pET-21b Plasmid

pET-21b

Search name

pET-21b,Plasmid pET-21b,pET-21b vector

pET-21b  Information

Promoter: T7/lac

Replicator: ColE1 ori, F1 ori

Terminator: T7 Terminator

Plasmid classification: Escherichia coli plasmid; large intestine expression plasmid; pET series plasmid.

Plasmid size: 5442bp

Plasmid label: N-T7, C-His

Prokaryotic resistance: ampicillin Amp (100 u g/ml)

Cloned strains of Escherichia coli, DH5 A and other Escherichia coli

Culture conditions: 37 centigrade, aerobic, LB

Expression of host: BL21 (DE3) and other Escherichia coli

Culture conditions: 37 centigrade, aerobic, LB

Inducement: IPTG or lactose and its analogues

5'sequencing primers: T7 (TAATACGACTCACTATAGGG)

3'sequencing primers: T7-ter (TGCTAGTTATTGCTCAGCGG)

pET-21b Description

pET-21b plasmid is a prokaryotic expression vector. The N terminal contains a T7 tag, and the C end contains a 6 x His tag. Expression of T7 RNA polymerase provided by the host cell was induced, and the target gene was cloned into plasmid vector, which was controlled by the strong transcription and translation signal of phage. PET system is the most powerful system to express recombinant protein in Escherichia coli, and 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 transcription.

The pET-21a-d(+) vectors carry an N-terminal T7?Tag? sequence plus an optional C-terminal His?Tag? sequence. These vectors differ from pET-24a-d(+) only by their selectable marker (ampicillin vs. kanamycin resistance). 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-21b Sequence

LOCUS       Exported                5442 bp ds-DNA     circular SYN 31-7-2015
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled 2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5442)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-7-31    
FEATURES             Location/Qualifiers
     source          1..5442
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          246..268
                     /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(206..238)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /note="T7 tag (gene 10 leader)"
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     RBS             246..268
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    283..307
                     /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(308..326)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        635..712
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             713..1795
                     /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             2604..2795
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    2897..3039
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3225..3813)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(3984..4844)
                     /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(4845..4949)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      complement(4976..5431)
                     /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 tcccgaccca tttgctgtcc accagtcatg ctagccatat
      241 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
      301 caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc
      361 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
      421 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
      481 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
      541 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
      601 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
      661 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
      721 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
      781 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
      841 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
      901 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
      961 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     1021 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     1081 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     1141 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     1201 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     1261 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     1321 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     1381 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     1441 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     1501 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     1561 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     1621 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     1681 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     1741 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     1801 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
     1861 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     1921 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
     1981 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
     2041 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     2101 gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc
     2161 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     2221 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     2281 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     2341 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     2401 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     2461 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     2521 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     2581 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     2641 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     2701 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     2761 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     2821 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     2881 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     2941 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa
     3001 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     3061 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3121 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3181 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3241 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3301 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3361 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3421 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3481 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3541 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3601 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3661 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3721 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3781 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3841 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3901 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3961 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4021 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4081 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4141 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4201 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4261 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
     4321 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4381 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4441 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4501 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4561 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4621 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4681 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4741 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4801 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4861 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4921 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaaatt
     4981 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5041 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5101 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5161 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     5221 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     5281 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5341 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5401 gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca
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