pET- 16b/ pET- 16b质粒/ pET- 16b Plasmid

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pET-16b Information

Promoter: T7/lac

Replicon: ColE1 ori

Terminator: T7 Terminator

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

Plasmid size: 5711bp

Plasmid tagging: N-His, N-Factor Xa

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)

pET-16b Description

The pET-16b plasmid is a prokaryotic expression vector. The N-terminal region of the plasmid contains a 10*His tag, a Factor Xa digestion site and a BamHI restriction endonuclease site. The expression was induced by T7 RNA polymerase from host cells. The target gene was cloned into plasmid vector and controlled by strong phage 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-16b vector carries an N-terminal His•Tag sequence followed by a Factor Xa site and three cloning sites. 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.

pET-16b Multiple cloning site

pET-16b Sequence

LOCUS       Exported                5711 bp ds-DNA     circular SYN 11-JUL-2017
DEFINITION  synthetic circular DNA.
KEYWORDS    pET-16b
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5711)
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, July 11, 2017 
FEATURES             Location/Qualifiers
     source          1..5711
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        104..208
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             209..1069
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(2258..2449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3761..4843)
                     /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"
     promoter        complement(4844..4921)
                     /gene="lacI"
                     /note="lacI promoter"
     promoter        5230..5248
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5249..5273
                     /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)."
     CDS             5323..5352
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /note="10xHis"
                     /translation="HHHHHHHHHH"
     CDS             5365..5376
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /note="Factor Xa site"
                     /translation="IEGR"
     terminator      5452..5499
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg