质粒名称: | pMSP1D12W/2Y |
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出品公司: | Addgene |
目录编号: | 124275 |
存储实验室: | Alexey S. Ladokhin |
载体骨架基本信息 |
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载体名称: | pET28b |
载体出品公司: | Novagen |
空载体大小: | 6000 bp |
完整质粒大小: | 6600 bp |
载体类型: | 大肠杆菌表达 |
载体抗性: | 卡那霉素 |
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: | Membrane scaffold protein |
物种来源: | 基因合成 |
插入大小: | 600 bp |
基因突变: | Two tryptophans changed to two tyrosines |
启动子: | T7 |
标签/融合蛋白: | 7xHis-tag (N terminal on insert) |
克隆方法: | 限制性内切酶 |
5’测序引物 | T7: TAATACGACTCACTATAGGG |
3’测序引物 | T7t: GCTAGTTATTGCTCAGCGG |
文献引用方法 |
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材料和方法部分: | pMSP1D12W/2Y was a gift from Alexey S. Ladokhin (Addgene plasmid # 124275 ; http://n2t.net/addgene:124275 ; RRID:Addgene_124275) |
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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 pMSP1D12W/2Y 5881 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Study of protein insertion into nanodisks by means of changes in tryptophan fluorescence. ACCESSION . VERSION . KEYWORDS pMSP1D12W/2Y SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5881) TITLE Bacterial expression plasmid for production of recombinant MSP1D12W/2Y protein where 2 tryptophan residues are substituted with 2 tyrosines JOURNAL Unpublished REFERENCE 2 (bases 1 to 5881) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5881 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 26..73 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind 69..87 /label=T7 Term /note="T7 terminator, reverse primer" CDS complement(140..157) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS complement(747..767) /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS complement(789..809) /codon_start=1 /product="6xHis affinity tag" /label=7xHis /translation="HHHHHHH" RBS 822..844 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind 859..883 /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 complement(883..902) /label=T7 /note="T7 promoter, forward primer" promoter complement(884..902) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(986..1005) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 1211..1288 /gene="lacI" /label=lacI promoter CDS 1289..2371 /codon_start=1 /gene="lacI" /product="lac repressor" /label=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" primer_bind complement(1308..1327) /label=LacI-R /note="LacI, reverse primer" CDS 3180..3371 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind complement(3365..3387) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 3473..3612 /label=bom /note="basis of mobility region from pBR322" primer_bind 3528..3547 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(3627..3644) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3798..4386) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3878..3897) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind complement(4497..4516) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 4508..5323 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(4581..4600) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin complement(5415..5870) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5552..5573) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5764..5783 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttac 181 tgggtattca gctttttagt atattcttcc agagctgaca gaaaactgac tttaaagctc 241 tcaagaacag gcaataagcc ctggcgtaga tcttccagcg ccggtttcgc tttttcgctc 301 aaggtgctca ggtgttctgt cgctttcgcg tggtactcag ccaagcgggc acccccgttt 361 tctttcagcg cctcgagacg tgcggccaaa cgctggcgaa gttcatccga atacggcgcc 421 aaatgagtcc ggagtgcatc aacatgtgca cgggcgcgat cgcgcatttc ttcgcctaat 481 gggctgagct tctcttggag ctcatggagt ttttggcgtg ccccctcttg cagttccgca 541 cgcagcggtt ccaccttttg acgatataat tccatctctt cctggtattt tttctgaaag 601 tcatcgagat atggctgaac cttggccttc acctcttcta aatctttgga catttcctga 661 cgcagtccct cggtttcttt ttccaggttg tcgtagaatt cctgcgtcac ggggcccagt 721 tgttcgcgaa gtttactgaa ggtagaaccc tgaaaataca aattctcagt agtaggaata 781 tcataatcgt gatgatgatg atgatgatga cccatggtat atctccttct taaagttaaa 841 caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat 901 taatttcgcg ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc 961 ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg 1021 gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg 1081 gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc 1141 aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt 1201 cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc 1261 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga 1321 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc 1381 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg 1441 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct 1501 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg 1561 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt 1621 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca 1681 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc 1741 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt 1801 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc 1861 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca 1921 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca 1981 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct 2041 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt 2101 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca 2161 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca 2221 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc 2281 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 2341 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca 2401 ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct 2461 tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc 2521 aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct 2581 ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc 2641 aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg 2701 gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct 2761 ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg 2821 actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg 2881 tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca 2941 tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc 3001 attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct 3061 cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct 3121 ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag 3181 tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa 3241 cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc 3301 ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg 3361 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3421 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3481 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 3541 gattgtactg agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa 3601 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 3661 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 3721 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 3781 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 3841 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 3901 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 3961 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 4021 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 4081 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 4141 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 4201 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 4261 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 4321 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 4381 tttcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 4441 cgttaaggga ttttggtcat gaacaataaa actgtctgct tacataaaca gtaatacaag 4501 gggtgttatg agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa 4561 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc 4621 gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa 4681 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt 4741 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac 4801 cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga 4861 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa 4921 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa 4981 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt 5041 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga 5101 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg 5161 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga 5221 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat 5281 gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg 5341 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 5401 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg 5461 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa 5521 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 5581 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 5641 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 5701 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 5761 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct 5821 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc 5881 a //
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