Source: http://www.patentsencyclopedia.com/app/20120083033
Timestamp: 2018-04-22 03:10:20
Document Index: 351683931

Matched Legal Cases: ['application No. 60', 'art: 3199', 'art: 5209', 'art: 2863', 'art: 1463', 'art: 6659', 'art: 8295', 'art: 13576', 'art 13576', 'art: 6706', 'art: 10010', 'art: 6659', 'art: 8295', 'art: 8731', 'art: 9363', 'art: 9892', 'art: 10968', 'art: 11442', 'art: 13180', 'art: 6659', 'art: 8312', 'art: 14204', 'art: 6627', 'art: 13858', 'art: 459', 'art: 3253', 'art: 5629', 'art: 6359', 'art.\n2919597']

PRODUCTION OF TRANSGENIC AVIANS USING IMPROVED RETROVIRAL VECTORS - Patent application
Patent application title: PRODUCTION OF TRANSGENIC AVIANS USING IMPROVED RETROVIRAL VECTORS
Inventors: Alex J. Harvey (Athens, GA, US) Jeffrey C. Rapp (Athens, GA, US)
USPC Class: 435349
Class name: Chemistry: molecular biology and microbiology animal cell, per se (e.g., cell lines, etc.); composition thereof; process of propagating, maintaining or preserving an animal cell or composition thereof; process of isolating or separating an animal cell or composition thereof; process of preparing a composition containing an animal cell; culture media therefore avian cell, per se
Patent application number: 20120083033
A transgenic avian containing in its genome an exogenous nucleotide sequence which includes a promoter component and a vector with reduced promoter interference wherein the exogenous nucleotide sequence is integrated into the genome and the avian.
1. An isolated nucleic acid molecule at least 95% identical to a nucleic acid molecule comprising: a 5' LTR; an OV promoter fragment comprising DHS I & II; a 5' UTR-5' portion from Exon L; and a 3' UTR, wherein the 5' LTR is the nucleotide sequence set forth in SEQ ID NO: 29; the 5' UTR-5' portion from Exon L is the nucleotide sequence 6659 through 6705 of SEQ ID NO: 22; the 3' UTR comprises the nucleotide sequence 13576 through 13996 of SEQ ID NO: 22; the DHS II is the nucleotide sequence 5629 through 6009 of SEQ ID NO: 22; and, the DHS I is the nucleotide sequence 6359 through 6659 of SEQ ID NO: 22.
2. The nucleic acid of claim 1, wherein the nucleic acid further comprises an Intron A sequence comprising nucleotides 6706 through 8294 of SEQ ID NO: 22.
3. The nucleic acid of claim 1, wherein the nucleic acid further comprises a 5' UTR-3' portion from Exon 1, wherein the 5' UTR-3' portion from Exon 1 is the nucleic acid sequence 8295 through 8311 of SEQ ID NO: 22.
4. The nucleic acid of claim 1, wherein the 3' UTR comprises the nucleotide sequence 13576 through 14209 of SEQ ID NO: 22.
5. The nucleic acid of claim 4, wherein said 3' UTR comprises DHS A, wherein said 3' UTR comprising the DHS is the nucleic acid sequence 13576 to 15163 of SEQ ID NO: 22.
6. The nucleic acid of claim 5, wherein the nucleic acid comprises 5' UTR-3' portion from Exon 1, wherein said 5' UTR-3' portion from Exon 1 is the nucleic acid sequence 8295 through 8311 of SEQ ID NO: 22.
7. The nucleic acid of claim 1, wherein the nucleic acid comprises Intron A, 5' UTR-3' portion from Exon 1 and RRE, wherein the Intron A is the nucleotide sequence 6706 through 8294 of SEQ ID NO: 22; the 5' UTR-3' portion from Exon 1 is the nucleotide sequence 8295 through 8311 of SEQ ID NO: 22; and the RRE is the nucleotide sequence set forth in SEQ ID NO: 25.
8. The nucleic acid of claim 1, wherein the nucleic acid further comprises ALV CTE, wherein the nucleotide sequence of the ALV CTE is set forth in SEQ ID NO: 26.
9. The nucleic acid of claim 8, wherein the nucleic acid comprises Intron A and 5' UTR-3' portion from Exon 1, wherein the Intron A is the nucleotide sequence 6706 through 8294 of SEQ ID NO: 22; and the 5' UTR-3' portion from Exon 1 is the nucleotide sequence 8295 through 8311 of SEQ ID NO: 22.
10. The nucleic acid of claim 1, wherein the OV fragment further comprises the DHS III, wherein the DHS III is the nucleotide sequence 3253 through 3559 of SEQ ID NO: 22.
11. The nucleic acid of claim 1, wherein the OV fragment is an 1.4 kb fragment that essentially consists of nucleotides 5209 through 6659 of SEQ ID NO: 22.
12. An isolated vector comprising the nucleic acid molecule of claim 1.
13. The vector of claim 12, wherein the vector is a self-inactivating vector derived from a retrovirus.
14. The vector of claim 13, wherein the retrovirus is selected from the group consisting of an avian leukosis virus (ALV), a murine leukemia virus (MLV), a moloney murine leukemia virus (MMLV) and a lentivirus.
15. The vector of claim 14, wherein the retrovirus is an avian leukosis virus.
16. The vector of claim 15, wherein the self-inactivation vector is a SC negative vector having no selectable marker.
13. A host cell containing the vector of claim 1.
[0001] This application is Divisional of U.S. patent application Ser. No. 11/978,360 which claims the benefit of U.S. provisional application Nos. 60/930,491, filed May 16, 2007 and 60/994,203, filed Sep. 18, 2007 and is a continuation-in-part of U.S. patent application Ser. No. 11/699,257, filed Jan. 26, 2007, now U.S. Pat. No. 7,541,512, issued Jun. 2, 2009, and is also a continuation-in-part of U.S. patent application Ser. No. 11/799,253, filed May 1, 2007, now abandoned, which is a continuation-in-part of U.S. patent application Ser. No. 11/210,165, filed Aug. 23, 2005, now abandoned which claims the benefit of U.S. provisional application No. 60/640,203, filed Dec. 29, 2004. The disclosures of each of these US patent applications and provisional applications are incorporated in their entirety herein by reference.
[0002] The present invention relates generally to the use of promoters which function in cells of a transgenic avian (e.g., oviduct cells) such as a transgenic chicken and vectors which contain such promoters. More specifically, the invention relates to recombinant nucleic acids and expression vectors, transfected cells and transgenic animals, for example, transgenic avians such as transgenic chickens, that contain vectors with gene expression controlling regions operably linked to coding sequences.
[0003] The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and the phenomena of gene activation, expression and interaction. Transgenics technology has also been used to produce models for various diseases in humans and other animals and is among the most powerful tools available for the study of genetics, and the understanding of genetic mechanisms and function. From an economic perspective, the use of transgenic technology to convert animals into "protein factories" for the production of specific proteins or other substances of pharmaceutical interest (Gordon et al., 1987, Biotechnology 5: 1183-1187; Wilmut et al., 1990, Theriogenology 33: 113-123) offers significant advantages over more conventional methods of protein production by gene expression.
[0004] One system useful for expressing foreign proteins is the avian reproductive system. The production of an avian egg begins with formation of a large yolk in the ovary of the hen. The unfertilized oocyte or ovum is positioned on top of the yolk sac. After ovulation, the ovum passes into the infundibulum of the oviduct where it is fertilized, if sperm are present, and then moves into the magnum of the oviduct, which is lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin, into the lumen of the magnum where they are deposited onto the avian embryo and yolk. In the past exogenous protein production has been performed in the avian reproductive system specifically targeting the avian oviduct.
[0005] Advantages of targeting the avian oviduct for exogenous protein expression can include proper folding and post-translation modification of the target protein, the ease of product recovery, and a shorter developmental period of birds such as chickens compared to other animal species.
[0006] Directing expression of a heterologous gene product in the oviduct of a transgenic avian can be significantly advantageous over ubiquitous expression in the bird. That is, the consequences of ubiquitous expression of a bioactive gene product in a host animal may be undesirable. For example, in certain instances the ubiquitous presence of the recombinant protein may be harmful to the development of the avian which can kill the bird. Additionally, the bird's health may be negatively effected leading to reduced levels of protein production.
[0007] By weight, approximately 60% of an avian egg is composed of albumen which is composed of four major protein components; ovalbumin, ovomucoid, lysozyme and ovotransferrin with ovalbumin and ovomucoid being present in the greatest quantities.
[0008] The ovalbumin promoter, ovomucoid promoter and lysozyme promoter have been successfully employed for the production of heterologous (exogenous) protein in the oviduct of transgenic avians in the past. See, for example, U.S. Pat. No. 6,875,588, issued Apr. 5, 2005; U.S. Pat. No. 7,176,300, issued Feb. 13, 2007; U.S. Pat. No. 7,199,279, issued Apr. 3, 2007; and US patent publication No. 2006/0130170, published Jun. 15, 2006 (the disclosures of each of these three issued patents and one published patent application are incorporated in their entirety herein by reference) which discloses the production of exogenous protein in the avian oviduct facilitated by various avian promoters which are primarily or exclusively expressed in the oviduct. Though expression levels in avians using the promoters and fragments of the promoters disclosed in these issued patents and published application have been at useful levels, the yields have typically been well below 0.1 mg/ml of egg white.
[0009] What is needed is a system that will provide for high level expression of an exogenous coding sequence in the cells of a transgenic avian, in particular, in the oviduct cells (e.g., tubular gland cells) of a transgenic avian.
[0010] The present invention meets this need and more. After years of exogenous protein production in transgenic avian oviduct tissue with modest yield the inventors of the present invention have discovered that such production levels can be boosted by about 10 fold to about 100 fold and more by employing new compositions and methods as disclosed herein.
[0011] In one aspect, the invention is directed to transgenic avians (e.g., chicken, turkey, quail) containing in their genome an exogenous nucleotide sequence which includes a promoter component and a SIN vector. Typically, the promoter component is linked to a coding sequence exogenous to the avian, i.e., the coding sequence is not normally or naturally present in the avian. Typically, the exogenous nucleotide sequence is integrated into the genome of the avian. In one particularly useful embodiment, the promoter component functions or expresses primarily in the oviduct (e.g., tubular gland cells) of an avian. For example, the promoter component may be an oviduct specific promoter. For example, the promoter component may be one of an avian ovomucoid promoter component, an avian ovalbumin promoter component, an avian lysozyme promoter component and an avian ovoinhibitor promoter component (i.e., conalbumin promoter component).
[0012] SIN vectors have been shown by the inventors to be particularly useful for increasing the quantity of exogenous protein produced in the avian oviduct. This effect can be further enhanced when the SIN vector is also an SC negative vector (i.e., a vector not containing a selectable marker cassette with a functional promoter).
[0013] The invention also includes methods of making the transgenic avians of the invention and methods of producing an exogenous protein using transgenic avians of the invention. In one embodiment, the transgenic avian has a nucleotide sequence in its genome comprising a vector which is at least one of a SIN vector and an SC negative vector. Typically, the nucleotide sequence includes a promoter component linked to an exogenous coding sequence.
[0014] In one useful embodiment, the exogenous coding sequence is expressed in avian oviduct cells and is secreted from the oviduct cells. For example, the exogenous coding sequence may be expressed in tubular gland cells. In one embodiment, the exogenous protein is deposited in a hard shell egg laid by the transgenic avian. In one embodiment, the exogenous protein is a human protein. In one embodiment, the exogenous protein is a therapeutic protein, e.g., a cytokine.
[0015] In one embodiment, the transgenic avian contains an exogenous nucleotide sequence in its genome which has a SC negative vector and a promoter component linked to an exogenous coding sequence encoding an exogenous protein. In one embodiment, the SC negative vector is also a SIN vector.
[0016] In one aspect, avian leukosis virus vector (ALV), a murine leukemia virus (MLV) retroviral vector, moloney murine leukemia Virus (MMLV) and a lentiviral vector can be used in accordance with the invention.
[0017] The invention includes chimeric transgenic avians and fully transgenic germline avians which can be obtained from germline chimeras as is understood by a practitioner of skill in the art of poultry breeding.
[0018] The invention also includes gene expression controlling regions or promoters having a nucleotide sequence (i.e., DNA sequence) similar or identical to the following sequences numbered 1 to 8. In a particularly useful embodiment of the invention, the fragments are listed top to bottom in the 5' to 3' linear order in which they are present on a single DNA molecule. For example, the 3' end of the 3.5 kb OV fragment of sequence 1 would be covalently linked to the 5' end of the 5' UTR-5' portion and the 3' end of the 5' UTR-5' portion would be covalently linked to the 5' end of 5' UTR-3' portion. However, the invention is not limited to any particular order of the fragments and intervening nucleotide sequences may be present between the fragments.
1. 3.5 kb OV fragment (includes DHS I, II & III)
[0019] 5' UTR-5' portion (from Exon L)
[0020] 5' UTR-3' portion (from Exon 1);
2. 3.5 kb OV fragment (includes DHS I, II & III)
[0021] 5' UTR-5' portion (from Exon L)
[0022] Intron A
[0023] 5' UTR-3' portion (from Exon 1)
[0024] 3' UTR;
3. 3.5 kb OV fragment (includes DHS I, II & III)
[0025] 5' UTR-5' portion (from Exon L)
[0026] Intron A
[0027] 5' UTR-3' portion (from Exon 1);
4. 3.5 kb OV fragment (includes DHS I, II & III)
[0028] 5' UTR-5' portion (from Exon L)
[0029] 5' UTR-3' portion (from Exon 1)
[0030] 3' UTR;
5. 3.5 kb OV fragment (includes DHS I, II & III)
[0031] 5' UTR-5' portion (from Exon L)
[0032] Intron A
[0033] 5' UTR-3' portion (from Exon 1)
[0034] 3' UTR/DHS A (bp 13576 to 15163 of SEQ ID NO: 22)
6. 3.5 kb OV fragment (includes DHS I, II & III)
[0035] 5' UTR-5' portion (from Exon L)
[0036] 5' UTR-3' portion (from Exon 1)
[0037] 3' UTR/DHS A (bp 13576 to 15163 of SEQ ID NO: 22)
7. 3.5 kb OV fragment (includes DHS I, II & III)
[0038] 5' UTR-5' portion (from Exon L)
[0039] Intron A
[0040] 5' UTR-3' portion (from Exon 1)
[0041] partial 3' UTR
[0042] RRE (Rev response element) FIG. 9a
8. ALV CTE (FIG. 9b) inserted 5' of 3.5 kb OV fragment
[0043] 3.5 kb OV fragment (includes DHS I, II & III)
[0044] 5' UTR-5' portion (from Exon L)
[0045] Intron A
[0046] 5' UTR-3' portion (from Exon 1)
[0047] partial 3' UTR;
[0048] Coordinates of some of the elements for specific ovalbumin constructs disclosed herein (e.g., constructs 1 to 8 described above) are shown in the 16051 bp ovalbumin DNA segment of SEQ ID NO: 22 as follows:
[0049] 3.5 kb OV fragment (includes DHS I, II & III): Start: 3199 End: 6659 of FIG. 8 (SEQ ID NO: 22);
[0050] 1.4 kb OV fragment (includes DHS I & II): Start: 5209 End: 6659 of FIG. 8 (SEQ ID NO: 22);
[0051] 3.8 kb OV fragment: Start: 2863 End: 6659 of FIG. 8 (SEQ ID NO: 22);
[0052] 5.2 kb OV fragment: Start: 1463 End: 6659 of FIG. 8 (SEQ ID NO: 22);
[0053] 5' UTR-5' portion (from Exon L): Start: 6659 End: 6705 of FIG. 8 (SEQ ID NO: 22);
[0054] 5' UTR-3' portion (from Exon 1): Start: 8295 End: 8311 of FIG. 8 (SEQ ID NO: 22);
[0055] 3' UTR: Start: 13576 End: 14209 of FIG. 8 (SEQ ID NO: 22);
[0056] partial 3' UTR: Start 13576 End: 13996 of FIG. 8 (SEQ ID NO: 22);
[0057] Intron A: Start: 6706 End: 8294 of FIG. 8 (SEQ ID NO: 22);
[0058] Intron E: Start: 10010 End: 10968 of FIG. 8 (SEQ ID NO: 22);
[0059] Exon L: Start: 6659 End: 6705 of FIG. 8 (SEQ ID NO: 22);
[0060] Exon 1: Start: 8295 End: 8478 of FIG. 8 (SEQ ID NO: 22);
[0061] Exon 2: Start: 8731 End: 8781 of FIG. 8 (SEQ ID NO: 22);
[0062] Exon 3: Start: 9363 End: 9491 of FIG. 8 (SEQ ID NO: 22);
[0063] Exon 4: Start: 9892 End: 10009 of FIG. 8 (SEQ ID NO: 22);
[0064] Exon 5: Start: 10968 End: 11110 of FIG. 8 (SEQ ID NO: 22);
[0065] Exon 6: Start: 11442 End: 11597 of FIG. 8 (SEQ ID NO: 22);
[0066] Exon 7: Start: 13180 End: 13575 of FIG. 8 (SEQ ID NO: 22);
[0067] +1 SITE: Start: 6659 End: 6659 of FIG. 8 (SEQ ID NO: 22);
[0068] ATG: Start: 8312 End: 8312 of FIG. 8 (SEQ ID NO: 22);
[0069] Poly A: Start: 14204 End: 14209 of FIG. 8 (SEQ ID NO: 22);
[0070] TATA: Start: 6627 End: 6632 of FIG. 8 (SEQ ID NO: 22);
[0071] DHS A: Start: 13858 End: 15163 of FIG. 8 (SEQ ID NO: 22);
[0072] DHS IV: Start: 459 End: 859 of FIG. 8 (SEQ ID NO: 22);
[0073] DHS III: Start: 3253 End: 3559 of FIG. 8 (SEQ ID NO: 22);
[0074] DHS II: Start: 5629 End: 6009 of FIG. 8 (SEQ ID NO: 22); and
[0075] DHS I: Start: 6359 End: 6659 of FIG. 8 (SEQ ID NO: 22).
[0076] Promoter constructs are also contemplated that have a nucleotide sequence 80% identical and 85% identical and 90% identical and 91% identical and 92% identical and 93% identical and 94% identical and 95% identical and 96% identical and 97% identical and 98% identical and 99% identical to each of the promoter constructs disclosed herein, such as those described above (i.e., 1 to 8 above).
[0077] The invention also contemplates promoter constructs which correspond to promoter constructs 1 through 8 above in which the 3.5 kb OV fragment is replaced with the 3.8 kb OV fragment. The invention also contemplates promoter constructs which correspond to promoter constructs 1 through 8 in which the 3.5 kb OV fragment is replaced with the 5.2 kb OV fragment.
[0078] Promoter constructs are also contemplated for each of the above specified recombinant promoters (i.e., 1 to 8) in which DHS III is omitted from the construct.
[0079] Promoter constructs are contemplated corresponding to each of constructs 2, 3, 5, 7 and 8 above in which Intron A is replaced with Intron E which may lead to increased levels of exogenous protein production. Intron A and E have DNA sequences that induce alignment of histones in surrounding DNA regions. Such alignment can provide for transcriptional regulation of the OV gene. Without wishing to be bound to any particular theory or mechanism of operation, substitution of Intron E with Intron A may provide a preferential spacing of histones that result from use of Intron E (i.e., the periodicity for Intron A is 202 bp+/-5 bp, for Intron E is 196 bp+/-5 bp). For example, it is believed that the packaging of DNA by histones leads to topological alteration of DNA the manipulation of which can lead to preferential alignment of binding sites for proteins responsible for the transcription regulation (e.g., transcription factors) leading to an enhanced level of transcription.
[0080] Also included in the invention are vector constructs, and other constructs and nucleotide sequences disclosed herein, having a nucleotide sequence 80% identical and 85% identical and 90% identical and 91% identical and 92% identical and 93% identical and 94% identical and 95% identical and 96% identical and 97% identical and 98% identical and 99% identical to each vector construct and other constructs and nucleotide sequences disclosed herein.
[0081] Any useful combination of features described herein is included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art
[0082] Additional objects and aspects of the present invention will become more apparent upon review of the detailed description set forth below when taken in conjunction with the accompanying figures, which are briefly described as follows.
[0083] FIG. 1 shows a circular map of the pALV-SIN-4.2-Lys-IFNa-2B vector. The sequence of pALV-SIN-4.2-Lys-IFNa-2B is shown in SEQ ID NO: 1.
[0084] FIG. 2 is a bar graph illustrating expression levels of IFNa in the egg white of a transgenic quail. G0 quail was produced by injection of pALV-SIN-4.0-Lys-IFNa-2B retroviral vector transduction particles into Japanese quail embryos.
[0085] FIG. 3 shows a circular map of the pSIN-OV-3.5-I-CTLA4-inv vector. The nucleotide sequence of pSIN-OV-3.5-I-CTLA4-inv is shown in SEQ ID NO: 19.
[0086] FIG. 4 shows a circular map of the pSIN-3.9-OM-CTLA4-Fc vector. The nucleotide sequence of pSIN-3.9-OM-CTLA4-Fc is shown in SEQ ID NO: 20.
[0087] FIG. 5 shows a circular map of the pBS-OM-4.4 vector. The nucleotide sequence of pBS-OM-4.4 is shown in SEQ ID NO: 23.
[0088] FIG. 6 shows a circular map of the pAVIJCR-A137.91.1.2 vector. The nucleotide sequence of pAVIJCR-A137.91.1.2 is shown in SEQ ID NO: 24.
[0089] FIG. 7 shows a circular map of the pSIN-1.8-OM-IFNa-2B plasmid vector. The nucleotide sequence of pSIN-1.8-OM-IFNa-2B is shown in SEQ ID NO: 21.
[0090] FIG. 8a-e (SEQ ID NO: 22) shows a segment of a chicken ovalbumin gene.
[0091] FIG. 9a (SEQ ID NO: 25) shows the RRE (rev responsive element) sequence of a lenti virus. FIG. 9b (SEQ ID NO: 26) shows the ALV CTE (constitutive transport element) sequence.
[0092] FIG. 10a shows a diagram of the segment deleted from an exemplary retroviral LTR (ALV) to make a SIN vector. FIG. 10b (SEQ ID NO: 29) shows the sequence of the LTR shown in 10a. The underlined sequence is the deleted sequence.
[0093] The term "animal" is used herein to include all vertebrate animals, including avians and may include humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
[0094] The term "antibody" as used herein refers to polyclonal and monoclonal antibodies and functional fragments thereof. An antibody includes modified or derivatised antibody variants that retain the ability to specifically bind an epitope.
[0095] Antibodies are capable of selectively binding to a target antigen or epitope. Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized and other chimeric antibodies, single chain antibodies (scFvs), Fab fragments, F(ab')2 fragments and disulfide-linked Fvs (sdFv) fragments.
[0096] The term "avian" as used herein refers to any species, subspecies or strain of organism of the taxonomic class ayes, such as, but not limited to, such organisms as chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary. The term includes the various known strains of Gallus gallus, or chickens, (for example, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, Italian Partridge-colored), as well as strains of turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred in commercial quantities.
[0097] The phrases "based on" or "derived from" as in a retroviral vector being based on or derived from a particular retrovirus or based on a nucleotide sequence of a particular retrovirus mean that the genome of the retroviral vector contains a substantial portion of the nucleotide sequence of the genome of the particular retrovirus. The substantial portion may be a particular gene or nucleotide sequence such as the nucleotide sequence encoding the gag, pol and/or env proteins or other structural or functional nucleotide sequence of the virus genome such as sequences encoding the LTRs or may be substantially the complete retrovirus genome, for example, most (e.g., more than 60% or more than 70% or more than 80% or more than 90%) or all of the retrovirus genome, as will be apparent from the context in the specification as the knowledge of one skilled in the art. Examples of retroviral vectors that are based on or derived from a retrovirus are the NL retroviral vectors (e.g., NLB) which are based on the ALV retrovirus as disclosed in Cosset et al, Journal of Virology (1991) vol 65, p 3388-3394.
[0098] The terms "coding sequence" and "coding region" as used herein refer to nucleotide sequences and nucleic acid sequences, including both RNA and DNA, that encode genetic information for the synthesis of an RNA, a protein, or any portion of an RNA or protein. Nucleotide sequences that are not naturally part of a particular organism's genome are referred to as "foreign nucleotide sequences," "heterologous nucleotide sequences" or "exogenous nucleotide sequences". "Heterologous proteins" are proteins encoded by foreign, heterologous or exogenous nucleotide sequences and therefore are often not naturally expressed in the cell. A nucleotide sequence that has been isolated and then reintroduced into the same type (e.g., same species) of organism is not considered to be a naturally occurring part of a particular organism's genome and is therefore considered exogenous or heterologous.
[0099] The term "construct" as used herein refers to a linear or circular nucleotide sequence such as DNA that has been assembled from more than one segments of nucleotide sequence which have been isolated from a natural source or have been chemically synthesized, or combinations thereof.
[0100] The term "complementary" as used herein refers to two nucleic acid molecules that can form specific interactions with one another. In the specific interactions, an adenine base within one strand of a nucleic acid can form two hydrogen bonds with thymine within a second nucleic acid strand when the two nucleic acid strands are in opposing polarities. Also in the specific interactions, a guanine base within one strand of a nucleic acid can form three hydrogen bonds with cytosine within a second nucleic acid strand when the two nucleic acid strands are in opposing polarities. Complementary nucleic acids as referred to herein, may further comprise modified bases wherein a modified adenine may form hydrogen bonds with a thymine or modified thymine, and a modified cytosine may form hydrogen bonds with a guanine or a modified guanine
[0101] The term "cytokine" as used herein refers to any secreted amino acid sequence that affects the functions of cells and is a molecule that modulates interactions between cells in the immune, inflammatory or hematopoietic responses. A cytokine includes, but is not limited to, monokines and lymphokines regardless of which cells produce them. For instance, a monokine is generally referred to as being produced and secreted by a mononuclear cell, such as a macrophage and/or monocyte. Many other cells however also produce monokines, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epideral keratinocytes and B-lymphocytes. Lymphokines are generally referred to as being produced by lymphocyte cells. Examples of cytokines include, but are not limited to, Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor Necrosis Factor-alpha (TNF-alpha) and Tumor Necrosis Factor beta (TNF-beta).
[0102] The term "expressed" or "expression" as used herein refers to the transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene. The term "expressed" or "expression" as used herein can also refer to the translation of RNA to produce a protein or peptide.
[0103] The term "expression vector" as used herein refers to a nucleic acid vector that comprises a gene expression controlling region, such as a promoter or promoter component, operably linked to a nucleotide sequence coding at least one polypeptide.
[0104] The term "fragment" as used herein can refer to, for example, an at least about 10, 20, 50, 75, 100, 150, 200, 250, 300, 500, 1,000, 2,000, 5,000, 6,000, 8,000, 10,000, 20,000, 30,000, 40,000, 50,000 or 60,000 nucleotide long portion of a nucleic acid that has been constructed artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or enzymatically, for example, by PCR or any other polymerizing technique known in the art, or expressed in a host cell by recombinant nucleic acid technology known to one of skill in the art. The term "fragment" as used herein may also refer to, for example, an at least about 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, 1,000, 2,000, 5,000, 6,000, 8,000 or 10,000 amino acid portion of an amino acid sequence, which portion is cleaved from a naturally occurring amino acid sequence by proteolytic cleavage by at least one protease, or is a portion of the naturally occurring amino acid sequence synthesized by chemical methods or using recombinant DNA technology (e.g., expressed from a portion of the nucleotide sequence encoding the naturally occurring amino acid sequence) known to one of skill in the art. "Fragment" may also refer to a portion, for example, of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% about 90% about 95% or about 99% of a particular nucleotide sequence or amino acid sequence.
[0105] "Functional portion" or "functional fragment" are used interchangeably and as used herein mean a portion or fragment of a whole capable of performing, in whole or in part, a function of the whole. For example, a biologically functional portion of a molecule means a portion of the molecule that performs a biological function of the whole or intact molecule. For example, a functional portion of a gene expression controlling region is a fragment or portion of the specified gene expression controlling region that, in whole or in part, regulates or controls gene expression (e.g., facilitates either in whole or in part) in a biological system (e.g., a promoter). Functional portions may be of any useful size. For example, a functional fragment may range in size from about 20 bases in length to a length equal to the entire length of the specified sequence minus one nucleotide. In another example, a functional fragment may range in size from about 50 bases in length to a length equal to the entire length of the specified sequence minus one nucleotide. In another example, a functional fragment may range in size from about 50 bases in length to about 20 kb in length. In another example, a functional fragment may range in size from about 500 bases in length to about 20 kb in length. In another example, a functional fragment may range in size from about 1 kb in length to about 20 kb in length. In another example, a functional fragment may range in size from about 0.1 kb in length to about 10 kb in length. In another example, a functional fragment may range in size from about 20 bases kb in length to about 10 kb in length.
[0106] The term "gene expression controlling region" as used herein refers to nucleotide sequences that are associated with a coding sequence and which regulate, in whole or in part, expression of the coding sequence, for example, regulate, in whole or in part, the transcription of the coding sequence. Gene expression controlling regions may be isolated from a naturally occurring source or may be chemically synthesized and can be incorporated into a nucleic acid vector to enable regulated transcription in appropriate cells. The "gene expression controlling regions" may precede, but is not limited to preceding, the region of a nucleic acid sequence that is in the region 5' of the end of a coding sequence that may be transcribed into mRNA.
[0107] The terms "heterologous", "exogenous" and "foreign" are used interchangeably herein and in general refer to a biomolecule such as a nucleic acid or a protein that is not normally found in a certain organism or in a certain cell, tissue or other component contained in or produced by an organism. For example, a protein that is heterologous or exogenous to an egg is a protein that is not normally found in the egg. As used herein, the terms "heterologous", "exogenous" and "foreign" with reference to nucleic acids, such as DNA and RNA, are used interchangeably and refer to nucleic acid that does not occur naturally as part of a chromosome, a genome or cell in which it is present or which is found in a location(s) and/or in amounts that differ from the location(s) and/or amounts in which it occurs in nature. It can be nucleic acid that is not endogenous to the genome, chromosome or cell and has been exogenously introduced into the genome, chromosome or cell. Examples of heterologous DNA include, but are not limited to, a DNA comprising a gene expression control region and DNA that encodes a product or products, for example, RNA or protein product. Examples of heterologous DNA include, but are not limited to, gene expression controlling regions or promoters disclosed herein once isolated from the avian and as used thereafter, e.g., after re-introduction into an avian genome.
[0108] The term "isolated nucleic acid" as used herein covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic molecule but is not flanked by at least one of the sequences that flank that part of the molecule in the genome of the species in which it naturally occurs; (b) a nucleic acid which has been incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting vector or genomic DNA is not identical to naturally occurring DNA from which the nucleic acid was obtained; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis, or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein, and (e) a recombinant nucleotide sequence that is part of a hybrid sequence that is not naturally occurring. Isolated nucleic acid molecules of the present invention can include, for example, natural allelic variants as well as nucleic acid molecules modified by nucleotide deletions, insertions, inversions, or substitutions.
[0109] The term "nucleic acid" as used herein refers to any linear or sequential array of nucleotides and nucleosides, for example cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides and derivatives thereof. For ease of discussion, non-naturally occurring nucleic acids may be referred to herein as constructs. Nucleic acids can include bacterial plasmid vectors including expression, cloning, cosmid and transformation vectors such as, animal viral vectors such as, but not limited to, modified adenovirus, influenza virus, polio virus, pox virus, retroviruses such as avian leukosis virus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviral vector, and a lentivirus vector, and the like and fragments thereof. In addition, the nucleic acid can be an LTR of an avian leukosis virus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviral vector, or a lentivirus vector and fragments thereof. Nucleic acids can also include NL vectors such as NLB, NLD, NLA and fragments thereof and synthetic oligonucleotides such as chemically synthesized DNA or RNA. Nucleic acids can include modified or derivatised nucleotides and nucleosides such as, but not limited to, halogenated nucleotides such as, but not only, 5-bromouracil, and derivatised nucleotides such as biotin-labeled nucleotides.
[0110] The term "vector" and "nucleic acid vector" as used herein refers to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule that can be transfected or transformed into cells and replicate independently of, or within, the host cell genome. A circular double stranded vector can be linearized by treatment with an appropriate restriction enzyme based on the nucleotide sequence of the vector. A nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the desired pieces together.
[0111] The term "operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Gene expression controlling regions or promoters (e.g., promoter components) operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The controlling sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
[0112] The term "oviduct specific promoter" as used herein refers to promoters and promoter components which are functional, i.e., provide for transcription of a coding sequence, to a large extent, for example, primarily (i.e., more than 50% of the transcription product produced in the animal by a particular promoter type being produced in oviduct cells) or exclusively in oviduct cells of a bird. Examples of oviduct specific promoters include, ovalbumin promoter, ovomucoid promoter, ovoinhibitor promoter, lysozyme promoter and ovotransferrin promoter and functional portions of these promoters, e.g., promoter components.
[0113] The terms "percent sequence identity" and "percent identity" as used in, for example, "% identical" and "percent sequence homology" and "percent homology", as used in, for example, "% homology" and "percent sequence similarity" each refer to the degree of sequence matching between two nucleic acid sequences or two amino acid sequences as determined using the algorithm of Karlin & Attschul (1990) Proc. Natl. Acad. Sci. 87: 2264-2268, modified as in Karlin & Attschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Attschul et al. (1990) T. Mol. Biol. Q15: 403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a reference amino acid sequence. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Attschul et al. (1997) Nucl. Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) are used. Other algorithms, programs and default settings may also be suitable such as, but not only, the GCG-Sequence Analysis Package of the U.K. Human Genome Mapping Project Resource Centre that includes programs for nucleotide or amino acid sequence comparisons.
[0114] The terms "polynucleotide," "oligonucleotide", "nucleotide sequence" and "nucleic acid sequence" can be used interchangeably herein and include, but are not limited to, coding sequences, i.e., polynucleotide(s) or nucleic acid sequence(s) which are transcribed and translated into polypeptide in vitro or in vivo when placed under the control of appropriate regulatory or control sequences; controlling sequences, e.g., translational start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, enhancers, silencers, DNA sequences to which a transcription factor(s) binds and alters the activity of a gene's promoter either positively (induction) or negatively (repression) and the like. No limitation as to length or to synthetic origin are suggested by the terms described herein.
[0115] As used herein the terms "polypeptide" and "protein" refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds. The term "polypeptide" includes proteins, protein fragments, protein analogues, oligopeptides and the like. The term "polypeptides" includes polypeptides as defined above that are encoded by nucleic acids, produced through recombinant technology (e.g., isolated from a transgenic bird), or synthesized. The term "polypeptides" further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids covalently or noncovalently linked to labeling ligands.
[0116] The term "promoter" as used herein refers to a DNA sequence useful to initiate transcription initiation by an RNA polymerase in an avian cell. A "promoter component" is a DNA sequence that can, by itself or, in combination with other DNA sequences effect or facilitate transcription. Specific promoter components such as ovalbumin promoter components, ovomucoid promoter components and lysozyme promoter components and other promoters and promoter components disclosed and claimed herein do not describe a specific promoter sequence. Rather, they encompass any sequence or sequence fragment of the respective promoter that is useful to effect or facilitate transcription of a coding sequence. For example, an ovomucoid promoter component includes, without limitation, the about 1.8 kb, the about 3.9 kb and the about 10 kb ovomucoid promoters disclosed in U.S. publication No. 11/649,543, published May 17, 2007, which is incorporated in its entirety herein by reference. "Promoter components" can also encompass rearranged gene expression controlling regions which function to initiate RNA transcription and hybrid DNA molecules composed of naturally occurring DNA sequences and/or synthetic DNA sequences which function to initiate RNA transcription.
[0117] The terms "recombinant nucleic acid" and "recombinant DNA" as used herein refer to combinations of at least two nucleic acid sequences that are not naturally found in a eukaryotic or prokaryotic cell. The nucleic acid sequences may include, but are not limited to, nucleic acid vectors, gene expression regulatory elements, origins of replication, suitable gene sequences that when expressed confer antibiotic resistance, protein-encoding sequences and the like. The term "recombinant polypeptide" is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location, purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
[0118] As used herein, the term "regulatory sequences" includes promoters, enhancers, and other elements that may control gene expression.
[0119] An "SC negative vector" is a vector that does not contain a selectable or screenable cassette marker having a functional promoter. The promoter may be deleted in whole or in part or may be inactivated by a nucleotide sequence insertion. Screenable cassettes include, without limitation, DNA sequences for antibiotic resistance markers such as neomycin resistance and DNA sequences for other selectable markers such as GFP or lacZ.
[0120] A "SIN vector" is a self-inactivating vector. In particular, a SIN vector is a retroviral vector having an altered genome such that upon integration into genomic DNA of the target cell (e.g., avian embryo cells) the 5' LTR of the integrated retroviral vector will not function as a promoter. For example, a portion or all of the nucleotide sequence of the retroviral vector that results in the U3 region of the 5' LTR of the retroviral vector once integrated may be deleted or altered in order to reduce or eliminate promoter activity of the 5' LTR. In certain examples, deletion of the CAAT box and/or the TAATA box from U3 of the 5' LTR can result in a SIN vector, as is understood in the art.
[0121] A "SIN/SC negative vector" is a vector, i.e., a retroviral vector, that is both a SIN vector and a SC negative vector.
[0122] The term "sense strand" as used herein refers to a single stranded DNA molecule from a genomic DNA that may be transcribed into RNA and translated into the natural polypeptide product of the gene. The term "antisense strand" as used herein refers to the single strand DNA molecule of a genomic DNA that is complementary with the sense strand of the gene.
[0123] A "therapeutic protein" or "pharmaceutical protein" is a substance that, in whole or in part, makes up a drug. In particular, "therapeutic proteins" and "pharmaceutical proteins" include an amino acid sequence which in whole or in part makes up a drug.
[0124] The terms "transcription regulatory sequences" and "gene expression control regions" and "promoter components" as used herein refer to nucleotide sequences that are associated with a nucleic acid sequence and which regulate the transcriptional expression of a coding sequence. Exemplary transcription regulatory sequences include enhancer elements, hormone response elements, steroid response elements, negative regulatory elements, and the like. The "transcription regulatory sequences" may be isolated and incorporated into a vector nucleic acid to enable regulated transcription in appropriate cells of portions of the vector DNA. The "transcription regulatory sequence" may precede, but is not limited to, the region of a nucleic acid sequence that is in the region 5' of the end of a protein coding sequence that may be transcribed into mRNA. Transcriptional regulatory sequences may also be located within a protein coding region, in regions of a gene that are identified as "intron" regions, or may be in regions of nucleic acid sequence that are in the region of nucleic acid.
[0125] The terms "transformation" and "transfection" as used herein refer to the process of inserting a nucleic acid into a host. Many techniques are well known to those skilled in the art to facilitate transformation or transfection of a nucleic acid into a prokaryotic or eukaryotic organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt such as, but not only a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection, to render the host cell competent for the uptake of the nucleic acid molecules.
[0126] As used herein, a "transgenic animal" is any non-human animal, such as an avian species, including the chicken, in which one or more of the cells of the avian may contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques known in the art (see, for example, US patent publication No. 2007/0243165, published Oct. 18, 2007, the disclosure of which is incorporated in its entirety herein by reference) including those disclosed herein. The nucleic acid is introduced into an animal, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the typical transgenic animal, the transgene can cause cells to express a recombinant form of the target protein or polypeptide. The terms "chimeric animal" or "mosaic animal" are used herein to refer to animals in which a transgene is found, or in which the recombinant nucleotide sequence is expressed in some but not all cells of the animal. A germ-line chimeric animal contains a transgene in its germ cells and can give rise to a transgenic animal in which most or all cells of the offspring animal will contain the transgene.
[0127] As used herein, the term "transgene" means a nucleic acid sequence (encoding, for example, a human protein) that is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene according to the present invention can include a vector of the invention (e.g., SIN vector) which contains sequences useful for exogenous protein production in an avian (e.g., in an avian oviduct).
[0128] Techniques useful for isolating and characterizing the nucleic acids and proteins of the present invention are well known to those of skill in the art and standard molecular biology and biochemical manuals may be consulted to select suitable protocols for use without undue experimentation. See, for example, Sambrook et al, 1989, "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor, the content of which is herein incorporated by reference in its entirety.
[0129] Abbreviations used herein may include the following: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to an RNA; nt, nucleotide(s); kb, 1000 base pairs; μg, microgram; ml, milliliter; ng, nanogram.
[0130] SIN vectors designed and used in accordance with the invention can reduce or eliminate promoter interference of promoters of interest which are employed in transgenic avians. In a particularly useful embodiment, the promoters (i.e., promoter components) of interest preferentially express their gene product in oviduct cells or oviduct tissue, e.g., oviduct specific promoters. Examples of such promoters (e.g., promoter components) include but are not limited to, functional portions of the ovalbumin, lysozyme, conalbumin (i.e., ovotransferrin), ovomucoid, ovomucin, and/or ovoinhibitor gene expression controlling regions or promoter regions. In one embodiment, the promoter of interest is a combination or a fusion of one or more promoters or a fusion of a fragment of one or more promoters such as ovalbumin, lysozyme, conalbumin (i.e., ovotransferrin), ovomucoid, ovomucin, and/or ovoinhibitor promoters with another promoter or promoter fragment such as a viral promoter (e.g., an LTR promoter).
[0131] SIN vectors have been shown to be particularly useful with oviduct specific promoters. Without wishing to limit the invention to any particular theory or mechanism of operation it is believed that oviduct specific promoters can be particularly susceptible to influences of a retroviral LTR promoter. As a result, SIN vectors are particularly useful when employed in combination with avian oviduct specific promoters.
[0132] In one particularly useful embodiment, a SIN vector is produced in which an interfering promoter (e.g., an LTR promoter) that can at least partially inhibit transcription of a coding sequence operably linked to an oviduct specific promoter of the invention is inactivated, for example, by a deletion, insertion or transposition of all or part of the interfering promoter sequence. For example, in the vector pALV-SIN-4.2-Lys-IFNa-2B, shown in FIG. 1, the 3' RAV2 LTR has a deletion in the enhancer such that when the retroviral region integrates, the 5' LTR is inactivated, as is understood in the art. For a detailed diagrammatic of an LTR deletion, see FIG. 10.
[0133] In one useful embodiment of the invention, a SIN vector is employed that is also an SC negative vector to produce a SIN/SC negative vector. The combination of negative vector and SIN vector can result in a vector with a substantially reduced amount of promoter interference compared to a vector that is only a SIN vector or only a SC negative vector. For example, pALV-SIN-4.2-Lys-IFNa-2B as well as other SIN vectors disclosed in the Examples also lacks an antibiotic resistance marker making it both a SC negative vector and a SIN vector.
[0134] SIN vectors, SC negative vectors and SIN/SC negative vectors are contemplated for use in accordance with the invention in any useful avian such as chicken, quail and turkey to produce chimeras including germ-line chimeras and progeny birds produced using breeding techniques such as those known to practitioners of ordinary skill in the art. In addition, it is contemplated that an SC negative retroviral vector (which is a non-SIN vector) will also enhance or increase the quantity of exogenous protein produced in a transgenic avian relative to a transgenic avian produced with essentially the same retroviral vector that is not a SC negative vector.
[0135] Without wishing to limit the invention to any particular theory or mechanism of operation it is believed that the lack of a selectable marker cassette decreases the presence of promoter elements such as enhancers which would otherwise be in cis and in close proximity to the promoter employed for exogenous protein production in avian oviduct cells (e.g., oviduct specific promoters). This close proximity may allow for interference by the transcription regulating elements of the marker gene with the promoter of interest, i.e., the promoter employed for exogenous protein production. However, the invention contemplates that marker gene coding sequences, for example, and without limitation, neomycin resistance coding sequence and beta lactamase coding sequence, may be operably linked to a promoter (i.e., second promoter) which does not interfere with the promoter employed for exogenous protein production in avian oviduct cells (i.e., first promoter). For example, it is contemplated that if the marker promoter and the promoter of interest are the same or similar promoters, interference by the selectable cassette will be minimized or eliminated. For example, a second ovalbumin promoter operably linked to a marker gene coding sequence may not interfere with a first ovalbumin promoter employed for exogenous protein production in avian oviduct cells.
[0136] The invention contemplates the employment of any useful oviduct specific promoter, and oviduct specific promoter fragments, in vectors of the invention for exogenous protein expression in avians. For example, promoters and useful (e.g., functional) fragments of promoters (e.g., promoter components) disclosed in US patent publication No. 2005/0176047, filed Jan. 31, 2005, the disclosure of which is incorporated in its entirety herein by reference, and US patent publication No. 2007/0124829, filed Jan. 26, 2007, the disclosure of which is incorporated in its entirety herein by reference, and US patent publication No. 2006/0130170, filed Dec. 11, 2003, the disclosure of which is incorporated in its entirety herein by reference, are contemplated for use in conjunction with SIN vectors and SC negative vectors and SIN/SC negative vectors in accordance with the invention.
[0137] The invention also contemplates other promoters and transcriptionally functional portions thereof (e.g., promoter components) for use as promoters of interest in accordance with the invention such as a cytomegalovirus (CMV) promoter, a rous-sarcoma virus (RSV) promoter, a β-actin promoter (e.g., a chicken β-actin promoter) a murine leukemia virus (MLV) promoter, and a mouse mammary tumor virus (MMTV) promoter.
[0138] The invention also includes various ovalbumin promoter components which are contemplated for use in producing exogenous proteins in transgenic avians. Each of the promoters disclosed herein are contemplated for use in vectors in accordance with the invention.
[0139] Examples of vectors of the invention which contain recombinant ovalbumin DNA are shown below. The fragments are listed top to bottom in the 5' to 3' linear order in which they are present on a single DNA molecule. For example, the 3' end of the 3.5 kb OV fragment of sequence 1 would be covalently linked to the 5' end of the 5' UTR-5' portion and the 3' end of the 5' UTR-5' portion would be covalently linked to the 5' end of 5' UTR-3' portion.
1. pSIN-OV-3.5-CSI
[0140] 3.5 kb OV fragment (includes DHS I, II & III)
[0141] 5' UTR-5' portion (from Exon L)
[0142] 5' UTR-3' portion (from Exon 1)
2. pSIN-OV-3.5-Int-CSI-inv
[0143] 3.5 kb OV fragment (includes DHS I, II & III)
[0144] 5' UTR-5' portion (from Exon L)
[0145] Intron A
[0146] 5' UTR-3' portion (from Exon 1)
[0147] 3' UTR
3. pSIN-OV-3.5-Int-CSI
[0148] 3.5 kb OV fragment (includes DHS I, II & III)
[0149] 5' UTR-5' portion (from Exon L)
[0150] Intron A
[0151] 5' UTR-3' portion (from Exon 1)
4. pSIN-OV-3.5-CSI-UTR-inv
[0152] 3.5 kb OV fragment (includes DHS I, II & III)
[0153] 5' UTR-5' portion (from Exon L)
[0154] 5' UTR-3' portion (from Exon 1)
[0155] 3' UTR
5. pSIN-OV-3.5-Int-CSI-LUTR-inv
[0156] 3.5 kb OV fragment (includes DHS I, II & III)
[0157] 5' UTR-5' portion (from Exon L)
[0158] Intron A
[0159] 5' UTR-3' portion (from Exon 1)
[0160] 3' UTR/DHS A (bp 13576 to 15163 of FIG. 8);
6. pSIN-OV-3.5-CSI-LUTR-inv
[0161] 3.5 kb OV fragment (includes DHS I, II & III)
[0162] 5' UTR-5' portion (from Exon L)
[0163] 5' UTR-3' portion (from Exon 1)
[0164] 3' UTR/DHS A (bp 13576 to 15163 of FIG. 8);
7. pSIN-OV-3.5-Int-CSI-RRE
[0165] 3.5 kb OV fragment (includes DHS I, II & III)
[0166] 5' UTR-5' portion (from Exon L)
[0167] Intron A
[0168] 5' UTR-3' portion (from Exon 1)
[0169] partial 3' UTR
[0170] RRE (Rev response element) FIG. 9a
[0171] Construct 7 includes RRE to allow transport of the unspliced RNA genome to the cytoplasm and thus may enhance packaging of intact retroviral RNA. RRE is only active in presence of the Rev protein. Rev activity is provided in the form of DNA encoding the Rev, RNA encoding the Rev, and/or the Rev protein, which is well known in the art and commercially available (e.g., Invitrogen, Inc.), during the transient transfection of retroviral components. Thus the intron will be present in the transgene contained in the genome of the transgenic bird produced by the virus particles (the rev protein is not present in the cells of the transgenic bird). As a result the RNA should be spliced in the oviduct cells of a laying hen resulting in an enhanced level of protein expression compared to a same transgenic bird having the same transgene without the intron.
8. pSIN-CTE-OV-3.5-Int-CSI
[0172] ALV CTE (FIG. 9b) inserted 5' of 3.5 kb OV fragment
[0173] 3.5 kb OV fragment (includes DHS I, II & III)
[0174] 5' UTR-5' portion (from Exon L)
[0175] Intron A
[0176] 5' UTR-3' portion (from Exon 1)
[0177] partial 3' UTR
[0178] Coordinates for some of the elements for the above eight vectors are described elsewhere in the application. For example, coordinates of sequences from the ovalbumin nucleotide sequence are described in the Summary section above. CSI means a coding sequence of interest, i.e., nucleotide sequence encoding the protein desired to be expressed in a transgenic avian oviduct.
[0179] SIN vectors, SIN/SC negative vectors and SC negative vectors for use in accordance with the invention include vectors such as Avian Leukemia/Leukosis Viruses (ALV), for example, and without limitation, RAV-0, RAV-1, RAV-2; Avian Sarcoma Viruses (ASV); Avian Sarcoma/Acute Leukemia Viruses (ASLV) including, without limitation, Rous Sarcoma Virus (RSV); Fujinami Sarcoma Viruses (FSV); Avian Myeloblastosis Viruses (AMV); Avian Erythroblastosis Viruses (AEV); Avian Myelocytomatosis Viruses (MCV), for example, and without limitation, MC29; Reticuloendotheliosis Viruses (REV), for example, and without limitation, Spleen Necrosis Virus (SNV). The invention also contemplates other useful retroviral vector, including, without limitation, retroviral vectors based upon Murine Leukemia Viruses (MLV); Molony Murine Sarcoma Viruses (MMSV); Moloney Murine Leukemia Viruses (MMLV); and lentiviruses (e.g., human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV) which are altered to be SIN vectors, SIN/SC negative vectors or SC negative vectors as is understood by a practitioner of ordinary skill in the art.
[0180] In one very specific embodiment, a portion of the 5' LTR of a modified ALV vector disclosed in Cosset et al, J of Virology (1991) vol 65, no. 6, p 3388-3394, the disclosure of which is incorporated in its entirety herein by reference, is deleted to produce a SIN vector. In particular, nucleotides 1 to 173 were deleted from the ALV based vector LTR sequence shown in SEQ ID NO: 29. Specific deletions from 5' LTR sequences useful to produce SIN vectors from other vectors which can be used in avian transgenesis can be determined by a practitioner of ordinary skill in the art.
[0181] In one particularly useful embodiment, the invention is drawn to the production of therapeutic proteins which may be produced in the oviduct of a transgenic avian, such as a chicken, in accordance with the invention. Exemplary proteins for production in accordance with the invention include, without limitation, erythropoietin, GM-CSF, interferon β, fusion protein, CTLA4-Fc fusion protein, growth hormones, cytokines, structural proteins, interferon, lysozyme, β-casein, albumin, α-1 antitrypsin, antithrombin III, collagen, factors VIII, IX, X (and the like), fibrinogen, lactoferrin, protein C, tissue-type plasminogen activator (tPA), somatotropin, and chymotrypsin, immunoglobulins, antibodies, immunotoxins, factor VIII, b-domain deleted factor VIII, factor VIIa, factor IX, anticoagulants, hirudin, alteplase, tpa, reteplase, tpa-3 of 5 domains deleted, insulin, insulin lispro, insulin aspart, insulin glargine, long-acting insulin analogs, glucagons, tsh, follitropin-beta, fsh, pdgh, ifn-beta, ifn-alpha 1, ifn-alpha 2, ifn-beta, ifn-beta 1b, ifn-beta 1a, ifn-gamma, ifn-gamma 1b, il-2, il-11, hbsag, ospa, dornase-alpha dnase, beta glucocerebrosidase, tnf-alpha, il-2-diptheria toxin fusion protein, tnfr-lgg fragment fusion protein laronidase, dnaases, alefacept, tositumomab, murine mab, alemtuzumab, rasburicase, agalsidase beta, teriparatide, parathyroid hormone derivatives, adalimumab (lgg1), anakinra, nesiritide, human b-type natriuretic peptide (hbnp), colony stimulating factors, pegvisomant, human growth hormone receptor antagonist, recombinant activated protein c, omalizumab, immunoglobulin e (lge) blocker, lbritumomab tiuxetan, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, etanercept, antidiuretic hormones, prolactin and thyroid stimulating hormone, an immunoglobulin polypeptide, immunoglobulin polypeptide D region, immunoglobulin polypeptide J region, immunoglobulin polypeptide C region, immunoglobulin light chain, immunoglobulin heavy chain, an immunoglobulin heavy chain variable region, an immunoglobulin light chain variable region and a linker peptide. Production of each of these, and other, proteins is contemplated using methods, vectors and promoters of the invention.
[0182] The present invention is further illustrated by the following examples, which are provided by way of illustration and should not be construed as limiting. The contents of all references, published patents and patents cited throughout the present application are hereby incorporated by reference in their entireties.
Production of pALV-SIN-4.2-Lys-IFNa-2B
[0183] The vector pALV-SIN-4.2-Lys-IFNa-2B (shown in FIG. 1) was constructed and is shown in FIG. 1. The sequence of pALV-SIN-4.2-Lys-IFNa-2B is shown in SEQ ID NO: 1. The 4.2 Kb lysozyme promoter spans from nucleotides 4810 to 9008 of SEQ ID NO: 1. The lysozyme signal peptide coding sequence spans from nucleotides 9037 to 9090 of SEQ ID NO: 1. The interferon alpha 2b coding sequence spans from nucleotides 9091 to 9585 of SEQ ID NO: 1. Other components of the sequence include LTRs spanning from nucleotides 4000 to 4345 and from nucleotides 725 to 897 of SEQ ID NO: 1.
[0184] pALV-SIN-4.2-Lys-IFNa-2B can be constructed by a variety of methods which are apparent to a practitioner of skill in the art. However, the method believed to be the most useful for making the vector is as follows: A 3427 bp region of pNLB-CMV-IFN-alpha2B (disclosed in U.S. patent application Ser. No. 11/167,052, filed Jun. 24, 2005, the disclosure of which is incorporated in its entirety herein by reference) is PCR amplified using primers ATGCGCGCATTGGTAATTGATCGGCTGG (Primer ALV-SIN-1, SEQ ID NO: 2) and ATATGCGGCCGCGGTACCGCCCGGGCATCGATATCAAGCTTACGGTTCACT A AACGAGCTCTGCTTATATAGACCTCCCA (Primer ALV-SIN-2, SEQ ID NO: 3). The product is digested with BssHII and Not I resulting in a 3428 bp fragment which can be isolated by gel purification. A 1436 bp region of pNLB-CMV-IFN-alpha2B is PCR amplified with primers ATATGCGGCCGCGTCGACGGCCGGCCAGATCTGCTGAGCCGGTCGCTACCA TTACCAGT (Primer ALV-SIN-3, SEQ ID NO: 4) and ATACGCGTATTCCCTAACGATCACGTCG (Primer ALV-SIN-4, SEQ ID NO: 5). The resulting product is digested with Not I and Mlu I yielding a 1438 bp fragment which is isolated by gel purification. A Bluescript II SK vector containing a BssHII stuffer fragment is digested with BssHII resulting in a linearized Bluescript vector of 2788 bp which is gel purified and then ligated to the 3428 bp and 1438 bp PCR products to yield JCR.A108.49.5.24.
[0185] JCR.A108.49.5.24 is digested with Hind III and the resulting 6823 bp fragment is circularized by ligation to yield JCR.A108.76.1.1.
[0186] A 1175 bp region of JCR.A108.76.1.1 is PCR amplified with primers CTGAAGTGTAAGGAATGTAAG (Primer ALV-SIN-5, SEQ ID NO: 6) and GCGCGTCTCATCCCCCTCCCTATGCAAAAG (Primer ALV-SIN-6, SEQ ID NO: 7) and the resulting fragment is digested with Blp I and Esp3I producing a 1030 bp fragment which is isolated by gel purification. A 660 bp region of JCR.A108.76.1.1 is PCR amplified with primers GGGCGTCTCAGGGACGGATTGGACGAACCACTGAATT (Primer ALV-SIN-7, SEQ ID NO: 8) and TTAGTGCTTTACGGCACCTC (Primer ALV-SIN-8, SEQ ID NO: 9) and digested with Esp3I and DraIII resulting in a 596 bp fragment which is isolated by gel purification. JCR.A108.76.1.1 is digested with DraIII and Blp I and the 5024 bp linear vector is ligated to the 1030 and 596 bp PCR fragments to produce pALV-SIN.
[0187] pALV-SIN is digested with BamHI and the 4795 bp linear vector is isolated by gel purification. A 4815 bp region of JCR.115.93.1.2 (disclosed in US patent publication No. 2007/0124829, published May 31, 2007) is PCR amplified with primers GACGGATCCGATACCGTCCCTATTTTTGTGTTTGCTTC (Primer ALV-SIN-9, SEQ ID NO: 10) and TAACGGATCCTAGACTTTTTACTCCTTAGA (Primer ALV-SIN-10, SEQ ID NO: 11) and is digested with BamHI. The resulting 4802 fragment is ligated to the 4795 bp linear pALV-SIN to produce pALV-SIN-4.0-Lys-IFNa-2B.
Production of Transgenic Quail Using pALV-SIN-4.2-Lys-IFNa-2B
[0188] Transduction particles of the vector pALV-SIN-4.2-Lys-IFNa-2B were produced in fibroblast cells as disclosed in US patent publication No. 2007/0077650, published Apr. 5, 2007, entitled: Rapid Production of High Titer Virus, the disclosure of which is incorporated in its entirety herein by reference.
[0189] Fertilized Japanese quail eggs were windowed essentially according to the Speksnijder procedure disclosed in U.S. Pat. No. 5,897,998, the disclosure of which is incorporated in its entirety herein by reference. Eighty eggs were injected in the subgerminal cavity with about 7 microliters (approximately 7×104 viral particles total) of pALV-SIN-4.2-Lys-IFNa-2B transducing particles per egg. Since no selectable marker is used in pALV-SIN-4.2-Lys-IFNa-2B, the concentration of viral particles is estimated based upon past results for viral particle production where a selectable cassette or marker was used in the vector which allowed for particle quantification. Sixteen chicks hatched about 18 days after injection and human IFN levels were measured by IFN ELISA from serum samples collected from chicks 12 weeks after hatch. None were positive for the IFN protein in the serum.
[0190] In order to identify G0 quail which contained the interferon alpha 2 coding sequence containing transgene in their genome, DNA was extracted from blood of the birds and the DNA samples were subjected to Taqman® analysis on a 7700 Sequence Detector (Perkin Elmer).
[0191] Eggs from eight G0 quail were tested for the presence of the IFN protein in the egg white by ELISA. Quail No. 4 was found to have significant levels of IFN in egg white from her eggs. FIG. 2 shows a bar graph illustrating expression levels of IFN in the egg white of Quail No. 4. Quail No. 4 expressed IFN-alpha-2 at 0.45 μg/ml of egg white, which is a high level of expression for a G0 avian. There was no interferon alpha 2 detected in the blood of Quail No. 4 which is particularly significant. For example, in certain instances the recombinant protein may be harmful to the development or health of the avian when present in the blood which can kill the bird or can lead to reduced levels of protein production.
Production of Transgenic Quail Using pALV-SIN-6.5-Lys-IFNa-2B
[0192] The 4.2 kb lysozyme promoter of vector pALV-SIN-4.2-Lys-IFNa-2B is removed and replaced with a 6.5 kb lysozyme promoter corresponding to about nucleotides 5363 to 11863 of SEQ ID NO: 12, using standard methodologies known to practitioners of skill in the art, resulting in pALV-SIN-6.5-Lys-IFNa-2B. Transduction particles of the new vector pALV-SIN-6.5-Lys-IFNa-2B are produced as disclosed in US patent publication No. 2007/0077650, published Apr. 5, 2007.
[0193] Fertilized chicken eggs or Japanese quail eggs are windowed and about 7×104 pALV-SIN-6.5-Lys-IFNa-2B transducing particles are injected into the subgerminal cavity of each egg. Eggs hatch 21 or 18 days after injection and chimeric birds are identified that contain the active transgene in their genome, as described in Example 2. Fully transgenic G1 birds which contain the transgene in their genome are produced from chimeras using methods known in the art, i.e., crossing male chimeras with non-transgenic females.
Production of Vector pSIN-OV-3.5-I-CTLA4-Fc-Inv
[0194] This vector includes the ovalbumin Dnase hypersensitive sites (DHS) I, II and III, the first exon (exon L), the first intron and the CTLA4-Fc fusion protein coding sequence inserted in frame with the ATG of second exon (exon 1) and with the 3' untranslated region (UTR). The expression cassette is inserted in the inverse orientation into an avian leukosis virus (ALV) vector, which was made self-inactivating (SIN) by deletion of nucleotides 1 to 173 of the ALV LTR sequence shown in SEQ ID NO: 29.
[0195] The vector was constructed as follows: pNLB-3.9-OM-CTLA4-Fc, disclosed in Example 20 of US patent publication No. 2007/0113299, published May 17, 2007, the disclosure of which is incorporated in its entirety herein by reference, was cut with Nae I and Not I. The Not I site was filled in by Klenow reaction. The resulting 8125 bp fragment was gel purified, religated, producing pOM-3.9-CTLA4-dSacI.
[0196] pOM-3.9-CTLA4-dSacl was cut with EcoRI and Kpn I and the 8115 bp fragment gel purified. The 3' UTR of the chicken ovalbumin gene was PCRed from BAC 26, disclosed in US patent publication No. 2006/0130170, published Jun. 15, 2006, with the primers 5'-GCGGAATTCAAAGAAGAAAGCTGAAAAAC-3' (SEQ ID NO: 13) and 5'-GCGGGTACCTTCAAATACTACAAGTGAAA-3' (SEQ ID NO: 14). The 3' UTR PCR was cut with Eco RI and Kpn I and the 684 bp fragment gel purified. The 8115 bp fragment of pOM-3.9-CTLA4-dSacI was ligated to the 684 bp fragment of 3' UTR PCR, producing pOM-3.9-CTLA4-OV3'UTR.
[0197] The 3.5 kb OV promoter region, exon L, first intron and the UTR of exon 1 was PCR amplified with BAC26 as a template and with primers 5'-GGCCTCGAGTCAAGTTCTGAGTAGGTTTTAGTG-3' (SEQ ID NO: 15) and 5'-GCGCGTCTCTGTCTAGAGCAAACAGCAGAACAGTGAAAATG-3' (SEQ ID NO: 16). The PCR product was cut with Xho I and Esp3I and the 5094 bp product was gel purified.
[0198] A 5' portion of the CTLA4-Fc gene was PCR amplified using pOM-3.9-CTLA4 as a template and primers 5'-GCGCGTCTCAAGACAACTCAGAGTTCACCATGGGTGTACTGCTCACACAG-3' (SEQ ID NO: 17) and 5'-GGCCCGGGAGTTTTGTCAGAAGATTTGGG-3' (SEQ ID NO: 18). The PCR product was cut with Esp3I and Sad and the 384 bp product gel purified.
[0199] pOM-3.9-CTLA4-OV3'UTR was cut with Sac I and Xho I, the 4473 bp product gel purified and ligated to the 5094 bp OV PCR fragment and 384 bp CTLA4-Fc fragment, producing pOV-3.5-I-CTLA4.
[0200] pALV-SIN, disclosed, for example, in Example 10 of parent case US patent publication No. 2007/0124829, published May 31, 2007, was cut with Mfe I and Xho I, filled in with Klenow and the 4911 bp fragment gel purified.
[0201] pOV-3.5-I-CTLA4 was cut with XhoI and BamHI, filled in with Klenow and the 6957 bp fragment gel purified. This fragment was ligated into the 4911 bp fragment of pAVI-SIN such that the CTLA4-Fc gene and flanking expression elements are in the opposite orientation of the ALV long terminal repeats, producing pSIN-OV-3.5-I-CTLA4-inv. See FIG. 3 and SEQ ID NO: 19. Such opposite orientation may be preferred if the coding sequence of interest (i.e., CSI) in the transgene contains one or more introns or splice sites.
Production of Transgenic Quail Using SIN-OV-3.5-I-CTLA4-inv
[0202] Retroviral particles containing the pSIN-OV-3.5-I-CTLA4-inv vector (FIG. 3) and pseudotyped with the VSV envelope protein were produced as described in US patent publication No. 2007/0077650, published Apr. 5, 2007. Virus particles were harvested at 48 hours post-transfection, concentrated and on the same day, approximately 4 microliters of the virus suspension containing about 1×105 particles was injected into the subgerminal cavity of stage X quail eggs. Eggs were resealed and hatched.
[0203] ALV has a CTE element in the 3' end of its genome that allows transport of unspliced retroviral RNA to the cytoplasm. In pSIN-OV-3.5-I-CTLA4-inv, due to the inverse orientation of the OV promoter relative to the LTRs, the CTE is upstream of the OV promoter such that the CTE element is only in RNAs derived from the 5' LTR promoter and not in RNAs transcribed by the OV promoter. Therefore, any RNA transcribed by the OV promoter should be spliced prior to being transported into the cytoplasm.
[0204] Egg whites from chimeric quail were assayed using an ELISA for CTLA4-Fc. One quail was found to have CTLA4-Fc in her egg white at approximately 16 μg/ml. The transgenesis level in these quail is estimated at about 5% or less. Thus the level in a G1 should be substantially greater. It is expected that similar levels would be seen in a chicken and other avians, as the quail and chicken ovalbumin genes, as well as ovalbumin genes of other avians, are very similar.
Construction of pSIN-3.9-OM-CTLA4-Fc
[0205] The 4907 bp Mfe I/Xho I fragment of pALV-SIN (disclosed, for example, in US patent publication No. 2007/0124829, published May 31, 2007) was ligated to the 5115 XhoI/EcoRI fragment of pOM-3.9-CTLA4 (shown in FIG. 15 of US patent publication No. 2007/0113299, published May 17, 2007), producing pSIN-3.9-OM-CTLA4-Fc shown in FIG. 4 and SEQ ID NO: 20.
Production of Transgenic Chickens Using pSIN-3.9-OM-CTLA4-Fc
[0206] Retroviral particles pseudotyped with the VSV envelope protein and containing the pSIN-3.9-OM-CTLA4-Fc (FIG. 4) vector were produced as described in US patent publication No. 2007/0077650, published Apr. 5, 2007. Virus was harvested at 48 hours post-transfection, concentrated and on the same day approximately 7 microliters injected into the subgerminal cavity of stage X eggs. Eggs were resealed and incubated until hatch.
[0207] Egg white from hens was assayed using an ELISA for CTLA4-Fc. One hen was found to have CTLA4-Fc in her egg white at approximately 0.37 μg/ml. The transgenesis level in these hens is estimated at 5% or less. Thus the levels in a G1 should be substantially greater.
[0208] Any useful coding sequence may be inserted in place of the CTLA4-Fc coding sequence for production of the corresponding product.
Construction of pSIN-1.8-OM-IFNa-2B
[0209] The 1051 bp Nco I-Nco I fragment from pBS-OM-4.4 (FIG. 5 SEQ ID NO: 23) was inserted into the Nco I site of pAVIJCR-A137.91.1.2 (FIG. 6 SEQ ID NO: 24), thereby inserting the 1 kb ovomucoid promoter in front of an IFN coding sequence and SV40 polyadenylation signal and producing plkb-OM-IFNMM. A 1816 bp Cla I-Sac I fragment of plkb-OM-IFNMM was inserted into the 6245 bp Cla I-Sac I fragment of pBS-OM-4.4, thereby fusing the 4.4 kb ovomucoid fragment with the IFN coding sequence and producing p4.4OM-IFNMM. The 8511 bp BamH I-Sal I fragment of pBS-OMUP-10 was ligated to the 5148 bp BamH I-Sal I fragment of p4.4OM-IFN, thereby placing the 10 kb ovomucoid promoter in front of the IFN coding sequence, producing p10-OM-IFN.
[0210] Region 2487-4889 of p10.0-OM-IFN was PCR amplified with primers 5'-GGCGTCGACGGATCCGTTAACCCTAGAACTAGTGGATCTCTGCCCTTGTGC TGAC-3' (SEQ ID NO: 27) and 5'-GGCCTCGAGCCTAGACTTTTTACTCCTTAGA-3' (SEQ ID NO: 28). The PCR product was digested with Sal I and Xho I and the 2435 bp isolated. pALV-SIN (disclosed, for example, in US patent publication No. 2007/0124829, published May 31, 2007) was digested with Xho I and the 4915 bp fragment isolated and ligated to the 2435 bp fragment, producing pSIN-1.8-OM-IFNa-2B, shown in FIG. 7 and SEQ ID NO: 21.
Production of Transgenic Chickens Using pSIN-1.8-OM-IFNa-2B
[0211] Retroviral particles having the pSIN-1.8-OM-IFNa-2B transgene and pseudotyped with the VSV envelope protein were produced as described in US patent publication No. 2007/0077650, published Apr. 5, 2007. Virus was harvested at 48 hours post-transfection, concentrated and, on the same day, approximately 7 microliters injected into the subgerminal cavity of stage X eggs. Eggs were resealed and incubated until hatch.
[0212] Egg whites from hens were assayed using an ELISA for IFNa-2B. Hens were found to have IFNa-2B in egg white at levels that ranged from 1.5 to 865.0 ng/ml with IFNa-2B levels at least about 600 fold lower in the serum. The transgenesis level in these hens is estimated at 5% or less.
[0213] Five G0 sperm positive roosters were bred to non-transgenic hens. Of 1251 offspring, 30 carried the pSIN-1.8-OM-IFNa-2B transgene. Six of the 30 hens expressed human IFN-α-2B at 34.1 to 165.6 μg/ml of egg white. Each of the six hens had a single copy of the transgene. Serum levels of human IFN-a-2B were 0.3 to 9.2 ng/ml which, on average, was 30,000 fold lower than egg white levels.
Production of Transgenic Chickens Using Lentivirus Vectors and Moloney Murine Leukemia Virus
[0214] The invention specifically contemplates the employment of other retroviral vectors that are useful in avian transgenesis to be used in accordance with the present invention. Such vectors can be employed to produce transgenic avians, for example, in the same way as ALV-SIN vectors have been used in Examples 1 to 9 above. For example, Moloney Murine Leukemia Virus (MMLV) and Lentiviral Vectors can be used in accordance with the invention, each, for example, by deleting one or more of the CAAT box; the TAATA box; and enhancer contained in the U3 region of the upstream LTR of each virus to produce a SIN vector. Alternatively, or in addition (i.e., in conjunction with a SIN vector) no transcriptionally active markers or selectable cassettes are included in each of the retroviral vectors.
[0215] Although preferred embodiments of the invention have been described using specific terms, devices, and methods, such description is for illustrative purposes only. The words used are words of description rather than of limitation. It is to be understood that changes and variations may be made by those of ordinary skill in the art without departing from the spirit or the scope of the present invention, which is set forth in the following claims. In addition, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part.
2919597DNAArtificial SequencepALV-SIN-4.2-Lys-IFNa-2B Vector 1gatcccccgt gctgcagaac cgagcggcta ttgacttctt gctcctagct cacggccatg 60gctgtgagga cattgcggga atgtgttgtt tcaatctgag tgatcacagt gagtctatac 120agaagaagtt ccagctaatg aaggaacatg tcaataagat cggcgtgaac aacgacccaa 180tcggaagttg gctgcgagga ttattcggag gaataggaga atgggccgta cacttgctga 240aaggactgct tttggggctt gtagttatct tgttgctagt agtatgcttg ccttgccttt 300tgcaatgtgt atctagtagt attcgaaaga tgattgataa ttcactcggc tatcgcgagg 360aatataaaaa aattacagga ggcttataag cagcccgaaa gaagagcgta ggcgagttct 420tgtattccgt gtgatagctg gttggattgg taattgatcg gctggcacgc ggaatatagg 480aggtcgctga atagtaaact tgtagacttg gctacagcat agagtatctt ctgtagctct 540gatgactgct aggaaataat gctacggata atgtggggag ggcaaggctt gcgaatcggg 600ttgtaacggg caaggcttga ctgaggggac aatagcatgt ttaggcgaaa agcggggctt 660cggttgtacg cggttaggag tcccctcagg atatagtagt ttcgcttttg catagggagg 720gggacggatt ggacgaacca ctgaattccg cattgcagag atattgtatt taagtgccta 780gctcgataca ataaacgcca tttgaccatt caccacattg gtgtgcacct gggttgatgg 840ccggaccgtt gattccctgr cgactacgag cacatgcatg aagcagaagg cttcatttgg 900tgaccccgac gtgatcgtta gggaatacgc gctcactggc cgtcgtttta caacgtcgtg 960actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 1020gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 1080atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1140aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 1200atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 1260cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 1320accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 1380taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 1440agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 1500cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt 1560tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 1620tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 1680gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 1740ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 1800agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 1860agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 1920tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 1980tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 2040cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 2100aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 2160tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 2220tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 2280ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 2340ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 2400cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 2460gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 2520actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 2580aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 2640caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 2700aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 2760accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 2820aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 2880ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 2940agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 3000accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 3060gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 3120tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 3180cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 3240cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 3300cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 3360ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 3420taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 3480gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 3540cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 3600cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 3660tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc 3720attggtaatt gatcggctgg cacgcggaat ataggaggtc gctgaatagt aaacttgtag 3780acttggctac agcatagagt atcttctgta gctctgatga ctgctaggaa ataatgctac 3840ggataatgtg gggagggcaa ggcttgcgaa tcgggttgta acgggcaagg cttgactgag 3900gggacaatag catgtttagg cgaaaagcgg ggcttcggtt gtacgcggtt aggagtcccc 3960tcaggatata gtagtttcgc ttttgcatag ggagggggaa atgtagtctt atgcaatact 4020cttgtagtct tgcaacatgc ttatgtaacg atgagttagc aacatgcctt ataaggagag 4080aaaaagcacc gtgcatgccg attggtggga gtaaggtggt atgatcgtgg tatgatcgtg 4140ccttgttagg aaggcaacag acgggtctaa cacggattgg acgaaccact gaattccgca 4200ttgcagagat attgtattta agtgcctagc tcgatacaat aaacgccatt tgaccattca 4260ccacattggt gtgcacctgg gttgatggcc ggaccgttga ttccctgrcg actacgagca 4320catgcatgaa gcagaaggct tcatttggtg accccgacgt gatcgttagg gaatagtggt 4380cggccacagg cggcgtggcg atcctgtcct catccgtctc gcttattcgg ggagcggacg 4440atgaccctag tagagggggc tgcggcttag gagggcagaa gctgagtggc gtcggaggga 4500gccctactgc agggggccaa cataccctac cgagaactca gagagtcgtt ggaagacggg 4560aaggaagccc gacgactgag cggtccaccc caggcgtgat tccggttgct ctgcgtgatt 4620ccggtcgccc ggtggatcaa gcatggaagc cgtcataaag gtgatttcgt ccgcgtgtaa 4680gacctattgc gggaaaacct ctccttctaa gaaggaaata ggggctatgt tgtccctgtt 4740acaaaaggaa gggttgctta cgtccccctc agacttatat tccccggggt cctgggatcc 4800gataccgtcc ctatttttgt gtttgcttca gcagccattt aattcttcag tgtcatcttg 4860ttctgttgat gccactggaa caggattttc agcagtcttg caaagaacat ctagctgaaa 4920actttctgcc attcaatatt cttaccagtt cttcttgttt gaggtgagcc ataaattact 4980agaacttcgt cactgacaag tttatgcatt ttattacttc tattatgtac ttactttgac 5040ataacacaga cacgcacata ttttgctggg atttccacag tgtctctgtg tccttcacat 5100ggttttactg tcatacttcc gttataacct tggcaatctg cccagctgcc catcacaaga 5160aaagagattc cttttttatt acttctcttc agccaataaa caaaatgtga gaagcccaaa 5220caagaacttg tggggcaggc tgccatcaag ggagagacag ctgaagggtt gtgtagctca 5280atagaattaa gaaataataa agctgtgtca gacagttttg cctgatttat acaggcacgc 5340cccaagccag agaggctgtc tgccaaggcc accttgcagt ccttggtttg taagataagt 5400cataggtaac ttttctggtg aattgcgtgg agaatcatga tggcagttct tgctgtttac 5460tatggtaaga tgctaaaata ggagacagca aagtaacact tgctgctgta ggtgctctgc 5520tatccagaca gcgatggcac tcgcacacca agatgaggga tgctcccagc tgacggatgc 5580tggggcagta acagtgggtc ccatgctgcc tgctcattag catcacctca gccctcacca 5640gcccatcaga aggatcatcc caagctgagg aaagttgctc atcttcttca catcatcaaa 5700cctttggcct gactgatgcc tcccggatgc ttaaatgtgg tcactgacat ctttattttt 5760ctatgatttc aagtcagaac ctccggatca ggagggaaca catagtggga atgtaccctc 5820agctccaagg ccagatcttc cttcaatgat catgcatgct acttaggaag gtgtgtgtgt 5880gtgaatgtag aattgccttt gttatttttt cttcctgctg tcaggaacat tttgaatacc 5940agagaaaaag aaaagtgctc ttcttggcat gggaggagtt gtcacacttg caaaataaag 6000gatgcagtcc caaatgttca taatctcagg gtctgaagga ggatcagaaa ctgtgtatac 6060aatttcaggc ttctctgaat gcagcttttg aaagctgttc ctggccgagg cagtactagt 6120cagaaccctc ggaaacagga acaaatgtct tcaaggtgca gcaggaggaa acaccttgcc 6180catcatgaaa gtgaataacc actgccgctg aaggaatcca gctcctgttt gagcaggtgc 6240tgcacactcc cacactgaaa caacagttca tttttatagg acttccagga aggatcttct 6300tcttaagctt cttaattatg gtacatctcc agttggcaga tgactatgac tactgacagg 6360agaatgagga actagctggg aatatttctg tttgaccacc atggagtcac ccatttcttt 6420actggtattt ggaaataata attctgaatt gcaaagcagg agttagcgaa gatcttcatt 6480tcttccatgt tggtgacagc acagttctgg ctatgaaagt ctgcttacaa ggaagaggat 6540aaaaatcata gggataataa atctaagttt gaagacaatg aggttttagc tgcatttgac 6600atgaagaaat tgagacctct actggatagc tatggtattt acgtgtcttt ttgcttagtt 6660acttattgac cccagctgag gtcaagtatg aactcaggtc tctcgggcta ctggcatgga 6720ttgattacat acaactgtaa ttttagcagt gatttagggt ttatgagtac ttttgcagta 6780aatcataggg ttagtaatgt taatctcagg gaaaaaaaaa aaaagccaac cctgacagac 6840atcccagctc aggtggaaat caaggatcac agctcagtgc ggtcccagag aacacaggga 6900ctcttctctt aggaccttta tgtacagggc ctcaagataa ctgatgttag tcagaagact 6960ttccattctg gccacagttc agctgaggca atcctggaat tttctctccg ctgcacagtt 7020ccagtcatcc cagtttgtac agttctggca ctttttgggt caggccgtga tccaaggagc 7080agaagttcca gctatggtca gggagtgcct gaccgtccca actcactgca ctcaaacaaa 7140ggcgaaacca caagagtggc ttttgttgaa attgcagtgt ggcccagagg ggctgcacca 7200gtactggatt gaccacgagg caacattaat cctcagcaag tgcaatttgc agccattaaa 7260ttgaactaac tgatactaca atgcaatcag tatcaacaag tggtttggct tggaagatgg 7320agtctagggg ctctacagga gtagctactc tctaatggag ttgcattttg aagcaggaca 7380ctgtgaaaag ctggcctcct aaagaggctg ctaaacatta gggtcaattt tccagtgcac 7440tttctgaagt gtctgcagtt ccccatgcaa agctgcccaa acatagcact tccaattgaa 7500tacaattata tgcaggcgta ctgcttcttg ccagcactgt ccttctcaaa tgaactcaac 7560aaacaatttc aaagtctagt agaaagtaac aagctttgaa tgtcattaaa aagtatatct 7620gctttcagta gttcagctta tttatgccca ctagaaacat cttgtacaag ctgaacactg 7680gggctccaga ttagtggtaa aacctacttt atacaatcat agaatcatag aatggcctgg 7740gttggaaggg accccaagga tcatgaagat ccaacacccc cgccacaggc agggccacca 7800acctccagat ctggtactag accaggcagc ccagggctcc atccaacctg gccatgaaca 7860cctccaggga tggagcatcc acaacctctc tgggcagcct gtgccagcac ctcaccaccc 7920tctctgtgaa gaacttttcc ctgacatcca atctaagcct tccctccttg aggttagatc 7980cactccccct tgtgctatca ctgtctactc ttgtaaaaag ttgattctcc tcctttttgg 8040aaggttgcaa tgaggtctcc ttgcagcctt cttctcttct gcaggatgaa caagcccagc 8100tccctcagcc tgtctttata ggagaggtgc tccagccctc tgatcatctt tgtggccctc 8160ctctggaccc gctccaagag ctccacatct ttcctgtact gggggcccca ggcctgaatg 8220cagtactcca gatggggcct caaaagagca gagtaaagag ggacaatcac cttcctcacc 8280ctgctggcca gccctcttct gatggagccc tggatacaac tggctttctg agctgcaact 8340tctccttatc agttccacta ttaaaacagg aacaatacaa caggtgctga tggccagtgc 8400agagtttttc acacttcttc atttcggtag atcttagatg aggaacgttg aagttgtgct 8460tctgcgtgtg cttcttcctc ctcaaatact cctgcctgat acctcacccc acctgccact 8520gaatggctcc atggccccct gcagccaggg ccctgatgaa cccggcactg cttcagatgc 8580tgtttaatag cacagtatga ccaagttgca cctatgaata cacaaacaat gtgttgcatc 8640cttcagcact tgagaagaag agccaaattt gcattgtcag gaaatggttt agtaattctg 8700ccaattaaaa cttgtttatc taccatggct gtttttatgg ctgttagtag tggtacactg 8760atgatgaaca atggctatgc agtaaaatca agactgtaga tattgcaaca gactataaaa 8820ttcctctgtg gcttagccaa tgtggtactt cccacattgt ataagaaatt tggcaagttt 8880agagcaatgt ttgaagtgtt gggaaatttc tgtatactca agagggcgtt tttgacaact 8940gtagaacaga ggaatcaaaa gggggtggga ggaagttaaa agaagaggca ggtgcaagag 9000agcttgcagt cccgctgtgt gtacgacact ggcaacatga ggtctttgct aatcttggtg 9060ctttgcttcc tgcccctggc tgccttaggg tgcgatctgc ctcagaccca cagcctgggc 9120agcaggagga ccctgatgct gctggctcag atgaggagaa tcagcctgtt tagctgcctg 9180aaggataggc acgattttgg ctttcctcaa gaggagtttg gcaaccagtt tcagaaggct 9240gagaccatcc ctgtgctgca cgagatgatc cagcagatct ttaacctgtt tagcaccaag 9300gatagcagcg ctgcttggga tgagaccctg ctggataagt tttacaccga gctgtaccag 9360cagctgaacg atctggaggc ttgcgtgatc cagggcgtgg gcgtgaccga gacccctctg 9420atgaaggagg atagcatcct ggctgtgagg aagtactttc agaggatcac cctgtacctg 9480aaggagaaga agtacagccc ctgcgcttgg gaagtcgtga gggctgagat catgaggagc 9540tttagcctga gcaccaacct gcaagagagc ttgaggtcta aggagtaaaa agtctag 9597228DNAArtificial SequencePrimer ALV-SIN-1 2atgcgcgcat tggtaattga tcggctgg 28380DNAArtificial SequencePrimer ALV-SIN-2 3atatgcggcc gcggtaccgc ccgggcatcg atatcaagct tacggttcac taaacgagct 60ctgcttatat agacctccca 80459DNAArtificial SequencePrimer ALV-SIN-3 4atatgcggcc gcgtcgacgg ccggccagat ctgctgagcc ggtcgctacc attaccagt 59528DNAArtificial SequencePrimer ALV-SIN-4 5atacgcgtat tccctaacga tcacgtcg 28621DNAArtificial SequencePrimer ALV-SIN-5 6ctgaagtgta aggaatgtaa g 21730DNAArtificial SequencePrimer ALV-SIN-6 7gcgcgtctca tccccctccc tatgcaaaag 30837DNAArtificial SequencePrimer ALV-SIN-7 8gggcgtctca gggacggatt ggacgaacca ctgaatt 37920DNAArtificial SequencePrimer ALV-SIN-8 9ttagtgcttt acggcacctc 201038DNAArtificial SequencePrimer ALV-SIN-9 10gacggatccg ataccgtccc tatttttgtg tttgcttc 381130DNAArtificial SequencePrimer ALV-SIN-10 11taacggatcc tagacttttt actccttaga 301211945DNAArtificial SequenceProximal promoter and lysozyme signal peptide 12tgccgccttc tttgatattc actctgttgt atttcatctc ttcttgccga tgaaaggata 60taacagtctg tataacagtc tgtgaggaaa tacttggtat ttcttctgat cagtgttttt 120ataagtaatg ttgaatattg gataaggctg tgtgtccttt gtcttgggag acaaagccca 180cagcaggtgg tggttggggt ggtggcagct cagtgacagg agaggttttt ttgcctgttt 240tttttttttt tttttttttt aagtaaggtg ttcttttttc ttagtaaatt ttctactgga 300ctgtatgttt tgacaggtca gaaacatttc ttcaaaagaa gaaccttttg gaaactgtac 360agcccttttc tttcattccc tttttgcttt ctgtgccaat gcctttggtt ctgattgcat 420tatggaaaac gttgatcgga acttgaggtt tttatttata gtgtggcttg aaagcttgga 480tagctgttgt tacacgagat accttattaa gtttaggcca gcttgatgct ttattttttc 540cctttgaagt agtgagcgtt ctctggtttt tttcctttga aactggtgag gcttagattt 600ttctaatggg attttttacc tgatgatcta gttgcatacc caaatgcttg taaatgtttt 660cctagttaac atgttgataa cttcggattt acatgttgta tatacttgtc atctgtgttt 720ctagtaaaaa tatatggcat ttatagaaat acgtaattcc tgatttcctt tttttttatc 780tctatgctct gtgtgtacag gtcaaacaga cttcactcct atttttattt atagaatttt 840atatgcagtc tgtcgttggt tcttgtgttg taaggataca gccttaaatt tcctagagcg 900atgctcagta aggcgggttg tcacatgggt tcaaatgtaa aacgggcacg tttggctgct 960gccttcccga gatccaggac actaaactgc ttctgcactg aggtataaat cgcttcagat 1020cccagggaag tgcagatcca cgtgcatatt cttaaagaag aatgaatact ttctaaaata 1080ttttggcata ggaagcaagc tgcatggatt tgtttgggac ttaaattatt ttggtaacgg 1140agtgcatagg ttttaaacac agttgcagca tgctaacgag tcacagcgtt tatgcagaag 1200tgatgcctgg atgcctgttg cagctgttta cggcactgcc ttgcagtgag cattgcagat 1260aggggtgggg tgctttgtgt cgtgttccca cacgctgcca cacagccacc tcccggaaca 1320catctcacct gctgggtact tttcaaacca tcttagcagt agtagatgag ttactatgaa 1380acagagaagt tcctcagttg gatattctca tgggatgtct tttttcccat gttgggcaaa 1440gtatgataaa gcatctctat ttgtaaatta tgcacttgtt agttcctgaa tcctttctat 1500agcaccactt attgcagcag gtgtaggctc tggtgtggcc tgtgtctgtg cttcaatctt 1560ttaaagcttc tttggaaata cactgacttg attgaagtct cttgaagata gtaaacagta 1620cttacctttg atcccaatga aatcgagcat ttcagttgta aaagaattcc gcctattcat 1680accatgtaat gtaattttac acccccagtg ctgacacttt ggaatatatt caagtaatag 1740actttggcct caccctcttg tgtactgtat tttgtaatag aaaatatttt aaactgtgca 1800tatgattatt acattatgaa agagacattc tgctgatctt caaatgtaag aaaatgagga 1860gtgcgtgtgc ttttataaat acaagtgatt gcaaattagt gcaggtgtcc ttaaaaaaaa 1920aaaaaaaaag taatataaaa aggaccaggt gttttacaag tgaaatacat tcctatttgg 1980taaacagtta catttttatg aagattacca gcgctgctga ctttctaaac ataaggctgt 2040attgtcttcc tgtaccattg catttcctca ttcccaattt gcacaaggat gtctgggtaa 2100actattcaag aaatggcttt gaaatacagc atgggagctt gtctgagttg gaatgcagag 2160ttgcactgca aaatgtcagg aaatggatgt ctctcagaat gcccaactcc aaaggatttt 2220atatgtgtat atagtaagca gtttcctgat tccagcaggc caaagagtct gctgaatgtt 2280gtgttgccgg agacctgtat ttctcaacaa ggtaagatgg tatcctagca actgcggatt 2340ttaatacatt ttcagcagaa gtacttagtt aatctctacc tttagggatc gtttcatcat 2400ttttagatgt tatacttgaa atactgcata acttttagct ttcatgggtt cctttttttc 2460agcctttagg agactgttaa gcaatttgct gtccaacttt tgtgttggtc ttaaactgca 2520atagtagttt accttgtatt gaagaaataa agaccatttt tatattaaaa aatacttttg 2580tctgtcttca ttttgacttg tctgatatcc ttgcagtgcc cattatgtca gttctgtcag 2640atattcagac atcaaaactt aacgtgagct cagtggagtt acagctgcgg ttttgatgct 2700gttattattt ctgaaactag aaatgatgtt gtcttcatct gctcatcaaa cacttcatgc 2760agagtgtaag gctagtgaga aatgcataca tttattgata cttttttaaa gtcaactttt 2820tatcagattt ttttttcatt tggaaatata ttgttttcta gactgcatag cttctgaatc 2880tgaaatgcag tctgattggc atgaagaagc acagcactct tcatcttact taaacttcat 2940tttggaatga aggaagttaa gcaagggcac aggtccatga aatagagaca gtgcgctcag 3000gagaaagtga acctggattt ctttggctag tgttctaaat ctgtagtgag gaaagtaaca 3060cccgattcct tgaaagggct ccagctttaa tgcttccaaa ttgaaggtgg caggcaactt 3120ggccactggt tatttactgc attatgtctc agtttcgcag ctaacctggc ttctccacta 3180ttgagcatgg actatagcct ggcttcagag gccaggtgaa ggttgggatg ggtggaagga 3240gtgctgggct gtggctgggg ggactgtggg gactccaagc tgagcttggg gtgggcagca 3300cagggaaaag tgtgggtaac tatttttaag tactgtgttg caaacgtctc atctgcaaat 3360acgtagggtg tgtactctcg aagattaaca gtgtgggttc agtaatatat ggatgaattc 3420acagtggaag cattcaaggg tagatcatct aacgacacca gatcatcaag ctatgattgg 3480aagcggtatc agaagagcga ggaaggtaag cagtcttcat atgttttccc tccacgtaaa 3540gcagtctggg aaagtagcac cccttgagca gagacaagga aataattcag gagcatgtgc 3600taggagaact ttcttgctga attctacttg caagagcttt gatgcctggc ttctggtgcc 3660ttctgcagca cctgcaaggc ccagagcctg tggtgagctg gagggaaaga ttctgctcaa 3720gtccaagctt cagcaggtca ttgtctttgc ttcttccccc agcactgtgc agcagagtgg 3780aactgatgtc gaagcctcct gtccactacc tgttgctgca ggcagactgc tctcagaaaa 3840agagagctaa ctctatgcca tagtctgaag gtaaaatggg ttttaaaaaa gaaaacacaa 3900aggcaaaacc ggctgcccca tgagaagaaa gcagtggtaa acatggtaga aaaggtgcag 3960aagcccccag gcagtgtgac aggcccctcc tgccacctag aggcgggaac aagcttccct 4020gcctagggct ctgcccgcga agtgcgtgtt tctttggtgg gttttgtttg gcgtttggtt 4080ttgagattta gacacaaggg aagcctgaaa ggaggtgttg ggcactattt tggtttgtaa 4140agcctgtact tcaaatatat attttgtgag ggagtgtagc gaattggcca atttaaaata 4200aagttgcaag agattgaagg ctgagtagtt gagagggtaa cacgtttaat gagatcttct 4260gaaactactg
cttctaaaca cttgtttgag tggtgagacc ttggataggt gagtgctctt 4320gttacatgtc tgatgcactt gcttgtcctt ttccatccac atccatgcat tccacatcca 4380cgcatttgtc acttatccca tatctgtcat atctgacata cctgtctctt cgtcacttgg 4440tcagaagaaa cagatgtgat aatccccagc cgccccaagt ttgagaagat ggcagttgct 4500tctttccctt tttcctgcta agtaaggatt ttctcctggc tttgacacct cacgaaatag 4560tcttcctgcc ttacattctg ggcattattt caaatatctt tggagtgcgc tgctctcaag 4620tttgtgtctt cctactctta gagtgaatgc tcttagagtg aaagagaagg aagagaagat 4680gttggccgca gttctctgat gaacacacct ctgaataatg gccaaaggtg ggtgggtttc 4740tctgaggaac gggcagcgtt tgcctctgaa agcaaggagc tctgcggagt tgcagttatt 4800ttgcaactga tggtggaact ggtgcttaaa gcagattccc taggttccct gctacttctt 4860ttccttcttg gcagtcagtt tatttctgac agacaaacag ccacccccac tgcaggctta 4920gaaagtatgt ggctctgcct gggtgtgtta cagctctgcc ctggtgaaag gggattaaaa 4980cgggcaccat tcatcccaaa caggatcctc attcatggat caagctgtaa ggaacttggg 5040ctccaacctc aaaacattaa ttggagtacg aatgtaatta aaactgcatt ctcgcattcc 5100taagtcattt agtctggact ctgcagcatg taggtcggca gctcccactt tctcaaagac 5160cactgatgga ggagtagtaa aaatggagac cgattcagaa caaccaacgg agtgttgccg 5220aagaaactga tggaaataat gcatgaattg tgtggtggac atttttttta aatacataaa 5280ctacttcaaa tgaggtcgga gaaggtcagt gttttattag cagccataaa accaggtgag 5340cgagtaccat ttttctctac aagaaaaacg attctgagct ctgcgtaagt ataagttctc 5400catagcggct gaagctcccc cctggctgcc tgccatctca gctggagtgc agtgccattt 5460ccttggggtt tctctcacag cagtaatggg acaatacttc acaaaaattc tttcttttcc 5520tgtcatgtgg gatccctact gtgccctcct ggttttacgt taccccctga ctgttccatt 5580cagcggtttg gaaagagaaa aagaatttgg aaataaaaca tgtctacgtt atcacctcct 5640ccagcatttt ggtttttaat tatgtcaata actggcttag atttggaaat gagagggggt 5700tgggtgtatt accgaggaac aaaggaaggc ttatataaac tcaagtcttt tatttagaga 5760actggcaagc tgtcaaaaac aaaaaggcct taccaccaaa ttaagtgaat agccgctata 5820gccagcaggg ccagcacgag ggatggtgca ctgctggcac tatgccacgg cctgcttgtg 5880actctgagag caactgcttt ggaaatgaca gcacttggtg caatttcctt tgtttcagaa 5940tgcgtagagc gtgtgcttgg cgacagtttt tctagttagg ccacttcttt tttccttctc 6000tcctcattct cctaagcatg tctccatgct ggtaatccca gtcaagtgaa cgttcaaaca 6060atgaatccat cactgtagga ttctcgtggt gatcaaatct ttgtgtgagg tctataaaat 6120atggaagctt atttattttt cgttcttcca tatcagtctt ctctatgaca attcacatcc 6180accacagcaa attaaaggtg aaggaggctg gtgggatgaa gagggtcttc tagctttacg 6240ttcttccttg caaggccaca ggaaaatgct gagagctgta gaatacagcc tggggtaaga 6300agttcagtct cctgctggga cagctaaccg catcttataa ccccttctga gactcatctt 6360aggaccaaat agggtctatc tggggttttt gttcctgctg ttcctcctgg aaggctatct 6420cactatttca ctgctcccac ggttacaaac caaagataca gcctgaattt tttctaggcc 6480acattacata aatttgacct ggtaccaata ttgttctcta tatagttatt tccttcccca 6540ctgtgtttaa ccccttaagg cattcagaac aactagaatc atagaatggt ttggattgga 6600aggggcctta aacatcatcc atttccaacc ctctgccatg ggctgcttgc cacccactgg 6660ctcaggctgc ccagggcccc atccagcctg gccttgagca cctccaggga tggggcaccc 6720acagcttctc tgggcagcct gtgccaacac ctcaccactc tctgggtaaa gaattctctt 6780ttaacatcta atctaaatct cttctctttt agtttaaagc cattcctctt tttcccgttg 6840ctatctgtcc aagaaatgtg tattggtctc cctcctgctt ataagcagga agtactggaa 6900ggctgcagtg aggtctcccc acagccttct cttctccagg ctgaacaagc ccagctcctt 6960cagcctgtct tcgtaggaga tcatcttagt ggccctcctc tggacccatt ccaacagttc 7020cacggctttc ttgtggagcc ccaggtctgg atgcagtact tcagatgggg ccttacaaag 7080gcagagcaga tggggacaat cgcttacccc tccctgctgg ctgcccctgt tttgatgcag 7140cccagggtac tgttggcctt tcaggctccc agaccccttg ctgatttgtg tcaagctttt 7200catccaccag aacccacgct tcctggttaa tacttctgcc ctcacttctg taagcttgtt 7260tcaggagact tccattcttt aggacagact gtgttacacc tacctgccct attcttgcat 7320atatacattt cagttcatgt ttcctgtaac aggacagaat atgtattcct ctaacaaaaa 7380tacatgcaga attcctagtg ccatctcagt agggttttca tggcagtatt agcacatagt 7440caatttgctg caagtacctt ccaagctgcg gcctcccata aatcctgtat ttgggatcag 7500ttaccttttg gggtaagctt ttgtatctgc agagaccctg ggggttctga tgtgcttcag 7560ctctgctctg ttctgactgc accattttct agatcaccca gttgttcctg tacaacttcc 7620ttgtcctcca tcctttccca gcttgtatct ttgacaaata caggcctatt tttgtgtttg 7680cttcagcagc catttaattc ttcagtgtca tcttgttctg ttgatgccac tggaacagga 7740ttttcagcag tcttgcaaag aacatctagc tgaaaacttt ctgccattca atattcttac 7800cagttcttct tgtttgaggt gagccataaa ttactagaac ttcgtcactg acaagtttat 7860gcattttatt acttctatta tgtacttact ttgacataac acagacacgc acatattttg 7920ctgggatttc cacagtgtct ctgtgtcctt cacatggttt tactgtcata cttccgttat 7980aaccttggca atctgcccag ctgcccatca caagaaaaga gattcctttt ttattacttc 8040tcttcagcca ataaacaaaa tgtgagaagc ccaaacaaga acttgtgggg caggctgcca 8100tcaagggaga gacagctgaa gggttgtgta gctcaataga attaagaaat aataaagctg 8160tgtcagacag ttttgcctga tttatacagg cacgccccaa gccagagagg ctgtctgcca 8220aggccacctt gcagtccttg gtttgtaaga taagtcatag gtaacttttc tggtgaattg 8280cgtggagaat catgatggca gttcttgctg tttactatgg taagatgcta aaataggaga 8340cagcaaagta acacttgctg ctgtaggtgc tctgctatcc agacagcgat ggcactcgca 8400caccaagatg agggatgctc ccagctgacg gatgctgggg cagtaacagt gggtcccatg 8460ctgcctgctc attagcatca cctcagccct caccagccca tcagaaggat catcccaagc 8520tgaggaaagt tgctcatctt cttcacatca tcaaaccttt ggcctgactg atgcctcccg 8580gatgcttaaa tgtggtcact gacatcttta tttttctatg atttcaagtc agaacctccg 8640gatcaggagg gaacacatag tgggaatgta ccctcagctc caaggccaga tcttccttca 8700atgatcatgc atgctactta ggaaggtgtg tgtgtgtgaa tgtagaattg cctttgttat 8760tttttcttcc tgctgtcagg aacattttga ataccagaga aaaagaaaag tgctcttctt 8820ggcatgggag gagttgtcac acttgcaaaa taaaggatgc agtcccaaat gttcataatc 8880tcagggtctg aaggaggatc agaaactgtg tatacaattt caggcttctc tgaatgcagc 8940ttttgaaagc tgttcctggc cgaggcagta ctagtcagaa ccctcggaaa caggaacaaa 9000tgtcttcaag gtgcagcagg aggaaacacc ttgcccatca tgaaagtgaa taaccactgc 9060cgctgaagga atccagctcc tgtttgagca ggtgctgcac actcccacac tgaaacaaca 9120gttcattttt ataggacttc caggaaggat cttcttctta agcttcttaa ttatggtaca 9180tctccagttg gcagatgact atgactactg acaggagaat gaggaactag ctgggaatat 9240ttctgtttga ccaccatgga gtcacccatt tctttactgg tatttggaaa taataattct 9300gaattgcaaa gcaggagtta gcgaagatct tcatttcttc catgttggtg acagcacagt 9360tctggctatg aaagtctgct tacaaggaag aggataaaaa tcatagggat aataaatcta 9420agtttgaaga caatgaggtt ttagctgcat ttgacatgaa gaaattgaga cctctactgg 9480atagctatgg tatttacgtg tctttttgct tagttactta ttgaccccag ctgaggtcaa 9540gtatgaactc aggtctctcg ggctactggc atggattgat tacatacaac tgtaatttta 9600gcagtgattt agggtttatg agtacttttg cagtaaatca tagggttagt aatgttaatc 9660tcagggaaaa aaaaaaaaag ccaaccctga cagacatccc agctcaggtg gaaatcaagg 9720atcacagctc agtgcggtcc cagagaacac agggactctt ctcttaggac ctttatgtac 9780agggcctcaa gataactgat gttagtcaga agactttcca ttctggccac agttcagctg 9840aggcaatcct ggaattttct ctccgctgca cagttccagt catcccagtt tgtacagttc 9900tggcactttt tgggtcaggc cgtgatccaa ggagcagaag ttccagctat ggtcagggag 9960tgcctgaccg tcccaactca ctgcactcaa acaaaggcga aaccacaaga gtggcttttg 10020ttgaaattgc agtgtggccc agaggggctg caccagtact ggattgacca cgaggcaaca 10080ttaatcctca gcaagtgcaa tttgcagcca ttaaattgaa ctaactgata ctacaatgca 10140atcagtatca acaagtggtt tggcttggaa gatggagtct aggggctcta caggagtagc 10200tactctctaa tggagttgca ttttgaagca ggacactgtg aaaagctggc ctcctaaaga 10260ggctgctaaa cattagggtc aattttccag tgcactttct gaagtgtctg cagttcccca 10320tgcaaagctg cccaaacata gcacttccaa ttgaatacaa ttatatgcag gcgtactgct 10380tcttgccagc actgtccttc tcaaatgaac tcaacaaaca atttcaaagt ctagtagaaa 10440gtaacaagct ttgaatgtca ttaaaaagta tatctgcttt cagtagttca gcttatttat 10500gcccactaga aacatcttgt acaagctgaa cactggggct ccagattagt ggtaaaacct 10560actttataca atcatagaat catagaatgg cctgggttgg aagggacccc aaggatcatg 10620aagatccaac acccccgcca caggcagggc caccaacctc cagatctggt actagaccag 10680gcagcccagg gctccatcca acctggccat gaacacctcc agggatggag catccacaac 10740ctctctgggc agcctgtgcc agcacctcac caccctctct gtgaagaact tttccctgac 10800atccaatcta agccttccct ccttgaggtt agatccactc ccccttgtgc tatcactgtc 10860tactcttgta aaaagttgat tctcctcctt tttggaaggt tgcaatgagg tctccttgca 10920gccttcttct cttctgcagg atgaacaagc ccagctccct cagcctgtct ttataggaga 10980ggtgctccag ccctctgatc atctttgtgg ccctcctctg gacccgctcc aagagctcca 11040catctttcct gtactggggg ccccaggcct gaatgcagta ctccagatgg ggcctcaaaa 11100gagcagagta aagagggaca atcaccttcc tcaccctgct ggccagccct cttctgatgg 11160agccctggat acaactggct ttctgagctg caacttctcc ttatcagttc cactattaaa 11220acaggaacaa tacaacaggt gctgatggcc agtgcagagt ttttcacact tcttcatttc 11280ggtagatctt agatgaggaa cgttgaagtt gtgcttctgc gtgtgcttct tcctcctcaa 11340atactcctgc ctgatacctc accccacctg ccactgaatg gctccatggc cccctgcagc 11400cagggccctg atgaacccgg cactgcttca gatgctgttt aatagcacag tatgaccaag 11460ttgcacctat gaatacacaa acaatgtgtt gcatccttca gcacttgaga agaagagcca 11520aatttgcatt gtcaggaaat ggtttagtaa ttctgccaat taaaacttgt ttatctacca 11580tggctgtttt tatggctgtt agtagtggta cactgatgat gaacaatggc tatgcagtaa 11640aatcaagact gtagatattg caacagacta taaaattcct ctgtggctta gccaatgtgg 11700tacttcccac attgtataag aaatttggca agtttagagc aatgtttgaa gtgttgggaa 11760atttctgtat actcaagagg gcgtttttga caactgtaga acagaggaat caaaaggggg 11820tgggaggaag ttaaaagaag aggcaggtgc aagagagctt gcagtcccgc tgtgtgtacg 11880acactggcaa catgaggtct ttgctaatct tggtgctttg cttcctgccc ctggctgcct 11940taggg 119451329DNAArtificial SequenceBAC 26 Primer-1 13gcggaattca aagaagaaag ctgaaaaac 291429DNAArtificial SequenceBAC 26 Primer-2 14gcgggtacct tcaaatacta caagtgaaa 291533DNAArtificial SequenceBAC 26-OV Primer 1 15ggcctcgagt caagttctga gtaggtttta gtg 331641DNAArtificial SequenceBAC 26-OV Primer 2 16gcgcgtctct gtctagagca aacagcagaa cagtgaaaat g 411750DNAArtificial SequenceCTLA-4-Fc Primer 1 17gcgcgtctca agacaactca gagttcacca tgggtgtact gctcacacag 501829DNAArtificial SequenceCTLA-4-Fc Primer 2 18ggcccgggag ttttgtcaga agatttggg 291911868DNAArtificial SequencepSIN-OV-3.5-I-CTLA4-inv Vector 19aattgctaga ctaggatccc ccgtgctgca gaaccgagcg gctattgact tcttgctcct 60agctcacggc catggctgtg aggacattgc gggaatgtgt tgtttcaatc tgagtgatca 120cagtgagtct atacagaaga agttccagct aatgaaggaa catgtcaata agatcggcgt 180gaacaacgac ccaatcggaa gttggctgcg aggattattc ggaggaatag gagaatgggc 240cgtacacttg ctgaaaggac tgcttttggg gcttgtagtt atcttgttgc tagtagtatg 300cttgccttgc cttttgcaat gtgtatctag tagtattcga aagatgattg ataattcact 360cggctatcgc gaggaatata aaaaaattac aggaggctta taagcagccc gaaagaagag 420cgtaggcgag ttcttgtatt ccgtgtgata gctggttgga ttggtaattg atcggctggc 480acgcggaata taggaggtcg ctgaatagta aacttgtaga cttggctaca gcatagagta 540tcttctgtag ctctgatgac tgctaggaaa taatgctacg gataatgtgg ggagggcaag 600gcttgcgaat cgggttgtaa cgggcaaggc ttgactgagg ggacaatagc atgtttaggc 660gaaaagcggg gcttcggttg tacgcggtta ggagtcccct caggatatag tagtttcgct 720tttgcatagg gagggggacg gattggacga accactgaat tccgcattgc agagatattg 780tatttaagtg cctagctcga tacaataaac gccatttgac cattcaccac attggtgtgc 840acctgggttg atggccggac cgttgattcc ctgrcgacta cgagcacatg catgaagcag 900aaggcttcat ttggtgaccc cgacgtgatc gttagggaat acgcgctcac tggccgtcgt 960tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 1020tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 1080gttgcgcagc ctgaatggcg aatggaaatt gtaagcgtta atattttgtt aaaattcgcg 1140ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct 1200tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt 1260ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat 1320ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca 1380ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac 1440gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta 1500gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg 1560tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 1620cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 1680aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 1740ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 1800cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 1860agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 1920gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 1980cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 2040gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 2100ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 2160gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 2220gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 2280cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 2340ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 2400gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 2460gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 2520gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 2580ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 2640gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 2700gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 2760caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 2820ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 2880tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 2940ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 3000tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 3060cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 3120gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 3180ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 3240gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 3300agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 3360tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 3420tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 3480gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 3540taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 3600aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 3660atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 3720tacgccaagc gcgcattggt aattgatcgg ctggcacgcg gaatatagga ggtcgctgaa 3780tagtaaactt gtagacttgg ctacagcata gagtatcttc tgtagctctg atgactgcta 3840ggaaataatg ctacggataa tgtggggagg gcaaggcttg cgaatcgggt tgtaacgggc 3900aaggcttgac tgaggggaca atagcatgtt taggcgaaaa gcggggcttc ggttgtacgc 3960ggttaggagt cccctcagga tatagtagtt tcgcttttgc atagggaggg ggaaatgtag 4020tcttatgcaa tactcttgta gtcttgcaac atgcttatgt aacgatgagt tagcaacatg 4080ccttataagg agagaaaaag caccgtgcat gccgattggt gggagtaagg tggtatgatc 4140gtggtatgat cgtgccttgt taggaaggca acagacgggt ctaacacgga ttggacgaac 4200cactgaattc cgcattgcag agatattgta tttaagtgcc tagctcgata caataaacgc 4260catttgacca ttcaccacat tggtgtgcac ctgggttgat ggccggaccg ttgattccct 4320grcgactacg agcacatgca tgaagcagaa ggcttcattt ggtgaccccg acgtgatcgt 4380tagggaatag tggtcggcca caggcggcgt ggcgatcctg tcctcatccg tctcgcttat 4440tcggggagcg gacgatgacc ctagtagagg gggctgcggc ttaggagggc agaagctgag 4500tggcgtcgga gggagcccta ctgcaggggg ccaacatacc ctaccgagaa ctcagagagt 4560cgttggaaga cgggaaggaa gcccgacgac tgagcggtcc accccaggcg tgattccggt 4620tgctctgcgt gattccggtc gcccggtgga tcaagcatgg aagccgtcat aaaggtgatt 4680tcgtccgcgt gtaagaccta ttgcgggaaa acctctcctt ctaagaagga aataggggct 4740atgttgtccc tgttacaaaa ggaagggttg cttacgtccc cctcagactt atattccccg 4800gggtcctggg atcccattac cgcggcgctc tctcagcggg ctatggtact tggaaaatcg 4860ggagagttaa aaacctgggg attggttttg ggggcattga aggcggctcg agatccggta 4920ccttcaaata ctacaagtga aaagtgtttg cttaaacatg tttttattat gattaaagga 4980acaaaagagc acattcacaa gacccattac atatgggtac aaggaaaaca atttgaatag 5040taatatacca tatttgccaa cataccatga ttgagtcaaa gtttagggag aaatgtgaat 5100tataagattt ttataatgca tctttaggaa gtcaggaaga gccttgtagt atcaggaaca 5160cagagaacaa gcaattgcct tgtcagcata ggaatggttg gtgacagttg ataatttaat 5220ctgagagatt ttgagtgact aattctggag cagcttggtc atacagatat ctggcttaat 5280tggaaggctg catttttccc ccataaacct tctgctgatg tatcaggttg catttttcag 5340tgtgatgact cagtactgtg agtccaattt cattccctta agccttcatc catgagttac 5400cagtattact ctgtgtaaag gaaaagtgaa ttgcacctgt tctcacagtg taatttcttt 5460ctgatttttt ttctagatta agctccagct tttatgaagt ctggatgcag cagataacat 5520acttttcatt ttacccctga tactacagtg ctctgggtct tgttggaagg gacagagttt 5580ttcagctttc ttctttgaat tcctcattta cccggagaca gggagaggct cttctgcgtg 5640tagtggttgt gcagagcctc atgcatcacg gagcatgaga agacgttccc ctgctgccac 5700ctgctcttgt ccacggtgag cttgctgtag aggaagaagg agccgtcgga gtccagcacg 5760ggaggcgtgg tcttgtagtt gttctccggc tgcccattgc tctcccactc cacggcgatg 5820tcgctgggat agaagccttt gaccaggcag gtcaggctga cctggttctt ggtcagctca 5880tcccgggatg ggggcagggt gtacacctgt ggttctcggg gctgcccttt ggctttggag 5940atggttttct cgatgggggc tgggagggct ttgttggaga ccttgcactt gtactccttg 6000ccattcagcc agtcctggtg caggacggtg aggacgctga ccacccggta cgtgctgttg 6060tactgctcct cccgcggctt tgtcttggca ttatgcacct ccacgccgtc cacgtaccag 6120ttgaacttga cctcagggtc ttcgtggctc acgtccacca ccacgcatgt gacctcaggg 6180gtccgggaga tcatgagggt gtccttgggt tttgggggga agaggaagac tgacgatccc 6240cccaggagtt caggtgctgg ggacggtggg gatgtgtgag ttttgtcaga agatttgggc 6300tcctgatcag aatctgggca cggttctgga tcaattacat aaatctgggt tccgttgcct 6360atgcccaggt agtatggcgg tgggtacatg agctccacct tgcagatgta gagtcccgtg 6420tccatggccc tcagtccttg gatagtgagg ttcacttgat ttccactgga ggtgcccgtg 6480cagatggaat catctaggaa ggtcaactca ttccccatca tgtaggttgc cgcacagact 6540tcagtcacct ggctgtcagc ctgccgaagc actgtcaccc ggacctcagt ggctttgcct 6600ggagatgcat actcacacac aaagctrgcg atgcctcggc tgctggccag taccacagca
6660ggctgggcca cgtgcattgc catgctcgcc atgcttggaa acaggagtgc aaggaccaga 6720ctgagcagcg tcctctgtgt gagcagtaca cccatggtga actctgagtt gtctagagca 6780aacagcagaa cagtgaaaat gtaaggatgg aatgctgtac atagtaccat gcagggtact 6840ctatggtagg ctacaacagt aaattacgag cagtttttag gcaattaaat gttaacaagt 6900agttttaaag taattctgtg gtaatgtgtc tgttgctata tccacctctc atgtgcatgt 6960tcaaaaccat attcataaat ctatttatgt atttgcattc agttgtcttt tgggtagcaa 7020actgtcccag aagccagttg cctctacata tttttgttca gtgaaagcta gaattcattg 7080atacttttca gtacctctga ttaaaacaca atctgatagg cttgcaaaac tggaaattca 7140aagagcaaat ttcagtaaac tttaggtttg gacagatata tgagaaagca gaggcttgct 7200gactatttta tttcttattt ttattcccta aaaataaatg tagagaaata tctgtttgtt 7260gcacactact tgctatgagt agatcttcaa aagtattttt acctttgttt tggtgatggc 7320agaatagata aggaatgtaa tttatatggg gtcatgtagt ctaggagaaa gacacgcatg 7380taattcatat tctgctctat tgcactttca ggtatggttt gctttgctca aagatatgca 7440tgtgtactgt agtataaact ttctgtggag ttaaatttta gtggtgacat tcagacagaa 7500gagaaatgca gacatgataa aatagcaatg tttactataa aacagagcca ctgaatgaat 7560tcttgttcat gacatagacc aatagaagat ttatacttgt tctgtctgtt tctattataa 7620agagctgaac tgtacaacta ttgtatagcc agtgtgctta tataaagcac agcttttgga 7680gccagcatga atctagttgc tttcctgaga tttatataat ctgtgaaagt cagaagtcct 7740tcagagccca gccctttata tgcgtactga gtgctggggc ctcaggattg gattttctgt 7800attaaacccc tcaaaagttt ttactgacca cgtgtgtgag tatacacaca cacatttttc 7860tcattttctt ttctgtatat aagttcacat gtatctatta ttgtaagaat atacgtttat 7920gcacccccca catttttatc ttgtgtagtg atcagcagct gcactttgca ggaattaaac 7980ttctagagaa ttttcacatt aaaataactc cccagaattc actgaacacc atgattttgc 8040tctctgtgca ctctgtaggg ctagaagtta atcaagcaaa ctgcaaagca tatcagatag 8100tgaacgacag gataagatgt tctgaaatta aaaacatatt ttaagcacaa agaataagcc 8160tcctgaaaac aaacacaaag cttttacaca taataaaata gtgcagaatg catacacagg 8220tgagaagttt ttataggggg tatcacgcag gtacttcacc cttaaagata caacacatag 8280cacaataatt gttaattttt taaagtttag gtgcaagtaa gagctaatat agagagaagg 8340taattccaga gagttgctta cctttcgagc ttgactgcta aaggcaatac agctttctag 8400ctgtatgtac agacactggc tgagccctgg ggaatatata gtctgaattg tgacccaccc 8460acaggttccc ttcagaagtt tgacctttga caccatagaa atcatttaat gggattgggt 8520tagattttag tttcaatagg tccattttgg attgaatgga gagcaaatat tagtttttaa 8580ttctgggtaa caatgtgttt tctgcctgtt ctgctaatcc atcaggactg ttggatggga 8640gagaagactg ggaaatattg ctcatgttcc attgagcttc agttacaacc agataatggg 8700atctttaaga aaacagaaaa atgtgggaac cttggagatg gaaaacataa ttagcaatta 8760ttagttagtg tgcttattac tatggttgta gtaacagacc agaagtctgt ttcatttgat 8820ccttcttgta tgtacaatgt gcatctgagc cacgctagac aggacataaa tgagaacaag 8880acttgaccta ttattttctt gacaaaatag gagaaataaa gaagcgtgca tgtgaaggag 8940ccaactgaga ctagagtgaa gagcagacac actttctttc ctatagttgg aatatttaaa 9000tctatctttt tatgggtgtg aatgctttat aacaaacttt tattctgagg atacagcaaa 9060acatagctcc atacaatgca aaacaatact caatttcaaa tgtgtttatg atatgaactt 9120gcagtgttcc tcaaagatct tccatgaata acttaatggc ctggcagatg acagaggaat 9180tgtgaaattc agctggagga gtgttcatgg ttcgagggac aatcataata tacaatagca 9240aatatatttc agttatagaa gctattgttc tgtattgaaa taatagaatt gacaaacagt 9300aaagaaacca ttctgacctc tgtaaagcac tgtttgattt aaaaatgggg gaaaaaagta 9360caacataatt cttcaggaca tacatagaga tcactgcaat ctctgttaag cagaattact 9420ttcctatacc actagctgaa gtttagtcag tgccattttc ttttgtttct ctccttcctt 9480ttgtgaaaac atatatactg tggaaatcta cattctcctt gccaagtctg aggacttaag 9540acaagatggt agtgcaaata atattttttt gctggatgtc tacaccacag gtatcaactg 9600attttttttg tttcattttg tttttaatca cgtcttttgc ttctatttca gccactaaga 9660aagtctgaaa atcttgcctg ctttttgtga tgatagatgt gcttcccagt aaatgttatc 9720tctacctatg aaatgcatgt cagtctgcag aaagagaaag gagattggga ataggttttc 9780tcagatgcac ttctctgtca tctggtgtca atcaaacact aataatttgt gtatagatat 9840cttatatata tatatatatt tggaatttgc aggttggcat agttcagata gtcctgtcac 9900attgtaatat cctggtgaga taacaaggaa aagagagacc gtttcggctc ttactaaggc 9960agggaactgc ttaccagaca gggaggttct ggagatgaca tccagcatga aaagcacact 10020tccaaatact taaaggtatc aagtctaact tgtcagacag gctccagaat aacttctgtc 10080ctaatgctac agaaaagggg gaaggtatcc accatggcca aaattgtcag ccattttgtc 10140tcagcaaaca gcagatctgg tcagtaagga caagattctt ccaaagcaac catgccatat 10200ataattaagc atgtgtaatt aattaataaa aaatataatt tagtgtattt cctcctttgg 10260atgttatgaa gaaatgcttt tattaacaat tcaccataat ctgtcctaag agtagtgaat 10320aacaacaggc tgcttctcac cctgtggttg ggtgtaccag tgagccagag ctaaacgcca 10380cgtttcctct tttgtatccc atagcagaga gggtctccat ttcatttctg tagctcagaa 10440agttgtagtg gatttacact acaagttgtg gtagtggagg tctgccggag tggcctctgt 10500gaacagagcc cagcagctgt cccgtgtcct caaaagggag ctgccactgg ccagagctga 10560gccagtgatc gatgctagat gtacctcagg aggagcaata tgtaagaaca actgctgtac 10620aatggtagtt gggagaggtg agtgagaaaa tgtgagagaa acagccctga tgacactgag 10680gtcagtgcgg aggagggcag gaggtgttcc aggtgtagaa cagaagttcc ctgcagccca 10740agagaggccc atggtggagc actctgaccc tctgcagccc atggtatatc atataaacct 10800cagttctgtg acattatttt aactccatat cccttttctg ttcagggtca ctttgagttc 10860acagccattt ctttatattt ctccaatatc agccttccat tgctacatat gagacttgga 10920cagtacatct gattcagtca aatctgcctt cagaacgtcc ctgaagccct tcttagacag 10980tctcaattct ccttcccttc atctctttta tcatacatgg accacggacc tgtccagacc 11040tgagtcatat gtccatcttt acgtccatct ctatgtcttg tactttaaga caaataaaat 11100atcaaggaaa ttgatgcagt tatgtcagtt atcactgtca tagtatcgtg ctgcaaatat 11160aagatgagaa tgatcccaaa ggctttttaa agctgctcta tttgacttcc acatagtgtc 11220ctgattccag acctacagaa cagttttgta tgcatttgac ttgcagagct ttgttttgtg 11280agtcttataa aagccatttt tcctctccaa gaagtagccg gtggtttaaa acaatgtaga 11340ttaagtgtgg agcatgagaa tttctgcttt tctgtcagat gagaaggata tactacactc 11400tttcccaatg gaagaccagc tgcaagcaac aaaaattgtc catgaacaaa tgagatcttg 11460atcagaacag gctgtcatca tagtgttgtc agcatacctg catagttggt ttgacttggg 11520ggtctagaga gagtaagcaa caatcttctt gcagttggaa ggttacctgg gataggtggc 11580aatggattgc cctgcccagc acagctgtgc aaagcagtac aaatagtttt gtcacacatt 11640gtttgacaat gcttgtccca agaaaaggtc agctaaggct ctgctgccct ttcctatgcc 11700aggcatttca ttgtgggtct gtccctaaac caacagtctc atgaataaag actcggagac 11760ctgaaagtta taaaagcact ttttatccaa aaggatatga agtccaggtg agctcacagg 11820tcaaagcctc ttatccaatc actaaaacct actcagaact tgactcga 118682010021DNAArtificial SequencepSIN-3.9-OM-CTLA4-Fc Vector 20ctagactagg atcccccgtg ctgcagaacc gagcggctat tgacttcttg ctcctagctc 60acggccatgg ctgtgaggac attgcgggaa tgtgttgttt caatctgagt gatcacagtg 120agtctataca gaagaagttc cagctaatga aggaacatgt caataagatc ggcgtgaaca 180acgacccaat cggaagttgg ctgcgaggat tattcggagg aataggagaa tgggccgtac 240acttgctgaa aggactgctt ttggggcttg tagttatctt gttgctagta gtatgcttgc 300cttgcctttt gcaatgtgta tctagtagta ttcgaaagat gattgataat tcactcggct 360atcgcgagga atataaaaaa attacaggag gcttataagc agcccgaaag aagagcgtag 420gcgagttctt gtattccgtg tgatagctgg ttggattggt aattgatcgg ctggcacgcg 480gaatatagga ggtcgctgaa tagtaaactt gtagacttgg ctacagcata gagtatcttc 540tgtagctctg atgactgcta ggaaataatg ctacggataa tgtggggagg gcaaggcttg 600cgaatcgggt tgtaacgggc aaggcttgac tgaggggaca atagcatgtt taggcgaaaa 660gcggggcttc ggttgtacgc ggttaggagt cccctcagga tatagtagtt tcgcttttgc 720atagggaggg ggacggattg gacgaaccac tgaattccgc attgcagaga tattgtattt 780aagtgcctag ctcgatacaa taaacgccat ttgaccattc accacattgg tgtgcacctg 840ggttgatggc cggaccgttg attccctgrc gactacgagc acatgcatga agcagaaggc 900ttcatttggt gaccccgacg tgatcgttag ggaatacgcg ctcactggcc gtcgttttac 960aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 1020ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 1080gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa 1140tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa 1200atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact 1260attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 1320actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa 1380tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 1440gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt 1500cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg 1560tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 1620aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 1680gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 1740ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 1800gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 1860tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 1920attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 1980tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 2040agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 2100aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 2160tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 2220cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 2280tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 2340tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 2400tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 2460tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 2520aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 2580gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 2640tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 2700aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 2760aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 2820tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 2880gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 2940cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 3000acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 3060cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 3120cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 3180aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 3240gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 3300atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 3360tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 3420gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 3480agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg 3540cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt 3600gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt 3660gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc 3720caagcgcgca ttggtaattg atcggctggc acgcggaata taggaggtcg ctgaatagta 3780aacttgtaga cttggctaca gcatagagta tcttctgtag ctctgatgac tgctaggaaa 3840taatgctacg gataatgtgg ggagggcaag gcttgcgaat cgggttgtaa cgggcaaggc 3900ttgactgagg ggacaatagc atgtttaggc gaaaagcggg gcttcggttg tacgcggtta 3960ggagtcccct caggatatag tagtttcgct tttgcatagg gagggggaaa tgtagtctta 4020tgcaatactc ttgtagtctt gcaacatgct tatgtaacga tgagttagca acatgcctta 4080taaggagaga aaaagcaccg tgcatgccga ttggtgggag taaggtggta tgatcgtggt 4140atgatcgtgc cttgttagga aggcaacaga cgggtctaac acggattgga cgaaccactg 4200aattccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgccattt 4260gaccattcac cacattggtg tgcacctggg ttgatggccg gaccgttgat tccctgrcga 4320ctacgagcac atgcatgaag cagaaggctt catttggtga ccccgacgtg atcgttaggg 4380aatagtggtc ggccacaggc ggcgtggcga tcctgtcctc atccgtctcg cttattcggg 4440gagcggacga tgaccctagt agagggggct gcggcttagg agggcagaag ctgagtggcg 4500tcggagggag ccctactgca gggggccaac ataccctacc gagaactcag agagtcgttg 4560gaagacggga aggaagcccg acgactgagc ggtccacccc aggcgtgatt ccggttgctc 4620tgcgtgattc cggtcgcccg gtggatcaag catggaagcc gtcataaagg tgatttcgtc 4680cgcgtgtaag acctattgcg ggaaaacctc tccttctaag aaggaaatag gggctatgtt 4740gtccctgtta caaaaggaag ggttgcttac gtccccctca gacttatatt ccccggggtc 4800ctgggatccc attaccgcgg cgctctctca gcgggctatg gtacttggaa aatcgggaga 4860gttaaaaacc tggggattgg ttttgggggc attgaaggcg gctcgaggtc gacggtatcg 4920ataagcttgc agtccaaggc tttgtctgtg tacccagtga aatccttcct ctgttacata 4980aagcccagat aggactcaga aatgtagtca ttccagcccc cctcttcctc agatctggag 5040cagcacttgt ttgcagccag tcctccccaa aatgcacaga cctcgccgag tggagggaga 5100tgtaaacagc gaaggttaat tacctccttg tcaaaaacac tttgtggtcc atagatgttt 5160ctgtcaatct tacaaaacag aaccgagagg cagcgagcac tgaagagcgt gttcccatgc 5220tgagttaatg agacttggca gctcgctgtg cagagatgat ccctgtgctt catgggaggc 5280tgtaacctgt ctccccatcg ccttcacacc gcagtgctgt cctggacacc tcaccctcca 5340taagctgtag gatgcagctg cccagggatc aagagacttt tcctaaggct cttaggactc 5400atctttgccg ctcagtagcg tgcagcaatt actcatccca actatactga atgggtttct 5460gccagctctg cttgtttgtc aataagcatt tcttcatttt gcctctaagt ttctctcagc 5520agcaccgctc tgggtgacct gagtggccac ctggaacccg aggggcacag ccaccacctc 5580cctgttgctg ctgctccagg gactcatgtg ctgctggatg gggggaagca tgaagttcct 5640cacccagaca cctgggttgc aatggctgca gcgtgctctt cttggtatgc agattgtttc 5700cagccattac ttgtagaaat gtgctgtgga agccctttgt atctctttct gtggcccttc 5760agcaaaagct gtgggaaagc tctgaggctg ctttcttggg tcgtggagga attgtatgtt 5820ccttctttaa caaaaattat ccttaggaga gagcactgtg caagcattgt gcacataaaa 5880caattcaggt tgaaagggct ctctggaggt ttccagcctg actactgctc gaagcaaggc 5940caggttcaaa gatggctcag gatgctgtgt gccttcctga ttatctgtgc caccaatgga 6000ggagattcac agccactctg cttcccgtgc cactcatgga gaggaatatt cccttatatt 6060cagatagaat gttatccttt agctcagcct tccctataac cccatgaggg agctgcagat 6120ccccatactc tccccttctc tggggtgaag gccgtgtccc ccagcccccc ttcccaccct 6180gtgccctaag cagcccgctg gcctctgctg gatgtgtgcc tatatgtcaa tgcctgtcct 6240tgcagtccag cctgggacat ttaattcatc accagggtaa tgtggaactg tgtcatcttc 6300ccctgcaggg tacaaagttc tgcacggggt cctttcggtt caggaaaacc ttcactggtg 6360ctacctgaat caagctctat ttaataagtt cataagcaca tggatgtgtt ttcctagaga 6420tacgttttaa tggtatcagt gatttttatt tgctttgttg cttacttcaa acagtgcctt 6480tgggcaggag gtgagggacg ggtctgccgt tggctctgca gtgatttctc caggcgtgtg 6540gctcaggtca gatagtggtc actctgtggc cagaagaagg acaaagatgg aaattgcaga 6600ttgagtcacg ttaagcaggc atcttggagt gatttgaggc agtttcatga aagagctacg 6660accacttatt gttgttttcc ccttttacaa cagaagtttt catcaaaata acgtggcaaa 6720gcccaggaat gtttgggaaa agtgtagtta aatgttttgt aattcatttg tcggagtgct 6780accagctaag aaaaaagtcc tacctttggt atggtagtcc tgcagagaat acaacatcaa 6840tattagtttg gaaaaaaaca ccaccaccac cagaaactgt aatggaaaat gtaaaccaag 6900aaattccttg ggtaagagag aaaggatgtc gtatactggc caagtcctgc ccagctgtca 6960gcctgctgac cctctgcagt tcaggaccat gaaacgtggc actgtaagac gtgtcccctg 7020cctttgcttg cccacagatc tctgcccttg tgctgactcc tgcacacaag agcatttccc 7080tgtagccaaa cagcgattag ccataagctg cacctgactt tgaggattaa gagtttgcaa 7140ttaagtggat tgcagcagga gatcagtggc agggttgcag atgaaatcct tttctagggg 7200tagctaaggg ctgagcaacc tgtcctacag cacaagccaa accagccaag ggttttcctg 7260tgctgttcac agaggcaggg ccagctggag ctggaggagg ttgtgctggg acccttctcc 7320ctgtgctgag aatggagtga tttctgggtg ctgttcctgt ggcttgcact gagcagctca 7380agggagatcg gtgctcctca tgcagtgcca aaactcgtgt ttgatgcaga aagatggatg 7440tgcacctccc tcctgctaat gcagccgtga gcttatgaag gcaatgagcc ctcagtgcag 7500caggagctgt agtgcactcc tgtaggtgct agggaaaatc tctggttccc agggatgcat 7560tcataagggc aatatatctt gaggctgcgc caaatctttc tgaaatattc atgcgtgttc 7620ccttaattta tagaaacaaa cacagcagaa taattattcc aatgcctccc ctcgaaggaa 7680acccatattt ccatgtagaa atgtaaccta tatacacaca gccatgctgc atccttcaga 7740acgtgccagt gctcatctcc catggcaaaa tactacaggt attctcacta tgttggacct 7800gtgaaaggaa ccatggtaag aaacttcggt taaaggtatg gctgcaaaac tactcatacc 7860aaaacagcag agctccagac ctcctcttag gaaagagcca cttggagagg gatggtgtga 7920aggctggagg tgagagacag agcctgtccc agttttcctg tctctatttt ctgaaacgtt 7980tgcaggagga aaggacaact gtactttcag gcatagctgg tgccctcacg taaataagtt 8040ccccgaactt ctgtgtcatt tgttcttaag atgctttggc agaacacttt gagtcaattc 8100gcttaactgt gactaggtct gtaaataagt gctccctgct gataaggttc aagtgacatt 8160tttagtggta tttgacagca tttaccttgc tttcaagtct tctaccaagc tcttctatac 8220ttaagcagtg aaaccgccaa gaaacccttc cttttatcaa gctagtgcta aataccatta 8280acttcatagg ttagatacgg tgctgccagc ttcacctggc agtggttggt cagttctgct 8340ggtgacaaag cctccctggc ctgtgctttt acctagaggt gaatatccaa gaatgcagaa 8400ctgcatggaa agcagagctg caggcacgat ggtgctgagc cttagctgct tcctgctggg 8460agatgtggat gcagagacga atgaaggacc tgtcccttac tcccctcagc attctgtgct 8520atttagggtt ctaccagagt ccttaagagg tttttttttt ttttggtcca aaagtctgtt 8580tgtttggttt tgaccactga gagcatgtga cacttgtctc aagctattaa ccaagtgtcc 8640agccaaaatc aattgcctgg gagacgcaga ccattacctg gaggtcagga cctcaataaa 8700tattaccagc ctcattgtgc cgctgacaga ttcagctggc tgctccgtgt tccagtccaa 8760cagttcggac gccacgtttg tatatatttg caggcagcct cggggggacc atctcaggag 8820cagagcaccg gcagccgcct gcagagccgg gcagtacctc aacatgggtg tactgctcac 8880acagaggacg ctgctcagtc tggtccttgc actcctgttt ccaagcatgg cgagcatggc 8940aatgcacgtg gcccagcctg ctgtggtact ggccagcagc cgaggcatcg cyagctttgt 9000gtgtgagtat gcatctccag gcaaagccac tgaggtccgg gtgacagtgc ttcggcaggc 9060tgacagccag gtgactgaag tctgtgcggc aacctacatg atggggaatg agttgacctt 9120cctagatgat tccatctgca cgggcacctc cagtggaaat caagtgaacc tcactatcca 9180aggactgagg gccatggaca cgggactcta catctgcaag gtggagctca tgtacccacc 9240gccatactac ctgggcatag gcaacggaac ccagatttat gtaattgatc cagaaccgtg 9300cccagattct gatcaggagc ccaaatcttc tgacaaaact cacacatccc caccgtcccc 9360agcacctgaa ctcctggggg gatcgtcagt cttcctcttc cccccaaaac ccaaggacac 9420cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga 9480ccctgaggtc aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa 9540gccgcgggag gagcagtaca acagcacgta ccgggtggtc agcgtcctca ccgtcctgca 9600ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc 9660ccccatcgag aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac 9720cctgccccca tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa
9780aggcttctat cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa 9840ctacaagacc acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct 9900caccgtggac aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga 9960ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta aatgaggaat 10020t 10021217350DNAArtificial SequencepSIN-1.8-OM-IFNa-2B Vector 21tcgagatcaa ttgctagact aggatccccc gtgctgcaga accgagcggc tattgacttc 60ttgctcctag ctcacggcca tggctgtgag gacattgcgg gaatgtgttg tttcaatctg 120agtgatcaca gtgagtctat acagaagaag ttccagctaa tgaaggaaca tgtcaataag 180atcggcgtga acaacgaccc aatcggaagt tggctgcgag gattattcgg aggaatagga 240gaatgggccg tacacttgct gaaaggactg cttttggggc ttgtagttat cttgttgcta 300gtagtatgct tgccttgcct tttgcaatgt gtatctagta gtattcgaaa gatgattgat 360aattcactcg gctatcgcga ggaatataaa aaaattacag gaggcttata agcagcccga 420aagaagagcg taggcgagtt cttgtattcc gtgtgatagc tggttggatt ggtaattgat 480cggctggcac gcggaatata ggaggtcgct gaatagtaaa cttgtagact tggctacagc 540atagagtatc ttctgtagct ctgatgactg ctaggaaata atgctacgga taatgtgggg 600agggcaaggc ttgcgaatcg ggttgtaacg ggcaaggctt gactgagggg acaatagcat 660gtttaggcga aaagcggggc ttcggttgta cgcggttagg agtcccctca ggatatagta 720gtttcgcttt tgcataggga gggggacgga ttggacgaac cactgaattc cgcattgcag 780agatattgta tttaagtgcc tagctcgata caataaacgc catttgacca ttcaccacat 840tggtgtgcac ctgggttgat ggccggaccg ttgattccct grcgactacg agcacatgca 900tgaagcagaa ggcttcattt ggtgaccccg acgtgatcgt tagggaatac gcgctcactg 960gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 1020gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 1080tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat attttgttaa 1140aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca 1200aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga 1260acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc 1320agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc 1380gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc 1440cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg 1500caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac 1560agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 1620tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 1680aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 1740ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 1800ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 1860tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 1920tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 1980actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 2040gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 2100acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 2160gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 2220acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 2280gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 2340ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 2400gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 2460cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 2520agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 2580catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 2640tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 2700cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 2760gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 2820taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 2880ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 2940tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 3000ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 3060cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 3120agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 3180gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 3240atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 3300gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 3360gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 3420ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 3480cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 3540cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 3600acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 3660cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 3720accatgatta cgccaagcgc gcattggtaa ttgatcggct ggcacgcgga atataggagg 3780tcgctgaata gtaaacttgt agacttggct acagcataga gtatcttctg tagctctgat 3840gactgctagg aaataatgct acggataatg tggggagggc aaggcttgcg aatcgggttg 3900taacgggcaa ggcttgactg aggggacaat agcatgttta ggcgaaaagc ggggcttcgg 3960ttgtacgcgg ttaggagtcc cctcaggata tagtagtttc gcttttgcat agggaggggg 4020aaatgtagtc ttatgcaata ctcttgtagt cttgcaacat gcttatgtaa cgatgagtta 4080gcaacatgcc ttataaggag agaaaaagca ccgtgcatgc cgattggtgg gagtaaggtg 4140gtatgatcgt ggtatgatcg tgccttgtta ggaaggcaac agacgggtct aacacggatt 4200ggacgaacca ctgaattccg cattgcagag atattgtatt taagtgccta gctcgataca 4260ataaacgcca tttgaccatt caccacattg gtgtgcacct gggttgatgg ccggaccgtt 4320gattccctgr cgactacgag cacatgcatg aagcagaagg cttcatttgg tgaccccgac 4380gtgatcgtta gggaatagtg gtcggccaca ggcggcgtgg cgatcctgtc ctcatccgtc 4440tcgcttattc ggggagcgga cgatgaccct agtagagggg gctgcggctt aggagggcag 4500aagctgagtg gcgtcggagg gagccctact gcagggggcc aacataccct accgagaact 4560cagagagtcg ttggaagacg ggaaggaagc ccgacgactg agcggtccac cccaggcgtg 4620attccggttg ctctgcgtga ttccggtcgc ccggtggatc aagcatggaa gccgtcataa 4680aggtgatttc gtccgcgtgt aagacctatt gcgggaaaac ctctccttct aagaaggaaa 4740taggggctat gttgtccctg ttacaaaagg aagggttgct tacgtccccc tcagacttat 4800attccccggg gtcctgggat cccattaccg cggcgctctc tcagcgggct atggtacttg 4860gaaaatcggg agagttaaaa acctggggat tggttttggg ggcattgaag gcggctcgac 4920ggatccgtta accctagaac tagtggatct ctgcccttgt gctgactcct gcacacaaga 4980gcatttccct gtagccaaac agcgattagc cataagctgc acctgacttt gaggattaag 5040agtttgcaat taagtggatt gcagcaggag atcagtggca gggttgcaga tgaaatcctt 5100ttctaggggt agctaagggc tgagcaacct gtcctacagc acaagccaaa ccagccaagg 5160gttttcctgt gctgttcaca gaggcagggc cagctggagc tggaggaggt tgtgctggga 5220cccttctccc tgtgctgaga atggagtgat ttctgggtgc tgttcctgtg gcttgcactg 5280agcagctcaa gggagatcgg tgctcctcat gcagtgccaa aactcgtgtt tgatgcagaa 5340agatggatgt gcacctccct cctgctaatg cagccgtgag cttatgaagg caatgagccc 5400tcagtgcagc aggagctgta gtgcactcct gtaggtgcta gggaaaatct ctggttccca 5460gggatgcatt cataagggca atatatcttg aggctgcgcc aaatctttct gaaatattca 5520tgcgtgttcc cttaatttat agaaacaaac acagcagaat aattattcca atgcctcccc 5580tcgaaggaaa cccatatttc catgtagaaa tgtaacctat atacacacag ccatgctgca 5640tccttcagaa cgtgccagtg ctcatctccc atggcaaaat actacaggta ttctcactat 5700gttggacctg tgaaaggaac catggtaaga aacttcggtt aaaggtatgg ctgcaaaact 5760actcatacca aaacagcaga gctccagacc tcctcttagg aaagagccac ttggagaggg 5820atggtgtgaa ggctggaggt gagagacaga gcctgtccca gttttcctgt ctctattttc 5880tgaaacgttt gcaggaggaa aggacaactg tactttcagg catagctggt gccctcacgt 5940aaataagttc cccgaacttc tgtgtcattt gttcttaaga tgctttggca gaacactttg 6000agtcaattcg cttaactgtg actaggtctg taaataagtg ctccctgctg ataaggttca 6060agtgacattt ttagtggtat ttgacagcat ttaccttgct ttcaagtctt ctaccaagct 6120cttctatact taagcagtga aaccgccaag aaacccttcc ttttatcaag ctagtgctaa 6180ataccattaa cttcataggt tagatacggt gctgccagct tcacctggca gtggttggtc 6240agttctgctg gtgacaaagc ctccctggcc tgtgctttta cctagaggtg aatatccaag 6300aatgcagaac tgcatggaaa gcagagctgc aggcacgatg gtgctgagcc ttagctgctt 6360cctgctggga gatgtggatg cagagacgaa tgaaggacct gtcccttact cccctcagca 6420ttctgtgcta tttagggttc taccagagtc cttaagaggt tttttttttt tttggtccaa 6480aagtctgttt gtttggtttt gaccactgag agcatgtgac acttgtctca agctattaac 6540caagtgtcca gccaaaatca attgcctggg agacgcagac cattacctgg aggtcaggac 6600ctcaataaat attaccagcc tcattgtgcc gctgacagat tcagctggct gctccgtgtt 6660ccagtccaac agttcggacg ccacgtttgt atatatttgc aggcagcctc ggggggacca 6720tctcaggagc agagcaccgg cagccgcctg cagagccggg cagtacctca ccatggcttt 6780gacctttgcc ttactggtgg ctctcctggt gctgagctgc aagagcagct gctctgtggg 6840ctgcgatctg cctcagaccc acagcctggg cagcaggagg accctgatgc tgctggctca 6900gatgaggaga atcagcctgt ttagctgcct gaaggatagg cacgattttg gctttcctca 6960agaggagttt ggcaaccagt ttcagaaggc tgagaccatc cctgtgctgc acgagatgat 7020ccagcagatc tttaacctgt ttagcaccaa ggatagcagc gctgcttggg atgagaccct 7080gctggataag ttttacaccg agctgtacca gcagctgaac gatctggagg cttgcgtgat 7140ccagggcgtg ggcgtgaccg agacccctct gatgaaggag gatagcatcc tggctgtgag 7200gaagtacttt cagaggatca ccctgtacct gaaggagaag aagtacagcc cctgcgcttg 7260ggaagtcgtg agggctgaga tcatgaggag ctttagcctg agcaccaacc tgcaagagag 7320cttgaggtct aaggagtaaa aagtctaggc 73502216051DNAArtificial Sequence16 kbp Ovalbumin Sequence 22ctgcagccca ggcagcacac tagagcagag aaatctagtt agcagcaacc actggcagac 60agaaatgatt atatagatta catactgacc ctagcctctt acactgccta ctgcatcact 120gaaaggactg ggaagaagag agtgcaataa cgaagctgaa gctaggagga aggcaaggag 180aactgaagct gactagggaa aagggggatt aaaggtttaa gtgtctattc catagtttgc 240tggtttgttt tttgtcaatt cctgaatcag taatttttat gttaattagc aaaaaattac 300aaacactccc caagtcagga ctgttaccta caacagaagc tcagatcagc tgagccttag 360tcttttggtc cctccctagg gaatgctgta tgtgtctctc tctccaggcc tgctcaaaat 420tgacctcaga cccaaacttt tgctgaatct ccagtaccac ctcttttgct cctaactaga 480taacaaagcc ctgagcgctt tgcttttagc aaagctttaa gtgccattac caactgcacc 540tggagccttt acctacccct atggacccag gctctatatt taagctctgc cctgaacctt 600cacttctttc ctgtcctaag ttagatgtac tagtatggtg tgtactatgt ctccagttca 660aacacagctg tgcccatacc tggccaagga ctcctagtat gacctgggct gtgccttgct 720gctaaggacc tgctgggtga ttgctggacc tgatcctaat cctgaattaa gaaatgattt 780cttggcttga ctggatgtgc cctgtggtat gatactgcct tatgatttgg actcttgttt 840gcagctgtgc aaatccctaa ggagcccagt ctctggccac ctggaatctt gtcactacca 900aacttcctga gggactggtc ttgctctggg ttctgatctc tggacagtac tcacccttta 960ctcagcccag gctcccagtt aagccccttt ccaccctgcc aggctctccg ctccatccct 1020agcaggggct ctcatgacag tgtgaccccc ccttactcag gtcagggcca cttgtgccac 1080gttcctttcc tgtcttctgt ccctgccttg gctctaaagc agtgtgctac catccacaac 1140cactgcatct ctctaaagta agcctctcct gagcccaagt ctctgtaacg aggaaggatg 1200cactttgctc agaaggatgc gaggctgctt ctgagctctg agggcactga cctcccatga 1260ggtacacccc atacccagga ccacaattca gcctgctgga accatcaact cctgctggag 1320taaggccata gcaagaccag catccacctc cctgcagccc tgccctgccc agatattggg 1380cctgctgatc tcaggatgca gacttgcttc tcagcttgac ctaagcattg ccctgtcttt 1440atggacccac ctggttagca agttcagtgc agaaggaggc tgttggcatc tagctaattt 1500tccacccaca ttactgtctg ctgactcatt ctacgtctct cccatcttgt tacaataata 1560atttgggaga tcatattgaa ggtcttaata aagtcaaggc atgtgatatt ctctgctttg 1620cctttgtttc tagaataagc cacttcatca tagaagatga aaatgctgat cagcagagat 1680ctgtgcttga taaatccatg ctggcttttc ctatcacctt atattccttc atatgccttg 1740agacacccaa ggaggccttg gatcagagct gtctgtagca gtcctaactg gtatacaatt 1800agttgtacaa caggtagtga tccgcataat agttggcgtg agaaagtggg cctgtgctgt 1860gtcaagcata gagtttgggt tccagtcctg ttctgcatgg cacatatgcc tgagcagctg 1920ggtaatctct gcattccaat tggaaggcag gggcctgtag gcagttccca cttggcatgg 1980gtgattgtac cacctgtgtc ctcatctgtg aagcatcatg ttttcattca aatatccttt 2040tgtttgacag tagaaatgaa cagaattgtt tttttttcct aagcaaattc tgcaagagct 2100ctgaagaaca aggtgtcagt gaacttctag ctccatagat aggacttgca tcacatgtca 2160tgccttgatt ggaggtctat ccgatactga acaacttgtg gttccctgag ggaatgtaag 2220attactgata ctactctctc tttatgttag ctacaataaa tggtaggtta agcaatagat 2280acagagtttg agtgcctttc ttacaagcat catagtgaac aaatccactg gtgatctacc 2340ttttcaataa ctacagagaa ttgtaatctc ttggattctc ctccttcccc gttctgaaaa 2400tgtgttcttt ttttccaaat cagaaacctt cctcaaccac cctgactatt ctttggacat 2460tgttttgttc ttgctcctaa ataggcttta taatttttgt aagtgaaagg ctttgcatgc 2520aggtgaggct acaactcatt cagtaacaat gaggaagact gtcagatttt ggggaaaatt 2580ctcccaccca accttttgct agccagtaag atgtaatcac tgaatgtcat gccacaaaga 2640ccataccaac atcagaccac atatctacag gaagctttaa ggaatcattg actgtacagt 2700gaagggtaaa tcaaattaaa atgaatgtga ggtctgatac gagatatcct catgggaatc 2760aagagcaaag acaaatagtt tttcacagtc ttgtcatgat ctgtcacaga ccaaggcagc 2820acagcaggca acaatgttgg tctcttcaga atggcacagc accgctgcag aaaaatgcca 2880ggtggactat gaactcacat ccaaaggagc ttgacctgat acctgatttt cttcaaacag 2940gggaaacaac acaatcccac aaaatagctc agagagaaac catcactgat ggctacagca 3000ccaaggtatg caatggcaat ccattcgaca ttcatctgtg acctgagcaa aatgatttat 3060ctctccatga atggttgctt ctttccctca tgaaaaggca atttccacac tcacaatatg 3120caacaaagac aaacagagaa caattaatgt gctccttcct aatgtcaaaa ttgtagtggc 3180aaagaggaga acaaaatctc aagttctgag taggttttag tgattggata agaggctttg 3240acctgtgagc tcacctggac ttcatatcct tttggataaa aagtgctttt ataactttca 3300ggtctccgag tctttattca tgagactgtt ggtttaggga cagacccaca atgaaatgcc 3360tggcatagga aagggcagca gagccttagc tgaccttttc ttgggacaag cattgtcaaa 3420caatgtgtga caaaactatt tgtactgctt tgcacagctg tgctgggcag ggcaatccat 3480tgccacctat cccaggtaac cttccaactg caagaagatt gttgcttact ctctctagac 3540ccccaagtca aaccaactat gcaggtatgc tgacaacgct atgatgacag cctgttctga 3600tcaagatctc atttgttcat ggacaatttt tgttgcttgc agctggtctt ccattgggaa 3660agagtgtagt atatccttct catctgacag aaaagcagaa attctcatgc tccacactta 3720atctacattg ttttaaacca ccagctactt cttggagagg aaaaatggct tttataagac 3780tcacaaaaca aagctctgca agtcaaatgc atacaaaact gttctgtagg tctggaatca 3840ggacactatg tggaagtcaa atagagaagc tttaaaaaaa cctttgggat cattctcatc 3900ttatatttgc agcacgatac tatgacagtg ataactgaca taactgcatc aatttccttg 3960atattttatt tgtcttaaag tacaagacat agagatggac gtaaagatgg acatatgact 4020caggtctgga caggtccgtg gtccatgtat gataaaagag atgaagggaa ggagaatgga 4080gactgtctaa gaagggcttc agggacgttc tgaaggcaga tttgactgaa tcagatgtac 4140tgtccaagtc tcatatgtag caatggaaga ctgatattgg agaaatataa agaaatggct 4200gtgaactcaa agtgaccctg aacagaaaag ggatatggag ttaaaataat ggcacagaac 4260tgaggtttat atgatatacc atgggctgca gagggtcaga gtgctccacc atgggcctct 4320cttgggctgc agggaacttc tgttctacac ctggaacacc tcctgccctc ctccgcactg 4380acctcagtgt catcagggct gtttctctca cattttctca ctcacctctc ccaactacca 4440ttgtacagca gttgttctta catcttgctc ctcctgaggt gcatctagca tcgatcactg 4500gctcagctct ggccagtggc agctcccttt tgaggacacg ggacagctgc tgggctctgt 4560tcacagaggc cactccagca gacctccact accacaactt gtagtgtaaa tccactacaa 4620ctttctgagc tacagaaatg aaatggagac cctctctgct atgggataca aaagaggaaa 4680cgtggcgttt agtgctctgg ctcactggta cacccaacca cagggtgaga agcagcctgt 4740tgttattcac tactcttagg acagattatg gtgaattgtt aataaaagca tttcttcata 4800acatccaaag gaggaaatac actaaattat attttttatt tattaattac acatgcttaa 4860ttatatatgg catggttgct ttgaaagaac cttgtcctta ctgaccagat ctgctgtttg 4920ctgagacaaa atggctgaca attttggcca tggtggatac cttccccctt ttctgtagca 4980ttaggacaga agttattctg gagcctgtct gacaagtcag acttgataac tttaagtatt 5040tggaagtgtg cttttcatgc tggatgtcat ctccagaacc tccctgtctg gtaagcagtt 5100ccctgcctta gtaagagccg aaacggtctc tcttttcctt gttatctcac caggatatta 5160caatgtgaca ggactatctg aactacgcca acctgcaaat tccaaatata tatatatata 5220tgtaagatat ctatacacaa attattagtg tttgattgac accagatgac agagaagtgc 5280atctgagaaa acctattccc aatctccttt ctctttctgc agactgacat gcatttcata 5340ggtagagata acatttactg ggaagcacat ctatcatcat aaaaagcagg caagattttc 5400agactttctt agtggctgaa atagaagcaa aagacgtgat taaaaacaaa atgaaacaaa 5460aaaaatcagt tgatacctgt ggtgtagaca tccagcaaaa aaatattatt tgcactacca 5520tcttgtctta agtcctcaga cttggcaagg agaatgtaga tttctacagt atatatgttt 5580tcacaaaagg aaggagagaa acaaaagaaa atggcactga ctaaacttca gctagtggta 5640taggaaagta attctgctta acagagattg cagtgatctc tatgtatgtc ctgaagaatt 5700atgttgtact tttttccccc atttttaaat caaacagtgc tttacagagg tcagaatggt 5760ttctttactg tttgtcaatt ctattatttc aatacagaac aatagcttct ataactgaaa 5820tatatttgct attgtatatt atgattgtcc ctcgaaccat gaacactcct ccagctgaat 5880ttcacaattc ctctgtcatc tgccaggcca ttaagttatt catggaagat ctttgaggaa 5940cactgcaagt tcatatcata aacacatttg aaattgagta ttgttttgca ttgtatggag 6000ctatgttttg ctgtatcctc agaaaaaaag tttgttataa agcattcaca cccataaaaa 6060gatagattta aatattccag ctataggaaa gaaagtgcgt ctgctcttca ctctagtctc 6120agttggctcc ttcacatgca tgcttcttta tttctcctat tttgtcaaga aaataatagg 6180tcacgtcttg ttctcactta tgtcctgcct agcatggctc agatgcacgt tgtagataca 6240agaaggatca aatgaaacag acttctggtc tgttactaca accatagtaa taagcacact 6300aactaataat tgctaattat gttttccatc tctaaggttc ccacattttt ctgttttctt 6360aaagatccca ttatctggtt gtaactgaag ctcaatggaa catgagcaat atttcccagt 6420cttctctccc atccaacagt cctgatggat tagcagaaca ggcagaaaac acattgttac 6480ccagaattaa aaactaatat ttgctctcca ttcaatccaa aatggaccta ttgaaactaa 6540aatctaaccc aatcccatta aatgatttct atggcgtcaa aggtcaaact tctgaaggga 6600acctgtgggt gggtcacaat tcaggctata tattccccag ggctcagcca gtgtctgtac 6660atacagctag aaagctgtat tgcctttagc agtcaagctc gaaaggtaag caactctctg 6720gaattacctt ctctctatat tagctcttac ttgcacctaa actttaaaaa attaacaatt 6780attgtgctat gtgttgtatc tttaagggtg aagtacctgc gtgatacccc ctataaaaac 6840ttctcacctg tgtatgcatt ctgcactatt ttattatgtg taaaagcttt gtgtttgttt 6900tcaggaggct tattctttgt gcttaaaata tgtttttaat ttcagaacat cttatcctgt 6960cgttcactat ctgatatgct ttgcagtttg cttgattaac ttctagccct acagagtgca 7020cagagagcaa aatcatggtg ttcagtgaat tctggggagt tattttaatg tgaaaattct 7080ctagaagttt aattcctgca aagtgcagct gctgatcact acacaagata aaaatgtggg 7140gggtgcataa acgtatattc ttacaataat agatacatgt gaacttatat acagaaaaga 7200aaatgagaaa aatgtgtgtg tgtatactca cacacgtggt cagtaaaaac ttttgagggg 7260tttaatacag
aaaatccaat cctgaggccc cagcactcag tacgcatata aagggctggg 7320ctctgaagga cttctgactt tcacagatta tataaatctc aggaaagcaa ctagattcat 7380gctggctcca aaagctgtgc tttatataag cacactggct atacaatagt tgtacagttc 7440agctctttat aatagaaaca gacagaacaa gtataaatct tctattggtc tatgtcatga 7500acaagaattc attcagtggc tctgttttat agtaaacatt gctattttat catgtctgca 7560tttctcttct gtctgaatgt caccactaaa atttaactcc acagaaagtt tatactacag 7620tacacatgca tatctttgag caaagcaaac catacctgaa agtgcaatag agcagaatat 7680gaattacatg cgtgtctttc tcctagacta catgacccca tataaattac attacttatc 7740tattctgcca tcaccaaaac aaaggtaaaa atacttttga agatctactc atagcaagta 7800gtgtgcaaca aacagatatt tctctacatt tatttttagg gaataaaaat aagaaataaa 7860atagtcagca agcctctgct ttctcatata tctgtccaaa cctaaagttt actgaaattt 7920gctctttgaa tttccagttt tgcaagccta tcagattgtg ttttaatcag aggtactgaa 7980aagtatcaat gaattctagc tttcactgaa caaaaatatg tagaggcaac tggcttctgg 8040gacagtttgc tacccaaaag acaactgaat gcaaatacat aaatagattt atgaatatgg 8100ttttgaacat gcacatgaga ggtggatata gcaacagaca cattaccaca gaattacttt 8160aaaactactt gttaacattt aattgcctaa aaactgctcg taatttactg ttgtagccta 8220ccatagagta ccctgcatgg tactatgtac agcattccat ccttacattt tcactgttct 8280gctgtttgct ctagacaact cagagttcac catgggctcc atcggtgcag caagcatgga 8340attttgtttt gatgtattca aggagctcaa agtccaccat gccaatgaga acatcttcta 8400ctgccccatt gccatcatgt cagctctagc catggtatac ctgggtgcaa aagacagcac 8460caggacacaa ataaataagg tgagcctaca gttaaagatt aaaacctttg ccctgctcaa 8520tggagccaca gcacttaatt gtatgataat gtcccttgga aactgcatag ctcagaggct 8580gaaaatctga aaccagagtt atctaaaagt gtggccacct ccaactccca gagtgttacc 8640caaatgcact agctagaaat cttgaaactg gattgcataa cttctttttg tcataaccat 8700tatttcagct actattattt tcaattacag gttgttcgct ttgataaact tccaggattc 8760ggagacagta ttgaagctca ggtacagaaa taatttcacc tccttctcta tgtccctttc 8820ctctggaagc aaaatacagc agatgaagca atctcttagc tgttccaagc cctctctgat 8880gagcagctag tgctctgcat ccagcagttg ggagaacact gttcataaga acagagaaaa 8940agaaggaagt aacaggggat tcagaacaaa cagaagataa aactcaggac aaaaataccg 9000tgtgaatgag gaaacttgtg gatatttgta cgcttaagca agacagctag atgattctgg 9060ataaatgggt ctggttggaa aagaaggaaa gcctggctga tctgctggag ctagattatt 9120gcagcaggta ggcaggagtt ccctagagaa aagtatgagg gaattacaga agaaaaacag 9180cacaaaattg taaatattgg aaaaggacca catcagtgta gttactagca gtaagacaga 9240caggatgaaa aatagttttg taaacagaag tatctaacta ctttactctg ttcatacact 9300acgtaaaact tactaagtaa taaaactaga ataacaacat ctttctttct ctttgtattc 9360agtgtggcac atctgtaaac gttcactctt cacttagaga catcctcaac caaatcacca 9420aaccaaatga tgtttattcg ttcagccttg ccagtagact ttatgctgaa gagagatacc 9480caatcctgcc agtaagttgc tctaaaatct gatctgagtg tattccatgc caaagctcta 9540ccattctgta atgcaaaaac agtcagagtt ccacatgttt cactaagaaa atttcttttt 9600ctcttgtttt tacaaatgaa agagaggaca aataacattt ctctatcacc gacctgaaac 9660tctacagtct tcagagaatg aatggcttgc taaaagaatg tcaaatctta ctatacagct 9720atttcatatt acactactaa atacactata aggcatagca tgtagtaata cagtgtaaaa 9780tagcttttta cactactata ttattaatat ctgttaattc cagtcttgca tttcacattt 9840gcaaaacgtt ttgaaattcg tatctgaaag ctgaatactc ttgctttaca ggaatacttg 9900cagtgtgtga aggaactgta tagaggaggc ttggaaccta tcaactttca aacagctgca 9960gatcaagcca gagagctcat caattcctgg gtagaaagtc agacaaatgg taaggtagaa 10020catgctttgt acatagtgag agttggttca ccctaatact gagaacttgg atatagctca 10080gccagcgtgc tttgcgttca agcttaccag agctgttgta tgcctgttaa gcagggcata 10140cagtcatgag gctcttgaaa aatcttaaca gacaaagggc aatggaaaat cggagttaag 10200ggatggtagg gataaaatgc atagaaagag gtaccacaat tttgattttt gccctaatgc 10260ctctctgcgt ggttcctcaa tttttctact tcattcctca tctcctcaga gcattccttt 10320ccctcatgct tgaaacacag atgaaagact gtgaattcta actgagatga aaacatccac 10380aaccacacaa cctctggtgt ggagtcacat tctgtgaagg caaaaactag gccacgtaat 10440ctatgcgtgc aagctacgcg taagctatgt gtgtgacagg acaatgtgag gaacatacta 10500tgtgcacaag gactgcagaa taaacaggag caaagttttt gaagaaaaca gagtaaaatc 10560ctgttttcct cttttgttac attctttaca tatatctcaa atttcctctt tggttagaag 10620caagtaatat ttatgtttct tggtactgtt tgggttgaag accattctgg gataagagaa 10680attccagtgg ttcttcccct aatcataaaa tgtcaggttt agtttttttg taacacagaa 10740atctcttcat cttttatctt ttgttgtgat tcttgataga gagagaaaca agacttactg 10800acaatagcag caagaaaatc aatcttggaa gaacaagatt gcaattgcaa aaacaaacca 10860atgtccttgc ccctacatcc tcttccccat aaattctaca ttctctatct accttgtgct 10920tgccaacatg atatacgtaa actctctttt cctattcatt cttaaaggaa ttatcagaaa 10980tgtccttcag ccaagctccg tggattctca aactgcaatg gttctggtta atgccattgt 11040cttcaaagga ctgtgggaga aagcatttaa ggatgaagac acacaagcaa tgcctttcag 11100agtgactgag gtatatgggc ataccttaga gatgtaatct agaatttatg aagagagtag 11160acatgttgtt atatgaacac tgcattagcg tatctgctca tttgtctgca tctctttcag 11220acactgtgtt aaaagcaggg aattttcctt atgtctctct cgtcacaata ttcctgacat 11280tgcaaagctc ctgagaaata acttcagatt ccacttttcc taggaaggct tctggatgag 11340aactaatcat cttaactgta actagacatt tctgcatcca agaataatct ttgttaaaac 11400tatattctct ctctcttttt tttttttttt tggttctcca gcaagaaagc aaacctgtgc 11460agatgatgta ccagattggt ttatttagag tggcatcaat ggcttctgag aaaatgaaga 11520tcctggagct tccatttgcc agtgggacaa tgagcatgtt ggtgctgttg cctgatgaag 11580tctcaggcct tgagcaggta tggccctaga agttggcttc agaatattaa aaacacatgg 11640aaatttagct gttgtaaagc tcttttcaac acagttatcc taaaacattt aaccagcaca 11700aatttcatca tgattcaata tgtgattgtt gcatagaagt gtagatttgt cccactgggt 11760cctgcaatag cccatgctga gcatggcttg ctgaaagaac tgctttagag ggtgaaaagt 11820ttgacacagc agacaagatg attctcacct aagcagctgt tactgtagtg gcttgaactc 11880taaaggtctt gtatctccat tcctgtgcac tgaggagctt cttggaaagt tcatataagg 11940tttactagtt ctaactatta tctcatttgg tggcactcaa tgtgctttgt tcacgtcttc 12000ataaattaat ctatctaaaa attggatgtg gttaaagcaa tttcagaaat aacatgtaca 12060taatgtacaa ttattgatat gaacagaaca caggcatagc atattgtaat taggaggact 12120gtagttattt tgaataggaa acacaatgta ataaatgaga attcattgaa atgttagtat 12180gctaactcaa tctaaattat aaagataaag aggcatttaa tcacagctag atttccatca 12240cttgtgacag acaggcatat gaatgattat gtacagctct aggaaaaaaa gtatgtagga 12300aaactagtac attttgatta gaaagtctga aaatgaggtg ccttgatcaa agagaatacg 12360tgtgtttgag aaaaaaaaag tttggataga ggtggtaaga gagaatatat tgaaatggtg 12420tttctacaaa ctgccatggc cagatttgtg taagagacat tcagtaagta ggcaaggaaa 12480gaaatattac taggtacaaa gcaacatcag taataccaaa agaaaccaat tattccagat 12540gccaatctcg taatagggtt aagagatttc cacccctcta gtggtcacca gtgcaaccag 12600taactttgct aatttacatt ttcttttttt aaatggcaga tatagctttg aactgagtga 12660tcatgaactg gtactgtgta atagatgaag acatacttga cgactaaact tctgattttt 12720aaaaactcaa attctcttga aagatcagtt cccagtctag taacagctga tagtttaagt 12780atcagtaatt ggctaccatt aacaactggc tcctgagagg tcttaaatgt agagacagct 12840ttaaactcaa aagcacagag tgatttttag aatagatttc ccaagcaaag aaaataaaca 12900gggaggagct ttaagggagt agccatctca ttattattat tatttaaaga aatggcagca 12960agcctacaaa agaaaaataa gacagagcag agaagaaaga gtcatggtat gcttttctat 13020cttagcaaaa ttaatctcta catgcctagg aaaaagccat gacaagagca atcagttcaa 13080aaggtgtatg caaaaaacca cataatagta actagtactg cattgccagg aaggaagtta 13140tgtcgccatt ccatggatct cattctcatt tccttgcagc ttgagagtat aatcaacttt 13200gaaaaactga ctgaatggac cagttctaat gttatggaag agaggaagat caaagtgtac 13260ttacctcgca tgaagatgga ggaaaaatac aacctcacat ctgtcttaat ggctatgggc 13320attactgacg tgtttagctc ttcagccaat ctgtctggca tctcctcagc agagagcctg 13380aagatatctc aagctgtcca tgcagcacat gcagaaatca atgaagcagg cagagaggtg 13440gtagggtcag cagaggctgg agtggatgct gcaagcgtct ctgaagaatt tagggctgac 13500catccattcc tcttctgtat caagcacatc gcaaccaacg ccgttctctt ctttggcaga 13560tgtgtttccc cttaaaaaga agaaagctga aaaactctgt cccttccaac aagacccaga 13620gcactgtagt atcaggggta aaatgaaaag tatgttatct gctgcatcca gacttcataa 13680aagctggagc ttaatctaga aaaaaaatca gaaagaaatt acactgtgag aacaggtgca 13740attcactttt cctttacaca gagtaatact ggtaactcat ggatgaaggc ttaagggaat 13800gaaattggac tcacagtact gagtcatcac actgaaaaat gcaacctgat acatcagcag 13860aaggtttatg ggggaaaaat gcagccttcc aattaagcca gatatctgta tgaccaagct 13920gctccagaat tagtcactca aaatctctca gattaaatta tcaactgtca ccaaccattc 13980ctatgctgac aaggcaattg cttgttctct gtgttcctga tactacaagg ctcttcctga 14040cttcctaaag atgcattata aaaatcttat aattcacatt tctccctaaa ctttgactca 14100atcatggtat gttggcaaat atggtatatt actattcaaa ttgttttcct tgtacccata 14160tgtaatgggt cttgtgaatg tgctcttttg ttcctttaat cataataaaa acatgtttaa 14220gcaaacactt ttcacttgta gtatttgaag tacagcaagg ttgtgtagca gggaaagaat 14280gacatgcaga ggaataagta tggacacaca ggctagcagc gactgtagaa caagtactag 14340tgggtgagaa gttgaacaag agtcccctac aagcaactta atctaataag ctagtggtct 14400acatcagcta aaagagcata gtgagggatg aaattggttc tcctttctaa gcatcacctg 14460ggacaactca tctggagcag tgtgtccaat ctgccgctgc cctgatctcg gctggggtga 14520tgggacagac cttggctgcc actgagacat ctgagacact gagatctgtc tcaactcaga 14580tttacccaag aacagctcat tgccaacaga acaaaatctc aaacttatgg ctagtgatga 14640cagcagtcag ttgtcccatc tgtgacccac caaggctggc atgctggaat gagcaggctt 14700tggtggcatg tagttactgg acagcaccac tgacatgggc aggggaaaaa ctgagcatgg 14760tgtaaatcac tgcctcaaag ccacttctct gtgcctgcac catgcttgaa agctcttcta 14820caggagctgg gtttgttcaa gaaagcttct gtttctccca tctgcttctt gtaccttcac 14880agggacagag ttagaagggt acagccatgg ctggaagggg ctgactttca aatgtgccta 14940attttccttt ggttgctgct gcagctgcag aagaaggggt tcagaagcca agagctttga 15000gataaggatg cctaacctat gttgaagaca tttgtgctga cacctcaggc cccaggatag 15060gacaactgct ggattgtggc taacccacta gctacagaac ctaatttata ttaccagatt 15120aggaagagca aaagaacatg tatttataac aggaggtctt ctgtgcttct ctactaaaag 15180gtgctgtgaa ggagcccaca gtgcagcagt gtatgaggcc tgaaagaggc cgcagcacac 15240gaagagccct ggtaggagca gcacacagag gggcaggagg gctgggggaa ctgccaccca 15300tggggacctg tgtgaagcag tgcactcctg aggggtggac tgcgtgggaa aggaaaagaa 15360agcaaacaga cctgtgatga actgtcacac agactgcaga gtgacagagg agggcacgag 15420gcagtgcgcc cactgcaggg agtggcgctc cttcctcaca gcagcgctaa cagcttggca 15480ccaatattca gtagtctgtg gtgatacttt ttccagtttc accacacagc atttcgcttg 15540ttctacttgt tttagctttc cccctccaca agataacaca tactttgcca gtcagtccct 15600aagaccttaa cttaacagtt agcaaacagg atcttgcaaa agaaggaaga taacatgaca 15660ccaccttcac tggtgtataa atagttcaaa tactttcctt cactttcccg taaattagtt 15720gattgcaggt caggagataa caggggaact tactgcaaga gagaaaatga tgtttaatat 15780tgtcttggac tttctggtgg tctgggcatg aaaatggggt actcaaaatc ctcgggacgt 15840ttatttttca cctgatttat tcccaaactg cactatttct aggccattgg agttcttatc 15900aattaaatta tactttggct ctctgctatc tcactccctt tcatcttcag catcactttc 15960agcacaatta caggagaaga cttagactca gagctttagg actcatcata agaggctttc 16020attgctctgt caccacaccc catatagatc t 16051237334DNAArtificial SequencepBS-OM-4.4 Vector 23atcaagctta tcgataccgt cgacctcgag ggggggcccg gtacccagct tttgttccct 60ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 120ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 180gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 240gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 300tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 360gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 420ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 480ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 540acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 600tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 660ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 720ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 780ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 840actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 900gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 960tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 1020caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 1080atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 1140acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 1200ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 1260ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 1320tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 1380tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 1440gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 1500tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 1560tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 1620ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 1680tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 1740ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 1800gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 1860ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 1920cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 1980ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 2040ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 2100gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 2160ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 2220gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc 2280gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc 2340ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag 2400agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc 2460gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa 2520gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg 2580aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt 2640gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc 2700gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 2760tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 2820ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca 2880ctatagggcg aattggagct ccaccgcggt ggcggccgct ctagaactag tggatccttc 2940ttaaaaagca gaccatcatt cactgcaaac ccagagcttc atgcctctcc ttccacaacc 3000gaaaacagcc ggcttcattt gtctttttta aatgctgttt tccaggtgaa ttttggccag 3060cgtgttggct gagatccagg agcacgtgtc agctttctgc tctcattgct cctgttctgc 3120attgcctctt tctggggttt ccaagagggg gggagacttt gcgcggggat gagataatgc 3180cccttttctt agggtggctg ctgggcagca gagtggctct gggtcactgt ggcaccaatg 3240ggaggcacca gtgggggtgt gttttgtgca ggggggaagc attcacagaa tggggctgat 3300cctgaagctt gcagtccaag gctttgtctg tgtacccagt gaaatccttc ctctgttaca 3360taaagcccag ataggactca gaaatgtagt cattccagcc cccctcttcc tcagatctgg 3420agcagcactt gtttgcagcc agtcctcccc aaaatgcaca gacctcgccg agtggaggga 3480gatgtaaaca gcgaaggtta attacctcct tgtcaaaaac actttgtggt ccatagatgt 3540ttctgtcaat cttacaaaac agaaccgaga ggcagcgagc actgaagagc gtgttcccat 3600gctgagttaa tgagacttgg cagctcgctg tgcagagatg atccctgtgc ttcatgggag 3660gctgtaacct gtctccccat cgccttcaca ccgcagtgct gtcctggaca cctcaccctc 3720cataagctgt aggatgcagc tgcccaggga tcaagagact tttcctaagg ctcttaggac 3780tcatctttgc cgctcagtag cgtgcagcaa ttactcatcc caactatact gaatgggttt 3840ctgccagctc tgcttgtttg tcaataagca tttcttcatt ttgcctctaa gtttctctca 3900gcagcaccgc tctgggtgac ctgagtggcc acctggaacc cgaggggcac agccaccacc 3960tccctgttgc tgctgctcca gggactcatg tgctgctgga tggggggaag catgaagttc 4020ctcacccaga cacctgggtt gcaatggctg cagcgtgctc ttcttggtat gcagattgtt 4080tccagccatt acttgtagaa atgtgctgtg gaagcccttt gtatctcttt ctgtggccct 4140tcagcaaaag ctgtgggaaa gctctgaggc tgctttcttg ggtcgtggag gaattgtatg 4200ttccttcttt aacaaaaatt atccttagga gagagcactg tgcaagcatt gtgcacataa 4260aacaattcag gttgaaaggg ctctctggag gtttccagcc tgactactgc tcgaagcaag 4320gccaggttca aagatggctc aggatgctgt gtgccttcct gattatctgt gccaccaatg 4380gaggagattc acagccactc tgcttcccgt gccactcatg gagaggaata ttcccttata 4440ttcagataga atgttatcct ttagctcagc cttccctata accccatgag ggagctgcag 4500atccccatac tctccccttc tctggggtga aggccgtgtc ccccagcccc ccttcccacc 4560ctgtgcccta agcagcccgc tggcctctgc tggatgtgtg cctatatgtc aatgcctgtc 4620cttgcagtcc agcctgggac atttaattca tcaccagggt aatgtggaac tgtgtcatct 4680tcccctgcag ggtacaaagt tctgcacggg gtcctttcgg ttcaggaaaa ccttcactgg 4740tgctacctga atcaagctct atttaataag ttcataagca catggatgtg ttttcctaga 4800gatacgtttt aatggtatca gtgattttta tttgctttgt tgcttacttc aaacagtgcc 4860tttgggcagg aggtgaggga cgggtctgcc gttggctctg cagtgatttc tccaggcgtg 4920tggctcaggt cagatagtgg tcactctgtg gccagaagaa ggacaaagat ggaaattgca 4980gattgagtca cgttaagcag gcatcttgga gtgatttgag gcagtttcat gaaagagcta 5040cgaccactta ttgttgtttt ccccttttac aacagaagtt ttcatcaaaa taacgtggca 5100aagcccagga atgtttggga aaagtgtagt taaatgtttt gtaattcatt tgtcggagtg 5160ctaccagcta agaaaaaagt cctacctttg gtatggtagt cctgcagaga atacaacatc 5220aatattagtt tggaaaaaaa caccaccacc accagaaact gtaatggaaa atgtaaacca 5280agaaattcct tgggtaagag agaaaggatg tcgtatactg gccaagtcct gcccagctgt 5340cagcctgctg accctctgca gttcaggacc atgaaacgtg gcactgtaag acgtgtcccc 5400tgcctttgct tgcccacaga tctctgccct tgtgctgact cctgcacaca agagcatttc 5460cctgtagcca aacagcgatt agccataagc tgcacctgac tttgaggatt aagagtttgc 5520aattaagtgg attgcagcag gagatcagtg gcagggttgc agatgaaatc cttttctagg 5580ggtagctaag ggctgagcaa cctgtcctac agcacaagcc aaaccagcca agggttttcc 5640tgtgctgttc acagaggcag ggccagctgg agctggagga ggttgtgctg ggacccttct 5700ccctgtgctg agaatggagt gatttctggg tgctgttcct gtggcttgca ctgagcagct 5760caagggagat cggtgctcct catgcagtgc caaaactcgt gtttgatgca gaaagatgga 5820tgtgcacctc cctcctgcta atgcagccgt gagcttatga aggcaatgag ccctcagtgc 5880agcaggagct gtagtgcact cctgtaggtg ctagggaaaa tctctggttc ccagggatgc 5940attcataagg gcaatatatc ttgaggctgc gccaaatctt tctgaaatat tcatgcgtgt 6000tcccttaatt tatagaaaca aacacagcag aataattatt ccaatgcctc ccctcgaagg 6060aaacccatat ttccatgtag aaatgtaacc tatatacaca cagccatgct gcatccttca 6120gaacgtgcca gtgctcatct cccatggcaa aatactacag gtattctcac tatgttggac 6180ctgtgaaagg
aaccatggta agaaacttcg gttaaaggta tggctgcaaa actactcata 6240ccaaaacagc agagctccag acctcctctt aggaaagagc cacttggaga gggatggtgt 6300gaaggctgga ggtgagagac agagcctgtc ccagttttcc tgtctctatt ttctgaaacg 6360tttgcaggag gaaaggacaa ctgtactttc aggcatagct ggtgccctca cgtaaataag 6420ttccccgaac ttctgtgtca tttgttctta agatgctttg gcagaacact ttgagtcaat 6480tcgcttaact gtgactaggt ctgtaaataa gtgctccctg ctgataaggt tcaagtgaca 6540tttttagtgg tatttgacag catttacctt gctttcaagt cttctaccaa gctcttctat 6600acttaagcag tgaaaccgcc aagaaaccct tccttttatc aagctagtgc taaataccat 6660taacttcata ggttagatac ggtgctgcca gcttcacctg gcagtggttg gtcagttctg 6720ctggtgacaa agcctccctg gcctgtgctt ttacctagag gtgaatatcc aagaatgcag 6780aactgcatgg aaagcagagc tgcaggcacg atggtgctga gccttagctg cttcctgctg 6840ggagatgtgg atgcagagac gaatgaagga cctgtccctt actcccctca gcattctgtg 6900ctatttaggg ttctaccaga gtccttaaga ggtttttttt ttttttggtc caaaagtctg 6960tttgtttggt tttgaccact gagagcatgt gacacttgtc tcaagctatt aaccaagtgt 7020ccagccaaaa tcaattgcct gggagacgca gaccattacc tggaggtcag gacctcaata 7080aatattacca gcctcattgt gccgctgaca gattcagctg gctgctccgt gttccagtcc 7140aacagttcgg acgccacgtt tgtatatatt tgcaggcagc ctcgggggga ccatctcagg 7200agcagagcac cggcagccgc ctgcagagcc gggcagtacc tcaccatggc catggcaggt 7260gtcttcgtgc tgttctcttt cgtgctttgt ggcttcctcc caggtgagta actcccagag 7320tgctgcagaa gctt 7334244327DNAArtificial SequencepAVIJCR-A137.91.1.2 Vector 24gccaatgtgg tacttcccac attgtataag aaatttggca agtttagagc aatgtttgaa 60gtgttgggaa atttctgtat actcaagagg gcgtttttga caactgtaga acagaggaat 120caaaaggggg tgggaggaag ttaaaagaag aggcaggtgc aagagagctt gcagtcccgc 180tgtgtgtacg acactggcac catggctttg acctttgcct tactggtggc tctcctggtg 240ctgagctgca agagcagctg ctctgtgggc tgcgatctgc ctcagaccca cagcctgggc 300agcaggagga ccctgatgct gctggctcag atgaggagaa tcagcctgtt tagctgcctg 360aaggataggc acgattttgg ctttcctcaa gaggagtttg gcaaccagtt tcagaaggct 420gagaccatcc ctgtgctgca cgagatgatc cagcagatct ttaacctgtt tagcaccaag 480gatagcagcg ctgcttggga tgagaccctg ctggataagt tttacaccga gctgtaccag 540cagctgaacg atctggaggc ttgcgtgatc cagggcgtgg gcgtgaccga gacccctctg 600atgaaggagg atagcatcct ggctgtgagg aagtactttc agaggatcac cctgtacctg 660aaggagaaga agtacagccc ctgcgcttgg gaagtcgtga gggctgagat catgaggagc 720tttagcctga gcaccaacct gcaagagagc ttgaggtcta aggagtaaaa agtctagagt 780cggggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc 840acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 900tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 960tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt 1020ggtaaaatcg ataaggatcc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc 1080cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat 1140gcaactcgta ggacaggtgc cggcagcgct cttccgcttc ctcgctcact gactcgctgc 1200gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 1260ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 1320ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 1380atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 1440aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 1500gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 1560ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 1620ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 1680acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 1740gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 1800ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 1860ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 1920gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 1980ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 2040agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 2100ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 2160gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 2220catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 2280cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 2340cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 2400gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 2460tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 2520gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 2580tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 2640gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 2700gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 2760taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 2820tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 2880ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 2940taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 3000tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 3060aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg 3120cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 3180tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 3240gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 3300accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 3360tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 3420gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 3480cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 3540tattaacgtt tacaatttcc cattcgccat tcaggctgcg caactgttgg gaagggcgat 3600cggtgcgggc ctcttcgcta ttacgccagc ccaagctacc atgataagta agtaatatta 3660aggtacggga ggtacttgga gcggccgcaa taaaatatct ttattttcat tacatctgtg 3720tgttggtttt ttgtgtgaat cgatagtact aacatacgct ctccatcaaa acaaaacgaa 3780acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagt gcaggtgcca gaacatttct 3840ctatcgatag gtaccgagct cttacgcgtg ctagccccga tgtacgggcc agatatacgc 3900gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 3960gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 4020ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 4080ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 4140atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 4200cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 4260tattagtcat cgctattacc atgcatggct ttgacctttg ccttactggt ggctctcctg 4320gtgctta 432725244DNAArtificial SequenceThe RRE (rev responsive element) sequence 25aattgaggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 60tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 120aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 180aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 240gtac 24426158DNAArtificial SequenceThe ALV CTE (constitutive transport element) sequence 26aatgtgggga gggcaaggct tgcgaatcgg gttgtaacgg gcaaggcttg actgagggga 60caatagcatg tttaggcgaa aagcggggct tcggttgtac gcggttagga gtcccctcag 120gatatagtag tttcgctttt gcatagggag ggggaaat 1582755DNAArtificial Sequencep10.0-OM-IFN-1 Primer 27ggcgtcgacg gatccgttaa ccctagaact agtggatctc tgcccttgtg ctgac 552831DNAArtificial Sequencep10.0-OM-IFN-2 28ggcctcgagc ctagactttt tactccttag a 3129346DNAArtificial SequenceALV vector 5' LTR sequence 29aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg cttatgtaac gatgagttag 60caacatgcct tataaggaga gaaaaagcac cgtgcatgcc gattggtggg agtaaggtgg 120tatgatcgtg gtatgatcgt gccttgttag gaaggcaaca gacgggtcta acacggattg 180gacgaaccac tgaattccgc attgcagaga tattgtattt aagtgcctag ctcgatacaa 240taaacgccat ttgaccattc accacattgg tgtgcacctg ggttgatggc cggaccgttg 300attccctgrc gactacgagc acatgcatga agcagaaggc ttcatt 346
Patent applications by Alex J. Harvey, Athens, GA US
Patent applications by Jeffrey C. Rapp, Athens, GA US
Patent applications in class Avian cell, per se
Patent applications in all subclasses Avian cell, per se
2011-08-25 Method for the production of adenoviral vectors
2010-01-07 Production of lentiviral vectors
2010-07-22 Targeting pseudotyped retroviral vectors
2011-11-24 Production of glycoproteins using manganese
2012-01-12 Restoration of nucleic acid from degraded or formalin-fixed and paraffin-embedded tissue and uses thereof
2016-01-07 Method for suppressing teratoma formation via selective cell death induction in undifferentiated human-induced pluripotent stem cells
2015-04-30 Influenza virus reassortment method
2015-03-12 Method for producing continuous cell lines
2014-07-03 Use of cellular extracts for obtaining pluripotent stem cells
2013-10-31 Engineered kinetochores and uses thereof
2015-01-29 Lysosomal storage disease enzyme
2015-01-22 Npp1 fusion proteins
2014-12-25 Npp1 fusion proteins
2014-11-13 Avian derived antibodies
2014-10-02 Glycosylation in avians