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基本信息

質粒圖譜和多克隆位點信息

注意：

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如要使用本公司保存的母源載體時，本公司不能保證所有母源載體完全符合客戶預期，也不承擔由此產生的任何責任。

載體描述

pGEX-4T-1-6*His與pGEX-4T-1一樣，是一種常用的原核表達載體，具有Amp抗性，質粒大小為4.9 kb。該載體多克隆位點區域含有多個常用的內切酶位點序列，便于不同基因的克隆；且全序列中含有tac啟動子、GST標簽序列，是一種高效的蛋白表達載體。Tac啟動子在沒有誘導物存在的情況下，質粒上攜帶的lacIq基因產物可以有效抑制tac啟動子的轉錄。可在BL21、Rosstea等表達菌株中高表達融合有GST標簽的蛋白，且用凝血酶可將目的蛋白的GST標簽切掉，同時C端帶有His標簽，便于下游蛋白純化。

載體序列

LOCUS       Exported                4990 bp ds-DNA     circular SYN 15-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    .

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4990)

  AUTHORS   .

  TITLE     Direct Submission

  JOURNAL   Exported Friday, Mar 15, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..4990

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     promoter        183..211

                     /label=tac promoter

                     /note="strong E. coli promoter; hybrid between the trp and

                     lac UV5 promoters"

     protein_bind    219..235

                     /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)."

     CDS             258..911

                     /codon_start=1

                     /product="glutathione S-transferase from Schistosoma

                     japonicum"

                     /label=GST

                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK

                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY

                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL

                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"

     primer_bind     869..891

                     /label=pGEX5'

     CDS             918..935

                     /codon_start=1

                     /product="thrombin recognition and cleavage site"

                     /label=thrombin site

                     /translation="LVPRGS"

     misc_feature    936..959

                     /label=MCS

                     /note="Multi clone site"

     CDS             960..977

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     primer_bind     complement(1040..1062)

                     /label=pGEX3'

     promoter        1293..1397

                     /gene="bla"

                     /label=AmpR promoter

     CDS             1398..2258

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /label=AmpR

                     /note="confers resistance to ampicillin, carbenicillin, and

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     rep_origin      2429..3017

                     /direction=RIGHT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of

                     replication"

     promoter        3261..3338

                     /gene="lacI (mutant)"

                     /label=lacIq promoter

                     /note="In the lacIq allele, a single base change in the

                     promoter boosts expression of the lacI gene about 10-fold."

     CDS             3339..4421

                     /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"

     protein_bind    4434..4455

                     /label=CAP binding site

                     /bound_moiety="E. coli catabolite activator protein"

                     /note="CAP binding activates transcription in the presence

                     of cAMP."

     promoter        4470..4500

                     /label=lac promoter

                     /note="promoter for the E. coli lac operon"

     protein_bind    4508..4524

                     /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     4532..4548

                     /label=M13 rev

                     /note="common sequencing primer, one of multiple similar

                     variants"

     CDS             4544..4990

                     /codon_start=1

                     /gene="lacZ fragment"

                     /product="LacZ-alpha fragment of beta-galactosidase"

                     /label=lacZ-alpha

                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART

                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV

                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGI"

     primer_bind     complement(4564..4580)

                     /label=M13 fwd

                     /note="common sequencing primer, one of multiple similar

                     variants"

ORIGIN

        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg

       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt

      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc

      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca

      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc

      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc

      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc

      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata

      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc

      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact

      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag

      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt

      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa

      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat

      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc

      901 atcctccaaa atcggatctg gttccgcgtg gatccccgga attcccgggt cgactcgagc

      961 accaccacca ccaccacgag cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt

     1021 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc

     1081 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt

     1141 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac

     1201 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt

     1261 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct

     1321 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat

     1381 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg

     1441 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg

     1501 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc

     1561 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat

     1621 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact

     1681 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca

     1741 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact

     1801 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg

     1861 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg

     1921 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg

     1981 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg

     2041 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag

     2101 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc

     2161 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga

     2221 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat

     2281 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc

     2341 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag

     2401 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct

     2461 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac

     2521 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc

     2581 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg

     2641 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt

     2701 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt

     2761 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc

     2821 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca

     2881 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata

     2941 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg

     3001 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct

     3061 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta

     3121 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag

     3181 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta

     3241 tttcacaccg cataaattcc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat

     3301 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg

     3361 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca

     3421 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca

     3481 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca

     3541 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg

     3601 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta

     3661 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc

     3721 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc

     3781 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc

     3841 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc

     3901 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca

     3961 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac

     4021 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc

     4081 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata

     4141 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca

     4201 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct

     4261 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa

     4321 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc

     4381 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg

     4441 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg

     4501 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga

     4561 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa

     4621 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga

     4681 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc

     4741 accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt

     4801 cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca acgtaaccta

     4861 tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct

     4921 cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg

     4981 cgttggaatt

//