pFB-ERV

价格:4500元

联系方式:I47-825O-882O

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载体基本信息

出品公司: Agilent
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: Neomycin_1.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: --
产品目录号: 217564
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

载体质粒图谱和多克隆位点信息

pFB-ERV载体图谱



pFB-ERV 载体特征

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 be packaged 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        

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

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