pT7HisAGK人源基 |
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基本信息名称:pT7-His-AGK人源基因质粒别称: BC022777.1 启动子: T7 复制子:pBR322 质粒分类: 基因库质粒 原核抗性: 卡那霉素Kan克隆菌株: DH5a培养条件: 37度质粒属性载体宿主:大肠杆菌载体用途:蛋白表达基因种属:人基因类型:ORF原核抗性:Kan真核抗性:荧光蛋白:质粒简介 pT7-His-AGK是一个人源基因大肠表达质粒。质粒图谱质粒序列LOCUS Exported 6550 bp ds-DNA circular SYN 21-十二月-2016FEATURES Location/Qualifiers source 1..6550 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 141..159 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 160..184 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /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)." RBS 199..221 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 240..257 /codon_start=1 /product="6xHis affinity tag" /note="6xHis" /translation="HHHHHH" CDS 276..1544 /codon_start=1 /note="AGK" /translation="MTVFFKTLRNHWKKTTAGLCLLTWGGHWLYGKHCDNLLRRAACQE AQVFGNQLIPPNAQVKKATVFLNPAACKGKARTLFEKNAAPILHLSGMDVTIVKTDYEG QAKKLLELMENTDVII-VAGGDGTLQEVVTGVLRRTDEATFSKIPIGFIPLGETSSLSHT LFAESGNKVQHITDATLAIVKGETVPLDVLQIKGEKEQPVFAMTGLRWGSFRDAGVKVS KYWYLGPLKIKAAHFFSTLKEWPQTHQASISYTGPTERPPNEPEETPVQRPSLYRRILR RLASYWAQPQDALSQEVSPEVWKDVQLSTIELSITTRNNQLDPTSKEDFLNICIEPDTI SKGDFITIGSRKVRNPKLHVEGTECLQASQCTLLIPEGAGGSFSIDSEEYEAMPVEVKL LPRKLQFFCDPRKREQMLTSPTQ" CDS complement(895..906) /codon_start=1 /product="Factor Xa recognition and cleavage site" /note="Factor Xa site" /translation="IEGR" CDS 1551..1568 /codon_start=1 /product="6xHis affinity tag" /note="6xHis" /translation="HHHHHH" terminator 1635..1682 /note="T7 terminator" /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1719..2174 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" CDS complement(2267..3082) /codon_start=1 /product="aminoglycoside phosphotransferase" /note="KanR" /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" rep_origin 3204..3792 /direction=RIGHT /note="pBR322 ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" misc_feature 3978..4120 /note="bom" /note="basis of mobility region from pBR322" CDS complement(4222..4413) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /note="rop" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5222..6304) /codon_start=1 /gene="lacI" /product="lac repressor" /note="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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" promoter complement(6305..6382) /gene="lacI" /note="lacI promoter"ORIGIN 1 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc 61 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg 121 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 181 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc 241 atcatcatca tcatcacagc agcggccgcg gatccatgac ggtgttcttt aaaacgcttc 301 gaaatcactg gaagaaaact acagctgggc tctgcctgct gacctgggga ggccattggc 361 tctatggaaa acactgtgat aacctcctaa ggagagcagc ctgtcaagaa gctcaggtgt 421 ttggcaatca actcattcct cccaatgcac aagtgaagaa ggccactgtt tttctcaatc 481 ctgcagcttg caaaggaaaa gctaggactc tatttgaaaa aaatgctgcc ccgattttac 541 atttatctgg catggatgtg actattgtta agacagatta tgagggacaa gccaagaaac 601 tcctggaact gatggaaaac acggatgtga tcattgttgc aggaggagat gggacactgc 661 aggaggttgt tactggtgtt cttcgacgaa cagatgaggc taccttcagt aagattccca 721 ttggatttat cccactggga gagaccagta gtttgagtca taccctcttt gccgaaagtg 781 gaaacaaagt ccaacatatt actgatgcca cacttgccat tgtgaaagga gagacagttc 841 cacttgatgt cttgcagatc aagggtgaaa aggaacagcc tgtatttgca atgaccggcc 901 ttcgatgggg atctttcaga gatgctggcg tcaaagttag caagtactgg tatcttgggc 961 ctctaaaaat caaagcagcc cactttttca gcactcttaa ggagtggcct cagactcatc 1021 aagcctctat ctcatacacg ggacctacag agagacctcc caatgaacca gaggagaccc 1081 ctgtacaaag gccttctttg tacaggagaa tattacgaag gcttgcgtcc tactgggcac 1141 aaccacagga tgccctttcc caagaggtga gcccggaggt ctggaaagat gtgcagctgt 1201 ccaccattga actgtccatc acaacacgga ataatcagct tgacccgaca agcaaagaag 1261 attttctgaa tatctgcatt gaacctgaca ccatcagcaa aggagacttt ataactatag 1321 gaagtcgaaa ggtgagaaac cccaagctgc acgtggaggg cacggagtgt ctccaagcca 1381 gccagtgcac tttgcttatc ccggagggag cagggggctc ttttagcatt gacagtgagg 1441 agtatgaagc gatgcctgtg gaggtgaaac tgctccccag gaagctgcag ttcttctgtg 1501 atcctaggaa gagagaacag atgctcacaa gccccaccca gtgactcgag caccaccacc 1561 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca 1621 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt 1681 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt 1741 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 1801 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 1861 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 1921 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 1981 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 2041 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc 2101 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt 2161 aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 2221 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat 2281 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa 2341 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat 2401 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct 2461 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga 2521 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt 2581 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac 2641 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca 2701 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct 2761 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga 2821 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct 2881 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat 2941 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc 3001 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg 3061 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt 3121 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 3181 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 3241 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 3301 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 3361 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 3421 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 3481 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 3541 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 3601 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 3661 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 3721 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 3781 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 3841 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 3901 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 3961 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 4021 caccgcatat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 4081 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc 4141 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 4201 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca 4261 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc 4321 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc 4381 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct 4441 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga 4501 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg 4561 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg 4621 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg 4681 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt 4741 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga 4801 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag 4861 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa 4921 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac 4981 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac 5041 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc 5101 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa 5161 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg 5221 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 5281 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg 5341 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt 5401 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat 5461 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca 5521 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca 5581 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg 5641 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat 5701 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga 5761 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg 5821 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat 5881 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca 5941 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc 6001 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg 6061 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca 6121 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca 6181 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc 6241 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt 6301 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg 6361 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt 6421 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca 6481 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg 6541 aaacaagcgc//质粒菌株产品操作说明书一、扩增流程 收到产品后,请先根据产品管壁标签来判断产品形式,并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。 1、质粒干粉(常温运输,存于-20度,90天保质期,请务必转化挑单克隆培养,不要直接使用和测序) ①收到质粒干粉后请先5000rpm离心1min,再加入20μl ddH2O去离子水溶解质粒; ②取1支100μl 感受态于冰上解冻10min,加入2μl质粒,再冰浴30min后,42℃热激60s,不要搅动,再冰浴2min;(从第二步开始均要在超净工作台中无菌操作) ③加入900μl无抗的LB液体培养基,180rpm震荡37℃培养45min; ④6000rpm离心5min,仅留100μl上清液重悬细菌沉淀,并涂布至目标质粒抗性的LB平板上;(可使用本平台的平板涂布专用玻璃珠进行涂布,可以比传统涂布方法获得更多转化子) ⑤将平板正向培养1h,再倒置37℃培养14h。如果要求是30度则培养20h;(菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态,要先转克隆感受态,重提质粒后再导入表达感受态) ⑥挑取单菌落至LB液体培养基中,加入对应抗生素,220rpm震荡培养14h,根据实验需要和质粒提取试剂盒说明书提取质粒。 2、甘油菌种(冰袋运输,存于-80℃,保质期90天,请务必划线挑单克隆培养) 四区划线后挑单菌落培养,酵母菌需要先液体复苏再四区划线,再挑单菌落液体培养。 3、穿刺菌种(冰袋运输,存于4℃,保质期7天) 穿刺接种,液体培养后四区划线,再挑单菌落液体培养。 4、菌落平板(冰袋运输,存于4℃,保质期7天) 直接挑取单菌落至液体培养基中。 5、液体质粒(冰袋运输,存于-20℃,保质期90天) 单独提取的液体质粒收到后可直接使用。 6、滤纸质粒(常温运输,存于-20度,90天保质期,请务必转化挑单克隆培养,不要直接使用和测序) 收到货后将滤纸画圈部分剪下放入EP管中,加100ul无菌水将滤纸浸湿并浸泡5min,吸取5ul质粒转化,离心全涂。二、转化图片 P1171/pECMV-3×FLAG-GSK3B人源基因质粒 P1172/pECMV-3×FLAG-CD1D人源基因质粒 P1719/pECMV-3×FLAG-CD4人源基因质粒 P1830/pECMV-3×FLAG-CD22人源基因质粒 P1195/pECMV-3×FLAG-AFP人源基因质粒 P4834/pCDNA3.1-AFP人源基因质粒 P4100/pECMV-3×FLAG-CCNE2人源基因质粒 P4270/pECMV-3×FLAG-AATF人源基因质粒 P4268/pECMV-3×FLAG-BLVRA(1同义突变)人源基因质粒 P4267/pECMV-3×FLAG-BTN2A2人源基因质粒 P4266/pECMV-3×FLAG-WDR63人源基因质粒 P1197/pECMV-3×FLAG-NRF2人源基因质粒
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