pCOLADuet-1

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

Sequence Author: MilliporeSigma (Novagen)

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HomePlasmidspET & Duet Vectors (Novagen)pCOLADuet-1
RBS lac operator 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) BtgI - NcoI (69) BamHI (106) EcoRI (112) Eco53kI (120) SacI (122) 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 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
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.
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
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.
EcoRI  (112)
1 site		 G A A T T C C T T A A G
Eco53kI  (120)
1 site		 G A G C T C C T C G A G
SacI  (122)
1 site		 G A G C T C C T C G A G
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 N A G C C G A N T C G
Sticky ends from different BlpI sites may not be compatible.
EcoO109I  (478)
1 site		 R G G N C C Y Y C C N G G R
Sticky ends from different EcoO109I sites may not be compatible.
Bsu36I  (517)
1 site		 C C T N A G G G G A N T C C
Sticky ends from different Bsu36I sites may not be compatible.
AgeI  (566)
1 site		 A C C G G T T G G C C A
DrdI  (637)
1 site		 G A C N N N N N N G T C C T G N N N N N N C A G
Sticky ends from different DrdI sites may not be compatible.
PflFI  (637)
1 site		 G A C N N N G T C C T G N N N C A G
The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (637)
1 site		 G A C N N N G T C C T G N N N C A G
The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BsaI  (645)
1 site		 G G T C T C N C C A G A G N ( N ) 4
Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
XmnI  (733)
1 site		 G A A N N N N T T C C T T N N N N A A G
Bpu10I  (1102)
1 site		 C C T N A G C G G A N T C G
Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
TspMI  (1246)
1 site		 C C C G G G G G G C C C
XmaI  (1246)
1 site		 C C C G G G G G G C C C
Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (1248)
1 site		 C C C G G G G G G C C C
SmaI can be used at 37°C for brief incubations.
BspDI  (1429)
1 site		 A T C G A T T A G C T A
ClaI  (1429)
1 site		 A T C G A T T A G C T A
NruI  (1465)
1 site		 T C G C G A A G C G C T
NheI  (1654)
1 site		 G C T A G C C G A T C G
NspI  (1654)
1 site		 R C A T G Y Y G T A C R
SphI  (1654)
1 site		 G C A T G C C G T A C G
BmtI  (1658)
1 site		 G C T A G C C G A T C G
ACYCDuetUP1 Primer
19-mer  /  63% GC
1 binding site
3660 .. 3678  =  19 annealed bases
Tm  =  60°C
DuetUP2 Primer
20-mer  /  50% GC
1 binding site
189 .. 208  =  20 annealed bases
Tm  =  57°C
DuetDOWN1 Primer
20-mer  /  50% GC
1 binding site
189 .. 208  =  20 annealed bases
Tm  =  57°C
T7 Terminator Primer
19-mer  /  53% GC
1 binding site
448 .. 466  =  19 annealed bases
Tm  =  57°C
lacI
2497 .. 3579  =  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
2497 .. 3579  =  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).
KanR
739 .. 1554  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
739 .. 1554  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
ColA ori
1664 .. 2299  =  636 bp
Plasmids containing the ColA origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins.
ColA ori
1664 .. 2299  =  636 bp
Plasmids containing the ColA origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins.
MCS-2
297 .. 438  =  142 bp
multiple cloning site 2
MCS-2
297 .. 438  =  142 bp
multiple cloning site 2
MCS-1
69 .. 168  =  100 bp
multiple cloning site 1
MCS-1
69 .. 168  =  100 bp
multiple cloning site 1
AmpR promoter
1555 .. 1646  =  92 bp
AmpR promoter
1555 .. 1646  =  92 bp
lacI promoter
3580 .. 3657  =  78 bp
lacI promoter
3580 .. 3657  =  78 bp
T7 terminator
462 .. 509  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
462 .. 509  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
233 .. 257  =  25 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
233 .. 257  =  25 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
3 .. 27  =  25 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
3 .. 27  =  25 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).
T7 promoter
3703 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3703 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
286 .. 291  =  6 bp
ribosome binding site
RBS
286 .. 291  =  6 bp
ribosome binding site
ATG
300 .. 302  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
300 .. 302  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
S-Tag
366 .. 410  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
366 .. 410  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
ATG
71 .. 73  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
71 .. 73  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
83 .. 100  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
83 .. 100  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ORF:  1833 .. 2069  =  237 bp
ORF:  78 amino acids  =  8.6 kDa
ORF:  2463 .. 2714  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  3329 .. 158  =  549 bp
ORF:  182 amino acids  =  19.6 kDa
ORF:  2477 .. 2740  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  739 .. 1554  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  2497 .. 3456  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
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Individual Sequences & Maps

pACYCDuet-1
pET-24a(+)
pET-3a
pET-51b(+) Ek_LIC (linearized)
pCDF-1b
pET-24b(+)
pET-3b
pET-52b(+)
pCDF-2 Ek_LIC
pET-24c(+)
pET-3c
pET-52b(+) 3C_LIC
pCDF-2 Ek_LIC (linearized)
pET-24d(+)
pET-3d
pET-52b(+) 3C_LIC (linearized)
pCDFDuet-1
pET-25b(+)
pET-40b(+)
pET-53-DEST
pCOLA-2-DEST
pET-26b(+)
pET-41 Ek_LIC
pET-54-DEST
pCOLA-3-DEST
pET-27b(+)
pET-41 Ek_LIC (linearized)
pET-55-DEST
pCOLADuet-1
pET-28a(+)
pET-41a(+)
pET-56-DEST
pET-11a
pET-28b(+)
pET-41b(+)
pET-57-DEST
pET-11b
pET-28c(+)
pET-41c(+)
pET-58-DEST
pET-11c
pET-29a(+)
pET-42a(+)
pET-59-DEST
pET-11d
pET-29b(+)
pET-42b(+)
pET-60-DEST
pET-14b
pET-29c(+)
pET-42c(+)
pET-61-DEST
pET-15b
pET-30 Ek_LIC
pET-43.1 Ek_LIC
pET-62-DEST
pET-16b
pET-30 Ek_LIC (linearized)
pET-43.1 Ek_LIC (linearized)
pET-9a
pET-17b
pET-30 Xa_LIC
pET-43.1a(+)
pET-9b
pET-19b
pET-30 Xa_LIC (linearized)
pET-43.1b(+)
pET-9c
pET-20b(+)
pET-30a(+)
pET-43.1c(+)
pET-9d
pET-21(+)
pET-30b(+)
pET-44a(+)
pETBlue-1
pET-21a(+)
pET-30c(+)
pET-44b(+)
pETBlue-2
pET-21b(+)
pET-31b(+)
pET-44c(+)
pETcoco-1
pET-21c(+)
pET-32 Ek_LIC
pET-45b(+)
pETcoco-2
pET-21d(+)
pET-32 Ek_LIC (linearized)
pET-46 Ek_LIC
pETDuet-1
pET-22b(+)
pET-32 Xa_LIC
pET-46 Ek_LIC (linearized)
pLacI
pET-23(+)
pET-32 Xa_LIC (linearized)
pET-47b(+)
pLysE
pET-23a(+)
pET-32a(+)
pET-48b(+)
pLysS
pET-23b(+)
pET-32b(+)
pET-49b(+)
pRSF-1b
pET-23c(+)
pET-32c(+)
pET-50b(+)
pRSF-2 Ek_LIC
pET-23d(+)
pET-33b(+)
pET-51b(+)
pRSF-2 Ek_LIC (linearized)
pET-24(+)
pET-39b(+)
pET-51b(+) Ek_LIC
pRSFDuet-1

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