pET-41a大肠表达质粒

基本信息

启动子:	T7/lac
复制子:	ColE1 ori，F1 ori
终止子:	T7 terminator
质粒分类:	大肠杆菌载体；PET系列表达质粒
质粒大小:	5933bp
质粒标签:	N-GST, N-6×His, N-Thrombin
原核抗性:	卡那霉素Kan
克隆菌株:	DH5α
培养条件:	37℃，有氧，LB
表达宿主:	BL21(DE3)
培养条件:	37℃，有氧，LB
诱导方式:	IPTG或乳糖及其类似物
5'测序引物:	T7:TAATACGACTCACTATAGGG
3'测序引物:	T7-ter:TGCTAGTTATTGCTCAGCGG

质粒简介

       pET41系列载体是设计用来克隆和高水平表达蛋白质的载体，载体融合表达一个含有220个氨基酸的GST标签蛋白纯化片段。pET41a载体的单一的多克隆位点见上面的环状质粒图谱。注意：载体序列是以pBR322质粒的编码规矩进行编码的，所以T7蛋白表达区在质粒图谱上面是反向的。
       T7 RNA聚合酶启动的克隆和表达区域在质粒图谱中也被标注了出来。质粒的F1复制子是被定向的，所以在T7噬菌体聚合酶的作用下，包含有蛋白编码序列的病毒 粒子能够产生，并启动蛋白表达，同时蛋白表达将被T7终止子序列的作用下终止蛋白翻译。pET-41a载体上面含有一个EK蛋白酶切位点，当将目的基因使用载体上面的PshAI限制性内切酶位点插入进去时，可以通过EK蛋白酶将载体上的融合的所有氨基酸序列包括GST标签序列，全部切除掉。

质粒图谱

质粒序列

LOCUS       Exported                5933 bp ds-DNA     circular SYN 10-8-2015
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5933)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-10   from MLCC
            http://www.miaolinhbio.com
FEATURES             Location/Qualifiers
     source          1..5933
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1103..1125
                     /organism="Enterobacteria phage T7"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:10760"
     terminator      26..73
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     CDS             complement(150..173)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /note="8xHis"
                     /translation="HHHHHHHH"
     CDS             complement(265..279)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /note="enterokinase site"
                     /translation="DDDDK"
     CDS             complement(310..354)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic
                     ribonuclease A"
                     /note="S-Tag"
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(370..387)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             complement(397..414)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(442..1095)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma
                     japonicum"
                     /note="GST"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     RBS             1103..1125
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1140..1164
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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(1165..1183)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1496..1573
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             1574..2656
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="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             3228..3419
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    3521..3663
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3849..4437)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             4559..5374
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin in bacteria or G418
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(5467..5922)
                     /direction=LEFT
                     /note="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 ggatccccga tatcccatgg gactcttgtc gtcgtcatca ccggagccac caccggtacc
      301 cagatctggg ctgtccatgt gctggcgttc gaatttagca gcagcggttt ctttcatacc
      361 aattgcagta ctaccgcgtg gcaccagacc cgcggagtga tggtgatggt gatgaccaga
      421 accactagtt gaaccatccg attttggagg atggtcgcca ccaccaaacg tggcttgcca
      481 gccctgcaaa ggccatgcta tatacttgct ggatttcaag tacttatcaa tttgtgggat
      541 agcttcaata cgttttttaa aacaaactaa ttttgggaac gcatccaggc acattgggtc
      601 catgtataaa acaacatcaa gagcgtcata caacatgaag tcaggatggg ttacatgatc
      661 accatttaaa tatgttttat gacataaacg atcttcgaac attttcagca tttcaggtag
      721 cttgctaaga aaatcaactt tgagagtttc aaagtcttta ctatatgcaa ttctcgaaac
      781 accgtatcta atatccaaaa ccgctccttc aagcattgaa atctctgcac gctcttttgg
      841 acaaccaccc aacatgttgt gcttgtcagc tatataacgt atgatggcca tagactgtgt
      901 taatttaaca tcaccatcaa tataataagg aagattggga aactccaaac ccaattcaaa
      961 ctttttgttt cgccatttat caccttcatc gcgctcatac aaatgctctt catatttttc
     1021 ttcaagatat tccaaaagaa gtcgagtggg ttgcacaagg cccttaattt tccaataacc
     1081 tagtataggg gacatatgta tatctccttc ttaaagttaa acaaaattat ttctagaggg
     1141 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
     1201 tcgatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     1261 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     1321 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     1381 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     1441 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
     1501 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     1561 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     1621 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     1681 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     1741 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     1801 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     1861 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     1921 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     1981 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     2041 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     2101 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     2161 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     2221 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     2281 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     2341 cattaccgag tccgggctgc gcgttggtgc ggacatctcg gtagtgggat acgacgatac
     2401 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     2461 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     2521 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     2581 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     2641 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
     2701 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     2761 gcatgactag catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc
     2821 ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca
     2881 aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc
     2941 tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct
     3001 gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag
     3061 tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca
     3121 gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg
     3181 gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg
     3241 aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga
     3301 aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg
     3361 ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     3421 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     3481 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     3541 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     3601 agtgcaccat atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3661 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3721 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3781 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3841 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3901 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3961 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4021 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4081 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4141 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4201 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4261 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4321 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4381 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4441 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4501 attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     4561 gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc
     4621 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     4681 tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     4741 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     4801 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     4861 ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     4921 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     4981 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     5041 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     5101 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     5161 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     5221 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     5281 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     5341 gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat
     5401 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5461 cacctgaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     5521 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     5581 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     5641 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     5701 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     5761 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     5821 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     5881 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca