pCOLADuet-1

Bacterial vector with a ColA origin for the co-expression of two genes.

Sequence Author: MilliporeSigma (Novagen)

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SacI (122) Eco53kI (120) EcoRI (112) BamHI (106) BtgI - NcoI (69) RBS lac operator T7 promoter ACYCDuetUP1 Primer (3660 .. 3678) PfoI (3656) AclI (3564) BstAPI (3548) ApaLI (3244) AflIII - MluI (3224) BclI * (3210) BstEII (3042) ApaI (3021) PspOMI (3017) HpaI (2722) PluTI (2589) SfoI (2587) NarI * (2586) KasI (2585) AcuI (2364) XbaI (2304) CsiI - SexAI * (2163) BmgBI (1908) BfuAI - BspMI (124) AscI (125) PstI - SbfI (135) SalI (137) HindIII (143) NotI (150) AflII (163) DuetUP2 Primer (189 .. 208) BsrGI - TatI (190) DuetDOWN1 Primer (189 .. 208) T7 promoter lac operator RBS NdeI (298) BglII (305) MfeI (311) NgoMIV (324) NaeI (326) FseI (328) AsiSI (337) ZraI (344) AatII (346) Acc65I (348) KpnI (352) PaeR7I - PspXI - XhoI (354) PacI (429) AvrII (433) BlpI (451) T7 Terminator Primer (448 .. 466) EcoO109I (478) T7 terminator Bsu36I (517) AgeI (566) DrdI - PflFI - Tth111I (637) BsaI (645) XmnI (733) Bpu10I (1102) TspMI - XmaI (1246) SmaI (1248) BspDI - ClaI (1429) NruI (1465) NheI - NspI - SphI (1654) BmtI (1658) pCOLADuet™-1 3719 bp
SacI  (122)
1 site
G A G C T C C T C G A G
Eco53kI  (120)
1 site
G A G C T C C T C G A G
EcoRI  (112)
1 site
G A A T T C C T T A A G
BamHI  (106)
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.
BtgI  (69)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (69)
1 site
C C A T G G G G T A C C
PfoI  (3656)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
AclI  (3564)
1 site
A A C G T T T T G C A A
BstAPI  (3548)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
ApaLI  (3244)
1 site
G T G C A C C A C G T G
AflIII  (3224)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
MluI  (3224)
1 site
A C G C G T T G C G C A
BclI  (3210)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BstEII  (3042)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
ApaI  (3021)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (3017)
1 site
G G G C C C C C C G G G
HpaI  (2722)
1 site
G T T A A C C A A T T G
PluTI  (2589)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
SfoI  (2587)
1 site
G G C G C C C C G C G G
NarI  (2586)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI recognition sequence.
KasI  (2585)
1 site
G G C G C C C C G C G G
AcuI  (2364)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Cleavage may be enhanced when more than one copy of the AcuI recognition sequence is present.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
XbaI  (2304)
1 site
T C T A G A A G A T C T
CsiI  (2163)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (2163)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
BmgBI  (1908)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
BfuAI  (124)
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  (124)
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.
AscI  (125)
1 site
G G C G C G C C C C G C G C G G
PstI  (135)
1 site
C T G C A G G A C G T C
SbfI  (135)
1 site
C C T G C A G G G G A C G T C C
SalI  (137)
1 site
G T C G A C C A G C T G
HindIII  (143)
1 site
A A G C T T T T C G A A
NotI  (150)
1 site
G C G G C C G C C G C C G G C G
AflII  (163)
1 site
C T T A A G G A A T T C
BsrGI  (190)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
TatI  (190)
1 site
W G T A C W W C A T G W
NdeI  (298)
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.
BglII  (305)
1 site
A G A T C T T C T A G A
MfeI  (311)
1 site
C A A T T G G T T A A C
NgoMIV  (324)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
NaeI  (326)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
FseI  (328)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
AsiSI  (337)
1 site
G C G A T C G C C G C T A G C G
ZraI  (344)
1 site
G A C G T C C T G C A G
AatII  (346)
1 site
G A C G T C C T G C A G
Acc65I  (348)
1 site
G G T A C C C C A T G G
KpnI  (352)
1 site
G G T A C C C C A T G G
PaeR7I  (354)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (354)
1 site
V C T C G A G B B G A G C T C V
XhoI  (354)
1 site
C T C G A G G A G C T C
PacI  (429)
1 site
T T A A T T A A A A T T A A T T
AvrII  (433)
1 site
C C T A G G G G A T C C
BlpI  (451)
1 site
G C T