pET- 20b Plasmid

PVT0317 2ug
 

pET-20b Information

Promoter: T7/lac promoter

Replicator: ColE1 ori, F1 ori

Terminator: T7 Terminator

Plasmid classification: Escherichia coli vector; PET series expression plasmid

Plasmid size: 3716bp

Plasmid label: N-pelB, C-His

Prokaryotic resistance: ampicillin Amp

Cloned strain: Escherichia coli DH5 alpha

Culture conditions: 37 centigrade, aerobic, LB

Expression host: Escherichia coli 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

Remarks: secretory expression

pET-20b Description

PET-20b plasmid is an Escherichia coli protein expression vector. T7 promoter drives the fusion expression of target gene and 6 x HIS tag, and secrete to the periplasmic substance under the action of pelB signal peptide.The pET-20b vector carries an N-terminal pelB signal sequence for potential periplasmic localization, plus optional C-terminal His•Tag sequence. 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- 20b Plasmid Reference

Clonaje, expresión y purificación de FrpAang, una nueva proteína de membrana receptora de sideróforos en Vibrio anguillarum
C Amil, J Manuel - 2019 - ruc.udc.es

Cloning, expression and purification of FrpAang, a novel membrane protein siderophore receptor in Vibrio anguillarum”
Máster en Biotecnología Avanzada
Trabajo de Fin de Máster
Febrero, 2019
 

pET-20b Sequence

LOCUS       Exported                3716 bp ds-DNA     circular SYN 30-7-2015
DEFINITION  synthetic circular DNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3716)
FEATURES             Location/Qualifiers
     source          1..3716
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          297..319
                     /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(224..289)
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /note="pelB signal sequence"
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     RBS             297..319
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        complement(351..369)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             878..1069
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    1171..1313
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(1499..2087)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(2258..3118)
                     /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(3119..3223)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      complement(3250..3705)
                     /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 tccgaattaa ttccgatatc catggccatc gccggctggg
      241 cagcgaggag cagcagacca gcagcagcgg tcggcagcag gtatttcata tgtatatctc
      301 cttcttaaag ttaaacaaaa ttatttctag agggaaaccg ttgtggtctc cctatagtga
      361 gtcgtattaa tttcgcggga tcgagatctc gggcagcgtt gggtcctggc cacgggtgcg
      421 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
      481 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
      541 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
      601 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
      661 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
      721 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
      781 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
      841 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
      901 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
      961 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     1021 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     1081 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     1141 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     1201 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     1261 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
     1321 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1381 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     1441 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     1501 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     1561 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     1621 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     1681 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     1741 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     1801 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     1861 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1921 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     1981 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2041 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2101 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2161 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2221 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2281 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2341 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     2401 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     2461 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     2521 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca
     2581 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2641 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2701 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2761 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     2821 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     2881 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     2941 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3001 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3061 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3121 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3181 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     3241 tgccacctga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     3301 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3361 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     3421 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     3481 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3541 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3601 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3661 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//

Search name

pET-20b,Plasmid pET-20b,pET-20b vector