pET-33b Plasmid

PVT0351    2ug

pET-33b Plasmid Information

Promoter: T7/lac

Replicator: ColE1 ori, F1 ori

Terminator: T7 Terminator

Plasmid classification: Escherichia coli vector; PET series expression plasmid

Plasmid size: 5383bp

Plasmid label: N-6 x His, N-Thrombin, C-6 x His

Prokaryotic resistance: kanamycin Kan

Cloned strain: DH5 alpha

Culture conditions: 37 centigrade, aerobic, LB

Expression host: BL21 (DE3)

Culture conditions: 37 centigrade, aerobic, LB

Inducement: IPTG or lactose and its analogues

5'sequencing primers: T7:TAATACGACTCACTATAGGG

3'sequencing primers: T7-ter:TGCTAGTTATTGCTCAGCGG

Note: protein kinase PKA identification site

pET-33b Plasmid Description

pET-33b(+) vector is derived from pET-28b(+) and carries a 15bp sequence encoding the protein kinase A (PKA) site RRASV, located between the thrombin cleavage and Nde I sites (1). Proteins expressed in pET-33b(+) can be easily purified by metal chelation chromatography (via either N- or C-terminal His•Tag® sequences) and efficiently labeled with 32P- or 33P-gATP and the catalytic subunit of cAMP-dependent protein kinase from heart muscle. Labeled proteins can be used as direct probes in protein-protein interaction studies. 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 .

pET-33b Plasmid Sequence

LOCUS       Exported                5383 bp ds-DNA     circular SYN 31-AUG-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pET-33b
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5383)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, August 31, 2016 from SnapGene Viewer 3.1.4
FEATURES             Location/Qualifiers
     source          1..5383
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     CDS             complement(257..274)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             complement(284..301)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     protein_bind    357..381
                     /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(382..400)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        709..786
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             787..1869
                     /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             2678..2869
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     rep_origin      complement(3299..3887)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             4009..4824
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(4917..5372)
                     /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 gaacagatgc acgacggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat
      301 ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa
      361 ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct
      421 cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg
      481 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
      541 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
      601 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
      661 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa
      721 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
      781 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
      841 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
      901 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
      961 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     1021 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     1081 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     1141 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     1201 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     1261 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     1321 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     1381 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     1441 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     1501 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     1561 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
     1621 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     1681 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     1741 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
     1801 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     1861 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg
     1921 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac
     1981 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc
     2041 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct
     2101 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc
     2161 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg
     2221 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
     2281 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
     2341 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
     2401 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
     2461 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
     2521 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
     2581 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
     2641 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
     2701 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
     2761 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     2821 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     2881 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     2941 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     3001 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     3061 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
     3121 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3181 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3241 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3301 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3361 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3421 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3481 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3541 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3601 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3661 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3721 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3781 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3841 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3901 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3961 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     4021 caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat
     4081 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat
     4141 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     4201 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     4261 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa
     4321 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     4381 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     4441 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     4501 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     4561 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     4621 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     4681 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     4741 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     4801 ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat
     4861 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaaat
     4921 tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     4981 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     5041 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5101 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5161 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     5221 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     5281 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     5341 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca
//

Caution:
Product is for research use only!