pTrcHisB Plasmid

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pTrcHisB

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pTrcHisB,Plasmid pTrcHisB,pTrcHisB vector

 

 

pTrcHisB Information

Promoter: Trc/lac

Replicon: PBR322 ori

Terminator: rrnB TT

Plasmid classification: Escherichia coli vector; pTrc series expression plasmid

Plasmid size: 4404bp

Plasmid tagging: N-His, N-EK

Prokaryotic resistance: ampicillin Amp

Clonal strain: DH5 alpha

Culture conditions: 37 C, aerobic, LB

Expression host: Escherichia coli

Culture conditions: 37 C, aerobic, LB

Induction: IPTG or lactose and its analogues.

5'sequencing primers: pTrcHis-F: GAGGTATATATTAATGTATCG

3'sequencing primers: pTrcHis-R:GATTTAATCTGTATCAGG

Remarks: pTrc expression plasmid

 

pTrcHisB Description

PTrcHis vector is an expression vector derived from pBR322 plasmid. It is designed to express and purify recombinant protein efficiently in E. coli.

Trc promoter and rrnB anti-termination region on the vector make it possible to express high level protein. Trc promoter consists of - 35 region of Trp promoter and - 10 region of Lac promoter. The pTrcHis vector also contains a LacIq gene, which encodes an inhibitor of the Lac promoter. The presence of LacIq gene makes it possible for both LacIq + and LacIq - hosts to effectively inhibit the transcription of inserted genes.

During protein expression, E. coli needs to grow to logarithmic phase, and then add 1 mM IPTG to remove repressor protein and induce expression. The Mini cis-trans structure on the vector can enhance the transcription level of the inserted gene, and thus express the target protein at a high level. DNA insertion fragment is located downstream of the vector expression fragment. Upstream of the fragment, there is an N-terminal fusion expression polypeptide sequence, which contains ATG initiation codon, 6xHis tag, translation enhancer of T7 bacteriophage 10 gene, X press epitope and enterokinase digestion recognition site.

 

pTrcHisB Multiple cloning site

pTrcHisB-vector

 

 

pTrcHisB Sequence

LOCUS       Exported                4404 bp ds-DNA     circular SYN 29-JUL-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pTrcHisB
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4404)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2016-7-29 from SnapGene Viewer 2.8.1
           
FEATURES             Location/Qualifiers
     source          1..4404
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    193..222
                     /note="trc promoter"
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    230..246
                     /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)."
     misc_feature    263..332
                     /note="rrnG antiterminator"
                     /note="antiterminator from the E. coli rrnG leader region
                     (Berg et al., 1989)"
     CDS             383..409
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /note="minicistron"
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             425..442
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             446..478
                     /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             482..505
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /note="Xpress(TM) tag"
                     /translation="DLYDDDDK"
     misc_feature    505..554
                     /note="MCS"
     terminator      757..843
                     /gene="Escherichia coli rrnB"
                     /note="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      935..962
                     /note="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        982..1073
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             1074..1934
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2105..2693
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     misc_feature    2879..3019
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     promoter        3205..3282
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             3283..4365
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
      

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