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Plasmid Files

pCold™ TF

Vector for cold shock-induced expression in E. coli of a protein fused to the Trigger Factor chaperone.

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pCold TF.dna
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Sequence Author:  TaKaRa
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pCold-F (208 .. 226) NdeI (1664) Eco53kI (1671) SacI (1673) Acc65I (1675) KpnI (1679) AbsI - PaeR7I - PspXI - XhoI (1681) BamHI (1687) EcoRI (1693) HindIII (1699) SalI (1705) PstI (1715) XbaI (1717) BfuAI - BspMI (1718) pCold-R (1746 .. 1765) pCold™ TF 5769 bp
NdeI  (1664)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
Eco53kI  (1671)
1 site
G A G C T C C T C G A G
SacI  (1673)
1 site
G A G C T C C T C G A G
Acc65I  (1675)
1 site
G G T A C C C C A T G G
KpnI  (1679)
1 site
G G T A C C C C A T G G
AbsI  (1681)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (1681)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1681)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1681)
1 site
C T C G A G G A G C T C
BamHI  (1687)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
EcoRI  (1693)
1 site
G A A T T C C T T A A G
HindIII  (1699)
1 site
A A G C T T T T C G A A
SalI  (1705)
1 site
G T C G A C C A G C T G
PstI  (1715)
1 site
C T G C A G G A C G T C
XbaI  (1717)
1 site
T C T A G A A G A T C T
BfuAI  (1718)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (1718)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
pCold-F
19-mer  /  58% GC
1 binding site
208 .. 226  =  19 annealed bases
Tm  =  60°C
pCold-R
20-mer  /  50% GC
1 binding site
1746 .. 1765  =  20 annealed bases
Tm  =  55°C
Trigger Factor
289 .. 1584  =  1296 bp
432 amino acids  =  48.2 kDa
Product: ribosome-associated chaperone from E. coli (Hoffmann et al., 2010)
Trigger Factor
289 .. 1584  =  1296 bp
432 amino acids  =  48.2 kDa
Product: ribosome-associated chaperone from E. coli (Hoffmann et al., 2010)
lacI
4524 .. 5606  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
4524 .. 5606  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
AmpR
2733 .. 3593  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2733 .. 2801  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2733 .. 3593  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2802 .. 3593  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2733 .. 3593  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3764 .. 4352  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3764 .. 4352  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2073 .. 2528  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2073 .. 2528  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
cspA 3'UTR
1730 .. 1874  =  145 bp
3'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
cspA 3'UTR
1730 .. 1874  =  145 bp
3'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
cspA 5'UTR
122 .. 255  =  134 bp
5'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
cspA 5'UTR
122 .. 255  =  134 bp
5'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
AmpR promoter
2628 .. 2732  =  105 bp
AmpR promoter
2628 .. 2732  =  105 bp
lacI promoter
5607 .. 5684  =  78 bp
lacI promoter
5607 .. 5684  =  78 bp
cspA promoter
15 .. 81  =  67 bp
promoter of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
cspA promoter
15 .. 81  =  67 bp
promoter of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
MCS
1663 .. 1722  =  60 bp
multiple cloning site
MCS
1663 .. 1722  =  60 bp
multiple cloning site
HRV 3C site
1594 .. 1617  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
HRV 3C site
1594 .. 1617  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
6xHis
271 .. 288  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
271 .. 288  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
thrombin site
1627 .. 1644  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
thrombin site
1627 .. 1644  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
lac operator
84 .. 100  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
84 .. 100  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
TEE
256 .. 270  =  15 bp
5 amino acids  =  627.8 Da
Product: translation enhancing element for E. coli (Qing et al., 2004)
TEE
256 .. 270  =  15 bp
5 amino acids  =  627.8 Da
Product: translation enhancing element for E. coli (Qing et al., 2004)
Factor Xa site
1651 .. 1662  =  12 bp
4 amino acids  =  473.5 Da
Product: Factor Xa recognition and cleavage site
Factor Xa site
1651 .. 1662  =  12 bp
4 amino acids  =  473.5 Da
Product: Factor Xa recognition and cleavage site
ORF:  256 .. 1725  =  1470 bp
ORF:  489 amino acids  =  54.4 kDa
ORF:  4322 .. 4741  =  420 bp
ORF:  139 amino acids  =  15.3 kDa
ORF:  2733 .. 3593  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  298 .. 1767  =  1470 bp
ORF:  489 amino acids  =  56.9 kDa
ORF:  4504 .. 4767  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  5356 .. 5721  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  621 .. 1133  =  513 bp
ORF:  170 amino acids  =  18.0 kDa
ORF:  4524 .. 5483  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3197 .. 3463  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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