pCFB-EGSH

pCFB-EGSH

pCFB-EGSH逆病毒载体基本信息：

pCFB-EGSH载体质粒图谱和多克隆位点信息：

pCFB-EGSH载体简介：

pCFB-EGSH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.

pCFB-EGSH载体限制性酶切位点

pCFB-EGSH, 6982 bp                       version 075003

Enzymes with 1-10 cleavage sites:

              #sites   --  Bp position of recognition site --

  AatII          7      328,    381,    464,    650,   1153

                       3244,   6908

  Acc65I         3      822,   2022,   4508

  AccI           4     2143,   2603,   2885,   4868

  AccIII         6     2258,   3442,   3979,   4115,   4228

                       4429

  AclI           2     6215,   6588

  AcuI           2     5624,   6672

  AflII          4      202,   1440,   2333,   4381

  AflIII         6      164,   2277,   2564,   3132,   4460

                       5097

  AgeI           1     2016

  AhdI           6      862,    908,   1449,   4548,   4594

                       5985

  Alw44I         6     1229,   3501,   3803,   4913,   5411

                       6657

  AlwNI          1     5508

  ApaI           1     1413

  ApoI           5       87,   1303,   2905,   3068,   3464

  AscI           1     4452

  AseI           1     6161

  AsiSI          1     3581

  AvaI          10      785,    818,   1416,   2194,   2647

                       2705,   2938,   3203,   4471,   4504

  BanII          4      939,   1413,   2306,   4625

  BbeI           3      790,   1831,   4476

  BbsI           1     6975

  BceAI          8     1174,   1574,   2147,   3498,   3928

                       4059,   4163,   5583

  BciVI          6      831,   2162,   2273,   4517,   5306

                       6833

  BclI           1     4423

  BfrBI          2      774,   4434

  BglI           4      291,    413,    484,   6104

  BglII          2     1853,   2879

  BlpI           2     2545,   2556

  Bme1580I       8     1229,   1413,   1865,   3501,   3803

                       4913,   5411,   6657

  BmgBI          1     2563

  BmrI           3      501,   4843,   6035

  BmtI           6        6,     16,     26,    197,   2346

                       4376

  BpmI           5     1974,   3868,   3922,   4329,   6075

  Bpu10I         2     1723,   2308

  BpuEI          4     5203,   5465,   5742,   6610

  BsaAI          4      545,   2176,   4439,   4849

  BsaBI          1     2915

  BsaI           9      869,    890,    957,   1589,   1977

                       4555,   4576,   4643,   6057

  BseRI          5      893,   1243,   1736,   1775,   4579

  BseYI          3     3048,   4217,   5401

  BsiHKAI        8     1229,   2306,   3501,   3803,   4913

                       5411,   6572,   6657

  BsiWI          2     2268,   2318

  BsmBI          7     1151,   1268,   1512,   1571,   1757

                       3283,   4746

  BspHI          3     5817,   6825,   6930

  BspMI          1     3525

  BsrBI          7     2935,   2941,   3610,   3968,   4285

                       5028,   6829

  BsrDI          3     3211,   6044,   6226

  BsrGI          1     1716

  BssHII         1     4453

  BssSI          5     3321,   3800,   5270,   6654,   6961

  Bst1107I       1     4868

  BstAPI         3     3520,   3796,   4915

  BstBI          1     3071

  BstEII         1     1521

  BstXI          1     2920

  Bsu36I         1     1451

  BtgI           7      151,    567,   1106,   2962,   3573

                       3929,   3998

  BtgZI          3      556,   2151,   3878

  BtsI           2     6383,   6411

  Cfr10I         3     2016,   3559,   6070

  DraI           4     2339,   5854,   5873,   6565

  DraIII         3     2049,   3504,   3797

  DrdI           4     3719,   4100,   4786,   5199

  EagI           8     1136,   2873,   2932,   2944,   3426

                       3591,   4161,   4263

  EarI           4     1509,   1739,   4975,   6779

  EciI           9      713,   1099,   1731,   1750,   2060

                       2652,   5169,   5315,   6143

  Eco57MI        7     1974,   3868,   3922,   4329,   5624

                       6075,   6672

  EcoICRI        1     2306

  EcoNI          1     1822

  EcoO109I       4     1652,   2100,   4445,   6965

  EcoRI          3     2905,   3068,   3464

  EcoRV          1     2915

  FspI           1     6210

  HaeII          6      790,   1831,   2611,   4476,   4971

                       5341

  HincII         4     2885,   3183,   4055,   6529

  HindIII        1     2330

  KasI           3      790,   1831,   4476

  KpnI           3      822,   2022,   4508

  MluI           3     2277,   3132,   4460

  MlyI          10      617,    812,   1195,   1220,   2039

                       4291,   4498,   4997,   5468,   5985

  MmeI           8      859,   1558,   2700,   3287,   3821

                       4545,   5287,   5471

  MscI           3     1002,   1543,   1843

  MslI           8      159,    568,   2957,   3077,   4427

                       6238,   6397,   6756

  MunI           2       11,     21

  NarI           3      790,   1831,   4476

  NcoI           2      567,   3573

  NdeI           6      440,   1839,   1847,   2295,   3670

                       4919

  NheI           6        6,     16,     26,    197,   2346

                       4376

  NotI           3     2931,   2943,   4262

  NsiI           2      774,   4434

  NspI           5      164,    244,   3014,   4732,   5097

  PacI           1     2324

  PciI           2      164,   5097

  PfoI           3      946,   4632,   4741

  PleI          10      617,    812,   1195,   1220,   2039

                       4291,   4498,   4997,   5468,   5985

  PmeI           1     2338

  PmlI           1     2176

  PpuMI          3     1652,   2100,   4445

  PshAI          2     1190,   3244

  PsiI           1     2892

  PspOMI         1     1413

  PstI           7     1353,   1535,   2505,   2910,   3161

                       3554,   6231

  PvuI           3     1209,   3582,   6357

  PvuII          1     2526

  RsrII          2     3101,   3628

  SacI           1     2306

  SacII          2      151,   3998

  SalI           1     2885

  SanDI          1     4445

  SapI           1     4974

  ScaI           3     2695,   4189,   6468

  SexAI          1     1649

  SfoI           3      790,   1831,   4476

  SmaI           7      818,   1416,   2194,   2647,   2705

                       3203,   4504

  SmlI           9      202,   1440,   2333,   2938,   4381

                       5203,   5465,   5742,   6610

  SnaBI          2      545,   4439

  SpeI           1     1072

  SphI           1      244

  SrfI           1     1415

  SspI           1     6792

  StuI           1     2312

  StyI           5      567,    881,   1679,   3573,   4567

  TatI           9      424,    504,    537,    588,   1716

                       2695,   4189,   4903,   6468

  TfiI           6     2482,   3317,   3439,   3639,   3890

                       5072

  Tsp45I         8     1457,   1666,   3129,   3512,   4752

                       4847,   6247,   6458

  TspDTI        10       34,    930,   2955,   3221,   3668

                       4347,   4616,   5866,   5968,   6271

  TspGWI         7     1296,   2271,   2600,   3737,   3836

                       6440,   6757

  Tth111I        6      808,   1648,   3278,   3722,   4494

                       4841

  XbaI           1     2212

  XhoI           1     2938

  XhoII          9     1853,   2879,   3199,   5738,   5749

                       5835,   5847,   6615,   6632

  XmaI           7      818,   1416,   2194,   2647,   2705

                       3203,   4504

  XmnI           2     2385,   6585

  ZraI           7      328,    381,    464,    650,   1153

                       3244,   6908

Enzymes that do NOT cut molecule:

AarI        AleI        BamHI       BbvCI       BlnI       

BsgI        BsmI        ClaI        Eco47III    FseI       

FspAI       HpaI        NaeI        NgoMIV      NruI       

SbfI        SfiI        SgrAI       SwaI        Van91I     

XcmI  

pCFB-EGSH载体序列：

pCFB-EGSH, 6982 bp                             version 075003

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 AACGCATGCC

  251  CCATATATGG AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG

  301  CTGACCGCCC AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC

  351  CCATAGTAAC GCCAATAGGG ACTTTCCATT GACGTCAATG GGTGGAGTAT

  401  TTACGGTAAA CTGCCCACTT GGCAGTACAT CAAGTGTATC ATATGCCAAG

  451  TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG

  501  CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA

  551  TTAGTCATCG CTATTACCAT GGTGATGCGG TTTTGGCAGT ACATCAATGG

  601  GCGTGGATAG CGGTTTGACT CACGGGGATT TCCAAGTCTC CACCCCATTG

  651  ACGTCAATGG GAGTTTGTTT TGGCACCAAA ATCAACGGGA CTTTCCAAAA

  701  TGTCGTAACA ACTCCGCCCC ATTGACGCAA ATGGGCGGTA GGCGTGTACG

  751  GTGGGAGGTC TATATAAGCA GAGATGCATC CTCACTCGGG GCGCCAGTCC

  801  TCCGATTGAC TGAGTCGCCC GGGTACCCGT GTATCCAATA AACCCTCTTG

  851  CAGTTGCATC CGACTTGTGG TCTCGCTGTT CCTTGGGAGG GTCTCCTCTG

  901  AGTGATTGAC TACCCGTCAG CGGGGGTCTT TCATTTGGGG GCTCGTCCGG

  951  GATCGGGAGA CCCCTGCCCA GGGACCACCG ACCCACCACC GGGAGGTAAG

 1001  CTGGCCAGCA ACTTATCTGT GTCTGTCCGA TTGTCTAGTG TCTATGACTG

 1051  ATTTTATGCG CCTGCGTCGG TACTAGTTAG CTAACTAGCT CTGTATCTGG

 1101  CGGACCCGTG GTGGAACTGA CGAGTTCGGA ACACCCGGCC GCAACCCTGG

 1151  GAGACGTCCC AGGGACTTCG GGGGCCGTTT TTGTGGCCCG ACCTGAGTCC

 1201  AAAAATCCCG ATCGTTTTGG ACTCTTTGGT GCACCCCCCT TAGAGGAGGG

 1251  ATATGTGGTT CTGGTAGGAG ACGAGAACCT AAAACAGTTC CCGCCTCCGT

 1301  CTGAATTTTT GCTTTCGGTT TGGGACCGAA GCCGCGCCGC GCGTCTTGTC

 1351  TGCTGCAGCA TCGTTCTGTG TTGTCTCTGT CTGACTGTGT TTCTGTATTT

 1401  GTCTGAAAAT ATGGGCCCGG GCCAGACTGT TACCACTCCC TTAAGTTTGA

 1451  CCTTAGGTCA CTGGAAAGAT GTCGAGCAGA TCGCTCACAA CCAGTCGGTA

 1501  GATGTCAAGA AGAGACGTTG GGTTACCTTC TGCTCTGCAG AATGGCCAAC

 1551  CTTTAACGTC GGATGGCCGC GAGACGGCAC CTTTAACCGA GACCTCATCA

 1601  CCCAGGTTAA GATCAAGGTC TTTTCACCTG GCCCGCATGG ACACCCAGAC

 1651  CAGGTCCCCT ACATCGTGAC CTGGGAAGCC TTGGCTTTTG ACCCCCCTCC

 1701  CTGGGTCAAG CCCTTTGTAC ACCCTAAGCC TCCGCCTCCT CTTCCTCCAT

 1751  CCGCCCCGTC TCTCCCCCTT GAACCTCCTC GTTCGACCCC GCCTCGATCC

 1801  TCCCTTTATC CAGCCCTCAC TCCTTCTCTA GGCGCCCCCA TATGGCCATA

 1851  TGAGATCTTA TATGGGGCAC CCCCGCCCCT TGTAAACTTC CCTGACCCTG

 1901  ACATGACAAG AGTTACTAAC AGCCCCTCTC TCCAAGCTCA CTTACAGGCT

 1951  CTCTACTTAG TCCAGCACGA AGTCTGGAGA CCTCTGGCGG CAGCCTACCA

 2001  AGAACAACTG GACCGACCGG TGGTACCTCA CCCTTACCGA GTCGGCGACA

 2051  CAGTGTGGGT CCGCCGACAC CAGACTAAGA ACCTAGAACC TCGCTGGAAA

 2101  GGACCTTACA CAGTCCTGCT GACCACCCCC ACCGCCCTCA AAGTAGACGG

 2151  CATCGCAGCT TGGATACACG CCGCCCACGT GAAGGCTGCC GACCCCGGGG

 2201  GTGGACCATC CTCTAGACTA TTAAGCGTAG TCAGGTACGT CGTAAGGGTA

 2251  AGCGTAATCC GGAACGTCGT ACGGATACGC GTAGTCTGGA ACGTCATATG

 2301  GGTACGAGCT CAGGCCTCGT ACGTTAATTA AGCTTAAGTT TAAACGCTAG

 2351  CTGTGTGTGA GTTCTTCTTT CTCGGTAACT TGTTGAAAGT ATTCAGAGTT

 2401  CTCGTCTTGT ATTCAATAAT TACTTCTTGG CAGATTTCAG TAGTTGCAGT

 2451  TGATTTACTT GGTTGCTGGT TACTTTTAAT TGATTCACTT TAACTTGCAC

 2501  TTTACTGCAG ATTGTTTAGC TTGTTCAGCT GCGCTTGTTT ATTTGCTTAG

 2551  CTTTCGCTTA GCGACGTGTT CACTTTGCTT GTTTGAATTG AATTGTCGCT

 2601  CCGTAGACGA AGCGCCTCTA TTTATACTCC GGCGGTCGAG GGTACTCCCG

 2651  GGGCGGAGCT ATGCGGGGCC GGGGCTAATC GCTAGGGGCG GGGCAGTACT

 2701  CCGACCCGGG TACTGAGCTT TCAGCAAGAG AACAATGCAC TTGTCCATCG

 2751  AGCTTTCAGC AAGAGAACAA TGCACTTGTC CATCGAGCTT TCAGCAAGAG

 2801  AACAATGCAC TTGTCCATCG AGCTAACGAG AACAATGCAC TTAGCGGTAT

 2851  CGAGAACAAT GCACTTAATA TGCGGCCGAG ATCTGTCGAC TTTATAATAA

 2901  GGGCGAATTC TGCAGATATC CATCACACTG GCGGCCGCTC GAGCGGCCGC

 2951  CTGCTTCATC CCCGTGGCCC GTTGCTCGCG TTTGCTGGCG GTGTCCCCGG

 3001  AAGAAATATA TTTGCATGTC TTTAGTTCTA TGATGACACA AACCCCGCCC

 3051  AGCGTCTTGT CATTGGCGAA TTCGAACACG CAGATGCAGT CGGGGCGGCG

 3101  CGGTCCGAGG TCCACTTCGC ATATTAAGGT GACGCGTGTG GCCTCGAACA

 3151  CCGAGCGACC CTGCAGCGAC CCGCTTAACA GCGTCAACAG CGTGCCGCAG

 3201  ATCCCGGGGG GCAATGAGAT ATGAAAAAGC CTGAACTCAC CGCGACGTCT

 3251  GTCGAGAAGT TTCTGATCGA AAAGTTCGAC AGCGTCTCCG ACCTGATGCA

 3301  GCTCTCGGAG GGCGAAGAAT CTCGTGCTTT CAGCTTCGAT GTAGGAGGGC

 3351  GTGGATATGT CCTGCGGGTA AATAGCTGCG CCGATGGTTT CTACAAAGAT

 3401  CGTTATGTTT ATCGGCACTT TGCATCGGCC GCGCTCCCGA TTCCGGAAGT

 3451  GCTTGACATT GGGGAATTCA GCGAGAGCCT GACCTATTGC ATCTCCCGCC

 3501  GTGCACAGGG TGTCACGTTG CAAGACCTGC CTGAAACCGA ACTGCCCGCT

 3551  GTTCTGCAGC CGGTCGCGGA GGCCATGGAT GCGATCGCTG CGGCCGATCT

 3601  TAGCCAGACG AGCGGGTTCG GCCCATTCGG ACCGCAAGGA ATCGGTCAAT

 3651  ACACTACATG GCGTGATTTC ATATGCGCGA TTGCTGATCC CCATGTGTAT

 3701  CACTGGCAAA CTGTGATGGA CGACACCGTC AGTGCGTCCG TCGCGCAGGC

 3751  TCTCGATGAG CTGATGCTTT GGGCCGAGGA CTGCCCCGAA GTCCGGCACC

 3801  TCGTGCACGC GGATTTCGGC TCCAACAATG TCCTGACGGA CAATGGCCGC

 3851  ATAACAGCGG TCATTGACTG GAGCGAGGCG ATGTTCGGGG ATTCCCAATA

 3901  CGAGGTCGCC AACATCTTCT TCTGGAGGCC GTGGTTGGCT TGTATGGAGC

 3951  AGCAGACGCG CTACTTCGAG CGGAGGCATC CGGAGCTTGC AGGATCGCCG

 4001  CGGCTCCGGG CGTATATGCT CCGCATTGGT CTTGACCAAC TCTATCAGAG

 4051  CTTGGTTGAC GGCAATTTCG ATGATGCAGC TTGGGCGCAG GGTCGATGCG

 4101  ACGCAATCGT CCGATCCGGA GCCGGGACTG TCGGGCGTAC ACAAATCGCC

 4151  CGCAGAAGCG CGGCCGTCTG GACCGATGGC TGTGTAGAAG TACTCGCCGA

 4201  TAGTGGAAAC CGACGCCCCA GCACTCGTCC GGATCGGGAG ATGGGGGAGG

 4251  CTAACTGATA AGCGGCCGCG ATCCGAGTTC TTCTGAGCGG GACTCTGGGG

 4301  TTCGATAAAA TAAAAGATTT TATTTAGTCT CCAGAAAAAG GGGGGAATGA

 4351  AAGACCCCAC CTGTAGGTTT GGCAAGCTAG CTTAAGTAAC GCCATTTTGC

 4401  AAGGCATGGA AAAATACATA ACTGATCATC CGGATGCATA CGTAGGGACC

 4451  CGGCGCGCCA CGCGTCCTCA CTCGGGGCGC CAGTCCTCCG ATTGACTGAG

 4501  TCGCCCGGGT ACCCGTGTAT CCAATAAACC CTCTTGCAGT TGCATCCGAC

 4551  TTGTGGTCTC GCTGTTCCTT GGGAGGGTCT CCTCTGAGTG ATTGACTACC

 4601  CGTCAGCGGG GGTCTTTCAT TTGGGGGCTC GTCCGGGATC GGGAGACCCC

 4651  TGCCCAGGGA CCACCGACCC ACCACCGGGA GGTAAGCTGG CTGCCTCGCG

 4701  CGTTTCGGTG ATGACGGTGA AAACCTCTGA CACATGCAGC TCCCGGAGAC

 4751  GGTCACAGCT TGTCTGTAAG CGGATGCCGG GAGCAGACAA GCCCGTCAGG

 4801  GCGCGTCAGC GGGTGTTGGC GGGTGTCGGG GCGCAGCCAT GACCCAGTCA

 4851  CGTAGCGATA GCGGAGTGTA TACTGGCTTA ACTATGCGGC ATCAGAGCAG

 4901  ATTGTACTGA GAGTGCACCA TATGCGGTGT GAAATACCGC ACAGATGCGT

 4951  AAGGAGAAAA TACCGCATCA GGCGCTCTTC CGCTTCCTCG CTCACTGACT

 5001  CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG

 5051  GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT

 5101  GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC

 5151  TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA

 5201  CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC

 5251  GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC

 5301  TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT

 5351  CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA

 5401  GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT

 5451  CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA

 5501  CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG

 5551  TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGGA

 5601  CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA

 5651  GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT

 5701  TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG

 5751  ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA

 5801  CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT

 5851  CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT

 5901  AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA

 5951  GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA

 6001  GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA

 6051  TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG

 6101  CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC

 6151  CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG

 6201  TTAATAGTTT GCGCAACGTT GTTGCCATTG CTGCAGGCAT CGTGGTGTCA

 6251  CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG

 6301  GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG

 6351  GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG

 6401  GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG

 6451  CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA

 6501  TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAACACGGGA TAATACCGCG

 6551  CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG

 6601  GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC

 6651  CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT

 6701  TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG

 6751  GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT

 6801  GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT

 6851  ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT

 6901  GCCACCTGAC GTCTAAGAAA CCATTATTAT CATGACATTA ACCTATAAAA

 6951  ATAGGCGTAT CACGAGGCCC TTTCGTCTTC AA

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