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Plasmid Files

pCOLA-2-DEST™

Gateway® Nova bacterial destination vector with a ColA origin for expressing proteins tagged at the C-terminus with Strep-Tag II.

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pCOLA-2-DEST Sequence and MappCOLA-2-DEST.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 EcoO109I (5094) BlpI (5067) AvrII (5049) BstBI (5030) SalI (4876) PstI (4874) BfuAI - BspMI (4863) BsaBI * (4632) SrfI (4619) AlwNI (4527) BbvCI (4473) BstZ17I (4283) ScaI (4165) PasI (3980) EcoRI (3748) BspEI (3744) EagI - NotI (3428) 6xHis BtgI - NcoI (3273) RBS T7 promoter PfoI (3141) BstAPI (3033) BclI * (2695) BstEII (2527) Bsu36I (2) AgeI (51) DrdI - PflFI - Tth111I (122) BsaI (130) XmnI (218) BspDI - ClaI (914) NruI (950) NheI - NspI - SphI (1139) BmtI (1143) EcoRV (1565) SexAI * (1648) PvuI (1740) XbaI (1789) AcuI (1849) KasI (2070) NarI * (2071) SfoI (2072) PluTI (2074) HpaI (2207) PspOMI (2502) ApaI (2506) NmeAIII (2509) pCOLA-2-DEST™ 5131 bp
EcoO109I  (5094)
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.
BlpI  (5067)
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.
AvrII  (5049)
1 site
C C T A G G G G A T C C
BstBI  (5030)
1 site
T T C G A A A A G C T T
SalI  (4876)
1 site
G T C G A C C A G C T G
PstI  (4874)
1 site
C T G C A G G A C G T C
BfuAI  (4863)
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  (4863)
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.
BsaBI  (4632)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
SrfI  (4619)
1 site
G C C C G G G C C G G G C C C G
AlwNI  (4527)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
BbvCI  (4473)
1 site
C C T C A G C G G A G T C G
BstZ17I  (4283)
1 site
G T A T A C C A T A T G
ScaI  (4165)
1 site
A G T A C T T C A T G A
PasI  (3980)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
EcoRI  (3748)
1 site
G A A T T C C T T A A G
BspEI  (3744)
1 site
T C C G G A A G G C C T
EagI  (3428)
1 site
C G G C C G G C C G G C
NotI  (3428)
1 site
G C G G C C G C C G C C G G C G
BtgI  (3273)
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  (3273)
1 site
C C A T G G G G T A C C
PfoI  (3141)
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.
BstAPI  (3033)
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.
BclI  (2695)
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  (2527)
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.
Bsu36I  (2)
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  (51)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
DrdI  (122)
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  (122)
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  (122)
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  (130)
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  (218)
1 site
G A A N N N N T T C C T T N N N N A A G
BspDI  (914)
1 site
A T C G A T T A G C T A
ClaI  (914)
1 site
A T C G A T T A G C T A
NruI  (950)
1 site
T C G C G A A G C G C T
NheI  (1139)
1 site
G C T A G C C G A T C G
NspI  (1139)
1 site
R C A T G Y Y G T A C R
SphI  (1139)
1 site
G C A T G C C G T A C G
BmtI  (1143)
1 site
G C T A G C C G A T C G
EcoRV  (1565)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
SexAI  (1648)
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.
PvuI  (1740)
1 site
C G A T C G G C T A G C
XbaI  (1789)
1 site
T C T A G A A G A T C T
AcuI  (1849)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Efficient cleavage requires at least two copies of the AcuI
recognition sequence.
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).
KasI  (2070)
1 site
G G C G C C C C G C G G
NarI  (2071)
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.
SfoI  (2072)
1 site
G G C G C C C C G C G G
PluTI  (2074)
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.
HpaI  (2207)
1 site
G T T A A C C A A T T G
PspOMI  (2502)
1 site
G G G C C C C C C G G G
ApaI  (2506)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
NmeAIII  (2509)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
lacI
1982 .. 3064  =  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
1982 .. 3064  =  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
224 .. 1039  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
224 .. 1039  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
CmR
3535 .. 4194  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
3535 .. 4194  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ColA ori
1149 .. 1784  =  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
1149 .. 1784  =  636 bp
Plasmids containing the ColA origin of replication
can be propagated in E. coli cells that contain
additional plasmids with compatible origins.
ccdB
4536 .. 4841  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
4536 .. 4841  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
attR1
3302 .. 3426  =  125 bp
recombination site for the Gateway® LR reaction
attR1
3302 .. 3426  =  125 bp
recombination site for the Gateway® LR reaction
attR2
4882 .. 5006  =  125 bp
recombination site for the Gateway® LR reaction
attR2
4882 .. 5006  =  125 bp
recombination site for the Gateway® LR reaction
AmpR promoter
1040 .. 1131  =  92 bp
AmpR promoter
1040 .. 1131  =  92 bp
lacI promoter
3065 .. 3142  =  78 bp
lacI promoter
3065 .. 3142  =  78 bp
T7 terminator
5078 .. 5125  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
5078 .. 5125  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac UV5 promoter
3451 .. 3481  =  31 bp
   Segment 1:  -35  
   3451 .. 3456  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
3451 .. 3481  =  31 bp
   Segment 2:  
   3457 .. 3474  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
3451 .. 3481  =  31 bp
   Segment 3:  -10  
   3475 .. 3481  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
3451 .. 3481  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
lac operator
3207 .. 3231  =  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
3207 .. 3231  =  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).
Strep-Tag II
5014 .. 5037  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin, an
engineered form of streptavidin
Strep-Tag II
5014 .. 5037  =  24 bp
8 amino acids  =  1.1 kDa
Product: peptide that binds Strep-Tactin, an
engineered form of streptavidin
T7 promoter
3188 .. 3206  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3188 .. 3206  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
3281 .. 3298  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
3281 .. 3298  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
3262 .. 3267  =  6 bp
ribosome binding site
RBS
3262 .. 3267  =  6 bp
ribosome binding site
ATG
3275 .. 3277  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3275 .. 3277  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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