pFB-ERV

pFB-ERV

pFB-ERV逆病毒载体基本信息：

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 number

of 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 the ponA-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.3

A 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 EXPRESSION

The 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.4

 When 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, represses transcription 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) VP16

OVERVIEW OF REPLICATION-DEFECTIVE RETROVIRAL GENE TRANSFER SYSTEMS

Non-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 bepackaged into 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 Vectors

The 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 a self-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. The pCFB-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 011006

Enzymes with 1-10 cleavage sites:

              #sites   --  Bp position of recognition site --

  AarI           3     5297,   6078,   7229

  AatII          7      978,   2200,   2253,   2336,   2522

                       3692,  10993

  Acc65I         8      647,   1847,   2966,   3609,   4792

                       5409,   7341,   8593

  AccI           6     1968,   2058,   3212,   3762,   6234

                       8953

  AccIII         1     8514

  AclI           3     4990,  10300,  10673

  AflII          3      202,   1265,   8466

  AflIII         9      164,   2820,   3249,   5283,   5458

                       7215,   7390,   8545,   9182

  AgeI           2     1841,   6125

  AhdI           6      687,    733,   1274,   8633,   8679

                      10070

  AleI           2     5607,   6402

  Alw44I         6     1054,   5445,   7377,   8998,   9496

                      10742

  AlwNI          3      323,    398,   9593

  ApaI           5     1238,   5081,   5754,   5787,   7013

  ApoI           4       87,   1128,   2064,   2986

  AscI           2     3231,   8537

  AseI           2     2085,  10246

  AvaI          10      577,    610,    643,   1241,   2019

                       4259,   4340,   5700,   8556,   8589

  BamHI          4     3167,   6272,   6425,   6539

  BbeI           7      615,   1656,   4376,   6598,   6939

                       7704,   8561

  BbsI          10     2714,   3947,   4148,   5101,   5201

                       5567,   7033,   7133,   7499,  11060

  BbvCI          6      453,   4448,   4454,   4526,   4538

                       6744

  BciVI          7      656,   1987,   3038,   7913,   8602

                       9391,  10918

  BclI           1     8508

  BfrBI          1     8519

  BglI          10     2163,   2285,   2356,   3926,   4122

                       4859,   5264,   5906,   7196,  10189

  BglII          2     1678,   4266

  BlnI           3     5119,   7051,   7542

  BlpI           4     4349,   5850,   6283,   6817

  BmgBI          4     2774,   5508,   6562,   7440

  BmrI           6     2373,   5318,   7250,   7644,   8928

                      10120

  BmtI           6        6,     16,     26,    197,   4772

                       8461

  BpmI           7     1799,   4049,   4592,   6277,   6934

                       8414,  10160

  Bpu10I        10      337,    412,    453,   1548,   4448

                       4454,   4526,   4538,   5730,   6744

  BpuEI          8     5370,   5920,   7302,   8096,   9288

                       9550,   9827,  10695

  BsaAI          8     2001,   2417,   5282,   6316,   7214

                       8007,   8524,   8934

  BsaBI          1     2807

  BsaI          10      694,    715,    782,   1414,   1802

                       6310,   8640,   8661,   8728,  10142

  BsgI           3     5668,   6184,   6559

  BsiWI          1     4187

  BsmBI          8      976,   1093,   1337,   1396,   1582

                       4337,   4630,   8831

  BsmI           7     3317,   4894,   5114,   5147,   5712

                       7046,   7079

  BspHI          3     9902,  10910,  11015

  BspMI          7     3085,   3951,   5298,   6079,   7230

                       7601,   7964

  BsrDI          6     3420,   5072,   7004,   7931,  10129

                      10311

  BsrGI          3     1541,   3252,   6017

  BssHII         6      563,   3232,   3551,   6681,   8102

                       8538

  BssSI          4     8297,   9355,  10739,  11046

  Bst1107I       1     8953

  BstAPI         4      322,    397,   4392,   9000

  BstEII         2     1346,   4961

  Bsu36I         3     1276,   5939,   5996

  BtsI           4     4015,   4124,  10468,  10496

  ClaI           2     5534,   7466

  DraI           3     9939,   9958,  10650

  DraIII         3     1874,   5326,   7258

  DrdI           4     6454,   7726,   8871,   9284

  EagI           7      961,   2679,   3311,   4040,   6728

                       6962,   7611

  EcoICRI        3      580,   3403,   5733

  EcoNI          2     1647,   4048

  EcoRI          1     2064

  EcoRV          2      308,    383

  FseI           1     2676

  FspI           3     6059,   7805,  10295

  HincII         5     2058,   3663,   4551,   6234,  10614

  HindIII        3     2957,   5192,   7124

  KasI           7      615,   1656,   4376,   6598,   6939

                       7704,   8561

  KpnI           8      647,   1847,   2966,   3609,   4792

                       5409,   7341,   8593

  MluI           1     8545

  MmeI          10      684,   1383,   3823,   4811,   4936

                       5336,   7268,   8630,   9372,   9556

  MscI           7      827,   1368,   1668,   3537,   6158

                       6548,   7785

  MunI           2       11,     21

  NaeI           5     2677,   4388,   4735,   6837,   8205

  NarI           7      615,   1656,   4376,   6598,   6939

                       7704,   8561

  NcoI           7     2439,   2688,   5651,   5783,   6161

                       7560,   8137

  NdeI           4     1664,   1672,   2312,   9004

  NgoMIV         5     2677,   4388,   4735,   6837,   8205

  NheI           6        6,     16,     26,    197,   4772

                       8461

  NotI           1     6961

  NruI           2     3004,   3885

  NsiI           1     8519

  PciI           6      164,   2820,   3249,   5458,   7390

                       9182

  PfoI           6      771,   2848,   3327,   6275,   8717

                       8826

  PmlI           3     2001,   5282,   7214

  PpuMI          8      499,   1477,   1925,   3082,   4723

                       5924,   6065,   8530

  PshAI          2     1015,   2731

  PspOMI         5     1238,   5081,   5754,   5787,   7013

  PstI           4     1178,   1360,   7754,  10316

  PvuI           4     1034,   5532,   7464,  10442

  PvuII          6      286,    361,   4541,   5707,   6021

                       7809

  RsrII          2     4035,   8220

  SacI           3      580,   3403,   5733

  SacII          3      151,   4431,   4677

  SalI           2     2058,   6234

  SanDI          1     8530

  SapI           5     3070,   3202,   8054,   8264,   9059

  ScaI           2     6134,  10553

  SexAI          3     1474,   4663,   6458

  SfcI          10      182,   1178,   1360,   5216,   7148

                       7754,   8446,   9447,   9638,  10316

  SfoI           7      615,   1656,   4376,   6598,   6939

                       7704,   8561

  SmaI           5      643,   1241,   2019,   5700,   8589

  SnaBI          2     2417,   8524

  SpeI           1      897

  SphI           3     2788,   3502,   8106

  SrfI           1     1240

  SspI           3     3532,   3877,  10877

  StuI           2     3813,   6773

  TatI          10     1541,   2296,   2376,   2409,   2460

                       3252,   6017,   6134,   8988,  10553

  TfiI           8     2837,   3469,   3911,   4493,   4603

                       8190,   8324,   9157

  Tsp45I         9     1282,   1491,   6364,   7826,   8132

                       8837,   8932,  10332,  10543

  Tth111I        8      633,   1473,   3992,   6358,   6457

                       7820,   8579,   8926

  Van91I         2     5415,   7347

  XbaI           3      464,   2037,   2972

  XcmI           2     5741,   5813

  XhoI           2     4259,   4340

  XmaI           5      643,   1241,   2019,   5700,   8589

  XmnI           3     5176,   7108,  10670

  ZraI           7      978,   2200,   2253,   2336,   2522

                       3692,  10993

Enzymes that do NOT cut molecule:

AsiSI       BstBI       BstXI       Eco47III    FspAI      

HpaI        PacI        PmeI        PsiI        SbfI       

SfiI        SgrAI       SwaI       

pFB-ERV载体序列：

pFB-ERV, 11067 bp                              version 011006

NOTE: The following sequence has been verified for accuracy

at the junctions. The remainder of the sequence has been

obtained from existing data.

     1  GAATTGCTAG CAATTGCTAG CAATTGCTAG CAATTCATAC CAGATCACCG

    51  AAAACTGTCC TCCAAATGTG TCCCCCTCAC ACTCCCAAAT TCGCGGGCTT

   101  CTGCCTCTTA GACCACTCTA CCCTATTCCC CACACTCACC GGAGCCAAAG

   151  CCGCGGGACA TATACATGTG AAAGACCCCA CCTGTAGGTT TGGCAAGCTA

   201  GCTTAAGTAA CGCCATTTTG CAAGGCATGG AAAAATACAT AACTGAGAAT

   251  AGAAAAGTTC AGATCAAGGT CAGGAACAGA TGGAACAGCT GAATATGGGC

   301  CAAAGCGGAT ATCTGTGGTA AGCAGTTCCT GCCCCGGCTC AGGGCCAAGA

   351  ACAGATGGAA CAGCTGAATA TGGGCCAAAC AGGATATCTG TGGTAAGCAG

   401  TTCCTGCCCC GGCTCAGGGC CAAGAACAGA TGGTCCCCAG ATGCGGTCCA

   451  GCCCTCAGCA GTTTCTAGAG AACCATCAGA TGTTTCCAGG GTGCCCCAAG

   501  GACCTGAAAT GACCCTGTGC CTTATTTGAA CTAACCAATC AGTTCGCTTC

   551  TCGCTTCTGT TCGCGCGCTT CTGCTCCCCG AGCTCAATAA AAGAGCCCAC

   601  AACCCCTCAC TCGGGGCGCC AGTCCTCCGA TTGACTGAGT CGCCCGGGTA

   651  CCCGTGTATC CAATAAACCC TCTTGCAGTT GCATCCGACT TGTGGTCTCG

   701  CTGTTCCTTG GGAGGGTCTC CTCTGAGTGA TTGACTACCC GTCAGCGGGG

   751  GTCTTTCATT TGGGGGCTCG TCCGGGATCG GGAGACCCCT GCCCAGGGAC

   801  CACCGACCCA CCACCGGGAG GTAAGCTGGC CAGCAACTTA TCTGTGTCTG

   851  TCCGATTGTC TAGTGTCTAT GACTGATTTT ATGCGCCTGC GTCGGTACTA

   901  GTTAGCTAAC TAGCTCTGTA TCTGGCGGAC CCGTGGTGGA ACTGACGAGT

   951  TCGGAACACC CGGCCGCAAC CCTGGGAGAC GTCCCAGGGA CTTCGGGGGC

  1001  CGTTTTTGTG GCCCGACCTG AGTCCAAAAA TCCCGATCGT TTTGGACTCT

  1051  TTGGTGCACC CCCCTTAGAG GAGGGATATG TGGTTCTGGT AGGAGACGAG

  1101  AACCTAAAAC AGTTCCCGCC TCCGTCTGAA TTTTTGCTTT CGGTTTGGGA

  1151  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 TGTGCGATGA

  3351  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 ATATGGAACA

  5551  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 TGCCCTGAAT

  7751  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

  9951  ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA

 10001  GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT

 10051  CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG

 10101  GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC

 10151  GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC

 10201  GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC AGTCTATTAA

 10251  TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA

 10301  ACGTTGTTGC CATTGCTGCA GGCATCGTGG TGTCACGCTC GTCGTTTGGT

 10351  ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC

 10401  CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG

 10451  TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG

 10501  CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT CTGTGACTGG

 10551  TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG CGACCGAGTT

 10601  GCTCTTGCCC GGCGTCAACA CGGGATAATA CCGCGCCACA TAGCAGAACT

 10651  TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG

 10701  GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA

 10751  ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA

 10801  ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG

 10851  TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTGAAGC ATTTATCAGG

 10901  GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA GAAAAATAAA

 10951  CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGACGTCTA

 11001  AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG CGTATCACGA

 11051  GGCCCTTTCG TCTTCAA

pFB-ERV其他相关逆病毒载体：