pRetroX-Tight-Hyg载体描述pRetroX-Tight-Hyg is an inducible, self-inactivating, retroviral expression vector designed to express a gene of interest under the control of PTight, a modifi ed Tet-responsive promoter. PTight consists of seven direct repeats of a 36 bp regulatory sequence that contains the 19 bp tet operator sequence (tetO), and a modifi ed minimal CMV promoter (1). This vector is designed for use with our Retro-XTM Tet-On Advanced and Tet-Off Advanced Inducible Expression Systems (Cat. Nos. 632104 and 632105). These systems provide ready access to the inducible gene expression strategy of Gossen & Bujard, and incorporate major improvements described by Urlinger, et al. (2-6).pRetroX-Tight-Hyg is optimized to eliminate promoter interference through LTR selfinactivation. The hybrid 5’ LTR consists of the cytomegalovirus (CMV) type I enhancer and the mouse sarcoma virus (MSV) promoter. This promoter drives high levels of viral genome transcription in HEK 293-based packaging cell lines due, in part, to the presence of adenoviral E1A (7-8) in these cells. The self-inactivating feature of the vector is provided by a deletion in the 3’ LTR enhancer region (U3). During reverse transcription of the retroviral RNA, a copy of the inactivated 3’ LTR U3 region replaces the corresponding region of the 5’ LTR, resulting in the inactivation of the 5’ LTR CMV enhancer sequence. This mechanism can reduce the phenomenon known as promoter interference (9-10) and allow more effi cient expression. In this way, pRetroX-Tight-Hyg supports high viral titers, yet eliminates potential downstream interference between the inducible PTight promoter and its adjacent viral LTR.pRetroX-Tight-Hyg contains all of the necessary viral RNA processing elements; these include the 5’ and 3’ LTRs, the packaging signal (Ψ+), and the tRNA primer binding site. For safety reasons, however, the vector lacks the structural genes (gag, pol, and env) necessary for retroviral particle formation and replication. pRetroX-Tight-Hyg contains a hygromycin resistance gene (Hygr) under the control of the murine phosphoglycerate kinase (PGK) promoter (PPGK) for the selection of stable transfectants. In addition, the vector contains a ColE1 origin of replication and an E. coli Ampr gene for propagation and selection in bacteria.pRetroX-Tight-Hyg can be used as either a plasmid or retroviral expression vector. When used as a retroviral expression vector, it must be transfected into a packaging cell line, such as GP2-293; we recommend the Retro-X Universal Packaging System (Cat. No. 631530). Packaging cell lines allow you to produce infectious, replication-incompetent retroviral particles. These retroviral particles can infect a wide range of target cells and transmit your gene of interest, but they cannot replicate within these cells due to the absence of viral structural genes. The separate introduction and integration of the structural genes into the packaging cell line minimizes the chance of producing replication-competent virus due to recombination events during cell proliferation.Propagation in E. coli Suitable host strains: DH5α and other general purpose strains. Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts. E. coli replication origin: ColE1 Copy number: highNotes:The attached sequence fi le has been compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by Clontech. This vector has not been completely sequence.The viral supernatants produced by this retroviral vector could contain potentially hazardous recombinant virus. Due caution must be exercised in the production and handling of recombinant retrovirus. Appropriate NIH, regional, and institutional guidelines apply.
LOCUS pRetroX-Tight-Hyg7419 bp DNASYNDEFINITION pRetroX-Tight-HygACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.FEATURES Location/Qualifiers source 1..7419 /organism="pRetroX-Tight-Hyg" /mol_type="other DNA" promoter 2..506 /label="CMV_immearly_promoter" misc_feature 6..688 /label="5_LTR" misc_feature 9..296 /label="CAG_enhancer" misc_feature 463..483 /label="CMV_fwd_primer" misc_feature 758..1567 /label="psi_plus_pack" misc_feature 1154..1561 /label="gag" misc_feature 1497..1519 /label="MSCV_primer" misc_feature 1497..1519 /label="pLXSN_5_primer" misc_feature 1535..1551 /label="pBABE_5_primer" CDS 1669..2196 /label="ORF frame 1" /translation="MSSLLPISDRERMSSLLPISDRERMSSLLPISDRERMSSLSLSV IENVCRVYSLSVIENVCRGRRVRWEAYISRARLVNRQIAWRRIRGRAGSRSRTREFYR VGEALFPRQSGACALAAPLGTWRYTSGLWPRTHSTSTGRRQPAPFFGGPFAPPSTPPL VRKFPPAPQLASCRT*" misc_feature 1888..1907 /label="pCEP_fwd_primer" /translation="MSSLLPISDRERMSSLLPISDRERMSSLLPISDRERMSSLSLSV IENVCRVYSLSVIENVCRGRRVRWEAYISRARLVNRQIAWRRIRGRAGSRSRTREFYR VGEALFPRQSGACALAAPLGTWRYTSGLWPRTHSTSTGRRQPAPFFGGPFAPPSTPPL VRKFPPAPQLASCRT*" misc_feature 1890..1914 /label="LNCX_primer" /translation="MSSLLPISDRERMSSLLPISDRERMSSLLPISDRERMSSLSLSV IENVCRVYSLSVIENVCRGRRVRWEAYISRARLVNRQIAWRRIRGRAGSRSRTREFYR VGEALFPRQSGACALAAPLGTWRYTSGLWPRTHSTSTGRRQPAPFFGGPFAPPSTPPL VRKFPPAPQLASCRT*" misc_feature complement(2008..2031) /label="MSCV_rev_primer" /translation="MSSLLPISDRERMSSLLPISDRERMSSLLPISDRERMSSLSLSV IENVCRVYSLSVIENVCRGRRVRWEAYISRARLVNRQIAWRRIRGRAGSRSRTREFYR VGEALFPRQSGACALAAPLGTWRYTSGLWPRTHSTSTGRRQPAPFFGGPFAPPSTPPL VRKFPPAPQLASCRT*" CDS 2253..3515 /label="ORF frame 3" /translation="MEAGRPLGQRPIAALLLRFLGSEAGKGWVRGRAQGRAQGRGGRP KVLRRPGILHASKAHVCRAVLLFLISGPFDLHPATMKKPELTATSVEKFLIEKFDSVS DLMQLSEGEESRAFSFDVGGRGYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIG EFSESLTYCISRRAQGVTLQDLPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQ GIGQYTTWRDFICAIADPHVYHWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHA DFGSNNVLTDNGRITAVIDWSEAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPE LAGSPRLRAYMLRIGLDQLYQSLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRS AAVWTDGCVEVLADSGNRRPSTRPRAKE*" misc_feature 2405..2424 /label="mPGK_F_primer" /translation="MEAGRPLGQRPIAALLLRFLGSEAGKGWVRGRAQGRAQGRGGRP KVLRRPGILHASKAHVCRAVLLFLISGPFDLHPATMKKPELTATSVEKFLIEKFDSVS DLMQLSEGEESRAFSFDVGGRGYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIG EFSESLTYCISRRAQGVTLQDLPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQ GIGQYTTWRDFICAIADPHVYHWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHA DFGSNNVLTDNGRITAVIDWSEAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPE LAGSPRLRAYMLRIGLDQLYQSLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRS AAVWTDGCVEVLADSGNRRPSTRPRAKE*" CDS complement(2418..3248) /label="ORF frame 2" /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT AGDAIGQALAELPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLFHGGGMQVERPGDEEEENSAADVRF *" gene 2505..3512 /label="hygroB" /gene="hygroB" /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT AGDAIGQALAELPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLFHGGGMQVERPGDEEEENSAADVRF *" misc_feature complement(4653..4675) /label="pGEX_3_primer" /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT AGDAIGQALAELPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLFHGGGMQVERPGDEEEENSAADVRF *" misc_feature complement(4811..4831) /label="pBABE_3_primer" /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT AGDAIGQALAELPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLFHGGGMQVERPGDEEEENSAADVRF *" misc_feature complement(4817..5032) /label="SV40_enhancer" /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT AGDAIGQALAELPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLFHGGGMQVERPGDEEEENSAADVRF *" promoter 4829..5097 /label="SV40_promoter" /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT AGDAIGQALAELPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLFHGGGMQVERPGDEEEENSAADVRF *" rep_origin 4996..5073 /label="SV40_origin" /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT AGDAIGQALAELPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLFHGGGMQVERPGDEEEENSAADVRF *" misc_feature 5058..5077 /label="SV40pro_F_primer" /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT AGDAIGQALAELPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLFHGGGMQVERPGDEEEENSAADVRF *" gene complement(6197..7057) /label="Ampicillin" /gene="Ampicillin" /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT AGDAIGQALAELPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLFHGGGMQVERPGDEEEENSAADVRF *" CDS complement(6197..7057) /label="ORF frame 3" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" promoter complement(7099..7127) /label="AmpR_promoter" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" promoter 7349..506 /label="CMV_immearly_promoter" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" misc_feature 7413..688 /label="5_LTR" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*"ORIGIN 1 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC 61 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC 121 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA 181 TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC 241 AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA 301 TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC 361 GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC 421 AACGGGACTT TCCAAAATGT CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC 481 GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCAATAAAA GAGCCCACAA CCCCTCACTC 541 GGCGCGCCAG TCTTCCGATA GACTGCGTCG CCCGGGTACC CGTATTCCCA ATAAAGCCTC 601 TTGCTGTTTG CATCCGAATC GTGGTCTCGC TGTTCCTTGG GAGGGTCTCC TCTGAGTGAT 661 TGACTACCCA CGACGGGGGT CTTTCATTTG GGGGCTCGTC CGGGATTTGG AGACCCCTGC 721 CCAGGGACCA CCGACCCACC ACCGGGAGGT AAGCTGGCCA GCAACTTATC TGTGTCTGTC 781 CGATTGTCTA GTGTCTATGT TTGATGTTAT GCGCCTGCGT CTGTACTAGT TAGCTAACTA 841 GCTCTGTATC TGGCGGACCC GTGGTGGAAC TGACGAGTTC TGAACACCCG GCCGCAACCC 901 TGGGAGACGT CCCAGGGACT TTGGGGGCCG TTTTTGTGGC CCGACCTGAG GAAGGGAGTC 961 GATGTGGAAT CCGACCCCGT CAGGATATGT GGTTCTGGTA GGAGACGAGA ACCTAAAACA 1021 GTTCCCGCCT CCGTCTGAAT TTTTGCTTTC GGTTTGGAAC CGAAGCCGCG CGTCTTGTCT 1081 GCTGCAGCGC TGCAGCATCG TTCTGTGTTG TCTCTGTCTG ACTGTGTTTC TGTATTTGTC 1141 TGAAAATTAG GGCCAGACTG TTACCACTCC CTTAAGTTTG ACCTTAGGTC ACTGGAAAGA 1201 TGTCGAGCGG ATCGCTCACA ACCAGTCGGT AGATGTCAAG AAGAGACGTT GGGTTACCTT 1261 CTGCTCTGCA GAATGGCCAA CCTTTAACGT CGGATGGCCG CGAGACGGCA CCTTTAACCG 1321 AGACCTCATC ACCCAGGTTA AGATCAAGGT CTTTTCACCT GGCCCGCATG GACACCCAGA 1381 CCAGGTCCCC TACATCGTGA CCTGGGAAGC CTTGGCTTTT GACCCCCCTC CCTGGGTCAA 1441 GCCCTTTGTA CACCCTAAGC CTCCGCCTCC TCTTCCTCCA TCCGCCCCGT CTCTCCCCCT 1501 TGAACCTCCT CGTTCGACCC CGCCTCGATC CTCCCTTTAT CCAGCCCTCA CTCCTTCTCT 1561 AGGCGCCGGA ATTGAAGATC TGAGGCCCTT TCGTCTTCAC TCGAGTTTAC TCCCTATCAG 1621 TGATAGAGAA CGTATGTCGA GTTTACTCCC TATCAGTGAT AGAGAACGAT GTCGAGTTTA 1681 CTCCCTATCA GTGATAGAGA ACGTATGTCG AGTTTACTCC CTATCAGTGA TAGAGAACGT 1741 ATGTCGAGTT TACTCCCTAT CAGTGATAGA GAACGTATGT CGAGTTTATC CCTATCAGTG 1801 ATAGAGAACG TATGTCGAGT TTACTCCCTA TCAGTGATAG AGAACGTATG TCGAGGTAGG 1861 CGTGTACGGT GGGAGGCCTA TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG 1921 AGAAGGATCC GCGGCCGCGC CGGCTCTAGA TCGCGAACGC GTGAATTCTA CCGGGTAGGG 1981 GAGGCGCTTT TCCCAAGGCA GTCTGGAGCA TGCGCTTTAG CAGCCCCGCT GGGCACTTGG 2041 CGCTACACAA GTGGCCTCTG GCCTCGCACA CATTCCACAT CCACCGGTAG GCGCCAACCG 2101 GCTCCGTTCT TTGGTGGCCC CTTCGCGCCA CCTTCTACTC CTCCCCTAGT CAGGAAGTTC 2161 CCCCCCGCCC CGCAGCTCGC GTCGTGCAGG ACGTGACAAA TGGAAGTAGC ACGTCTCACT 2221 AGTCTCGTGC AGATGGACAG CACCGCTGAG CAATGGAAGC GGGTAGGCCT TTGGGGCAGC 2281 GGCCAATAGC AGCTTTGCTC CTTCGCTTTC TGGGCTCAGA GGCTGGGAAG GGGTGGGTCC 2341 GGGGGCGGGC TCAGGGGCGG GCTCAGGGGC GGGGCGGGCG CCCGAAGGTC CTCCGGAGGC 2401 CCGGCATTCT GCACGCTTCA AAAGCGCACG TCTGCCGCGC TGTTCTCCTC TTCCTCATCT 2461 CCGGGCCTTT CGACCTGCAT CCCGCCACCA TGAAAAAGCC TGAACTCACC GCGACGTCTG 2521 TCGAGAAGTT TCTGATCGAA AAGTTCGACA GCGTCTCCGA CCTGATGCAG CTCTCGGAGG 2581 GCGAAGAATC TCGTGCTTTC AGCTTCGATG TAGGAGGGCG TGGATATGTC CTGCGGGTAA 2641 ATAGCTGCGC CGATGGTTTC TACAAAGATC GTTATGTTTA TCGGCACTTT GCATCGGCCG 2701 CGCTCCCGAT TCCGGAAGTG CTTGACATTG GGGAGTTCAG CGAGAGCCTG ACCTATTGCA 2761 TCTCCCGCCG TGCACAGGGT GTCACGTTGC AAGACCTGCC TGAAACCGAA CTGCCCGCTG 2821 TTCTGCAGCC GGTCGCGGAG GCCATGGATG CGATCGCTGC GGCCGATCTT AGCCAGACGA 2881 GCGGGTTCGG CCCATTCGGA CCGCAAGGAA TCGGTCAATA CACTACATGG CGTGATTTCA 2941 TATGCGCGAT TGCTGATCCC CATGTGTATC ACTGGCAAAC TGTGATGGAC GACACCGTCA 3001 GTGCGTCCGT CGCGCAGGCT CTCGATGAGC TGATGCTTTG GGCCGAGGAC TGCCCCGAAG 3061 TCCGGCACCT CGTGCACGCG GATTTCGGCT CCAACAATGT CCTGACGGAC AATGGCCGCA 3121 TAACAGCGGT CATTGACTGG AGCGAGGCGA TGTTCGGGGA TTCCCAATAC GAGGTCGCCA 3181 ACATCTTCTT CTGGAGGCCG TGGTTGGCTT GTATGGAGCA GCAGACGCGC TACTTCGAGC 3241 GGAGGCATCC GGAGCTTGCA GGATCGCCGC GGCTCCGGGC GTATATGCTC CGCATTGGTC 3301 TTGACCAACT CTATCAGAGC TTGGTTGACG GCAATTTCGA TGATGCAGCT TGGGCGCAGG 3361 GTCGATGCGA CGCAATCGTC CGATCCGGAG CCGGGACTGT CGGGCGTACA CAAATCGCCC 3421 GCAGAAGCGC GGCCGTCTGG ACCGATGGCT GTGTAGAAGT ACTCGCCGAT AGTGGAAACC 3481 GACGCCCCAG CACTCGTCCG AGGGCAAAGG AATAGAGTAG ATGCCGACCG AACAAGAGCT 3541 GATTTCGAGA ACGCCTCAGC CAGCAACTCG CGCGAGCCTA GCAAGGCAAA TGCGAGAGAA 3601 CGGCCTTACG CTTGGTGGCA CAGTTCTCGT CCACAGTTCG CTAAGCTCGC TCGGCTGGGT 3661 CGCGGGAGGG CCGGTCGCAG TGATTCAGGC CCTTCTGGAT TGTGTTGGTC CCCAGGGCAC 3721 GATTGTCATG CCCACGCACT CGGGTGATCT GACTGATCCC GCAGATTGGA GATCGCCGCC 3781 CGTGCCTGCC GATTGGGTGC AGATCCGTCG ACCTGCAGCC AAGCTTATCG ATAAAATAAA 3841 AGATTTTATT TAGTCTCCAG AAAAAGGGGG GAATGAAAGA CCCCACCTGT AGGTTTGGCA 3901 AGCTAGCTTA AGTAACGCCA TTTTGCAAGG CATGGAAAAT ACATAACTGA GAATAGAGAA 3961 GTTCAGATCA AGGTTAGGAA CAGAGAGACA GCAGAATATG GGCCAAACAG GATATCAGTG 4021 GTCCAGGCTC TAGTTTTGAC TCAACAATAT CACCAGCTGA AGCCTATAGA GTACGAGCCA 4081 TAGATAAAAT AAAAGATTTT ATTTAGTCTC CAGAAAAAGG GGGGAATGAA AGACCCCACC 4141 TGTAGGTTTG GCAAGCTAGC TTAAGTAACG CCATTTTGCA AGGCATGGAA AAATACATAA 4201 CTGAGAATAG AGAAGTTCAG ATCAAGGTCA GGAACAGATG GAACAGGGTC GACCCTAGAG 4261 AACCATCAGA TGTTTCCAGG GTGCCCCAAG GACCTGAAAT GACCCTGTGC CTTATTTGAA 4321 CTAACCAATC AGTTCGCTTC TCGCTTCTGT TCGCGCGCTT CTGCTCCCCG AGCTCAATAA 4381 AAGAGCCCAC AACCCCTCAC TCGGGGCGCC AGTCCTCCGA TTGACTGAGT CGCCCGGGTA 4441 CCCGTGTATC CAATAAACCC TCTTGCAGTT GCATCCGACT TGTGGTCTCG CTGTTCCTTG 4501 GGAGGGTCTC CTCTGAGTGA TTGACTACCC GTCAGCGGGG GTCTTTCATT TGGGGGCTCG 4561 TCCGGGATCG GGAGACCCCT GCCCAGGGAC CACCGACCCA CCACCGGGAG GTAAGCTGGC 4621 TGCCTCGCGC GTTTCGGTGA TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG 4681 GTCACAGCTT GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG CGCGTCAGCG 4741 GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG CGGAGTGTAG 4801 ATCCGGCTGT GGAATGTGTG TCAGTTAGGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC 4861 AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA GGTGTGGAAA GTCCCCAGGC 4921 TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG 4981 CCCCTAACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT 5041 GGCTGACTAA TTTTTTTTAT TTATGCAGAG GCCGAGGCCG CCTCGGCCTC TGAGCTATTC 5101 CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT GCAAAAAGCT TACTGGCTTA 5161 ACTATGCGGC ATCAGAGCAG ATTGTACTGA GAGTGCACCA TATGCGGTGT GAAATACCGC 5221 ACAGATGCGT AAGGAGAAAA TACCGCATCA GGCGCTCTTC CGCTTCCTCG CTCACTGACT 5281 CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC 5341 GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA 5401 AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG 5461 ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA 5521 GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC 5581 TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC 5641 GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC 5701 CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG 5761 TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT 5821 ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGGA 5881 CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT 5941 CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA 6001 TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG 6061 CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT 6121 TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT 6181 AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC 6241 TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG 6301 GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG 6361 ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT 6421 TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG 6481 TTAATAGTTT GCGCAACGTT GTTGCCATTG CTGCAGGCAT CGTGGTGTCA CGCTCGTCGT 6541 TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA 6601 TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG 6661 CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT 6721 CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA 6781 TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAACACGGGA TAATACCGCG CCACATAGCA 6841 GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT 6901 TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT 6961 CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA 7021 AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT 7081 GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA 7141 ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA 7201 CCATTATTAT CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTCTTC 7261 AAGAATTAGC TTGGCCATTG CATACGTTGT ATCCATATCA TAATATGTAC ATTTATATTG 7321 GCTCATGTCC AACATTACCG CCATGTTGAC ATTGATTATT GACTAGTTAT TAATAGTAAT 7381 CAATTACGGG GTCATTAGTT CATAGCCCAT ATATGGAGT//
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