pET- 42b

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  • Model: PVT0100
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pET-42b

PVT0100       2ug

 

pET-42b Informaiton

Promoter: T7/lac

Replicon: ColE1 ori, F1 ori

Terminator: T7 Terminator

Plasmid classification: Escherichia coli vector; PET series expression plasmid

Plasmid size: 5929bp

Plasmid tagging: N-GST, N-6 x His, N-S, C-6 x His, N-Thrombin, N-Factor Xa

Prokaryotic resistance: kanamycin Kan

Clonal strain: DH5 alpha

Culture conditions: 37 C, aerobic, LB

Expression host: BL21 (DE3)

Culture conditions: 37 C, aerobic, LB

Induction: IPTG or lactose and its analogues.

5'sequencing primers: T7:TAATACGACTCACTATAGGG

3'sequencing primers: T7-ter:TGCTAGTTATTGCTCAGCGG

 

pET-42b Description

The pET42 series carriers are used for cloning and high-level expression of GST fusion tags containing 220 amino acids.

The single polyclonal site of the pET42a vector is seen above the circular plasmid map. Note: The vector sequence is encoded according to the coding rules of the pBR322 plasmid, so the T7 protein expression region is reversed on the plasmid map. The cloning and expression regions initiated by T7 RNA polymerase were also labelled in plasmid profiles. The F1 replicon of the plasmid is directed, so the viral particles containing the protein coding sequence can be produced by the T7 phage polymerase, and the protein expression can be initiated. The protein expression will be terminated by the T7 terminator sequence.

When the expressed gene is inserted into the vector's PshAI restriction endonuclease site, the fused GST tag can be completely removed by Factor Xa protease.

 

pET-42b Description

pET-42b-vector

 

 

pET-42b Sequence

LOCUS       Exported                5929 bp ds-DNA     circular SYN 10-8-2015
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled 4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5929)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-10 
FEATURES             Location/Qualifiers
     source          1..5929
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1099..1121
                     /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(264..275)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /note="Factor Xa site"
                     /translation="IEGR"
     CDS             complement(306..350)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic
                     ribonuclease A"
                     /note="S-Tag"
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(366..383)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             complement(393..410)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(438..1091)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma
                     japonicum"
                     /note="GST"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     RBS             1099..1121
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1136..1160
                     /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(1161..1179)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1492..1569
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             1570..2652
                     /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             3224..3415
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    3517..3659
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3845..4433)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             4555..5370
                     /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(5463..5918)
                     /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 tatccatgga cccgcgtccc tcaataccgg agccaccacc ggtacccaga
      301 tctgggctgt ccatgtgctg gcgttcgaat ttagcagcag cggtttcttt cataccaatt
      361 gcagtactac cgcgtggcac cagacccgcg gagtgatggt gatggtgatg accagaacca
      421 ctagttgaac catccgattt tggaggatgg tcgccaccac caaacgtggc ttgccagccc
      481 tgcaaaggcc atgctatata cttgctggat ttcaagtact tatcaatttg tgggatagct
      541 tcaatacgtt ttttaaaaca aactaatttt gggaacgcat ccaggcacat tgggtccatg
      601 tataaaacaa catcaagagc gtcatacaac atgaagtcag gatgggttac atgatcacca
      661 tttaaatatg ttttatgaca taaacgatct tcgaacattt tcagcatttc aggtagcttg
      721 ctaagaaaat caactttgag agtttcaaag tctttactat atgcaattct cgaaacaccg
      781 tatctaatat ccaaaaccgc tccttcaagc attgaaatct ctgcacgctc ttttggacaa
      841 ccacccaaca tgttgtgctt gtcagctata taacgtatga tggccataga ctgtgttaat
      901 ttaacatcac catcaatata ataaggaaga ttgggaaact ccaaacccaa ttcaaacttt
      961 ttgtttcgcc atttatcacc ttcatcgcgc tcatacaaat gctcttcata tttttcttca
     1021 agatattcca aaagaagtcg agtgggttgc acaaggccct taattttcca ataacctagt
     1081 ataggggaca tatgtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
     1141 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatcga
     1201 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
     1261 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     1321 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     1381 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     1441 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc
     1501 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     1561 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     1621 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     1681 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     1741 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     1801 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     1861 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     1921 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     1981 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     2041 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     2101 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     2161 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     2221 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     2281 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     2341 accgagtccg ggctgcgcgt tggtgcggac atctcggtag tgggatacga cgataccgaa
     2401 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     2461 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     2521 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     2581 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     2641 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc
     2701 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     2761 gactagcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     2821 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     2881 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     2941 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     3001 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     3061 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     3121 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     3181 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     3241 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     3301 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     3361 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     3421 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3481 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     3541 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     3601 caccatatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     3661 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3721 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3781 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3841 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3901 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3961 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4021 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4081 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4141 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4201 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4261 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     4321 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4381 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4441 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4501 tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     4561 catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat
     4621 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga
     4681 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     4741 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     4801 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     4861 gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     4921 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     4981 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     5041 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     5101 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     5161 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     5221 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     5281 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     5341 tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga
     5401 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5461 tgaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     5521 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     5581 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     5641 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5701 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5761 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5821 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5881 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//


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