质粒名称: | pXD023 - pLKO-EF1a-Puro-P2A-FLuc-WPRE |
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目录编号: | 192203 |
存储实验室: | Sidi Chen |
载体骨架基本信息 |
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载体名称: | pLenti |
空载体大小: | 4038 bp |
完整质粒大小: | 9668 bp |
载体类型: | Lentiviral ; Mammalian Expression |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | NEB Stable |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | Lenti-PL(Puro-Luciferase) |
物种来源: | Synthetic |
克隆方法: | 限制性内切酶 |
5’克隆位点: | 未知 |
文献引用方法 |
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材料和方法部分: | pXD023 - pLKO-EF1a-Puro-P2A-FLuc-WPRE was a gift from Sidi Chen (Addgene plasmid # 192203 ; http://n2t.net/addgene:192203 ; RRID:Addgene_192203) |
参考文献部分: | A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy. Ye L, Park JJ, Peng L, Yang Q, Chow RD, Dong MB, Lam SZ, Guo J, Tang E, Zhang Y, Wang G, Dai X, Du Y, Kim HR, Cao H, Errami Y, Clark P, Bersenev A, Montgomery RR, Chen S. Cell Metab. 2022 Apr 5;34(4):595-614.e14. doi: 10.1016/j.cmet.2022.02.009. Epub 2022 Mar 10. 10.1016/j.cmet.2022.02.009 PubMed 35276062 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pXD023 - pLKO-EF1a-Puro-P2A-FLuc-WPRE 9668 bp ds-DNA circular SYN 21-OCT-2022 DEFINITION Lenti-PL(Puro-Luciferase). ACCESSION . VERSION . KEYWORDS pXD023 - pLKO-EF1a-Puro-P2A-FLuc-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9668) AUTHORS Ye L, Park JJ, Peng L, Yang Q, Chow RD, Dong MB, Lam SZ, Guo J, Tang E, Zhang Y, Wang G, Dai X, Du Y, Kim HR, Cao H, Errami Y, Clark P, Bersenev A, Montgomery RR, Chen S TITLE A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy. JOURNAL Cell Metab. 2022 Apr 5;34(4):595-614.e14. doi: 10.1016/j.cmet.2022.02.009. Epub 2022 Mar 10. PUBMED 35276062 REFERENCE 2 (bases 1 to 9668) AUTHORS . TITLE Direct Submission JOURNAL Exported Oct 21, 2022 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..9668 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 5..231 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 232..412 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 459..584 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1077..1310 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1495..1539 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 1741..1858 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1997..3175 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2228..3166 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3123..3143 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 3188..3784 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(3188..3207) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3684..3704 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 3794..3850 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 3851..5503 /codon_start=1 /gene="luc+" /product="firefly luciferase" /label=luciferase /note="enhanced luc+ version of the luciferase gene" /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV" primer_bind complement(3917..3935) /label=LucNrev /note="Firefly luciferase, reverse primer" misc_feature 5525..6113 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5578..5598) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5996..6007) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 6185..6418 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 6496..6617 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6533..6552) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6587..6606 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 6657..6792 /label=SV40 ori /note="SV40 origin of replication" primer_bind 6719..6738 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(6812..6831) /label=T7 /note="T7 promoter, forward primer" promoter complement(6813..6831) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6841..6858) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6841..6857) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6850..6872) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 6999..7454 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7086..7105) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7296..7317 /label=F1ori-F /note="F1 origin, forward primer" promoter 7480..7584 /gene="bla" /label=AmpR promoter CDS 7585..8445 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(7803..7822) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8616..9204 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9105..9124 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 9358..9375 /label=L4440 /note="L4440 vector, forward primer" protein_bind 9492..9513 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 9528..9558 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9566..9582 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 9571..9593 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 9590..9606 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 9590..9606 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 9625..9645 /label=T3 /note="T3 promoter, forward primer" promoter 9627..9645 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa 61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac 121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa 181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc 241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc 421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc 481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga 601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat 661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt 721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag 781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa 841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa 901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg 961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta 1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga 1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc 1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat 1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac 1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat 1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca 1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga 1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg 1681 tttcagaccc acctcccaac cccgagggga cccgagacaa atggcagtat tcatccacaa 1741 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 1801 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg 1861 ggtttattac agggacagca gagatccact ttggcgccgg ctcgaggggg cccgggtgca 1921 aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt 1981 gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt 2041 ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg 2101 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga 2161 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag 2221 aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc 2281 ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg 2341 ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc 2401 ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg 2461 cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc 2521 gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat 2581 ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc 2641 gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg 2701 gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct 2761 ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg 2821 gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag 2881 gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc 2941 gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga 3001 ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc 3061 ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat 3121 tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgacgtac 3181 ggccaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag 3241 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga 3301 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg 3361 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gccgtggcgg tctggaccac 3421 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt 3481 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc 3541 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg 3601 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc 3661 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt 3721 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg 3781 tgccggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa 3841 tcctggaccg atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatccgct 3901 ggaagatgga accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc 3961 tggaacaatt gcttttacag atgcacatat cgaggtggac atcacttacg ctgagtactt 4021 cgaaatgtcc gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag 4081 aatcgtcgta tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt 4141 tatcggagtt gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag 4201 tatgggcatt tcgcagccta ccgtggtgtt cgtttccaaa aaggggttgc aaaaaatttt 4261 gaacgtgcaa aaaaagctcc caatcatcca aaaaattatt atcatggatt ctaaaacgga 4321 ttaccaggga tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa 4381 tgaatacgat tttgtgccag agtccttcga tagggacaag acaattgcac tgatcatgaa 4441 ctcctctgga tctactggtc tgcctaaagg tgtcgctctg cctcatagaa ctgcctgcgt 4501 gagattctcg catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat 4561 tttaagtgtt gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat 4621 atgtggattt cgagtcgtct taatgtatag atttgaagag gagctgtttc tgaggagcct 4681 tcaggattac aagattcaaa gtgcgctgct ggtgccaacc ctattctcct tcttcgccaa 4741 aagcactctg attgacaaat acgatttatc taatttacac gaaattgctt ctggtggcgc 4801 tcccctctct aaggaagtcg gggaagcggt tgccaagagg ttccatctgc caggtatcag 4861 gcaaggatat gggctcactg agactacatc agctattctg attacacccg agggggatga 4921 taaaccgggc gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga 4981 taccgggaaa acgctgggcg ttaatcaaag aggcgaactg tgtgtgagag gtcctatgat 5041 tatgtccggt tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg 5101 gctacattct ggagacatag cttactggga cgaagacgaa cacttcttca tcgttgaccg 5161 cctgaagtct ctgattaagt acaaaggcta tcaggtggct cccgctgaat tggaatccat 5221 cttgctccaa caccccaaca tcttcgacgc aggtgtcgca ggtcttcccg acgatgacgc 5281 cggtgaactt cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga 5341 gatcgtggat tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt 5401 gtttgtggac gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga 5461 gatcctcata aaggccaaga agggcggaaa gatcgccgtg taagaattca cgcgttaagt 5521 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 5581 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 5641 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga 5701 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc 5761 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct 5821 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg 5881 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct 5941 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc 6001 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg 6061 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact 6121 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg 6181 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg 6241 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 6301 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 6361 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 6421 acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata 6481 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc 6541 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 6601 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc 6661 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta 6721 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct 6781 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc 6841 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 6901 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 6961 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt 7021 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 7081 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 7141 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 7201 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 7261 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 7321 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc 7381 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt 7441 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 7501 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 7561 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 7621 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 7681 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 7741 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 7801 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 7861 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 7921 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 7981 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 8041 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 8101 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 8161 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 8221 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 8281 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 8341 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 8401 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 8461 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 8521 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 8581 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 8641 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 8701 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 8761 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 8821 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 8881 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 8941 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 9001 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 9061 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 9121 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 9181 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 9241 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 9301 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 9361 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 9421 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 9481 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 9541 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 9601 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag 9661 ctgcaagc //