pET-32b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:	5899bp
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
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

       The pET-32a-c 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                5899 bp ds-DNA     circular SYN 05-8-2015
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled 9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5899)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-5  from MLCC
FEATURES             Location/Qualifiers
     source          1..5899
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          699..721
                     /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(218..232)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /note="enterokinase site"
                     /translation="DDDDK"
     CDS             complement(248..292)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic
                     ribonuclease A"
                     /note="S-Tag"
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(299..316)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             complement(326..343)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(365..691)
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /note="TrxA"
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     RBS             699..721
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    736..760
                     /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(761..779)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1092..1169
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             1170..2252
                     /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             3061..3252
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    3354..3496
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3682..4270)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(4441..5301)
                     /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(5302..5406)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      complement(5433..5888)
                     /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 tccgatatcg ccatggcctt gtcgtcgtcg tcggtaccca
      241 gatctgggct gtccatgtgc tggcgttcga atttagcagc agcggtttct ttcataccag
      301 aaccgcgtgg caccagacca gaagaatgat gatgatgatg gtgcatatgg ccagaaccag
      361 aaccggccag gttagcgtcg aggaactctt tcaactgacc tttagacagt gcacccactt
      421 tggttgccgc cacttcaccg tttttgaaca gcagcagagt cgggatacca cggatgccat
      481 atttcggcgc agtgccaggg ttttgatcga tgttcagttt tgcaacggtc agtttgccct
      541 gatattcgtc agcgatttca tccagaatcg gggcgatcat tttgcacgga ccgcaccact
      601 ctgcccagaa atcgacgagg atcgccccgt ccgctttgag tacatccgtg tcaaaactgt
      661 cgtcagtcag gtgaataatt ttatcgctca tatgtatatc tccttcttaa agttaaacaa
      721 aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa
      781 tttcgcggga tcgagatcga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac
      841 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
      901 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
      961 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
     1021 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     1081 tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     1141 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     1201 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     1261 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     1321 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     1381 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     1441 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     1501 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     1561 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     1621 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     1681 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     1741 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     1801 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     1861 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     1921 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggac atctcggtag
     1981 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     2041 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     2101 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     2161 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     2221 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     2281 actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     2341 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     2401 caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc
     2461 tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct
     2521 caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc
     2581 ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc
     2641 tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc
     2701 gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt
     2761 gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc
     2821 atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg
     2881 cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc
     2941 tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc
     3001 tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca
     3061 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta
     3121 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg
     3181 cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt
     3241 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     3301 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     3361 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     3421 agattgtact gagagtgcac catatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     3481 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3541 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3601 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3661 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3721 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3781 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3841 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3901 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3961 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4021 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4081 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     4141 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4201 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4261 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4321 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4381 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4441 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4501 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4561 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     4621 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     4681 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     4741 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     4801 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     4861 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     4921 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     4981 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5041 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5101 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5161 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5221 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5281 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     5341 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5401 tccgcgcaca tttccccgaa aagtgccacc tgaaattgta aacgttaata ttttgttaaa
     5461 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     5521 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     5581 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     5641 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     5701 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     5761 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     5821 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     5881 gggcgcgtcc cattcgcca