DK1896590T3 - Serumfri stabil transfektion og fremstilling af rekombinante humane proteiner i humane cellelinjer - Google Patents

Description

DESCRIPTION

[0001] The present invention relates to an improved method for the serum-free production of an immortalized human cell line stably transfected under serum-free conditions with a specific vector carrying the gene coding for the protein of interest. Furthermore the invention relates to a production cell line obtained by said method, a production method for said protein of interest utilizing said production cell line, and the specific vector carrying the gene of interest itself.

Background [0002] The recombinant production of human proteins is generally performed by cultivation of stably transfected eukaryotic and preferably mammalian cell lines and isolation of the protein from the culture broth. In case the recombinant proteins are intended for pharmaceutical applications, it was for a long time general practice to employ non-human cell lines in order to exclude the risk of copurifying infectious agents which may be harbored and expressed by human cells.

[0003] In the production of some human proteins, such as human blood clotting factor VIII, the use of non-human cell lines were found to entail certain disadvantages, e.g. unsatisfactory secretion levels of the expressed protein into the medium. It is believed that this may be due to slight differences within different types of mammalian cells concerning intracellular pathways for protein translation and modification, which also might have an effect on the biological activity of the expressed polypeptide. Apart from this, there were concerns that therapeutic proteins purified from non-human expression systems are contaminated with cellular components which can give rise to antigenic reactions in the patients. Also a concern was the non-human glycosylation pattern found on human proteins recombinantly produced in non-human expression systems. It is thought that this increases the likelihood of antigenic reactions in the patient. Furthermore, biological stability and efficacy of blood proteins such as clotting factors is substantially influenced by their N-glycosylation pattern. Especially peripheral and terminal monosaccharides are important, because they are detected by specific receptors from cells which are responsible for their degradation. Clotting factors, for example, carry sialic acid residues as terminal monosaccharides. Modification on the composition of sialic acids in the antennae of glycoproteins can result in heterogenous glycosylation patterns. Thus, biological stability and efficacy is crucially involved when modification occurs. Hence, it is an important consideration in the production of recombinant clotting factors to evaluate the influence of glycosylation from non-human production cell lines versus human cell lines.

[0004] On the other hand, general methods for high level protein expression of a desired gene comprising immortalized, stably transfected mammalian cell lines expressing viral transcription activator proteins were made available (e.g. U.S. Patent 5,712,119). These cell lines can be transformed with a vector construct where a suitable viral transcription promoter is operatively associated with a DNAsequence defining a gene of interest, the transcription activator proteins provided by the cell lines activate the viral transcription promoter and hence initiate the expression of the gene of interest.

[0005] As important as the cell line is the vector used for the introduction of the recombinant gene into an immobilized production cell line. A wide variety of vectors were utilized for translation of mammalian proteins, (for example, Witsch-Baumgartner, M et al. Am. J. Genet (2000). 66, 402-412 cloned DHCR7 cDNAinto pCI-neo mammalian expression vector and expressed in the HEK 293 cells; McGarvey, T. W. et. al. Oncogene (2001) 20, 1041-1051 cloned TERE1 gene into the pTARGET mammalian expression vector and expressed in the human bladder transitional cell carcinomas; and Lin Lin et. al. J Biol Chem (2002) 277 (44) 41872-8 cloned the AchR gene into mammalian cell expression vector pEF6/myc-His vector and expressed it in 293 cells). A recently developed very potent vector which has proven to be capable of over-expression of recombinant proteins is the so-called pcDNA™3.1 vector of Invitrogen. Li J. et al., Life Sci. 2004 Apr 16; 74(22):2693-705 have successfully over-expressed histone deacetylases using pcDNA3.1 in HEK 293 cells. The cells were stably transfected and cultured in the presence of serum. Yuan-Gen Fu. et al., World J Gastroenterol 2003 have produced recombinant Caspase-3 using a pcDNA 3.1(+) based eukaryotic vector on gastric cancer cell line SGC7901 transiently transfected with said vector and cultured in the presence of serum. Ma H. et al., Invest Ophthalmol Vis Sci. 2000 Dec;41(13):4232-9 examined the lack of stable protein and loss of enzymatic activity expressing Lp82 and Lp82-related proteins subcloned into pcDNA3.1 vector using COS-7 as cell line. The cells were transiently transfected and cultured in the presence of serum in the medium. Thioredoxin overexpression prevents NO-induced reduction of NO synthase activity in lung endothelial cells. Zhang J. et al., Am J Physiol. 1998 Aug; 275(2 Pt 1): L288-93 disclose the overexpression of thioredoxin gene in cultured porcine pulmonary artery endothelial cells by transient transfection of these cells with pcDNA3.1 vector. The transfected cells were cultured in medium supplemented with serum. Shinki T. et al., Proc Natl. Acad. Sci. USA 1997 Nov 25; 94(24):12920-5 compared a full length cDNAfor the rat kidney mitochondrial cytrochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1 alpha-hydroxylase with vitamin D-deficient rat kidney cDNA and subcloned it into mammalian expression vector pcDNA 3.1 (+) and transiently transfected the vector into COS-7 transformed monkey kidney cells.

The transfected cells were cultured in medium supplemented with serum. Zhang et al., Acta Biochimica et Biophysica Sinica 2004, 36(10): 707-712 disclose the transfection of human embryonic kidney 293 cells with pcDNA containing a gene coding for the humanized 520C9 single chain Fv antibody/human interleukin-2 fusion protein. Supernatant was taken after having cultured the cells for three days in serum-free SFM II media. The resultant fusion protein possessed binding specificity against p185 (promising target for antibody therapy in breast cancer) and retained the important immuno-stimulatory activities of IL-2. Chen, J.Z. et al., Int J Biochem Cell Biol. 2004 Aug; 36(8):1554-61 over-expressed Bim proteins, which are essential factors for apoptosis, using HEK 293 cells transfected with pcDNA-Bim alpha3.

[0006] A further measure for increasing the safety of recombinant proteins for pharmaceutical applications is the use of serum-free medium in the culturing process, as the use of serum represents a safety hazard as well as a source of unwanted contaminations. Such serum-free cultivation has the drawback that the yields of the production process are generally significantly reduced. Afurther safety concern is the use of serum when transfecting the host cells as a regular way in the practice, as the use of serum in the transfection procedure may cause unwanted biological material to be integrated into the cells which later on contaminated the product expressed by the cells in the production process. While some of the available methods for the production of recombinant proteins (including those mentioned above) do allow serum-free cultivation, serum-free stable transfection of human cells is not known. In the 19th ESACT Meeting, Harrogate, 5th-8th June 2005 the serum free transfection of CHO cells was suggested by Kuchenbecker et al. Thus, it is desirable to develop an effective and safe method to produce human recombinant proteins.

Summary of the invention [0007] Surprisingly, it was found that a non-contaminated human protein (i.e. a protein preparation free of unwanted protein byproducts) can be obtained in good yield from immortalized human cell lines stably transfected, under serum-free conditions, with the gene encoding the protein of interest. In more detail, the present invention provides: 1. (1) a method for preparing an immortalized human cell line stably transfected with a nucleic acid sequence comprising a gene encoding a human target protein or a derivative or mutant thereof, a promoter and a bovine growth hormone polyadenylation (polyA) signal, said promoter and polyA signal being linked to the 5' and 3' end of the gene encoding said human target protein, respectively, which method comprises transfecting an immortalized human host cell line under serum-free conditions with a transfection vector comprising said nucleic acid sequence and an origin of replication; 2. (2) the method of (1) above wherein the transfection vector is derived from pcDNA3.1 vector having the sequence of SEQ ID NO:4 or 5; 3. (3) the method of (1) or (2) above, wherein the human cell line is a human embryonic kidney cell selected from 293 cells (ATCC CRL-1573; DSM ACC 305), Freestyle 293 cells (hereinafter "293F" cells; Invitrogen R79007), and 293T cells (ATCC CRL 11268; DSM ACC 2494); 4. (4) the method of (1) to (3) above, wherein the human protein is blood clotting factor IX, (e.g. as encoded by in bps 939 to 2324 of SEQ ID NO:1), alpha-1-antitrypsin (hereinafter "A1AT"; e.g. as encoded by bps 913 to 2259 of SEQ ID NO:2), blood clotting factor VIII (including wt factor VIII as shown in SEQ ID NO:8 or a B-domain deleted factor VIII mutant as encoded by bps 783 to 5162 of SEQ ID NO:3), factor Vll/Vlla (including the a and b form thereof encoded by SEQ ID NOs:13 and 14), G-CSF (including the G-CSF a, b and c form shown in SEQ ID NOs:15, 16 and 17, respectively), or von Willebrand factor (vWF); 5. (5) a transfection vector comprising an origin of replication and a gene encoding a human protein as defined in (1) and (2) above, preferably said transfection vector being a pcDNA3.1 vector comprising the gene for a human protein as defined in (4) above; 6. (6) an immortalized human cell line obtainable by the method as defined in (1) to (5) above, preferably said human cell line being as defined in (3) or (4) above; and 7. (7) a method for the recombinant production of a human target protein or a derivative or mutant thereof which comprises culturing an immortalized human cell line as defined in (6) above, preferably under serum-free conditions.

Description of the Figures [0008]

Figure 1: The wild-type human clotting factor IX (FIX) protein. A schematic drawing of the FIX gene with its 5' untranslated (5' UTR) region and its 3' UTR region. The eight domains of the unprocessed 461 amino acid protein are indicated: Si signal peptide; P: propeptide; Gla domain: γ-carboxyglutamyl domain; H domain: hydrophobic sequence; EGF domain: epidermal growth factor-like domain; L. linking sequence: AP: activation peptide: Catalytic domain. The FIX mature protein has a length of 415 amino acids and an approximate molecular weight of 55 kDa.

Figure 2: The vector pcDNA3.1 -FIX The circular DNA vector comprises 6,960 base pairs, the exact sequence thereof being given in SEQ ID NO: 1. In the schematic drawing the CMV promoter (CMV), the human FIX gene (hFIX), the f1 origin (f1), the hygromycin (Hyg) gene under control of the SV40 promoter (SV40), a poly A region (SV40 poly A), the pUC origin and the ampicillin (Amp) resistance gene are indicated, as well as numerous restriction sites. This vector is derived from the resequenced pcDNA 3.1 vector pcDNA3.1 Hygro(+)-zz of SEQ ID NO:5.

Figure 3: The vector pcDNA3.1-A1AT. The circular DNA vector comprises 6,914 bps, the exact sequence thereof being given in SEQ ID NO:2. In the schematic drawing the CMV enhancer promoter, the A1AT cDNA, bovine growth hormone polyadenylation (polyA) signal including a transcription termination sequence for enhanced mRNAstability, the f1 origin (f1), the hygromycin (Hyg) gene under control of the SV40 promoter (SV40), the SV40 poly A region (SV40 polyA), the pUC origin and the ampicillin (Amp) resistance gene are indicated, as well as numerous restriction sites. This vector is derived from vector pcDNA3.1Hygro(+)-zz of SEQ ID NO:5.

Figure 4: Transient transfection of different human embryonic kidney cells with vectors coding for alpha-1-antitrypsin. A cell line comparison in transient transfection studies is shown. The amount (%) of alpha-1-antitrypsin (A1AT) expressed per 106 cells is shown for 293, 293T and 293F cells. The A1AT amount expressed in 293F cells transiently transfected with pcDNA3.1-A1AT has been set as 100%.

Figure 5: Transient transfection of 293F cell line with different vectors coding for alpha-1-antitrypsin. A comparison of A1AT concentrations expressed from different vectors using transiently transfected freestyle 293F cell line is shown. The expression level of A1AT pcDNA3.1-A1AT has been set as 100%. Various other vectors carrying the A1AT gene were also tested: An in-house vector pTG1-A1AT (an in-house vector for producing human recombinant A1AT as shown in Fig. 10), the pCMV Script® A1AT (Stratagene) and pd neo-A1AT (Promega) were compared against the pcDNA3.1-A1AT (pcDNA3.1). None of the other vectors came close to the high expression levels observed with pcDNA3.1-A1AT.

Figure 6: SDS-PAGE and Western blot of cell culture supernatant. Aliquots of the supernatant of freestyle 293F cells transiently transfected with pcDNA3.1-A1AT (lanes 1-3 and 6-8) or with a GFP-expressing control plasmid (lanes 4 and 8) were analysed both by SDS-PAGE and Western blot. Lane 1 contains a size marker, and lane 5 is empty. The band for A1AT is marked with an arrow. Also visible is the 27 kDa band corresponding to GFP in lanes 4 and 8.

Figure 7 shows the vector pcDNA 3.1-F.VIII. The vector comprises 9,975 bps, the exact sequence thereof being shown in SEQ ID NO:3. The factor VIII protein encoded by bps 783 to 5162 is a B-domain deleted factor VIII mutant as disclosed in WO 01/70968. Again this vector is derived from vector pcDNA 3.1 Hygro(+)-zz of SEQ ID NO:5.

Figure 8: Comparison of the average amount of produced factor VIII of the best three stably transfected clones, transfecting 293 and 293F cells with pcDNA3.1-FVIII and the in-house vector pTGF8-2hyg-s, the exact sequence thereof being given in SEQ ID NO:7.

Figure 9 shows the vector pTG1-A1AT. The vector comprises 5,610 bps, the exact sequence thereof being shown in SEQ ID NO:6.

Figure 10 shows the vector pCR2.1d2-GCSFb. The vector comprises 4,542 bps, the exact sequence thereof being shown in SEQ ID NO:21.

Figure 11 shows the vector pDNA3.1-GCSFb. The vector comprises 6,237 bps, the exact sequence thereof being shown in SEQ ID NO:22.

Figure 12 shows the vector pCINeo-GCSFb. The vector comprises 6,101 bps, the exact sequence thereof being shown in SEQ ID NO:23.

Figure 13 shows the vector pCMVScript-GCSFb. The vector comprises 4,920 bps, the exact sequence thereof being shown in SEQ ID NO:24.

Figure 14 shows the vector pTG2-GCSFb-hyg-as. The vector comprises 6,917 bps, the exact sequence thereof being shown in SEQ ID NO:25.

Figure 15 compares the amount of rhG-CSF produced by different expression constructs according to Example 9.

Figure 16 shows a western blot of rhG-CSF produced according to Example 9.

Detailed Description of Invention [0009] The present invention provides an improved method for the transfection and production of human recombinant proteins in human immortalized cell lines completely under serum- and protein-free conditions. It allows the serum-free transfection and production of human proteins. The method may include one or more purification step(s) including viral inactivation procedures, which reduces the risk for contamination of the recombinant protein with human pathogens. Since human recombinant proteins produced in human cell lines carry a human glycosylation pattern, they are also less susceptible to degradation in comparison to human proteins lacking their natural glycosylation pattern. In summary the method of the invention offers various advantages over the prior art.

[0010] In particular, the method of embodiment (7) of the invention provides an effective system to produce safe and highly active human recombinant blood clotting factors, for example factors IX and FVIII for therapeutic application for Hemophilia B and A in humans. The method is suitable for expression of those wild-type proteins, but can also be used for mutants of those proteins, for example of factor VIII, which are exceptionally stable against proteolytic inactivation and thus allow to be subjected to vigorous virus inactivation protocols.

[0011] A preferred mode the method of embodiment (7) of the invention comprises serum-free culturing an immortalized cell line carrying a vector having a promoter linked to the 5' end of a DNA sequence encoding said human blood protein. The 3' end of the DNA sequence encoding said human blood protein is functionally linked to a bovine growth hormone polyA signal. According to the invention the immortalized human cell line is stably transfected with the vector. To detect stable transfection, the vector may further comprise, in addition to the gene for the human blood protein, at least one gene for a selection marker system which is functionally linked to a promoter.

[0012] Suitable promoters include viral promoters, housekeeping gene promoters, tissue specific promoters, etc. In case the promoter is a viral promoter, the cell line does not comprise the matching viral transcription activator protein for said promoter. However, the cell may comprise a viral transcription activator protein such as the T antigen which complements another viral promoter which is not functionally linked to the gene encoding the human blood protein. Preferably the promoter is a SV40 promoter, CMV promoter, EF-1alpha promoter, HSV TK promoter etc., most preferably the promoter is a CMV promoter, i.e. the constitutive, major intermediate early promoter of cytomegalovirus.

[0013] The expressions "transfection" or "transfected" refers to the introduction of a nucleic acid into a cell under conditions allowing expression of the protein. In general the nucleic acid is a DNA sequence, in particular a vector or a plasmid carrying a gene of interest under a suitable promoter, whose expression is controlled by said promoter. However, the term transfection also comprises RNA transfection. The skilled artisan is familiar with the various transfection methods such those using carrier molecules like cationic lipids such as DOTAP (Roche), DOSPER (Roche), Fugene (Roche), Transfectam® (Promega), TransFast™ (Promega) and Tfx™ (Promega), Lipofectamine (Invitrogene) and 293fectin™ (Invitrogene), or calcium phosphate and DEAE dextran. He is also familiar with brute-force transfection techniques. These include electroporation, bombardment with nucleic-acid-coated carrier particles (gene gun), and microinjection. Finally the skilled artisan is also familiar with nucleic acid transfection using viral vectors.

[0014] "Transiently transfected" or "transient transfection" refer to the transient, i.e. non-permanent expression of the gene of interest due to the episomal nature of the introduced nucleic acid. By its very nature, RNA transfection or cytolytic viruses can only be used for transient expression. Episomal nucleic acids, including DNA (plasmids or vectors), is degraded by the cells after two to four days, and hence the expression of the gene of interest ceases then.

[0015] "Stably transfected" or "stable transfection" refers to the permanent expression of the gene of interest due to the integration of the transfected DNA into the genome of the cell. Most if not all cells have the potential to incorporate episomal DNA into their genome albeit at a very low rate. However, sophisticated selection strategies are employed to expand those cells that have integrated the transfected DNA. For that the vector must contain at least one gene for a selection marker such as e.g. hygromycin.

[0016] The term "stable transfection" or "stably transfected" is here also used to refer to cells carrying plasmids that can autonomously replicate and thus can be used for long-term expression of foreign genes. One particularly gene transfer system applicable for "stably transfecting" cells is based on recombinant retroviruses. Since integration of the proviral DNA is an obligatory step during the retroviral replication cycle, infection of cells with a recombinant retrovirus will give rise to a very high proportion of cells that have integrated the gene of interest and are thus stably transfected.

[0017] The term "culturing" refers to the maintaince of cells/cell lines in vitro in containers with medium supporting their proliferation and gene expression. Thus the culturing causes accumulation of the expressed secretable proteins in the culture medium. The medium normally contains supplements stabilizing the pH, as well as amino acids, lipids, trace elements, vitamins and other growth enhancing components. The "serum-free", "serum-free transfection" or "serum-free cultivation" refers to the transfection and culturing of cells in medium containing suitable supplements except any kind of serum. Supplements are selected from amino acids, lipids, trace elements, vitamins and other growth enhancing components. Often the "serum-free" culture conditions are even more stringent and, if no exogeneous protein is added, or already included in the medium, the medium is called "protein-free".

[0018] The term "immortalized human cell line" refers to human cells that are not primary cells taken directly from an organism. In particular it refers to permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space, and thus have escaped the Hayflick limit.

[0019] The term "concentration" refers to the concentration of the produced recombinant protein from the culture medium. Inherently it also results in a concentration of the protein. The person skilled in the art is familiar with concentration techniques such as filtration, including ultra filtration, centrifugation, precipitation, etc. The concentration does not necessarily result in a pure protein, and the isolated protein may still comprise non-protein and protein contaminants. Additional purification steps are often required.

[0020] The term "purification" refers to steps applied to the isolated protein is subjected to in order to obtain a substantially pure (at least 60% pure, preferably at least 75% pure, more preferably over 90% pure and most preferably over 99.9% pure) human recombinant protein. Purity can be measured by an appropriate method. The person skilled in the art is familiar with techniques employable for the purification of a recombinant protein such as immuno-affinity chromatography, affinity chromatography, protein precipitation, buffer exchanges, ionic exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, electrophoresis. In addition, the purification may comprise a virus inactivation step such as heat treatment and/or solvent detergent (SD)-treatment, at either dry or liquid state, in the presence or without chemical substances including protease inhibitors. Further, the purification may include one or more steps for prion removal, such as protein precipitation, filtration, chromatography steps, in particular affinity chromatography steps (see e.g. "Partitioning of TSE infectivity during ethanol fractionation of human plasma", Gregori, L. et al., Biologicals 32 1-10; 2 (2004); and "Removal of TSE agents from blood products", Foster, P.R., Vax Sanguinis 87 (Suppl. 2), S7-S10 (2004 )). After virus inactivation a further purification step selected from anyone of the above listed ones may be necessary for removal of the chemical substances used for virus inactivation.

[0021] The term “vector" refers to any genetic construct, such as a plasmid, phage, cosmid, etc., which is capable of replication when associated with the proper control elements, into which fragments of DNA may be inserted or cloned. A vector comprises unique restriction sites and may be capable of autonomous replication in a host cell. The term includes cloning and expression vehicles. The "vector" may further carry one or more further regulatory elements, said regulatory elements preferably being selected from splice sites, recombination sites, polyA sites, enhancers, multicloning site and prokaryotic plasmid sequences.

[0022] The term "functionally linked" refers to the configuration of the vector where the promoter is located within the vector in such a manner that it can stimulate transcription of the DNA sequence coding for the protein of interest, in particular for the human blood protein.

[0023] The term "mature" refers to the molecular structure of a given protein of the processed protein, i.e. a protein which lacks the N-terminal export signal.

[0024] The term "promoter" refers to a region of a regulatory DNA sequence bound by RNA polymerase and transcription factors during the initiation of transcription.

[0025] The term "enhancer" refers to a cis-acting sequence that increases the utilization of an eukaryotic promoter, and can function in either orientation and in any location (upstream or downstream) relative to the promoter.

[0026] The term "polyadenylation (polyA) signal" refers to a specialized termination sequence. It signals the addition of a "tail" of adenines to the end of the mRNAthat enables export of the mRNAto the cytoplasm. Upon reaching the cytoplasm, the polyA tail of the mRNA is maintained during protein translation and stabilizes the mRNA during protein expression.

[0027] The term "encodes" or "encoding" refers to a property of the nucleic acid sequence to be transcribed (in case of DNA) or translated (in case of mRNA) into a polypeptide (protein) in vitro or in vivo when placed under the control of an appropriate regulatory sequence.

[0028] For the purpose of the present application the term "express", "expressing" or "expression" refers to the transcription and translation of a gene encoding a protein.

[0029] The "human proteins" of the invention include, but are not limited to human proteins, polypeptides, mutations and modifications thereof. In particular the human proteins include recombinant plasma proteins, e.g. blood clotting factors (such as factor VIII, Factor Vll/Vlla, Factor V, factor IX, Factor XI, von Willebrand factor, etc.), growth factors (such as erythropoietin, etc.), colony-stimulating factors (CSFs) (such as granulocyte stimulating factor (G-CSF), macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), cytokines (such as interleukins including interleukin 3, etc.), protease inhibitors (such as alpha-1-antitrypsin (A1AT), chymotrypsin, etc.), transport proteins (such as hormones, etc.), inhibitory or regulatory acting proteins, and the like. Furthermore mutations and modifications of these proteins or polypeptides are included, specifically mutations or modifications providing for a better stability of the recombinant protein, an elongated half-life, or a better recovery and include deletion, substitution or insertion mutants and chemical mutations of functional groups, respectively. Particularly preferred proteins which can be produced by the method of the invention of the application are human factor VIII (including B-domain deleted or wild-type), human factor IX, human G-CSF, human A1 AT, human factor Vll/Vlla and von Willebrand factor.

[0030] The recombinant production of the factor VIII and IX is known in the art (EP-A-160457; WO-A-86/01961, U.S. Patents 4,770,999, 5,521,070 and 5,521,070).

[0031] In the case of factor VIII recombinant expression of subunits for the production of complexes showing coagulant activity is known in the art (e.g., from EP-A-150735, EP-A-232112, EP-A-0500734, WO-91/07490, WO-95/13300 U.S. Patents 5,045,455 and 5,789,203). Moreover, the expression of truncated cDNA-versions partially or entirely lacking the sequence coding for the highly glycosylated B-domain have been described (e.g. in WO-86/06101, WO-87/04187, WO-87/07144, WO-88/00381, EP-A-251843, EP-A-253455, EP-A-254076, U.S. Patents 4,868,112 and 4,980,456, EP-A-294910, EP-A-265778, EP-A-303540 and WO-91/09122). A particular factor VIII mutant in which the B-domain between positions Arg740 and Glu1649 has been replaced by an Arg-rich linker peptide having at least 3 Arg residues and comprising 10 to 25 amino acid residues (wherein said factor VIII numbering is relative to the mature wild-type factor VIII sequence shown in SEQ ID NO:9) is disclosed in WO 01/70968 which is herewith incorporated in its entirety. In particular, the Arg-rich linker peptide has 14 to 20 amino acid residues, while a linker comprising: the amino acid sequence SFSQNSRFI (SEQ ID NO:10), and/or the amino acid sequence QAYRYRRG (SEQ ID NO:11), and/or the amino acid sequence SFSQNSRHQAYRYRRG (SEQ ID NO:12) is particularly preferred. Such B-domain factor VIII mutein is encoded by nt 783 to 5162 of SEQ ID NO:3.

[0032] G-CSF is a lineage specific, small molecule in human blood that stimulates the production of a type of white blood cell from the bone marrow, known as neutrophils. Neutrophils play a cental role in the body's immune system and defend infections. G-CSF (particular cDNA sequences of the a, b and c form thereof being given in SEQ ID NOs:15, 16 and 17, respectively; the protein of the G-CSF b form (hereinafter "G-CSFb" protein) is shown in SEQ ID NO:27) is naturally produced by monocytes, fibroblasts, and endothelial cells. Normally the concentration in blood is about 40 pg/ml in healthy persons. In patient plasma, the level of G-CSF can drop more than ten-fold. G-CSF is also produced in cancer cell lines like 5637 cells which secrete about 70 ng/ml. For therapy, recombinant human G-CSF is produced in E. coli as a N-terminal methylated, non-glycosylated form by Amgen Inc. (Filgrastim/Neupogen®), which is also available as a PEGylated product (Pegfilgrastim/Neulasta®). Another drug is produced in CHO cells by Chugai Pharmaceuticals Co, which results in a glycosylated product (Lenograstim/Granocyte®). G-CSF is used as a drug to treat neutropenia either inherited or caused by chemotherapy (cancer), AIDS or bone marrow transplantation. For this, a typical dose is 5 pg/kg and day.

[0033] A particular A1AT cDNA sequence suitable with the invention of the present application is given in bps 973 to 2259 of SEQ ID NO:2. Particular factor Vll/Vlla cDNA sequences are given in SEQ ID NOs:13 and 14 corresponding to the a and b form thereof. A particular vWF cDNA is given in SEQ ID NO: 18.

[0034] The selection marker system includes hygromycin resistance, puromycin resistance, neomycin resistance, adenosine deaminase (ADA) resistance, aminoglycoside phosphotransferase (neo, G418, APH) resistance, bleomycin (phleo, bleo, zeocin) resistance, cytosine deaminase (CDA, CD) resistance, cytosine deaminase (CDA, CD) resistance, dihydrofolate reductase (DHFR) resistance, histidinol dehydrogenase (hisD) resistance, hygromycin-B-phosphotransferase (HPH) resistance, puromycin-N-acetyl transferase (PAC, puro) resistance, thymidine kinase (TK) resistance, and Xanthine-guanine phosphoribosyltransferase (XGPRT, gpt) resistance. Particularly preferred is the hygromycin resistance gene. Also the gene for the selection marker may be functionally linked with a polyA signal such as the one derived from the bovine growth hormone (BGH) or the SV40 polyadenylation signal.

[0035] The transfected cells are constantly exposed in their culture medium to the protein of the selection marker system, such as hygromycin during the selection phase, resulting in the survival of only those cells carrying the vector. A person skilled in the art is familiar with alternative selection markers suitable for the establishment of stably transfected cells, as well with the concentrations of the chosen selective agents wfnich needs to be applied.

[0036] A particularly preferred vector of the invention carries a CMV promoter, a hygromycin gene, a polyA sequence and the gene of interest and preferably is the pcDNA3.1 vector of Invitrogen having the sequence of SEQ ID NO:4 wherein resequencing said vector it was found that it in fact has the sequence shown in SEQ ID NO: 5.

[0037] The immortalized cell lines suitable for the method of the invention are selected from the group of kidney, bladder, liver, lung, cardiac muscle, smooth muscle, ovary or gastrointestinal cells. Those cells may carry in their genome adenoviral DNA sequences, in particular the first 4344 nucleotides of Ad5 sequences. Preferred are human foetal kidney cells (HEK) selected from the group consisting of 293 cells (ATCC CRL-1573; DSM ACC 305; ECACC ref.: 85120602), 293T cells (DSM ACC 2494; ECACC: tsa201, ref. 96121229), and Freestyle 293 cells (293F cells; Invitrogen R79007). Most preferred are 293F cells. Those immortalized cell lines carrying said vector are cultured under conditions allowing expression of the recombinant gene. Essentially those are standard culturing conditions known to the person skilled in the art, however in case of cells carrying the gene for human factor IX, vitamin K should be included in the medium.

[0038] A particular embodiment of the present invention is the serum-free production of the recombinant protein in serum-free culture of the stably immortalized cells, which are also transfected under serum-free conditions. For that anyone of the above described immortalized human cell lines, preferably the 293F cell line is transfected and cultured under serum-free conditions. The cells are stably transfected in suspension culture in the absence of serum and then adapted to adherent cell growth for selection of single cell clones. Once individual clones are obtained, they are expanded adherently. After selection of best producing clones the cells are transferred to suspension culture. During the whole stable cell line procedure and in further up-scaling for production, cells are grown in serum-free medium and are never in touch with serum or human or animal proteins. The recombinant blood protein such as anyone of the blood clotting factors or a protease inhibitor such as A1AT or growth factors (such as G-CSF and GM-CSF) are isolated from the culture broth, and standard purification steps do follow. In more detail, the particular embodiment of serum-free production of the recombinant human blood protein , in particular human factor VIII or factor IX or A1AT or G-CSFb comprises the following steps: 1. (1) Transfection of human immortalized cells, preferably 293F cells in suspension culture without serum. Cells are cultured in disposable, sterile polycarbonate Erlenmeyer flasks. Cells are transfected with a density of e.g. 1 x 106 viable cells per ml with a transfection agent, preferably a cationic transfection agent, more preferred lipofectamine 2000 CD reagent (Invitrogen) or a reagent for the calcium phosphate transfection method); a vector is transfected encoding the human blood protein, preferably the vector is pcDNA3.1-FIX, pcDNA3.1-FVIII, pcDNA3.1-A1AT or pcDNA3.1-GCSFb; 2. (2) 24 to 120 h, preferably 36-96, more preferably 48 h post-transfection a suitable number of cells (103 to 1010; preferably 105 to 108, most preferably 106 cells) are transferred into a flat culture dish for sedimentation to establish adherent growth. Preferably the culture dish is a 10 cm-dish and cells are cultured in serum- and protein-free media, preferably Freestyle 293 Expression medium (12338-018, Invitrogen) or the serum-free in-house medium (Octapharma Stockholm). 3. (3) Selection pressure is started at 2 to 50 h, preferably 48 h post transfer into the flat culture dish. The medium is supplemented with a suitable selective agent selected from the group consisting of selection markers, e.g. hygromycin, neomycin, G418 and Zeocin. The preferred selective agent is hygromicin with a concentration of 10 to 300 pg/ml, preferably 50 to 200 pg/ml, most preferably 50 pg/ml. The pressure is maintained for at least 10 to 20 days, preferably for 14 days, whereby the hygromycin supplemented medium is exchanged every other day. Only stably transfected cells survive these selection conditions and form adherent cell clones which can be individually picked. Additionally an attachment factor can be used in order to stick cells on the dish and prevent cells floating from one clone to an other cell clone. This attachment factors could be f. e. poly-D-Lysine, synthetic medium supplements without human or animal proteins, or other substances. Alternatively, cloning rings could be used for picking of clones. 4. (4) Individual cell clones are picked and transferred into separate culture containers for serum-free expansion of cells (scaling up) while the selective pressure is omitted. Any culture container is suitably, but preferably the individual clones are first transferred into 96 well plates with a sufficient amount of medium, and then to 48-, to 24-, to 12- and to 6-well plates and then to spintubes. At the spintube stage, the cells are cultured in serum-free medium softly shaking in order to bring cells back into suspension growth. Once the cells have reached the 6-cell well plate or the spintube stage, it is optional to select the best cell clones according to some selection criteria, i.e. the growth rate of the cells, faster growing cells are preferred, and the amount of the recombinant protein they do produce. However, said selection can also be performed at any later stage. 5. (5) Cells obtained from the spintube culture were seeded into Erlenmeyer culture vessels with a sufficient amount of serum free medium. Additional selection criteria for up-scaled, in suspension growing serum- and protein-free cell clones are as follows: viability, cell morphology, no aggregation, robustness concerning centrifugation and no cell debris.

[0039] The method of the present invention works particularly well if the vector is pcDNA3.1-hygro(+)-zz. It is preferred that the gene encoding the human protein, in particular human FIX, FVIII, A1AT or G-CSFb is inserted in such a way that it is under the control of the CMV promoter as it is shown in Figs. 2, 3, 7, and 11 respectively. Preferably the wild-type sequences of said genes are inserted, such that the recombinantly expressed protein is without any mutation and is structurally identical to the wild-type protein isolated from blood plasma. A schematic drawing of the wild-type human factor IX is shown in Fig. 1. SEQ ID NOs:1, 2, 3 and 22 provide the nucleic acid sequence for pcDNA3.1-FIX, pcDNA3.1-A1AT, pcDNA3.1-FVIII and pcDNA3.1-GCSFb, respectively. The respective proteins are encoded by nucleotides 939 to 2224, 913 to 2259, 679 to 5055 and 970 to 1584, respectively.

[0040] The present invention thus provides a method for the recombinant production of human factor IX, A1AT, factor VIII and G-CSFb cloned into pcDNA3.1™ giving rise to pcDNA3.1-FIX, pcDNA3.1-A1AT, pcDNA3.1-FVIII and pcDNA3.1-GCSFb, respectively, which are integrated into the genome of immortalized human cells, preferably human embryonic kidney cells such as 293 cells (ATCC CRL-1573; DSM ACC 305; ECACC ref.: 85120602), Freestyle 293 cells (293F cells; Invitrogen R79007) or 293T cells (DSM ACC 2494; ECACC: tsa201, ref. 96121229).

[0041] Those cells carrying either pcDNA3.1-FIX or pcDNA3.1-A1AT or pcDNA 3.1-FVIII or pcDNA 3.1-GCSFb are cultured in medium under standard conditions enabling gene expression or alternatively they are cultured under serum-free conditions to minimize the risk of contamination with human pathogens. One or more prion removal steps may be included, such as protein precipitation, filtration, chromatography steps, in particular affinity chromatography steps. Alternatively/additionally a prion knockout cell line can be used as expression cell. This can be obtained by complete genomic knock-out or antisense technology. In case of the production for human factor IX the cells are preferably cultured in the presence vitamin K. The human blood protein is isolated from the culture supernatant and subjected to subsequent purification steps known in the art to maximize the yield of a pure, stable and highly active product and are selected from immunoaffinity chromatography, anion exchange chromatography, size exclusion chromatography, etc., and combinations thereof. They can easily be adapted to the specific requirements needed to isolate recombinant factor IX G-CSFb or A1AT. Quantity and activity of the purified protein during and after the purification procedure may be monitored by ELISA and/or one-stage coagulation time assays (aPTT).

[0042] To overcome the problems of possible infectious contaminations in the purified protein samples or in the product directly obtained from the cell culture supernatant containing the secreted recombinant protein of choice, the culture supernatant might be treated with procedures for virus inactivation including heat treatment and/or SD-treatment (dry or in liquid state, with or without the addition of chemical substances including protease inhibitors). A person skilled in the art is familiar with purification procedures. For example, the isolation and purification and recovery of high purity virus-inactivated factor VIII from blood plasma by anion exchange chromatography was described I (WO93/15105). In addition several processes for the production of high-purity, non-infectious coagulation factors from blood plasma or other biological sources have been reported. Lipid coated viruses are effectively inactivated by treating the potentially infectious material with a hydrophobic phase forming a two-phase system from which the water insoluble part is subsequently removed. A further advantage has been proven to complement the hydrophobic phase treatment simultaneously or sequentially with a treatment with a non-ionic biocompatible detergents and dialkyl or trialkyl phosphates (WO 96/36369, EP 0131740, US 6, 007,979). Non-lipid coated viruses require inactivation protocols consisting in treatment with non-ionic detergents followed by a heating step (60-65 °C) for several hours (WO 94/17834). After virus inactivation, a further purifying step for removing the chemical substances may be necessary. In summary, the present invention provides an effective protein production method based on a human cell line linked to approved methods of protein purification and for inactivation of potentially dangerous infectious agents. A safe and easy to use-system for production of recombinant proteins, for example the blood clotting factor IX or VIII, A1 AT and G-CSFb has been established. The activity of the recombinantly produced proteins can be examined with standard tests. In case of the human factor IX for example with an activated partial thromboplastin time assay using Dapptin TC (Kaolin/Sulfatid-Phospholipid Cat. No. 5035090, Technoclone GmbH) activation with a manual coagulation instrument. Finally the thus obtained recombinant proteins, such as the blood protein described hereinbefore, in particular the human factor IX may be used in a pharmaceutical composition.

[0043] The invention is further described in the following examples. Said examples are however not to be construed as to limit the invention.

Examples

Materials and Methods [0044] Human cell lines for protein expression: Preferred cell lines are HEK293 (ECACC Ref. 85120602), Freestyle 293 (293F; Invitrogen R79007) and 293T (tsA201, ECACC Ref. 96121229) which is a transformed embryonic human kidney cell line stably expressing an SV40 temperature-sensitive T antigen. These epithelial-like cell lines have been used in a variety of functional expression assays and been reported to produce high levels of recombinant proteins. The 293F cell line (Invitrogen), which is derived from the 293 cell line was preferably used in the Examples below. The parental cell line 293 is a permanent line established from primary embryonal human kidney transformed with sheared human adenovirus type 5 DNA (Graham et al., 1977; Harrison et al., 1977). The 293F cell line is a variant of the 293 cell line that has been adapted to suspension growth in Freestyle™ 293 (293F) Expression Medium(12338-018, Invitrogen). The 293F cell line was obtained from Robert Horlick at Pharmacopeia. The 293F cell line was originally prepared from low passage Master Cell Bank cultures derived from the parental 293F cells that were re-cloned by limiting dilution. Cells have been constantly grown in the serum-free Freestyle 293 Expression medium or a serum-free medium (Octapharma Stockholm) with good viability and good morphology for more than one year during the development of the present invention.

[0045] For efficient production of human factor IX the medium can be modified by addition of vitamin K. These cell lines are capable of being cultivated in serum-free and/or protein-free medium containing suitable supplements.

Determination and measurement of target proteins [0046] Determination of human factor IX concentration bv ELISA: Human recombinant factor IX levels in the supernatant were determined by ELISA using a goat anti-human FIX (GAFIX-AP, Affinity Biologicals) as capture antibody according to standard procedure. All incubations were performed in a humid chamber at RT. Both standards, Octanyne (plasma-derived FIX Octapharma) and BeneFIX (recombinant FIX, Genetics Institute) were used. The detecting antibody was a peroxidase conjugated goat anti-human FIX (GAFIX-APHRP, Affinity Biologicals). ABTS (Cat.No. 1682008, Roche Diagnostics) was added to each well as substrate, colorimetric reaction was detected at 405 nm in 15 minutes. Results were calculated by linear regression of standard concentration versus standard absorbance.

[0047] Detection of Human Clotting factor IX Activity; The clotting activity of human recombinant factor IX in supernatants was determined as follows: The clotting activity was assayed based on an activated partial thromboplastin time assay using Dapptin TC (Kaolin/Sulfatid-Phospholipid, Cat.No. 5035090, Technoclone GmbH) activation with a manual coagulation instrument (Amelung KC 4A micro, Amelung GmbH). For the study, 50 pi supernatant from transfected cells, 50 μΙ FIX-deficient plasma (Progen) and 50 μΙ Dapptin TC were incubated for 2 minutes at 37°C. Coagulation was started by adding 50 μΙ CaCl2 (Cat.No. 84687-22F, Instumentation Laboratory). Sample coagulation time was compared to both Octanyne or/and BeneFIX

[0048] Determination of BDDrhFVIII with COAMATIC:FVIII assay fChromoaenixt: The commercial chromogenic assay kit COAMATIC:FVIII (Chromogenix, cat. No. 82 25 85) contains FIX, FXand a chromogen which is turned into a yellow water soluble dye by FXa cleavage. FVIII containing samples complete this system: FVIII activates FIX by complexing, this complex activates FX by proteolytic cleavage to become FXa. FXa turns the chromogen into a dye which subsequently is determined photometrically at 405 nm. This test is designed for determination of FVIII from patient plasmas. The following procedure was set up in order to make this test applicable for the factor VIII measurement in diluted culture media. As control standards, full length recombinant human clotting factor VIII (NIBSC, order no. 57814F) and normal control plasma (Instrumentation Laboratory Company) was used.

[0049] Sample preparation: Samples were diluted with dilution buffer delivered with COAMATIC reagents to a prospective final FVIII activity between 2 and 20 mlU/ml and are compared to the WHO No. 6 standard curve.

[0050] Method: On a 96-well array placed on the thermobloc, both standards and samples are measured in triple.

Operation scheme fper welh:

[0051] Determination of A1AT activity with elastase activity test: After transfection of A1AT cDNA, A1AT was expressed and secreted into cell culture medium. After removal of cells by centrifugation (5 minutes, 1000 rpm), A1AT activity was measured in culture supernatant. In this activity test, A1AT activity was determined by its inhibitory effect upon elastase. Elastase cleaves pNA from the substrate N-succinyl-(Ala)3-pNA. pNA release is measured photometrically at 405 nm. By comparison with standard samples with defined A1AT activity, the activity of the respective samples is determined. As proven in other experiments, the test is valid in serum-free Freestyle medium. To confirm the fact that Freestyle medium has no influence on the test, two standard curves were prepared: standard human plasma was diluted in T+ buffer or in Freestyle medium.

[0052] Dilution of samples: All samples were tested undiluted, 1:10 and 1:50 diluted in Freestyle medium and are compared with standard dilutions of human plasma.

[0053] Method: 50 μΙ of each standard dilution and sample dilution, respectively was pipetted into a well of the 96-well micro titer plate. After adding 150 μΙ of Elastase working solution to each well, the 96-well plate was shaken for 1 minute on the ELISA reader and incubated for 30 minutes at 37°C. 100 μΙ of substrate working solution was added to each well with the multipette. Absorption at 405 nm was measured immediately after addition of substrate solution and after 7 minutes incubation at 37°C in the dark. The first value represents the basis absorption without elastase-catalised reaction and is substacted from the second one which represents the absorption after elastase cleaved pNA from the substrate. Using the result after the substraction, the A1AT activities of the samples are calculated according to the standard curve.

[0054] Determination of G-CSF activity bv ELISA: Human recombinant G-CSF levels in the cell culture supernatants were determined by ELISA using a mouse anti-human G-CSF antibody (MAB-214, R&D System) as capture antibody according to standard procedure. All incubations were performed in a humid chamber at room temperature. The G-CSF standard (recombinant hG-CSF, E.coli, 214-CS-025, R&D Systems) was used. The detection antibody was a biotinylated goat anti-human G-CSF (BAF-214, R&D Systems). Streptavidin was conjugated to horseradish-peroxidase (DY998, R&D Systems) linked to the detection antibody. The QuantaBlu™ Fluorogenic Peroxidase Substrate (15169, Pierce) was added to each well as substrate, fluorometic reaction was detected at extinction 320 nm/Emission 420 nm within 60 min. Results were calculated by linear regression of standard concentration versus standard relative fluorescence units (RFU).

Example 1: Cloning of target proteins.

[0055] A. Cloning of human factor IX From the vector pTG36 as disclosed in W001/70968, a 1.4 kb fragment containing the open reading frame of the human clotting factor IX was cut out by double-digestion with Hind III and Notl. This fragment was ligated to the 5.6 kb fragment of the Hindlll and Notl double-digested vector pcDNA3.1Hygro(+)-zz (derived from V870-20, Invitrogen) resulting in the vector pcDNA3.1-FIX shown in Fig. 2. The DNA sequence of pcDNA3.1-FIX is shown in SEQ ID NO.1. Three additional nucleotide insertions in the vector backbone were found in the pcDNA3.1Hygro(+)-zz vector (see SEQ ID NO:5) compared to the sequence published by Invitrogen (as shown in SEQ ID NO:4). The vector pcDNA3.1-FIX contains a cassette hygromycin-resistance gene to enable a selection method for a stably transfected cell clone (see Figs. 2, 3 and 7). The vector allows the establishment of stably expressing cell lines by calcium phosphate transfection or others, and subsequent selection for hygromycine resistants. B. Cloning of human factor VIII: A 4380 bp FVIIIcDNA containing the open reading frame of a B-domain deleted human clotting factor VIII was isolated from the vector pTGF8-2hyg-s (SEQ ID NO:7; the production of which being disclosed in W001/70968) with Notl+Xhol digestion and ligated with pcDNA3.1 Hygro(+)-zz, which was linearized with Xhol+PspOMI resulting in the vector pcDNA3.1-FVIII shown in Fig. 7. C. Cloning of human A1 AT: A1AT mRNAwas isolated directly from the HepG-2 cells (DSMZ#ACC 180) using mRNAMiniprep Kit (Sigma, Cat# MRN-10). In the following step mRNAwas captured on oligo (dT) beads. Afterwards, mRNAwill be transcribed into double-stranded cDNAwith Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT, Promega, Cat# M5101) following RT-PCR (reverse Transcription- Polymerase Chain Reaction). A1AT cDNAwas amplified with PCR reaction. The PCR product was loaded on agarose gel. The appropriate DNA- band was isolated and afterwards purified with the Qiaquik Gel Extraction Kit (Qiagen, Cat# 28704). Then A1AT fragment was subcloned into a commercial Vector (TOPO® Invitrogen, Cat# K4650-01). For cloning of pCMV-Script: PCR II TOPO-A1AT was digested with EcoRI, the A1AT 1370 bp fragment was ligated with pCMV-Script linearized with EcoRI.

For cloning of pCI-neo-A1AT PCR II TOPO-A1AT was digested with EcoRI, the A1AT 1370 bp fragment was ligated with pCI-neo linearized with EcoRI.

For cloning of pcDNA3.1-FVIII 1370 bp A1AT was isolated with PCR II TOPO-A1AT digested with Xhol+Hindlll and ligated with pcDNA3.1 linearized by Xhol+Hindlll. The resulting vector is shown in Fig. 3.

For cloning of pTG1-A1AT PCR II TOPO-A1AT was digested with Hindlll and Notl. The A1AT 1370 bp fragment was ligated with pTG1 (no PRE), linearized with Hindlll and Notl. The resulting vector is shown in Fig. 9. D. Cloning of human G-CSF cDNA: Total RNAwas isolated directly from natural 5637 human urinary bladder carcinoma cells with RNeasy mini kit (QIAGEN, cat. No. 74104). Afterwards, the isolated total RNAwas incubated with DNase I to digested possibly mixed genomic DNA of 5637 cells. To get DNase-free total RNA the reaction mixture was treated with RNeasy clean-up kit (QIAGEN, cat. No. 74204). RT-PCR with the total RNA as template was performed with oligo(dT)12-18 primer (Invitrogen, Cat. No. 18418-012) and Superscript™ II RNase H- Reverse Transcriptase (Invitrogen, Cat. No. 18064-022) in the presence of RNase inhibitor (Roche, Cat. No. 799-017) to synthesize ds cDNA pool from 5637 cells. G-CSF cDNAwas amplified then with PCR reaction. G-CSF cDNAwas isolated from agarose gel with QIAquick Gel Extraction kit (QIAGEN, Cat. No. 28704) and sequenced with both of the following G-CSF PCR primers: • G-CSF Forward: 5'- ATG GCT GGA CCT GCC ACC CAG AGC -3 '(SEQ ID NO: 19) • G-CSF Reverse: 5'- TCAGGG CTG GGC AAG GTG GCG TAG-3' (SEQ ID NO:20) The sequence of the DNA synthesized from 5637 cells was confirmed by sequence analysis to be a GCSF-b form (having the sequence shown in SEQ ID NO:26).

[0056] The cDNA of GCSF-b form isolated as described above was then directly ligated into the commercial vector pCR2.1 (Invitrogen). The resulting plasmid was designated pCR2.1d2-GCSFb and is shown in Fig. 10.

[0057] PCR2.1d2-GCSFb was digested with Hindlll and Notl, the 705 bp GCSFb cDNA fragment was isolated and ligated into the vector pcDNA3.1Hygro(+)-zz, which was linearized with Hindlll and Notl. The resulting pDNA3.1-GCSFb vector is shown in Fig. 11.

[0058] PCR2.1d2-GCSFb was digested with EcoRI, the 629 bp GCSFb cDNA fragment was isolated and ligated into the pCINeo vector, which was linearized with EcoRI. The resulting pCINeo-GCSFb vector is shown in Fig. 12.

[0059] PCR2.1d2-GCSFb was digested with BamHI and Xhol, the 693 bp GCSFb cDNA fragment was isolated and ligated into the pCMVScript vector, which was linearized with BamHI and Xhol. The resulting pCMVScript-GCSFb vector is shown in Fig. 13. PCR2.1d2-GCSFb was digested with Hindlll and Notl, the 705 bp GCSFb cDNA fragment was isolated and ligated into pTG2-hyg-as vector, which was linearized with Hindlll and Notl. The resulting pTG2-GCSFb-hyg-as vector is shown in Fig. 14.

[0060] Example 2: Expression of target proteins in different cell lines and different vectors: When optimizing the present method for recombinant protein production the ability for high levels of expression of different cell lines- all carrying a vector comprising the recombinant gene for Alpha-1-antitrypsin (A1AT) - was tested. CHO, BHK and other cell lines were found to produce less recombinant protein in transient transfection assays compared to the 293T cell line. Therefore other human embryonic kidney cell line derivates were examined. The results are shown in Figs. 4 to 6.

[0061] Example 3: Transient transfection of 293T and 293 cells in serum containinn medium as comparison to 293F cells, which were transfected and cultured under serum-free conditions: 0.1 -0.2 x 106 viable cells of 293T or 293 cells were plated into 6-well.

On the next day cells were transfected using Calcium phosphate method (Biotechniques 6:7 632-638 (1988)): 4pg of plasmid DNA were diluted in 0.1 xTE buffer (ad 200 pi transfection mix), mixed gently, 20 μΙ 2.5 M CaCl2 and 100 μΙ 2x HBS were added to the transfection sample. The transfection sample was incubated for 20 min at room temperature. After 6 h incubation medium was exchanged and cells were then incubated for 48 h.

[0062] Example 4: Serum-free Transfection and expression of target proteins in 293F cells in serum-free medium: 28 ml suspension culture was prepared with a cell density of 106 viable 293F cells (on the same day of the transfection experiment). A lipid-DNA complex was prepared by diluting 30 pg of plasmid DNA in Opti-MEM® I (Invitrogen) to a total volume of 1 ml, and 40 μΙ of 293fectin®was diluted in Opti-MEM® I to a total volume of 1ml. After the 5min incubation at room temperature, diluted DNA was added to 293fectin® to obtain a total volume of 2 ml. The transfected samples have been incubated for 20 min at room temperature in the dark. 2 ml of the transfection mix was added to the 28 ml 293F suspension culture (final cell density is 1x106 cells/ml). The transfected 293F cells were incubated at 37 °C/humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm for 72 h. 1. A: Transfection and expression of A1AT: The results of those experiments comparing 293F with 293 and 293T cells are shown in Fig. 4. In all experiments a defined quantity of cells (106 cells) were transfected with pcDNA3.1-A1AT. The amount of A1AT expressed in these different cell lines was compared. The expressed amount of A1AT in 293 F cells was set as 100 %. As can be seen from Fig. 4, 293 and 293 T cells produced only 12 - 13 % of the amount of A1AT than 293F cells. Moreover, it was tested whether different vector backbones influence the amount of recombinant protein produced in 293F cells. The coding sequence for human Alpha-1-antitrypsin (A1AT) was inserted into pTG (in-house vectors), pCMV Script® (Stratagene), pel neo (Promega) as well as into the pcDNA3.1 ™ vector.

The expression level of A1AT from pcDNA3.1-A1AT was set as 100%. None of the other vectors came close to the high expression observed with pcDNA™3.1-A1AT. It was found that pcDNA3.1-A1AT produced the greatest amount of A1AT as detected with ELISA (see Fig. 5). The in-house vector expressed only an amount of 20% and the other commercial vectors revealed a range of 15-30% compared to the amount of A1AT expressed from pcDNA3.1. Therefore pcDNA3.1 was chosen for all further experiments.

In summary, different cell lines had been transiently transfected with pcDNA3.1 ™ carrying the A1 AT gene. It was shown that the serum-free 293F cell line expresses 7-times more A1AT per 106 cells than 293T and 293 cells. Therefore freestyle 293 F cell line were chosen for stable transfection experiments.

The results of transient transfection experiments are shown in Fig. 6. The top panel (A) shows the SDS-PAGE analysis of the supernatant of 6 different transfection trials using different transfection vectors. Derived from analytical Figures it can be concluded that the a1-antitrypsin present in the analysed cell culture supernatants is of good quality as can be deduced from the ratio of activity to antigen being 1 (data not shown). Validity of the test results can be deduced from the fact that the negative control does not showa1-antitrypsin activity nor antigen.

The molecular weight distribution analysed by SDS-PAGE shows well comparable pictures for the three a1-antitrypsin containing samples. In the negative control, besides the lack in the a1-antitrypsin representing band, an additional band at a molecular weight of 27 kD is visible as expected.

By analysis using western blotting (using an anti human a1-antitrypsin primary antibody) the protein can be identified in the expected molecular weight region under reducing conditions. Split products are not visible.

The black arrow points to the prominent band which corresponds to the 52 kDa recombinant protein alpha-1-antitrypsin. Also visible is a band corresponding to the 27 kDa GFP protein in lanes 4 and 8, which was transiently expressed as control in cell line freestyle 293 F cells. The additional bands in lane 1,2,3 and 5,6 and 7 are host cell proteins (from freestyle 293 F cells). The lower panel (B) shows the Western Blot analysis. The results are identical except that due to the higher stringency of the assay, the results appear cleaner, and only the band corresponding to the A1 AT is visible. 2. B: Transfection and expression of FVIII: In Fig. 8 the average amount of factor VIII of the best three stably transfected clones is shown. The average amount of factor VIII of the three best clones expressed with pcDNA-FVIII vector is set as 100% productivity. A comparison with the in-house vector pTGF8-2hyg-s reveals almost 3-fold higher productivity of factor VIII with pcDNA3.1-FVIII vector in 293F cells. 3. C: Transfection and expression of FIX In stably transfected 293F cells using pcDNA3.1-FIX and a pUC 19/X based vector pTGF36 (see WO 01/70968) expressing factor IX almost 3-fold higher productivity could be shown with the use of pcDNA3.1 vector in 293F cells as can be seen in the following Table 1.

Table 1: FIX productivity in 293 and 293F cells after stable transfection with pcDNA3.1 and pTG2 vector

Example 5: Production of G-CSFb [0063] 1. A. Transfection of 293F cells in serum-free medium, transient transfection: 28 ml suspension culture of 293F cells with a cell density of 1.1x 106 viable 293F cells per ml was prepared on the same day of the transfection experiment. Alipid-DNA complex was prepared by diluting 30 pg of plasmid DNA(pcDNA3.1-G-CSFb) in Opti-MEM® I (Invitrogen) to a total volume of 1 ml and 30 pi of Lipofectamine 2000 CD was diluted in Opti-MEM® I to a total volume of 1ml. After 5 min incubation at room temperature, diluted DNA was added to Lipofectamine 2000 CD to obtain a total volume of 2 ml. The transfection samples were incubated for 20 min at room temperature in the dark. 2 ml of the transfection mix was added to the 28 ml 293F suspension culture (final cell density is 1x 106 cells per ml). The transfected 293F cells were incubated at 37 °C/humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm for 72 h. 2. B. Stable transfection: 72 h after transient transfection as set forth in A. above, a suitable number of cells (105 and 106 cells) were transferred into a flat dish for sedimentation to establish adherent growth. Selection pressure was started after 2 to 50 h, preferably 48 h post transfer into the flat dish. The preferred selective agent was hygromycine with a concentration of 75 pg/ml. The pressure was maintained for at least 10 to 20 days, preferably for 14 days, whereby the hygromcine supplemented medium was exchanged all 2 to 3 days. 3. C. Selection of best G-CSF producer clones using the analysis and picking robot ClonePixFL fGenetixl: Freestyle 293F cells stably transfected as described in B. above were seeded in semi-solid methyl-celluloses based medium containing an appropriate antibiotic for selection of clones after about two days and a labelled antibody for detection of the highest producer clones via fluorescence. Large numbers (thousands) of clones were analyzed using ClonePixFL (Genetix) with respect to the cell number and to G-CSF secretion in order to subsequently pick only a few hundred G-CSF best producer clones. In contrast to other know/n methods, where non-producer clones and mixed clones are randomly picked as well, the use of ClonePixFL allows picking of fast growing clones, which are high producers only, originated from single cells. The picked cells are expanded in microtiter plates and later in spin tubes, cell culture flasks and fermenters under serum-free conditions for the complete procedure.

Here as well the whole stable transfection procedure is generated under serum-free conditions. Additionally, during the whole following expansion and cell culture procedure, the cell did not have any contact to serum or animal derived proteins. During expansion, the best clones are selected with respect to robustness, high growth rate, viability and production of active G-CSF as measured in ELISA format. After this selection phase, the picked clones are cultured under serum-free conditions without antibiotic supplements. 293F cells were cultured completely serum-free during the whole procedure, medium was exchanged every other day. Up-scaling of the cells was performed under completely serum-free conditions from Erlenmeyer flasks in Kiihner Shakers to higher volumes in wave reactor (Wave Biotech Europe). During this selection the number is reduced again to only a few best producing clones. Correct cDNA sequence, mRNA content and behaviour upon fermentation are the criteria to identify the best clone(s) for subcloning. For this, cells of the selected clone(s) are plated, analyzed and picked with ClonePixFL, and then expanded and selected as described before. Subcloning is an essential step in order to select again for better producer clones to eliminate possible genetic variations in the plated subpopulation of the clone. After subcloning, the selected clone(s) are banked again under serum-free conditions. The expressed recombinant human G-CSF protein is characterized biochemically in more detail. 4. D. Determination of human G-CSF concentration by ELISA: The quantity of the rhG-CSF expressed by the FeeStyle 293F cell lines thus obtained was determined by ELISA, and the yield of protein obtained with cells transfected with different vectors was compared (see Fig. 15). As expected from the expression experiments with FIX and A1AT described in Examples 2 and 3, here again a combination of the vector pcDNA 3.1-G-CSFb with the 293F cell line showed the highest productivity and was therefore used for production of recombinant human G-CSFb. 5. E. Western blot of rhG-CSF in reducing SDS PAGE: 10 μΙ G-CSF produced in supernatants from HEK293 and HEK293F cells was analyzed on 15% SDS PAGE and western blot. Detection of G-CSF was done via BAF214-bio/SA-HRP/DAB (see Fig. 16). The arrow indicates the monomeric band of rhG-CSF of the correct molecular mass.

Sequence Listing, Free text [0064] SEP ID NO:1: dcDNA3.1-FIX.

SEQ ID NO:2: dcDNA3.1-A1AT

SEQ ID NO:3: pcDNA3.1-FVIII_

SEQ ID N0:4: pcDNA3.1 sequence published by Invitrogen

SEQ ID NO:5: pcDNA3.1 Hygro(+)-zz, having 3 additonal nt "GGT" at position 4380 compared to SEQ ID NO:4

SEQ ID NO:6: vector pTG 1 -A1 AT SEQ ID NO:7: vector pFGF8-hva-s

SEQ ID N0:8: human wild-type factor VIII cDNA

SEQ ID N0:9: human wild-type factor VIII SEQ ID N0s:1Q-12: linker peptides SEQ ID N0:13: cDNA of hFVII a-form SEQ ID NO: 14: cDNA of hFVII b-form SEQ ID NO: 15: cDNA of human GCSF a-form, CDS: 41-661 SEQ ID NO: 16: cDNA of human GCSF b-form, CDS: 41-652 SEQ ID NO: 17: cDNA of human GCSF c-form, CDS: 229-828

SEQ ID NO: 18: cDNA of hvWF SEQ ID NOs:19 and 20: G-CSF forward and reverse primer SEQ ID NO:21: vector PCR2.1d2-GCSFb

SEQ ID NO:22: vector pcDNA3.1-hyg(+)-GCSFb

SEQ ID NO:23: vector pCINeo-GCSFb

SEQ ID NO:24: vector pCMVScript_GCSFb,

SEQ ID NO:25: pTG2-GCSFb-hyg-as_

SEQ ID NO:26: cDNA of human GCSF b-form SEQ ID NO:27: human GCSF b-form protein

SEQUENCE LISTING

[0065]

<110> Octapharma AG <120> Serum-Free Stabile Transfrection and Production of Recombinant Human Proteins in Human Cell Lines

<130> 060523wo/JH <150> EP05105965.7 <151 >2005-06-30 <160> 27 <170> Patentln version 3.3

<210> 1 <211 >6960 <212> DNA <213> Artificial Sequence <220> <223> vector pcDNA3.1-FIX <400> 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaactt aagcttgcat gccaattccg caaaggttat gcagcgcgtg aacatgatca 960 tggcagaatc accaggcctc atcaccatct gccttttagg atatctactc agtgctgaat 1020 gtacagtttt tcttgatcat gaaaacgcca acaaaattct gaatcggcca aagaggtata 1080 attcaggtaa attggaagag tttgttcaag ggaaccttga gagagaatgt atggaagaaa 1140 agtgtagttt tgaagaagca cgagaagttt ttgaaaacac tgaaagaaca actgaatttt 1200 ggaagcagta tgttgatgga gatcagtgtg agtccaatcc atgtttaaat ggcggcagtt 1260 gcaaggatga cattaattcc tatgaatgtt ggtgtccctt tggatttgaa ggaaagaact 1320 gtgaattaga tgtaacatgt aacattaaga atggcagatg cgagcagttt tgtaaaaata 1380 gtgctgataa caaggtggtt tgctcctgta ctgagggata tcgacttgca gaaaaccaga 1440 agtcctgtga accagcagtg ccatttccat gtggaagagt ttctgtttca caaacttcta 1500 agctcacccg tgctgagact gtttttcctg atgtggacta tgtaaattct actgaagctg 1560 aaaccatttt ggataacatc actcaaagca cccaatcatt taatgacttc actcgggttg 1620 ttggtggaga agatgccaaa ccaggtcaat tcccttggca ggttgttttg aatggtaaag 1680 ttgatgcatt ctgtggaggc tctatcgtta atgaaaaatg gattgtaact gctgcccact 1740 gtgttgaaac tggtgttaaa attacagttg tcgcaggtga acataatatt gaggagacag 1800 aacatacaga gcaaaagcga aatgtgattc gaattattcc tcaccacaac tacaatgcag 1860 ctattaataa gtacaaccat gacattgccc ttctggaact ggacgaaccc ttagtgctaa 1920 acagctacgt tacacctatt tgcattgctg acaaggaata cacgaacatc ttcctcaaat 1980 ttggatctgg ctatgtaagt ggctggggaa gagtcttcca caaagggaga tcagctttag 2040 ttcttcagta ccttagagtt ccacttgttg accgagccac atgtcttcga tctacaaagt 2100 tcaccatcta taacaacatg ttctgtgctg gcttccatga aggaggtaga gattcatgtc 2160 aaggagatag tgggggaccc catgttactg aagtggaagg gaccagtttc ttaactggaa 2220 ttattagctg gggtgaagag tgtgcaatga aaggcaaata tggaatatat accaaggtat 2280 cccggtatgt caactggatt aaggaaaaaa caaagctcac ttaatgggat cggtcgagcg 2340 gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag 2400 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 2460 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 2520 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 2580 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg 2640 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 2700 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 2760 cccttccttt ctcgccacgL tcgccggctt tccccgtcaa gctctaaatc gggggctccc 2820 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 2880 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 2940 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 3000 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 3060 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga 3120 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 3180 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 3240 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc 3300 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 3360 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc 3420 tLLLgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgatg 3480 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc 3540 gtctccgacc tgatgcagct ctcggagggc gaagaaLclc gtgctttcag cLtcgatgta 3600 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt 3660 tatgtttatc ggcactttgc atcggccgcg cLcccgaLtc cggaaglgcL tgacattggg 3720 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa 3780 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg 3840 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc 3900 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac 3960 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg 4020 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc 4080 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg 4140 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt 4200 aLggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg 4260 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc 4320 aatttcgatg atgcagcttg ggcgcagggt cgaLgcgacg caatcglccg aLccggagcc 4380 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt 4440 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa 4500 tagcacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga 4560 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc 4620 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 4680 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 4740 atcaatgtat cttatcalgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca 4800 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 4860 gccggaagca taaagLgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 4920 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 4980 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcLLccgc ttcctcgctc 5040 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 5100 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 5160 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 5220 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 5280 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 5340 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 5400 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 5460 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 5520 aacccggtaa gacacgactt atcgccacLg gcagcagcca ctggtaacag gattagcaga 5580 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 5640 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccLtcgg aaaaagagtt 5700 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 5760 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 5820 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 5880 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 5940 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 6000 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 6060 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 6120 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 6180 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 6240 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 6300 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 6360 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 6420 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 6480 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 6540 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 6600 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 6660 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 6720 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 6780 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 6840 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 6900 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 6960

<2102 <211> 6914 <212> DNA <213> Artificial Sequence <220> <223> vector pcDNA3.1-A1AT <400>2 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaactt aagcttgtga atcgacaatg ccgtcttctg tctcgtgggg catcctcctg 960 ctggcaggcc tgtgctgcct ggtccctgtc tccctggctg aggatcccca gggagatgct 1020 gcccagaaga cagatacatc ccaccatgat caggatcacc caaccttcaa caagatcacc 1080 cccaacctgg ctgagttcgc cttcagccta taccgccagc tggcacacca gtccaacagc 1140 accaatatct tcttctcccc agtgagcatc gctacagcct ttgcaatgct ctccctgggg 1200 accaaggctg acactcacga tgaaatcctg gagggcctga atttcaacct cacggagatt 1260 ccggaggctc agatccatga aggcttccag gaactcctcc gtaccctcaa ccagccagac 1320 agccagctcc agctgaccac cggcaatggc ctgttcctca gcgagggcct gaagctagtg 1380 gataagtttt tggaggatgt taaaaagttg taccactcag aagccttcac tgtcaacttc 1440 ggggacaccg aagaggccaa gaaacagatc aacgattacg tggagaaggg tactcaaggg 1500 aaaattgtgg atttggtcaa ggagcttgac agagacacag tttttgctct ggtgaattac 1560 atcttcttta aaggcaaatg ggagagaccc tttgaagtca aggacaccga ggaagaggac 1620 ttccacgtgg accaggtgac caccgtgaag gtgcctatga tgaagcgttt aggcatgttt 1680 aacatccagc actgtaagaa gctgtccagc tgggtgctgc tgatgaaata cctgggcaat 1740 gccaccgcca tcttcttcct gcctgatgag gggaaactac agcacctgga aaatgaactc 1800 acccacgata tcatcaccaa gttcctggaa aatgaagaca gaaggtctgc cagcttacat 1860 ttacccaaac tgtccattac tggaacctat gatctgaaga gcgtcctggg tcaactgggc 1920 atcactaagg tcttcagcaa tggggctgac ctctccgggg tcacagagga ggcacccctg 1980 aagctctcca aggccgtgca taaggctgtg ctgaccatcg acgagaaagg gactgaagct 2040 gctggggcca tgtttttaga ggccataccc atgtctatcc cccccgaggt caagttcaac 2100 aaaccctttg tcttcttaat gattgaacaa aataccaagt ctcccctctt catgggaaaa 2160 gtggtgaatc ccacccaaaa ataactgcct ctcgctcctc aacccctccc ctccatccct 2220 ggccccctcc ctggatgaca ttaaagaagg gggtaccgca agggcgaatt ctgcagatat 2280 ccatcacact ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg 2340 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 2400 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 2460 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 2520 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa 2580 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg 2640 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 2700 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 2760 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 2820 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 2880 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt Lccaaactgg aacaacactc 2940 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 3000 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc 3060 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 3120 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 3180 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 3240 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 3300 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 3360 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 3420 atcagcacgt gatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg 3480 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt 3540 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt 3600 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag 3660 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg 3720 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg 3780 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg 3840 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc 3900 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg 3960 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg 4020 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact 4080 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc 4140 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg 4200 caggatcgcc qcqgctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga 4260 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg 4320 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct 4380 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc 4440 cgagggcaaa ggaatagcac gtgctacgag atttcgattc caccgccgcc ttctatgaaa 4500 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 4560 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 4620 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 4680 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 4740 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 4800 cacacaacat acqagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 4860 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 4920 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 4980 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 5040 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 5100 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5160 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 5220 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5280 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5340 tggcgctttc tcatagctca cgctgtåggt atctcagttc ggtgtaggtc gttcgctcca 5400 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5460 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5520 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5580 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 5640 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccqctggt agcggtggtt 5700 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5760 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5820 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 5880 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 5940 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 6000 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 6060 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 6120 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 6180 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 6240 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 6300 ggcgagttac atgatocccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 6360 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 6420 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 6480 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 6540 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 6600 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 6660 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 6720 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 6780 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 6840 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 6900 tgccacctga cgtc 6914

<210> 3 <211> 9975 <212> DNA <213> Artificial Sequence <220> <223> vector pcDNA3.1-FVII <400>3 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc 60 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 120 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 180 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 240 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cocctattga 300 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 360 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 420 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 480 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 540 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 600 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac 660 gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttgg taccgagctc 720 ggatccacta gtccagtgtg gtggaattct gcagatatcc agcacagtgg cggccgctcg 780 agatgcaaat agagctctcc acctgcttct ttctgtgcct tttgcgattc tgctttagtg 840 ccaccagaag atactacctg ggtgcagtgg aactgtcatg ggactatatg caaagtgatc 900 tcggtgagct gcctgtggac gcaagatttc ctcctagagt gccaaaatct tttccattca 960 acacctcagt cgtgtacaaa aagactctgt ttgtagaatt cacggatcac cttttcaaca 1020 tcgctaagcc aaggccaccc tggatgggtc tgctaggtcc taccatccag gctgaggttt 1080 atgatacagt ggtcattaca cttaagaaca tggcttccca tcctgtcagt cttcatgctg 1140 ttggtgtatc ctactggaaa gcttctgagg gagctgaata tgatgatcag accagtcaaa 1200 gggagaaaga agatgataaa gtcttccctg gtggaagcca tacatatgtc tggcaggtcc 1260 tgaaagagaa tggtccaatg gcctctgacc cactgtgcct tacctactca tatctttctc 1320 atgtggacct ggtaaaagac ttgaattcag gcctcattgg agccctacta gtatgtagag 1380 aagggagtct ggccaaggaa aagacacaga ccttgcacaa atttatacta ctttttgctg 1440 tatttgatga agggaaaagt tggcactcag aaacaaagaa ctccttgatg caggataggg 1500 atgctgcatc tgctcgggcc tggcctaaaa tgcacacagt caatggttat gtaaacaggt 1560 ctctgccagg tctgattgga tgccacagga aatcagtcta ttggcatgtg attggaatgg 1620 gcaccactcc tgaagtgcac tcaatattcc tcgaaggtca cacatttctt gtgaggaacc 1680 atcgccaggc gtccttggaa atctcgccaa taactttcct tactgctcaa acactcttga 1740 tggaccttgg acagtttcta ctgttttgtc atatctcttc ccaccaacat gatggcatgg 1800 aagcttatgt caaagtagac agctgtccag aggaacccca actacgaatg aaaaataatg 1860 aagaagcgga, agactatgat gatgatctta ctgattctga aatggatgtg gtcaggtttg 1920 atgatgacaa ctctccttcc tttatccaaa ttcgctcagt tgccaagaag catcctaaaa 1980 cttgggtaca ttacattgct gctgaagagg aggactggga ctatgctccc ttagtcctcg 2040 cccccgatga cagaagttat aaaagtcaat atttgaacaa tggccctcag cggattggta 2100 ggaagtacaa aaaagtccga tttatggcat acacagatga aacctttaag actcgtgaag 2160 ctattcagca tgaatcagga atcttgggac ctttacttta tggggaagtt ggagacacac 2220 tgttgattat atttaagaat caagcaagca gaccatataa catctaccct cacggaatca 2280 ctgatgtccg tcctttgtat tcaaggagat taceaaaagg tgtaaaacat ttgaaggatt 2340 ttccaattct gccaggagaa atattcaaat. ataaatggac agtgac.tgta gaagatgggc 2400 caactaaatc agatcctcgg tgcctgaccc gctattactc tagtttcgtt aatatggaga 2460 gagatetage ttcaggactc attggccctc tcctcatctg ctacaaagaa tetgtagate 2520 aaagaggaaa ccagataatg tcagacaaga ggaatgtcat cctgttttct gtatttgatg 2580 agaaccgaag ctggtacctc aeagagaata tacaacgctt tctccccaat- ccagctggag 2640 tgcagcttga ggatccagag ttccaagcct ccaacatcat gcacagcatc aatggctatg 2700 tttttgatag tttgcagttg tcagtttgtt tgeatgaggt ggcatactgg tacattctaa 2760 gcattggagc aeagaetgae ttcctttctg tcttcttctc tggatatacc ttcaaacaca 2820 aaatggtcta tgaagacaca ctcaccctat tcccattctc aggagaaact gtcttcatgt 2880 cgatggaaaa cccaggtcta tggattctgg ggtgccacaa eteagaettt eggaaeagag 2940 gcatgaccgc ettaetgaag gtttctagtt gtgacaagaa cactggtgat tattaegagg 3000 aeagttatga agatatttea gcatacttgc tgagtaaaaa caatgccatt gaaccaagaa 3060 gcttctccca gaattcaaga catcaagctt ategataeeg tcgaggggaa ataactcgta 3120 ctactcttca gtcagatcaa gaggaiaattg actatgatga taccatatca gttgaaatga 3180 agaaggaaga ttttgacatt tatgatgagg atgaaaatca gagcccccgc agctttcaaa 3240 agaaaacacg acactatttt attgctgcag tggagagget ctgggattat gggatgagta 3300 gctccccaca tgttetaaga aacagggctc agagtggcag tgtccctcag ttcaagaaag 3360 ttgttttcca ggaatttact gatggctcct ttactcaqcc cttataccqt qgagaactaa 3420 atgaacattt gggactcctg gggccatata taagageaga agttgaagat aatatcatgg 3480 taactttcag aaatcaggcc tctcgtccct atteetteta ttctagcctt atttettatg 3540 aggaagatca gaggcaagga gcagaaccta gaaaaaactt tgtcaagcct aatgaaacca 3600 aaaettaett ttggaaagtg caacatcata tggcacccac taaagatgag tttgactgca 3660 aagcctgggc ttatttetet gatgttgacc tggaaaaaga tgtgcactca ggcctgattg 3720 gaccccttct ggtctgccac actaacaeac tgaaccctgc tcatgggaga caagtgacag 3780 taeaggaatt tgctctgttt ttcaccatct ttgatgagac caaaagctgg taetteaetg 3840 aaaatatgga aagaaactgc agggctccct gcaatatcca gatggaagat cccactttta 3900 aagagaatta tcgcttccat gcaatcaatg gctacataat ggatacacta cctggcttag 3960 taatggctca ggatcaaagg attegatggt atetgeteag catgggcagc aatgaaaaca 4020 tccattctat teattteagt ggacatgtgt teaetgtaeg aaaaaaagag gagtataaaa 4080 tggcactgta caatctctat ccaggtgttt ttgagaeagt ggaaatgtta ccatccaaag 4140 ctggaatttg gcgggtggaa tgccttattg gegageatet acatgctggg atgagcacac 4200 ttLttcLggL gtacagcaal aagLglcaga cLccccLggg aatggcLLcL ggacacalta 4260 gogattttea gattaeaget tcaggacaat atggacagtg ggccccaaag ctggccagac 4320 tteattatte cggatcaatc aatgcctgga gcaccaagga gcccttttct tggatcaagg 4380 tggatctgtt ggcaccaatg attatteaeg gcatcaagac ccagggtgcc egteagaagt 4440 tctccagcct ctacatctct eagtttatea tcatgtatag tcttgatggg aagaagtgge 4500 agaettateg aggaaattcc actggaacct taatggtctt ctttggcaat gtggattcat 4560 ctgggataaa acacaatatt tttaaccctc caattattgc tcgatacatc cgtttgcacc 4620 caactcatta tageattege agcactcttc gcatggagtt gatgggctgt gatttaaata 4680 gttgcagcat gccattggga atggagagta aagcaatatc agatgeaeag attaetgett 4740 catcctactt taccaatatg tttgccacct ggtctccttc aaaagctcga cttcacctcc 4800 aagggaggag LaaLgccLgg agacctcagg LgaaLaatcc aaaagagtgg ctgcaagtgg 4860 acttccagaa gacaatgaaa gtcacaggag taactactca gggagtaaaa tetetgetta 4920 ccagcatgta tgtgaaggag ttcctcatct ccagcagtca agatggccat cagtggaccc 4980 tettttttea gaatggcaaa gtaaaggttt ttcagggaaa tcaagactcc ttcacacctg 5040 tggtgaactc tctagaccca ccgttactga ctcgctacct tegaatteae ccccagagtt 5100 gggtgcacca gattgccctg aggatggagg ttctgggctg egaggeaeag gacctctact 5160 gagcggcccg tttaaacccg ctgatcagcc tcgactgtgc ettetagttg ccagccatct 5220 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5280 tcctaataaa atgaggaaat tgeategeat tgtctgagta ggtgtcattc tattctgggg 5340 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 5400 gatgeggtgg gctctatggc ttetgaggeg gaaagaacca gctggggctc tagggggtat 5460 ccccacgcgc cctgtagcgg egeattaage gcggcgggtg tggtggttac gcgcagcgtg 5520 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 5580 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggtteega 5640 tttagtgett tacggcacct cgaccccaaa aaacttgatt agggtgatgg tteaegtagt 5700 gggccatcgc cctgatagac ggtttttege cctttgacgt tggagtccac gttctttaat 5760 agtggactct tgttccaaac tggaacaaca ctcaacccta teteggteta ttcttttgat 5820 ttataaggga ttttgccga.t ttcggcctat tggttaaaaa atgagetgat ttaacaaaaa 5880 tttaaegega attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 5940 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 6000 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 6060 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 6120 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 6130 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 6240 tcccgggagc LLgtatatcc attttcggat ctigaLcagca cgtgatgaaa aagcctgaac 6300 tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga 6360 tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat 6420 atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc 6480 actttgcatc. ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga 6540 gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa 6600 ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgaLc gctgcggccg 6660 atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta 6720 catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga 6780 tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg 6840 aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga 6900 cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc 6960 aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga 7020 cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata 7080 tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg 7140 cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc 7200 gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg 7260 ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtgctac 7320 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg 7380 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca 7440 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 7500 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 7560 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt 7620 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 7680 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 7740 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 7800 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 7860 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 7920 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 7980 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 8040 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 8100 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 8160 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 8220 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 8280 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 8340 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 8400 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 8460 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 8520 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 8580 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 8640 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 8700 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 8760 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 8820 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 8880 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 8940 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 9000 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 9060 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 9120 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 9180 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 9240 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 9300 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 9360 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 9420 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 9480 tgttgagatc caqttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 9540 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 9600 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 9660 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 9720 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag 9780 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag 9840 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta 9900 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg 9960 ttttgcgctg cttcg 9975

<2104 <211 > 5597 <212> DNA <213> Artificial Sequence <220 <223> vector pcDNA3.1 <400 4 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat c'tgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg .660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc 960 agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca 1020 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 1080 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 1140 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 1200 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 1260 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta 1320 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 1380 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 1440 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 1500 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 1560 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 1620 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 1680 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 1740 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 1800 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 1860 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 1920 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 1980 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 2040 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg 2100 gatctgatca gcacgtgatg aaaaagcc.tg aactcaccgc gacgtctgtc gagaagtttc 2160 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc 2220 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg 2280 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc 2340 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg 2400 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcc.cgctgtt ctgcagccgg 2460 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc 2520 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg 2580 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg 2640 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg 2700 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca 2760 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct 2820 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg 2880 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct 2940 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg 3000 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg 3060 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca 3120 ctcgtccgag ggcaaaggaa tagcacgtgc tacgagattt cgattccacc gccgccttct 3180 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 3240 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt 3300 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 3360 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta 3420 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 3480 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 3540 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 3600 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 3660 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 3720 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 3780 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 3840 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 3900 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 3960 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 4020 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 4080 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 4140 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 4200 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 4260 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 4320 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 4380 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 4440 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 4500 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 4560 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 4620 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 4680 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 4740 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 4800 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 4860 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 4920 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 4980 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 5040 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 5100 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 5160 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaata'c 5220 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 5280 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 5340 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 5400 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 5460 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 5520 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 5580 aagtgccacc tgacgtc 5597

<210>5 <211> 5600 <212> DNA <213> Artificial Sequence <220> <223> vector cDNA3.1 Hygro(+)-zz <400>5 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc 960 agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca 1020 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 1080 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 1140 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 1200 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 1260 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta 1320 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 1380 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 1440 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 1500 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 1560 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 1620 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 1680 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 1740 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 1800 tgcatctcaa ttagtcagca accaqgtgtg gaaagtcccc aqgctcccca gcaqqcagaa 1860 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 1920 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 1980 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 2040 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg 2100 gatctgatca gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc 2160 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc 2220 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg 2280 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc 2340 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg 2400 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg 2460 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc 2520 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg 2580 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg 2640 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg 2700 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca 2760 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct 2820 gqaggccgtg gttggcttgt atggagcagc agacqcgcta cttcqagcgg aggcatccgg 2880 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct 2940 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg 3000 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg 3060 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca 3120 ctcgtccgag ggcaaaggaa tagcacgtgc tacgagattt cgattccacc gccgccttct 3180 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 3240 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt 3300 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 3360 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta 3420 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 3480 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 3540 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 3600 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 3660 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 3720 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 3780 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 3840 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 3900 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 3960 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 4020 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 4080 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 4140 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 4200 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 4260 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 4320 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 4380 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 4440 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 4500 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 4560 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 4620 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catågttgcc tgactccccg 4680 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 4740 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 4800 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 4860 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 4920 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 4980 gatcaaggcg agttacatga tcccccatgt tgtgcaaiaaa agcggttagc tccttcggtc 5040 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 5100 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 5160 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 5220 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 5280 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 5340 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 5400 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 5460 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 5520 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 5580 gaaaagtgcc acctgacgtc 5600

<210> 6 <211 > 5610 <212> DNA <213> Artificial Sequence <220> <223> vector pTG1-A1AT <400>6 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180 tagggacttt ccattgacgt caatgggtgg actatttacg gtaaactgcc cacttggcag 240 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600 ctagagaacc cactgcttaa ctggcttatc gaaattaata cgactcacta tagggagacc 660 ggaagcttgt gaatcgacaa tgccgtcttc tgtctcgtgg ggcatcctcc tgctggcagg 720 cctgtgctgc ctggtccctg tctccctggc tgaggatccc cagggagatg ctgcccagaa 780 gacagataca tcccaccatg atcaggatca cccaaccttc aacaagatca cccccaacct 840 ggctgagttc gccttcagcc tataccgcca gctggcacac cagtccaaca gcaccaatat 900 cttcttctcc ccagtgagca tcgctacagc ctttgcaatg ctctccctgg ggaccaaggc 960 tgacactcac gatgaaatcc tggagggcct gaatttcaac ctcacggaga ttccggaggc 1020 tcagatccat gaaggcttcc aggaactcct ccgtaccctc aaccagccag acagccagct 1080 ccagctgacc accggcaatg gcctgttcct cagcgagggc ctgaagctag tggataagtt 1140 tttggaggat gttaaaaagt tgtaccactc agaagccttc actgtcaact tcggggacac 1200 cgaagaggcc aagaaacaga tcaacgatta cgtggagaag ggtactcaag ggaaaattgt 1260 ggatttggtc aaggagcttg acagagacac agtttttgct ctggtgaatt acatcttctt 1320 taaaggcaaa tgggagagac cctttgaagt caaggacacc gaggaagagg acttccacgt 1380 ggaccaggtg accaccgtga aggtgcctat gatgaagcgt ttaggcatgt ttaacatcca 1440 gcactgtaag aagctgtcca gctgggtgct gctgatgaaa tacctgggca atgccaccgc 1500 catcttcttc ctgcctgatg aggggaaact acagcacctg gaaaatgaac tcacccacga 1560 tatcatcacc aagttcctgg aaaatgaaga cagaaggtct gccagcttac atttacccaa 1620 actgtccatt actggaacct atgatctgaa gagcgtcctg ggtcaactgg gcatcactaa 1680 ggtcttcagc aatggggctg acctctccgg ggtcacagag gaggcacccc tgaagctctc 1740 caaggccgtg cataaggctg tgctgaccat cgacgagaaa gggactgaag ctgctggggc 1800 catgttttta gaggccatac ccatgtctat cccccccgag gtcaagttca acaaaccctt 1860 tgtcttctta atgattgaac aaaataccaa gtctcccctc ttcatgggaa aagtggtgaa 1920 tcccacccaa aaataactgc ctctcgctcc tcaacccctc ccctccatcc ctggccccct 1980 ccctggatga cattaaagaa gggggtaccg caagggcgaa ttctgcagat atccatcaca 2040 ctggcggccg cgactctagc tagaggatct ttgtgaagga accttacttc tgtggtgtga 2100 cataattgga caaactacct acagagattt aaagctctaa ggtaaatata aaatttLlaa 2160 gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt ccaacctatg 2220 gaacLgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg ttttgctcag 2280 aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct actcctccaa 2340 aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta agttttttga 2400 gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc acaaaggaaa 2460 aagctgcact gctatacaag aaaattatgg aaaaatattc tgtaaccttt ataagtaggc 2520 ataacagtta taatcataac atactgtttt ttcttactcc acacaggcat agagtgtctg 2580 ctattaataa ctatgctcaa aaattgtgta cctttagctt tttaatttgt aaaggggtta 2640 ataaggaata tttgatgtat agtgccttga ctagagatca taatcagcca taccacactt 2700 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa 2760 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc 2820 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 2880 tccaaaetca tcaatgtatc ttatcatgtc tggatccccg ggtaccgctc tagagcgaat 2940 taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact 3000 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac 3060 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt 3120 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg 3180 ctctqatgcc gcataqttaa gccagccccg acacccgcca acacccgctg acgcgccctg 3240 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 3300 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat 3360 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 3420 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 3480 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 3540 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 3600 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 3660 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 3720 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 3780 ccgtattqac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 3840 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 3900 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 3960 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 4020 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 4080 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 4140 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 4200 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 4260 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 4320 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 4380 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 4440 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 4500 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 4560 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 4620 accaccgcta ccagcggtgg tttgtttgcc ggaLcaagag ctaccaactc tttttccgaa 4680 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt 4740 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 4800 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 4860 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 4920 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 4980 qcttcccgaa qqgagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaqa 5040 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 5100 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 5160 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 5220 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 5280 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 5340 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 5400 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 5460 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 5520 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct 5580 ctctagagag cttgcatgcc tgcaggtcga 5610

<210>7 <211 > 10705 <212> DNA <213> Artificial Sequence <220> <223> vector pFGF8-hyg-s <400>7 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660 aagcttgacc tcgagatgca aatagagctc tccacctgct tctttctgtg ccttttgcga 720 ttctgcttta gtgccaccag aagatactac ctgggtgcag tggaactgtc atgggactat 780 atgcaaagtg atctcggtga gctgcctgtg gacgcaagat ttcctcctag agtgccaaaa 840 tcttttccat tcaacacctc agtcgtgtac aaaaagactc tgtttgtaga attcacggat 900 caccttttca acatcgctaa gccaaggcca ccctggatgg gtctgctagg tcctaccatc 960 caggctgagg tttatgatac agtggtcatt acacttaaga acatggcttc ccatcctgtc 1020 agtcttcatg ctgttggtgt atcctactgg aaagcttctg agggagctga atatgatgat 1080 cagaccagtc aaagggagaa agaagatgat aaagtcttcc ctggtggaag ccatacatat 1140 gtctggcagg tcctgaaaga gaatggtcca atggcctctg acccactgtg ccttacctac 1200 tcatatcttt ctcatgtgga cctggtaaaa gacttgaatt caggcctcat tggagcccta 1260 ctagtatgta gagaagggag tctggccaag gaaaagacac agaccttgca caaatttata 1320 ctactttttg ctgtatttga tgaagggaaa agttggcact cagaaacaaa gaactccttg 1380 atgcaggata gggatgctgc atctgctcgg gcctggccta aaatgcacac agtcaatggt 1440 tatgtaaaca ggtctctgcc aggtctgatt ggatgccaca ggaaatcagt ctattggcat 1500 gtgattggaa tgggcaccac tcctgaagtg cactcaatat tcctcgaagg tcacacattt 1560 cttgtgagga accatcgcca ggcgtccttg gaaatctcgc caataacttt ccttactgct 1620 caaacactct tgatggacct tggacagttt ctactgtttt gtcatatctc ttcccaccaa 1680 catgatggca tggaagctta tgtcaaagta gacagctgtc cagaggaacc ccaactacga 1740 atgaaaaata atgaagaagc ggaagactat gatgatgatc ttactgattc tgaaatggat 1800 gtggtcaggt ttgatgatga caactctcct tcctttatcc aaattcgctc agttgccaag 1860 aagcatccta aaacttgggt acattacatt gctgctgaag aggaggactg ggactatgct 1920 cccttagtcc tcgcccccga tgacagaagt tataaaagtc aatatttgaa caatggccct 1980 cagcggattg gtaggaagta caaaaaagtc cgatttatgg catacacaga tgaaaccttt 2040 aagactcgtg aagctattca gcatgaatca ggaatcttgg gacctttact ttatggggaa 2100 gttggagaca cactgttgat tatatttaag aatcaagcaa gcagaccata taacatctac 2160 cctcacggaa tcactgatgt ccgtcctttg tattcaagga gattaccaaa aggtgtaaaa 2220 catttgaagg attttccaat tctgccagga gaaatattca aatataaatg gacagtgact 2280 gtagaagatg ggccaactaa atcagatcct cggtgcctga cccgctatta ctctagtttc 2340 gttaatatgg agagagatct agcttcagga ctcattggcc ctctcctcat ctgctacaaa 2400 gaatctgtag atcaaagagg aaaccagata atgtcagaca agaggaatgt catcctgttt 2460 tctgtatttg atgagaaccg aagctggtac ctcacagaga atatacaacg ctttctcccc 2520 aatccagctg gagtgcagct tgaggatcca gagttccaag cctccaacat catgcacagc 2580 atcaatggct atgtttttga tagtttgcag ttgtcagttt gtttgcatga ggtggcatac 2640 tggtacattc taagcattgg agcacagact gacttccttt ctgtcttctt ctctggatat 2700 accttcaaac acaaaatggt ctatgaagac acactcaccc tattcccatt ctcaggagaa 2760 actgtcttca tgtcgatgga aaacccaggt ctatggattc tggggtgcca caactcagac 2820 tttcggaaca gaggcatgac cgccttactg aaggtttcta gttgtgacaa gaacactggt 2880 gattattacg aggacagtta tgaagatatt tcagcatact tgctgagtaa aaacaatgcc 2940 attgaaccaa gaagcttctc ccagaattca agacatcaag cttatcgata ccgtcgaggg 3000 gaaataactc gtactactct tcagtcagat caagaggaaa ttgactatga tgataccata 3060 tcagttgaaa tgaagaagga agattttgac atttatgatg aggatgaaaa tcagagcccc 3120 cgcagctttc aaaagaaaac acgacactat tttattgctg cagtggagag gctctgggat 3180 LatgggaLga gtagctcccc acatgttcta agaaacaggg ctcagagtgg cagtgtccct 3240 cagttcaaga aagttgtttt ccaggaattt actgatggct cctttactca gcccttatac 3300 cgtggagaac taaatgaaca tttgggactc ctggggccat atataagagc agaagttgaa 3360 gataatatca tggtaacttt cagaaatcag gcctctcgtc cctattcctt ctattctagc 3420 cttatttctt atgaggaaga tcagaggcaa ggagcagaac ctagaaaaaa ctttgtcaag 3480 cctaatgaaa ccaaaactta cttttggaaa gtgcaacatc atatggcacc cactaaagat 3540 gagtttgact gcaaagcctg ggcttatttc tctgatgttg acctggaaaa agatgtgcac 3600 tcaggcctga ttggacccct tctggtctgc cacactaaca cactgaaccc tgctcatggg 3660 agacaagtga cagtacagga atttgctctg LLUttcacca tctttgatga gaccaaaagc 3720 tggtacttca ctgaaaatat ggaaagaaac tgcagggctc cctgcaatat ccagatggaa 3780 gatcccactt ttaaagagaa ttatcgcttc catgcaatca atggctacat aaLggataca 3840 ctacctggct tagtaatggc tcaggatcaa aggattcgat ggtatctgct cagcatgggc 3900 agcaatgaaa acatccattc tattcatttc agtggacatg tgttcactgt acgaaaaaaa 3960 gaggagtata aaatggcact gtacaatctc tatccaggtg tttttgagac agtggaaatg 4020 ttaccatcca aagctggaat ttggcgggtg gaatgcctta ttggcgagca tctacatgct 4080 gggatqagca cactttttct ggtgtacagc aataagtgtc agactcccct gggaatggct 4140 tctggacaca ttagagattt tcagattaca gcttcaggac aatatggaca gtgggcccca 4200 aagctggcca gacttcatta ttccggatca atcaatgcct ggagcaccaa ggagcccttt 4260 tcttggatca aggtggatct gttggcacca atgattattc acggcatcaa gacccagggt 4320 gcccgtcaga agttctccag cctctacatc tctcagttta tcatcatgta tagtcttgat 4380 gggaagaagt ggcagactta tcgaggaaat tccactggaa ccttaatggt cttctttggc 4440 aatgtggatt catctgggat aaaacacaat atttttaacc ctccaattat tgctcgatac 4500 atccgtttgc acccaactca ttatagcatt cgcagcactc ttcgcatgga gttgatgggc 4560 tgtgatttaa atagttgcag catgccattg ggaatggaga gtaaagcaat atcagatgca 4620 cagattactg cttcatccta ctttaccaat atgtttgcca cctggtctcc ttcaaaagct 4680 cgacttcacc tccaagggag gagtaaLgcc Lggagacctc aggtgaataa tccaaaagag 4740 tggctgcaag tggacttcca gaagacaatg aaagtcacag gagtaactac tcagggagta 4800 aaatctctgc ttaccagcat gtatgtgaag gagLLccLca tctccagcag tcaagatggc 4860 catcagtgga ccctcttttt tcagaatggc aaagtaaagg tttttcaggg aaatcaagac 4920 tccttcacac ctgtggtgaa ctctctagac ccaccgttac tgactcgcta ccttcgaatt 4980 cacccccaga gttgggtgca ccagattgcc ctgaggatgg aggttctggg ctgcgaggca 5040 caggacctct actgagcggc cgcgactcta ctagaggatc tttgtgaagg aaccttactt 5100 ctgtggtgtg acataattgg acaaactacc tacagagatt taaagctcta aggtaaatat 5160 aaaattttta agtgtataat gtgttaaact actgattcta attgtttgtg tattttagat 5220 tccaacctat ggaactgatg aatgggagca gtqgtggaat gcctttaatg aggaaaacct 5280 gttttgctca gaagaaatgc catctagtga tgatgaggct actgctgact ctcaacattc 5340 tactcctcca aaaaagaaga gaaaggtaga agaccccaag gactttcctt cagaattgct 5400 aagttttttg agtcatgctg tgtttagtaa tagaactctt gcttgctttg ctatttacac 5460 cacaaaggaa aaagctgcac tgctatacaa gaaaattatg gaaaaatatt ctgtaacctt 5520 tataagtagg cataacagtt ataatcataa catactgttt tttcttactc cacacaggca 5580 tagagtgtct gctattaata actatgclca aaaattgtgt acctttagct ttttaatttg 5640 taaaggggtt aataaggaat atttgatgta tagtgccttg actagagatc ataatcagcc 5700 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc 5760 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt 5820 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca cLgcattcta 5880 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcccc cgaacgccag 5940 caagacgtag cccagcgcgt cggccccgag atgcgccgcg tgcggctgct ggagatggcg 6000 gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct ccgcaagaat 6060 tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg ggctgcttca 6120 tccccgtggc ccgttgctcg cgtttgctgg cggtgtcccc ggaagaaata tatttgcatg 6180 tctttagttc tatgatgaca caaaccccgc ccagcgtctt gtcattggcg aaLLcgaaca 6240 cgcagatgca gtcggggcgg cgcggtccca ggtccacttc gcatattaag gtgacgcgtg 6300 tggcctcgaa caccgagcga ccctgcagcg acccgcttaa cagcgtcaac agcgtgccgc 6360 aagatcagct tgatatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga 6420 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg 6480 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg 6540 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg 6600 aagtgcctga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac 6660 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg 6720 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat 6780 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg 6840 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc 6900 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc 6960 acgcggattt cqgctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg 7020 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga 7080 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc 7140 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc 7200 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa 7260 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg 7320 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc 7380 gtggggatcg ggagatgggg gaggctaact gaaacacgga aggagacaat accggaagga 7440 acccgcgcta tgacggcaat aaaaagacag aataaaacgc a-cgggtgttg ggtcgtttgt 7500 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca 7560 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caacccccaa gttcgggtga 7620 aggcccaggg ctcgcagcca acgtcggggc ggcaagcccg ccatagccac gggccccgtg 7680 ggttagggac ggggtccccc atggggaatg gtttatggtt cgtgggggtt attcttttgg 7740 gcgttgcgtg gggtcaggtc cacgactgga ctgagcagac agacccatgg tttttggatg 7800 gcctgggcat ggaccgcatg tactggcgcg acacgaacac cgggcgtctg tggctgccaa 7860 acacccccga cccccaaaaa ccaccgcgcg gatttctggc gccagtgcca agctgggtac 7920 cctctagagc gaattaattc actggccgtc gttttacaac gtcgtgactg.ggaaaaccct 7980 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcataatagc 8040 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc 8100 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact 8160 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc 8220 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc 8280 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga 8340 aagggggggt accagcttcg tagctagaac atcatgttct gggatatcag cttcgtagct 8400 agaacatcat gttctggtac ccccctcgtg atacgcctat ttttataggt taatgtcatg 8460 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 8520 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca alaaccctga 8580 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 8640 cttattccct tLLLLgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 8700 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 8760 aacagcggta agatccttga gagtLLLcgc cccgaagaac gttttccaat gatgagcact 8820 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 8880 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 8940 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 9000 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 9060 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 9120 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 9180 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 9240 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 9300 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 9360 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 9420 g.aacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 9480 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 9540 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 9600 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 9660 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 9720 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 9780 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 9840 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 9900 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 9960 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 10020 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 10080 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 10140 gcctggtatc tttatagtce tgtcgggttt cgccacctct gacttgagcg tcgatttttg 10200 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 10260 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 10320 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 10380 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 10440 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 10500 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 10560 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 10620 ggaaacagct atgaccatga ttacgccaag ctctctagag ctctagagct ctagagctct 10680 agagagcttg catgcctgca ggtcg 10705 <2108 <211 >6996

<212> DNA <213> Homo sapiens <220> <221 > CDS <222> (1)..(6996) <400>8 gcc acc aga aga tac tac ctg ggt gca gtg gaa ctg tea tgg gac tat 48

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 15 10 15 atg caa agt gat ctc ggt gag ctg cct gtg gac gca aga ttt eet eet 96

Met Gin Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30 aga gtg cca aaa tet ttt cca tte aac acc tea gtc gtg tac aaa aag 144

Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 act ctg ttt gta gaa tte aeg gtt cac ett tte aac ate get aag cca 192

Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro 50 55 60 agg cca ccc tgg atg ggt ctg eta ggt cct acc ate cag get gag gtt 240

Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gin Ala Glu Val 65 70 75 80 tat gat aca gtg gtc att aca ett aag aac atg get tee cat cct gtc 288

Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95 agt ett cat get gtt ggt gta tee tac tgg aaa get tet gag gga get 336

Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110 gaa tat gat gat cag acc agt caa agg gag aaa gaa gat gat aaa gtc 384

Glu Tyr Asp Asp Gin Thr Ser Gin Arg Glu Lys Glu Asp Asp Lys Val 115 120 125 tte cct ggt gga age cat aca tat gtc tgg cag gtc ctg aaa gag aat 432

Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gin Val Leu Lys Glu Asn 130 135 140 ggt cca atg gee tet gac cca ctg tgc ett acc tac tea tat ett tet 480

Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 cat gtg gac ctg gta aaa gac ttg aat tea ggc etc att gga gcc eta 528

His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175 eta gta tgt aga gaa ggg agt ctg gcc aag gaa aag aca cag acc ttg 576

Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gin Thr Leu 180 185 190 cac aaa ttt ata eta ett ttt get gta ttt gat gaa ggg aaa agt tgg 624

His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205 cac tea gaa aca aag aac tee ttg atg cag gat agg gat get gea tet 672

His Ser Glu Thr Lys Asn Ser Leu Met Gin Asp Arg Asp Ala Ala Ser 210 215 220 get egg gcc tgg eet aaa atg cac aca gtc aat ggt tat gta aac agg 720

Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 225 230 235 240 tet ctg cca ggt ctg att gga tgc cac agg aaa tea gtc tat tgg cat 768

Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255 gtg att gga atg ggc acc act eet gaa gtg cac tea ata tte etc gaa 816

Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270 ggt cac aca ttt ett gtg agg aac cat ege cag geg tee ttg gaa atc 864

Gly His Thr Phe Leu Val Arg Asn His Arg Gin Ala Ser Leu Glu Ile 275 280 285 teg cca ata act tte ett act get caa aca etc ttg atg gac ett gga 912

Ser Pro Ile Thr Phe Leu Thr Ala Gin Thr Leu Leu Met Asp Leu Gly 290 295 300 cag ttt eta ctg ttt tgt cat ate tet tee cac caa cat gat ggc atg 960

Gin Phe Leu Leu Phe Cys His Ile Ser Ser His Gin His Asp Gly Met 305 310 315 320 gaa get tat gtc aaa gta gac age tgt cca gag gaa ccc caa eta ega 1008

Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gin Leu Arg 325 330 335 atg aaa aat aat gaa gaa geg gaa gac tat gat gat gat ett act gat 1056

Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350 tet gaa atg gat gtg gtc agg ttt gat gat gac aac tet eet tee ttt 1104

Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365 atc caa att ege tea gtt gee aag aag cat eet aaa act tgg gta cat 1152

Ile Gin Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380 tac att get get gaa gag gag gac tgg gac tat get ccc tta gtc etc 1200

Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400 gcc ccc gat gac aga agt tat aaa agt caa tat ttg aac aat ggc eet 1248

Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gin Tyr Leu Asn Asn Gly Pro 405 410 415 cag egg att ggt agg aag tac aaa aaa gtc ega ttt atg gea tac aca 1296

Gin Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430 gat gaa acc ttt aag act cgt gaa get att cag cat gaa tea gga atc 1344

Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gin His Glu Ser Gly Ile 435 440' 445 ttg gga cct tta ett tat ggg gaa gtt gga gac aca ctg ttg att ata 1392

Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460 ttt aag aat caa gea age aga cca tat aae atc tac cct cac gga atc 1440

Phe Lys Asn Gin Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465 470 475 480 act gat gtc egt cct ttg tat tea agg aga tta cca aaa ggt gta aaa 1488

Thr Asp val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495 cat ttg aag gat ttt cca att ctg cca gga gaa ata tte aaa tat aaa 1536

His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510 tgg aca gtg act gta gaa gat ggg cca act aaa tea gat cct egg tgc 1584

Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525 ctg acc ege tat tac tet agt tte gtt aat atg gag aga gat eta get 1632

Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540 tea gga etc att ggc cct etc etc atc tgc tac aaa gaa tet gta gat 1680

Ser Gly Leu Ile Gly Pro Leu Leu ile Cys Tyr Lys Glu Ser val Asp 545 550 555 560 caa aga gga aac cag ata atg tea gac aag agg aat gtc atc ctg ttt 1728

Gin Arg Gly Asn Gin Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575 tet gta ttt gat gag aac ega age tgg tac etc aca gag aat ata caa 1776

Ser val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gin 580 585 590 cgc ttt ctc ccc aat cca get gga gtg cag ett gag gat cca gag tte 1824

Arg Phe Leu Pro Asn Pro Ala Gly Val Gin Leu Glu Asp Pro Glu Phe 595 600 605 caa gee tee aac atc atg cac age atc aat ggc tat gtt ttt gat agt 1872

Gin Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620 ttg cag ttg tea gtt tgt ttg cat gag gtg gea tac tgg tac att eta 1920

Leu Gin Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 625 630 635 640 age att gga gea cag act gac tte ett tet gtc tte tte tet gga tat 1968

Ser Ile Gly Ala Gin Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655 acc tte aaa cac aaa atg gtc tat gaa gac aca etc acc eta tte cca 2016

Thr Phe Lys His Lys Met val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670 tte tea gga gaa act gtc tte atg teg atg gaa aac cca ggt eta tgg 2064

Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685 att ctg ggg tgc cac aac tea gac ttt egg aac aga ggc atg ace gcc 2112 lie Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700 tta ctg aag gtt tet agt tgt gac aag aac act ggt gat tat tac gag 2160

Lqu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720 gac agt tat gaa gat att tea gca tac ttg ctg agt aaa aac aat gcc 2208

Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735 att gaa cca aga age ttc tcc cag aat tea aga cac cct age act agg 2256 lie Glu Pro Arg Ser Phe Ser Gin Asn Ser Arg His Pro Ser Thr Arg 740 745 750 caa aag caa ttt aat gcc acc aca att cca gaa aat gac ata gag aag 2304

Gin Lys Gin Phe Asn Ala Thr Thr lie Pro Glu Asn Asp He Glu Lys 755 760 765 act gac cct tgg ttt gca cac aga aca cct atg cct aaa ata caa aat 2352

Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys He Gin Asn 770 775 780 gtc tcc tet agt gat ttg ttg atg etc ttg ega cag agt cct act cca 2400

Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gin Ser Pro Thr Pro 785 . 790 795 800 cat ggg eta tcc tta tet gat etc caa gaa gcc aaa tat gag act ttt 2448

His Gly Leu Ser Leu Ser Asp Leu Gin Glu Ala Lys Tyr Glu Thr Phe 805 810 815 tet gat gat cca tea cct gga gca ata gac agt aat aac age ctg tet 2496

Ser Asp Asp Pro Ser Pro Gly Ala He Asp Ser Asn Asn Ser Leu Ser 820 825 830 gaa atg aca cac ttc agg cca cag etc cat cac agt ggg gac atg gta 2544

Glu Met Thr His Phe Arg Pro Gin Leu His His Ser Gly Asp Met Val 835 840 845 ttt acc cct gag tea ggc etc caa tta aga tta aat gag aaa ctg ggg 2592.

Phe Thr Pro Glu Ser Gly Leu Gin Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860 aca act gca gca aca gag ttg aag aaa ett gat ttc aaa gtt tet agt 2640

Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys val Ser Ser 865 870 875 880 aca tea aat aat ctg att tea aca att cca tea gac aat ttg gca gca 2688

Thr Ser Asn Asn Leu lie Ser Thr lie Pro Ser Asp Asn Leu Ala Ala 885 890 895 ggt act gat aat aca agt tcc tta gga ccc cca agt atg cca gtt cat 2736

Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905 910 tat gat agt caa tta gat acc act eta ttt ggc aaa aag tea tet ccc 2784

Tyr Asp Ser Gin Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925 ctt act gag tct ggt gga cct ctg age ttg agt gaa gaa aat aat gat 2832

Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940 tea aag ttg tta gaa tea ggt tta atg aat age caa gaa agt tea tgg 2880

Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gin Glu Ser Ser Trp 945 950 955 960 gga aaa aat gta teg tea aca gag agt ggt agg tta ttt aaa ggg aaa 2928

Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975 aga get cat gga cct get ttg ttg act aaa gat aat gcc tta ttc aaa 2976

Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 965 990 gtt age ate tct ttg tta aag aca aac aaa act tee aat aat tea gea 3024

Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005 act aat aga aag act cac att gat ggc cca tea tta tta att gag aat 3072

Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu Asn 1010 1015 1020 agt cca tea gtc tgg caa aat ata tta gaa agt gac act gag ttt aaa 3120

Ser Pro Ser val Trp Gin Asn Ile Leu Glu Ser Asp Thr Glu Phe Lys 1025 1030 1035 1040 aaa gtg aca cct ttg att cat gac aga atg ctt atg gac aaa aat get 3166

Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala 1045 1050 1055 aca get ttg agg eta aat cat atg tea aat aaa act act tea tea aaa 3216

Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys 1060 1065 1070 aac atg gaa atg gtc caa cag aaa aaa gag ggc ccc att cca cca gat 3264

Asn Met Glu Met Val Gin Gin Lys Lys Glu Gly Pro lie Pro Pro Asp 1075 1080 1085 gca caa aat cca gat atg teg ttc ttt aag atg eta ttc ttg cca gaa 3312

Ala Gin Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu 1090 1095 1100 tea gca agg tgg ata caa agg act cat gga aag aac tct ctg aac tct 3360

Ser Ala Arg Trp lie Gin Arg Thr His Gly Lys Asn Ser Leu Asn Ser 1105 1110 1115 1120 ggg caa ggc ccc agt cca aag caa tta gta tcc tta gga cca gaa aaa 3408

Gly Gin Gly Pro Ser Pro Lys Gin Leu Val Ser Leu Gly Pro Glu Lys 1125 1130 1135 tct gtg gaa ggt cag aat ttc ttg tct gag aaa aac aaa gtg gta gta 3456

Ser Val Glu Gly Gin Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val 1140 1145 1150 gga aag ggt gaa ttt aca aag gac gta gga etc aaa gag atg gtt ttt 3504

Gly Lys Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met val Phe 1155 1160 1165 cca age age aga aac eta ttt ctt act aac ttg gat aat tta cat gaa 3552

Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu 1170 1175 1180 aat aat aca cac aat caa gaa aaa aaa att cag gaa gaa ata gaa aag 3600

Asn Asn Thr His Asn Gin Glu Lys Lys Ile Gin Glu Glu Ile Glu Lys 1185 1190 1195 1200 aag gaa aca tta atc caa gag aat gta gtt ttg cct cag ata cat aca 3648

Lys Glu Thr Leu Ile Gin Glu Asn Val Val Leu Pro Gin Ile His Thr 1205 1210 1215 gtg act ggc act aag aat ttc atg aag aac ctt ttc tta ctg age act 3696

Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr 1220 1225 1230 agg caa aat gta gaa ggt tea tat gag ggg gea tat get cca gta ctt 3744

Arg Gin Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245 caa gat ttt agg tea tta aat gat tea aca aat aga aca aag aaa cac 3792

Gin Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His 1250 1255 1260 aca get cat ttc tea aaa aaa ggg gag gaa gaa aac ttg gaa ggc ttg 3840

Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu Gly Leu 1265 1270 1275 1280 gga aat caa acc aag caa att gta gag aaa tat gea tgc acc aca agg 3888

Gly Asn Gin Thr Lys Gin Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg 1285 1290 1295 ata tet cct aat aca age cag cag aat ttt gtc aeg caa egt agt aag 3936

Ile Ser Pro Asn Thr Ser Gin Gin Asn Phe Val Thr Gin Arg Ser Lys 1300 1305 1310 aga get ttg aaa caa ttc aga ctc cca eta gaa gaa aca gaa ctt gaa 3984

Arg Ala Leu Lys Gin Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu 1315 1320 1325 aaa agg ata att gtg gat gac acc tea acc cag tgg tee aaa aac atg 4032

Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gin Trp Ser Lys Asn Met 1330 1335 1340 aaa cat ttg acc ccg age acc ctc aca cag ata gac tac aat gag aag 4080

Lys His Leu Thr Pro Ser Thr Leu Thr Gin Ile Asp Tyr Asn Glu Lys 1345 1350 1355 1360 gag aaa ggg gcc att act cag tet ccc tta tea gat tgc ctt aeg agg 4128

Glu Lys Gly Ala Ile Thr Gin Ser Pro Leu Ser Asp Cys Leu Thr Arg 1365 1370 1375 agt cat age atc cct caa gea aat aga tet cca tta ccc att gea aag 4176

Ser His Ser Ile Pro Gin Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys 1380 1385 1390 gta tea tea ttt cca tet att aga cct ata tat ctg acc agg gtc eta 4224

Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg val Leu 1395 1400 1405 ttc caa gac aac tet tet cat ctt cca gea gea tet tat aga aag aaa 4272

Phe Gin Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys 1410 1415 1420 gat tet ggg gtc caa gaa age agt cat ttc tta caa gga gcc aaa aaa 4320

Asp Ser Gly Val Gin Glu Ser Ser His Phe Leu Gin Gly Ala Lys Lys 1425 1430 1435 1440 aat aac ctt tct tta gcc att eta ace ttg gag atg act ggt gat caa 4368

Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gin 1445 1450 1455 aga gag gtt ggc tee ctg ggg aca agt gcc aca aat tea gtc aca tac 4416

Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr 1460 1465 1470 aag aaa gtt gag aac act gtt ctc ccg aaa cca gac ttg ccc aaa aca 4464

Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485 tct ggc aaa gtt gaa ttg ctt cca aaa gtt cac att tat cag aag gac 4512

Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gin Lys Asp 1490 1495 1500 eta ttc cct aeg gaa act age aat ggg tct eet ggc cat ctg gat etc 4560

Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu 1505 1510 1515 1520 gtg gaa ggg age ctt ctt cag gga aca gag gga gcg att aag tgg aat 4608 val Glu Gly Ser Leu Leu Gin Gly Thr Glu Gly Ala Ile Lys Trp Asn 1525 1530 1535 gaa gea aac aga cct gga aaa gtt ccc ttt ctg aga gta gea aca gaa 4656

Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu 1540 1545 1550 age tct gea aag act ccc tee aag eta ttg gat cct ctt get tgg gat 4704

Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp 1555 1560 1565 aac cac tat ggt act cag ata cca aaa gaa gag tgg aaa tee caa gag 4752

Asn His Tyr Gly Thr Gin Ile Pro Lys Glu Glu Trp Lys Ser Gin Glu 1570 1575 1580 aag tea cca gaa aaa aca get ttt aag aaa aag gat acc att ttg tee 4800

Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser 1585 1590 1595 1600 ctg aac get tgt gaa age aat cat gea ata gea gea ata aat gag gga 4848

Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly 1605 1610 1615· caa aat aag ccc gaa ata gaa gtc acc tgg gea aag caa ggt agg act 4896

Gin Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gin Gly Arg Thr 1620 1625 1630 gaa agg ctg tgc tct caa aac cca cca gtc ttg aaa ege cat caa egg 4944

Glu Arg Leu Cys Ser Gin Asn Pro Pro Val Leu Lys Arg His Gin Arg 1635 1640 1645 gaa ata act egt act act ctt cag tea gat caa gag gaa att gac tat 4992

Glu Ile Thr Arg Thr Thr Leu Gin Ser Asp Gin Glu Glu Ile Asp Tyr 1650 1655 1660 gat gat acc ata tea gtt gaa atg aag aag gaa gat ttt gac att tat 5040

Asp Asp Thr ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr 1665 1670 1675 1680 gat gag gat gaa aat cag age ccc ege age ttt caa aag aaa aca ega 5088

Asp Glu Asp Glu Asn Gin Ser Pro Arg Ser Phe Gin Lys Lys Thr Arg 1685 1690 1695 cac tat ttt att get gca gtg gag agg etc tgg gat tat ggg atg agt 5136

His Tyr Phe lie Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser 1700 1705 1710 age tee cca cat gtt eta aga aac agg get eag agt ggc agt gtc cct 5184

Ser Ser Pro His Val Leu Arg Asn Arg Ala Gin Ser Gly Ser Val Pro 1715 1720 1725 eag ttc aag aaa gtt gtt ttc cag gaa ttt act gat ggc tec ttt act 5232

Gin Phe Lys Lys Val Val Phe Gin Glu Phe Thr Asp Gly Ser Phe Thr 1730 1735 1740 cag ccc tta tac cgt gga gaa eta aat gaa cat ttg gga etc ctg ggg 5280

Gin Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly 1745 1750 1755 1760 cca tat ata aga gca gaa gtt gaa gat aat ate atg gta act ttc aga 5328

Pro Tyr lie Arg Ala Glu Val Glu Asp Asn lie Met Val Thr Phe Arg 1765 1770 1775 aat cag gcc tet cgt ccc tat tcc ttc tat tet age ett att tet tat 5376

Asn Gin Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu lie Ser Tyr 1780 1785 1790 gag gaa gat cag agg caa gga gca gaa cct aga aaa aac ttt gtc aag 5424

Glu Glu Asp Gin Arg Gin Gly Ala Glu Pro Arg Lys Asn Phe Val Lys 1795 1800 1805 cct aat gaa acc aaa act tac ttt tgg aaa gtg caa cat cat atg gca 5472

Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gin His His Met Ala 1810 1815 1820 ccc act aaa gat gag ttt gac tgc aaa gcc tgg get tat ttc tet gat 5520

Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp 1825 1830 1835 1840 gtt gac ctg gaa aaa gat gtg cac tea ggc ctg att gga ccc ett ctg 5568

Val Asp Leu Glu Lys Asp Val His Ser Giy Leu lie Gly Pro Leu Leu 1845 1850 1855 gtc tgc cac act aac aca ctg aac cct get cat ggg aga caa gtg aca 5616

Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gin Val Thr 1860 1865 1870 gta cag gaa ttt get ctg ttt ttc acc ate ttt gat gag acc aaa age 5664

Val Gin Glu Phe Ala Leu Phe Phe Thr lie Phe Asp Glu Thr Lys Ser 1875 1880 1885 tgg tac ttc act gaa aat atg gaa aga aac tgc agg get ccc tgc aat 5712

Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1890 1895 1900 ate cag atg gaa gat ccc act ttt aaa gag aat tat ege ttc cat gca 5760 lie Gin Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala 1905 1910 1915 1920 ate aat ggc tac ata atg gat aca eta cct ggc tta gta atg get cag 5808 lie Asn Gly Tyr lie Met Asp Thr Leu Pro Gly Leu Val Met Ala Gin 1925 1930 1935 gat caa agg att cga tgg tat ctg ctc age atg ggc age aat gaa aac 5856

Asp Gin Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn 1940 1945 1950 ate cat tet att cat ttc agt gga cat gtg ttc act gta cga aaa aaa 5904

Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960 1965 gag gag tat aaa atg gea ctg tac aat ctc tat cca ggt gtt ttt gag 5952

Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu 1970 1975 1980 aca gtg gaa atg tta cca tee aaa get gga att tgg egg gtg gaa tgc 6000

Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys 1985 1990 1995 2000 ett att ggc gag cat eta cat get ggg atg age aca ett ttt ctg gtg 6048

Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val 2005 2010 2015 tac age aat aag tgt cag act ccc ctg gga atg get tet gga cac att 6096

Tyr Ser Asn Lys Cys Gin Thr Pro Leu Gly Met Ala Ser Gly His Ile 2020 2025 2030 aga gat ttt cag att aca get tea gga caa tat gga cag tgg gcc cca 6144

Arg Asp Phe Gin Ile Thr Ala Ser Gly Gin Tyr Gly Gin Trp Ala Pro 2035 2040 2045 aag ctg gee aga ett cat tat tee gga tea ate aat gcc tgg age acc 6192

Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr 2050 2055 2060 aag gag ccc ttt tet tgg atc aag gtg gat ctg ttg gea cca atg att 6240

Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile 2065 2070 2075 2080 att cac ggc atc aag acc cag ggt gcc egt cag aag ttc tee age ctc 6288

Ile His Gly Ile Lys Thr Gin Gly Ala Arg Gin Lys Phe Ser Ser Leu 2085 2090 2095 tac atc tet cag ttt atc atc atg tat agt ett gat ggg aag aag tgg 6336

Tyr Ile Ser Gin Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp 2100 2105 2110 cag act tat cga gga aat tee act gga acc tta atg gtc ttc ttt ggc 6384

Gin Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe.Gly 2115 2120 2125 aat gtg gat tea tet ggg ata aaa cac aat att ttt aac eet cca att 6432

Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 2130 2135 2140 att get cga tac atc egt ttg cac cca act cat tat age att ege age 6480

Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser 2145 2150 2155 2160 act ett ege atg gag ttg atg ggc tgt gat tta aat agt tgc age atg 6528

Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met 2165 .2170 2175 cca ttg gga atg gag agt aaa gea ata tea gat gea cag att act get 6576

Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gin lie Thr Ala 2180 2185 2190 tea tee tac ttt acc aat atg ttt gcc acc tgg tet cct tea aaa get 6624

Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205 ega ett cac etc caa ggg agg agt aat gcc tgg aga cct cag gtg aat 6672

Arg Leu His Leu Gin Gly Arg Ser Asn Ala Trp Arg Pro Gin Val Asn 2210 2215 2220 aat cca aaa gag tgg ctg caa gtg gac ttc cag aag aca atg aaa gtc 6720

Asn Pro Lys Glu Trp Leu Gin Val Asp Phe Gin Lys Thr Met Lys Val 2225 2230 2235 2240 aca gga gta act act cag gga gta aaa tet ctg ett acc age atg tat 6768

Thr Gly Val Thr Thr Gin Gly Val Lys Ser Leu Leu Thr Ser Met Tyr 2245 2250 2255 gtg aag gag ttc etc ate tee age agt caa gat ggc cat cag tgg act 6816

Val Lys Glu Phe Leu He Ser Ser Ser Gin Asp Gly His Gin Trp Thr 2260 2265 2270 etc ttt ttt cag aat ggc aaa gta aag gtt ttt cag gga aat caa gac 6864

Leu Phe Phe Gin Asn Gly Lys Val Lys Val Phe Gin Gly Asn Gin Asp 2275 2280 2285 tcc ttc aca cct gtg gtg aac tet eta gac cca ccg tta ctg act ege 6912

Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg 2290 2295 2300 tac ett ega att cac ccc cag agt tgg gtg cac cag att gcc ctg agg 6960

Tyr Leu Arg lie His Pro Gin Ser Trp Val His Gin lie Ala Leu Arg 2305 2310 2315 2320 atg gag gtt ctg ggc tgc gag gca cag gac etc tac 6996

Met Glu Val Leu Gly Cys Glu Ala Gin Asp Leu Tyr 2325 2330 <210>9 <211 >2332

<212> PRT <213> Homo sapiens <400>9

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 15 10 15

Met Gin Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30

Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45

Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn He Ala Lys Pro 50 55 60

Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr lie Gin Ala Glu Val 65 70 75 80

Tyr Asp Thr Val val lie Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95

Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110

Glu Tyr Asp Asp Gin Thr Ser Gin Arg Glu Lys Glu Asp Asp Lys Val 115 120 125

Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gin Val Leu Lys Glu Asn 130 135 140

Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160

His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175

Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gin Thr Leu 180 185 190

His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205

His Ser Glu Thr Lys Asn Ser Leu Met Gin Asp Arg Asp Ala Ala Ser 210 215 220

Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg 225 230 235 240

Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser val Tyr Trp His 245 250 255

Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270

Gly His Thr Phe Leu Val Arg Asn His Arg Gin Ala Ser Leu Glu Ile 275 280 285

Ser Pro Ile Thr Phe Leu Thr Ala Gin Thr Leu Leu Met Asp Leu Gly 290 295 300

Gin Phe Leu Leu Phe Cys His Ile Ser Ser His Gin His Asp Gly Met 305 310 315 320

Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gin Leu Arg 325 330 335

Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350

Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365

Ile Gin Ile Arg Ser val Ala Lys Lys His Pro Lys Thr Trp val His 370 375 380

Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu 385 390 395 400

Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gin Tyr Leu Asn Asn Gly Pro 405 410 415

Gin Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430

Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gin His Glu Ser Gly Ile 435 440 445

Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp .Thr Leu Leu Ile Ile 450 455 460

Phe Lys Asn Gin Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465 470 475 480

Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495

His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510

Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525

Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540

Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr I.ys Glu Ser Val Asp 545 550 555 560

Gin Arg Gly Asn Gin Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575

Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gin 580 585 590

Arg Phe Leu Pro Asn Pro Ala Gly Val Gin Leu Glu Asp Pro Glu Phe 595 600 605

Gin Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620

Leu Gin Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu 625 630 635 640

Ser Ile Gly Ala Gin Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655

Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670

Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685

Ile Lea Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala • 690 695 700

Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720

Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735

Ile Glu Pro Arg Ser Phe Ser Gin Asn Ser Arg His Pro Ser Thr Arg 740 745 750

Gin Lys Gin Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755 760 765

Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gin Asn 770 775 780

Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gin Ser Pro Thr Pro 785 790 795 800

His Gly Leu Ser Leu Ser Asp Leu Gin Glu Ala Lys Tyr Glu Thr Phe 805 810 815

Ser Asp Asp Pro Ser Pro Gly Ala lie Asp Ser Asn Asn Ser Leu Ser 820 825 830

Glu Met Thr His Phe Arg Pro Gin Leu His His Ser Gly Asp Met Val 835 840 845

Phe Thr Pro Glu Ser Gly Leu Gin Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860

Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser 865 870 875 880

Thr Ser Asn Asn Leu lie Ser Thr lie Pro Ser Asp Asn Leu Ala Ala 885 890 895

Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905 910

Tyr Asp Ser Gin Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925

Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940

Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gin Glu Ser Ser Trp 945 950 955 960

Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975

Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990

Val Ser lie Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005

Thr Asn Arg Lys Thr His lie Asp Gly Pro Ser Leu Leu lie Glu Asn 1010 1015 1020

Ser Pro Ser Val Trp Gin Asn lie Leu Glu Ser Asp Thr Glu Phe Lys 025 1030 1035 1040

Lys Val Thr Pro Leu lie His Asp Arg Met Leu Met Asp Lys Asn Ala 1045 1050 1055

Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys 1060 1065 1070

Asn Met Glu Met Val Gin Gin Lys Lys Glu Gly Pro lie Pro Pro Asp 1075 1080 1085

Ala Gin Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu 1090 1095 1100

Ser Ala Arg Trp Ile Gin Arg Thr His Gly Lys Asn Ser Leu Asn Ser 105 1110 1115 1120

Gly Gin Gly Pro Ser Pro Lys Gin Leu Val Ser Leu Gly Pro Glu Lys 1125 1130 1135

Ser Val Glu Gly Gin Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val 1140 ' 1145 1150

Gly Lys Gly Glu Phe Thr Lys Asp vai Gly Leu Lys Glu Met val Phe 3155 1160 1165

Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu 1170 1175 1180

Asn Asn Thr His Asn Gin Glu Lys Lys lie Gin Glu Glu lie Glu Lys 185 1190 1195 1200

Lys Glu Thr Leu lie Gin Glu Asn Val Val Leu Pro Gin lie His Thr 1205 1210 1215

Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr 1220 1225 1230

Arg Gin Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245

Gin Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His 1250 1255 1260

Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu Gly Leu 265 1270 1275 1280

Gly Asn Gin Thr Lys Gin lie Val Glu Lys Tyr Ala Cys Thr Thr Arg 1285 1290 1295 lie Ser Pro Asn Thr Ser Gin Gin Asn Phe Val Thr Gin Arg Ser Lys 1300 1305 1310

Arg Ala Leu Lys Gin Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu 1315 1320 1325

Lys Arg lie lie Val Asp Asp Thr Ser Thr Gin Trp Ser Lys Asn Met 1330 1335 1340

Lys His Leu Thr Pro Ser Thr Leu Thr Gin lie Asp Tyr Asn Glu Lys 345 1350 1355 1360

Glu Lys Gly Ala He Thr Gin Ser Pro Leu Ser Asp Cys Leu Thr Arg 1365 1370 1375

Ser His Ser lie Pro Gin Ala Asn Arg Ser Pro Leu Pro He Ala Lys 1380 1385 1390

Val Ser Ser Phe Pro Ser He Arg Pro He Tyr Leu Thr Arg Val Leu 1395 1400 1405

Phe Gin Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys 1410 1415 1420

Asp Ser Gly Val Gin Glu Ser Ser His Phe Leu Gin Gly Ala Lys Lys 425 1430 1435 1440

Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gin 1445 1450 1455

Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser val Thr Tyr 1460 1465 1470

Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485

Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gin Lys Asp 1490 1495 1500

Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu 505 1510 1515 1520

Val Glu Gly Ser Leu Leu Gin Gly Thr Glu Gly Ala Ile Lys Trp Asn 1525 1530 1535

Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu 1540 1545 1550

Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp 1555 1560 1565

Asn His Tyr Gly Thr Gin Ile Pro Lys Glu Glu Trp Lys Ser Gin Glu 1570 1575 1580

Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser 585 1590 1595 1600

Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly 1605 1610 1615

Gin Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gin Gly Arg Thr 1620 1625 1630

Glu Arg Leu Cys Ser Gin Asn Pro Pro val Leu Lys Arg His Gin Arg 1635 1640 1645

Glu Ile Thr Arg Thr Thr Leu Gin Ser Asp Gin Glu Glu Ile Asp Tyr 1650 1655 1660

Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr 665 1670 1675 1680

Asp Glu Asp Glu Asn Gin Ser Pro Arg Ser Phe Gin Lys Lys Thr Arg 1685 1690 1695

His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser 1700 1705 1710

Ser Ser Pro His Val Leu Arg Asn Arg Ala Gin Ser Gly Ser Val Pro 1715 ·- 1720 1725

Gin Phe Lys Lys Val Val Phe Gin Glu Phe Thr Asp Gly Ser Phe Thr 1730 1735 1740

Gin Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly 745 1750 1755 1760

Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg 1'7 65 1770 1775

Asn Gin Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr 1780 1785 1790

Glu Glu Asp Gin Arg Gin Gly Ala Glu Pro Arg Lya Asn Phe Val Lys 1795 1800 1805

Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gin His His Met Ala •1810 1815 1820

Pro Thr Lys Asp Glu Phe Asp Cys Lys Al‘a Trp Ala Tyr Phe Ser Asp 825 1830 1835 1840

Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu 1845 1850 1855

Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gin Val Thr 1860 1865 1870

Val Gin Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser 1875 1880 1885

Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1890 1895 1900

Ile Gin Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala 905 1910 1915 1920

Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gin 1925 1930 1935

Asp Gin Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn G]u Asn 1940 1945 1950

Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960 1965

Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu 1970 1975 1980

Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys 985 1990 1995 2000

Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val 2005 2010 2015

Tyr Ser Asn Lys Cys Gin Thr Pro Leu Gly Met Ala Ser Gly His Ile 2020 2025 2030

Arg Asp Phe Gin Ile Thr Ala Ser Gly Gin Tyr Gly Gin Trp Ala Pro 2035 2040 2045

Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr 2050 2055 2060

Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile 065 2070 2075 2080

Ile His Gly Ile Lys Thr Gin Gly Ala Arg Gin Lys Phe Ser Ser Leu 2085 2090 2095

Tyr Ile Ser Gin Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp 2100 2105 2110

Gin Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly 2115 2120 2125

Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 2130 2135 2140

Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser 145 '2150 2155 2160

Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met 2165 2170 2175

Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gin Ile Thr Ala 2180 2185 2190

Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205

Arg Leu His Leu Gin Gly Arg Ser Asn Ala Trp Arg Pro Gin Val Asn 2210 2215 2220

Asn Pro Lys Glu Trp Leu Gin Val Asp Phe Gin Lys Thr Met Lys Val 225 2230 2235 2240

Thr Gly Val Thr Thr Gin Gly Val Lys Ser Leu Leu Thr Ser Met Tyr 2245 2250 2255

Val Lys Glu Phe Leu Ile Ser Ser Ser Gin Asp Gly His Gin Trp Thr 2260 2265 2270

Leu Phe Phe Gin Asn Gly Lys Val Lys Val Phe Gin Gly Asn Gin Asp 2275 2280 2285 .

Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg 2290 2295 2300

Tyr Leu Arg Ile His Pro Gin Ser Trp Val His Gin Ile Ala Leu Arg 305 2310 2315 2320

Met Glu Val Leu Gly Cys Glu Ala Gin Asp Leu Tyr 2325 2330

<210> 10 <211 > 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: B-domain linker peptide <400> 10

Ser Phe Ser Gin Asn Ser Arg His 1 5

<210 11 <211 > 8 <212> PRT <213> Artificial Sequence <220 <223> Description of Artificial Sequence: B-domain linker peptide <400 11

Gin Ala Tyr Arg Tyr Arg Arg Gly 1 5

<210> 12 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: B-domain linker peptide <400> 12

Ser Phe Ser Gin Asn Ser Arg His Gin Ala Tyr Arg Tyr Arg. Arg Gly 15 10 15 <210> 13 <211 >2478

<212> DNA <213> Homo sapiens <400> 13 agtcccatgg ggaatgtcaa caggcagggg cagcactgca gagatttcat catggtctcc 60 caggccctca ggctcctctg ccttctgctt gggcttcagg gctgcctggc tgcaggcggg 120 gtcgctaagg cctcaggagg agaaacacgg gacatgccgt ggaagccggg gcctcacaga 180 gtcttcgtaa cccaggagga agcccacggc gtcctgcacc ggcgccggcg cgccaacgcg 240 ttcctggagg agctgcggcc gggctccctg gagagggagt gcaaggagga gcagtgctcc 300 ttcgaggagg cccgggagat cttcaaggac gcggagagga cgaagctgtt ctggatttct 360 tacagtgatg gggaccagtg tgcctcaagt ccatgccaga atgggggctc ctgcaaggac 420 cagctccagt cctatatctg cttctgcctc cctgccttcg agggccggaa ctgtgagacg 480 cacaaggatg accagctgat ctgtgtgaac gagaacggcg gctgtgagca gtactgcagt 540 gaccacacgg gcaccaagcg ctcctgtcgg tgccacgagg ggtactctct gctggcagac 600 ggggtgtcct gcacacccac agttgaatat ccatgtggaa aaatacctat tctagaaaaa 660 agaaatgcca gcaaacccca aggccgaatt gtggggggca aggtgtgccc caaaggggag 720 tgtccatggc aggtcctgtt gttggtgaat ggagctcagt tgtgtggggg gaccctgatc 780 aacaccatct gggtggtctc cgcggcccac tgtttcgaca aaatcaagaa ctggaggaac 840 ctgatcgcgg tgctgggcga gcacgacctc agcgagcacg acggggatga gcagagccgg 900 cgggtggcgc aggtcatcat ccccagcacg tacgtcccgg gcaccaccaa ccacgacatc 960 gcgctgctcc gcctgcacca gcccgtggtc ctcactgacc atgtggtgcc cctctgcctg 1020 cccgaacgga cgttctctga gaggacgctg gccttcgtgc gcttctcatt ggtcagcggc 1080 tggggccagc tgctggaccg tggcgccacg gccctggagc tcatggtgct caacgtgccc 1140 cggctgatga cccaggactg cctgcagcag tcacggaagg tgggagactc cccaaatatc 1200 acggagtaca tgttctgtgc cggctactcg gatggcagca aggactcctg caagggggac 1260 agtggaggcc cacatgccac ccactaccgg ggcacgtggt acctgacggg catcgtcagc 1320 tggggccagg gctgcgcaac cgtgggccac tttggggtgt acaccagggt ctcccagtac 1380 atcgagtggc tgcaaaagct catgcgctca gagccacgcc caggagtcct cctgcgagcc 1440 ccatttccct agcccagcag ccctggcctg tggagagaaa gccaaggctg cgtcgaactg 1500 tcctggcacc aaatcccata tattcttctg cagttaatgg ggtagaggag ggcatgggag 1560 ggagggagag gtggggaggg agacagagac agaaacagag agagacagag acagagagag 1620 actgagggag agactctgag gacatggaga gagactcaaa gagactccaa gattcaaaga 1680 gactaataga gacacagaga tggaatagaa aagatgagag gcagaggcag acaggcgctg 1740 gacagagggg caggggagtg ccaaggttgt cctggaggca gacagcccag ctgagcctcc 1800 ttacctccct tcagccaagc cccacctgca cgtgatctgc tggccctcag gctgctgctc 1860 tgccttcatt gctggagaca gtagaggcat gaacacacat ggatgcacac acacacacgc 1920 caatgcacac acacagagat atgcacacac acggatgcac acacagatgg tcacacagag 1980 atacgcaaac acaccgatgc acacgcacat agagatatgc acacacagat gcacacacag 2040 atatacacat ggatgcacgc acatgccaat gcacgcacac. atcagtgcac acggatgcac 2100 agagatatgc acacåccg'at gtgcgcacac acagatatgc acacacatgg atgagcacac 2160 acacaccaag tgcgcacaca caccgatgta cacacacaga tgcacacaca gatgcacaca 2220 caccgatgct gactccatgt gtgctgtcct ctgaaggcgg ttgtttagct ctcacttttc 2280 tggttcttat ccattatcat cttcacttca gacaattcag aagcatcacc atgcatggtg 2340 gcgaatgccc ccaaactctc ccccaaatgt atttctccct tcgctgggtg ccgggctgca 2400 cagactattc cccacctgct tcccagcttc acaataaacg gctgcgtctc ctccgcacac 2460 ctgtggtgcc tgccaccc 2478 <210> 14 <211> 2412

<212> DNA <213> Homo sapiens <400> 14 agtcccatgg ggaatgtcaa caggcagggg cagcactgca gagatttcat catggtctcc 60 caggccctca ggctcctctg ccttctgctt gggcttcagg gctgcctggc tgcagtcttc 120 gtaacccagg aggaagccca cggcgtcctg caccggcgcc ggcgcgccaa cgcgttcctg 180 gaggagctgc ggccgggctc cctggagagg gagtgcaagg aggagcagtg ctccttcgag 240 gaggcccggg agatcttcaa ggacgcggag aggacgaagc tgtt'ctggat ttcttacagt 300 gatggggacc agtgtgcctc aagtccatgc cagaatgggg gctcctgcaa ggaccagctc 360 cagtcctata tctgcttctg cctccctgcc ttcgagggcc ggaactgtga gacgcacaag 420 gatgaccagc tgatctgtgt gaacgagaac ggcggctgtg agcagtactg cagtgaccac 480 acgggcacca agcgctcctg tcggtgccac gaggggtact ctctgctggc agacggggtg 540 tcctgcacac ccacagttga atatccatgt ggaaaaatac ctattctaga aaaaagaaat 600 gccagcaaac cccaaggccg aattgtgggg ggcaaggtgt gccccaaagg ggagtgtcca 660 tggcaggtcc tgttgttggt gaatggagct cagttgtgtg gggggaccct gatcaacacc 720 atctgggtgg tctccgcggc ccactgtttc gacaaaatca agaactggag gaacctgatc 780 gcggtgctgg gcgagcacga cctcagcgag cacgacgggg atgagcagag ccggcgggtg 840 gcgcaggtca tcatccccag cacgtacgtc ccgggcacca ccaaccacga catcgcgctg 900 ctccgcctgc accagcccgt ggtcctcact gaccatgtgg tgcccctctg cctgcccgaa 960 cggacgttct ctgagaggac gctggccttc gtgcgcttct cattggtcag cggctggggc 1020 cagctgctgg accgtggcgc cacggccctg gagctcatgg tgctcaacgt gccccggctg 1080 atgacccagg actgcctgca gcagtcacgg aaggtgggag actccccaaa tatcacggag 1140 tacatgttct gtgccggcta ctcggatggc agcaaggact cctgcaaggg ggacagtgga 1200 ggcccacatg ccacccacta ccggggcacg tggtacctga cgggcatcgt cagctggggc 1260 cagggctgcg caaccgtggg ccactttggg gtgtacacca gggtctccca gtacatcgag 1320 tggctgcaaa agctcatgcg ctcagagcca cgcccaggag tcctcctgcg agccccattt 1380 ccctagccca gcagccctgg cctgtggaga gaaagccaag gctgcgtcga actgtcctgg 1440 caccaaatcc catatattct tctgcagtta atggggtaga ggagggcatg ggagggaggg 1500 agaggtgggg agggagacag agacagaaac agagagagac agagacagag agagactgag 1560 ggagagactc tgaggacatg gagagagact caaagagact ccaagattca aagagactaa 1620 tagagacaca gagatggaat agaaaagatg agaggcagag gcagacaggc gctggacaga 1680 ggggcagggg agtgccaagg ttgtcctgga ggcagacagc ccagctgagc ctccttacct 1740 cccttcagcc aagccccacc tgcacgtgat ctgctggccc tcaggctgct gctctgcctt 1800 cattgctgga gacagtagag gcatgaacac acatggatgc acacacacac acgccaatgc i860 acacacacag agatatgcac acacacggat gcacacacag atggtcacac agagatacgc 1920 aaacacaccg atgcacacgc acatagagat atgcacacac agatgcacac acagatatac 1980 acatggatgc acgcacatgc caatgcacgc acacatcagt gcacacggat gcacagagat 2040 atgcacacac cgatgtgcgc acacacagat atgcacacac atggatgagc acacacacac 2100 caagtgcgca cacacaccga tgtacacaca cagatgcaca cacagatgca cacacaccga 2160 tgctgactcc atgtgtgctg tcctctgaag gcggttgttt agctctcact tttctggttc 2220 ttatccatta tcatcttcac ttcagacaat tcagaagcat caccatgcat ggtggcgaat 2280 gcccccaaac tctcccccaa atgtatttct cccttcgctg ggtgccgggc tgcacagact 2340 attccccacc tgcttcccag· cttcacaata aacggctgcg tctcctccgc acacctgtgg 2400 tgcctgccac cc 2412 <210> 15 <211> 1518

<212> DNA <213> Homo sapiens <400> 15 åaaacagccc ggagcctgca gcccagcccc acccagaccc atggctggac ctgccaccca 60 gagccccatg aagctgatgg ccctgcagct gctgctgtgg cacagtgcac tctggacagt 120 gcaggaagcc acccccctgg gccctgccag ctccctgccc cagagcttcc tgctcaagtg 180 cttagagcaa gtgaggaaga tccagggcga tggcgcagcg ctccaggaga agctggtgag 240 tgagtgtgcc acctacaagc tgtgccaccc cgaggagctg cjtgctgctcg gacactctct 300 gggcatcccc tgggctcccc tgagcagctg ccccagccag gccctgcagc tggcaggctg 360 cttgagccaa ctccatagcg gccttttcct ctaccagggg ctcctgcagg ccctggaagg 420 gatctccccc gagttgggtc ccaccttgga cacactgcag ctggacgtcg ccgactttgc 480 caccaccatc tggcagcaga tggaagaact gggaatggcc cctgccctgc agcccaccca 540 gggtgccatg ccggccttcg cctctgcttt ccagcgccgg gcaggagggg tcctggttgc 600 ctcccatctg cagagcttcc tggaggtgtc gtaccgcgtt ctacgccacc ttgcccagcc 660 ctgagccaag ccctccccat cccatgtatt tatctctatt taatatttat gtctatttaa 720 gcctcatatt taaagacagg gaagagcaga acggagcccc aggcctctgt gtccttccct 780 gcatttctga gtttcattct cctgcctgta gcagtgagaa aaagctcctg tcctcccatc 840 ccctggactg ggaggtagat aggtaaatac caagtattta ttactatgac tgctccccag 900 ccctggctct gcaatgggca ctgggatgag ccgctgtgag cccctggtcc tgagggtccc 960 cacctgggac ccttgagagt atcaggtctc ccacgtggga gacaagaaat ccctgtttaa 1020 tatttaaaca gcagtgttcc ccatctgggt ccttgcaccc ctcactctgg cctcagccga 1080 ctgcacagcg gcccctgcat ccccttggct gtgaggcccc tggacaagca gaggtggcca 1140 gagctgggag gcatggccct ggggtcccac gaatttgctg gggaatctcg tttttcttct 1200 taagactttt gggacatggt ttgactcccg aacatcaccg acgtgtctcc tgtttttctg 1260 ggtggcctcg ggacacctgc cctgccccca cgagggtcag gactgtgact ctttttaggg 1320 ccaggcaggt gcctggacat ttgccttgct ggacggggac tggggatgtg ggagggagca 1380 gacaggagga atcatgtcag gcctgtgtgt gaaaggaagc tccactgtca ccctccacct 1440 cttcaccccc cactcaccag tgtcccctcc actgtcacat tgtaactgaa cttcaggata 1500 ataaagtgtt tgcctcca 1518 <210> 16 <211 > 1509 <212> DNA <213> Homo sapiens <400> 16 aaaacagccc ggagcctgca gcccagcccc acccagaccc atggctggac ctgccaccca 60 gagccccatg aagctgatgg ccctgcagct gctgctgtgg cacagtgcac tctggacagt 120 gcaggaagcc acccccctgg gccctgccag ctccctgccc cagagcttcc tgctcaagtg 180 cttagagcaa gtgaggaaga tccagggcga tggcgcagcg ctccaggaga agctgtgtgc 240 cacctacaag ctgtgccacc ccgaggagct ggtgctgctc ggacactctc tgggcatccc 300 ctgggctccc ctgagcagct gccccagcca ggccctgcag ctggcaggct gcttgagcca 360 actccatagc ggccttttcc tctaccaggg gctcctgcag gccctggaag ggatctcccc 420 cgagttgggt cccaccttgg acacactgca gctggacgtc gccgactttg ccaccaccat 480 ctggcagcag atggaagaac tgggaatggc ccctgccctg cagcccaccc agggtgccat 540 gccggccttc gcctctgctt tccagcgccg ggcaggaggg gtcctggttg cctcccatct 600 gcagagcttc ctggaggtgt cgtaccgcgt tctacgccac cttgcccagc cctgagccaa 660 gccctcccca tcccatgtat ttatctctat ttaatattta tgtctattta agcctcatat 720 ttaaagacag ggaagagcag aacggagccc caggcctctg tgtccttccc tgcatttctg 780 agtttcattc tcctgcctgt agcagtgaga aaaagctcct gtcctcccat cccctggact 840 gggaggtaga taggtaaata ccaagtattt attactatga ctgctcccca gccctggctc 900 tgcaatgggc actgggatga gccgctgtga gcccctggtc ctgagggtcc ccacctggga 960 cccttgagag tatcaggtct cccacgtggg agacaagaaa tccctgttta atatttaaac 1020 agcagtg'ttc cccatctggg tccttgcacc cctcactctg gcctcagccg actgcacagc 1080 ggcccctgca tccccttggc tgtgaggccc ctggacaagc agaggtggcc agagctggga 1140 ggcatggccc tggggtccca cgaatttgct ggggaatctc gtttttcttc ttaagacttt 1200 tgggacatgg tttgactccc gaacatcacc gacgtgtctc ctgtttttct gggtggcctc 1260 gggacacctg ccctgccccc acgagggtca ggactgtgac tctttttagg gccaggcagg 1320 tgcctggaca tttgccttgc tggacgggga ctggggatgt gggagggagc agacaggagg 1380 aatcatgtca ggcctgtgtg tgaaaggaag ctccactgtc accctccacc tcttcacccc 1440 ccactcacca gtgtcccctc cactgtcaca ttgtaactga acttcaggat aataaagtgt 1500 ttgcctcca 1509 <210> 17 <211> 1703

<212> DNA <213> Homo sapiens <400> 17 aaaacagccc ggagcctgca gcccagcccc acccagaccc atggctggac ctgccaccca 60 gagccccatg aagctgatgg gtgagtgtct tggcccagga tgggagagcc gcctgccctg 120 gcatgggagg gaggctggtg tgacagaggg gctggggatc cccgttctgg gaatggggat 180 taaaggcacc cagtgtcccc gagagggcct caggtggtag ggaacagcat gtctcctgag 240 cccgctctgt ccccagccct gcagctgctg ctgtggcaca gtgcactctg gacagtgcag 300 gaagccaccc ccctgggccc tgccagctcc ctgccccaga gcttcctgct caagtgctta 360 gagcaagtga ggaagatcca gggcgatggc gcagcgctcc aggagaagct gtgtgccacc 420 tacaagctgt gccaccccga ggagctggtg ctgctcggac actctctggg catcccctgg 480 gctcccctga gcagctgccc cagccaggcc ctgcagctgg caggctgctt gagccaactc 540 catagcggcc ttttcctcta ccaggggctc ctgcaggccc tggaagggat ctcccccgag 600 ttgggtccca ccttggacac actgcagctg gacgtcgccg actttgccac caccatctgg 660 cagcagatgg aagaactggg aatggcccct gccctgcagc ccacccaggg tgccatgccg 720 gccttcgcct ctgctttcca gcgccgggca ggaggggtcc tggttgcctc ccatctgcag 780 agcttcctgg aggtgtcgta ccgcgttcta cgccaccttg cccagccctg agccaagccc 840 tccccatccc atgtatttat ctctatttaa tatttatgtc tatttaagcc tcatatttaa 900 agacagggaa gagcagaacg gagccccagg cctctgtgtc cttccctgca tttctgagtt 960 tcattctcct gcctgtagca gtgagaaaaa gctcctgtcc tcccatcccc tggactggga 1020 ggtagatagg taaataccaa gtatttatta ctatgactgc tccccagccc tggctctgca 1080 atgggcactg ggatgagccg ctgtgagccc ctggtcctga gggtccccac ctgggaccct 1140 tgagagtatc aggtctccca cgtgggagac aagaaatccc tgtttaatat ttaaacagca 1200 gtgttcccca tctgggtcct tgcacccctc actctggcct cagccgactg cacagcggcc 1260 cctgcatccc cttggctgtg aggcccctgg acaagcagag gtggccagag ctgggaggca 1320 tggccctggg gtcccacgaa tttgctgggg aatctcgttt ttcttcttaa gacttttggg 1380 acatggtttg actcccgaac atcaccgacg tgtctcctgt ttttctgggt ggcctcggga 1440 cacctgccct gcccccacga gggtcaggac tgtgactctt tttagggcca ggcaggtgcc 1500 tggacatttg ccttgctgga cggggactgg ggatgtggga gggagcagac aggaggaatc 1560 atgtcaggcc tgtgtgtgaa aggaagctcc actgtcaccc tccacctctt caccccccac 1620 tcaccagtgt cccctccact gtcacattgt aactgaactt caggataata aagtgtttgc 1680 ctccaaaaaa aaaaaaaaaa aaa 1703 <210> 18 <211 >8923

<212> DNA <213> Homo sapiens <400> 18 agctcacagc tattgtggtg ggaaagggag ggtggttggt ggatgtcaca gcttgggctt 60 tatctccccc agcagtgggg actccacagc ccctgggcta cataacagca agacagtccg 120 gagctgtagc agacctgatt gagcctttgc agcagctgag agcatggcct agggtgggcg 180 gcaccattgt ccagcagctg agtttcccag ggaccttgga gatagccgca gccctcattt 240 gcaggggaag gcaccattgt ccagcagctg agtttcccag ggaccttgga gatagccgca 300 gccctcattt atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt 360 gccagggacc ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct 420 tttcggaagt gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg 480 cagttacctc ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca 540 gaatggcaag agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt 600 tgtcaatggt accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg 660 gctgtatcta gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt 720 ggccaggatc gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa 780 gacctgcggg ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga 840 agggaccttg acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga 900 acagtggtgt gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat 960 gcagaagggc ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg 1020 ccaccctctg gtggaccccg agccttttgt'ggccctgtgt gagaagactt tgtqtgagtg 1080 tgctgggggg ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca 1140 ggagggaatg gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc 1200 tggtatggag tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat 1260 caatgaaatg tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct 1320 ggatgaaggc ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta 1380 ccctcccggc acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg 1440 gatctgcagc aatgaagaat gtccagggga gtgccttgtc actggtcaat cccacttcaa 1500 gagctttgac aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga 1560 ttgccaggac cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga 1620 cgctgtgtgc acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa 1680 actgaagcat ggggcaggag ttgccatgga tggccaggac atccagctcc ccctcctgaa 1740 aggtgacctc cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga 1800 cctgcagatg gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctacgc 1860 cgggaagacc tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac 1920 cccctctggg ctggcagagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg 1980 ggactgccag gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac 2040 caggttctcc gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg 2100 tgccgtcagc ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga 2160 cggccgcgag tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg 2220 cgtgcgcgtc gcgtggcgcg agccaggccg ctgtgaqctg aactqcccga aaggccaggt 2280 gtacctgcag tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga 2340 gqaatgcaat gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga 2400 gaggggggac tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca 2460 gccagaagac atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca 2520 ctgtaccatg agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct 2580 gtctcatcgc agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc 2640 cgctgacaac ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct 2700 ggagtgcatg agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca 2760 tgagaacaga tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc 2820 ccctggagaa acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa 2880 ctgcacagac catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac 2940 cttcgacggg ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta 3000 ctgcggcagt aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc 3060 ctcagtgaaa tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt 3120 tgacggggag gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga 3180 gtctggccgg tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca 3240 cctgagcatc tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg 3300 gaattttgat ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga 3360 ccctgtggac tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt 3420 gcclctggac tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga 3480 ttcctcctgt agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc 3540 cgagccatat ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg 3600 cgcctgcttc tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt 3660 ggtgacctgg aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga 3720 gaacgggtat gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg 3780 tcagcaccct gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg 3840 ccctccaggg aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc 3900 agtgtgtgag gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag 3960 tgaccctgag cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg 4020 ccaggagccg ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct 4080 gtatgtggag gacatctcgg aaccgccgtt gcacgatttc taccgcagca ggctactgga 4140 cctggtcttc ctgctggatg gctcctccag gctgtccgag gctgagtttg aagtgctgaa 4200 ggcctttgtq gtggacatga tggagcggct gcgcatctcc cagaagtggg tccgcgtggc 4260 cgtggtggag taccacgacg gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc 4320 gtcagagctg cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac 4380 cagcgaggtc ttgaaataca cactgttcca aatcttcagc aagatcgacc gccctgaagc 4440 ctcccgcatc gccctgctcc tgatggccag ccaggagccc caacggatgt cccggaactt 4500 tgtccgctae gteeagggcc tgaagaagaa gaaggtcatt gtgatcccgg tgggcattgg 4560 gccccatgcc aacctcaagc agatccgcct catcgagaag caggcccctg agaacaaggc 4620 cttcgtgctg agcagtgtgg atgagctgga gcagcaaagg gacgagatcg ttagctacct 4680 ctgtgacctt gcccctgaag cccctcctcc tactctgccc ccccacatgg cacaagtcac 4740 tgtgggcccg gggctcttgg gggtttcgac cctggggccc aagaggaact ccatggttct,4800 ggatgtggcg ttcgtcctgg aaggatcgga caaaattggt gaagccgact tcaacaggag 4860 caaggagttc atggaggagg tgattcagcg gatggatgtg ggccaggaca gcatccacgt 4920 cacggtgctg cagtactcct acatggtgac cgtggagtac cccttcagcg aggcacagtc 4980 caaaggggac atcctgcagc gggtgcgaga gatccgctac cagggcggca acaggaccaa 5040 cactgggctg gccctgcggt acctctctga ccacagcttc ttggtcagcc agggtgaccg 5100 ggagcaggcg cccaacctgg tctacatggt caccggaaat cctgcctctg atgagatcaa 5160 gaggctgcct ggagacatcc aggtggtgcc cattggagtg ggccctaatg ccaacgtgca 5220 ggagctggag aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct 5280 cccccgagag gctcctgacc tggtgctgca gaggtgctgc tccggagagg ggctgcagat 5340 ccccaccctc tcccctgcac ctgactgcag ccagcccctg gacgtgatcc ttctcctgga 5400 tggctcctcc agtttcccag cttcttattt tgatgaaatg aagagtttcg ccaaggcttt 5460 catttcaaaa gccaatatag ggcctcgtct cactcaggtg tcagtgctgc agtatggaag 5520 catcaccacc attgacgtgc catggaacgt ggtcccggag aaagcccatt tgctgagcct 5580 tgtggacgtc atgcagcggg agggaggccc cagccaaatc ggggatgcct tgggctttgc 5640 tgtgcgatac ttgacttcag aaatgcatgg tgccaggccg ggagcctcaa aggcggtggt 5700 catcctggtc acggacgtct ctgtggattc agtggatgca gcagctgatg ccgccaggtc 5760 caacagagtg acagtglLcc cLaLlggaat tggagatcgc tacgatgcag cccagctacg 5820 gatcttggca ggcccagcag gcgactccaa cgtggtgaag ctccagcgaa tcgaagacct 5880 ccctaccatg gtcaccttgg gcaattcctt cctccacaaa ctgtgctctg gatttgttag 5940 gatttgcatg gatgaggatg ggaatgagaa gaggcccggg gacgtctgga ccttgccaga 6000 ccagtgccac accgtgactt gccagccaga tggccagacc ttgctgaaga gtcatcgggt 6060 caactgtgac cgggggctga ggccttcgtg ccctaacagc cagtcccctg ttaaagtgga 6120 agagacctgL ggcLgccgct ggacctgccc ctgcgtgtgc acaggcagct ccactcggca 6180 catcgtgacc tttgatgggc agaatttcaa gctgactggc agctgttctt atgtcctatt 6240 tcaaaacaag gagcaggacc tggaggtgat tctccataat ggtgcctgca gccctggagc 6300 aaggcagggc tgcatgaaat ccatcgaggt gaagcacagt gccctctccg tcgagctgca 6360 caqtgacatg gaggtgacgg tgaatgggag actggtctct gttccttacg tgggtgggaa 6420 catggaagtc aacgtttatg gtgccatcat gcatgaggtc agattcaatc accttggtca 6480 catcttcaca ttcactccac aaaacaatga gttccaactg cagctcagcc ccaagacttt 6540 tgcttcaaag acgtatggtc tgtgtgggat ctgtgatgag aacggagcca atgacttcat 6600 gctgagggat ggcacagtca ccacagactg gaaaacactt gttcaggaat ggactgtgca 6660 gcggccaggg cagacgtgcc agcccatcct ggaggagcag tgtcttgtcc ccgacagctc 6720 ccactgccag gtcctcctct taccactgtt tgctgaatgc cacaaggtcc tggctccagc 6780 cacattctat gccatctgcc agcaggacag ttgccaccag gagcaagtgt gtgaggtgat 6840 cgcctcttat gcccacctct gtcggaccaa cgqqgtctgc gttgactgga ggacacctga 6900 tttctgtgct atgtcatgcc caccatctct ggtctacaac cactgtgagc atggctgtcc 6960 ccggcactgt gatggcaacg tgagctcctg tggggaccat ccctccgaag gctgtttctg 7020 ccctccagat aaagtcatgt tggaaggcag ctgtgtccct gaagaggcct gcactcagtg 7080 cattggtgag gatggagtcc agcaccagtt cctggaagcc tgggtcccgg accaccagcc 7140 ctgtcagatc tgcacatgcc tcagcgggcg gaaggtcaac tgcacaacgc agccctgccc 7200 cacggccaaa gctcccacgt gtggcctgtg tgaagtagcc cgcctccgcc agaatgcaga 7260 ccagtgctgc cccgagtatg agtgtgtgtg tgacccagtg agctgtgacc tgcccccagt 7320 gcctcactgt gaacgtggcc tccagcccac actgaccaac cctggcgagt gcagacccaa 7380 cttcacctgc gcctgcagga aggaggagtg caaaagagtg tccccaccct cctgcccccc 7440 gcaccgtttg cccacccttc ggaagaccca gtgctgtgat gagtatgagt gtgcctgcaa 7500 ctgtgtcaac tccacagtga gctgtcccct tgggtacttg gcctcaaccg ccaccaatga 7560 ctgtggctgt accacaacca cctgccttcc cgacaaggtg tgtgtccacc gaagcaccat 7620 clacccLglg ggccagttct gggaggaggg ctgcgatgtg tgcacctgca ccgacatgga 7680 ggatgccgtg atgggcctcc gcgtggccca gtgctcccag aagccctgtg aggacagctg 7740 tcggtcgggc ttcacttacg ttctgcatga aggcgagtgc tgtggaaggt gcctgccatc 7800 tgcctgtgag gtggtgactg gctcaccgcg gggggactcc cagtcttcct ggaagagtgt 7860 cggctcccag tgggcctccc cggagaaccc ctgccLcatc aaLgagtgtg tccgagtgaa 7920 ggaggaggtc tttatacaac aaaggaacgt ctcctgcccc cagctggagg tccctgtctg 7980 cccctcgggc tttcagctga gctgtaagac ctcagcgtgc tgcccaagct gtcgctgtga 8040 .gcgcatggag gcctgcatgc tcaatggcac tgtcattggg cccgggaaga ctgtgatgat 8100 cgatgtgtgc acgacctgcc gctgcatggt gcaggtgggg gtcatctctg gattcaagct 8160 ggagtgcagg aagaccacct gcaacccctg ccccctgggt tacaaggaag aaaataacac 8220 aggtgaatgt tgtgggagat gtttgcctac ggcttgcacc attcagctaa gaggaggaca 8280 gatcatgaca ctgaagcgtg atgagacgct ccaggatggc tgtgatactc acttctgcaa 8340 ggtcaatgag agaggagagt acttctggga gaagagggtc acaggctgcc caccctttga 8400 tgaacacaag tgtctggctg agggaggtaa aattatgaaa attccaggca cctgctgtga 8460 cacatgtgag gagcctgagt gcaacgacat cactgccagg ctgcagtatg tcaaggtggg 8520 aagctgtaag tctgaagtag aggtggatat ccactactgc cagggcaaat gtgccagcaa 8580 agccatgtac tccattgaca tcaacgatgt gcaggaccag tgctcctgct gctctccgac 8640 acggacggag cccatgcagg tggccctgca ctgcaccaat ggctctgttg tgtaccatga 8700 ggttctcaat gccatggagt gcaaatgctc ccccaggaag tgcagcaagt gaggctgctg 8760 cagctgcatg ggtgcctgct gctgcctgcc ttggcctgat ggccaggcca gagtgctgcc 8820 agtcctctgc atgttctgct cttgtgccct tctgagccca caataaaggc tgagctctta 8880 tcttgctgca tgttctgctc ttgtgccctt ctgagcccac aat 8923 <210> 19 <211> 24 <212> DNA <213> artificial <220> <223> primer <400> 19 atggctggac ctgccaccca gage 24 <210> 20 <211> 24 <212> DNA <213> artificial <220> <223> primer <400> 20 tcagggctgg gcaaggtggc gtag 24 <210> 21 <211 >4542 <212> DNA <213> artificial <220> <223> vector PCR2.1d2-GCSFb <400> 21 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg 240 gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgg ctatggctgg 300 acctgccacc cagagcccca tgaagctgat ggccctgcag ctgctgctgt ggcacagtgc 360 actctggaca gtgcaggaag ccacccccct gggccctgcc agctccctgc cccagagctt 420 cctgctcaag tgcttagagc aagtgaggaa gatccagggc gatggcgcag cgctccagga 480 gaagctgtgt gccacctaca agctgtgcca ccccgaggag ctggtgctgc tcggacactc 540 tctgggcatc ccctgggctc ccctgagcag ctgccccagc caggccctgc agctggcagg 600 ctgcttgagc caactccata gcggcctttt cctctaccag gggctcctgc aggccctgga 660 agggatctcc cccgagttgg gtcccacctt ggacacactg cagctggacg tcgccgactt 720 tgccaccacc atctggcagc agatggaaga actgggaatg gcccctgccc tgcagcccac 780 ccagggtgcc atgccggcct tcgcctctgc tttccagcgc cgggcaggag gggtcctggt 840 tgcctcccat ctgcagagct tcctggaggt gtcgtaccgc gttctacgcc accttgccca 900 gccctgaagc cgaattctgc agatatccat cacactggcg gccgctcgag catgcatcta 960 gagggcccaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac 1020 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 1080 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 1140 gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 1200 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 1260 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 1320 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 1380 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 1440 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 1500 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 1560 gatttaacaa aaatttaacg cgaattttaa caaaattcag ggcgcaaggg ctgctaaagg 1620 aagcggaaca cgtagaaagc cagtccgcag aaacggtgct gaccccggat gaatgtcagc 1680 tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt 1740 gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga accggaattg 1800 ccagctgggg cgccctctgg taaggttggg aagccctgca aagtaaactg gatggctttc 1860 ttgccgccaa ggatctgatg gcgcagggga tcaagatctg atcaagagac aggatgagga 1920 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag 1980 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc 2040 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg 2100 'aa’tgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 2160 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg 2220 ccggggcagg atctcctgtc atcccacctt gctcctgccg agaaagtatc catcatggct 2280 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg 2340 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat 2400 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc 2460 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg 2520 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc 2580 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct 2640 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat 2700 cgccttcttg acgagttctt ctgaattgaa aaaggaagag tatgagtatt caacatttcc 2760 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 2820 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 2880 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 2940 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 3000 agcaactcgg.tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 3060 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 3120 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 3180 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 3240 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 3300 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 3360 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 3420 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 3480 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 3540 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 3600 aactgtcaga ccaagtttac tcatatatac tt.tagattga tttaaaactt catttttaat 3660 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 3720 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 3780 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 3840 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 3900 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 3960 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 4020 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 4080 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 4140 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 4200 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 4260 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 4320 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 4380 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 4440 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 4500 .gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga ag 4542 <210> 22 <211> 6237 <212> DNA <213> artificial <220> <223> vector pcDNA3.1-hyg(+)-GCSFb <400> 22 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctat'tgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaactt aagcttggta ccgagctcgg atccactagt aacggccgcc agtgtgctgg 960 aattcggota tggctggacc tgccacccag agccccatga agctgatggc cctgcagctg 1020 ctgctgtggc acagtgcact ctggacagtg caggaagcca cccccctggg ccctgccagc 1080 tccctgcccc agagcttcct gctcaagtgc ttagagcaag tgaggaagat ccagggcgat 1140 ggcgcagcgc tccaggagaa gctgtgtgcc acctacaagc tgtgccaccc cgaggagctg 1200 gtgctgctcg gacactctct gggcatcccc LgggcLcccc tgagcagctg ccccagccag 1260 gccctgcagc tggcaggctg cttgagccaa ctccatagcg gccttttcct ctaccagggg 1320 ctcctgcagg ccctggaagg gatctccccc gagttgggtc ccaccttgga cacactgcag 1380 ctggacgtcg ccgactttgc caccaccatc tggcagcaga tggaagaact gggaatggcc 1440 cctgccctgc agcccaccca gggtgccatg ccggccttcg cctctgcttt ccagcgccgg 1500 gcaggagggg tcctggttgc ctcccatctg cagagcttcc tggaggtgtc gtaccgcgtt 1560 ctacgccacc ttgcccagcc ctgaagccga attctgcaga tatccatcac actggcggcc 1620 gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg 1680 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 1740 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 1800 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 1860 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 1920 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 1980 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 2040 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 2100 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 2160 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 2220 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 2280 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 2340 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag 2400 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 2460 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 2520 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 2580 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 2640 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 2700 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgatgaaa 2760 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc 2820 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga 2880 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat 2940 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa 3000 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac 3060 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc 3120 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt 3180 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg 3240 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg 3300 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac 3360 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc 3420 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg 3480 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc 3540 cgggcgtata tgctccgcat'tggtcttgac caactctatc agagcttggt tgacggcaat 3600 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg 3660 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta 3720 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag 3780 cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc 3840 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc 3900 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 3960 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 4020 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg 4080 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa ca'tacgagcc 4140 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 4200 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 4260 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 4320 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 4380 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 4440 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 4500 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 4560 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 4620 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 4680 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 4740 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 4800 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 4860 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 4920 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 4980 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 5040 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 5100 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 5160 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 5220 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 5280 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 5340 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 5400 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 5460 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 5520 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 5580 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 5640 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 5700 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 5760 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 5820 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 5880 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 5940 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 6000 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 6060 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 6120 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 6180 aaaaataaac aaataggggt tccgcgcaea tttceccgaa aagtgccacc tgacgtc 6237 <210> 23 <211 > 6101 <212> DNA <213> artificial <220> <223> vector pCINeo-GCSFb <400> 23 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg 660 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 720 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac 780 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt 840 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa 900 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact 960 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac 1020 aqgtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact 1080 ataggctagc ctcgagaatt cggctatggc tggacctgcc acccagagcc ccatgaagct 1140 gatggccctg cagctgctgc tgtqgcacag tgcactctgg acagtgcagg aagccacccc 1200 cctgggccct gccagctccc tgccccagag cttcctgctc aagtgcttag agcaagtgag 1260 gaagatccag ggcgatggcg cagcgctcca ggagaagctg tgtgccacct acaagctgtg 1320 ccaccccgag gagctggtgc tgctcggaca ctctctgggc atcccctggg ctcccctgag 1380 cagctgcccc agccaggccc tgcagctggc aggctgcttg agccaactcc atagcggcct 1440 tttcctctac caggggctcc tgcaggccct ggaagggatc tcccccgagt tgggtcccac 1500 cttggacaca ctgcagctgg acgtcgccga ctttgccacc accatctggc agcagatgga 1560 agaactggga atggcccctg ccctgcagcc cacccagggt gccatgccgg ccttcgcctc 1620 tgctttccag cgccgggcag gaggggtcct ggttgcctcc catctgcaga gcttcctgga 1680 ggLglcglac cgcgttctac gccaccttgc ccagccctga agccgaattc acgcgtggta 1740 cctctagagt cgacccgggc ggccgcttcc ctttagtgag ggttaatgct tcgagcagac 1800 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 1860 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 1920 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag 1980 gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg atcgatccgg 2040 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 2100 atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 2160 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 2220 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 2280 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 2340 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 2400 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 2460 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 2520 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat 2580 tttctcctta cgcatctgtg cggtattLca caccgcatac gcggatctgc gcagcaccat 2640 ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc 2700 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 2760 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 2820 cagcaggcag aagtatgcaa agcatgcatc LcaaLLagLc agcaaccaLa gLcccgcccc 2880 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 2940 gactaatttt. ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 3000 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagcttgat tcttctgaca 3060 caacagtctc gaacttaagg ctagagccac catgattgaa caagatggat tgcacgcagg 3120 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 3180 ctgctctgat gccgccgtgt tccggctgtc agcgcagqgg cgcccggttc tttttgtcaa 3240 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct 3300 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 3360 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 3420 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 3480 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 3540 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 3600 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 3660 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 3720 ccggctgggt gLggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 3780 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 3840 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg 3900 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gatggccgca ataaaatatc 3960 tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga taaggatccg 4020 cgtatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 4080 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 4140 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 4200 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 4260 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 4320 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 4380 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 4440 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 4500 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 4560 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 4620 agttctgcta.tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 4680 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 4740 tacggatggc a.tgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 4800 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 4860 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 4920 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 4980 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 5040 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 5100 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 5160 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 5220 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 5280 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 5340 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 5400 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 5460 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 5520 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 5580 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 5640 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 5700 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 5760 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 5820 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 5880 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 5940 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 6000 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 6060 ggccttttgc tggccttttg ctcacatggc tcgacagatc t 6101 <210> 24 <211 >4920 <212> DNA <213> artificial <220> <223> vector pCMVScript_GCSFb <400> 24 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga 60 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 120 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 180 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 240 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 300 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 360 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc 420 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaåaa 480 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 540 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta 600 gcgattacgc caagctcgaa attaaccctc actaaaggga acaaaagctg gagctccacc 660 gcggtggcgg ccgctctagc ccgggcggat ccactagtaa cggccgccag tgtgctggaa 720 ttcggctatg gctggacctg ccacccagag ccccatgaag ctgatggccc tgcagctgct 780 gctgtggcac agtgcactct ggacagtgca ggaagccacc cccctgggcc ctgccagctc 840 cctgccccag agcttcctgc tcaagtgctt agagcaagtg aggaagatec agggcgatgg 900 cgcagcgctc caggagaagc tgtgtgccac ctacaagctg tgccaccccg aggagctggt 960 gctgctcgga cactctctgg gcatcccctg ggctcccctg agcagctgcc ccagccaggc 1020 cctgcagctg gcaggctgct tgagccaact ccatagcggc cttttcctct accaggggct 1080 cctgcaggcc ctggaaggga tctcccccga gttgggtccc accttggaca cactgcagct 1140 ggacgtcgcc gactttgcca ccaccatctg gcagcagatg gaagaactgg gaatggcccc 1200 tgccctgcag cccacccagg gtgccatgcc ggccttcgcc tctgctttcc agcgccgggc 1260 aggaggggtc ctggttgcct cccatctgca gagcttcctg gaggtgtcgt accgcgttct 1320 acgccacctt gcccagccct gaagccgaat tctgcagata tccatcacac tggcggccgc 1380 tcgagggggg gcccggtacc aggtaagtgt acccaattcg ccctatagtg agtcgtatta 1440 caattcactc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggagatccaa 1500 tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt ttagattcac 1560 agtcccaagg ctcatttcag gcccctcagt cctcacagtc tgttcatgat cataatcagc 1620 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac 1680 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt 1740 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 1800 agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat 1860 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 1920 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 1980 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac 2040 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 2100 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 2160 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cggqcgctag 2220 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 2280 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 2340 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 2400 gcttcaataa tattgaaaaa ggaagaatcc tgaggcggaa agaaccagct gtggaatgtg 2460 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 2520 catctcaatt agtcagcaac caggtgtqqa aagtccccaq qctccccagc aggcagaagt 2580 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc 2640 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt 2700 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc 2760 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg 2820 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt 2880 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc 2940 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact 3000 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctL gcgcagcLgt 3060 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca 3120 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat 3180 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg 3240 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga 3300 agaacatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga 3360 cggcgaggat cLcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa 3420 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga 3480 catagcgttg gctacccgtg atattgctga agaacttggc ggcgaatggg ctgaccgctt 3540 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct 3600 tgacgagttc ttctgagcgg gactctgggg LLcgaaaLga ccgaccaagc gacgcccaac 3660 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc 3720 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc 3780 gcccacccto gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc 3840 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa 3900 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc 3960 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca 4020 gggctcgcag ccaacgtcgg gqcggcaggc cctgccatag cctcaggtta ctcatatata 4080 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 4140 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 4200 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 4260 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 4320 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 4380 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 4440 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 4500 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 4560 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 4620 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 4680 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 4740 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 4800 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 4860 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 4920 <210> 25 <211> 6917 <212> DNA <213> artificial <220> <223> vector pTG2-GCSFb-hyg-as <400> 25 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180 tagggacttt ccattgacgt caatgggtgg actatttacg gtaaactgcc cacttggcag 240 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600 ctagagaacc cactgcttaa ctggcttatc gaaattaata cgactcacta tagggagacc 660 ggaagcttgg taccgagctc ggatccacta gtaacggccg ccagtgtgct ggaattcggc 720 tatggctgga cctgccaccc agagccccat gaagctgatg gccctgcagc tgctgctgtg 780 gcacagtgca ctctggacag tgcaggaagc cacccccctg ggccctgcca gctccctgcc 840 ccagagcttc ctgctcaagt gcttagagca agtgaggaag atccagggcg atggcgcagc 900 gctccaggag aagctgtgtg ccacctacaa gctgtgccac cccgaggagc tggtgctgct 960 cggacactct ctgggcatcc cctgggctcc cctgagcagc tgccccagcc aggccctgca 1020 gctggcaggc tgcttgagcc aactccatag cggccttttc ctctaccagg ggctcctgca 1080 ggccctggaa gggatctccc ccgagttggg tcccaccttg gacacactgc agctggacgt 1140 cgccgacttt gccaccacca tctggcagca gatggaagaa ctgggaatgg cccctgccct 1200 gcagcccacc cagggtgcca tgccggcctt cgcctctgct ttccagcgcc gggcaggagg 1260 ggtcctggtt gcctcccatc tgcagagctt cctggaggtg tcgtaccgcg ttctacgcca 1320 ccttgcccag ccctgaagcc gaattctgca gatatccatc acactggcgg ccgcgactct 1380 agctagagga tctttgtgaa ggaaccttac ttctgtggtg tgacataatt ggacaaacta 1440 cctacagaga tttaaagctc taaggtaaat ataaaatttt taagtgtata atgtgttaaa 1500 ctactgattc taattgtttg tgtattttag attccaacct atggaactga tgaatgggag 1560 cagtggtgga atgcctttaa tgaggaaaac ctgttttgct cagaagaaat gccatctagt 1620 gatgatgagg ctactgctga ctctcaacat tctactcctc caaaaaagaa gagaaaggta 1680 gaagacccca aggactttcc ttcagaattg ctaagttttt tgagtcatgc tgtgtttagt 1740 aatagaactc ttgcttgctt tgctatttac accacaaagg aaaaagctgc actgctatac 1800 aagaaaatta tggaaaaata ttctgtaacc tttataagta ggcataacag ttataatcat 1860 aacatactgt tttttcttac tccacacagg catagagtgt ctgctattaa taactatgct 1920 caaaaattgt gtacctttag ctttttaatt tgtaaagggg ttaataagga atatttgatg 1980 tatagtgcct tgactagaga tcataatcag ccataccaca tttgtagagg ttttacttgc 2040 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 2100 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 2160 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 2220 atcttatcat gtctggatcc ccagcttggc actggcgcca gaaatccgcg cggtggtttt 2280 tgggggtcgg gggtgtttgg cagccacaga cgcccggtgt tcgtgtcgcg ccagtacatg 2340 cggtccatgc'ccaggccatc caaaaaccat gggtctgtct gctcagtcca gtcgtggacc 2400 tgaccccacg caacgcccaa aagaataacc cccacgaacc ataaaccatt ccccatgggg 2460 gaccccgtcc ctaacccacg gggcccgtgg ctatggcggg cttgccgccc cgacgttggc 2520 tgcgagccct gggccttcac ccgaacttgg gggttggggt ggggaaaagg aagaaacgcg 2580 ggcgtattgg ccccaatggg gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg 2640 aaccccgcgt ttatgaacaa acgacccaac acccgtgcgt tttattctgt ctttttattg 2700 ccgtcatagc gcgggttcct tccggtattg tctccttccg tgtttcagtt agcctccccc 2760 atctcccgat ccccacgagt gctggggcgt cggtttccac tatcggcgag tacttctaca 2820 cagccatcgg tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg 2880 gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg 2940 ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata cgcccggagc 3000 cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata 3060 caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga atccccgaac 3120 atcgcctcgc tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg 3180 gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc 3240 agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac agtttgccag 3300 tgatacacat ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg 3360 attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc cgcagcgatc 3420 gcatccatgg cctccgcgac cggctgcaga acagcgggca gttcggtttc aggcaggtct 3480 tgcaacgtga caccctgtgc acggcgggag atgcaatagg tcaggctctc gctgaattcc 3540 ccaaUgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg ataaacataa 3600 cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatcc acgccctcct 3660 acatcgaagc tgaaagcacg agattcttcg ccctccgaga gctgcatcag gtcggagacg 3720 ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc aggctttttc 3780 alatcaagct gatcttgcgg cacgctgttg acgctgttaa gcgggtcgct gcagggtcgc 3840 tcggtgttcg aggccacacg cgtcacctta atatgcgaag tggacctggg accgcgccgc 3900 cccgactgca tctgcgtgtt cgaattcgcc aatgacaaga cgctgggcgg ggtttgtgtc 3960 atcatagaac taaagacatg caaatatatt tcttccgggg acaccgccag caaacgcgag 4020 caacgggcca cggggatgaa gcagcccggc ggcacctcgc taacggattc accactccaa 4080 gaattggagc caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga 4140 acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg gggccgacgc 4200 gctgggctac gtcttgctgg cgttcggggt accgctctag agcgaattaa ttcactggcc 4260 gtcgttttac aacgtcgtga cLgggaaaac ccLggcgtta cccaacttaa tcgccttgca. 4320 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 4380 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat 4440 ctgtgeggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca 4500 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 4560 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 4620 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 4680 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 4740 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 4800 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 4860 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 4920 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 4980 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 5040 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 5100 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 5160 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 5220 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 5280 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 5340 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 5400 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 5460 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 5520 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 5580 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 5640 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 5700 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 5760 ttttaattta aaaggatcta ggtgaagatc cLttttgaLa alcLcatgac caaaatccct 5820 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 5880

Igagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 5940 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 6000 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 6060 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 6120 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 6180 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 6240 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 6300 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 6360 cttccagggg qaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 6420 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 6480 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 6540 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 6600 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata 6660 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 6720 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag 6780 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga 6840 taacaatttc acacaggaaa cagctatgac catgattacg ccaagctctc tagagagctt 6900 gcatgcctgc aggtcga 6917 <210> 26 <211> 615

<212> DNA <213> Homo sapiens <220> <221 > CDS <222> (1)..(612) <400> 26 atg get gga cct gee ace cag age ccc atg aag ctg atg gee ctg cag 48

Met Ala Gly Pro Ala Thr Gin Ser Pro Met Lys Leu Met Ala Leu Gin 15 10 15 ctg ctg ctg tgg cac agt gca etc tgg aca gtg cag gaa gee acc ccc 96

Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gin Glu Ala Thr Pro 20 25 30 ctg ggc cct gee age tee ctg ccc cag age ttc ctg etc aag tgc tta 144

Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Cys Leu 35 40 45 gag caa gtg agg aag ate cag ggc gat ggc gca geg etc cag gag aag 192

Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 50 55 60 ctg tgt gee acc tac aag ctg tgc cac ccc gag gag ctg gtg ctg etc 240

Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu 65 70 75 80 gga cac tet ctg ggc ate ccc tgg get ccc ctg age age tgc ccc age 288

Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser 85 90 95 cag gee ctg cag ctg gca ggc tgc ttg age caa etc cat age ggc ett 336

Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu 100 105 110 ttc etc tac cag ggg etc ctg cag gee ctg gaa ggg ate tee ccc gag 384

Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie Ser Pro Glu 115 120 125 ttg ggt ccc acc ttg gac aca ctg cag ctg gac gtc gee gac ttt gee 432

Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala 130 135 140 acc acc ate tgg cag cag atg gaa gaa ctg gga atg gee cct gee ctg 480

Thr Thr lie Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu 145 150 155 160 cag ccc acc cag ggt gee atg ccg gee ttc gee tet get ttc cag ege 528

Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg 165 170 175 egg gca gga ggg gtc ctg gtt gee tee cat ctg cag age ttc ctg gag 576

Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu 180 185 190 gtg teg tac ege gtt eta ege cac ett gee cag ccc tga 615

Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 195 200 <210> 27 <211 > 204

<212> PRT <213> Homo sapiens <400> 27

Met Ala Gly Pro Ala Thr Gin Ser Pro Met Lys Leu Met Ala Leu Gin 15 10 15

Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gin Glu Ala Thr Pro 20 25 30

Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Cys Leu 35 40 45 '

Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 50 55 60

Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu 65 70 75 80

Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser 85 90 95

Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu 100 105 110

Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie Ser Pro Glu 115 120 125

Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala 130 135 140

Thr Thr lie Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu 145 150 155 160

Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg 165 170 175

Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu 180 185 190

Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 195 200

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description • US5712119A Γ00041 • WQ0170968A Γ00081 Γ003Π f0055i fOOSSi Γ0062Ί • EP180457A Γ00301 • WQ8601961A [0030] • US4770999A [0030] • US5521070A [00301 f00301 • EP150735A Γ00311 • EP232112A Γ0031Ί • EPO500734 A rø031] . WQ9107490A F00311 • WQ9513300A Γ00311 • US5045455A F0831] • US5789203A f0031l • WQ860eiQ1A f00311 • WQ8704187A Γ00311 • WQ8707144A Γ00311 • VVQ88Q0381A F00311 • EP251843A Γ00311 • EP253455A Γ0031Ί • EP254076A Γ003Π • US4B68112Α Γ00311 • US49B0458A F00311 • FP29491OÅ Γ0031ΐ • FP26S77SA r0031l • FP303540A f003tf • WQ9109122A F00311 • WQ9315105A Γ00421 • WQ9636369A Γ00421 • FPO 31740A i00421 • USe007973A F0D421 • WQ9417834A TO0421 • ΕΡ05105965ΑΓ00651

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Claims (20)

1. Fremgangsmåde til fremstilling af en immortaliseret human cellelinje, der er stabilt transficeret med en nukleinsyresekvens omfattende et gen kodende for et humant målprotein eller et derivat eller en mutant deraf, en promotor og et bovint væksthormon-polyadenylerings(polyA)signal, hvor promotoren og polyA-signalet er forbundet til 5'- og 3'-enden, henholdsvis, af genet kodende for det humane målprotein, hvilken fremgangsmåde omfatter at transficere en immortaliseret human værtscellelinje under serumfrie betingelser med en transfektionsvektor omfattende nukleinsyresekvensen og et replika-tionsbegyndelsessted, hvor den immortaliserede humane cellelinje dyrkes under serumfrie betingelser.

2. Fremgangsmåde ifølge krav 1, hvor det humane målprotein er et humant plasmaprotein udvalgt blandt blodkoaguleringsfaktorer, vækstfaktorer, kolonistimulerende faktorer (CSF'er), cytokiner, proteasehæmmere, chymotrypsin, transportproteiner, hormoner, hæmmende eller regulatorisk virkende proteiner samt derivater og mutanter deraf.

3. Fremgangsmåde ifølge krav 2, hvor det humane målprotein er udvalgt blandt faktor IX, faktor VIII (vildtype og B-domæne-deleteret), faktor VII/VIIa og von Willebrand-faktor (vWF), erythropoietin, granulocytstimulerende faktor (G-CSF) makrofag-CSF (M-CSF) og granulocyt-makrofag-CSF (GM-CSF), interleukiner, alfa-l-antitrypsin (A1AT).

4. Fremgangsmåde ifølge krav 2, hvor det humane målprotein er udvalgt blandt faktor IX, faktor VIII indbefattende vildtype-faktor VIII og B-domæne-deleteret faktor VIII, faktor VII/VIIa, G-CSF, vWF og A1AT.

5. Fremgangsmåde ifølge krav 2, hvor det humane målprotein er blodkoaguleringsfaktor IX som kodet for af baseparrene 939 til 2324 i SEQ ID NO:l, human A1AT som kodet for af baseparrene 973 til 2259 i SEQ ID NO:2, vildtype-faktor VIII som vist i SEQ ID NO:9, B-domæne-deleteret human faktor VIII som kodet for af baseparrene 783 til 5162 i SEQ ID NO:3, faktor VII/VIIa som kodet for af SEQ ID NO: 13 og 14, G-CSF som kodet for af SEQ ID NO:15, 16 og 17, eller vWF som kodet for af SEQ ID NO:18.

6. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 5, hvor det for trans-fektionsvektoren gælder, at (i) promotoren er udvalgt blandt virale promotorer, husholdningsgenpromotorer og vævsspecifikke promotorer, fortrinsvis er promotoren en CMV-promotor med eller uden intron A, SV40-promotor, EF-l-alfa-promotor, HSV TK-promotor, mest foretrukket er promotoren en CMV-promotor; og/eller (ii) replikationsbegyndelsesstedet tillader replikation og opformering af plasmidet i bakterien; (iii) vektoren bærer yderligere mindst et gen for en selektionsmarkør, fortrinsvis udvalgt blandt hygromycinresistens, neomycinresistens, aminoglykosidphosphotransferasere-sistens, bleomycinresistens og xanthin-guaninphosphoribosyltransferaseresistens og/eller er under kontrol af en promotor som defineret i (i) ovenfor; og/eller (iv) vektoren bærer yderligere et eller flere regulatoriske elementer, hvilke regulato- riske elementer fortrinsvis er udvalgt blandt splejsningssteder, rekombinationssteder, po-lyA-steder, forstærkere, multikloningssteder og prokaryote plasmidsekvenser.

7. Fremgangsmåde ifølge krav 6, hvor transfektionsvektoren bærer en CMV-promotor, et hygromycingen, en polyA-sekvens og genet af interesse og fortrinsvis er afledt fra pcDNA3.1-vektoren med sekvensen ifølge SEQ ID NO:4 eller 5.

8. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 7, hvor den immortali-serede humane cellelinje (i) er i stand til at blive transficeret og dyrket under serumfrie betingelser; og/eller (ii) har adenovirussekvenser integreret i sit genom; og/eller (iii) er udvalgt fra gruppen bestående af nyre-, blære-, lever-, lunge-, hjertemuskel, glat muskel, æggestok- og mave/tarm-celler, fortrinsvis en human nyrecellelinje; og/eller (iv) ikke er i stand til at udtrykke et humant prionprotein.

9. Fremgangsmåde ifølge krav 8, hvor nyrecellerne er humane fosternyreceller, fortrinsvis de humane fosternyreceller udvalgt blandt 293-celler, 293T-celler, Freestyle 293-celler, og fortrinsvis er 293F-celler.

10. Fremgangsmåde ifølge krav 9, hvor cellelinjen er 293F, og transfektionsvektoren er afledt fra pcDNA3.1, vektoren koder fortrinsvis for human blodkoaguleringsfaktor IX som vist ved baseparrene 939 til 2324 i SEQ ID NO:l, human A1AT som kodet for af baseparrene 973 til 2259 i SEQ ID NO:2, vildtype-human faktor VIII som vist i SEQ ID NO:9 eller B-domæne-deleteret human faktor VIII som vist i SEQ ID NO:3, FVII/FVIIa, G-CSF indbefattende G-CSFb vist i SEQ ID NO:27 eller von Willebrand-faktor.

11. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 10, hvor den serumfrie transfektion sker i opslæmningskultur uden serum med et kationisk transfek-tionsmiddel eller calciumphosphat, fortrinsvis lipofectamine 2000 CD-reagens, der kan erhverves fra Invitrogen.

12. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 11, hvilken fremgangsmåde yderligere omfatter at selektere for stabilt transficerede celler, fortrinsvis efter produktivitet, aktivitet og produktkvalitet hos det dannede rekombinante protein.

13. Stabilt transficeret immortaliseret human cellelinje, som kan opnås ved fremgangsmåden ifølge krav 1 til 12.

14. Cellelinje ifølge krav 13, som kan opnås ved fremgangsmåden ifølge kravene 7 til 12, fortrinsvis er cellelinjen 293F, og transfektionsvektoren er afledt fra pcDNA3.1

15. Fremgangsmåde til rekombinant fremstilling af et humant målprotein eller et derivat eller en mutant deraf, som omfatter at dyrke en immortaliseret human cellelinje som defineret i krav 13 eller 14.

16. Fremgangsmåde ifølge krav 15, hvor det humane målprotein er human blodkoaguleringsfaktor IX som vist ved baseparrene 939 til 2324 i SEQ ID NO:l, human A1AT som kodet for af baseparrene 973 til 2259 i SEQ ID NO:2, vildtype-human faktor VIII som vist i SEQ ID NO:9 eller B-domæne-deleteret human faktor VIII som vist i SEQ ID NO:3, FVII/FVIIa, G-CSF indbefattende G-CSFb vist i SEQ ID NO:27 eller von Willebrand-faktor.

17. Fremgangsmåde ifølge krav 15 eller 16, hvilken fremgangsmåde yderligere omfatter trinnene at opkoncentrere det rekombinante humane protein fra kultursuppen og/eller at oprense proteinet og/eller prionfjernelse.

18. Fremgangsmåde ifølge et hvilket som helst af kravene 15 til 17, hvor fremstillingen af det humane protein sker under serumfrie betingelser.

19. Anvendelse af en transfektionsvektor omfattende et replikationsbegyndelsessted og en nukleinsyresekvens omfattende et gen kodende for et humant målprotein eller et derivat eller en mutant deraf som defineret i kravene 1 til 7 i en fremgangsmåde ifølge krav 1 til 12.

20. Anvendelse ifølge krav 19, hvor transfektionsvektoren er en pcDNA3.1-vektor omfattende genet kodende for et målprotein udvalgt fra gruppen bestående af human pro-teinblodkoaguleringsfaktor IX, human A1AT, human blodkoaguleringsfaktor VIII indbefattende vildtype og B-domæne-deleterede mutanter deraf, G-CSF indbefattende a-, b- og c-formen deraf, FVII/VIIa indbefattende a- og b-formen deraf, og vWF, fortrinsvis har transfektionsvektoren nukleinsyresekvensen vist i SEQ ID NO:l, 2, 3 eller 22.