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基本信息

質(zhì)粒圖譜和多克隆位點信息

注意：

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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(huán)狀質(zhì)粒圖譜。注意：載體序列是以pBR322質(zhì)粒的編碼規(guī)矩進行編碼的，所以T7蛋白表達區(qū)在質(zhì)粒圖譜上面是反向的。T7 RNA聚合酶啟動的克隆和表達區(qū)域在質(zhì)粒圖譜中也被標注了出來。質(zhì)粒的F1復(fù)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(chǎn)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(zhuǎn)導(dǎo)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

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

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