pET-32a大肠表达质粒

基本信息

别名:	pET32a，pET-32a(+)
启动子:	T7/lac promotor
复制子:	ColE1 ori，F1 ori
终止子:	T7 terminator
质粒分类:	大肠系列质粒；大肠表达质粒；pET系列质粒
质粒大小:	5900bp
质粒标签:	N-Trx，N-6×His，N-thrombin，N-S，N-enterokinase，C-6×His
原核抗性:	氨苄青霉素Amp（100μg/ml）
克隆菌株:	DH5α等大肠杆菌
培养条件:	37℃，有氧，LB
表达宿主:	BL21(DE3)等大肠杆菌
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

        pET-32a质粒是一种原核表达载体，C端含有一个6×His标签，N端含有一个thrombin位点、6×His标签、TrXA位点、Ek位点和S标签。该质粒含有多个常用的酶切位点，便于不同基因克隆。表达由宿主细胞提供的T7 RNA聚合酶诱导，目的基因被克隆到质粒载体上，受噬菌体强转录及翻译信号控制。
        pET系统是有史以来在大肠杆菌中表达重组蛋白的功能最强大的系统，也是现今原核表达方面使用最广泛的系统。该系列质粒能很容易的通过降低诱导物的浓度来削弱蛋白表达。在非诱导条件下，可以使目的基因处于完全沉默状态而不转录。
     The pET-32 series is designed for cloning and high-level expression of peptide sequences fused with the 109aa Trx•Tag™ thioredoxin protein (1). Cloning sites are available for producing fusion proteins also containing cleavable His•Tag® and S•Tag™ sequences for detection and purification. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circle map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore, single-stranded sequencing should be performed using the T7 terminator primer.

质粒图谱

 

质粒序列

LOCUS       Exported                5900 bp ds-DNA     circular SYN 05-8-2015
DEFINITION  synthetic circular DNA
KEYWORDS    Untitled 8
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5900)
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-5  from Plasmid
FEATURES             Location/Qualifiers
     source          1..5900
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          700..722
                     /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(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(219..233)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /note="enterokinase site"
                     /translation="DDDDK"
     CDS             complement(249..293)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic
                     ribonuclease A"
                     /note="S-Tag"
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(300..317)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             complement(327..344)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(366..692)
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /note="TrxA"
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     RBS             700..722
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    737..761
                     /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(762..780)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1093..1170
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             1171..2253
                     /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             3062..3253
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    3355..3497
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3683..4271)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(4442..5302)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5303..5407)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      complement(5434..5889)
                     /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 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgatatca gccatggcct tgtcgtcgtc gtcggtaccc
      241 agatctgggc tgtccatgtg ctggcgttcg aatttagcag cagcggtttc tttcatacca
      301 gaaccgcgtg gcaccagacc agaagaatga tgatgatgat ggtgcatatg gccagaacca
      361 gaaccggcca ggttagcgtc gaggaactct ttcaactgac ctttagacag tgcacccact
      421 ttggttgccg ccacttcacc gtttttgaac agcagcagag tcgggatacc acggatgcca
      481 tatttcggcg cagtgccagg gttttgatcg atgttcagtt ttgcaacggt cagtttgccc
      541 tgatattcgt cagcgatttc atccagaatc ggggcgatca ttttgcacgg accgcaccac
      601 tctgcccaga aatcgacgag gatcgccccg tccgctttga gtacatccgt gtcaaaactg
      661 tcgtcagtca ggtgaataat tttatcgctc atatgtatat ctccttctta aagttaaaca
      721 aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta
      781 atttcgcggg atcgagatcg atctcgatcc tctacgccgg acgcatcgtg gccggcatca
      841 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
      901 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
      961 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     1021 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     1081 gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     1141 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     1201 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     1261 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     1321 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     1381 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     1441 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     1501 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     1561 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     1621 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     1681 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     1741 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     1801 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     1861 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     1921 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga catctcggta
     1981 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     2041 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     2101 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     2161 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     2221 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     2281 cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     2341 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     2401 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     2461 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     2521 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     2581 cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     2641 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     2701 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     2761 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     2821 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     2881 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     2941 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     3001 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     3061 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     3121 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     3181 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     3241 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     3301 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     3361 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     3421 cagattgtac tgagagtgca ccatatatgc ggtgtgaaat accgcacaga tgcgtaagga
     3481 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3541 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3601 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3661 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3721 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3781 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3841 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3901 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3961 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4021 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4081 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4141 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4201 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4261 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4321 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4381 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4441 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4501 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4561 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4621 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4681 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4741 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4801 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4861 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4921 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4981 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5041 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5101 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5161 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5221 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5281 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5341 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5401 ttccgcgcac atttccccga aaagtgccac ctgaaattgt aaacgttaat attttgttaa
     5461 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     5521 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     5581 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     5641 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     5701 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     5761 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     5821 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     5881 agggcgcgtc ccattcgcca