pET-61-DEST

Gateway® Nova bacterial destination vector for expressing proteins tagged at the N-terminus with 6xHis and Strep-Tag II.

Sequence Author: MilliporeSigma (Novagen)

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AvrII (6745) SalI (6594) PstI (6592) BfuAI - BspMI (6581) BmgBI (6371) SmaI - SrfI (6337) AvaI - BsoBI - TspMI - XmaI (6335) BbvCI (6191) PasI (5698) EcoRI (5466) lac UV5 promoter EagI - NotI (5146) BstBI (5003) Strep-Tag II 6xHis ATG NcoI (4955) XbaI (4916) lac operator T7 promoter BspDI * - ClaI * (4849) SgrAI (4805) SphI (4657) EcoNI (4592) BstAPI (4448) BclI * (4110) BstEII (3942) ApaI (3921) PspOMI (3917) HpaI (3622) BlpI (6763) DraIII (245) PsiI (370) AhdI (655) BsaI (716) BglI (775) FspI (877) PvuI (1025) PciI (2260) BspQI - SapI (2377) PflFI - Tth111I (2518) PpuMI (3255) pET-61-DEST™ 6846 bp
AvrII  (6745)
1 site
C C T A G G G G A T C C
SalI  (6594)
1 site
G T C G A C C A G C T G
PstI  (6592)
1 site
C T G C A G G A C G T C
BfuAI  (6581)
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  (6581)
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.
BmgBI  (6371)
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.
SmaI  (6337)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (6337)
1 site
G C C C G G G C C G G G C C C G
AvaI  (6335)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (6335)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
TspMI  (6335)
1 site
C C C G G G G G G C C C
XmaI  (6335)
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.
BbvCI  (6191)
1 site
C C T C A G C G G A G T C G
PasI  (5698)
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  (5466)
1 site
G A A T T C C T T A A G
EagI  (5146)
1 site
C G G C C G G C C G G C
NotI  (5146)
1 site
G C G G C C G C C G C C G G C G
BstBI  (5003)
1 site
T T C G A A A A G C T T
NcoI  (4955)
1 site
C C A T G G G G T A C C
XbaI  (4916)
1 site
T C T A G A A G A T C T
BspDI  (4849)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (4849)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (4805)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
SphI  (4657)
1 site
G C A T G C C G T A C G
EcoNI  (4592)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
BstAPI  (4448)
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  (4110)
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  (3942)
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  (3921)
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  (3917)
1 site
G G G C C C C C C G G G
HpaI  (3622)
1 site
G T T A A C C A A T T G
BlpI  (6763)
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.
DraIII  (245)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
PsiI  (370)
1 site
T T A T A A A A T A T T
AhdI  (655)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BsaI  (716)
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.
BglI  (775)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
FspI  (877)
1 site
T G C G C A A C G C G T
PvuI  (1025)
1 site
C G A T C G G C T A G C
PciI  (2260)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2377)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (2377)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
PflFI  (2518)
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  (2518)
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.
PpuMI  (3255)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
lacI
3397 .. 4479  =  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
3397 .. 4479  =  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
582 .. 1442  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   582 .. 1373  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
582 .. 1442  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   1374 .. 1442  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
582 .. 1442  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
5253 .. 5912  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
5253 .. 5912  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
1616 .. 2204  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1616 .. 2204  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
12 .. 467  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
12 .. 467  =  456 bp