质粒名称: | pHR-OptoCAR-mScarlet |
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出品公司: | Addgene |
目录编号: | 170463 |
存储实验室: | John James |
载体骨架基本信息 |
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载体名称: | pHR-SIN |
载体出品公司: | Clontech |
完整质粒大小: | 11966bp |
载体类型: | 哺乳细胞表达, Lentiviral |
筛选标记: | 新霉素(G418) |
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: | OptoCAR |
物种来源: | Synthetic |
插入大小: | 3000bp |
启动子: | SFFV |
标签/融合蛋白: | mScarlet (C terminal on insert) |
克隆方法: | 限制性内切酶 |
5’克隆位点: | AsiSI (not destroyed) |
3’克隆位点: | NotI (not destroyed) |
5’测序引物 | cttctgttcgcgcgcttctgcttc |
3’测序引物 | CCAGAGGTTGATTATCGATAAGC |
文献引用方法 |
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材料和方法部分: |
pHR-OptoCAR-mScarlet was a gift from John James (Addgene plasmid # 170463 ; http://n2t.net/addgene:170463 ; RRID:Addgene_170463)
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参考文献部分: | Quantifying persistence in the T-cell signaling network using an optically controllable antigen receptor. Harris MJ, Fuyal M, James JR. Mol Syst Biol. 2021 May;17(5):e10091. doi: 10.15252/msb.202010091. 10.15252/msb.202010091 PubMed 33988299 |
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载体基本信息
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载体序列
LOCUS pHR-OptoCAR-mScarlet 11966 bp ds-DNA circular SYN 04-AUG-2021 DEFINITION Expresses an optically-controllable chimeric antigen receptor that is dependent on dimerizer drug, fused to mScarlet fluorophore. ACCESSION . VERSION . KEYWORDS pHR-OptoCAR-mScarlet SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11966) AUTHORS Harris MJ, Fuyal M, James JR TITLE Quantifying persistence in the T-cell signaling network using an optically controllable antigen receptor. JOURNAL Mol Syst Biol. 2021 May;17(5):e10091. doi: 10.15252/msb.202010091. PUBMED 33988299 REFERENCE 2 (bases 1 to 11966) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 4, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..11966 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..408 /label=SFFV promoter /note="spleen focus-forming virus long terminal repeat (LTR) promoter" regulatory 446..455 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 515..691 /codon_start=1 /product="""Zdark"" protein, derived from the Z subunit of protein A, that binds to the dark state of LOV2 and is released upon irradiation (Wang et al., 2016)" /label=Zdk1 /translation="MVDNKFNKEKTRAGAEIHSLPNLNVEQKFAFIVSLFDDPSQSANL LAEAKKLNDAQAPK" regulatory 1028..1037 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1034..1729 /codon_start=1 /product="bright monomeric red fluorescent protein evolved from a synthetic template (Bindels et al., 2016)" /label=mScarlet /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH STGGMDELYK" primer_bind complement(1167..1185) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1435..1454 /label=mCherry-F /note="mCherry, forward primer" CDS 1751..1807 /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 1868..1894 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1895..2215 /codon_start=1 /product="F36V mutant of FK506-binding protein FKBP12" /label=DmrB /note="binds synthetic ligands that do not bind wild-type FKBP" /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL KLE" misc_feature 3511..4099 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3564..3584) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3982..3993) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(4102..4118) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4103..4119) /label=pBluescriptKS /note="For pBluescript vector" LTR 4209..4442 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" promoter complement(4467..4485) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(4468..4485) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(4775..4794) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind complement(5008..5027) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5127..5149 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5187..5205) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5273..5377 /gene="bla" /label=AmpR promoter CDS 5378..6238 /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(5596..5615) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6409..6997 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6898..6917 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7151..7168 /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(7238..7258) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 7243..7572 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 7423..7558 /label=SV40 ori /note="SV40 origin of replication" primer_bind 7485..7504 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" intron 8706..8771 /label=small t intron /note="SV40 (simian virus 40) small t antigen intron" CDS 8901..8921 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 9346..9480 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(9383..9402) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 9437..9456 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" LTR 9648..10281 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 10328..10453 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 10950..11183 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 11368..11412 /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 11697..11814 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 gtaacgccat tttgcaaggc atggaaaaat accaaaccaa gaatagagaa gttcagatca 61 agggcgggta catgaaaata gctaacgttg ggccaaacag gatatctgcg gtgagcagtt 121 tcggccccgg cccggggcca agaacagatg gtcaccgcag tttcggcccc ggcccgaggc 181 caagaacaga tggtccccag atatggccca accctcagca gtttcttaag acccatcaga 241 tgtttccagg ctcccccaag gacctgaaat gaccctgcgc cttatttgaa ttaaccaatc 301 agcctgcttc tcgcttctgt tcgcgcgctt ctgcttcccg agctctataa aagagctcac 361 aacccctcac tcggcgcgcc agtcctccga cagactgagt cgcccggggg ggatctggag 421 ctctcgagaa ttctcacgcg cgatcgccac catgggctgt ggctgcagct cacacccgga 481 agatgacggc ggatctggag gctccggcgg atctatggtg gataacaaat tcaataaaga 541 aaagacacgt gccggtgcgg aaatccattc tctgccaaac ctgaatgttg agcagaagtt 601 tgccttcatc gtgagcctgt ttgatgatcc atctcagagc gccaatctgc tggccgaagc 661 caaaaaactg aacgatgccc aggccccaaa aactagcgca gacgcccccg cgtaccagca 721 gggccagaac cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt 781 ggacaagaga cgtggccggg accctgagat ggggggaaag ccgcagagaa ggaagaaccc 841 tcaggaaggc ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat 901 tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag 961 tacagccacc aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgggatcc 1021 accggtcgcc accatggtga gcaagggcga ggcagtgatc aaggagttca tgcggttcaa 1081 ggtgcacatg gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg 1141 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc 1201 cttctcctgg gacatcctgt cccctcagtt catgtacggc tccagggcct tcaccaagca 1261 ccccgccgac atccccgact actataagca gtccttcccc gagggcttca agtgggagcg 1321 cgtgatgaac ttcgaggacg gcggcgccgt gaccgtgacc caggacacct ccctggagga 1381 cggcaccctg atctacaagg tgaagctccg cggcaccaac ttccctcctg acggccccgt 1441 aatgcagaag aagacaatgg gctgggaagc gtccaccgag cggttgtacc ccgaggacgg 1501 cgtgctgaag ggcgacatta agatggccct gcgcctgaag gacggaggcc gctacctggc 1561 ggacttcaag accacctaca aggccaagaa gcccgtgcag atgcccggcg cctacaacgt 1621 cgaccgcaag ttggacatca cctcccacaa cgaggactac accgtggtgg aacagtacga 1681 acgctccgag ggccgccact ccaccggcgg catggacgag ctgtacaaga ccagttacca 1741 gggccctggc gccacgaact tctctctgtt aaagcaagca ggagacgtgg aagaaaaccc 1801 cggtcccacg cgtatgggag tcaaagttct gtttgccctg atctgcatcg ctgtggccga 1861 ggccaaatat ccgtacgacg taccagacta cgcaggcgtc caagtcgaaa ccattagtcc 1921 cggcgatggc agaacatttc ctaaaagggg acaaacatgt gtcgtccatt atacaggcat 1981 gttggaggac ggcaaaaagg tggacagtag tagagatcgc aataaacctt tcaaattcat 2041 gttgggaaaa caagaagtca ttaggggatg ggaggagggc gtggctcaaa tgtccgtcgg 2101 ccaacgcgct aagctcacca tcagccccga ctacgcatac ggcgctaccg gacatcccgg 2161 aattattccc cctcacgcta ccttggtgtt tgacgtcgaa ctgttgaagc tcgagggcgg 2221 aagcggaggt agtggtggaa gcctgaagat tcaagcttat ttcaatgaga ctgcagacct 2281 gccatgccaa tttgcaaact ctcaaaacca aagcctgagt gagctagtag tattttggca 2341 ggaccaggaa aacttggttc tgaatgaggt atacttaggc aaagagaaat ttgacagtgt 2401 tcattccaag tatatgggcc gcacaagttt tgattcggac agttggaccc tgagacttca 2461 caatcttcag atcaaggaca agggcttgta tcaatgtatc atccatcaca aaaagcccac 2521 aggaatgatt cgcatccacc agatgaattc tgaactgtca gtgcttgcta acttcagtca 2581 acctgaaata gtaccaattt ctaatataac agaaaatgtg tacataaatt tgacctgctc 2641 atctatacac ggttacccag aacctaagaa gatgagtgtt ttgctaagaa ccaagaattc 2701 aactatcgag tatgatggta ttatgcagaa atctcaagat aatgtcacag aactgtacga 2761 cgtttccatc agcttgtctg tttcattccc tgatgttacg agcaatatga ccatcttctg 2821 tattctggaa actgacaaga cgcggctttt atcttcacct ttctctatag agcttgagga 2881 ccctcagcct cccccagacc acattccttg gattacagct gtacttccaa cagttattat 2941 atgtgtgatg gttttctgtc taattctatg gaaatggaag aagaagaagc ggcctcggac 3001 tagtggcgga agcggaagtt tggctactac acttgaacgt attgagaaga actttgtcat 3061 tactgaccca agattgccag ataatcccat tatattcgcg tccgatagtt tcttgcagtt 3121 gacagaatat agccgtgaag aaattttggg aagaaactgc aggtttctac aaggtcctga 3181 aactgatcgc gcgacagtga gaaaaattag agatgccata gataaccaaa cagaggtcac 3241 tgttcagctg attaattata caaagagtgg taaaaagttc tggaacctct ttcacttgca 3301 gcctatgcga gatcagaagg gagatgtcca gtactttatt ggggttcagt tggatggaac 3361 tgagcatgtc cgagatgctg ccgagagaga gggagtcatg ctgattaaga aaactgcaga 3421 aaatattgat gaggcggcaa aagaactttg agcggccgcg actctagagt cgacctgcag 3481 gcatgcaagc ttgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa 3541 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta 3601 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa 3661 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg 3721 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc 3781 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc 3841 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg 3901 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg 3961 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg 4021 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc 4081 ctttgggccg cctccccgca tcgataccgt cgacctcgag ggaattaatt cgagctcggt 4141 acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa 4201 ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac 4261 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc 4321 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt 4381 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag 4441 cagcatctag aattaattcc gtgtattcta tagtgtcacc taaatcgtat gtgtatgata 4501 cataaggtta tgtattaatt gtagccgcgt tctaacgaca atatgtacaa gcctaattgt 4561 gtagcatctg gcttactgaa gcagacccta tcatctctct cgtaaactgc cgtcagagtc 4621 ggtttggttg gacgaacctt ctgagtttct ggtaacgccg tcccgcaccc ggaaatggtc 4681 agcgaaccaa tcagcagggt catcgctagc cagatcctct acgccggacg catcgtggcc 4741 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg 4801 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca 4861 ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg 4921 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag 4981 ggagagcgtc gaatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 5041 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 5101 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 5161 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 5221 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 5281 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 5341 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 5401 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 5461 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 5521 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 5581 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 5641 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 5701 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 5761 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 5821 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 5881 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 5941 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 6001 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 6061 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg 6121 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 6181 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 6241 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 6301 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 6361 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 6421 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 6481 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 6541 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 6601 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 6661 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 6721 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 6781 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 6841 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 6901 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 6961 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 7021 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 7081 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac 7141 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc 7201 tctccccgcg cgttggccga ttcattaatg cagctgtgga atgtgtgtca gttagggtgt 7261 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 7321 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 7381 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 7441 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 7501 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 7561 ggcttttgca aaaagcttgg acacaagaca ggcttgcgag atatgtttga gaataccact 7621 ttatcccgcg tcagggagag gcagtgcgta aaaagacgcg gactcatgtg aaatactggt 7681 ttttagtgcg ccagatctct ataatctcgc gcaacctatt ttcccctcga acacttttta 7741 agccgtagat aaacaggctg ggacacttca catgagcgaa aaatacatcg tcacctggga 7801 catgttgcag atccatgcac gtaaactcgc aagccgactg atgccttctg aacaatggaa 7861 aggcattatt gccgtaagcc gtggcggtct gtaccgggtg cgttactggc gcgtgaactg 7921 ggtattcgtc atgtcgatac cgtttgtatt tccagctacg atcacgacaa ccagcgcgag 7981 cttaaagtgc tgaaacgcgc agaaggcgat ggcgaaggct tcatcgttat tgatgacctg 8041 gtggataccg gtggtactgc ggttgcgatt cgtgaaatgt atccaaaagc gcactttgtc 8101 accatcttcg caaaaccggc tggtcgtccg ctggttgatg actatgttgt tgatatcccg 8161 caagatacct ggattgaaca gccgtgggat atgggcgtcg tattcgtccc gccaatctcc 8221 ggtcgctaat cttttcaacg cctggcactg ccgggcgttg ttctttttaa cttcaggcgg 8281 gttacaatag tttccagtaa gtattctgga ggctgcatcc atgacacagg caaacctgag 8341 cgaaaccctg ttcaaacccc gctttaaaca tcctgaaacc tcgacgctag tccgccgctt 8401 taatcacggc gcacaaccgc ctgtgcagtc ggcccttgat ggtaaaacca tccctcactg 8461 gtatcgcatg attaaccgtc tgatgtggat ctggcgcggc attgacccac gcgaaatcct 8521 cgacgtccag gcacgtattg tgatgagcga tgccgaacgt accgacgatg atttatacga 8581 tacggtgatt ggctaccgtg gcggcaactg gatttatgag tgggccccgg atctttgtga 8641 aggaacctta cttctgtggt gtgacataat tggacaaact acctacagag atttaaagct 8701 ctaaggtaaa tataaaattt ttaagtgtat aatgtgttaa actactgatt ctaattgttt 8761 gtgtatttta gattccaacc tatggaactg atgaatggga gcagtggtgg aatgccttta 8821 atgaggaaaa cctgttttgc tcagaagaaa tgccatctag tgatgatgag gctactgctg 8881 actctcaaca ttctactcct ccaaaaaaga agagaaaggt agaagacccc aaggactttc 8941 cttcagaatt gctaagtttt ttgagtcatg ctgtgtttag taatagaact cttgcttgct 9001 ttgctattta caccacaaag gaaaaagctg cactgctata caagaaaatt atggaaaaat 9061 attctgtaac ctttataagt aggcataaca gttataatca taacatactg ttttttctta 9121 ctccacacag gcatagagtg tctgctatta ataactatgc tcaaaaattg tgtaccttta 9181 gctttttaat ttgtaaaggg gttaataagg aatatttgat gtatagtgcc ttgactagag 9241 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac 9301 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca 9361 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 9421 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 9481 aactggataa ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc 9541 actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga attattttca 9601 ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagttgg aagggctaat 9661 tcactcccaa agaagacaag atatccttga tctgtggatc taccacacac aaggctactt 9721 ccctgattag cagaactaca caccagggcc aggggtcaga tatccactga cctttggatg 9781 gtgctacaag ctagtaccag ttgagccaga taaggtagaa gaggccaata aaggagagaa 9841 caccagcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtgtt 9901 agagtggagg tttgacagcc gcctagcatt tcatcacgtg gcccgagagc tgcatccgga 9961 gtacttcaag aactgctgat atcgagcttg ctacaaggga ctttccgctg gggactttcc 10021 agggaggcgt ggcctgggcg ggactgggga gtggcgagcc ctcagatcct gcatataagc 10081 agctgctttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct 10141 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag 10201 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt 10261 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc 10321 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg 10381 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg 10441 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt 10501 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct 10561 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact 10621 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac 10681 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt 10741 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg 10801 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa 10861 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg 10921 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca 10981 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg 11041 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg 11101 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga 11161 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc 11221 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac 11281 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta 11341 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa 11401 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc 11461 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg 11521 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg 11581 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc 11641 attcgattag tgaacggatc tcgacggtat cgccaaatgg cagtattcat ccacaatttt 11701 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca 11761 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt 11821 tattacaggg acagcagaga tccagtttgg atcgataagc ttgatatcga attcctgcag 11881 ccccgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca 11941 cctgtaggtt tggcaagcta gctgca //
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