pFB-ERV逆病毒载体使用说明

pFB-ERVpFB-ERV 逆病毒载体基本信息：载体名称:pFB-ERV 质粒类型: 逆病毒载体；双顺反子载体高拷贝/低拷贝: 低拷贝克隆方法: 限制性内切酶，多克隆位点启动子:CMV 载体大小:11067 bp 5'测序引物及序列:-- 3' 测序引物及序列:-- 载体标签:-- 载体抗性: 卡那霉素筛选标记: Neomycin1.html' target='_blank'>新霉素（Neomycin ） 克隆菌株: DH5α 等宿主细胞（系）: 常规细胞系（293、CV-1、CHO 等）备注:-- 稳定性: 稳表达组成型/诱导型: 组成型病毒/非病毒: 逆转录病毒pFB-ERV 载体质粒图谱和多克隆位点信息：pFB-ERV载体简介：pFB-ERV载体描述DNA vector-based systems that allow precise control of gene expression in vivo have become invaluable for the study of gene function in a variety of organisms, particularly when applied to the study of developmental and other biological processes for which the timing or dosage of gene expression is critical to gene function. Such systems have also been successfully used to overexpress toxic or disease-causing genes, to induce gene targeting, and to express antisense RNA. Inducible systems are currently being used by pharmaceutical companies to facilitate screening for inhibitors of clinically relevant biological pathways, and potential applications for gene therapy are being explored.The Agilent Complete Control ecdysone-inducible plasmid vectors are based on the insect molting hormone ecdysone, which can stimulate transcriptional activation in mammalian cells harboring the ecdysone receptor protein from Drosophila melanogaster.2 The system has a numberof advantages over alternative systems. Firstly, the lipophilic nature and short in vivo half-life of the ecdysone analog ponasterone A (ponA) allows efficient penetrance into all tissues including brain, resulting in rapid and potent inductions and rapid clearance.Secondly, ecdysteroids are not known, nor are they expected, to affect mammalian physiology in any measurable way. Thirdly, the heterodimeric ponA responsive receptor and receptor DNA recognition element have been genetically altered such that trans-activation of endogenous genes by the ecdysone receptor, or of theponA-responsive expression cassette by endogenous transcription factors, is extremely unlikely. In addition, it has been found that in the absence of inducer the heterodimer remains bound at the promoter in a complex with corepressors and histone deacetylase, and is thus tightly repressed until ligand binding, at which time high-level transcriptional activation occurs (i.e., the heterodimer is converted from a tight repressor to a transactivator). In transient assays and stable cell lines harboring receptor expression plasmids in combination with a plasmid bearing an inducible luciferase expression cassette, induction ratios of 1,000-fold have been achieved.3A limitation to the use of plasmid-based vectors for controlled gene expression is the fact that many cell types of academic, industrial or clinical interest are difficult or virtually impossible to transfect using current transfection methods. In particular, primary human cell lines, lymphocytes, neurons and other nondividing cells are best transduced using viral delivery systems. The most popular and user-friendly of these are the retroviral vectors. Infection with retroviruses often yields transduction efficiencies close to 100%, and the proviral copy number can be easily controlled by varying the multiplicity of infection (MOI). This latter feature is particularly important for inducible systems, for which low basal expression and high induction ratios are affected by copy number. Thus infection of the target cell with virus at an optimal MOI should yield a high frequency of clones capable of mediating desirable expression profiles without exhaustive colony screening.With the vectors pFB-ERV and pCFB-EGSH, we have adapted the ecdysone inducible components of the Complete Control System for retroviral delivery. Used together, we have attained induction ratios of >1,000-fold with these vectors in tissue culture cells.OVERVIEW OF ECDYSONE-REGULATABLE GENE EXPRESSIONThe ecdysone receptor (EcR) is a member of the retinoid-X-receptor (RXR) family of nuclear receptors and is composed of three domains: an N-terminal activation domain (AD), a central DNA-binding domain (DBD), and a C-terminal ligand-binding and dimerization domain (LBD). In insect cells, EcR and the nuclear receptor ultraspiracle (USP) form a promoterbound heterodimer, which regulates transcription (see Figure 1). In the absence of ecdysone, the receptor heterodimer binds to corepressors and tightly represses transcription.4When ecdysone binds to the EcR LBD, the corepressors are released, coactivators are recruited to the complex, and transcriptional activation is enabled.In mammalian cells harboring the EcR gene, EcR heterodimerizes with RXR, the mammalian homologue of USP. The EcR–RXR heterodimer binds to multiple copies of the ecdysone-responsive element (EcRE), and in the absence of ponA, repressestranscription of an expression cassette. When ponA binds to the receptor, the receptor complex activates transcription of a reporter gene or a gene of interest. To avoid pleiotropic interactions with endogenous pathways in mammalian host cells, both the EcRE recognition sequence and the EcR protein were modified.The EcRE sequence was modified to create a synthetic recognition site that does not bind any endogenous transcription factors. The wild-type EcRE sequence consists of two inverted repeat sequences separated by a single nucleotide: AGTGCA N TGCACT. The EcRE sequence was changed to AGTGCA N1 TGTTCT (and renamed E/GRE). Recognition of the synthetic E/GRE recognition sequence by either a steroid receptor or a wild-type RXR heterodimer receptor is extremely unlikely, as these receptors recognized only the wild-type perfect inverted repeat. The E/GRE recognition sequence has imperfect inverted half sites separated by one nucleotide. A wild-type RXR heterodimer requires single nucleotide separation of the inverted repeats, and the majority bind to direct repeats rather than inverted repeats (EcRE is an exception).The EcR protein was modified to create a synthetic ecdysone-binding receptor that does not transactivate any host genes. Three amino acids in the EcR DBD were mutated to change its DNA-binding specificity to that of the glucocorticoid receptor (GR), which recognizes the half-site AGAACA.2 Like all steroid receptors and unlike RXR receptors, the GR protein homodimerizes and recognizes two inverted repeat sequences separated by three nucleotides. The GR–EcR fusion protein (GEcR) retains the ability to dimerize with RXR and activate, with ponA-dependence, reporter genes that contain the synthetic E/GRE recognition sequence.The GEcR receptor was further modified by replacing the EcR AD with the more potent VP16 AD. The result of all the modifications is the synthetic ecdysone-binding receptor VgEcR. VgEcR is a fusion of the ligand-binding and dimerization domain of the D. melanogaster ecdysone receptor, the DNA-binding domain of the glucocorticoid receptor, and the transcription activation domain of herpes simplex virus (HSV) VP16OVERVIEW OF REPLICATION-DEFECTIVE RETROVIRAL GENE TRANSFER SYSTEMSNon-replicating retroviral vectors contain all of the cis elements required for transcription of mRNA molecules encoding a gene of interest, and packaging of these transcripts into infectious virus particles (Figure 2). The vectors are typically comprised of an E. coli plasmid backbone containing a pair of 600 base pair viral long terminal repeats (LTRs) between which the gene of interest is inserted. The LTR is divided into 3 regions. The U3 region contains the retroviral promoter/enhancer. The U3 region is flanked in the 3′ direction by the R region, which contains the viral polyadenylation signal (pA), followed by the U5 region which, along with R, contains sequences that are critical for reverse transcription. Expression of the viral RNA is initiated within the U3 region of the 5′ LTR and is terminated in the R region of the 3′ LTR. Between the 5′ LTR and the coding sequence for the gene of interest resides an extended version of the viral packaging signal (ψ+), which is required in cis for the viral RNA to be packagedinto virion particles.In order to generate infectious virus particles that carry the gene of interest, specialized packaging cell lines have been generated that contain chromosomally integrated expression cassettes for viral Gag, Pol and Env proteins, all of which are required in trans to make virus. The gag gene encodes internal structural proteins, pol encodes reverse transcriptase (RT) and integrase, and the env gene encodes the viral envelope protein, which resides on the viral surface and facilitates infection of the target cell by direct interaction with cell type-specific receptors; thus the host range of the virus is dictated not by the DNA vector but by the choice of the env gene used to construct the packaging cell. The packaging cell line is transfected with the vector DNA, and at this point either stable viral producer cell lines may be selected (providing the vector has an appropriate selectable marker), or mRNAs that are transiently transcribed from the vector are encapsidated and bud off into the cell supernatant. These supernatants are collected, and used to infect target cells. Upon infection of the target cell, the viral RNA molecule is reverse transcribed by RT (which is present in the virion particle), and the cDNA of the gene of interest, flanked by the LTRs, is integrated into the host DNA. Because the vector itself carries none of the viral proteins, once a target cell is infected the LTR expression cassette is incapable of proceeding through another round of virus production. Recent advances in transfection technology have allowed the production of high titer viral supernatants following transient cotransfection of the viral vector together with expression vectors encoding the gag, pol and env genes (Figure 2),5, 6 obviating the need for the production and maintenance of stable packaging cell lines. For example, Agilent pVPack gag-pol and env-expressing packaging vectors consistently give rise to titers of >107 infectious units (IU)/ml when cotransfected with the pFB-hrGFP control vector (Agilent Catalog #240027), using a 293-derived cell line for virus production.Description of the VectorsThe pFB-ERV vector was derived from the high-titer MoMLV vector pFBNeo5 for efficient delivery of the ecdysone receptor proteins VgEcR and RXR (Figure 3). In the vector pFB-ERV the ecdysone receptor and the neomycin-resistance open reading frame (ORF) are expressed from a tricistronic message with the neomycin resistance ORF expressed at the end of the message. Thus, maintenance of infected cell lines in G418 ensures expression of the transcript encoding the receptor genes. The tricistronic transcript is expressed from the CMV promoter, which is flanked by unique EcoR I and Fse I sites so that a cell type-specific promoter of interest may be substituted. The viral promoter within the 3′ LTR has been deleted to make this aself-inactivating (SIN) vector. Upon infection and chromosomal integration into the target cell genome, the SIN deletion is transferred to the 5′ LTR, resulting in an integrated expression cassette in which only the CMV promoter is active. Cells containing an estimated single integrated viral expression cassette can be selected in as high as 1 mg/ml G418, although 600 μg/ml is routinely used.The vector pCFB-EGSH contains an ecdysone-inducible expression cassette inserted between the viral LTRs in the antisense orientation relative to that for the viral promoter (see Figure 4). The U3 promoter within the 5′ LTR of the vector has been replaced with the CMV promoter to increase production of viral RNA in packaging cells, thereby increasing the titer of the viral supernatants. Potential interference from the proviral 5′ LTR is obviated due to the SIN deletion. The inducible expression cassette contains a multiple cloning site that contains three contiguous copies of the HA epitope(3× HA) positioned for fusion at the C-terminus of the protein of interest. A second expression cassette in which the hygromycin-resistance gene is expressed from the TK promoter is located downstream (relative to transcription from the LTRs) of the inducible cassette. A pBR322 origin and ampicillin-resistance gene allow pCFB-EGSH to be propagated in prokaryotes.The pCFB-EGSH-Luc vector contains the luciferase reporter gene and is intended for use as a positive control vector to test the expression of the VgEcR and RXR receptors in pFB-ERV-containing cell lines. The pCFB-EGSH-Luc vector is derived from the pCFB-EGSH vector and has the luciferase gene inserted in the MCS. ThepCFB-EGSH-Luc vector does not contain the HA epitope sequence.pFB-ERV载体限制性酶切位点pFB-ERV, 11067 bp version 011006Enzymes with 1-10 cleavage sites:#sites -- Bp position of recognition site --AarI 3 5297, 6078, 7229AatII 7 978, 2200, 2253, 2336, 25223692, 10993Acc65I 8 647, 1847, 2966, 3609, 47925409, 7341, 8593AccI 6 1968, 2058, 3212, 3762, 62348953AccIII 1 8514AclI 3 4990, 10300, 10673AflII 3 202, 1265, 8466AflIII 9 164, 2820, 3249, 5283, 54587215, 7390, 8545, 9182AgeI 2 1841, 6125AhdI 6 687, 733, 1274, 8633, 867910070AleI 2 5607, 6402Alw44I 6 1054, 5445, 7377, 8998, 949610742AlwNI 3 323, 398, 9593ApaI 5 1238, 5081, 5754, 5787, 7013ApoI 4 87, 1128, 2064, 2986AscI 2 3231, 8537AseI 2 2085, 10246AvaI 10 577, 610, 643, 1241, 20194259, 4340, 5700, 8556, 8589 BamHI 4 3167, 6272, 6425, 6539BbeI 7 615, 1656, 4376, 6598, 69397704, 8561BbsI 10 2714, 3947, 4148, 5101, 52015567, 7033, 7133, 7499, 11060 BbvCI 6 453, 4448, 4454, 4526, 45386744BciVI 7 656, 1987, 3038, 7913, 86029391, 10918BclI 1 8508BfrBI 1 8519BglI 10 2163, 2285, 2356, 3926, 41224859, 5264, 5906, 7196, 10189 BglII 2 1678, 4266BlnI 3 5119, 7051, 7542BlpI 4 4349, 5850, 6283, 6817BmgBI 4 2774, 5508, 6562, 7440BmrI 6 2373, 5318, 7250, 7644, 892810120BmtI 6 6, 16, 26, 197, 47728461BpmI 7 1799, 4049, 4592, 6277, 69348414, 10160Bpu10I 10 337, 412, 453, 1548, 44484454, 4526, 4538, 5730, 6744 BpuEI 8 5370, 5920, 7302, 8096, 92889550, 9827, 10695BsaAI 8 2001, 2417, 5282, 6316, 72148007, 8524, 8934BsaBI 1 2807BsaI 10 694, 715, 782, 1414, 18026310, 8640, 8661, 8728, 10142 BsgI 3 5668, 6184, 6559BsiWI 1 4187BsmBI 8 976, 1093, 1337, 1396, 15824337, 4630, 8831BsmI 7 3317, 4894, 5114, 5147, 57127046, 7079BspHI 3 9902, 10910, 11015BspMI 7 3085, 3951, 5298, 6079, 72307601, 7964BsrDI 6 3420, 5072, 7004, 7931, 1012910311BsrGI 3 1541, 3252, 6017BssHII 6 563, 3232, 3551, 6681, 81028538BssSI 4 8297, 9355, 10739, 11046Bst1107I 1 8953BstAPI 4 322, 397, 4392, 9000BstEII 2 1346, 4961Bsu36I 3 1276, 5939, 5996BtsI 4 4015, 4124, 10468, 10496ClaI 2 5534, 7466DraI 3 9939, 9958, 10650DraIII 3 1874, 5326, 7258DrdI 4 6454, 7726, 8871, 9284EagI 7 961, 2679, 3311, 4040, 67286962, 7611EcoICRI 3 580, 3403, 5733EcoNI 2 1647, 4048EcoRI 1 2064EcoRV 2 308, 383FseI 1 2676FspI 3 6059, 7805, 10295HincII 5 2058, 3663, 4551, 6234, 10614 HindIII 3 2957, 5192, 7124KasI 7 615, 1656, 4376, 6598, 69397704, 8561KpnI 8 647, 1847, 2966, 3609, 47925409, 7341, 8593MluI 1 8545MmeI 10 684, 1383, 3823, 4811, 49365336, 7268, 8630, 9372, 9556 MscI 7 827, 1368, 1668, 3537, 61586548, 7785MunI 2 11, 21NaeI 5 2677, 4388, 4735, 6837, 8205 NarI 7 615, 1656, 4376, 6598, 69397704, 8561NcoI 7 2439, 2688, 5651, 5783, 61617560, 8137NdeI 4 1664, 1672, 2312, 9004 NgoMIV 5 2677, 4388, 4735, 6837, 8205NheI 6 6, 16, 26, 197, 47728461NotI 1 6961NruI 2 3004, 3885NsiI 1 8519PciI 6 164, 2820, 3249, 5458, 73909182PfoI 6 771, 2848, 3327, 6275, 87178826PmlI 3 2001, 5282, 7214PpuMI 8 499, 1477, 1925, 3082, 47235924, 6065, 8530PshAI 2 1015, 2731PspOMI 5 1238, 5081, 5754, 5787, 7013 PstI 4 1178, 1360, 7754, 10316PvuI 4 1034, 5532, 7464, 10442PvuII 6 286, 361, 4541, 5707, 60217809RsrII 2 4035, 8220SacI 3 580, 3403, 5733SacII 3 151, 4431, 4677SalI 2 2058, 6234SanDI 1 8530SapI 5 3070, 3202, 8054, 8264, 9059 ScaI 2 6134, 10553SexAI 3 1474, 4663, 6458SfcI 10 182, 1178, 1360, 5216, 71487754, 8446, 9447, 9638, 10316 SfoI 7 615, 1656, 4376, 6598, 69397704, 8561SmaI 5 643, 1241, 2019, 5700, 8589 SnaBI 2 2417, 8524SpeI 1 897SphI 3 2788, 3502, 8106SrfI 1 1240SspI 3 3532, 3877, 10877StuI 2 3813, 6773TatI 10 1541, 2296, 2376, 2409, 24603252, 6017, 6134, 8988, 10553 TfiI 8 2837, 3469, 3911, 4493, 46038190, 8324, 9157Tsp45I 9 1282, 1491, 6364, 7826, 81328837, 8932, 10332, 10543Tth111I 8 633, 1473, 3992, 6358, 64577820, 8579, 8926Van91I 2 5415, 7347XbaI 3 464, 2037, 2972XcmI 2 5741, 5813XhoI 2 4259, 4340XmaI 5 643, 1241, 2019, 5700, 8589 XmnI 3 5176, 7108, 10670ZraI 7 978, 2200, 2253, 2336, 25223692, 10993Enzymes that do NOT cut molecule:AsiSI BstBI BstXI Eco47III FspAIHpaI PacI PmeI PsiI SbfISfiI SgrAI SwaIpFB-ERV载体序列：pFB-ERV, 11067 bp version 011006 NOTE: The following sequence has been verified for accuracyat the junctions. The remainder of the sequence has beenobtained from existing data.1 GAATTGCTAG CAATTGCTAG CAATTGCTAG CAATTCATAC CAGATCACCG51 AAAACTGTCC TCCAAATGTG TCCCCCTCAC ACTCCCAAAT TCGCGGGCTT101 CTGCCTCTTA GACCACTCTA CCCTATTCCC CACACTCACC GGAGCCAAAG151 CCGCGGGACA TATACATGTG AAAGACCCCA CCTGTAGGTT TGGCAAGCTA201 GCTTAAGTAA CGCCATTTTG CAAGGCATGG AAAAATACAT AACTGAGAAT251 AGAAAAGTTC AGATCAAGGT CAGGAACAGA TGGAACAGCT GAATATGGGC301 CAAAGCGGAT ATCTGTGGTA AGCAGTTCCT GCCCCGGCTC AGGGCCAAGA351 ACAGATGGAA CAGCTGAATA TGGGCCAAAC AGGATATCTG TGGTAAGCAG401 TTCCTGCCCC GGCTCAGGGC CAAGAACAGA TGGTCCCCAG ATGCGGTCCA451 GCCCTCAGCA GTTTCTAGAG AACCATCAGA TGTTTCCAGG GTGCCCCAAG501 GACCTGAAAT GACCCTGTGC CTTATTTGAA CTAACCAATC AGTTCGCTTC551 TCGCTTCTGT TCGCGCGCTT CTGCTCCCCG AGCTCAATAA AAGAGCCCAC601 AACCCCTCAC TCGGGGCGCC AGTCCTCCGA TTGACTGAGT CGCCCGGGTA651 CCCGTGTATC CAATAAACCC TCTTGCAGTT GCATCCGACT TGTGGTCTCG701 CTGTTCCTTG GGAGGGTCTC CTCTGAGTGA TTGACTACCC GTCAGCGGGG751 GTCTTTCATT TGGGGGCTCG TCCGGGATCG GGAGACCCCT GCCCAGGGAC801 CACCGACCCA CCACCGGGAG GTAAGCTGGC CAGCAACTTA TCTGTGTCTG851 TCCGATTGTC TAGTGTCTAT GACTGATTTT ATGCGCCTGC GTCGGTACTA901 GTTAGCTAAC TAGCTCTGTA TCTGGCGGAC CCGTGGTGGA ACTGACGAGT951 TCGGAACACC CGGCCGCAAC CCTGGGAGAC GTCCCAGGGA CTTCGGGGGC 1001 CGTTTTTGTG GCCCGACCTG AGTCCAAAAA TCCCGATCGT TTTGGACTCT1051 TTGGTGCACC CCCCTTAGAG GAGGGATATG TGGTTCTGGT AGGAGACGAG1101 AACCTAAAAC AGTTCCCGCC TCCGTCTGAA TTTTTGCTTT CGGTTTGGGA1151 CCGAAGCCGC GCCGCGCGTC TTGTCTGCTG CAGCATCGTT CTGTGTTGTC 1201 TCTGTCTGAC TGTGTTTCTG TATTTGTCTG AAAATATGGG CCCGGGCCAG 1251 ACTGTTACCA CTCCCTTAAG TTTGACCTTA GGTCACTGGA AAGATGTCGA 1301 GCAGATCGCT CACAACCAGT CGGTAGATGT CAAGAAGAGA CGTTGGGTTA 1351 CCTTCTGCTC TGCAGAATGG CCAACCTTTA ACGTCGGATG GCCGCGAGAC 1401 GGCACCTTTA ACCGAGACCT CATCACCCAG GTTAAGATCA AGGTCTTTTC 1451 ACCTGGCCCG CATGGACACC CAGACCAGGT CCCCTACATC GTGACCTGGG 1501 AAGCCTTGGC TTTTGACCCC CCTCCCTGGG TCAAGCCCTT TGTACACCCT 1551 AAGCCTCCGC CTCCTCTTCC TCCATCCGCC CCGTCTCTCC CCCTTGAACC 1601 TCCTCGTTCG ACCCCGCCTC GATCCTCCCT TTATCCAGCC CTCACTCCTT 1651 CTCTAGGCGC CCCCATATGG CCATATGAGA TCTTATATGG GGCACCCCCG 1701 CCCCTTGTAA ACTTCCCTGA CCCTGACATG ACAAGAGTTA CTAACAGCCC 1751 CTCTCTCCAA GCTCACTTAC AGGCTCTCTA CTTAGTCCAG CACGAAGTCT 1801 GGAGACCTCT GGCGGCAGCC TACCAAGAAC AACTGGACCG ACCGGTGGTA 1851 CCTCACCCTT ACCGAGTCGG CGACACAGTG TGGGTCCGCC GACACCAGAC 1901 TAAGAACCTA GAACCTCGCT GGAAAGGACC TTACACAGTC CTGCTGACCA 1951 CCCCCACCGC CCTCAAAGTA GACGGCATCG CAGCTTGGAT ACACGCCGCC 2001 CACGTGAAGG CTGCCGACCC CGGGGGTGGA CCATCCTCTA GACTGCCGGA 2051 TCGAATTGTC GACGAATTCG CCGTTGCATT AGTTATTAAT AGTAATCAAT 2101 TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC 2151 TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG 2201 ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA 2251 TTGACGTCAA TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC 2301 ATCAAGTGTA TCATATGCCA AGTACGCCCC CTATTGACGT CAATGACGGT 2351 AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT GGGACTTTCC 2401 TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC 2451 GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA 2501 TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA 2551 AAATCAACGG GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC 2601 AAATGGGCGG TAGGCGTGTA CGGTGGGAGG TCTATATAAG CAGAGCTGGT 2651 TTAGTGAACC GTCAGATCCG CTAGTGGCCG GCCGCCACCA TGGAACAAAA 2701 ACTTATTTCT GAAGAAGACT TGGCCCCCCC GACCGATGTC AGCCTGGGGG 2751 ACGAACTCCA CTTAGACGGC GAGGACGTGG CGATGGCGCA TGCCGACGCG 2801 CTAGACGATT TCGATCTGGA CATGTTGGGG GACGGGGATT CCCCAGGTCC 2851 GGGATTTACC CCCCACGACT CCGCCCCCTA CGGCGCTCTG GATATGGCCG 2901 ACTTCGAGTT TGAGCAGATG TTTACCGATG CCCTTGGAAT TGACGAGTAC 2951 GGTGGGAAGC TTCTAGGTAC CTCTAGAAGA ATATCAAATT CTATATCTTC 3001 AGGTCGCGAT GATCTCTCGC CTTCGAGCAG CTTGAACGGA TACTCGGCGA 3051 ACGAAAGCTG CGATGTGAAG AAGAGCAAGA AGGGACCTGC GCCACGGGTG 3101 CAAGAGGAGC TGTGCCTGGT TTGCGGCGAC AGGGCCTCCG GCTACCACTA 3151 CAACGCCCTC ACCTGTGGAT CCTGCAAGGT GTTCTTTCGA CGCAGCGTTA 3201 CGAAGAGCGC CGTCTACTGC TGCAAGTTCG GGCGCGCCTG CGAAATGGAC 3251 ATGTACATGA GGCGAAAGTG TCAGGAGTGC CGCCTGAAAA AGTGCCTGGC 3301 CGTGGGTATG CGGCCGGAAT GCGTCGTCCC GGAGAACCAA TGTGCGATGA3351 AGCGGCGCGA AGAGAAGGCC CAGAAGGAGA AGGACAAAAT GACCACTTCG 3401 CCGAGCTCTC AGCATGGCGG CAATGGCAGC TTGGCCTCTG GTGGCGGCCA 3451 AGACTTTGTT AAGAAGGAGA TTCTTGACCT TATGACATGC GAGCCGCCCC 3501 AGCATGCCAC TATTCCGCTA CTACCTGATG AAATATTGGC CAAGTGTCAA 3551 GCGCGCAATA TACCTTCCTT AACGTACAAT CAGTTGGCCG TTATATACAA 3601 GTTAATTTGG TACCAGGATG GCTATGAGCA GCCATCTGAA GAGGATCTCA 3651 GGCGTATAAT GAGTCAACCC GATGAGAACG AGAGCCAAAC GGACGTCAGC 3701 TTTCGGCATA TAACCGAGAT AACCATACTC ACGGTCCAGT TGATTGTTGA 3751 GTTTGCTAAA GGTCTACCAG CGTTTACAAA GATACCCCAG GAGGACCAGA 3801 TCACGTTACT AAAGGCCTGC TCGTCGGAGG TGATGATGCT GCGTATGGCA 3851 CGACGCTATG ACCACAGCTC GGACTCAATA TTCTTCGCGA ATAATAGATC 3901 ATATACGCGG GATTCTTACA AAATGGCCGG AATGGCTGAT AACATTGAAG 3951 ACCTGCTGCA TTTCTGCCGC CAAATGTTCT CGATGAAGGT GGACAACGTC 4001 GAATACGCGC TTCTCACTGC CATTGTGATC TTCTCGGACC GGCCGGGCCT 4051 GGAGAAGGCC CAGCTAGTCG AAGCGATCCA GAGCTACTAC ATCGACACGC 4101 TACGCATTTA TATACTCAAC CGCCACTGCG GCGACTCAAT GAGCCTCGTC 4151 TTCTACGCAA AGCTGCTCTC GATCCTCACC GAGCTGCGTA CGCTGGGCAA 4201 CCAGAACGCC GAGATGTGTT TCTCACTAAA GCTCAAAAAC CGCAAACTGC 4251 CCAAGTTCCT CGAGGAGATC TGGGACGTTC ATGCCATCCC GCCATCGGTC 4301 CAGTCGCACC TTCAGATTAC CCAGGAGGAG AACGAGCGTC TCGAGCGGGC 4351 TGAGCGTATG CGGGCATCGG TTGGGGGCGC CATTACCGCC GGCATTGATT 4401 GCGACTCTGC CTCCACTTCG GCGGCGGCAG CCGCGGCCCA GCATCAGCCT 4451 CAGCCTCAGC CCCAGCCCCA ACCCTCCTCC CTGACCCAGA ACGATTCCCA 4501 GCACCAGACA CAGCCGCAGC TACAACCTCA GCTACCACCT CAGCTGCAAG 4551 GTCAACTGCA ACCCCAGCTC CACCCACAGC TTCAGACGCA ACTCCAGCCA 4601 CAGATTCAAC CACAGCCACA GCTCCTTCCC GTCTCCGCTC CCGTGCCCGC 4651 CTCCGTAACC GCACCTGGTT CCTTGTCCGC GGTCAGTACG AGCAGCGAAT 4701 ACATGGGCGG AAGTGCGGCC ATAGGACCCA TCACGCCGGC AACCACCAGC 4751 AGTATCACGG CTGCCGTTAC CGCTAGCTCC ACCACATCAG CGGTACCGAT 4801 GGGCAACGGA GTTGGAGTCG GTGTTGGGGT GGGCGGCAAC GTCAGCATGT 4851 ATGCGAACGC CCAGACGGCG ATGGCCTTGA TGGGTGTAGC CCTGCATTCG 4901 CACCAAGAGC AGCTTATCGG GGGAGTGGCG GTTAAGTCGG AGCACTCGAC 4951 GACTGCATAG GGTTACCCCC CTCTCCCTCC CCCCCCCCTA ACGTTACTGG 5001 CCGAAGCCGC TTGGAATAAG GCCGGTGTGC GTTTGTCTAT ATGTTATTTT 5051 CCACCATATT GCCGTCTTTT GGCAATGTGA GGGCCCGGAA ACCTGGCCCT 5101 GTCTTCTTGA CGAGCATTCC TAGGGGTCTT TCCCCTCTCG CCAAAGGAAT 5151 GCAAGGTCTG TTGAATGTCG TGAAGGAAGC AGTTCCTCTG GAAGCTTCTT 5201 GAAGACAAAC AACGTCTGTA GCGACCCTTT GCAGGCAGCG GAACCCCCCA 5251 CCTGGCGACA GGTGCCTCTG CGGCCAAAAG CCACGTGTAT AAGATACACC 5301 TGCAAAGGCG GCACAACCCC AGTGCCACGT TGTGAGTTGG ATAGTTGTGG 5351 AAAGAGTCAA ATGGCTCTCC TCAAGCGTAT TCAACAAGGG GCTGAAGGAT 5401 GCCCAGAAGG TACCCCATTG TATGGGATCT GATCTGGGGC CTCGGTGCAC 5451 ATGCTTTACA TGTGTTTAGT CGAGGTTAAA AAACGTCTAG GCCCCCCGAA 5501 CCACGGGGAC GTGGTTTTCC GTTGAAAAAC ACGATCGATA ATATGGAACA5551 AAAACTTATT TCTGAAGAAG ACTTGGACAC CAAACTTTCC TGCCGCTCGA 5601 TTTCTCCACC CAGGTGAACT CCTCCCTCAC CTCCCCGACG GGGCGAGGCT 5651 CCATGGCTGC CCCCTCGCTG CACCCGTCCC TGGGGCCTGG CATCGGCTCC 5701 CCGGGACAGC TGCATTCTCC CATCAGCACC CTGAGCTCCC CCATCAACGG 5751 CATGGGCCCG CCTTTCTCGG TCATCAGCTC CCCCATGGGC CCCCACTCCA 5801 TGTCGGTGCC CACCACACCC ACCCTGGGCT TCAGCACTGG CAGCCCCCAG 5851 CTCAGCTCAC CTATGAACCC CGTCAGCAGC AGCGAGGACA TCAAGCCCCC 5901 CCTGGGCCTC AATGGCGTCC TCAAGGTCCC CGCCCACCCC TCAGGAAACA 5951 TGGCTTCCTT CACCAAGCAC ATCTGCGCCA TCTGCGGGGA CCGCTCCTCA 6001 GGCAAGCACT ATGGAGTGTA CAGCTGCGAG GGGTGCAAGG GCTTCTTCAA 6051 GCGGACGGTG CGCAAGGACC TGACCTACAC CTGCCGCGAC AACAAGGACT 6101 GCCTGATTGA CAAGCGGCAG CGGAACCGGT GCCAGTACTG CCGCTACCAG 6151 AAGTGCCTGG CCATGGGCAT GAAGCGGGAA GCCGTGCAGG AGGAGCGGCA 6201 GCGTGGCAAG GACCGGAACG AGAATGAGGT GGAGTCGACC AGCAGCGCCA 6251 ACGAGGACAT GCCGGTGGAG AGGATCCTGG AGGCTGAGCT GGCCGTGGAG 6301 CCCAAGACCG AGACCTACGT GGAGGCAAAC ATGGGGCTGA ACCCCAGCTC 6351 GCCGAACGAC CCTGTCACCA ACATTTGCCA AGCAGCCGAC AAACAGCTTT 6401 TCACCCTGGT GGAGTGGGCC AAGCGGATCC CACGCTTCTC AGAGCTGCCC 6451 CTGGACGACC AGGTCATCCT GCTGCGGGCA GGCTGGAATG AGCTGCTCAT 6501 CGCCTCCTTC TCCCACCGCT CCATCGCCGT GAAGGACGGG ATCCTCCTGG 6551 CCACCGGGCT GCACGTCCAC CGGAACAGCG CCCACAGCGC AGGGGTGGGC 6601 GCCATCTTTG ACAGGGTGCT GACGGAGCTT GTGTCCAAGA TGCGGGACAT 6651 GCAGATGGAC AAGACGGAGC TGGGCTGCCT GCGCGCCATC GTCCTCTTTA 6701 ACCCTGACTC CAAGGGGCTC TCGAACCCGG CCGAGGTGGA GGCGCTGAGG 6751 GAGAAGGTCT ATGCGTCCTT GGAGGCCTAC TGCAAGCACA AGTACCCAGA 6801 GCAGCCGGGA AGGTTCGCTA AGCTCTTGCT CCGCCTGCCG GCTCTGCGCT 6851 CCATCGGGCT CAAATGCCTG GAACATCTCT TCTTCTTCAA GCTCATCGGG 6901 GACACACCCA TTGACACCTT CCTTATGGAG ATGCTGGAGG CGCCGCACCA 6951 AATGACTTAG GCGGCCGCGA TCCGGTTATT TTCCACCATA TTGCCGTCTT 7001 TTGGCAATGT GAGGGCCCGG AAACCTGGCC CTGTCTTCTT GACGAGCATT 7051 CCTAGGGGTC TTTCCCCTCT CGCCAAAGGA ATGCAAGGTC TGTTGAATGT 7101 CGTGAAGGAA GCAGTTCCTC TGGAAGCTTC TTGAAGACAA ACAACGTCTG 7151 TAGCGACCCT TTGCAGGCAG CGGAACCCCC CACCTGGCGA CAGGTGCCTC 7201 TGCGGCCAAA AGCCACGTGT ATAAGATACA CCTGCAAAGG CGGCACAACC 7251 CCAGTGCCAC GTTGTGAGTT GGATAGTTGT GGAAAGAGTC AAATGGCTCT 7301 CCTCAAGCGT ATTCAACAAG GGGCTGAAGG ATGCCCAGAA GGTACCCCAT 7351 TGTATGGGAT CTGATCTGGG GCCTCGGTGC ACATGCTTTA CATGTGTTTA 7401 GTCGAGGTTA AAAAACGTCT AGGCCCCCCG AACCACGGGG ACGTGGTTTT 7451 CCGTTGAAAA ACACGATCGA TAATATGGAA CAAAAACTTA TTTCTGAAGA 7501 AGACTTGGAC ACCAAACTTT CCTGCCGCTC GATTTCTCCA CCCTAGGTGC 7551 CACGCGGTTC CATGGGATCG TTTCGCATGA TTGAACAAGA TGGATTGCAC 7601 GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG CTATTCGGCT ATGACTGGGC 7651 ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG CTGTCAGCGC 7701 AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT7751 GAACTGCAGG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT 7801 TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC 7851 TGCTATTGGG CGAAGTGCCG GGGCAGGATC TCCTGTCATC TCACCTTGCT 7901 CCTGCCGAGA AAGTATCCAT CATGGCTGAT GCAATGCGGC GGCTGCATAC 7951 GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA CATCGCATCG 8001 AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG 8051 GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA 8101 GGCGCGCATG CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT 8151 GCTTGCCGAA TATCATGGTG GAAAATGGCC GCTTTTCTGG ATTCATCGAC 8201 TGTGGCCGGC TGGGTGTGGC GGACCGCTAT CAGGACATAG CGTTGGCTAC 8251 CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG 8301 TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC 8351 CTTCTTGACG AGTTCTTCTG AGCGGGACTC TGGGGTTCGA TAAAATAAAA 8401 GATTTTATTT AGTCTCCAGA AAAAGGGGGG AATGAAAGAC CCCACCTGTA 8451 GGTTTGGCAA GCTAGCTTAA GTAACGCCAT TTTGCAAGGC ATGGAAAAAT 8501 ACATAACTGA TCATCCGGAT GCATACGTAG GGACCCGGCG CGCCACGCGT 8551 CCTCACTCGG GGCGCCAGTC CTCCGATTGA CTGAGTCGCC CGGGTACCCG 8601 TGTATCCAAT AAACCCTCTT GCAGTTGCAT CCGACTTGTG GTCTCGCTGT 8651 TCCTTGGGAG GGTCTCCTCT GAGTGATTGA CTACCCGTCA GCGGGGGTCT 8701 TTCATTTGGG GGCTCGTCCG GGATCGGGAG ACCCCTGCCC AGGGACCACC 8751 GACCCACCAC CGGGAGGTAA GCTGGCTGCC TCGCGCGTTT CGGTGATGAC 8801 GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA CAGCTTGTCT 8851 GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG 8901 TTGGCGGGTG TCGGGGCGCA GCCATGACCC AGTCACGTAG CGATAGCGGA 8951 GTGTATACTG GCTTAACTAT GCGGCATCAG AGCAGATTGT ACTGAGAGTG 9001 CACCATATGC GGTGTGAAAT ACCGCACAGA TGCGTAAGGA GAAAATACCG 9051 CATCAGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG CGCTCGGTCG 9101 TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA 9151 TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA 9201 GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA 9251 GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG 9301 TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC CCCCTGGAAG 9351 CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC GGATACCTGT 9401 CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG CTCACGCTGT 9451 AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA 9501 CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC 9551 TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT 9601 GGTAACAGGA TTAGCAGAGC GAGGTATGTA GGCGGTGCTA CAGAGTTCTT 9651 GAAGTGGTGG CCTAACTACG GCTACACTAG AAGGACAGTA TTTGGTATCT 9701 GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA 9751 TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA 9801 GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT 9851 CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG 9901 GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA。