质粒名称: | Flag Depdc5 pRK5 |
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出品公司: | Addgene |
目录编号: | 46340 |
存储实验室: | David Sabatini |
载体骨架基本信息 |
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载体名称: | pRK5 |
载体出品公司: | -- |
空载体大小: | -- |
完整质粒大小: | 9464 bp |
载体修饰: | -- |
载体类型: | 哺乳动物表达 |
筛选标记: | -- |
载体抗性: | 氨苄青霉素,100μg/mL |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | -- |
插入基因信息 |
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插入基因名称: | Depdc5 |
物种来源: | H. sapiens (human) |
插入大小: | 4746 bp |
基因突变: | 与Depdc5同种型1的区别在于从724位开始的10个氨基酸:ILTLSAPPVV |
GenBank编号 | -- |
启动子: | -- |
标签/融合蛋白: | 标志(主干上的N终端) |
克隆方法: | 限制性内切酶 |
5’克隆位点: | SaI (未破坏) |
3’克隆位点: | NotI (未破坏) |
5’测序引物 | SP6 |
3’测序引物 | SV40pA-R |
文献引用方法 |
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材料和方法部分: | Flag Depdc5 pRK5 was a gift from David Sabatini (Addgene plasmid # 46340 ; http://n2t.net/addgene:46340 ; RRID:Addgene_46340) |
参考文献部分: | A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Bar-Peled L, Chantranupong L, Cherniack AD, Chen WW, Ottina KA, Grabiner BC, Spear ED, Carter SL, Meyerson M, Sabatini DM. Science. 2013 May 31;340(6136):1100-6. doi: 10.1126/science.1232044. 10.1126/science.1232044 PubMed 23723238 |
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LOCUS Flag Depdc5 pRK5 9464 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS Flag Depdc5 pRK5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9464) AUTHORS Bar-Peled L, Chantranupong L, Cherniack AD, Chen WW, Ottina KA, Grabiner BC, Spear ED, Carter SL, Meyerson M, Sabatini DM TITLE A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. JOURNAL Science. 2013 May 31;340(6136):1100-6. doi: 10.1126/science.1232044. PUBMED 23723238 REFERENCE 2 (bases 1 to 9464) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..9464 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /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 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" enhancer 250..629 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 630..833 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 784..804 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 830..854 /label=LNCX /note="Human CMV promoter, forward primer" promoter 1064..1082 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 1064..1081 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 1095..1114 /label=pMT2-F /note="Synthetic intron, forward primer" CDS 1173..1196 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" regulatory 6000..6009 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 6013..6147 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6050..6069) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6104..6123 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" promoter 6216..6573 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(6239..6259) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 6424..6559 /label=SV40 ori /note="SV40 origin of replication" primer_bind 6486..6505 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(6593..6610) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6593..6609) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6602..6624) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 6822..7277 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6909..6928) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7119..7140 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7294..7313) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7413..7435 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7473..7491) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7559..7663 /gene="bla" /label=AmpR promoter CDS 7664..8524 /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(7882..7901) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8695..9283 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9184..9203 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 9437..9454 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga catgattacg aattaattcg 241 agctcgcccg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 301 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 361 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 421 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 481 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 541 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 601 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 661 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 721 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 781 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag 841 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc 901 gggaccgatc cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca 961 agagtgacgt aagtaccgcc tatagagtct ataggcccac ccccttggct tcgttagaac 1021 gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg tgacactata 1081 gaataacatc cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcacct 1141 cggttctatc gattgaattc accatggaaa tggactacaa ggatgacgat gacaagggtg 1201 gtggtggtgg tgcgtcgacg atgagaacaa caaaggtcta caaactcgtc atccacaaga 1261 agggctttgg gggcagtgat gatgagctag ttgtgaaccc caaagtgttc cctcacatca 1321 agcttggaga cattgtagag attgcacacc ccaacgatga atacagccct ctgcttttgc 1381 aggtcaagtc tcttaaggaa gatttacaga aggaaactat cagtgtggac cagactgtga 1441 ctcaagtgtt ccggctgaga ccttatcagg atgtctatgt taatgtcgta gaccctaagg 1501 atgtgaccct tgacctagtg gaattaactt ttaaggatca gtatattggc cgtggggata 1561 tgtggcgact aaagaaaagt ttggtcagca catgtgccta tatcacccag aaggtggagt 1621 ttgctggcat cagagcacag gctggtgaac tgtgggttaa gaatgagaag gtcatgtgtg 1681 gctacatcag tgaagatacc agggtggtgt ttcgttctac gtcggctatg gtttacatat 1741 ttattcagat gagctgtgaa atgtgggatt ttgatattta tggggatttg tattttgaga 1801 aagctgtgaa tggtttcctt gctgatctat ttaccaagtg gaaggagaag aactgtagtc 1861 atgaagtgac agtggtcctg ttttctagaa ctttctatga tgcaaaatct gttgatgaat 1921 ttcctgaaat aaaccgagcc tcaattcgac aggatcacaa ggggagattc tatgaagact 1981 tttacaaagt ggtggtgcag aatgagagaa gagaagaatg gacttcactt ctcgtaacca 2041 ttaaaaaact cttcatccag tatccagtgt tggtgcgact ggaacaggca gagggctttc 2101 ctcaaggaga taattctacc tcagcacaag gaaactacct ggaggccatc aatctgtcat 2161 tcaatgtgtt tgataagcac tacatcaacc gcaactttga ccgaactggg cagatgtcag 2221 tggtgatcac gcccggggtg ggtgtctttg aagtggaccg cctactcatg atcctgacca 2281 agcagcggat gatagataat ggaattggtg tggatttggt gtgcatggga gagcaaccgt 2341 tacatgctgt cccattgttc aagctccata atcggagtgc tccccgtgat tctcgtctgg 2401 gcgatgacta taatatccct cactggataa accacagttt ctacacatcc aaaagccagc 2461 tcttttgtaa tagtttcacc ccacgaataa aactggcagg aaagaagccc gcctctgaga 2521 aagcaaaaaa tggccgtgat acatctctcg ggagtccaaa agaatctgag aacgcccttc 2581 ccatccaagt agattatgac gcctatgacg ctcaagtgtt caggctgccc ggcccatccc 2641 gggcccagtg cctcaccacc tgcagatctg tgcgagagcg agagagtcac agtcgaaaga 2701 gtgccagctc ctgtgatgtt tcatccagcc cttccctacc aagccgcaca ctgcccactg 2761 aggaagtgag gagccaggct tctgacgaca gctccctagg caagagtgcc aacatcctga 2821 tgatcccaca cccccacctg caccagtatg aagtcagcag ctccttggga tacaccagca 2881 ctcgagatgt cctggagaac atgatggagc caccacagcg agactccagt gcaccaggga 2941 ggtttcacgt tggcagtgca gaatccatgc tgcatgttcg acctggtgga tacacgcccc 3001 agagagcact gattaacccc ttcgctccct ctcggatgcc catgaagctt acgtccaaca 3061 gaaggcgctg gatgcacact tttcctgtgg ggccatccgg agaagccatc cagatccacc 3121 accagacccg acagaatatg gcggagctac aaggcagcgg gcagagggat ccaactcact 3181 cctctgcaga gctgctggag ttagcatatc atgaagctgc tggaaggcac agcaattccc 3241 gccagcctgg tgacggcatg tccttcttga acttcagtgg aacagaggag ctttctgtcg 3301 gcctgcttag caacagtggt gcaggtatga atcctaggac ccagaataag gattctctag 3361 aggacagtgt ttctacctct ccagacccaa ttctgacact gtctgctccc cctgtagtgc 3421 caggcttctg ttgcacagtt ggagtggact ggaagtctct cactactccg gcgtgcctcc 3481 cccttaccac cgactacttc cctgaccgcc agggcctgca gaatgactac acagagggct 3541 gttatgatct ccttccagaa gcagacatcg acaggaggga cgaagatggt gtgcagatga 3601 cagcccagca ggtatttgaa gagtttattt gccaacgtct catgcagggc taccaaatca 3661 tagtgcagcc caagacacag aaacccaatc ctgctgtccc gcccccgctg agcagtagcc 3721 cactctatag ccgaggcctt gtgtcccgaa accgccctga ggaggaggac cagtattggc 3781 tgagtatggg cagaacgttc cacaaagtga cgctgaagga taagatgatc acagtgacgc 3841 gataccttcc caagtatcct tatgaatctg cccagatcca ctacacctac agcctctgtc 3901 cttcccactc agactcagag ttcgtctcct gctgggtgga attctcccac gaacggctgg 3961 aggagtacaa gtggaattac ttagatcagt atatctgttc tgccggctct gaagacttca 4021 gcttaattga gtccctgaag ttctggagga cccgcttcct gctgctgcca gcctgtgtca 4081 ccgccaccaa gcgcatcacg gagggggagg cccactgcga catctatggg gacaggcccc 4141 gtgcagacga ggacgagtgg caactcctgg atggttttgt ccgctttgtg gagggcttga 4201 atcgcattcg caggcggcat cgctcggatc gcatgatgcg gaaagggacc gccatgaaag 4261 gcttgcagat gactgggccc atttccacgc attctctgga gtcaactgca cccccagtgg 4321 ggaagaaggg aacctcagct ctctctgccc tgttggagat ggaggccagt cagaagtgcc 4381 tgggagaaca gcaggcagct gtgcatggtg ggaagagctc cgcccagtca gccgagagca 4441 gcagcgttgc catgactccc acctacatgg acagcccacg aaaggtatct gtggaccaaa 4501 cagccactcc tatgttggac ggcaccagtt tgggcatatg cacaggccaa tccatggaca 4561 gaggcaacag ccagaccttt gggaactccc agaacatagg agaacagggc tactcctcca 4621 caaactccag tgacagcagc tctcagcagc tggtggcaag ctccttgacc tcatcctcta 4681 ccctgacaga gatcctggaa gccatgaagc acccctcgac aggagtccag ctgctctctg 4741 aacagaaggg cctctcaccg tactgcttca tcagcgcgga ggtggtacac tggttggtga 4801 accacgtgga ggggatccag acacaggcga tggccattga catcatgcag aaaatgctgg 4861 aagagcagct catcacacat gcatctggcg aagcctggcg gaccttcatc tacggcttct 4921 atttctacaa gatagtaacg gacaaagagc ccgaccgagt ggccatgcag cagcccgcca 4981 ccacctggca cacagcagga gtggacgact tcgccagctt ccagcgcaag tggtttgagg 5041 tggcctttgt ggcagaagag ctcgtgcact ctgagattcc tgcctttctc ctgccctggc 5101 tgcctagccg gccagcctcc tatgcaagta ggcacagctc ctttagccga agttttggag 5161 gacggagcca ggcggcagca cttttagctg ccactgtccc agagcagagg actgtgaccc 5221 tggatgttga cgtgaacaac cgcacagacc ggctggagtg gtgcagctgt tattaccatg 5281 gcaacttttc tctgaatgca gcctttgaga tcaagctgca ctggatggcg gtgaccgcag 5341 cagtactctt cgagatggtc caaggttggc atcggaaagc cacctcctgt ggcttcttgt 5401 tagtcccagt tttggagggg ccttttgcac tgcccagtta cctgtatggc gacccccttc 5461 gtgcccagct cttcatccca ctcaacatca gctgcttgct caaggagggc agcgagcacc 5521 tgtttgatag ctttgaaccc gaaacgtact gggatcgaat gcacctcttc caggaagcca 5581 ttgcacacag gtttgggttt gtacaagata aatattctgc ctctgctttt aacttccctg 5641 ctgagaacaa gcctcagtat atccacgtta caggaacagt gtttctgcag ctgccctact 5701 ccaagcgcaa gttctcaggg cagcagcggc ggcggcggaa ctccaccagc tccaccaacc 5761 agaacatgtt ctgcgaggag cgggtcggct acaactgggc ctacaacacc atgctcacca 5821 aaacatggcg ctccagcgcc acaggggatg aaaagtttgc tgatcggctg ctgaaggact 5881 tcacggactt ctgcatcaac cgtgacaacc ggctggtcac gttctggaca agttgcctgg 5941 agaagatgca tgccagtgcc ccgtgagcgg ccgctaagta agtaaggatc cccagcttgg 6001 ccgccatggc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 6061 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 6121 atcaatgtat cttatcatgt ctggatcggg aattaattcg gcgcagcacc atggcctgaa 6181 ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa ccagctgtgg 6241 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 6301 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 6361 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 6421 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 6481 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga 6541 ggaggctttt ttggaggcct aggcttttgc aaaaagctgt taacagcttg gcactggccg 6601 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 6661 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 6721 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc 6781 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc 6841 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 6901 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 6961 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 7021 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt 7081 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 7141 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc 7201 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 7261 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 7321 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 7381 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 7441 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 7501 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 7561 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 7621 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 7681 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 7741 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 7801 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 7861 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 7921 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 7981 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 8041 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 8101 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 8161 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 8221 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 8281 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 8341 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 8401 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 8461 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 8521 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 8581 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 8641 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 8701 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 8761 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 8821 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 8881 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 8941 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 9001 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 9061 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 9121 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 9181 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 9241 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 9301 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 9361 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 9421 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag //
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