pET-31b
PVT0088 2ug
pET-31b Information
Replicon: ColE1 ori, F1 ori
Terminator: T7 Terminator
Plasmid classification: large intestine plasmid; large intestine expression plasmid; pET series plasmid.
Plasmid size: 5742bp
Plasmid tagging: C-6 * His
Prokaryotic resistance: ampicillin Amp (100 g/ml)
Cloning strains: E. coli DH5 and E.
Culture conditions: 37 C, aerobic, LB
Expression host: E. coli 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)
Remarks: ketosterol isomerase expression
pET-31b Description
pET-31b is a prokaryotic expression vector with a 6*His tag at the C-terminal. The target gene was cloned into the plasmid vector and controlled by phage strong transcription and translation signals.
The pET system is the most powerful system ever used to express recombinant protein in E. coli. It is also the most widely used system in prokaryotic expression. The plasmids can easily reduce protein expression by decreasing the concentration of inducers. Under non inducible conditions, the target gene can be completely silenced without transcribing.
The pET-31b(+) vector is designed for cloning and high-level expression of peptide sequences fused with the 125aa ketosteroid isomerase protein (1). The unique AlwN I cloning site allows the unidirectional insertion of of tandemly repeated peptide coding regions separated by methionine codons. 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 circular 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 .
pET-31b Multiple cloning site
pET-31b Sequence
LOCUS Exported 5742 bp ds-DNA circular SYN 05-8-2015
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 7
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5742)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported 2015-8-5
FEATURES Location/Qualifiers
source 1..5742
/organism="synthetic DNA construct"
/mol_type="other DNA"
source 552..574
/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(170..544)
/codon_start=1
/product="ketosteroid isomerase"
/note="KSI"
/translation="MHTPEHITAVVQRFVAALNAGDLDGIVALFADDATVEDPVGSEPR
SGTAAIREFYANSLKLPLAVELTQEVRAVANEAAFAFTVSFEYQGRKTVVAPIDHFRFN
GAGKVVSIRALFGEKNIHACQ"
RBS 552..574
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind 589..613
/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(614..632)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 941..1018
/gene="lacI"
/note="lacI promoter"
CDS 1019..2101
/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 2910..3101
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
misc_feature 3203..3345
/note="bom"
/note="basis of mobility region from pBR322"
CDS complement(4284..5144)
/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(5145..5249)
/gene="bla"
/note="AmpR promoter"
rep_origin complement(5276..5731)
/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 gagcagcatc tggcatgcgt
181 gaatattctt ctcgccaaac aaggcgcgga tgctcaccac cttgccggcg ccattgaagc
241 gaaagtgatc gatgggcgca actacggtct tgcggccctg atactcgaag ctgacggtga
301 aagcgaaggc cgcttcgttg gcgaccgcgc gtacctcctg cgtcagctcc accgccaaag
361 gcagtttgag cgagttggcg taaaactcac gaatcgcagc cgtaccggac ctgggctcgg
421 aacccacggg gtcttccacc gtggcgtcat cggcaaacag cgcgacgatg ccgtccagat
481 cgccggcatt gagcgcagcc acaaagcgct gtaccacggc ggtgatgtgt tctggggtat
541 gcatatgtat atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc
601 cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc
661 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
721 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt
781 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg
841 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc
901 ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa
961 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
1021 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
1081 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
1141 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
1201 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
1261 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
1321 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
1381 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
1441 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
1501 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
1561 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
1621 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
1681 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
1741 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
1801 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
1861 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
1921 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
1981 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
2041 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
2101 agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc
2161 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
2221 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
2281 gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg
2341 cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac
2401 gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg
2461 tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg
2521 aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag
2581 caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag
2641 cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa
2701 cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc
2761 gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat
2821 catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga
2881 acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca
2941 tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg
3001 cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca
3061 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
3121 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
3181 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
3241 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatatgcgg
3301 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
3361 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
3421 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
3481 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
3541 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
3601 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
3661 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
3721 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
3781 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
3841 gagtccaacc cggtaagaca cgacttatcg ccactggcag ctggtaacag gattagcaga
3901 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
3961 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
4021 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
4081 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
4141 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
4201 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
4261 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
4321 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
4381 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
4441 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
4501 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
4561 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
4621 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
4681 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
4741 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
4801 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
4861 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
4921 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
4981 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
5041 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
5101 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
5161 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
5221 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaaatt
5281 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
5341 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
5401 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
5461 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
5521 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
5581 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
5641 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
5701 gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca
//
Caution:
Product is for research use only!
Search name
pET-31b,Plasmid pET-31b,pET-31b vector
