pCold II

Vector for cold shock-induced expression in E. coli of a protein with a translation enhancing element followed by a 6xHis tag.

Sequence Author: TaKaRa

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cspA promoter lac operator pCold-F (208 .. 226) TEE NdeI (287) Eco53kI (294) SacI (296) Acc65I (298) KpnI (302) AbsI - PaeR7I - PspXI - XhoI (304) BamHI (310) EcoRI (316) HindIII (322) SalI (328) AccI (329) PstI (338) XbaI (340) BfuAI - BspMI (341) pCold-R (369 .. 388) pCold™ II 4392 bp
NdeI  (287)
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  (294)
1 site
G A G C T C C T C G A G
SacI  (296)
1 site
G A G C T C C T C G A G
Acc65I  (298)
1 site
G G T A C C C C A T G G
KpnI  (302)
1 site
G G T A C C C C A T G G
AbsI  (304)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (304)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (304)
1 site
V C T C G A G B B G A G C T C V
XhoI  (304)
1 site
C T C G A G G A G C T C
BamHI  (310)
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  (316)
1 site
G A A T T C C T T A A G
HindIII  (322)
1 site
A A G C T T T T C G A A
SalI  (328)
1 site
G T C G A C C A G C T G
AccI  (329)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
PstI  (338)
1 site
C T G C A G G A C G T C
XbaI  (340)
1 site
T C T A G A A G A T C T
BfuAI  (341)
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  (341)
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
369 .. 388  =  20 annealed bases
Tm  =  56°C
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)
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
lacI
3147 .. 4229  =  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
3147 .. 4229  =  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
1356 .. 2216  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   1356 .. 1424  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1356 .. 2216  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   1425 .. 2216  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1356 .. 2216  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2387 .. 2975  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2387 .. 2975  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
696 .. 1151  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
696 .. 1151  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
cspA 3'UTR
353 .. 497  =  145 bp
3'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)
cspA 3'UTR
353 .. 497  =  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
1251 .. 1355  =  105 bp
AmpR promoter
1251 .. 1355  =  105 bp
lacI promoter
4230 .. 4307  =  78 bp
lacI promoter
4230 .. 4307  =  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)
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).
MCS
286 .. 345  =  60 bp
multiple cloning site
MCS
286 .. 345  =  60 bp
multiple cloning site
ORF:  2945 .. 3364  =  420 bp
ORF:  139 amino acids  =  15.3 kDa
ORF:  1356 .. 2216  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3127 .. 3390  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  3979 .. 4344  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  3147 .. 4106  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  1820 .. 2086  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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