| 质粒名称: | pet28a-His6-Keap1 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 62454 |
| 存储实验室: | Yimon Aye |
|
载体骨架基本信息 |
|
| 载体名称: | pet28a |
| 载体出品公司: | EMD Biosciences |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 5369 bp |
| 完整质粒大小: | 7300 bp |
| 载体修饰: | -- |
| 载体类型: | 潮杆菌表达 |
| 筛选标记: | 新霉素（G418） |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
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| 插入基因名称: | Keap1 |
| 物种来源: | -- |
| 插入大小: | 1935 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | AgeI(未破坏) |
| 3' 克隆位点: | NotI (未破坏) |
| 5' 测序引物 | CMV-F |
| 3' 测序引物 | SV40pA-R |
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文献引用方法 |
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| 材料和方法部分: | pet28a-His6-Keap1 was a gift from Yimon Aye (Addgene plasmid # 62454 ; http://n2t.net/addgene:62454 ; RRID:Addgene_62454) |
| 参考文献部分: | Substoichiometric Hydroxynonenylation of a Single Protein Recapitulates Whole-Cell-Stimulated Antioxidant Response. Parvez S, Fu Y, Li J, Long MJ, Lin H, Lee DK, Hu GS, Aye Y. J Am Chem Soc. 2014 Dec 31. 10.1021/ja5084249 PubMed 25544059 |
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载体基本信息
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载体简介
载体序列
LOCUS pet28a-His6-Keap1 7141 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION His6 tagged Keap1 for E.coli expression. ACCESSION . VERSION . KEYWORDS pet28a-His6-Keap1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7141) AUTHORS Parvez S, Fu Y, Li J, Long MJ, Lin H, Lee DK, Hu GS, Aye Y TITLE Substoichiometric Hydroxynonenylation of a Single Protein Recapitulates Whole-Cell-Stimulated Antioxidant Response. JOURNAL J Am Chem Soc. 2014 Dec 31. PUBMED 25544059 REFERENCE 2 (bases 1 to 7141) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7141 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 20..159 /label=bom /note="basis of mobility region from pBR322" primer_bind 75..94 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(174..191) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(345..933) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(425..444) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind complement(1044..1063) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 1055..1870 /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(1128..1147) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin complement(1962..2417) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2099..2120) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2311..2330 /label=F1ori-R /note="F1 origin, reverse primer" terminator 2454..2501 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind 2497..2515 /label=T7 Term /note="T7 terminator, reverse primer" CDS complement(4435..4455) /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS complement(4474..4491) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" RBS 4510..4532 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind 4547..4571 /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(4571..4590) /label=T7 /note="T7 promoter, forward primer" promoter complement(4572..4590) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4674..4693) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 4899..4976 /gene="lacI" /label=lacI promoter CDS 4977..6059 /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(4996..5015) /label=LacI-R /note="LacI, reverse primer" CDS 6868..7059 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind complement(7053..7075) /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata 61 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa 121 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc 181 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 241 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 301 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 361 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 421 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 481 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 541 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 601 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 661 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 721 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 781 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 841 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 901 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 961 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact 1021 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc 1081 ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc 1141 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc 1201 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat 1261 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg 1321 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt 1381 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg 1441 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct 1501 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga 1561 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc 1621 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg 1681 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct 1741 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca 1801 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga 1861 gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac 1921 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat 1981 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 2041 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 2101 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac 2161 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 2221 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 2281 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag 2341 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 2401 gccgctacag ggcgcgtccc attcgccaat ccggatatag ttcctccttt cagcaaaaaa 2461 cccctcaaga cccgtttaga ggccccgagg ggttatgcta gttattgctc agcggtggca 2521 gcagccaact cagcttcctt tcgggctttg ttatcaacag gtacagttct gctggtcaat 2581 ctgcttccgg cagggctcca tggtgacagc cacgcccacc ccactccggc ccgatgtcat 2641 tcgggtcacc tcgctccagg tgtctgtatc tgggtcgtaa cactccacac tgtccaggaa 2701 cgtgtgacca tcatagcctc caaggacgta gattctcccc tggtggacag tgatccccag 2761 ggcacttcgc cggtgcttca tgggggctac gaaagtccac gtctctgttt ccacatcgta 2821 gcgctccacg ctgttcagct ggtcctgacc atcatagccc ccagcagcat agatacagtt 2881 gtgcaggacg cagacgcctg ccccgcttcg gatggtgttc attgctgtga tcattcgcca 2941 ctcgttcctc tctgggtagt aacactcagc tgaattaagg cggtttgtcc cgtcaaagcc 3001 ccccacggca taaaggagac gattgaggac agccacgccc accccgatcc ttcgtgtcag 3061 cattggggcc accaagtgcc actcatcccg ctctggctca tacctctcca cactgttgtg 3121 gtggatgcag ccgtgggagc cgccgacggc atagatgtgg ccatcgatga cccccacccc 3181 gatgcggtta cggggcacgc tcatgggggc gcagggcgac cactgattgg tcatggggtt 3241 gtaacagtcc agggcgctgg agtcggtgtt gccgtcgggc gagttgttcc tgccgcccac 3301 ggcgtacaac agcccgccca ccacgcagcc ggccaggccg ctccgcggca cctgcaggtc 3361 cgccaaccgg agccaggtgc cgtcactggg gttgtaagcc tccaggtagc tgagcgactg 3421 tcggaagtag ccgcccgcgg tgtagatcag gcggcccacc ttgggcgccc ggcagggcat 3481 cacctgcgtg ggcttgtgca gggtgagctc ctcgaagatc ttgaccaggt agtccttgca 3541 gcgggagtcg gactgcagga tctcgcactt ctgcagctgc atctgcagga agttcggcgt 3601 caacgagtgg cagcgcacgg cccgcagcag cgcctggacg tagaaccgtc gctgttcgca 3661 gtcgtacttg acccagttga tgcaggcgtg gaagacctcg gactcgcagc gcacgttcag 3721 gtcgtcccgg ctgatgaggg tcaccagttg gcagtgggac aggttgaaga actcctcttg 3781 cttggccacc tccccaaaat gcatgtagat gtactcccgg gcacgctggt gcaactccac 3841 acagccaatc tgctcagcga agttggcgat gccgatggca ttgctggggt ccagctgctg 3901 caccaggaag tcactgcagg cacggacaac gctgtcgatc tggtacatga cagcaccgtt 3961 catgacgtgg aggacacact tctcgcccat ggagatggag gccgtgtagg cgaattcaat 4021 gaggcgctcc atgaccttgg ggtggatacc ctcaatggac accacctcca tgccctgctc 4081 ccgcagcccg ttggtgaaca tggccttgaa gacagggctg gatgaggcca gcaccacctt 4141 gtgggccatg aactgggcgg ccggtgcatc ctggtacttg acctgcagtg tgacgtcaca 4201 cagctgctgg ctgagccgca gctcgttcat gatgccaaag gcctgcttgg tatgatcctc 4261 cagggtgtag ctgaaggtgc ggttgccatg ctgggagggc gtcacctccg ccttgcactc 4321 agtggaggcg tacatcaccg cgtcccctgc cccctcaggg cactgtgact gcaggggcag 4381 gaatcggcag caggccccag ccccgctagg cctgggatct ggctgcatcc ctgagccctg 4441 gaaatacaag ttttctccgg agccggagct gctgtgatga tgatgatgat ggctgctgcc 4501 catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg 4561 ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta 4621 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat 4681 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt 4741 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca 4801 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct 4861 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa 4921 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga 4981 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc 5041 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga 5101 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt 5161 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg 5221 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa 5281 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc 5341 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta 5401 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct 5461 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa 5521 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc 5581 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg 5641 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga 5701 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc 5761 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt 5821 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg 5881 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct 5941 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt 6001 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 6061 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc 6121 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc 6181 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg 6241 gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg 6301 gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt 6361 ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg 6421 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa 6481 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca 6541 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg 6601 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca 6661 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc 6721 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca 6781 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac 6841 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg 6901 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg 6961 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc 7021 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 7081 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 7141 g //
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