质粒名称: | Flag-HA-USP20 |
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出品公司: | Addgene |
目录编号: | 22573 |
存储实验室: | Wade Harper |
载体骨架基本信息 |
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载体名称: | pDEST_LTR_N_FLAG_HA_IRES_puro |
载体出品公司: | Harper_Lab |
空载体大小: | 6521bp |
完整质粒大小: | 9170 bp |
载体类型: | 哺乳细胞表达, Retroviral |
筛选标记: | Puromycin |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | USP20 / ubiquitin specific peptidase 20 /KIAA1003, LSFR3A, VDU2 |
物种来源: | H. sapiens (human) |
基因突变: | 无 |
GenBank编号 | NM_006676 |
启动子: | CMV |
标签/融合蛋白: | HA (N terminal on backbone) |
克隆方法: | Gateway Cloning |
5’克隆位点: | GATEWAY_LR (unknown if destroyed) |
3’克隆位点: | GATEWAY_LR (unknown if destroyed) |
5’测序引物 | CAGCCCTCACTCCTTCTCTAGG |
3’测序引物 | CAAGCGGCTTCGGCCAGTAAC |
文献引用方法 |
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材料和方法部分: |
Flag-HA-USP20 was a gift from Wade Harper (Addgene plasmid # 22573 ; http://n2t.net/addgene:22573 ; RRID:Addgene_22573)
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参考文献部分: | Defining the human deubiquitinating enzyme interaction landscape. Sowa ME, Bennett EJ, Gygi SP, Harper JW. Cell. 2009 Jul 23. 138(2):389-403. 10.1016/j.cell.2009.04.042 PubMed 19615732 |
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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 Flag-HA-USP20 9170 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS Flag-HA-USP20 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9170) AUTHORS Sowa ME, Bennett EJ, Gygi SP, Harper JW TITLE Defining the human deubiquitinating enzyme interaction landscape. JOURNAL Cell. 2009 Jul 23. 138(2):389-403. PUBMED 19615732 REFERENCE 2 (bases 1 to 9170) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..9170 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(63..80) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(234..822) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(314..333) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(993..1853) /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 1616..1635 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1854..1958) /gene="bla" /label=AmpR promoter primer_bind 2026..2044 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(2082..2104) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 2204..2223 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2417..2439 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2567..2586 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" LTR 2786..3302 /label=5' LTR /note="5' long terminal repeat from murine embryonic stem cell virus" misc_feature 3366..3707 /label=MESV Psi /note="packaging signal of murine embryonic stem cell virus" CDS 3774..4190 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 4120..4142 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 4158..4174 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" regulatory 4203..4212 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4212..4235 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 4248..4274 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" protein_bind 4290..4314 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(7057..7081) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" misc_feature 7111..7663 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(7255..7272) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 7482..7501 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 7675..8274 /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(7675..7694) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 8171..8191 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 8350..8864 /label=3' LTR /note="3' long terminal repeat from murine embryonic stem cell virus" protein_bind 9026..9042 /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)." promoter complement(9050..9080) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9095..9116 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." ORIGIN 1 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 61 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 121 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 181 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 241 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 301 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 361 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 421 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 481 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 541 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 601 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 661 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 721 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 781 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 841 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 901 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 961 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 1021 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 1081 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 1141 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 1201 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 1261 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 1321 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 1381 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 1441 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 1501 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 1561 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 1621 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 1681 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 1741 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 1801 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 1861 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 1921 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 1981 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 2041 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc 2101 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc 2161 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt 2221 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 2281 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 2341 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 2401 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggcgcaagg aatggtgcat 2461 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga 2521 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga 2581 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt 2641 agaggcgatt agtccaattt gttaaagaca ggatatcagt ggtccaggct ctagttttga 2701 ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa taaaagattt 2761 tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag 2821 cttaagtaac gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag 2881 atcaaggtta ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc 2941 agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag 3001 cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt 3061 gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc 3121 cgagctcaat aaaagagccc acaacccctc actcggcgcg ccagtcctcc gatagactgc 3181 gtcgcccggg tacccgtatt cccaataaag cctcttgctg tttgcatccg aatcgtggac 3241 tcgctgatcc ttgggagggt ctcctcagat tgattgactg cccacctcgg gggtctttca 3301 tttggaggtt ccaccgagat ttggagaccc ctgcccaggg accaccgacc cccccgccgg 3361 gaggtaagct ggccagcggt cgtttcgtgt ctgtctctgt ctttgtgcgt gtttgtgccg 3421 gcatctaatg tttgcgcctg cgtctgtact agttagctaa ctagctctgt atctggcgga 3481 cccgtggtgg aactgacgag ttctgaacac ccggccgcaa ccctgggaga cgtcccaggg 3541 actttggggg ccgtttttgt ggcccgacct gaggaaggga gtcgatgtgg aatccgaccc 3601 cgtcaggata tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg 3661 aatttttgct ttcggtttgg aaccgaagcc gcgcgtcttg tctgctgcag cgctgcagca 3721 tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagggccaga 3781 ctgttaccac tcccttaagt ttgaccttag gtcactggaa agatgtcgag cggatcgctc 3841 acaaccagtc ggtagatgtc aagaagagac gttgggttac cttctgctct gcagaatggc 3901 caacctttaa cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc atcacccagg 3961 ttaagatcaa ggtcttttca cctggcccgc atggacaccc agaccaggtc ccctacatcg 4021 tgacctggga agccttggct tttgaccccc ctccctgggt caagcccttt gtacacccta 4081 agcctccgcc tcctcttcct ccatccgccc cgtctctccc ccttgaacct cctcgttcga 4141 ccccgcctcg atcctccctt tatccagccc tcactccttc tctaggcgcc ggaattagat 4201 ctgccaccat ggactacaag gatgacgatg acaagctcga tggaggatac ccatacgatg 4261 ttccagatta cgctgctcga ggttatcaaa caagtttgta caaaaaagtt ggcatggggg 4321 actccaggga cctttgccct caccttgact ccataggaga ggtgaccaaa gaggacttgc 4381 tgctcaaatc taagggaacc tgtcagtcgt gtggggtcac cggaccaaac ctatgggcct 4441 gtctgcaggt tgcctgcccc tatgttggct gcggagaatc cttcgctgac cacagcacca 4501 ttcatgcaca ggcaaaaaag cacaacttga ccgtgaacct gaccacgttc cgactgtggt 4561 gttacgcctg tgagaaggag gtattcctgg agcagcggct ggcagcccct ctgctgggct 4621 cctcttccaa gttctctgaa caggactccc cgccaccctc ccaccctctg aaagctgttc 4681 ctattgctgt ggctgatgaa ggagagtctg agtcagagga tgatgacctg aaacctcgag 4741 gcctcacggg catgaagaac ctcgggaact cctgctacat gaacgccgcc ctgcaggccc 4801 tgtccaattg cccgccgctg actcagttct tcttggagtg tggcggcctg gtgcgcacag 4861 ataagaagcc agccctgtgc aagagctacc agaagctggt ctctgaggtc tggcataaga 4921 aacggccaag ctacgtggtc cccaccagtc tgtctcatgg gatcaagttg gtcaacccaa 4981 tgttccgagg ctatgcccag caggacaccc aagagttcct tcgctgcctg atggaccagc 5041 tgcacgagga gctcaaggag ccggtggtgg ccacggtggc gctgacggag gctcgggact 5101 cagattcgag tgacacggat gagaaacggg agggtgaccg gagcccatca gaagatgagt 5161 tcttgtcctg tgactcgagc agtgaccggg gtgagggtga cgggcagggg cgtggcgggg 5221 gcagctcgca ggccgagacg gagctgctga tcccagatga ggcgggccga gccatctctg 5281 agaaggagcg gatgaaggac cgcaagttct cctggggcca gcagcgtaca aactcggagc 5341 aagtggacga ggacgctgat gtggacactg ccatggctgc ccttgaccag cccgcggagg 5401 cccagccccc gtcaccacgg tcctccagcc cctgccggac gccagagccg gacaatgatg 5461 ctcacctacg cagctcctct cgcccctgca gccccgtcca ccaccacgag ggccatgcca 5521 agctgtctag cagcccccct cgtgcaagcc ccgtgaggat ggcaccgtcg tacgtgctca 5581 agaaagccca ggtattgagt gctggcagcc ggaggcggaa ggagcagcgc taccgcagcg 5641 tcatctcaga catctttgac ggctccattc tcagcctcgt gcagtgtctc acctgtgacc 5701 gggtatccac cacagtggaa acgttccagg acttatcact gcccattcct ggaaaggagg 5761 acctggccaa gctccattca gccatctacc agaatgtgcc ggccaagcca ggcgcctgtg 5821 gggacagcta tgccgcccag ggctggctgg ccttcattgt ggagtacatc cgacggtttg 5881 tggtatcctg tacccccagc tggttttggg ggcctgtcgt caccctggaa gactgccttg 5941 ctgccttctt tgccgctgat gagttaaagg gtgacaacat gtacagctgt gagcggtgta 6001 agaagctgcg gaacggagtg aagtactgca aagtcctgcg gttgcccgag atcctgtgca 6061 ttcacctaaa gcgctttcgg cacgaggtga tgtactcatt caagatcaac agccacgtct 6121 ccttccccct cgaggggctc gacctgcgcc ccttccttgc caaggagtgc acatcccaga 6181 tcaccaccta cgacctcctc tcggtcatct gccaccacgg cacggcaggc agtgggcact 6241 acatcgccta ctgccagaac gtgatcaatg ggcagtggta cgagtttgat gaccagtacg 6301 tcacagaagt ccacgagacg gtggtgcaga acgccgaggg ctacgtactc ttctacagga 6361 agagcagcga ggaggccatg cgggagcgac agcaggtggt gtccctggcc gccatgcggg 6421 agcccagcct gctgcggttc tacgtgtccc gcgagtggct caacaagttc aacaccttcg 6481 cggagccagg ccccatcacc aaccagacct tcctctgctc ccacggaggc atcccgcccc 6541 acaaatacca ctacatcgac gacctggtgg tcatcctgcc ccagaacgtc tgggagcacc 6601 tgtacaacag attcgggggt ggccccgccg tgaaccacct gtacgtgtgc tccatctgcc 6661 aggtggagat cgaggcactg gccaagcgca ggaggatcga gatcgacacc ttcatcaagt 6721 tgaacaaggc cttccaggcc gaggagtcgc cgggcgtcat ctactgcatc agcatgcagt 6781 ggttccggga gtgggaggcg ttcgtcaagg ggaaggacaa cgagcccccc gggcccattg 6841 acaacagcag gattgcacag gtcaaaggaa gcggccatgt ccagctgaag cagggagctg 6901 actacgggca gatttcggag gagacctgga cctacctgaa cagcctgtat ggaggtggcc 6961 ccgagattgc catccgccag agtgtggcgc agccgctggg cccagagaac ctgcacgggg 7021 agcagaagat cgaagccgag acgcgggccg tgtgataccc aactttcttg tacaaagtgg 7081 ttcgataacg aattccgccc ccccccccta acgttactgg ccgaagccgc ttggaataag 7141 gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga 7201 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg 7261 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt 7321 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca 7381 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc 7441 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat 7501 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc 7561 ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga 7621 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatgacc 7681 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc 7741 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac 7801 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc 7861 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc 7921 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg 7981 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg 8041 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc 8101 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc 8161 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc 8221 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgatgcatc 8281 gatgtcgacc tgcagccaag cttatcgata aaataaaaga ttttatttag tctccagaaa 8341 aaggggggaa tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt 8401 tgcaaggcat ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag 8461 agagacagca gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc 8521 agggccaaga acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca 8581 tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac 8641 caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag 8701 cccacaaccc ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt 8761 gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg 8821 gtctcctctg agtgattgac tacccgtcag cgggggtctt tcatgggtaa cagtttcttg 8881 aagttggaga acaacattct gagggtagga gtcgaatatt aagtaatcct gactcaatta 8941 gccactgttt tgaatccaca tactccaata ctcctgaaat agttcattat ggacagcgca 9001 gaaagagctg gggagaattg tgaaattgtt atccgctcac aattccacac aacatacgag 9061 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 9121 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg //
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