质粒名称: | NL4-3 mCherry Luciferase |
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出品公司: | 爱迪基因 |
目录编号: | 44965 |
存储实验室: | Warner Greene |
载体骨架基本信息 |
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载体名称: | pNLENG1 |
载体出品公司: | AIDS Research and Reference Reagent Program |
载体抗性: | 氨苄青霉素 |
空载体大小: | -- |
完整质粒大小: | 12424 bp |
载体修饰: | -- |
载体类型: | 慢病毒载体 |
筛选标记: | mCherry, Luciferase |
高拷贝/低拷贝: | -- |
生长条件: | 30 ℃ |
插入基因信息 |
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插入基因名称: | mCherry, Luciferase, IRES,HIV 1 reporter virus |
物种来源: | Lentivirus, Human specific |
插入大小: | -- |
基因突变: | S34C mutation in Nef |
GenBank编号 | -- |
启动子 | EF-1 alpha– driven mCherry:Puromycin |
标签/融合蛋白: |
带D212G的mCherry(插接件上的C端子)
荧光素酶,IRES(C端子插入)
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克隆方法: | 限制性内切酶 |
5' 克隆位点: | BamHI (未破坏) |
3' 克隆位点: | XhoI(在克隆过程中被破坏) |
5' 测序引物 | from pNLENG1 5' |
3' 测序引物 | from pNLENG1 3' |
文献引用方法 |
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材料和方法部分: | NL4-3 mCherry Luciferase was a gift from Warner Greene (Addgene plasmid # 44965 ; http://n2t.net/addgene:44965 ; RRID:Addgene_44965) |
参考文献部分: | A flexible model of HIV-1 latency permitting evaluation of many primary CD4 T-cell reservoirs. Lassen KG, Hebbeler AM, Bhattacharyya D, Lobritz MA, Greene WC. PLoS One. 2012;7(1):e30176. doi: 10.1371/journal.pone.0030176. Epub 2012 Jan 24. 10.1371/journal.pone.0030176 PubMed 22291913 |
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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 NL4-3 mCherry Luciferase 17833 bp ds-DNA circular SYN 16-DEC-2019 DEFINITION HIV dual reporter vector expressing mCh. ACCESSION . VERSION . KEYWORDS NL4-3 mCherry Luciferase SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 17833) AUTHORS Lassen KG, Hebbeler AM, Bhattacharyya D, Lobritz MA, Greene WC TITLE A flexible model of HIV-1 latency permitting evaluation of many primary CD4 T-cell reservoirs. JOURNAL PLoS One. 2012;7(1):e30176. doi: 10.1371/journal.pone.0030176. Epub 2012 Jan 24. PUBMED 22291913 REFERENCE 2 (bases 1 to 17833) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 16, 2019 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..17833 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 377..634 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" CDS 790..2292 /codon_start=1 /gene="gag" /product="gag protein from human immunodeficiency virus 1" /label=HIV-1 gag /translation="MGARASVLSGGELDKWEKIRLRPGGKKQYKLKHIVWASRELERFA VNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTIAVLYCVHQRIDVKDTKEALDKI EEEQNKSKKKAQQAAADTGNNSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVVEEKA FSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGP IAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTS ILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPG ATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAP PEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ" CDS 2085..5096 /codon_start=1 /gene="pol" /product="pol protein from human immunodeficiency virus 1" /label=HIV-1 pol /translation="FFREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAG ADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIG GIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETV PVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPVFAIKKKD STKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKQKKSVTVLDVGDAYFSVPLDKDFRK YTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCSMTKILEPFRKQNPDIVIYQYMD DLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPI VLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVRQLCKLLRGTKALTEVVPLTEEAELEL AENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMKGAHT NDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPP LVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPLTDTTNQKTELQ AIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAH KGIGGNEQVDKLVSAGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVA SCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQET AYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNK ELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQK QITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQ MAGDDCVASRQDED" misc_feature 4781..4898 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" misc_feature 7759..7992 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8515..8541 /codon_start=1 /product="nuclear export signal from the HIV Rev protein (Fischer et al., 1995)" /label=NES /translation="LPPLERLTL" CDS 8787..9494 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEGYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(8932..8950) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 9200..9219 /label=mCherry-F /note="mCherry, forward primer" CDS 9501..9554 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 9558..11207 /codon_start=1 /gene="luc+" /product="firefly luciferase" /label=luciferase /note="enhanced luc+ version of the luciferase gene" /translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF VDEVPKGLTGKLDARKIREILIKAKKGGKIAV" primer_bind complement(9621..9639) /label=LucNrev /note="Firefly luciferase, reverse primer" misc_feature 11212..11794 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(11378..11395) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 11605..11624 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" LTR 12084..12717 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" primer_bind complement(14357..14374) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(14528..15116) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(14608..14627) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(15287..16147) /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 15910..15929 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(16148..16252) /gene="bla" /label=AmpR promoter primer_bind 16320..16338 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(16376..16398) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 16498..16517 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 tggaagggct aatttggtcc caaaaaagac aagagatcct tgatctgtgg atctaccaca 61 cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac 121 tgacctttgg atggtgcttc aagttagtac cagttgaacc agagcaagta gaagaggcca 181 atgaaggaga gaacaacagc ttgttacacc ctatgagcca gcatgggatg gaggacccgg 241 agggagaagt attagtgtgg aagtttgaca gcctcctagc atttcgtcac atggcccgag 301 agctgcatcc ggagtactac aaagactgct gacatcgagc tttctacaag ggactttccg 361 ctggggactt tccagggagg tgtggcctgg gcgggactgg ggagtggcga gccctcagat 421 gctacatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 541 tgagtgctca aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 661 cgaaagtaaa gccagaggag atctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 781 aggagagaga tgggtgcgag agcgtcggta ttaagcgggg gagaattaga taaatgggaa 841 aaaattcggt taaggccagg gggaaagaaa caatataaac taaaacatat agtatgggca 901 agcagggagc tagaacgatt cgcagttaat cctggccttt tagagacatc agaaggctgt 961 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca 1021 ttatataata caatagcagt cctctattgt gtgcatcaaa ggatagatgt aaaagacacc 1081 aaggaagcct tagataagat agaggaagag caaaacaaaa gtaagaaaaa ggcacagcaa 1141 gcagcagctg acacaggaaa caacagccag gtcagccaaa attaccctat agtgcagaac 1201 ctccaggggc aaatggtaca tcaggccata tcacctagaa ctttaaatgc atgggtaaaa 1261 gtagtagaag agaaggcttt cagcccagaa gtaataccca tgttttcagc attatcagaa 1321 ggagccaccc cacaagattt aaataccatg ctaaacacag tggggggaca tcaagcagcc 1381 atgcaaatgt taaaagagac catcaatgag gaagctgcag aatgggatag attgcatcca 1441 gtgcatgcag ggcctattgc accaggccag atgagagaac caaggggaag tgacatagca 1501 ggaactacta gtacccttca ggaacaaata ggatggatga cacataatcc acctatccca 1561 gtaggagaaa tctataaaag atggataatc ctgggattaa ataaaatagt aagaatgtat 1621 agccctacca gcattctgga cataagacaa ggaccaaagg aaccctttag agactatgta 1681 gaccgattct ataaaactct aagagccgag caagcttcac aagaggtaaa aaattggatg 1741 acagaaacct tgttggtcca aaatgcgaac ccagattgta agactatttt aaaagcattg 1801 ggaccaggag cgacactaga agaaatgatg acagcatgtc agggagtggg gggacccggc 1861 cataaagcaa gagttttggc tgaagcaatg agccaagtaa caaatccagc taccataatg 1921 atacagaaag gcaattttag gaaccaaaga aagactgtta agtgtttcaa ttgtggcaaa 1981 gaagggcaca tagccaaaaa ttgcagggcc cctaggaaaa agggctgttg gaaatgtgga 2041 aaggaaggac accaaatgaa agattgtact gagagacagg ctaatttttt agggaagatc 2101 tggccttccc acaagggaag gccagggaat tttcttcaga gcagaccaga gccaacagcc 2161 ccaccagaag agagcttcag gtttggggaa gagacaacaa ctccctctca gaagcaggag 2221 ccgatagaca aggaactgta tcctttagct tccctcagat cactctttgg cagcgacccc 2281 tcgtcacaat aaagataggg gggcaattaa aggaagctct attagataca ggagcagatg 2341 atacagtatt agaagaaatg aatttgccag gaagatggaa accaaaaatg atagggggaa 2401 ttggaggttt tatcaaagta agacagtatg atcagatact catagaaatc tgcggacata 2461 aagctatagg tacagtatta gtaggaccta cacctgtcaa cataattgga agaaatctgt 2521 tgactcagat tggctgcact ttaaattttc ccattagtcc tattgagact gtaccagtaa 2581 aattaaagcc aggaatggat ggcccaaaag ttaaacaatg gccattgaca gaagaaaaaa 2641 taaaagcatt agtagaaatt tgtacagaaa tggaaaagga aggaaaaatt tcaaaaattg 2701 ggcctgaaaa tccatacaat actccagtat ttgccataaa gaaaaaagac agtactaaat 2761 ggagaaaatt agtagatttc agagaactta ataagagaac tcaagatttc tgggaagttc 2821 aattaggaat accacatcct gcagggttaa aacagaaaaa atcagtaaca gtactggatg 2881 tgggcgatgc atatttttca gttcccttag ataaagactt caggaagtat actgcattta 2941 ccatacctag tataaacaat gagacaccag ggattagata tcagtacaat gtgcttccac 3001 agggatggaa aggatcacca gcaatattcc agtgtagcat gacaaaaatc ttagagcctt 3061 ttagaaaaca aaatccagac atagtcatct atcaatacat ggatgatttg tatgtaggat 3121 ctgacttaga aatagggcag catagaacaa aaatagagga actgagacaa catctgttga 3181 ggtggggatt taccacacca gacaaaaaac atcagaaaga acctccattc ctttggatgg 3241 gttatgaact ccatcctgat aaatggacag tacagcctat agtgctgcca gaaaaggaca 3301 gctggactgt caatgacata cagaaattag tgggaaaatt gaattgggca agtcagattt 3361 atgcagggat taaagtaagg caattatgta aacttcttag gggaaccaaa gcactaacag 3421 aagtagtacc actaacagaa gaagcagagc tagaactggc agaaaacagg gagattctaa 3481 aagaaccggt acatggagtg tattatgacc catcaaaaga cttaatagca gaaatacaga 3541 agcaggggca aggccaatgg acatatcaaa tttatcaaga gccatttaaa aatctgaaaa 3601 caggaaagta tgcaagaatg aagggtgccc acactaatga tgtgaaacaa ttaacagagg 3661 cagtacaaaa aatagccaca gaaagcatag taatatgggg aaagactcct aaatttaaat 3721 tacccataca aaaggaaaca tgggaagcat ggtggacaga gtattggcaa gccacctgga 3781 ttcctgagtg ggagtttgtc aatacccctc ccttagtgaa gttatggtac cagttagaga 3841 aagaacccat aataggagca gaaactttct atgtagatgg ggcagccaat agggaaacta 3901 aattaggaaa agcaggatat gtaactgaca gaggaagaca aaaagttgtc cccctaacgg 3961 acacaacaaa tcagaagact gagttacaag caattcatct agctttgcag gattcgggat 4021 tagaagtaaa catagtgaca gactcacaat atgcattggg aatcattcaa gcacaaccag 4081 ataagagtga atcagagtta gtcagtcaaa taatagagca gttaataaaa aaggaaaaag 4141 tctacctggc atgggtacca gcacacaaag gaattggagg aaatgaacaa gtagataaat 4201 tggtcagtgc tggaatcagg aaagtactat ttttagatgg aatagataag gcccaagaag 4261 aacatgagaa atatcacagt aattggagag caatggctag tgattttaac ctaccacctg 4321 tagtagcaaa agaaatagta gccagctgtg ataaatgtca gctaaaaggg gaagccatgc 4381 atggacaagt agactgtagc ccaggaatat ggcagctaga ttgtacacat ttagaaggaa 4441 aagttatctt ggtagcagtt catgtagcca gtggatatat agaagcagaa gtaattccag 4501 cagagacagg gcaagaaaca gcatacttcc tcttaaaatt agcaggaaga tggccagtaa 4561 aaacagtaca tacagacaat ggcagcaatt tcaccagtac tacagttaag gccgcctgtt 4621 ggtgggcggg gatcaagcag gaatttggca ttccctacaa tccccaaagt caaggagtaa 4681 tagaatctat gaataaagaa ttaaagaaaa ttataggaca ggtaagagat caggctgaac 4741 atcttaagac agcagtacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga 4801 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta 4861 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca 4921 gagatccagt ttggaaagga ccagcaaagc tcctctggaa aggtgaaggg gcagtagtaa 4981 tacaagataa tagtgacata aaagtagtgc caagaagaaa agcaaagatc atcagggatt 5041 atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag gattaacaca 5101 tggaaaagat tagtaaaaca ccatatgtat atttcaagga aagctaagga ctggttttat 5161 agacatcact atgaaagtac taatccaaaa ataagttcag aagtacacat cccactaggg 5221 gatgctaaat tagtaataac aacatattgg ggtctgcata caggagaaag agactggcat 5281 ttgggtcagg gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct 5341 gacctagcag accaactaat tcatctgcac tattttgatt gtttttcaga atctgctata 5401 agaaatacca tattaggacg tatagttagt cctaggtgtg aatatcaagc aggacataac 5461 aaggtaggat ctctacagta cttggcacta gcagcattaa taaaaccaaa acagataaag 5521 ccacctttgc ctagtgttag gaaactgaca gaggacagat ggaacaagcc ccagaagacc 5581 aagggccaca gagggagcca tacaatgaat ggacactaga gcttttagag gaacttaaga 5641 gtgaagctgt tagacatttt cctaggatat ggctccataa cttaggacaa catatctatg 5701 aaacttacgg ggatacttgg gcaggagtgg aagccataat aagaattctg caacaactgc 5761 tgtttatcca tttcagaatt gggtgtcgac atagcagaat aggcgttact cgacagagga 5821 gagcaagaaa tggagccagt agatcctaga ctagagccct ggaagcatcc aggaagtcag 5881 cctaaaactg cttgtaccaa ttgctattgt aaaaagtgtt gctttcattg ccaagtttgt 5941 ttcatgacaa aagccttagg catctcctat ggcaggaaga agcggagaca gcgacgaaga 6001 gctcatcaga acagtcagac tcatcaagct tctctatcaa agcagtaagt agtacatgta 6061 atgcaaccta taatagtagc aatagtagca ttagtagtag caataataat agcaatagtt 6121 gtgtggtcca tagtaatcat agaatatagg aaaatattaa gacaaagaaa aatagacagg 6181 ttaattgata gactaataga aagagcagaa gacagtggca atgagagtga aggagaagta 6241 tcagcacttg tggagatggg ggtggaaatg gggcaccatg ctccttggga tattgatgat 6301 ctgtagtgct acagaaaaat tgtgggtcac agtctattat ggggtacctg tgtggaagga 6361 agcaaccacc actctatttt gtgcatcaga tgctaaagca tatgatacag aggtacataa 6421 tgtttgggcc acacatgcct gtgtacccac agaccccaac ccacaagaag tagtattggt 6481 aaatgtgaca gaaaatttta acatgtggaa aaatgacatg gtagaacaga tgcatgagga 6541 tataatcagt ttatgggatc aaagcctaaa gccatgtgta aaattaaccc cactctgtgt 6601 tagtttaaag tgcactgatt tgaagaatga tactaatacc aatagtagta gcgggagaat 6661 gataatggag aaaggagaga taaaaaactg ctctttcaat atcagcacaa gcataagaga 6721 taaggtgcag aaagaatatg cattctttta taaacttgat atagtaccaa tagataatac 6781 cagctatagg ttgataagtt gtaacacctc agtcattaca caggcctgtc caaaggtatc 6841 ctttgagcca attcccatac attattgtgc cccggctggt tttgcgattc taaaatgtaa 6901 taataagacg ttcaatggaa caggaccatg tacaaatgtc agcacagtac aatgtacaca 6961 tggaatcagg ccagtagtat caactcaact gctgttaaat ggcagtctag cagaagaaga 7021 tgtagtaatt agatctgcca atttcacaga caatgctaaa accataatag tacagctgaa 7081 cacatctgta gaaattaatt gtacaagacc caacaacaat acaagaaaaa gtatccgtat 7141 ccagagggga ccagggagag catttgttac aataggaaaa ataggaaata tgagacaagc 7201 acattgtaac attagtagag caaaatggaa tgccacttta aaacagatag ctagcaaatt 7261 aagagaacaa tttggaaata ataaaacaat aatctttaag caatcctcag gaggggaccc 7321 agaaattgta acgcacagtt ttaattgtgg aggggaattt ttctactgta attcaacaca 7381 actgtttaat agtacttggt ttaatagtac ttggagtact gaagggtcaa ataacactga 7441 aggaagtgac acaatcacac tcccatgcag aataaaacaa tttataaaca tgtggcagga 7501 agtaggaaaa gcaatgtatg cccctcccat cagtggacaa attagatgtt catcaaatat 7561 tactgggctg ctattaacaa gagatggtgg taataacaac aatgggtccg agatcttcag 7621 acctggagga ggcgatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt 7681 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga 7741 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac 7801 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctgatatagt 7861 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac 7921 agtctggggc atcaaacagc tccaggcaag aatcctggct gtggaaagat acctaaagga 7981 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc 8041 ttggaatgct agttggagta ataaatctct ggaacagatt tggaataaca tgacctggat 8101 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc 8161 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt 8221 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt 8281 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag 8341 gcagggatat tcaccattat cgtttcagac ccacctccca atcccgaggg gacccgacag 8401 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt 8461 gaacggatcc ttagcactta tctgggacga tctgcggagc ctgtgcctct tcagctacca 8521 ccgcttgaga gacttactct tgattgtaac gaggattgtg gaacttctgg gacgcagggg 8581 gtgggaagcc ctcaaatatt ggtggaatct cctacagtat tggagtcagg aactaaagaa 8641 tagtgctgtt aacttgctca atgccacagc catagcagta gctgagggga cagatagggt 8701 tatagaagta ttacaagcag cttatagagc tattcgccac atacctagaa gaataagaca 8761 gggcttggaa aggattttgc tataagatgg tgagcaaggg cgaggaggat aacatggcca 8821 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt 8881 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga 8941 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt 9001 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct 9061 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg 9121 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca 9181 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct 9241 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc 9301 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg 9361 tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg 9421 gctacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg 9481 acgagctgta caagccccgg gagggcagag gaagtcttct aacatgcggt gacgtggagg 9541 agaatcccgg ccctcgagaa gacgccaaaa acataaagaa aggcccggcg ccattctatc 9601 cgctggaaga tggaaccgct ggagagcaac tgcataaggc tatgaagaga tacgccctgg 9661 ttcctggaac aattgctttt acagatgcac atatcgaggt ggacatcact tacgctgagt 9721 acttcgaaat gtccgttcgg ttggcagaag ctatgaaacg atatgggctg aatacaaatc 9781 acagaatcgt cgtatgcagt gaaaactctc ttcaattctt tatgccggtg ttgggcgcgt 9841 tatttatcgg agttgcagtt gcgcccgcga acgacattta taatgaacgt gaattgctca 9901 acagtatggg catttcgcag cctaccgtgg tgttcgtttc caaaaagggg ttgcaaaaaa 9961 ttttgaacgt gcaaaaaaag ctcccaatca tccaaaaaat tattatcatg gattctaaaa 10021 cggattacca gggatttcag tcgatgtaca cgttcgtcac atctcatcta cctcccggtt 10081 ttaatgaata cgattttgtg ccagagtcct tcgataggga caagacaatt gcactgatca 10141 tgaactcctc tggatctact ggtctgccta aaggtgtcgc tctgcctcat agaactgcct 10201 gcgtgagatt ctcgcatgcc agagatccta tttttggcaa tcaaatcatt ccggatactg 10261 cgattttaag tgttgttcca ttccatcacg gttttggaat gtttactaca ctcggatatt 10321 tgatatgtgg atttcgagtc gtcttaatgt atagatttga agaagagctg tttctgagga 10381 gccttcagga ttacaagatt caaagtgcgc tgctggtgcc aaccctattc tccttcttcg 10441 ccaaaagcac tctgattgac aaatacgatt tatctaattt acacgaaatt gcttctggtg 10501 gcgctcccct ctctaaggaa gtcggggaag cggttgccaa gaggttccat ctgccaggta 10561 tcaggcaagg atatgggctc actgagacta catcagctat tctgattacg cccgaggggg 10621 atgataaacc gggcgcggtc ggtaaagttg ttccattttt tgaagcgaag gttgtggatc 10681 tggataccgg gaaaacgctg ggcgttaatc aaagaggcga actgtgtgtg agaggtccta 10741 tgattatgtc cggttatgta aacaatccgg aagcgaccaa cgccttgatt gacaaggatg 10801 gatggctaca ttctggagac atagcttact gggacgaaga cgaacacttc ttcatcgttg 10861 accgcctgaa gtctctgatt aagtacaaag gctatcaggt ggctcccgct gaattggaat 10921 ccatcttgct ccaacacccc aacatcttcg acgcaggtgt cgcaggtctt cccgacgatg 10981 acgccggtga acttcccgcc gccgttgttg ttttggagca cggaaagacg atgacggaaa 11041 aagagatcgt ggattacgtc gccagtcaag taacaaccgc gaaaaagttg cgcggaggag 11101 ttgtgtttgt ggacgaagta ccgaaaggtc ttaccggaaa actcgacgca agaaaaatca 11161 gagagatcct cataaaggcc aagaagggcg gaaagatcgc cgtgtaagga gcccctctcc 11221 ctcccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc 11281 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc 11341 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt 11401 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct 11461 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa 11521 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt 11581 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag 11641 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt 11701 acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc gaaccacggg gacgtggttt 11761 tcctttgaaa aacacgatga taatggccac aaccatgggt ggcaagtggt caaaaagtag 11821 tgtgattgga tggcctgctg taagggaaag aatgagacga gctgagccag cagcagatgg 11881 ggtgggagca gtatgtcgag acctagaaaa acatggagca atcacaagta gcaatacagc 11941 agctaacaat gctgcttgtg cctggctaga agcacaagag gaggaagagg tgggttttcc 12001 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 12061 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaaagaa gacaagatat 12121 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc 12181 agggccaggg gtcagatatc cactgacctt tggatggtgc tacaagctag taccagttga 12241 gccagataag gtagaagagg ccaataaagg agagaacacc agcttgttac accctgtgag 12301 cctgcatgga atggatgacc ctgagagaga agtgttagag tggaggtttg acagccgcct 12361 agcatttcat cacgtggccc gagagctgca tccggagtac ttcaagaact gctgacatcg 12421 agcttgctac aagggacttt ccgctgggga ctttccaggg aggcgtggcc tgggcgggac 12481 tggggagtgg cgagccctca gatgctgcat ataagcagct gctttttgcc tgtactgggt 12541 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 12601 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 12661 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaccc 12721 aggaggtaga ggttgcagtg agccaagatc gcgccactgc attccagcct gggcaagaaa 12781 acaagactgt ctaaaataat aataataagt taagggtatt aaatatattt atacatggag 12841 gtcataaaaa tatatatatt tgggctgggc gcagtggctc acacctgcgc ccggcccttt 12901 gggaggccga ggcaggtgga tcacctgagt ttgggagttc cagaccagcc tgaccaacat 12961 ggagaaaccc cttctctgtg tatttttagt agattttatt ttatgtgtat tttattcaca 13021 ggtatttctg gaaaactgaa actgtttttc ctctactctg ataccacaag aatcatcagc 13081 acagaggaag acttctgtga tcaaatgtgg tgggagaggg aggttttcac cagcacatga 13141 gcagtcagtt ctgccgcaga ctcggcgggt gtccttcggt tcagttccaa caccgcctgc 13201 ctggagagag gtcagaccac agggtgaggg ctcagtcccc aagacataaa cacccaagac 13261 ataaacaccc aacaggtcca ccccgcctgc tgcccaggca gagccgattc accaagacgg 13321 gaattaggat agagaaagag taagtcacac agagccggct gtgcgggaga acggagttct 13381 attatgactc aaatcagtct ccccaagcat tcggggatca gagtttttaa ggataactta 13441 gtgtgtaggg ggccagtgag ttggagatga aagcgtaggg agtcgaaggt gtccttttgc 13501 gccgagtcag ttcctgggtg ggggccacaa gatcggatga gccagtttat caatccgggg 13561 gtgccagctg atccatggag tgcagggtct gcaaaatatc tcaagcactg attgatctta 13621 ggttttacaa tagtgatgtt accccaggaa caatttgggg aaggtcagaa tcttgtagcc 13681 tgtagctgca tgactcctaa accataattt cttttttgtt tttttttttt tatttttgag 13741 acagggtctc actctgtcac ctaggctgga gtgcagtggt gcaatcacag ctcactgcag 13801 cctcaacgtc gtaagctcaa gcgatcctcc cacctcagcc tgcctggtag ctgagactac 13861 aagcgacgcc ccagttaatt tttgtatttt tggtagaggc agcgttttgc cgtgtggccc 13921 tggctggtct cgaactcctg ggctcaagtg atccagcctc agcctcccaa agtgctggga 13981 caaccggggc cagtcactgc acctggccct aaaccataat ttctaatctt ttggctaatt 14041 tgttagtcct acaaaggcag tctagtcccc aggcaaaaag ggggtttgtt tcgggaaagg 14101 gctgttactg tctttgtttc aaactataaa ctaagttcct cctaaactta gttcggccta 14161 cacccaggaa tgaacaagga gagcttggag gttagaagca cgatggaatt ggttaggtca 14221 gatctctttc actgtctgag ttataatttt gcaatggtgg ttcaaagact gcccgcttct 14281 gacaccagtc gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 14341 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 14401 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 14461 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 14521 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 14581 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 14641 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 14701 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 14761 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 14821 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 14881 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 14941 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 15001 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 15061 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 15121 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 15181 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 15241 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 15301 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 15361 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 15421 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 15481 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 15541 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 15601 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 15661 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 15721 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 15781 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 15841 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 15901 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 15961 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 16021 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 16081 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 16141 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 16201 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 16261 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 16321 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 16381 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 16441 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 16501 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg 16561 taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag 16621 ggcgatcggt gcgggcctct tcgctattac gccaggggag gcagagattg cagtaagctg 16681 agatcgcagc actgcactcc agcctgggcg acagagtaag actctgtctc aaaaataaaa 16741 taaataaatc aatcagatat tccaatcttt tcctttattt atttatttat tttctatttt 16801 ggaaacacag tccttcctta ttccagaatt acacatatat tctatttttc tttatatgct 16861 ccagtttttt ttagaccttc acctgaaatg tgtgtataca aaatctaggc cagtccagca 16921 gagcctaaag gtaaaaaata aaataataaa aaataaataa aatctagctc actccttcac 16981 atcaaaatgg agatacagct gttagcatta aataccaaat aacccatctt gtcctcaata 17041 attttaagcg cctctctcca ccacatctaa ctcctgtcaa aggcatgtgc cccttccggg 17101 cgctctgctg tgctgccaac caactggcat gtggactctg cagggtccct aactgccaag 17161 ccccacagtg tgccctgagg ctgccccttc cttctagcgg ctgcccccac tcggctttgc 17221 tttccctagt ttcagttact tgcgttcagc caaggtctga aactaggtgc gcacagagcg 17281 gtaagactgc gagagaaaga gaccagcttt acagggggtt tatcacagtg caccctgaca 17341 gtcgtcagcc tcacaggggg tttatcacat tgcaccctga cagtcgtcag cctcacaggg 17401 ggtttatcac agtgcaccct tacaatcatt ccatttgatt cacaattttt ttagtctcta 17461 ctgtgcctaa cttgtaagtt aaatttgatc agaggtgtgt tcccagaggg gaaaacagta 17521 tatacagggt tcagtactat cgcatttcag gcctccacct gggtcttgga atgtgtcccc 17581 cgaggggtga tgactacctc agttggatct ccacaggtca cagtgacaca agataaccaa 17641 gacacctccc aaggctacca caatgggccg ccctccacgt gcacatggcc ggaggaactg 17701 ccatgtcgga ggtgcaagca cacctgcgca tcagagtcct tggtgtggag ggagggacca 17761 gcgcagcttc cagccatcca cctgatgaac agaacctagg gaaagcccca gttctactta 17821 caccaggaaa ggc //
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