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基本信息

質粒圖譜和多克隆位點信息

注意：

客戶在我公司選擇構建載體服務項目時，請自行提供母源載體。

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

pET42系列載體是用來進行克隆和高水平表達含有220個氨基酸的GST融合純化標簽的蛋白表達載體。

pET42a載體的單一的多克隆位點見上面的環狀質粒圖譜。注意：載體序列是以pBR322質粒的編碼規矩進行編碼的，所以T7蛋白表達區在質粒圖譜上面是反向的。T7 RNA聚合酶啟動的克隆和表達區域在質粒圖譜中也被標注了出來。質粒的F1復制子是被定向的，所以在T7噬菌體聚合酶的作用下，包含有蛋白編碼序列的病毒 粒子能夠產生，并啟動蛋白表達，同時蛋白表達將被T7終止子序列的作用下終止蛋白翻譯。

轉導生物公司pET42a載體，在BamHI和KpnI酶切位點間，用HRV 3C蛋白酶切位點代替Factor Xa，可以將融合表達的GST標簽完全切除。

The recombinant human rhinovirus (HRV 3C) protease is used to remove fusion tags from proteins with the HRV 3C cleavage sequence (PreScission Site): Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro or LEVLFQ↓GP.

The recombinant HRV 3C protease is a fusion protein of 22-kDa size, with a his tag. The enzyme retains high activity at 4°C, and can cleave the target protein in solution, in dialysis or on column.

載體序列

LOCUS       Exported                5912 bp ds-DNA     circular SYN 14-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    pET42a

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 5912)

  AUTHORS   .

  TITLE     Direct Submission

  JOURNAL   Exported Thursday, Mar 14, 2019 from SnapGene Viewer 4.3.4

COMMENT     Alias: pET42A

FEATURES             Location/Qualifiers

     source          1..5912

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     terminator      26..73

                     /label=T7 terminator

                     /note="transcription terminator for bacteriophage T7 RNA

                     polymerase"

     CDS             complement(150..173)

                     /codon_start=1

                     /product="8xHis affinity tag"

                     /label=8xHis

                     /translation="HHHHHHHH"

     misc_feature    174..246

                     /label=MCS

     CDS             complement(250..273)

                     /codon_start=1

                     /product="recognition and cleavage site for human

                     rhinovirus 3C and PreScission proteases, in place of the

                     original?Commercial plasmid pET42a? Factor Xa protease

                     cleavage site?"

                     /label=HRV 3C site

                     /translation="LEVLFQGP"

     CDS             complement(289..333)

                     /codon_start=1

                     /product="affinity and epitope tag derived from pancreatic

                     ribonuclease A"

                     /label=S-Tag

                     /translation="KETAAAKFERQHMDS"

     CDS             complement(349..366)

                     /codon_start=1

                     /product="thrombin recognition and cleavage site"

                     /label=thrombin site

                     /translation="LVPRGS"

     CDS             complement(376..393)

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     CDS             complement(421..1074)

                     /codon_start=1

                     /product="glutathione S-transferase from Schistosoma

                     japonicum"

                     /label=GST

                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK

                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY

                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL

                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"

     RBS             1082..1104

                     /note="efficient ribosome binding site from bacteriophage

                     T7 gene 10 (Olins and Rangwala, 1989)"

     protein_bind    1119..1143

                     /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)."

     promoter        complement(1144..1162)

                     /label=T7 promoter

                     /note="promoter for bacteriophage T7 RNA polymerase"

     promoter        1475..1552

                     /gene="lacI"

                     /label=lacI promoter

     CDS             1553..2635

                     /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"

     CDS             3207..3398

                     /codon_start=1

                     /gene="rop"

                     /product="Rop protein, which maintains plasmids at low copy

                     number"

                     /label=rop

                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA

                     DELYRSCLARFGDDGENL"

     misc_feature    3500..3642

                     /label=bom

                     /note="basis of mobility region from pBR322"

     rep_origin      complement(3828..4416)

                     /direction=LEFT

                     /label=ori

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

                     replication"

     CDS             4538..5353

                     /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"

     rep_origin      complement(5446..5901)

                     /direction=LEFT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow

                     indicates direction of (+) strand synthesis"

ORIGIN

        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa

       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt

      121 tgtttagcag cctaggtatt aatcaattag tggtggtggt ggtggtggtg gtgctcgagt

      181 gcggccgcaa gcttgtcgac ggagctcgcc tgcaggcgcg ccaaggcctg tacagaattc

      241 ggatcccagg ggcccctgga acagaacttc cagggtaccc agatctgggc tgtccatgtg

      301 ctggcgttcg aatttagcag cagcggtttc tttcatacca attgcagtac taccgcgtgg

      361 caccagaccc gcggagtgat ggtgatggtg atgaccagaa ccactagttg aaccatccga

      421 ttttggagga tggtcgccac caccaaacgt ggcttgccag ccctgcaaag gccatgctat

      481 atacttgctg gatttcaagt acttatcaat ttgtgggata gcttcaatac gttttttaaa

      541 acaaactaat tttgggaacg catccaggca cattgggtcc atgtataaaa caacatcaag

      601 agcgtcatac aacatgaagt caggatgggt tacatgatca ccatttaaat atgttttatg

      661 acataaacga tcttcgaaca ttttcagcat ttcaggtagc ttgctaagaa aatcaacttt

      721 gagagtttca aagtctttac tatatgcaat tctcgaaaca ccgtatctaa tatccaaaac

      781 cgctccttca agcattgaaa tctctgcacg ctcttttgga caaccaccca acatgttgtg

      841 cttgtcagct atataacgta tgatggccat agactgtgtt aatttaacat caccatcaat

      901 ataataagga agattgggaa actccaaacc caattcaaac tttttgtttc gccatttatc

      961 accttcatcg cgctcataca aatgctcttc atatttttct tcaagatatt ccaaaagaag

     1021 tcgagtgggt tgcacaaggc ccttaatttt ccaataacct agtatagggg acatatgtat

     1081 atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat

     1141 tcccctatag tgagtcgtat taatttcgcg ggatcgagat cgatctcgat cctctacgcc

     1201 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc

     1261 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc

     1321 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca

     1381 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg

     1441 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt

     1501 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc

     1561 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt

     1621 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc

     1681 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct

     1741 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat

     1801 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg

     1861 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat

     1921 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt

     1981 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca

     2041 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc

     2101 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa

     2161 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat

     2221 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct

     2281 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg

     2341 cgttggtgcg gacatctcgg tagtgggata cgacgatacc gaagacagct catgttatat

     2401 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg

     2461 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact

     2521 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc

     2581 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca

     2641 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga

     2701 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactagc atgatcgtgc

     2761 tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat

     2821 caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa

     2881 caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc

     2941 cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac

     3001 ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc

     3061 gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat

     3121 cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca

     3181 gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg

     3241 cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg

     3301 atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct

     3361 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg

     3421 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg

     3481 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata

     3541 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tatgcggtgt

     3601 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg

     3661 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag

     3721 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa

     3781 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc

     3841 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca

     3901 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg

     3961 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct

     4021 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt

     4081 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag

     4141 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc

     4201 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac

     4261 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga

     4321 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc

     4381 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg

     4441 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa

     4501 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac

     4561 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg

     4621 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga

     4681 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga

     4741 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat

     4801 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca

     4861 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct

     4921 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg

     4981 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga

     5041 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt

     5101 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga

     5161 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga

     5221 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt

     5281 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga

     5341 tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata

     5401 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaaatt gtaaacgtta

     5461 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg

     5521 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg

     5581 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa

     5641 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg

     5701 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt

     5761 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg

     5821 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta

     5881 atgcgccgct acagggcgcg tcccattcgc ca

//