pRetroX-Tight-Pur载体描述pRetroX-Tight-Pur is an inducible, self-inactivating, retroviral expression vector designed to express a gene of interest under the control of PTight, a modified 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 modified minimal CMV promoter (1). This vector is supplied 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-Pur is optimized to eliminate promoter interference through LTR self-inactivation. 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 efficient expression. In this way, pRetroX-Tight-Pur supports high viral titers, yet eliminates potential downstream interference between the inducible PTight promoter and its adjacent viral LTR.pRetroX-Tight-Pur 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-Pur contains a puromycin resistance gene (Purr) 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-Pur 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, DH10B 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 vector sequence was 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 sequenced.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-Pur6567 bp DNASYNDEFINITION pRetroX-Tight-PurACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.FEATURES Location/Qualifiers source 1..6567 /organism="pRetroX-Tight-Pur" /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..3092 /label="ORF frame 3" /translation="MEAGRPLGQRPIAALLLRFLGSEAGKGWVRGRAQGRAQGRGGRP KVLRRPGILHASKAHVCRAVLLFLISGPFDLQPKLTMTEYKPTVRLATRDDVPRAVRT LAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPE SVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKG LGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRK PGA*" misc_feature 2405..2424 /label="mPGK_F_primer" /translation="MEAGRPLGQRPIAALLLRFLGSEAGKGWVRGRAQGRAQGRGGRP KVLRRPGILHASKAHVCRAVLLFLISGPFDLQPKLTMTEYKPTVRLATRDDVPRAVRT LAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPE SVEAGAVFAEIGPRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKG LGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRK PGA*" CDS complement(2418..3143) /label="ORF frame 2" /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG KLGLQVERPGDEEEENSAADVRF*" gene 2493..3092 /label="puro (variant)" /gene="puro (variant)" /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG KLGLQVERPGDEEEENSAADVRF*" misc_feature complement(3801..3823) /label="pGEX_3_primer" /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG KLGLQVERPGDEEEENSAADVRF*" misc_feature complement(3959..3979) /label="pBABE_3_primer" /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG KLGLQVERPGDEEEENSAADVRF*" misc_feature complement(3965..4180) /label="SV40_enhancer" /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG KLGLQVERPGDEEEENSAADVRF*" promoter 3977..4245 /label="SV40_promoter" /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG KLGLQVERPGDEEEENSAADVRF*" rep_origin 4144..4221 /label="SV40_origin" /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG KLGLQVERPGDEEEENSAADVRF*" misc_feature 4206..4225 /label="SV40pro_F_primer" /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG KLGLQVERPGDEEEENSAADVRF*" gene complement(5345..6205) /label="Ampicillin" /gene="Ampicillin" /translation="MGSCAPFGRALRVVGRASGTGLAGHAPGARSFGHLDVGGDGEAE PLVEGEVAGRGGLQEGGHPGALGRLHSGEHDGAAQTLALVVGRDADGGQEPRGLLGPV RRQEAFHLLLRGQPGTAQLGHARADLGEHRPRFDALRRGPDRHRGAVVRDPHLADVEP DAREEEFLQLGDPLDVAVRIDGVARGGVVGERGGEGAYGPGDVVAGGEAHRGLVLGHG KLGLQVERPGDEEEENSAADVRF*" CDS complement(5345..6205) /label="ORF frame 3" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" promoter complement(6247..6275) /label="AmpR_promoter" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" promoter 6497..506 /label="CMV_immearly_promoter" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW*" misc_feature 6561..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 cgacctgcag cccaagctta ccatgaccga gtacaagccc acggtgcgcc 2521 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact 2581 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc 2641 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg 2701 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg 2761 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg 2821 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg 2881 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg 2941 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct 3001 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca 3061 cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga 3121 ccgaaaggag cgcacgaccc catgcatcgg cgtctcgaga tatcagtggt ccaggctcta 3181 gttttgactc aacaatatca ccagctgaag cctatagagt acgagccata gataaaataa 3241 aagattttat ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc 3301 aagctagctt aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagag 3361 aagttcagat caaggtcagg aacagatgga acagggtcga ccctagagaa ccatcagatg 3421 tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag 3481 ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa 3541 cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca 3601 ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct 3661 ctgagtgatt gactacccgt cagcgggggt ctttcatttg ggggctcgtc cgggatcggg 3721 agacccctgc ccagggacca ccgacccacc accgggaggt aagctggctg cctcgcgcgt 3781 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 3841 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 3901 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtagat ccggctgtgg 3961 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 4021 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 4081 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 4141 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 4201 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga 4261 ggaggctttt ttggaggcct aggcttttgc aaaaagctta ctggcttaac tatgcggcat 4321 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa 4381 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 4441 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 4501 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 4561 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 4621 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 4681 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4741 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 4801 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4861 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 4921 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 4981 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 5041 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 5101 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 5161 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 5221 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 5281 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 5341 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 5401 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 5461 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 5521 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 5581 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 5641 gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 5701 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 5761 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 5821 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 5881 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 5941 gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag 6001 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 6061 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 6121 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 6181 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 6241 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 6301 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 6361 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattagctt 6421 ggccattgca tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa 6481 cattaccgcc atgttgacat tgattattga ctagttatta atagtaatca attacggggt 6541 cattagttca tagcccatat atggagt//
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