pCold TF/ pCold TF质粒/ pCold TF Plasmid

pCold TF

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pCold TF,Plasmid pCold TF,pCold TF vector

 

pCold TF Information

Promoter: CspA promoter
Replicator: ColE1 ori, F1 ori
Plasmid classification: Escherichia coli plasmids; large intestine expression plasmids; pCold series plasmids
Plasmid size: 5769bp
Plasmid Tags: N-His, N-Trigger factor, N-HRV 3C, N-Thrombin, N-Factor Xa
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 and other Escherichia coli
Culture conditions: 37 centigrade, aerobic, LB
Inducement: IPTG or lactose and its analogues
5'sequencing primers: pCold-TF-F1 (CCACTTTCAACGAGCTGATG)
3'sequencing primers: pCold-R (GGCAGGGATCTTAGATTCTG)
Remarks: cold start expression plasmid

 

pCold TF Description

pCold TF vector is a cold shock carrier that combines the soluble label "Trigger Factor (TF) companion". The "trigger factor" (48KD) is a prokaryotic ribosome related molecular chaperone protein (48 kDa), which is beneficial to the co translational folding of the newly expressed peptides. It is because it is derived from Escherichia coli that the trigger factor can be highly expressed in the Escherichia coli expression system. PCold TF vector is composed of CSPA promoter and its downstream sequence. Its downstream sequence mainly includes 5'untranslated region (5' non coding region), translation enhancer element (TEE), His tag sequence and polyclonal site (MCS). The Lac operon is inserted in the downstream of the CSPA promoter to ensure strict regulation of expression. In addition, HRV 3C protease, thrombin and Factor Xa recognition sites were located between Trigger Factor and polyclonal sites (MCS), so that labels could be removed for fusion proteins expressed. Most Escherichia coli strains can be used as the host for the expression of this vector. PCold TF vector provides a cold shock inducing method to express target protein at high level, plus the expression of triggering factor (partner) to improve the correct protein folding, so as to achieve efficient and soluble expression of other proteins in other systems.
The pColdTF plasmid is an efficient protein expression vector designed by using the cold shock gene CSPA promoter. At the lower reaches of the E.COLI promoter, the lac operon is inserted to strictly control the expression. In addition, the 5 'untranslated region (5' UTR), the translation enhancer element (TEE), the His tag sequence, the Xa factor cleavage site and the polyclonal site (MCS) are located downstream of the CAPS promoter. When the culture temperature was switched to low temperature, the growth of Escherichia coli stopped temporarily, and the protein expression of most Escherichia coli decreased. However, a group of proteins called cold shock protein were specifically induced to express.

 

 

pCold TF Sequence

LOCUS       Exported                5769 bp ds-DNA     circular SYN 13-SEP-2016
DEFINITION  synthetic circular DNA
KEYWORDS    Untitled 9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5769)
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, September 13, 2016 

FEATURES             Location/Qualifiers
     source          1..5769
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    84..100
                     /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             271..288
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             1594..1617
                     /codon_start=1
                     /product="recognition and cleavage site for human
                     rhinovirus 3C and PreScission proteases"
                     /note="HRV 3C site"
                     /translation="LEVLFQGP"
     CDS             1627..1644
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             1651..1662
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /note="Factor Xa site"
                     /translation="IEGR"
     rep_origin      complement(2073..2528)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2628..2732
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             2733..3593
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3764..4352
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(4524..5606)
                     /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(5607..5684)
                     /gene="lacI"
                     /note="lacI promoter"
ORIGIN
        1 aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa
       61 tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat
      121 ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt
      181 cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt