质粒名称: | pMDIAI |
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出品公司: | Addgene |
目录编号: | 51655 |
存储实验室: | Sheng Yang |
载体骨架基本信息 |
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载体名称: | pMD18-T Simple |
载体出品公司: | Takara |
空载体大小: | 2600 bp |
完整质粒大小: | 4104bp |
载体类型: | genetic modification |
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: | apramycin antibiotic resistance gene |
插入大小: | 1500 bp |
启动子: | 未知 |
克隆方法: | TOPO Cloning |
5’测序引物 | M13F |
3’测序引物 | M13R |
文献引用方法 |
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材料和方法部分: | pMDIAI was a gift from Sheng Yang (Addgene plasmid # 51655 ; http://n2t.net/addgene:51655 ; RRID:Addgene_51655) |
参考文献部分: | High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage. Yang J, Sun B, Huang H, Jiang Y, Diao L, Chen B, Xu C, Wang X, Liu J, Jiang W, Yang S. Appl Environ Microbiol. 2014 Jul 1;80(13):3826-3834. Epub 2014 Apr 18. 10.1128/AEM.00313-14 PubMed 24747889 |
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载体序列
LOCUS pMDIAI 4104 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION E coli genetic modification. ACCESSION . VERSION . KEYWORDS pMDIAI SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4104) AUTHORS Yang J, Sun B, Huang H, Jiang Y, Diao L, Chen B, Xu C, Wang X, Liu J, Jiang W, Yang S TITLE High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage. JOURNAL Appl Environ Microbiol. 2014 Jul 1;80(13):3826-3834. Epub 2014 Apr 18. PUBMED 24747889 REFERENCE 2 (bases 1 to 4104) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..4104 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(10..27) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(10..26) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(19..41) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(235..254) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 354..376 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(414..432) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 500..604 /gene="bla" /label=AmpR promoter CDS 605..1465 /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(823..842) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1636..2224 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2125..2144 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2378..2395 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2512..2533 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2548..2578 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2586..2602 /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 2591..2613 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2610..2626 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2610..2626 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" protein_bind 2721..2754 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" oriT 2761..2870 /note="incP origin of transfer" CDS 3186..3986 /codon_start=1 /gene="aac(3)IV" /product="aminoglycoside 3-N-acetyltransferase type IV " /label=ApmR /note="confers resistance to apramycin (Paget and Davies, 1996)" /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG" protein_bind 3996..4042 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." ORIGIN 1 ttctttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca 61 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 121 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta 181 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat 241 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc 301 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 361 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt 421 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 481 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 541 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 601 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 661 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 721 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 781 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 841 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 901 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 961 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 1021 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 1081 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 1141 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 1201 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 1261 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 1321 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 1381 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 1441 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 1501 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 1561 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 1621 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 1681 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 1741 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 1801 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 1861 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 1921 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 1981 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 2041 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 2101 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 2161 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 2221 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 2281 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 2341 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 2401 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 2461 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 2521 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 2581 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgaact 2641 cgtcctcggt tcccggcgat cctctggaga ttatattacc ctgttatccc taattccggg 2701 gatccgtcga cctgcagttc gaagttccta ttctctagaa agtataggaa cttcgaagtt 2761 cccgccagcc tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag 2821 tgaagaagga acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc 2881 gttggataca ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga 2941 aaaaggatgg atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg 3001 aaaatgcagc tcacggtaac tgatgccgta tttgcagtac cagcgtacgg cccacagaat 3061 gatgtcacgc tgaaaatgcc ggcctttgaa tgggttcatg tgcagctcca taagcaaaag 3121 gggatgataa gtttatcacc accgactatt tgcaacagtg ccgttgatcg tgctatgatc 3181 gactgatgtc atcagcggtg gagtgcaatg tcgtgcaata cgaatggcga aaagccgagc 3241 tcatcggtca gcttctcaac cttggggtta cccccggcgg tgtgctgctg gtccacagct 3301 ccttccgtag cgtccggccc ctcgaagatg ggccacttgg actgatcgag gccctgcgtg 3361 ctgcgctggg tccgggaggg acgctcgtca tgccctcgtg gtcaggtctg gacgacgagc 3421 cgttcgatcc tgccacgtcg cccgttacac cggaccttgg agttgtctct gacacattct 3481 ggcgcctgcc aaatgtaaag cgcagcgccc atccatttgc ctttgcggca gcggggccac 3541 aggcagagca gatcatctct gatccattgc ccctgccacc tcactcgcct gcaagcccgg 3601 tcgcccgtgt ccatgaactc gatgggcagg tacttctcct cggcgtggga cacgatgcca 3661 acacgacgct gcatcttgcc gagttgatgg caaaggttcc ctatggggtg ccgagacact 3721 gcaccattct tcaggatggc aagttggtac gcgtcgatta tctcgagaat gaccactgct 3781 gtgagcgctt tgccttggcg gacaggtggc tcaaggagaa gagccttcag aaggaaggtc 3841 cagtcggtca tgcctttgct cggttgatcc gctcccgcga cattgtggcg acagccctgg 3901 gtcaactggg ccgagatccg ttgatcttcc tgcatccgcc agaggcggga tgcgaagaat 3961 gcgatgccgc tcgccagtcg attggctgag ctcataagtt cctattccga agttcctatt 4021 ctctagaaag tataggaact tcgaagcagc tccagcctac acattaccct gttatcccta 4081 ataatcgtcc acttgccgtc atgc //