procedure in relation to conventional mammalian expression vectors. Furthermore, a significant percentage of the clones obtained using this system are high expressing clones. These results are apparently attributable to the modifications made to the neo selectable marker. Due to the translational impairment of the neo gene, transfected cells will not produce enough neo protein to survive drug selection, thereby decreasing the overall number of drug resistant colonies. Additionally, a higher percentage of the surviving clones will contain the expression vector integrated into sites in the genome where basal transcription levels are high, resulting in overproduction of neo, thereby allowing the cells to overcome the impairment of the neo gene. Concomitantly, the genes of interest linked to neo will be subject to similar elevated levels of transcription. This same advantage is also true as a result of the artificial intron created within neo; survival is dependent on the synthesis of a functional neo gene, which is in turn dependent on correct and efficient splicing of the neo introns. Moreover, these criteria are more likely to be met if the vector DNA has integrated into a region which is already highly transcriptionally active.

[0008]Following integration of the vector into a transcriptionally active region, gene amplification is performed by selection for the DHFR gene. Using this system, it has been possible to obtain clones selected using low levels of methotrexate (50 nM), containing few (<10) copies of the vector which secrete high levels of protein (>55 pg/cell/day). Furthermore, this can be. achieved in a relatively short period of time. However, the success in amplification is variable. Some transcriptionally active sites cannot be amplified and therefore the frequency and extent of amplification from a particular site is not predictable.

[0009]Overall, the use of these translationally impaired vectors represents a significant improvement over other methods of random integration. However, as discussed, the problem of lack of control over the integration site remains a significant concern.

[0010]One approach to overcome the problems of random integration is by means of gene targeting, whereby the exogenbus DNA is directed to a specific locus within the host genome. The exogenous DNA is inserted by means of homologous recombination occurring between sequences of DNA in the expression vector and the corresponding homologous sequence in the genome. However, while this type of recombination occurs at a high frequency naturally in yeast and other fungal organisms, in higher eukaryotic organisms it is an extremely rare event. In mammalian cells, the frequency of homologous versus non-homologous (random integration) recombination is reported to range from 1/100 to 1/5000 (for example, see Capecchi, Science, 244:1288-1292 .(1989); Morrow and Kucherlapati, Curr. Op. Biotech., 4:577-582 (1993)).

[0011]One of the earliest reports describing homologous recombination in mammalian cells comprised an artificial system created in mouse fibroblasts (Thomas et al, Cell, 44:419-428 (1986)). A cell line containing a mutated, non-functional version of the neo gene integrated into the host genome was created, and subsequently targeted with a second non-functional copy of neo containing a different mutation. Reconstruction of a functional neo gene could occur only by gene targeting. Homologous recombinants were identified by selecting for G418 resistant cells, and confirmed by analysis of genomic DNA isolated from the resistant clones.

[0012]Recently, the use of homologous recombination to replace the heavy and light immunoglobulin genes at endogenous loci in antibody secreting cells has been reported. (U.S. Pat. No. 5,202,238, Fell et al, (1993).) However, this particular approach is not widely applicable, because it is limited to the production of immunoglobulins in cells which endogenously express immunoglobulins, e.g., B cells and myeloma cells. Also, expression is limited to single copy gene levels because co-amplification after homologous recombination is not included. The method is further complicated by the fact that two separate integration events are required to produce a functional immunoglobulin: one for the light chain gene followed by one for the heavy chain gene.

[0013]An additional example of this type of system has been reported in NS/0 cells, where recombinant immunoglobulins are expressed by homologous recombination into the immunoglobulin gamma 2A locus (Hollis et al, international patent application # PCT/IB95 (00014).) Expression levels obtained from this site were extremely high--on the order of 20 pg/cell/day from a single copy integrant. However, as in the above example, expression is limited to this level because an amplifiable gene is not contegrated in this system. Also, other researchers have reported aberrant glycosylation of recombinant proteins expressed in NS/0 cells (for example, see Flesher et al, Biotech. and Bioeng., 48:399-407 (1995)), thereby limiting the applicability of this approach.

[0014]The cre-loxP recombination system from bacteriophage P1 has recently been adapted and used as a means of gene targeting in eukaryotic cells. Specifically, the site specific integration of exogenous DNA into the Chinese hamster ovary (CHO) cell genome using cre recombinase and a series of lox containing vectors have been described. (Fukushige and Sauer, Proc. Natl. Acad. Sci. USA, 89:7905-7909 (1992).) This system is attractive in that it provides for reproducible expression at the same chromosomal location. However, no effort was made to identify a chromosomal site from which gene expression is optimal, and as in the above example, expression is limited to single copy levels in this system. Also, it is complicated by the fact that one needs to provide for expression of a functional recombinase enzyme in the mammalian cell.

[0015]The use of homologous recombination between an introduced DNA sequence and its endogenous chromosomal locus has also been reported to provide a useful means of genetic manipulation in mammalian cells, as well as in yeast cells. (See e.g., Bradley et al, Meth. Enzymol., 223:855-879 (1993); Capecchi, Science, 244:1288-1292 (1989); Rothstein et al, Meth. Enzymol., 194:281-301 (1991)). To date, most mammalian gene targeting studies have been directed toward gene disruption ("knockout") or site-specific mutagenesis of selected target gene loci in mouse embryonic stem (ES) cells. The creation of these "knockout" mouse models has enabled scientists to examine specific structure-function issues and examine the biological importance of a myriad of mouse genes. This field of research also has important implications in terms of potential gene therapy applications.

[0016]Also, vectors have recently been reported by Cell-tech (Kent, U.K.) which purportedly are targeted to transcriptionally active sites in NSO cells, which do not require gene amplification (Peakman et al, Hum. Antibod. Hybridomas, 5:65-74 (1994)). However, levels of immunoglobulin secretion in these unamplified cells have not been reported to exceed 20 pg/cell/day, while in amplified CHO cells, levels as high as 100 pg/cell/day can be obtained (Id.).

[0017]It would be highly desirable to develop a gene targeting system which reproducibly provided for the integration of exogenous DNA into a predetermined site in the genome known to be transcriptionally active. Also, it would be desirable if such a gene targeting system would further facilitate co-amplification of the inserted DNA after integration. The design of such a system would allow for the reproducible and high level expression of any cloned gene of interest in a mammalian cell, and undoubtedly would be of significant interest to many researchers.

[0018]In this application, we provide a novel mammalian expression system, based on homologous recombination occurring between two artificial substrates contained in two different vectors. Specifically, this system uses a combination of two novel mammalian expression vectors, referred to as a "marking" vector and a "targeting" vector.

[0019]Essentially, the marking vector enables the identification and marking of a site in the mammalian genome which is transcriptionally active, i.e., a site at which gene expression levels are high. This site can be regarded as a "hot spot" in the genome. After integration of the marking vector, the subject expression system enables another DNA to be integrated at this site, i.e., the targeting vector, by means of homologous recombination occurring between DNA sequences common to both vectors. This system affords significant advantages over other homologous recombination systems.

[0020]Unlike most other homologous systems employed in mammalian cells, this system exhibits no background. Therefore, cells which have only undergone random integration of the vector do not survive the selection. Thus, any gene of interest cloned into the targeting plasmid is expressed at high levels from the marked hot spot. Accordingly, the subject method of gene expression substantially or completely eliminates the problems inherent to systems of random integration, discussed in detail above. Moreover, this system provides reproducible and high level expression of any recombinant protein at the same transcriptionally active site in the mammalian genome. In addition, gene amplification may be effected at this particular transcriptionally active site by including an amplifiable dominant selectable marker (e.g. DHFR) as part of the marking vector.

OBJECTS OF THE INVENTION

[0021]Thus, it is an object of the invention to provide an improved method for targeting a desired DNA to a specific site in a mammalian cell.

[0022]It is a more specific object of the invention to provide a novel method for targeting a desired DNA to a specific site in a mammalian cell via homologous recombination.

[0023]It is another specific object of the invention to provide novel vectors for achieving site specific integration of a desired DNA in a mammalian cell.

[0024]It is still another object of the invention to provide novel mammalian cell lines which contain a desired DNA integrated at a predetermined site which provides for high expression.

[0025]It is a more specific object of the invention to provide a novel method for achieving site specific integration of a desired DNA in a Chinese hamster ovary (CHO) cell.

[0026]It is another more specific object of the invention to provide a novel method for integrating immunoglobulin genes, or any other genes, in mammalian cells at predetermined chromosomal sites that provide for high expression.

[0027]It is another specific object of the invention to provide novel vectors and vector combinations suitable for integrating immunoglobulin genes into mammalian cells at predetermined sites that provide for high expression.

[0028]It is another object of the invention to provide mammalian cell lines which contain immunoglobulin genes integrated at predetermined sites that provide for high expression.

[0029]It is an even more specific object of the invention to provide a novel method for integrating immunoglobulin genes into CHO cells that provide for high expression, as well as novel vectors and vector combinations that provide for such integration of immunoglobulin genes into CHO cells.

[0030]In addition, it is a specific object of the invention to provide novel CHO cell lines which contain immunoglobulin genes integrated at predetermined sites that provide for high expression, and have been amplified by methotrexate selection to secrete even greater amounts of functional immunoglobulins.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1 depicts a map of a marking plasmid according to the invention referred to as Desmond. The plasmid is shown in circular form (1a) as well as a linearized version used for transfection (1b).

[0032]FIG. 2(a) shows a map of a targeting plasmid referred to "Molly". Molly is shown here encoding the anti-CD20 immunoglobulin genes, expression of which is described in Example 1.

[0033]FIG. 2(b) shows a linearized version of Molly, after digestion with the restriction enzymes Kpn1 and Pac1. This linearized form was used for transfection.

[0034]FIG. 3 depicts the potential alignment between Desmond sequences integrated into the CHO genome, and incoming targeting Molly sequences. One potential arrangement of Molly integrated into Desmond after homologous recombination is also presented.

[0035]FIG. 4 shows a Southern analysis of single copy Desmond clones. Samples are as follows:

Lane 1: λHindIII DNA size markerLane 2: Desmond clone 10F3Lane 3: Desmond clone 10C12Lane 4: Desmond clone 15C9Lane 4: Desmond clone 14B5Lane 6: Desmond clone 9B2

[0036]FIG. 5 shows a Northern analysis of single copy Desmond clones. Samples are as follows: Panel A: northern probed with CAD and DHFR probes, as indicated on the figure. Panel B: duplicate northern, probed with CAD and HisD probes, as indicated. The RNA samples loaded in panels A and B are as follows:

Lane 1: clone 9B2, lane 2; clone 10C12, lane 3; clone 14B5, lane 4; clone 15C9, lane 5; control RNA from CHO transfected with a HisD and DHFR containing plasmid, lane 6; untransfected CHO.

[0037]FIG. 6 shows a Southern analysis of clones resulting from the homologous integration of Molly into Desmond. Samples are as follows:

Lane 1: λHindIII DNA size markers, Lane 2: 20F4, lane 3; 5F9, lane 4; 21C7, lane 5; 24G2, lane 6; 25E1, lane 7; 28C9, lane 8; 29F9, lane 9; 39G11, lane 10; 42F9, lane 11; 50G10, lane 12; Molly plasmid DNA, linearized with BglII(top band) and cut with BglII and KpnI (lower band), lane 13; untransfected Desmond.

[0038]FIGS. 7A through 7G contain the Sequence Listing for Desmond.

[0039]FIGS. 8A through 8I contain the Sequence Listing for Molly-containing anti-CD20.

[0040]FIG. 9 contains a map of the targeting plasmid, "Mandy," shown here encoding anti-CD23 genes, the expression of which is disclosed in Example 5.

[0041]FIGS. 10A through 10N contain the sequence listing of "Mandy" containing the anti-CD23 genes as disclosed in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

[0042]The invention provides a novel method for integrating a desired exogenous DNA at a target site within the genome of a mammalian cell via homologous recombination. Also, the invention provides novel vectors for achieving the site specific integration of a DNA at a target site in the genome of a mammalian cell.

[0043]More specifically, the subject cloning method provides for site specific integration of a desired DNA in a mammalian cell by transfection of such cell with a "marker plasmid" which contains a unique sequence that is foreign to the mammalian cell genome and which provides a substrate for homologous recombination, followed by transfection with a "target plasmid" containing a sequence which provides for homologous recombination with the unique sequence contained in the marker plasmid, and further comprising a desired DNA that is to be integrated into the mammalian cell. Typically, the integrated DNA will encode a protein of interest, such as an immunoglobulin or other secreted mammalian glycoprotein.

[0044]The exemplified homologous recombination system uses the neomycin phosphotransferase gene as a dominant selectable marker. This particular marker was utilized based on the following previously published observations;

[0045](i) the demonstrated ability to target and restore function to a mutated version of the neo gene (cited earlier) and

[0046](ii) our development of translationally impaired expression vectors, in which the neo gene has been artificially created as two exons with a gene of interest inserted in the intervening intron; neo exons are correctly spliced and translated in vivo, producing a functional protein and thereby conferring G418 resistance on the resultant cell population. In this application, the neo gene is split into three exons. The third exon of neo is present on the "marker" plasmid and becomes integrated into the host cell genome upon integration of the marker plasmid into the mammalian cells. Exons 1 and 2 are present on the targeting plasmid, and are separated by an intervening intron into which at least one gene of interest is cloned. Homologous recombination of the targeting vector with the integrated marking vector results in correct splicing of all three exons of the neo gene and thereby expression of a functional neo protein (as determined by selection for G418 resistant colonies). Prior to designing the current expression system, we had experimentally tested the functionality of such a triply spliced neo construct in mammalian cells. The results of this control experiment indicated that all three neo exons were properly spliced and therefore suggested the feasibility of the subject invention.

[0047]However, while the present invention is exemplified using the neo gene, and more specifically a triple split neo gene, the general methodology should be efficacious with other dominant selectable markers.

[0048]As discussed in greater detail infra, the present invention affords numerous advantages to conventional gene expression methods, including both random integration and gene targeting methods. Specifically, the subject invention provides a method which reproducibly allows for site-specific integration of a desired DNA into a transcriptionally active domain of a mammalian cell. Moreover, because the subject method introduces an artificial region of "homology" which acts as a unique substrate for homologous recombination and the insertion of a desired DNA, the efficacy of subject invention does not require that the cell endogenously contain or express a specific DNA. Thus, the method is generically applicable to all mammalian cells, and can be used to express any type of recombinant protein.

[0049]The use of a triply spliced selectable marker, e.g., the exemplified triply spliced neo construct, guarantees that all G418 resistant colonies produced will arise from a homologous recombination event (random integrants will not produce a functional neo gene and consequently will not survive G418 selection). Thus, the subject invention makes it easy to screen for the desired homologous event. Furthermore, the frequency of additional random integrations in a cell that has undergone a homologous recombination event appears to be low.

[0050]Based on the foregoing, it is apparent that a significant advantage of the invention is that it substantially reduces the number of colonies that need be screened to identify high producer clones, i.e., cell lines containing a desired DNA which secrete the corresponding protein at high levels. On average, clones containing integrated desired DNA may be identified by screening about 5 to 20 colonies (compared to several thousand which must be screened when using standard random integration techniques, or several hundred using the previously described intronic insertion vectors) Additionally, as the site of integration was preselected and comprises a transcriptionally active domain, all exogenous DNA expressed at this site should produce comparable, i.e. high levels of the protein of interest.

[0051]Moreover, the subject invention is further advantageous in that it enables an amplifiable gene to be inserted on integration of the marking vector. Thus, when a desired gene is targeted to this site via. homologous recombination, the subject invention allows for expression of the gene to be further enhanced by gene amplification. In this regard, it has been reported in from the literature that different genomic sites e different capacities for gene amplification (Meinkoth et al, Mol. Cell Biol., 7:1415-1424 (1987)). Therefore, this technique is further advantageous as it allows for the placement of a desired gene of interest at a specific site that is both transcriptionally active and easily amplified. Therefore, this should significantly reduce the amount of time required to isolate such high producers.

[0052]Specifically, while conventional methods for the construction of high expressing mammalian cell lines can take 6 to 9 months, the present invention allows for such clones to be isolated on average after only about 3-6 months. This is due to the fact that conventionally isolated clones typically must be subjected to at least three rounds of drug resistant gene amplification in order to reach satisfactory levels of gene expression. As the homologously produced clones are generated from a preselected site which is a high expression site, fewer rounds of amplification should be required before reaching a satisfactory level of production.

[0053]Still further, the subject invention enables the reproducible selection of high producer clones wherein the vector is integrated at low copy number, typically single copy. This is advantageous as it enhances the stability of the clones and avoids other potential adverse-side-effects associated with high copy number. As described supra, the subject homologous recombination system uses the combination of a "marker plasmid" and a "targeting plasmid" which are described in more detail below.

[0054]The "marker plasmid" which is used to mark and identify a transcriptionally hot spot will comprise at least the following sequences:

[0055](i) a region of DNA that is heterologous or unique to the genome of the mammalian cell, which functions as a source of homology, allows for homologous recombination (with a DNA contained in a second target plasmid). More specifically, the unique region of DNA (i) will generally comprise a bacterial, viral, yeast synthetic, or other DNA which is not normally present in the mammalian cell genome and which further does not comprise significant homology or sequence identity to DNA contained in the genome of the mammalian cell. Essentially, this sequence should be sufficiently different to mammalian DNA that it will not significantly recombine with the host cell genome via homologous recombination. The size of such unique DNA will generally be at least about 2 to 10 kilobases in size, or higher, more preferably at least about 10 kb, as several other investigators have noted an increased frequency of targeted recombination as the size of the homology region is increased (Capecchi, Science, 244:1288-1292 (1989)).

[0056]The upper size limit of the unique DNA which acts as a site for homologous recombination with a sequence in the second target vector is largely dictated by potential stability constraints (if DNA is too large it may not be easily integrated into a chromosome and the difficulties in working with very large DNAs.

[0057](ii) a DNA including a fragment of a selectable marker DNA, typically an exon of a dominant selectable marker gene. The only essential feature of this DNA is that it not encode a functional selectable marker protein unless it is expressed in association with a sequence contained in the target plasmid. Typically, the target plasmid will comprise the remaining exons of the dominant selectable marker gene (those not comprised in "targeting" plasmid). Essentially, a functional selectable marker should only be produced if homologous recombination occurs (resulting in the association and expression of this marker DNA (i) sequence together with the portion(s) of the selectable marker DNA fragment which is (are) contained in the target plasmid).

[0058]As noted, the current invention exemplifies the use of the neomycin phosphotransferase gene as the dominant selectable marker which is "split" in the two vectors. However, other selectable markers should also be suitable, e.g., the Salmonella histidinol dehydrogenase gene, hygromycin phosphotransferase gene, herpes simplex virus thymidine kinase gene, adenosine deaminase gene, glutamine synthetase gene and hypoxanthine-guanine phosphoribosyl transferase gene.

[0059](iii) a DNA which encodes a functional selectable marker protein, which selectable marker is different from the selectable marker DNA (ii). This selectable marker provides for the successful selection of mammalian cells wherein the marker plasmid is successfully integrated into the cellular DNA. More preferably, it is desirable that the marker plasmid comprise two such dominant selectable marker DNAS, situated at opposite ends of the vector. This is advantageous as it enables integrants to be selected using different selection agents and further enables cells which contain the entire vector to be selected. Additionally, one marker can be an amplifiable marker to facilitate gene dominant selectable marker listed in (ii) can be used as well as others generally known in the art.

[0060]Moreover, the marker plasmid may optionally further comprise a rare endonuclease restriction site. This is potentially desirable as this may facilitate cleavage. If present, such rare restriction site should be situated close to the middle of the unique region that acts as a substrate for homologous recombination. Preferably such sequence will be at least about 12 nucleotides. The introduction of a double stranded break by similar methodology has been reported to enhance the frequency of homologous recombination. (Choulika et al, Mol. Cell. Biol., 15:1968-1973 (1995)). However, the presence of such sequence is not essential.

[0061]The "targeting plasmid" will comprise at least the following sequences:

[0062](1) the same unique region of DNA contained in the marker plasmid or one having sufficient homology or sequence identity therewith that said DNA is capable of combining via homologous recombination with the unique region (i) in the marker plasmid. Suitable types of DNAs are described supra in the description of the unique region of DNA (1) in the marker plasmid.

[0063](2) The remaining exons of the dominant selectable marker, one exon of which is included as (ii) in the marker plasmid listed above. The essential features of this DNA fragment is that it result in a functional (selectable) marker protein only if the target plasmid integrates via homologous recombination (wherein such recombination results in the association of this DNA with the other fragment of the selectable marker DNA contained in the marker plasmid) and further that it allow for insertion of a desired exogenous DNA. Typically, this DNA will comprise the remaining exons of the selectable marker DNA which are separated by an intron. For example, this DNA may comprise the first two exons of the neo gene and the marker plasmid may comprise the third exon (back third of neo).

[0064](3) The target plasmid will also comprise a desired DNA, e.g., one encoding a desired polypeptide, preferably inserted within the selectable marker DNA fragment contained in the plasmid. Typically, the DNA will be inserted in an intron which is comprised between the exons of the selectable marker DNA. This ensures that the desired DNA is also integrated if homologous recombination of the target plasmid and the marker plasmid occurs. This intron may be naturally occurring or it may be engineered into the dominant selectable marker DNA fragment.

[0065]This DNA will encode any desired protein, preferably one having pharmaceutical or other desirable properties. Most typically the DNA will encode a mammalian protein, and in the current examples provided, an immunoglobulin or an immunoadhesin. However the invention is not in any way limited to the production of immunoglobulins.

[0066]As discussed previously, the subject cloning method is suitable for any mammalian cell as it does not require for efficacy that any specific mammalian sequence or sequences be present. In general, such mammalian cells will comprise those typically used for protein expression, e.g., CHO cells, myeloma cells, COS cells, BHK cells, Sp2/0 cells, NIH 3T3 and HeLa cells. In the. examples which follow, CHO cells were utilized. The advantages thereof include the availability of suitable growth medium, their ability to crow efficiently and to high density in culture, and their ability to express mammalian proteins such as immunoglobulins in biologically active form.

[0067]Further, CHO cells were selected in large part because of previous usage of such cells by the inventors for the expression of immunoglobulins (using the translationally impaired dominant selectable marker containing vectors described previously). Thus, the present laboratory has considerable experience in using such cells for expression. However, based on the examples. which follow, it is reasonable to expect similar results will be obtained with other mammalian cells.

[0068]In general, transformation or transfection of mammalian cells according to the subject invention will be effected according to conventional methods. So that the invention may be better understood, the construction of exemplary vectors and their usage in producing integrants is described in the examples below.

EXAMPLE 1

Design and Preparation of Marker and Targeting Plasmid DNA Vectors

[0069]The marker plasmid herein referred to as "Desmond" was assembled from the following DNA elements:

[0070](a) Murine dihydrofolate reductase gene (DHFR), incorporated into a transcription cassette, comprising the mouse beta globin promoter 5'' to the DHFR start site, and bovine growth hormone poly adenylation signal 3'' to the stop codon. The DHFR transcriptional cassette was isolated from TCAE6, an expression vector created previously in this laboratory (Newman et al, 1992, Biotechnology, 10:1455-1460).

[0071](b) E. coli β-galactosidase gene--commercially available, obtained from Promega as pSV-b-galactosidase control vector, catalog # E1081.

[0072](c) Baculovirus DNA, commercially available, purchased from Clontech as pBAKPAK8, cat # 6145-1.

[0073](d) Cassette comprising promoter and enhancer elements from Cytomegalovirus and SV40 virus. The cassette was generated by PCR using a derivative of expression vector TCAE8 (Reff et al, Blood, 83:435-445 (1994)). The enhancer cassette was inserted within the baculovirus sequence, which was first modified by the insertion of a multiple cloning site.

[0074](e) E. coli GUS (glucuronidase) gene, commercially available, purchased from Clontech as pB101, cat. # 6017-1.

[0075](f) Firefly luciferase gene, commercially available, obtained from Promega as pGEM-Luc (catalog # E1541).

[0076](g) S. typhimurium histidinol dehydrogenase gene (HisD). This gene was originally a gift from (Donahue et el, Gene, 18:47-59 (1982)), and has subsequently been incorporated into a transcription cassette comprising the mouse beta globin major promoter 5' to the gene, and the SV40 polyadenylation signal 3' to the gene.

[0077]The DNA elements described in (a)-(g) were combined into a pBR derived plasmid backbone to produce a 7.7 kb contiguous stretch of DNA referred to in the attached figures as "homology". Homology in this sense refers to sequences of DNA which are not part of the mammalian genome and are used to promote homologous recombination between transfected plasmids sharing the same homology DNA sequences.

[0078](h) Neomycin phosphotransferase gene from TN5 (Davis and Smith, Ann. Rev. Micro., 32:469-518 (1978)). The complete neo gene was subcloned into pBluescript SK-(Stratagene catalog # 212205) to facilitate genetic manipulation. A synthetic linker was then inserted into a unique Pst1 site occurring across the codons for amino acid 51 and 52 of neo. This linker encoded the necessary DNA elements to create an artificial splice donor site, intervening intron and splice acceptor site within the neo gene, thus creating two separate exons, presently referred to as neo exon 1 and 2. Neo exon 1 encodes the first 51 amino acids of neo, while exon 2 encodes the remaining 203 amino acids plus the stop codon of the protein A Not1 cloning site was also created within the intron.

[0079]Neo exon 2 was further subdivided to produce neo exons 2 and 3. This was achieved as follows: A set of PCR primers were designed to amplify a region of DNA encoding neo exon 1, intron and the first 111 2/3 amino acids of exon2. The 3' PCR primer resulted in the introduction of a new 5' splice site immediately after the second nucleotide of the codon for amino acid 111 in exon 2, therefore generating a new smaller exon 2. The DNA fragment now encoding the original exon 1, intron and new exon 2 was then subcloned and propagated in a pBR based vector. The remainder of the original exon 2 was used as a template for another round of PCR amplification, which generated "exon3". The 5' primer for this round of amplification introduced a new splice acceptor site at the 5' side of the newly created exon 3, i.e. before the final nucleotide of the codon for amino acid 111. The resultant 3 exons of neo encode the following information: exon 1--the first 51 amino acids of neo; exon 2--the next 111 2/3 amino acids, and exon 3 the final 91 1/3 amino acids plus the translational stop codon of the neo gene.

[0080]Neo exon 3 was incorporated along with the above mentioned DNA elements into the marking plasmid "Desmond". Neo exons 1 and 2 were incorporated into the targeting plasmid "Molly". The Not1 cloning site created within the intron between exons 1 and 2 was used in subsequent cloning steps to insert genes of interest into the targeting plasmid.

[0081]A second targeting plasmid "Mandy" was also generated. This plasmid is almost identical to "Molly" (some restriction sites on the vector have been changed) except that the original HisD and DHFR genes contained in "Molly" were inactivated. These changes were incorporated because the Desmond cell line was no longer being cultured in the presence of Histidinol, therefore it seemed unnecessary to include a second copy of the HisD gene. Additionally, the DHFR gene was inactivated to ensure that only a single DHFR gene, namely the one present in the Desmond marked site, would be amplifiable in any resulting cell lines. "Mandy" was derived from "Molly" by the following modifications:

[0082](i) A synthetic linker was inserted in the middle of the DHFR coding region. This linker created a stop codon and shifted the remainder of the DHFR coding region out of frame, therefore rendering the gene nonfunctional.

[0083](ii) A portion of the HisD gene was deleted and replaced with a PCR generated HisD fragment lacking the promoter and start codon of the gene.

[0084]FIG. 1 depicts the arrangement of these DNA elements in the marker plasmid "Desmond". FIG. 2 depicts the arrangement of these elements in the first targeting plasmid, "Molly". FIG. 3 illustrates the possible arrangement in the CHO genome, of the various DNA elements after targeting and integration of Molly DNA into Desmond marked CHO cells. FIG. 9 depicts the targeting plasmid "Mandy."

[0085]Construction of the marking and targeting plasmids from the above listed DNA elements was carried out following conventional cloning techniques (see, e.g., Molecular Cloning, A Laboratory Manual, J. Sambrook et al, 1987, Cold Spring Harbor Laboratory Press, and Current Protocols in Molecular Biology, F. M. Ausubel et al, eds., 1987, John Wiley and Sons). All plasmids were propagated and maintained in E. coli XLI blue (Stratagene, cat. # 200236). Large scale plasmid preparations were prepared using Promega Wizard Maxiprep DNA Purification System®, according to the manufacturer's directions.

EXAMPLE 2

Construction of a Marked CHO Cell Line

1. Cell Culture and Transfection Procedures to Produced Marked CHO Cell Line

[0086]Marker plasmid DNA was linearized by digestion overnight at 37° C. with Bst1107I. Linearized vector was ethanol precipitated and resuspended in sterile TE to a concentration of 1 mg/ml. Linearized vector was introduced into DHFR-Chinese hamster ovary cells (CHO cells) DG44 cells (Urlaub et al, Som. Cell and Mol. Gen., 12:555-566 (1986)) by electroporation as follows.

[0087]Exponentially growing cells were harvested by centrifugation, washed once in ice cold SBS (sucrose buffered solution, 272 mM sucrose, 7 mM sodium phosphate, pH 7.4, 1 mM magnesium chloride) then resuspended in SBS to a concentration of 10⁷ cells/ml. After a 15 minute incubation on ice, 0.4 ml of the cell suspension was mixed with 40 μg linearized DNA in a disposable electroporation cuvette. Cells were shocked using a BTX electrocell manipulator (San Diego, Calif.) set at 230 volts, 400 microfaraday capacitance, 13 ohm resistance. Shocked cells were then mixed with 20 ml of prewarmed CHO growth media (CHO-S-SFMII, Gibco/BRL, catalog # 31033-012) and plated in 96 well tissue culture plates. Forty eight hours after electroporation, plates were fed with selection media (in the case of transfection with Desmond, selection media is CHO-S-SFMII without hypoxanthine or thymidine, supplemented with 2 mM Histidinol (Sigma catalog # H6647)). Plates were maintained in selection media for up to 30 days, or until some of the wells exhibited cell growth. These cells were then removed from the 96 well plates and expanded ultimately to 120 ml spinner flasks where they were maintained in selection media at all times.

EXAMPLE 3

Characterization of Marked CHO Cell Lines

(a) Southern Analysis

[0088]Genomic DNA was isolated from all stably growing Desmond marked CHO cells. DNA was isolated using the Invitrogen Easy® DNA kit, according to the manufacturer's directions. Genomic DNA was then digested with HindIII overnight at 37° C., and subjected to Southern analysis using a PCR generated digoxygenin labelled probe specific to the DHFR gene. Hybridizations and washes were carried out using Boehringer Mannheim's DIG easy hyb (catalog # 1603 558) and DIG Wash and Block Buffer Set (catalog # 1585 762) according to the manufacturer's directions. DNA samples containing a single band hybridizing to the DHFR probe were assumed to be Desmond clones arising from a single cell which had integrated a single copy of the plasmid. These clones were retained for further analysis. Out of a total of 45 HisD resistant cell lines isolated, only 5 were single copy integrants. FIG. 4 shows a Southern blot containing all 5 of these single copy Desmond clones. Clone names are provided in the figure legend.

(b) Northern Analysis

[0089]Total RNA was isolated from all single copy Desmond clones using TRIzol reagent (Gibco/BRL cat # 15596-026) according to the manufacturer's directions. 10-20 μg RNA from each clone was analyzed on duplicate formaldehyde gels. The resulting blots were probed with PCR generated digoxygenin labelled DNA probes to (i) DHFR message, (ii) HisD message and (iii) CAD message. CAD is a trifunctional protein involved in uridine biosynthesis (Wahl et al, J. Biol. Chem., 254, 17:8679-8689 (1979)), and is expressed equally in all cell types. It is used here as an internal control to help quantitate RNA loading. Hybridizations and washes were carried out using the above mentioned Boehringer Mannheim reagents. The results of the Northern analysis are shown in FIG. 5. The single copy Desmond clone exhibiting the highest levels of both the His D and DHFR message is clone 15C9, shown in lane 4 in both panels of the figure. This clone was designated as the "marked cell line" and used in future targeting experiments in CHO, examples of which are presented in the following sections.

EXAMPLE 4

Expression of Anti-CD20 Antibody in Desmond Marked CHO Cells

[0090]C2B8, a chimeric antibody which recognizes B-cell surface antigen CD20, has been cloned and expressed previously in our laboratory. (Reff et al, Blood, 83:434-45 (1994)). A 4.1 kb DNA fragment comprising the C2B8 light and heavy chain genes, along with the necessary regulatory elements (eukaryotic promoter and polyadenylation signals) was inserted into the artificial intron created between exons 1 and 2 of the neo gene contained in a pBR derived cloning vector. This newly generated 5 kb DNA fragment (comprising neo exon 1, C2B8 and neo exon 2) was excised and used to assemble the targeting plasmid Molly. The other DNA elements used in the construction of Molly are identical to those used to construct the marking plasmid Desmond, identified previously. A complete map of Molly is shown in FIG. 2.

[0091]The targeting vector Molly was linearized prior to transfection by digestion with Kpn1 and Pac1, ethanol precipitated and resuspended in sterile TE to a concentration of 1.5 mg/mL. Linearized plasmid was introduced into exponentially growing Desmond marked cells essentially as described, except that 80 μg DNA was used in each electroporation. Forty eight hours postelectroporation, 96 well plates were supplemented with selection medium--CHO-SSFMII supplemented with 400 μg/mL Geneticin (G418, Gibco/BRL catalog # 10131-019). Plates were maintained in selection medium for up to 30 days, or until cell growth occurred in some of the wells. Such growth was assumed to be the result of clonal expansion of a single G418 resistant cell. The supernatants from all G418 resistant wells were assayed for C2B8 production by standard ELISA techniques, and all productive clones were eventually expanded to 120 mL spinner flasks and further analyzed.

Characterization of Antibody Secreting Targeted Cells

[0092]A total of 50 electroporations with Molly targeting plasmid were carried out in this experiment, each of which was plated into separate 96 well plates. A total of 10 viable, anti-CD20 antibody secreting clones were obtained and expanded to 120 ml spinner flasks. Genomic DNA was isolated from all clones, and Southern analyses were subsequently performed to determine whether the clones represented single homologous recombination events or whether additional random integrations had occurred in the same cells. The methods for DNA isolation and Southern hybridization were as described in the previous section. Genomic DNA was digested with EcoRI and probed with a PCR generated digoxygenin labelled probe to a segment of the CD20 heavy chain constant region. The results of this Southern analysis are presented in FIG. 6. As can be seen in the figure, 8 of the 10 clones show a single band hybridizing to the CD20 probe, indicating a single homologous recombination event has occurred in these cells. Two of the ten, clones 24G2 and 28C9, show the presence of additional band(s), indicative of an additional random integration elsewhere in the genome.

[0093]We examined the expression -levels of anti-CD20 antibody in all ten of these clones, the data for which is shown in Table 1, below.

TABLE-US-00001 TABLE 1 Expression Level of Anti-CD20 Secreting Homologous Integrants Clone Anti-CD20, pg/c/d 20F4 3.5 25E1 2.4 42F9 1.8 39G11 1.5 21C7 1.3 50G10 0.9 29F9 0.8 5F9 0.3 28C9* 4.5 24G2* 2.1 *These clones contained additional randomly integrated copies of anti-CD20. Expression levels of these clones therefore reflect a contribution from both the homologous and random sites.

Expression levels are reported as picogram per cell per day (pg/c/d) secreted by the individual clones, and represented the mean levels obtained from three separate ELISAs on samples taken from 120 mL spinner flasks.

[0094]As can be seen from the data, there is a variation in antibody secretion of approximately ten fold between the highest and lowest clones. This was somewhat unexpected as we anticipated similar expression levels from all clones due to the fact the anti-CD20 genes are all integrated into the same Desmond marked site. Nevertheless, this observed range in expression extremely small in comparison to that seen using any traditional random integration method or with our translationally impaired vector system.

[0095]Clone 20F4, the highest producing single copy integrant was selected for further study. Table 2 (below) presents ELISA and cell culture data from seven day production runs of this clone.

TABLE-US-00002 TABLE 2 7 Day Production Run Data for 20F4 % Viable/ml Day Viable (×10⁵) Tx2 (hr) mg/L pg/c/d 1 96 3.4 31 1.3 4.9 2 94 6 29 2.5 3.4 3 94 9.9 33 4.7 3.2 4 90 17.4 30 6.8 3 5 73 14 8.3 6 17 3.5 9.5 Clone 20F4 was seeded at 2 × 10⁵ ml in a 120 ml spinner flask on day 0. On the following six days, cell counts were taken, doubling times calculated and 1 ml samples of supernatant removed from the flask and analyzed for secreted anti-CD20 by ELISA.

This clone is secreting on average, 3-5 pg antibody/cell/day, based on this ELISA data. This is the same level as obtained from other high expressing single copy clones obtained previously in our laboratory using the previously developed translationally impaired random integration vectors. This result indicates the following:

[0096](1) that the site in the CHO genome marked by the Desmond marking vector is highly transcriptionally active, and therefore represents an excellent site from which to express recombinant proteins, and

[0097](2) that targeting by means of homologous recombination can be accomplished using the subject vectors and occurs at a frequency high enough to make this system a viable and desirable alternative to random integration methods.

[0098]To further demonstrate the efficacy of this system, we have also demonstrated that this site is amplifiable, resulting in even higher levels of gene expression and protein secretion. Amplification was achieved by plating serial dilutions of 20F4 cells, starting at a density of 2.5×10⁴ cells/ml, in 96 well tissue culture dishes, and culturing these cells in media (CHO-SSFMII) supplemented with 5, 10, 15 or 20 nM methotrexate. Antibody secreting clones were screened using standard ELISA techniques, and the highest producing clones were expanded nd further analyzed. A summary of this amplification experiment is presented in Table 3 below.

TABLE-US-00003 TABLE 3 Summary of 20F4 Amplification Expression Level # Wells Expression Level # Wells pg/c/d from nM MTX Assayed mg/l 96 well Expanded spinner 10 56 3-13 4 10-15 15 27 2-14 3 15-18 20 17 4-11 1 ND Methotrexate amplification of 20F4 was set up as described in the text, using the concentrations of methotrexate indicated in the above table. Supernatants from all surviving 96 well colonies were assayed by ELISA, and the range of anti-CD20 expressed by these clones is indicated in column 3. Based on these results, the highest producing clones were expanded to 120 ml spinners and several ELISAs conducted on the spinner supernatants to determine the pg/cell/day expression levels, reported in column 5.

The data here clearly demonstrates that this site can be amplified in the presence of methotrexate. Clones from the 10 and 15 nM amplifications were found to produce on the order of 15-20 pg/cell/day.

[0099]A 15 nM clone, designated 20F4-15A5, was selected as the highest expressing cell line. This clone originated from a 96 well plate in which only 22 wells grew, and was therefore assumed to have arisen from a single cell. A 15 nM clone, designated 20F4-15A5, was selected as the highest expressing cell line. This clone originated from a 96 well plate in which only 22 wells grew, and was therefore assumed to have arisen from a single cell. The clone was then subjected to a further round of methotrexate amplification. As described above, serial dilutions of the culture were plated into 96 well dishes and cultured in CHO-SS-FMII medium supplemented with 200, 300 or 400 nM methotrexate. Surviving clones were screened by ELISA, and several high producing clones were expanded to spinner cultures and further analyzed. A summary of this second amplification experiment is presented in Table 4.

TABLE-US-00004 TABLE 4 Summary of 20F4-15A5 Amplification # Wells Expression Level # Wells Expression Level nM MTX Assayed mg/l 96 well Expanded pg/c/d, spinner 200 67 23-70 1 50-60 250 86 21-70 4 55-60 300 81 15-75 3 40-50 Methotrexate amplifications of 20F4-15A5 were set up and assayed as described in the text. The highest producing wells, the numbers of which are indicated in column 4, were expanded to 120 ml spinner flasks. The expression levels of the cell lines derived from these wells is recorded as pg/c/d in column 5.

The highest producing clone came from the 250 nM methotrexate amplification. The 250 nM clone, 20F4-15A5-250A6 originated from a 96 well plate in which only wells grew, and therefore is assumed to have arisen from a single cell. Taken together, the data in Tables 3 and 4 strongly indicates that two rounds of methotrexate amplification are sufficient to reach expression levels of 60 pg/cell/day, which is approaching the maximum secretion capacity of immunoglobulin in mammalian cells (Reff, M. E., Curr. Opin. Biotech., 4:573-576 (1993)). The ability to reach this secretion capacity with just two amplification steps further enhances the utility of this homologous recombination system. Typically, random integration methods require more than two amplification steps to reach this expression level and are generally less reliable in terms of the ease of amplification. Thus, the homologous system offers a more efficient and time saving method of achieving high level gene expression in mammalian cells.

EXAMPLE 5

Expression of Anti-Human CD23 Antibody in Desmond Marked CHO Cells

[0100]CD23 is low affinity IgE receptor which mediates binding of IgE to B and T lymphocytes (Sutton, B. J., and Gould, H. J., Nature, 366:421-428 (1993)). Anti-human CD23 monoclonal antibody 5E8 is a human gamma-1 monoclonal antibody recently cloned and expressed in our laboratory. This antibody is disclosed in commonly assigned Ser. No. 08/803,085, filed on Feb. 20, 1997.

[0101]The heavy and light chain genes of 5E8 were cloned into the mammalian expression vector NSKG1, a derivative of the vector NEOSPLA (Barnett et al, in Antibody Expression and Engineering, H. Y Yang and T. Imanaka, eds., pp 27-40 (1995)) and two modifications were then made to the genes. We have recently observed somewhat higher secretion of immunoglobulin light chains compared to heavy chains in other expression constructs in the laboratory (Reff et al, 1997, unpublished observations). In an attempt to compensate for this deficit, we altered the 5E8 heavy chain gene by the addition of a stronger promoter/enhancer element immediately upstream of the start site. In subsequent steps, a 2.9 kb DNA fragment comprising the 5E8 modified light and heavy chain genes was isolated from the NSKG1 vector and inserted into the targeting vector Mandy. Preparation of 5E8-containing Molly and electroporation into Desmond 15C9 CHO cells was essentially as described in the preceding section.

[0102]One modification to the previously described protocol was in the type of culture medium used. Desmond marked CHO cells were cultured in protein-free CD-CHO medium (Gibco-BRL, catalog # AS21206) supplemented with 3 mg/L recombinant insulin (3 mg/mL stock, Gibco-BRL, catalog # AS22057) and 8 mM L-glutamine (200 mM stock, Gibco-BRL, catalog # 25030-081). Subsequently, transfected cells were selected in the above medium supplemented with 400 μg/mL geneticin. In this experiment, 20 electroporations were performed and plated into 96 well tissue culture dishes. Cells grew and secreted anti-CD23 in a total of 68 wells, all of which were assumed to be clones originating from a single G418 cell. Twelve of these wells were expanded to 120 ml spinner flasks for further analysis. We believe the increased number of clones isolated in this experiment (68 compared with 10 for anti-CD20 as described in Example 4) is due to a higher cloning efficiency and survival rate of cells grown in CD-CHO medium compared with CHO-SS-FMII medium. Expression levels for those clones analyzed in spinner culture ranged from 0.5-3 pg/c/d, in close agreement with the levels seen for the anti-CD20 clones. The highest producing anti-CD23 clone, designated 4H12, was subjected to methotrexate amplification in order to increase its expression levels. This amplification was set up in a manner similar to that described for the anti-CD20 clone in Example 4. Serial dilutions of exponentially growing 4H12 cells were plated into 96 well tissue culture dishes and grown in CD-CHO medium supplemented with 3 mg/L insulin, 8 mM glutamine and 30, 35 or 40 nM methotrexate. A summary of this amplification experiment is presented in Table 5.

TABLE-US-00005 TABLE 5 Summary of 2H12 Amplification Expression Level # Wells Expression Level # Wells pg/c/d from nM MTX Assayed mg/l 96 well Expanded spinner 30 100 6-24 8 10-25 35 64 4-27 2 10-15 40 96 4-20 1 ND The highest expressing clone obtained was a 30 nM clone, isolated from a plate on which 22 wells had grown. This clone, designated 4H12-30G5, was reproducibly secreting 18-22 pg antibody per cell per day. This is the same range of expression seen for the first amplification of the anti CD20 clone 20F4 (clone 20F4-15A5 which produced 15-18 pg/c/d, as described in Example 4). This data serves to further support the observation that amplification at this marked site in CHO is reproducible and efficient. A second amplification of this 30 nM cell line is currently underway. It is anticipated that saturation levels of expression will be achievable for the anti-CD23 antibody in just two amplification steps, as was the case for anti-CD20.

EXAMPLE 6

Expression of Immunoadhesin in Desmond Marked CHO Cells

[0103]CTLA-4, a member of the Ig superfamily, is found on the surface of T lymphocytes and is thought to play a role in antigen-specific T-cell activation (Dariavach et al, Eur. J. Immunol., 18:1901-1905 (1988); and Linsley et al, J. Exp. Med., 174:561-569 (1991)). In order to further study the precise role of the CTLA-4 molecule in the activation pathway, a soluble fusion protein comprising the extracellular domain of CTLA-4 linked to a truncated form of the human IgG1 constant region was created (Linsley et al (Id.). We have recently expressed this CTLA-4 Ig fusion protein in the mammalian expression vector BLECH1, a derivative of the plasmid NEOSPLA (Barnett et al, in Antibody Expression and Engineering, H. Y Yang and T. Imanaka, eds., pp 27-40 (1995)). An 800 bp fragment encoding the CTLA-4 Ig was isolated from this vector and inserted between the SacII and BglII sites in Molly.

[0104]Preparation of CTLA-4Ig-Molly and electroporation into Desmond clone 15C9 CHO cells was performed as described in the previous example relating to anti-CD20. Twenty electroporations were carried out, and plated into 96 well culture dishes as described previously. Eighteen CTLA-4 expressing wells were isolated from the 96 well plates and carried forward to the 120 ml spinner. stage. Southern analyses on genomic DNA isolated from each of these clones were then carried out to determine how many of the homologous clones contained additional random integrants. Genomic DNA was digested with BglII and probed with a PCR generated digoxygenin labelled probe to the human IgC-1 constant region. The results of this analysis indicated that 85% of the CTLA-4 clones are homologous integrants only; the remaining 15% contained one additional random integrant. This result corroborates the findings from the expression of anti-CD20 discussed above, where 80% of the clones were single homologous integrants. Therefore, we can conclude that this expression system reproducibly yields single targeted homologous integrants in at least 80% of all clones produced.

[0105]Expression levels for the homologous CT1A4-Ig clones ranged from 8-12 pg/cell/day. This is somewhat higher than the range reported for anti-CD20 antibody and anti-CD23 antibody clones discussed above. However, we have previously observed that expression of this molecule using the intronic insertion vector system also resulted in significantly higher expression levels than are obtained for immunoglobulins. We are currently unable to provide an explanation for this observation.

EXAMPLE 7

Targeting Anti-CD20 to an Alternate Desmond Marked CHO Cell Line

[0106]As we described in a preceding section, we obtained 5 single copy Desmond marked CHO cell lines (see FIGS. 4 and 5.). In order to demonstrate that the success of our targeting strategy is not due to some unique property of Desmond clone 15C9 and limited only to this clone, we introduced anti-CD20 Molly into Desmond clone 9B2 (lane 6 in FIG. 4, lane 1 in FIG. 5). Preparation of Molly DNA and electroporation into Desmond 9B2 was exactly as described in the previous example pertaining to anti-CD20. We obtained one homologous integrant from this experiment. This clone was expanded to a 120 ml spinner flask, where it produced on average 1.2 pg anti-CD20/cell/day. This is considerably lower expression than we observed with Molly targeted into Desmond 15C9. However, this was the anticipated result, based on our northern analysis of the Desmond clones. As can be seen in FIG. 5, mRNA levels from clone 9B2 are considerably lower than those from 15C9, indicating the site in this clone is not as transcriptionally active as that in 15C9. Therefore, this experiment not only demonstrates the reproducibility of the system--presumably any marked Desmond site can be targeted with Molly--it also confirms the northern data that the site in Desmond 15C9 is the most transcriptionally active.

[0107]From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without diverting from the scope of the invention. Accordingly, the invention is not limited by the appended claims.

Sequence CWU 1

3114683DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA referred to as "Desmond" 1tttctagacc tagggcggcc agctagtagc tttgcttctc aatttcttat ttgcataatg 60agaaaaaaag gaaaattaat tttaacacca attcagtagt tgattgagca aatgcgttgc 120caaaaaggat gctttagaga cagtgttctc tgcacagata aggacaaaca ttattcagag 180ggagtaccca gagctgagac tcctaagcca gtgagtggca cagcattcta gggagaaata 240tgcttgtcat caccgaagcc tgattccgta gagccacacc ttggtaaggg ccaatctgct 300cacacaggat agagagggca ggagccaggg cagagcatat aaggtgaggt aggatcagtt 360gctcctcaca tttgcttctg acatagttgt gttgggagct tggatagctt ggacagctca 420gggctgcgat ttcgcgccaa acttgacggc aatcctagcg tgaaggctgg taggatttta 480tccccgctgc catcatggtt cgaccattga actgcatcgt cgccgtgtcc caaaatatgg 540ggattggcaa gaacggagac ctaccctggc ctccgctcag gaacgagttc aagtacttcc 600aaagaatgac cacaacctct tcagtggaag gtaaacagaa tctggtgatt atgggtagga 660aaacctggtt ctccattcct gagaagaatc gacctttaaa ggacagaatt aatatagttc 720tcagtagaga actcaaagaa ccaccacgag gagctcattt tcttgccaaa agtttggatg 780atgccttaag acttattgaa caaccggaat tggcaagtaa agtagacatg gtttggatag 840tcggaggcag ttctgtttac caggaagcca tgaatcaacc aggccacctt agactctttg 900tgacaaggat catgcaggaa tttgaaagtg acacgttttt cccagaaatt gatttgggga 960aatataaact tctcccagaa tacccaggcg tcctctctga ggtccaggag gaaaaaggca 1020tcaagtataa gtttgaagtc tacgagaaga aagactaaca ggaagatgct ttcaagttct 1080ctgctcccct cctaaagcta tgcattttta taagaccatg ggacttttgc tggctttaga 1140tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 1200tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 1260tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 1320ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggaacca 1380gctggggctc gaagcggccg cccatttcgc tggtggtcag atgcgggatg gcgtgggacg 1440cggcggggac cgtcacactg aggttttccg ccagacgcca ctgctgccag gcgctgatgt 1500gcccggcttc tgaccatgcg gtcgcgttcg gttgcactac gcgtactgtg agccagagtt 1560gcccggcgct ctccggctgc ggtagttcag gcagttcaat caactgttta ccttgtggag 1620cgacatccag aggcacttca ccgcttgcta gcggcttacc atccagcgcc accatccagt 1680gcaggagctc gttatcgcta tgacggaaca ggtattcgct ggtcacttcg atggtttgcc 1740cggataaacg gaactggaaa aactgctgct ggtgttttgc ttccgtcagc gctggatgcg 1800gcgtgcggtc ggcaaagacc agaccgttca tacagaactg gcgatcgttc ggcgtatcac 1860caaaatcacc gccgtaagcc gaccacgggt tgccgttttc atcatattta atcagcgact 1920gatccaccca gtcccagacg aagccgccct gtaaacgggg atactgacga aacgcctgcc 1980agtatttagc gaaaccgcca agactgttac ccatcgcgtg ggcgtattcg caaaggatca 2040gcgggcgcgt ctctccgggt agcgaaagcc attttttgat ggaccatttc ggaccagccg 2100ggaagggctg gtcttcatcc acgcgcgcgt acatcgggca aataatatcg gtggccgtgg 2160tgtcggctcc gccgccttca tactgcaccg ggcgggaagg atcgacagat ttgatccagc 2220gatacagcgc gtcgtgatta gcgccgtggc ctgattcatt ccccagcgac cagatgatca 2280cactcgggtg attacgatcg cgctgcacca ttcgcgttac gcgttcgctc atcgccggta 2340gccagcgcgg atcatcggtc agacgattca ttggcaccat gccgtgggtt tcaatattgg 2400cttcatccac cacatacagg ccgtagcggt cgcacagcgt gtaccacagc ggatggttcg 2460gataatgcga acagcgcacg gcgttaaagt tgttctgctt catcagcagg atatcctgca 2520ccatcgtctg ctcatccatg acctgaccat gcagaggatg atgctcgtga cggttaacgc 2580ctcgaatcag caacggcttg ccgttcagca gcagcagacc atttccaatc cgcacctcgc 2640ggaaaccgac atcgcaggct tctgcttcaa tcagcgtgcc gtcggcggtg tgcagttcaa 2700ccaccgcacg atagagattc gggatttcgg cgctccacag tttcgggttt tcgacgttca 2760gacgcagtgt gacgcgatcg gcataaccac caggctcatc gataatttca ccgccgaaag 2820gcgcggtgcc gctggcgacc tgcgtttcac cctgccataa agaaactgtt acccgtaggt 2880agtcacgcaa ctcgccgcac atctgaactt cagcctccag tacagcgcgg ctgaaatcat 2940cattaaagcg agtggcaaca tggaaatcgc tgatttgtgt agtcggttta tgcagcaacg 3000agacgtcacg gaaaatgccg ctcatccgcc acatatcctg atcttccaga taactgccgt 3060cactccaacg cagcaccatc accgcgaggc ggttttctcc ggcgcgtaaa aatgcgctca 3120ggtcaaattc agacggcaaa cgactgtcct ggctgtaacc gacccacgcc ccgttgcacc 3180acagatgaaa cgccgagtta acgccatcaa aaataattcg cgtctggcct tcctgtagcc 3240agctttcatc aacattaaat gtgagcgagt aacaacccgt cggattctcc gtgggaacaa 3300acggcggatt gaccgtaatg ggataggtta cgttggtgta gatgggcgca tcgtaaccgt 3360gcatctgcca gtttgagggg acgacgacag tatcggcctc aggaagatcg cactccagcc 3420agctttccgg cactgcttct ggtgccggaa accaggcaaa gcgccattcg ccattcaggc 3480tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 3540aagcgggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 3600gttgtaaaac gacttaatcc gtcgaggggc tgcctcgaag cagacgacct tccgttgtgc 3660agccagcggc gcctgcgccg gtgcccacaa tcgtgcgcga acaaactaaa ccagaacaaa 3720tcataccggc ggcaccgccg ccaccacctt ctcctgtgcc taacattcca gcgcctccac 3780cactaccacc accatcgatg tctgaattgc cgcccgctcc accaatgccg acggaacctc 3840aacccgctgc acctttagac gacagacaac aattgttgga agctattaga aacgaaaaaa 3900atcgcactcg tctcagaccg gctctcttaa ggtagctcaa accaaaaacg gcgcccgaaa 3960ccagtacaat agttgaggtg ccgactgtgt tgcctaaaga gacatttgag cttaaaccgc 4020cgtctgcacc accgccacca cctccgcctc cgcctccgcc gccagccccg cctgcgcctc 4080caccgatggt agattcatca tcagctccac caccgccgcc attagtagat ttgccgtctg 4140aaatgttacc accgcctgca ccatcgcttt ctaacgtgtt gtctgaatta aaatcgggca 4200cagttagatt gaaacccgcc caaaaacgcc cgcaatcaga aataattcca aaaagctcaa 4260ctacaaattt gatcgcggac gtgttagccg acacaattaa taggcgtcgt gtggctatgg 4320caaaatcgtc ttcggaagca acttctaacg acgagggttg ggacgacgac gataatcggc 4380ctaataaagc taacacgccc gatgttaaat atgtccaagc tactagtggt accttaatta 4440aggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc 4500gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag 4560cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg 4620cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca cagaattaat 4680tcccctagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 4740ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgctcaa cgacccccgc 4800ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 4860cgtcaatggg tggactattt acggtaaact gcccacttgg cagtacatca agtgtatcat 4920atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 4980cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 5040attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 5100cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg aagcttggcc 5160ggccatataa acggcggcca gctttattta acgtgtttac gtcgagtcaa ttgtacacta 5220acgacagtga tgaaagaaat acaaaagcgc ataatatttt gaacgacgtc gaacctttat 5280tacaaaacaa aacacaaacg aatatcgaca aagctagatt gctgctacaa gatttggcaa 5340gttttgtggc gttgagcgaa aatccattag atagtccagc catcggttcg gaaaaacaac 5400ccttgtttga aactaatcga aacctatttt acaaatctat tgaggattta atatttaaat 5460tcagatataa agacgctgaa aatcatttga ttttcgctct aacataccac cctaaagatt 5520ataaatttaa tgaattatta aaatacatca gcaactatat attgatagac atttccagtt 5580tgtgatatta gtttgtgcgt ctcattacaa tggctgttat ttttaacaac aaacaactgc 5640tcgcagacaa tagtatagaa aagggaggtg aactgttttt gtttaacggt tcgtacaaca 5700ttttggaaag ttatgttaat ccggtgctgc taaaaaatgg tgtaattgaa ctagaagaag 5760ctgcgtacta tgccggcaac atattgtaca aaaccgacga tcccaaattc attgattata 5820taaatttaat aattaaagca acacactccg aagaactacc agaaaatagc actgttgtaa 5880attacagaaa aactatgcgc agcggtacta tacaccccat taaaaaagac atatatattt 5940atgacaacaa aaaatttact ctatacgata gatacatata tggatacgat aataactatg 6000ttaattttta tgaggagaaa aatgaaaaag agaaggaata cgaagaagaa gacgacaagg 6060cgtctagttt atgtgaaaat aaaattatat tgtcgcaaat taactgtgaa tcatttgaaa 6120atgattttaa atattacctc agcgattata actacgcgtt ttcaattata gataacacta 6180caaatgttct tgttgcgttt ggtttgtatc gttaataaaa aacaaattta gcatttataa 6240ttgttttatt attcaataat tacaaatagg attgagaccc ttgcagttgc cagcaaacgg 6300acagagcttg tcgaggagag ttgttgattc attgtttgcc tccctgctgc ggtttttgac 6360cgaagttcat gccagtccag cgtttttgca gcagaaaagc cgccgacttc ggtttgcggt 6420cgcgagtgaa gatccctttc ttgttaccgc caacgcgcaa tatgccttgc gaggtcgcaa 6480aatcggcgaa attccatacc tgttcaccga cgacggcgct gacgcgatca aagacgcggt 6540gatacatatc cagccatgca cactgatact cttcactcca catgtcggtg tacattgagt 6600gcagcccggc taacgtatcc acgccgtatt cggtgatgat aatcggctga tgcagtttct 6660cctgccaggc cagaagttct ttttccagta ccttctctgc cgtttccaaa tcgccgcttt 6720ggacatacca tccgtaataa cggttcaggc acagcacatc aaagagatcg ctgatggtat 6780cggtgtgagc gtcgcagaac attacattga cgcaggtgat cggacgcgtc gggtcgagtt 6840tacgcgttgc ttccgccagt ggcgcgaaat attcccgtgc accttgcgga cgggtatccg 6900gttcgttggc aatactccac atcaccacgc ttgggtggtt tttgtcacgc gctatcagct 6960ctttaatcgc ctgtaagtgc gcttggtgag tttccccgtt gactgcctct tcgttgtaca 7020gttctttcgg cttgttgccc gcttcgaaac caatgcctaa agagaggtta aagccgacag 7080cagcagtttc atcaatcacc acgatgccat gttcatctgc ccagtcgagc atctcttcag 7140cgtaagggta atgcgaggta cggtaggagt tggccctaat ccagtccatt aatgcgtggt 7200cgtgcaccat cagcacgtta tcgaatcctt tgccacgcaa gtccgcatct tcatgacgac 7260caaagccagt aaagtagaac ggtttgtggt taatcaggaa ctgttcgccc ttcactgcca 7320ctgaccggat gccgacgcga agcgggtaga tatcacactc tgtctggctt ttggctgtga 7380cgcacagttc atagagataa ccttcacccg gttgccagag gtgcggattc accacttgca 7440aagtcccgct agtgccttgt ccagttgcaa ccacctgttg atccgcatca cgcagttcaa 7500cgctgacatc accattggcc accacctgcc agtcaacaga cgcgtggtta cagtcttgcg 7560cgacatgcgt cactacggtg atatcgtcca cccaggtgtt cggcgtggtg tagagcatta 7620cgctgcgatg gattccggca tagttaaaga aatcatggaa gtaagattgc tttttcttgc 7680cgttttcgtt ggtaatcacc attcccggcg ggatagtctg ccagttcagt tcgttgttca 7740cacaaacggt gatacccctc gacggattaa agacttcaag cggtcaacta tgaagaagtg 7800ttcgtcttcg tcccagtaag ctatgtctct agaatgtagc catccatcct tgtcaatcaa 7860ggcgttggtc gcttccggat tgtttacata accggacata atcataggtc ctctgacaca 7920taatacgcct ctctgattaa cgcccagcgt tttcccggta tccagatcca caaccttcgc 7980ttcaaaaaat ggaacaactt taccgaccgc gcccggttta tcatccccct cgggtgtaat 8040cagaatagct gatgtagtct cagtgagccc atatccttgt cgtatccctg gaagatggaa 8100gcgttttgca accgcttccc cgacttcttt cgaaagaggt gcgcccccag aagcaatttc 8160gtgtaaatta gataaatcgt atttgtcaat cagagtgctt ttggcgaaga atgaaaatag 8220ggttggtact agcaacgcac tttgaatttt gtaatcctga agggatcgta aaaacagctc 8280ttcttcaaat ctatacatta agacgactcg aaatctacat atcaaatatc cgagtgtagt 8340aaacattcca aaaccgtgat ggaatggaac aacacttaaa atcgcagtat ccggaatgat 8400ttgattgcca aaaataggat ctctggcatg cgagaatcta gcgcaggcag ttctatgcgg 8460aagggccaca cccttaggta acccagtaga tccagaggaa ttgttttgtc acgatcaaag 8520gactctggta caaaatcgta ttcattaaaa ccgggaggta gatgagatgt gacgaaggtg 8580tacatcgact gaaatccctg gtaatccgtt ttagaatcca tgataataat tttctggatt 8640attggtaatt ttttttgcac gttcaaaatt ttttgcaacc cctttttgga aacaaacact 8700acggtaggct gcgaaatgtt catactgttg agcaattcac gttcattata aatgtcgttc 8760gcgggcgcaa ctgcaactcc gataaataac gcgcccaaca ccggcataaa gaattgaaga 8820gagttttcac tgcatacgac gattctgtga tttgtattca gcccatatcg tttcatagct 8880tctgccaacc gaacggacat ttcgaagtat tccgcgtacg tgatgttcac ctcgatatgt 8940gcatctgtaa aaggaattgt tccaggaacc agggcgtatc tcttcatagc cttatgcagt 9000tgctctccag cggttccatt ctctagcttt gcttctcaat ttcttatttg cataatgaga 9060aaaaaaggaa aattaatttt aacaccaatt cagtagttga ttgagcaaat gcgttgccaa 9120aaaggatgct ttagagacag tgttctctgc acagataagg acaaacatca ttcagaggga 9180gtacccagag ctgagactcc taagccagtg agtggcacag cattctaggg agaaatatgc 9240ttgtcatcac cgaagcctga ttccgtagag ccacaccttg gtaagggcca atctgctcac 9300acaggataga gagggcagga gccagggcag agcatataag gtgaggtagg atcagttgct 9360cctcacattt gcttctgaca tagttgtgtt gggagcttgg atcgatccac catgggcttc 9420aataccctga ttgactggaa cagctgtagc cctgaacagc agcgtgcgct gctgacgcgt 9480ccggcgattt ccgcctctga cagtattacc cggacggtca gcgatattct ggataatgca 9540aaaacgcgcg gtgacgatgc cctgcgtgaa tacagcgcta aatttgataa aacagaagtg 9600acagcgctac gcgtcacccc tgaagagatc gccgccgccg gcgcgcgtct gagcgacgaa 9660ttaaaacagg cgatgaccgc tgccgtcaaa aatattgaaa cgttccattc cgcgcagacg 9720ctaccgcttg tagatgtgga aacccagcca ggcgtgcgtt gccagcaggt tacgcgtccc 9780gtctcgtctg tcggtctgta tattcccggc ggctcggctc cgctcttctc aacggtgctg 9840atgctggcga cgccggcgcg cattgcggga tgctagaagg tggttctgtg ctcgccgccg 9900cccatcgctg atgaaatcct ctatgcggcg caactgtgtg gcgtgcagga attctttaac 9960ctcggcggcg cgcaggcgat tgccgctctg gccttcggca gcgagtccgt accgaaagtg 10020gataaaattt ttggccccgg caacgccttt gtaaccgaag ccaaacgtca ggtcagccag 10080cgtctcgacg gcgcggctat cgatatgcca gccgagccgt ctgaagtact ggtgatcgca 10140gacagcggcg caacaccgga tttcgtcgct tctgacctgc tctcccagac tgagcacggc 10200ccggattccc aggtgatcct gctgacgcct gatgctgaca ttgcccgcaa ggtggcggag 10260gcggtagaac gtcaactggc ggaactgccg cgcgcggaca ccgcctggca ggccctgagc 10320gccagtcgtc tgattgtgac caaagattta gcgcagtgcg tcgccatctc taatcagtat 10380gggccggaac acttaatcat ccagacgcgc aatgcgcgcg atttggtgga tgcgattacc 10440agcgcaggct cggtatttct cggcgactgg tcgccggaat ccgccggtga ttacgcttcc 10500ggaaccaacc atgttttacc gacctatggc catactgcta cctgttccag ccttgggtta 10560gcggatttcc agaaacggat gaccgttcag gaactgtcga aagcgggctt ttccgctctg 10620gcatcaacca ttgaaacatt ggcgggggca gaacgtctga ccgcccataa aaatgccgtg 10680accctgcgcg taaacgccct caaggagcaa gcatgagcac tgaaaacact ctcagcgtcg 10740ctgacttagc ccgtgaaaat gtccgcaacc tggagatcca gacatgataa gatacattga 10800tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg 10860tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa 10920ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta 10980aaacctctac aaatgtggta tggctgatta tgatctctag ctcgacgggg cgcctggccg 11040ctactaactc tctcctccct cctttttcct gcaggctcaa ggcgcgcatg cccgacggcg 11100aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 11160gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 11220cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 11280tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 11340agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 11400atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 11460ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 11520ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 11580cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatct 11640atcttatcat gtctggatcg cggccggtct ctctctagcc ctaggtctag acttggcaga 11700acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg 11760ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc 11820ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact 11880gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt 11940cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg 12000caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt 12060gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac 12120aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc 12180gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga 12240ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc 12300ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc 12360acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa 12420acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 12480gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga 12540cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat 12600tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata 12660ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 12720gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 12780taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 12840cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 12900ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 12960aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 13020tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 13080gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 13140cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 13200ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 13260cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 13320gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 13380cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 13440tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 13500ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 13560aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 13620atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 13680ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 13740tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 13800agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 13860taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 13920tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 13980cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 14040ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 14100tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 14160tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 14220cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 14280tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 14340gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 14400tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 14460atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 14520tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 14580cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac 14640ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaa 14683218986DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA referred to as "Molly" 2ttaattaagg ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt 60taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc 120tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca

tgctttgcat 180acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca cattccacag 240aattaattcc cctagttatt aatagtaatc aattacgggg tcattaggtc atagcccata 300tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 360cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 420ccattgacgt caatgggtgg actatttacg gtaaactgcc cacttggcag tacatcaagt 480gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 540ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 600catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 660tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttgaag 720cttggccggc catataaacg gcggccagct ttatttaacg tgtttacgtc gagtcaattg 780tacactaacg acagtgatga aagaaataca aaagcgcata atattttgaa cgacgtcgaa 840cctttattac aaaacaaaac acaaacgaat atcgacaaag ctagattgct gctacaagat 900ttggcaagtt ttgtggcgtt gagcgaaaat ccattagata gtccagccat cggttcggaa 960aaacaaccct tgtttgaaac taatcgaaac ctattttaca aatctattga ggatttaata 1020tttaaattca gatataaaga cgctgaaaat catttgattt tcgctctaac ataccaccct 1080aaagattata aatttaatga attattaaaa tacatcagca actatatatt gatagacatt 1140tccagtttgt gatattagtt tgtgcgtctc attacaatgg ctgttatttt taacaacaaa 1200caactgctcg cagacaatag tatagaaaag ggaggtgaac tgtttttgtt taacggttcg 1260tacaacattt tggaaagtta tgttaatccg gtgctgctaa aaaatggtgt aattgaacta 1320gaagaagctg cgtactatgc cggcaacata ttgtacaaaa ccgacgatcc caaattcatt 1380gattatataa atttaataat taaagcaaca cactccgaag aactaccaga aaatagcact 1440gttgtaaatt acagaaaaac tatgcgcagc ggtactatac accccattaa aaaagacata 1500tatatttatg acaacaaaaa atttactcta tacgatagat acatatatgg atacgataat 1560aactatgtta atttttatga ggagaaaaat gaaaaagaga aggaatacga agaagaagac 1620gacaaggcgt ctagtttatg tgaaaataaa attatattgt cgcaaattaa ctgtgaatca 1680tttgaaaatg attttaaata ttacctcagc gattataact acgcgttttc aattatagat 1740aatactacaa atgttcttgt tgcgtttggt ttgtatcgtt aataaaaaac aaatttagca 1800tttataattg ttttattatt caataattac aaataggatt gagacccttg cagttgccag 1860caaacggaca gagcttgtcg aggagagttg ttgattcatt gtttgcctcc ctgctgcggt 1920ttttcaccga agttcatgcc agtccagcgt ttttgcagca gaaaagccgc cgacttcggt 1980ttgcggtcgc gagtgaagat ccctttcttg ttaccgccaa cgcgcaatat gccttgcgag 2040gtcgcaaaat cggcgaaatt ccatacctgt tcaccgacga cggcgctgac gcgatcaaag 2100acgcggtgat acatatccag ccatgcacac tgatactctt cactccacat gtcggtgtac 2160attgagtgca gcccggctaa cgtatccacg ccgtattcgg tgatgataat cggctgatgc 2220agtttctcct gccaggccag aagttctttt tccagtacct tctctgccgt ttccaaatcg 2280ccgctttgga cataccatcc gtaataacgg ttcaggcaca gcacatcaaa gagatcgctg 2340atggtatcgg tgtgagcgtc gcagaacatt acattgacgc aggtgatcgg acgcgtcggg 2400tcgagtttac gcgttgcttc cgccagtggc gcgaaatatt cccgtgcacc ttgcggacgg 2460gtatccggtt cgttggcaat actccacatc accacgcttg ggtggttttt gtcacgcgct 2520atcagctctt taatcgcctg taagtgcgct tgctgagttt ccccgttgac tgcctcttcg 2580ctgtacagtt ctttcggctt gttgcccgct tcgaaaccaa tgcctaaaga gaggttaaag 2640ccgacagcag cagtttcatc aatcaccacg atgccatgtt catctgccca gtcgagcatc 2700tcttcagcgt aagggtaatg cgaggtacgg taggagttgg ccccaatcca gtccattaat 2760gcgtggtcgt gcaccatcag cacgttatcg aatcctttgc cacgcaagtc cgcatcttca 2820tgacgaccaa agccagtaaa gtagaacggt ttgtggttaa tcaggaactg ttcgcccttc 2880actgccactg accggatgcc gacgcgaagc gggtagatat cacactctgt ctggcttttg 2940gctgtgacgc acagttcata gagataacct tcacccggtt gccagaggtg cggattcacc 3000acttgcaaag tcccgctagt gccttgtcca gttgcaacca cctgttgatc cgcatcacgc 3060agttcaacgc tgacatcacc attggccacc acctgccagt caacagacgc gtggttacag 3120tcttgcgcga catgcgtcac cacggtgata tcgtccaccc aggtgttcgg cgtggtgtag 3180agcattacgc tgcgatggat tccggcatag ttaaagaaat catggaagta agactgcttt 3240ttcttgccgt tttcgtcggt aatcaccatt cccggcggga tagtctgcca gttcagttcg 3300ttgttcacac aaacggtgat acccctcgac ggattaaaga cttcaagcgg tcaactatga 3360agaagtgttc gtcttcgtcc cagtaagcta tgtctccaga atgtagccat ccatccttgt 3420caatcaaggc gttggtcgct tccggattgt ttacataacc ggacataatc ataggtcctc 3480tgacacataa ttcgcctctc tgattaacgc ccagcgtttt cccggtatcc agatccacaa 3540ccttcgcttc aaaaaatgga acaactttac cgaccgcgcc cggtttatca tccccctcgg 3600gtgtaatcag aatagctgat gtagtctcag tgagcccata tccttgtcgt atccctggaa 3660gatggaagcg ttttgcaacc gcttccccga cttctttcga aagaggtgcg cccccagaag 3720caatttcgtg taaattagat aaatcgtatt tgtcaatcag agtgcttttg gcgaagaatg 3780aaaatagggt tggtactagc aacgcacttt gaattttgta atcctgaagg gatcgtaaaa 3840acagctcttc ttcaaatcta tacattaaga cgactcgaaa tccacatatc aaatatccga 3900gtgtagtaaa cattccaaaa ccgtgatgga atggaacaac acttaaaatc gcagtatccg 3960gaatgatttg attgccaaaa ataggatctc tggcatgcga gaatctagcg caggcagttc 4020tatgcggaag ggccacaccc ttaggtaacc cagtagatcc agaggaattg ttttgtcacg 4080atcaaaggac tctggtacaa aatcgtattc attaaaaccg ggaggtagat gagatgtgac 4140gaacgtgtac atcgactgaa atccctggta atccgtttta gaatccatga taataatttt 4200ctggattatt ggtaattttt tttgcacgtt caaaattttt tgcaacccct ttttggaaac 4260aaacactacg gtaggctgcg aaatgttcat actgttgagc aattcacgtt cattataaat 4320gtcgttcgcg ggcgcaactg caactccgat aaataacgcg cccaacaccg gcataaagaa 4380ttgaagagag ttttcactgc atacgacgat tctgtgattt gtattcagcc catatcgttt 4440catagcttct gccaaccgaa cggacatttc gaagtattcc gcgtacgtga tgttcacctc 4500gatatgtgca tctgtaaaag gaattgttcc aggaaccagg gcgtatctct tcatagcctt 4560atgcagttgc tctccagcgg ttccatcctc tagctttgct tctcaatttc ttatttgcat 4620aatgagaaaa aaaggaaaat taattttaac accaattcag tagttgattg agcaaatgcg 4680ttgccaaaaa ggatgcttta gagacagtgt tctctgcaca gataaggaca aacattattc 4740agagggagta cccagagctg agactcctaa gccagtgagt ggcacagcat tctagggaga 4800aatatgcttg tcatcaccga agcctgattc cgtagagcca caccttggta agggccaatc 4860tgctcacaca ggatagagag ggcaggagcc agggcagagc atataaggtg aggtaggatc 4920agttgctcct cacatttgct tctgacatag ttgtgttggg agcttggatc gatccaccat 4980gggcttcaat accctgattg actggaacag ctgtagccct gaacagcagc gtgcgctgct 5040gacgcgtccg gcgatttccg cctctgacag tattacccgg acggtcagcg atattctgga 5100taatgtaaaa acgcgcggtg acgatgccct gcgtgaatac agcgctaaat ttgataaaac 5160agaagtgaca gcgctacgcg tcacccctga agagatcgcc gccgccggcg cgcgtctgag 5220cgacgaatta aaacaggcga tgaccgctgc cgtcaaaaat attgaaacgt tccattccgc 5280gcagacgcta ccgcctgtag atgtggaaac ccagccaggc gtgcgttgcc agcaggttac 5340gcgtcccgtc tcgtctgtcg gtctgtatat tcccggcggc tcggctccgc tcttctcaac 5400ggtgctgatg ctggcgacgc cggcgcgcat tgcgggatgc cagaaggtgg ttctgtgctc 5460gccgccgccc atcgctgatg aaatcctcta tgcggcgcaa ctgtgtggcg tgcaggaaat 5520ctttaacgtc ggcggcgcgc aggcgattgc cgctctggcc ttcggcagcg agtccgtacc 5580gaaagtggat aaaatttttg gccccggcaa cgcctttgta accgaagcca aacgtcaggt 5640cagccagcgt ctcgacggcg cggctatcga tatgccagcc gggccgtctg aagtactggt 5700gatcgcagac agcggcgcaa caccggattt cgtcgcttct gacctgctct cccaggctga 5760gcacggcccg gattcccagg tgatcctgct gacgcctgat gctgacattg cccgcaaggt 5820ggcggaggcg gtagaacgta aactggcgga actgccgcgc gcggacaccg cccggcaggc 5880cctgagcgcc agtcgtctga ttgtgaccaa agatttagcg cagtgcgtcg ccatctctaa 5940tcagtatggg ccggaacact taatcatcca gacgcgcaat gcgcgcgatt tggtggatgc 6000gattaccagc gcaggctcgg tatttctcgg cgactggtcg ccggaatccg ccggtgatta 6060cgcttccgga accaaccatg ttttaccgac ctatggctat actgctacct gttccagcct 6120tgggttagcg gatttccaga aacggatgac cgttcaggaa ctgtcgaaag cgggcttttc 6180cgctctggca tcaaccattg aaacattggc ggcggcagaa cgtctgaccg cccataaaaa 6240tgccgtgacc ctgcgcgtaa acgccctcaa ggagcaagca tgagcactga aaacactctc 6300agcgtcgctg acttagcccg tgaaaatgtc cgcaacctgg agatccagac atgataagat 6360acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg 6420aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca 6480acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa 6540gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tctctagctc gacggcgcgc 6600ctctagagca gtgtggtttt gcaagaggaa gcaaaaagcc tctccaccca ggcctggaat 6660gtttccaccc aatgtcgagc agtgtggttt tgcaagagga agcaaaaagc ctctccaccc 6720aggcctggaa tgtttccacc caatgtcgag caaaccccgc ccagcgtctt gtcattggcg 6780aattcgaaca cgcagatgca gtcggggcgg cgcggtccca gtcccacttc gcatattaag 6840gtgacgcgtg tggcctcgaa caccgagcga ccctgcagcc aatatgggat cggccattga 6900acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga 6960ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg 7020gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggtaag 7080tgcggccgtc gatggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc 7140catgcatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt 7200aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct 7260ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata 7320cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac attccacaga 7380attaattccc ctagttatta atagtaatca attacggggt cattagttca tagcccatat 7440atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 7500ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 7560cattgacgtc aatgggtgga ctatttacgg taaactgccc acttggcagt acatcaagtg 7620tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 7680tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta gctattagtc 7740atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 7800gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 7860caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 7920ggtaggcgtg tacggtggga ggtctatata agcagagctg ggtacgtgaa ccgtcagatc 7980gcctggagac gccatcacag atctctcact atggattttc aggtgcagat tatcagcttc 8040ctgctaatca gtgcttcagt cataatgtcc agaggacaaa ttgttctctc ccagtctcca 8100gcaatcctgt ctgcatctcc aggggagaag gtcacaatga cttgcagggc cagctcaagt 8160gtaagttaca tccactggtt ccagcagaag ccaggatcct cccccaaacc ctggatttat 8220gccacatcca acctggcttc tggagtccct gttcgcttca gtggcagtgg gtctgggact 8280tcttactctc tcacaatcag cagagtggag gctgaagatg ctgccactta ttactgccag 8340cagtggacta gtaacccacc cacgttcgga ggggggacca agctggaaat caaacgtacg 8400gtggctgcac catctgtctt catcttcccg ccatctgatg agcagttgaa atctggaact 8460gcctctgttg tgtgcctgct gaataacttc tatcccagag aggccaaagt acagtggaag 8520gtggataacg ccctccaatc gggtaactcc caggagagtg tcacagagca ggacagcaag 8580gacagcacct acagcctcag cagcaccctg acgctgagca aagcagacta cgagaaacac 8640aaagtctacg cctgcgaagt cacccatcag ggcctgagct cgcccgtcac aaagagcttc 8700aacaggggag agtgttgaat tcagatccgt taacggttac caactaccta gactggattc 8760gtgacaacat gcggccgtga tatctacgta tgatcagcct cgactgtgcc ttctagttgc 8820cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 8880actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 8940attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 9000catgctgggg atgcggtggg ctctatggaa ccagctgggg ctcgacagct atgccaagta 9060cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga 9120ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg 9180tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 9240caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 9300ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 9360gggaggtcta tataagcaga gctgggtacg tcctcacatt cagtgatcag cactgaacac 9420agacccgtcg acatgggttg gagcctcatc ttgctcttcc ttgtcgctgt tgctacgcgt 9480gtcctgtccc aggtacaact gcagcagcct ggggctgagc tggtgaagcc tggggcctca 9540gtgaagatgt cctgcaaggc ttctggctac acatttacca gttacaatat gcactgggta 9600aaacagacac ctggtcgggg cctggaatgg attggagcta tttatcccgg aaatggtgat 9660acttcctaca atcagaagtt caaaggcaag gccacattga ctgcagacaa atcctccagc 9720acagcctaca tgcagctcag cagcctgaca tctgaggact ctgcggtcta ttactgtgca 9780agatcgactt actacggcgg tgactggtac ttcaatgtct ggggcgcagg gaccacggtc 9840accgtctctg cagctagcac caagggccca tcggtcttcc ccctggcacc ctcctccaag 9900agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 9960gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 10020ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 10080ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 10140aaagcagagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 10200ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 10260tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 10320aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 10380gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 10440ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 10500aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 10560tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 10620cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 10680acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 10740aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 10800aaccactaca cgcagaagag cctctccctg tctccgggta aatgaggatc cgttaacggt 10860taccaactac ctagactgga ttcgtgacaa catgcggccg tgatatctac gtatgatcag 10920cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 10980tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 11040attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 11100aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gaaccagctg 11160gggctcgaca gcaacgctag gtcgaggccg ctactaactc tctcctccct cctttttcct 11220gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt 11280gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca 11340ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat 11400gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg 11460catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga 11520agagcatcag gggctcgcgc cagccgaact gttcgccagg taagtgagct ccaattcaag 11580cttcctaggg cggccagcta gtagctttgc ttctcaattt cttatttgca taatgagaaa 11640aaaaggaaaa ttaattttaa caccaattca gtagttgatt gagcaaatgc gttgccaaaa 11700aggatgcttt agagacagtg ttctctgcac agataaggac aaacattatt cagagggagt 11760acccagagct gagactccta agccagtgag tggcacagca ttctagggag aaatatgctt 11820gtcatcaccg aagcctgatt ccgtagagcc acaccttggt aagggccaat ctgctcacac 11880aggatagaga gggcaggagc cagggcagag catataaggt gaggtaggat cagttgctcc 11940tcacatttgc ttctgacata gttgtgttgg gagcttggat agcttggaca gctcagggct 12000gcgatttcgc gccaaacttg acggcaatcc tagcgtgaag gctggtagga ttttatcccc 12060gctgccatca tggttcgacc attgaactgc atcgtcgccg tgtcccaaaa tatggggatt 12120ggcaagaacg gagacctacc ctggcctccg ctcaggaacg agttcaagta cttccaaaga 12180atgaccacaa cctcttcagt ggaaggtaaa cagaatctgg tgattatggg taggaaaacc 12240tggttctcca ttcctgagaa gaatcgacct ttaaaggaca gaattaatat agttctcagt 12300agagaactca aagaaccacc acgaggagct cattttcttg ccaaaagttt ggatgatgcc 12360ttaagactta ttgaacaacc ggaattggca agtaaagtag acatggtttg gatagtcgga 12420ggcagttctg tttaccagga agccatgaat caaccaggcc accttagact ctttgtgaca 12480aggatcatgc aggaatttga aagtgacacg tttttcccag aaattgattt ggggaaatat 12540aaacttctcc cagaataccc aggcgtcctc tctgaggtcc aggaggaaaa aggcatcaag 12600tataagtttg aagtctacga gaagaaagac taacaggaag atgctttcaa gttctctgct 12660cccctcctaa agctatgcat ttttataaga ccatgggact tttgctggct ttagatcagc 12720ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 12780gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 12840ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 12900ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg aaccagctgg 12960ggctcgaagc ggccgcccat ttcgctggtg gtcagatgcg ggatggcgtg ggacgcggcg 13020gggagcgtca cactgaggtt ttccgccaga cgccactgct gccaggcgct gatgtgcccg 13080gcttctgacc atgcggtcgc gttcggttgc actacgcgta ctgtgagcca gagttgcccg 13140gcgctctccg gctgcggtag ttcaggcagt tcaatcaact gtttaccttg tggaccgaca 13200tccagaggca cttcaccgct tgccagcggc ttaccatcca gcgccaccat ccagtgcagg 13260agctcgttat cgctatgacg gaacaggtat tcgctggtca cttcgatggt ttgcccggat 13320aaacggaact ggaaaaactg ctgctggtgt tttgcttccg tcagcgctgg atgcggcgtg 13380cggtcggcaa agaccagacc gttcatacag aactggcgat cgttcggcgt atcgccaaaa 13440tcaccgccgt aagccgacca cgggttgccg ttttcatcat atttaatcag cgactgatcc 13500acccagtccc agacgaagcc gccctgtaaa cggggatact gacgaaacgc ctgccagtat 13560ttagcgaaac cgccaagact gttacccatc gctggggcgt attcgcaaag gatcagcggg 13620cgcgtctctc cgggtagcga aagccatttt ttgatggacc atttcggacc agccgggaag 13680ggctggtctt catccacgcg cgcgtacatc gggcaaataa tatcggtggc cgtggtgtcg 13740gctccgccgc cttcatactg caccgggcgg gaaggatcga cagatttgat ccagcgatac 13800agcgcgtcgt gattagcgcc gtggcctgat tcattcccca gcgaccagat gatcacactc 13860gggtgattac gatcgcgctg caccattcgc gttacgcgtt cgctcatcgc cggtagccag 13920cgcggatcat cggtcagacg attcattggc accatgccgt gggtttcaat attggcttca 13980tccaccacat acaggccgta gcggtcgcac agcgtgtacc acagcggatg gttcggataa 14040tgccaacagc gcacggcgtt aaagttgttc tgcttcatca gcaggatatc ctgcaccatc 14100gtctgctcat ccatgacctg accatgcaga ggatgatgct cgtgacggtt aacgcctcga 14160atcagcaacg gcttgccgtt cagcagcagc agaccatttt caatccgcac ctcgcggaaa 14220ccgacatcgc aggcttctgc ttcaatcagc gtgccgtcgg cggtgtgcag ttcaaccacc 14280gcacgataga gattcgggat ttcggcgctc cacagtttcg ggttttcgac gttcagacgc 14340agtgtgacgc gatcggcata accaccacgc tcatcgataa tttcaccgcc gaaaggcgcg 14400gtgccgctgg cgacctgcgt ttcaccctgc cataaagaaa ctgttacccg taggtagtca 14460cgcaactcgc cgcacatctg aacttcagcc tccagtacag cgcggctgaa atcatcatta 14520aagcgagtgg caacatggaa atcgctgatt tgtgtagtcg gtttatgcag caacgagacg 14580tcacggaaaa tgccgctcat ccgccacata tcctgatctt ccagataact gccgtcactc 14640caacgcagca ccatcaccgc gaggcggttt tctccggcgc gtaaaaatgc gctcaggtca 14700aattcagacg gcaaacgact gtcctggccg taaccgaccc acgccccgtt gcaccacaga 14760tgaaacgccg agttaacgcc atcaaaaata attcgcgtct ggccttcctg tagccagctt 14820tcatcaacat taaatgtgag cgagtaacaa cccgtcggat tctccgtggg aacaaacggc 14880ggattgaccg taatgggata ggttacgttg gtgtagatgg gcgcatcgta accgtgcatc 14940tgccagtttg aggggacgac gacagtatcg gcctcaggaa gatcgcactc cagccagctt 15000tccggcaccg cttctggtgc cggaaaccag gcaaagcgcc attcgccatt caggctgcgc 15060aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg 15120ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt 15180aaaacgactt aatccgtcga ggggctgcct cgaagcagac gaccttccgt

tgtgcagcca 15240gcggcgcctg cgccggtgcc cacaatcgtg cgcgaacaaa ctaaaccaga acaaattata 15300ccggcggcac cgccgccacc accttctccc gtgcctaaca ttccagcgcc tccaccacca 15360ccaccaccat cgatgtctga attgccgccc gctccaccaa tgccgacgga acctcaaccc 15420gctgcacctt tagacgacag acaacaattg ttggaagcta ttagaaacga aaaaaatcgc 15480actcgtctca gaccggtcaa accaaaaacg gcgcccgaaa ccagtacaat agttgaggtg 15540ccgactgtgt tgcctaaaga gacatttgag cctaaaccgc cgtctgcatc accgccacca 15600cctccgcctc cgcctccgcc gccagccccg cctgcgcctc caccgatggt agatttatca 15660tcagctccac caccgccgcc attagtagat ttgccgtctg aaatgttacc accgcctgca 15720ccatcgcttt ctaacgtgtt gtctgaatta aaatcgggca cagttagatt gaaacccgcc 15780caaaaacgcc cgcaatcaga aataattcca aaaagctcaa ctacaaattt gatcgcggac 15840gtgttagccg acacaattaa taggcgtcgt gtggctatgg caaaatcgtc ttcggaagca 15900acttctaacg acgagggttg ggacgacgac gataatcggc ctaataaagc taacacgccc 15960gatgttaaat atgtccaagc tactagtggt accgcttggc agaacatatc catcgcgtcc 16020gccatctcca gcagccgcac gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg 16080cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt 16140tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct 16200gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg 16260cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta 16320ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt 16380ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg 16440gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt 16500acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc 16560gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac 16620gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag 16680ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag 16740ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag 16800ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat 16860agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc 16920atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt 16980ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 17040ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 17100tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 17160tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 17220gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 17280ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 17340tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 17400agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 17460atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 17520acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 17580actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 17640tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 17700tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 17760tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 17820tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 17880caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 17940cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 18000agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 18060acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 18120gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 18180ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca 18240tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 18300ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 18360tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 18420attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 18480agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg 18540ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 18600ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 18660cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 18720gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 18780tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 18840tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 18900tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta 18960tcacgaggcc ctttcgtctt caagaa 18986319040DNAArtificial SequenceDescription of Artificial Sequence Synthetic DNA referred to as "Mandy" 3ttaattaagg ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt 60taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc 120tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat 180acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca cattccacag 240aattaattcc cctagttatt aatagtaatc aattacgggg tcattagttc atagcccata 300tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 360cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 420ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 480gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 540ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 600catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 660tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttgaag 720ctgtttaaac agcttggccg gccagcttta tttaacgtgt ttacgtcgag tcaattgtac 780actaacgaca gtgatgaaag aaatacaaaa gcgcataata ttttgaacga cgtcgaacct 840ttattacaaa acaaaacaca aacgaatatc gacaaagcta gattgctgct acaagatttg 900gcaagttttg tggcgttgag cgaaaatcca ttagatagtc cagccatcgg ttcggaaaaa 960caacccttgt ttgaaactaa tcgaaaccta ttttacaaat ctattgagga tttaatattt 1020aaattcagat ataaagacgc tgaaaatcat ttgattttcg ctctaacata ccaccctaaa 1080gattataaat ttaatgaatt attaaaatac atcagcaact atatattgat agacatttcc 1140agtttgtgat attagtttgt gcgtctcatt acaatggctg ttatttttaa caacaaacaa 1200ctgctcgcag acaatagtat agaaaaggga ggtgaactgt ttttgtttaa cggttcgtac 1260aacattttgg aaagttatgt taatccggtg ctgctaaaaa atggtgtaat tgaactagaa 1320gaagctgcgt actatgccgg caacatattg tacaaaaccg acgatcccaa attcattgat 1380tatataaatt taataattaa agcaacacac tccgaagaac taccagaaaa tagcactgtt 1440gtaaattaca gaaaaactat gcgcagcggt actatacacc ccattaaaaa agacatatat 1500atttatgaca acaaaaaatt tactctatac gatagataca tatatggata cgataataac 1560tatgttaatt tttatgagga gaaaaatgaa aaagagaagg aatacgaaga agaagacgac 1620aaggcgtcta gtttatgtga aaataaaatt atattgtcgc aaattaactg tgaatcattt 1680gaaaatgatt ttaaatatta cctcagcgat tataactacg cgttttcaat tatagataat 1740actacaaatg ttcttgttgc gtttggtttg tatcgttaat aaaaaacaaa tttgacattt 1800ataattgttt tattattcaa taattacaaa taggattgag acccttgcag ttgccagcaa 1860acggacagag cttgtcgagg agagttgttg attcattgtt tgcctccctg ctgcggtttt 1920tcaccgaagt tcatgccagt ccagcgtttt tgcagcagaa aagccgccga cttcggtttg 1980cggtcggcga gtgaagatcc ctttcttgtt accgccaacg cgcaatatgc cttgcgaggt 2040cgcaaaatcg gcgaaattcc atacctgttc accgacgacg gcgctgacgc gatcaaagac 2100gcggtgatac atatccagcc atgcacactg atactcttca ctccacatgt cggtgtacat 2160tgagtgcagc ccggctaacg tatccacgcc gtattcggtg atgataatcg gctgatgcag 2220tttctcctgc caggccagaa gttctttttc cagtaccttc tctgccgttt ccaaatcgcc 2280gctttgggac ataccatccg taataacggt tcaggcacag cacatcaaag agatcgctga 2340tggtatcggt gtgagcgtcg cagaacatta cattgacgca ggtgatcgga cgcgtcgggt 2400cgagtttacg cgttgcttcc gccagtggcg cgaaatattc ccgtgcacct tgcggacggg 2460tatccggttc gttggcaata ctccacatca ccacgcttgg gtggtttttg tcacgcgcta 2520tcagctcttt aatcgcctgt aagtgcgctt gctgagtttc cccgttgact gcctcttcgc 2580tgtacagttc tttcggcttg ttgcccgctt cgaaaccaat gcctaaagag aggttaaagc 2640cgacagcagc agtttcatca atcaccacga tgccatgttc atctgcccag tcgagcatct 2700cttcagcgta agggtaatgc gaggtacggt aggagttggc cccaatccag tccattaatg 2760cgtggtcgtg caccatcagc acgttatcga atcctttgcc acgcaagtcc gcatcttcat 2820gacgaccaaa gccagtaaag tagaacggtt tgtggttaat caggaactgt tcgcccttca 2880ctgccactga ccggatgccg acgcgaagcg ggtagatatc acactctgtc tggcttttgg 2940ctgtgacgca cagttcatag agataacctt cacccggttg ccagaggtgc ggattcacca 3000cttgcaaagt cccgctagtg ccttgtccag ttgcaaccac ctgttgatcc gcatcacgca 3060gttcaacgct gacatcacca ttggccacca cctgccagtc aacagacgcg tggttacagt 3120cttgcgcgac atgcgtcacc acggtgatat cgtccaccca ggtgttcggc gtggtgtaga 3180gcattacgct gcgatggatt ccggcatagt taaagaaatc atggaagtaa gactgctttt 3240tcttgccgtt ttcgtcggta atcaccattc ccggcgggat agtctgccag ttcagttcgt 3300tgttcacaca aacggtgata cccctcgacg gattaaagac ttcaagcggt caactatgaa 3360gaagtgttcg tcttcgtccc agtaagctat gtctccagaa tgtagccatc catccttgtc 3420aatcaaggcg ttggtcgctt ccggattgtt tacataaccg gacataatca taggtcctct 3480gacacataat tcgcctctct gattaacgcc cagcgttttc ccggtatcca gatccacaac 3540cttcgcttca aaaaatggaa caactttacc gaccgcgccc ggtttatcat ccccctcggg 3600tgtaatcaga atagctgatg tagtctcagt gagcccatat ccttgtcgta tccctggaag 3660atggaagcgt tttgcaaccg cttccccgac ttctttcgaa agaggtgcgc ccccagaagc 3720aatttcgtgt aaattagata aatcgtattt gtcaatcaga gtgcttttgg cgaagaatga 3780aaatagggtt ggtactagca acgcactttg aattttgtaa tcctgaaggg atcgtaaaaa 3840cagctcttct tcaaatctat acattaagac gactcgaaat ccacatatca aatatccgag 3900tgtagtaaac attccaaaac cgtgatggaa tggaacaaca cttaaaatcg cagtatccgg 3960aatgatttga ttgccaaaaa taggatctct ggcatgcgag aatctgacgc aggcagttct 4020atgcggaagg gccacaccct taggtaaccc agtagatcca gaggaattgt tttgtcacga 4080tcaaaggact ctggtacaaa atcgtattca ttaaaaccgg gaggtagatg agatgtgacg 4140aacgtgtaca tcgactgaaa tccctggtaa tccgttttag aatccatgat aataattttc 4200tggattattg gtaatttttt ttgcacgttc aaaatttttt gcaacccctt tttggaaaca 4260aacactacgg taggctgcga aatgttcata ctgttgagca attcacgttc attataaatg 4320tcgttcgcgg gcgcaactgc aactccgata aataacgcgc ccaacaccgg cataaagaat 4380tgaagagagt tttcactgca tacgacgatt ctgtgatttg tattcagccc atatcgtttc 4440atagcttctg ccaaccgaac ggacatttcg aagtattccg cgtacagccc ggccgtttaa 4500acggccgggc ttcaataccc tgattgactg gaacagctgt agccctgaac agcagcgtgc 4560gctgctgacg cgtccggcga tttccgcctc tgacagtatt acccggacgg tcagcgatat 4620tctggataat gtaaaaacgc gcggtgacga tgccctgcgt gaatacagcg ctaaatttga 4680taaaacagaa gtgacagcgc tacgcgtcac ccctgaagag atcgccgccg ccggcgcgcg 4740tctgagcgac gaattaaaac aggcgatgac cgctgccgtc aaaaatattg aaacgttcca 4800ttccgcgcag acgctaccgc ctgtagatgt ggaaacccag ccaggcgtgc gttgccagca 4860ggttacgcgt cccgtctcgt ctgtcggtct gtatattccc ggcggctcgg ctccgctctt 4920ctcaacggtg ctgatgctgg cgacgccggc gcgcattgcg ggatgccaga aggtggttct 4980gtgctcgccg ccgcccatcg ctgatgaaat cctctatgcg gcgcaactgt gtggcgtgca 5040ggaaatcttt aacgtcggcg gcgcgcaggc gatttgccgc tctggccttc ggcagcgagt 5100ccgtaccgaa agtggataaa atttttggcc ccggcaacgc ctttgtaacc gaagccaaac 5160gtcaggtcag ccagcgtctc gacggcgcgg ctatcgatat gccagccggg cggtctgaag 5220tactggtgat cgcagacagc ggcgcaacac cggatttcgt cgcttctgac ctgctcttcc 5280caggctgagc acggcccgga ttcccaggtg atcctgctga cgcctgatgc tgacattgcc 5340cgcaaggtgg cggaggcggt agaacgtcaa ctggcggaac tgccgcgcgc ggacaccgcc 5400cggcaggccc tgagcgccag tcgtctgatt gtgaccaaag atttagcgca gtgcgtcgcc 5460atctctaatc agtatgggcc ggaacactta atcatccaga cgcgcaatgc gcgcgatttg 5520gtggatgcga ttaccagcgc aggctcggta tttctcggcg actggtcgcc ggaatccgcc 5580ggtgattacg cttccggaac caaccatgtt ttaccgacct atggctatac tgctacctgt 5640tccagccttg ggttagcgga tttccagaaa cggatgaccg ttcaggaact gtcgaaagcg 5700ggcttttccg ctctggcatc aaccattgaa acattggcgg cggcagaacg tctgaccgcc 5760cataaaaatg ccgtgaccct gcgcgtaaac gccctcaagg agcaagcatg agcactgaaa 5820acactctcag cgtcgctgac ttagcccgtg aaaatgtccg caacctggag atccagacat 5880ggataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 5940ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 6000aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg 6060ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat ctctagggcc 6120ggccctcgac ggcgcgtcta gagcagtgtg gttttcaaga ggaagcaaaa agcctctcca 6180cccaggcctg gaatgtttcc acccaatgtc gagcagtgtg gttttgcaag aggaagcaaa 6240aagcctctcc acccaggcct ggaatgtttc cacccaatgt cgagcaaacc ccgcccagcg 6300tcttgtcatt ggcgaattgg aacacgcata tgcagtcggg gcggcgcggt cccaggtcca 6360cttcgcatat taaggtggcg cgtgtggcct cgaacaccga gcgaccctgc agccaatatg 6420ggatcggcca ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 6480ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 6540ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 6600gaactgcagg taagtgcggc cgtcgatggc cgaggcggcc tcggcctctg cataaataaa 6660aaaaattagt cagccatgca tggggcggag aatgggcgga actgggcgga gttaggggcg 6720ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca 6780tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac taattgagat 6840gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac 6900acacattcca cagaattaat tcccctagtt attaatagta atcaattacg gggtcattag 6960ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 7020gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 7080caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 7140cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 7200ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tgccagtaca 7260tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 7320gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 7380gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 7440tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctgggtacg 7500tgaaccgtca gatcgcctgg agacgccatc acagatctct caccatggac atgagggtcc 7560ccgctcagct cctggggctc cttctgctct ggctcccagg tgccagatgt gacatccaga 7620tgacccagtc tccatcttcc ctgtctgcat ctgtagggga cagagtcacc atcacttgca 7680gggcaagtca ggacattagg tattatttaa attggtatca gcagaaacca ggaaaagctc 7740ctaagctcct gatctatgtt gcatccagtt tgcaaagtgg ggtcccatca aggttcagcg 7800gcagtggatc tgggacagag ttcactctca ccgtcagcag cctgcagcct gaagattttg 7860cgacttatta ctgtctacag gtttatagta cccctcggac gttcggccaa gggaccaagg 7920tggaaatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca tctgatgagc 7980agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat cccagagagg 8040ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag gagagtgtca 8100cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg ctgagcaaag 8160cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc ctgagctcgc 8220ccgtcacaaa gagcttcaac aggggagagt gttgaattca gatccgttaa cggttaccaa 8280ctacctagac tggattcgtg acaacatgcg gccgtgatat ctacgtatga tcagcctcga 8340ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 8400tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 8460tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 8520gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 8580gaaccagctg ggactagtcg caattgggcg gagttagggg cgggatgggc ggagttaggg 8640gcggggacta tggtgctgac taattgagat gcatgctttg catacttctg cctgctgggg 8700agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc 8760tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc cacagaatta 8820attcccctag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 8880agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 8940gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 9000gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 9060atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 9120cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 9180ctgttaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 9240cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 9300atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 9360ggcgtgtacg gtgggaggtc tatataagca gagctgggta cgtgaaccgt cagatcgcct 9420ggagacgccg tcgacatggg ttggagcctc atcttgctct tccttgtcgc tgttgctacg 9480cgtgtcctgt ccgaggtgca gctggtggag tctgggggcg gcttggcaaa gcctgggggg 9540tccctgagac tctcctgcgc agcctccggg ttcaggttca ccttcaataa ctactacatg 9600gactgggtcc gccaggctcc agggcagggg ctggagtggg tctcacgtat tagtagtagt 9660ggtgatccca catggtacgc agactccgtg aagggcagat tcaccatctc cagagagaac 9720gccaagaaca cactgtttct tcaaatgaac agcctgagag ctgaggacac ggctgtctat 9780tactgtgcga gcttgactac agggtctgac tccctggggc cagggagtcc tggtcaccgt 9840ctcctcagct agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac 9900ctctgggggc acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac 9960ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca 10020gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac 10080ccagacctac atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt 10140tgagcccaaa tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct 10200ggggggaccg tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg 10260gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt 10320caactggtac gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca 10380gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa 10440tggcaaggag tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac 10500catctccaaa gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg 10560ggatgagctg accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag 10620cgacatcgcc gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc 10680tcccgtgctg gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag 10740caggtggcag caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca 10800ctacacgcag aagagcctct ccctgtctcc gggtaaatga ggatccgtta acggttacca 10860actacctaga ctggattcgt gacaacatgc ggccgtgata tctacgtatg atcagcctcg 10920actgtgcctt ctagttgcca gccatctgtt gtttgccccc tcccccgtgc cttccttgac 10980cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 11040tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 11100ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga 11160aagaaccagc tggggctcga

cagcaacgct aggtcgaggc cgctactaac tctctcctcc 11220ctcctttttc ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc 11280ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga 11340agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat 11400ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca 11460agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga 11520tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggtaagtgag 11580ctccaattca agctctcgag ctagggcggc cagctagtag ctttgcttct caatttctta 11640tttgcataat gagaaaaaaa ggaaaattaa ttttaacacc aattcagtag ttgattgagc 11700aaatgcgttg ccaaaaagga tgctttagag acagtgttct ctgcacagat aaggacaaac 11760attattcaga gggagtaccc agagctgaga ctcctaagcc agtgagtggc acagcatcca 11820gggagaaata tgcttgtcat caccgaagcc tgattccgta gagccacacc ctggtaaggg 11880ccaatctgct cacacaggat agagagggca ggagccaggc agagcatata aggtgaggta 11940ggatcagttg ctcctcacat ttgcttctga catagttgtg ttgggagctt ggatagcttg 12000ggggggggac agctcagggc tgcgatttcg cgccaaactt gacggcaatc ctagcgtgaa 12060ggctggtagg attttatccc cgctgccatc atggttcgac cattgaactg catcgtcgcc 12120gtgtcccaaa atatggggat tggcaagaac ggagacctac cctggcctcc gctcaggaac 12180gagttcaagt acttccaaag aatgaccaca acctcttcag tggaaggtaa acagaatctg 12240gtgattatgg gtaggaaaac ctggttctcc attcctgaga agaatcgacc tttaaaggac 12300agaattaata tagttctcag tagagaactc aaagaaccac cacgaggagc tcattttctt 12360gccaaaagtt tggatgatgc cttaacgtag gcgcgccatt aagacttatt gaacaaccgg 12420aattggcaag taaagtagac atggtttgga tagtcggagg cagttctgtt taccaggaag 12480ccatgaatca accaggcaac ctcagactct ttgtgacaag gatcatgcag gaatttgaaa 12540gtgacacgtt tttcccagaa attgatttgg ggaaatataa acttctccca gaatacccag 12600gcgtcctctc tgaggtcaag gaggaaaaag gcatcaagta taagtttgaa gtctacgaga 12660agaaagacta acaggaagat gctttcaagt tctctgctcc cctcctaaag ctatgcattt 12720ttataagacc atgggacttt tgctggcttt agatcagcct cgactgtgcc ttctagttgc 12780cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 12840actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 12900attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 12960catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg 13020aagcggccgc ccatttcgct ggtggtcaga tgcgggatgg cgtgggacgc ggcggggagc 13080gtcacactga ggttttccgc cagacgccac tgctgccagg cgctgatgtg cccggcttct 13140gaccatgcgg tcgcgttcgg ttgcactacg cgtactgtga gccagagttg cccggcgctc 13200tccggctgcg gtagttcagg cagttcaatc aactgtttac cttgtggagc gacatccaga 13260ggcacttcac cgcttgccag cggcttacca tccagcgcca ccatccagtg caggagctcg 13320ttatcgctat gacggaacag gtattcgctg gtcacttcga tggtttgccc ggataaacgg 13380aactggaaaa actgctgctg gtgttttgct tccgtcagcg ctggatgcgg cgtgcggtcg 13440gcaaagacca gaccgttcat acagaactgg cgatccgttc ggctatcgcc aaaatcaccg 13500ccgtaagccg accacgggtt gccgttttca tcatatttaa tcagcgactg atccacccag 13560tcccagacga agccgccctg taaacgggga tactgacgaa acgcctgcca gtatttagcg 13620aaaccgccaa gactgttacc catcgcgtgg gcgtattcgc aaaggatcag cgggcgcgtc 13680tctccaggta gcgaaagcca ttttttgatg gaccatttcg gcacagccgg gaagggctgg 13740tcttcatcca cgcgcgcgta catcgggcaa ataatatcgg tggccgtggt gtcggctccg 13800ccgccttcat actgcaccgg gcgggaagga tcgacagatt tgatccagcg atacagcgcg 13860tcgtgattag cgccgtggcc tgattcattc cccagcgacc agatgatcac actcgggtga 13920ttacgatcgc gctgcaccat tcgcgttacg cgttcgctca tcgccggtag ccagcgcgga 13980tcatcggtca gacgattcat tggcaccatg ccgtgggttt caatattggc ttcatccacc 14040acatacaggc cgtagcggtc gcacagcgtg taccacagcg gatggttcgg ataatgcgaa 14100cagcgcacgg cgttaaagtt gttctgcttc atcagcagga tatcctgcac catcgtctgc 14160tcatccatga cctgaccatg cagaggatga tgctcgtgac ggttaacgcc tcgaatcagc 14220aacggcttgc cgttcagcag cagcagacca ttttcaatcc gcacctcgcg gaaaccgaca 14280tcgcaggctt ctgcttcaat cagcgtgccg tcggcggtgt gcagttcaac caccgcacga 14340tagagattcg ggatttcggc gctccacagt ttcgggtttt cgacgttcag acgtagtgtg 14400acgcgatcgg cataaccacc acgctcatcg ataatttcac cgccgaaagg cgcggtgccg 14460ctggcgacct gcgtttcacc ctgccataaa gaaactgtta cccgtaggta gtcacgcaac 14520tcgccgcaca tctgaacttc agcctccagt acagcgcggc tgaaatcatc attaaagcga 14580gtggcaacat ggaaatcgct gatttgtgta gtcggtttat gcagcaacga gacgtcacgg 14640aaaatgccgc tcatccgcca catatcctga tcttccagat aactgccgtc actccagcgc 14700agcaccatca ccgcgaggcg gttttctccg gcgcgtaaaa atgcgctcag gtcaaattca 14760gacggcaaac gactgtcctg gccgtaaccg acccagcgcc cgttgcacca cagatgaaac 14820gccgagttaa cgccatcaaa aataattcgc gtctggcctt cctgtagcca gctttcatca 14880acattaaatg tgagcgagta acaacccgtc ggattctccg tgggaacaaa cggcggattg 14940accgtaatgg gataggtcac gttggtgtag atgggcgcat cgtaaccgtg catctgccag 15000tttgagggga cgacgacagt atcggcctca ggaagatcgc actccagcca gctttccggc 15060accgcttctg gtgccggaaa ccagggcaag cgccattcgc cattcaggct gcgcaactgt 15120tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 15180gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 15240acttaatccg tcgaggggct gcctcgaagc agacgacctt ccgttgtgca gccagcggcg 15300cctgcgccgg tgcccacaat cgtgcgcgaa caaactaaac cagaacaaat tataccggcg 15360gcaccgccgc caccaccttc tcccgtgcct aacattccag cgcctccacc accaccacca 15420ccatcgatgt ctgaattgcc gcccgctcca ccaatgccga cggaacctca acccgctgca 15480cctttagacg acagacaaca attgttggaa gctattagaa acgaaaaaaa tcgcactcgt 15540ctcagaccgg tcaaaccaaa aacggcgccc gaaaccagta caatagttga ggtgccgact 15600gtgttgccta aagagacatt tgagcctaaa ccgccgtctg catcaccgcc accacctccg 15660cctccgcctc cgccgccagc cccgcctgcg cctccaccga tggtagattt atcatcagct 15720ccaccaccgc cgccattagt agatttgccg tctgaaatgt taccaccgcc tgcaccatcg 15780ctttctaacg tgttgtctga attaaaatcg ggcacagtta gattgaaacc cgcccaaaaa 15840cgcccgcaat cagaaataat tccaaaaagc tcaactacaa atttgatcgc ggacgtgtta 15900gccgacacaa ttaataggcg tcgtgtggct atggcaaaat cgtcttcgga agcaacttct 15960aacgacgagg gttgggacga cgacgataat cggcctaata aagctaacac gcccgatgtt 16020aaatatgtcc aagctactag tggtaccgct tggcagaaca tatccatcgc gtccgccatc 16080tccagcagcc gcacgcggcg catctcgggc agcgttgggt cctggccacg ggtgcgcatg 16140atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag 16200aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc 16260tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag 16320tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt 16380ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg 16440tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt 16500tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc 16560atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt 16620aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg 16680gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac 16740cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg 16800gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg 16860tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga 16920gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc 16980ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt 17040cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 17100caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 17160caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 17220ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 17280cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 17340ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 17400tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 17460gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 17520ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 17580ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 17640gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 17700aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 17760tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 17820ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 17880tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 17940aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 18000tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 18060ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 18120gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 18180gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 18240taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg 18300tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 18360ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 18420tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 18480ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 18540tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata 18600ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 18660aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 18720actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 18780aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 18840tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 18900aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 18960ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 19020ggccctttcg tcttcaagaa 19040

Claims

1-41. (canceled)

42. A method for selecting a mammalian cell that has high expression of an immunoglobulin of interest comprising:(i) transfecting mammalian cells with a marking vector, wherein said marking vector comprises(a) a first region of DNA that is heterologous to the mammalian cell genome which when integrated in the mammalian cell genome provides a unique site for homologous recombination,(b) a first DNA fragment encoding a selectable marker protein that provides for selection of mammalian cells which have been successfully integrated with the marking vector wherein the selectable marker protein is not a neomycin phosphotransferase, and(c) a second DNA fragment comprising at least one, but not all, exons of a neomycin phosphotransferase gene;(ii) selecting in vitro for the mammalian cells which contain a single marking vector integrated into the genome wherein said cells have high expression of the selectable marker;(iii) transfecting the mammalian cells that have high expression of the selectable marker with a targeting vector comprising(a) a third DNA fragment comprising the remaining neomycin phosphotransferase exon or exons that are not present the marking vector,(b) an immunoglobulin DNA that encodes at least one immunoglobulin of interest, and(c) a second region of DNA that is identical or is sufficiently homologous to the unique site in said marking plasmid is integrated into the genome by homologous recombination at said unique site; and(iv) selecting in vitro for the mammalian cells which express said selectable marker, said neomycin phosphotransferase and said immunoglobulin of interest; thereby selecting a mammalian cell that has high expression of said immunoglobulin of interest.

43. The method of claim 42, wherein the selectable marker DNA encodes a protein that is selected from the group consisting of histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine guanine phosphoribosyl transferase.

44. The method of claim 43, wherein the selectable marker DNA encodes a dihydrofolate reductase protein.

45. The method of claim 42, which further comprises determining the RNA levels of the selectable marker.

46. The method of claim 42, wherein said second DNA fragment encoding the remaining neomycin phosphotransferase exons comprise two neomycin phosphotransferase exons which are separated by the immunoglobulin DNA that encodes at least one immunoglobulin of interest.

47. The method of claim 42, wherein said immunoglobulin DNA encodes a heavy chain and a light chain.

48. A eukaryotic cell which comprises a marking vector integrated into the genome for identifying a transcriptionally active site in the genome of a mammalian cell, wherein said marking vector comprises:(i) a first region of DNA that is heterologous to the mammalian cell genome which when integrated in the mammalian cell genome provides a unique site for homologous recombination,(ii) a first selectable marker DNA encoding for a first selectable marker that provides for selection of mammalian cells which have been successfully integrated with the marking vector, and(iii) a first DNA fragment encoding a portion of a second selectable marker that is different from the first selectable marker, wherein said second selectable marker is only expressed in said mammalian cell when a targeting vector comprising (a) a second DNA fragment encoding a second portion of said second selectable marker DNA and (b) a second region of DNA that is identical or is sufficiently homologous to the unique site in said marking plasmid is integrated into the genome by homologous recombination at said unique site.

49. The eukaryotic cell of claim 48, wherein said cell is a mammalian cell.

50. The eukaryotic cell of claim 49, wherein said cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, myeloma cells, baby hamster kidney cells, COS cells, NSO cells, HeLa cells, and NIH 3T3 cells.

51. The eukaryotic cell of claim 50, wherein the cell is a CHO cell.

52. The eukaryotic cell of claim 51, wherein the cell is clone 15C9.

53. A method for marking a transcriptionally active site in the genome of a mammalian cell comprising:(i) transfecting said mammalian cell with a marking vector, wherein said marking vector comprises (a) a first region of DNA that is heterologous to the mammalian cell genome which when integrated in the mammalian cell genome provides a unique site for homologous recombination, (b) a first selectable marker DNA encoding for a first selectable marker that provides for selection of mammalian cells which have been successfully integrated with the marking vector, and (c) a first DNA fragment encoding a portion of a second selectable marker that is different from the first selectable marker, wherein said second selectable marker is only expressed in said mammalian cell when a targeting vector comprising (a) a second DNA fragment encoding a second portion of said second selectable marker and (b) a second region of DNA that is identical or is sufficiently homologous to the unique site in said marking plasmid is integrated into the genome by homologous recombination at said unique site; and(ii) selecting in vitro for the mammalian cells which contain a single marking vector integrated into the genome wherein said cells have high expression of said first selectable marker; thereby marking the transcriptionally active site in the genome of said mammalian cell.

54. The method of claim 53, wherein the in vitro screening comprises determining the protein levels of the first selectable marker.

55. The method of claim 54, wherein the in vitro screening further comprises determining the RNA levels of the first selectable marker.

56. The method of claim 53, wherein the first selectable marker DNA encodes a protein that is selected from the group consisting of neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine guanine phosphoribosyl transferase.

57. The method of claim 56, wherein the first selectable marker DNA encodes a dihydrofolate reductase protein.

58. The method of claim 53, wherein the second selectable marker DNA encodes a protein that is selected from the group consisting of neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine guanine phosphoribosyl transferase.

59. The method of claim 58, wherein the second selectable marker DNA encodes a neomycin phosphotransferase protein.

60. The method of claim 53, wherein the DNA region that is heterologous to the mammalian cell genome is a bacterial DNA, an insect DNA, a viral DNA or a synthetic DNA.

61. The method of claim 53, wherein the region of DNA that is heterologous to the mammalian cell genome does not contain any functional genes.

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