pET104.1-DEST

Gateway® destination vector for high-level inducible expression of biotinylated proteins in bacteria.

Sequence Author: Thermo Fisher (Invitrogen)

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EcoNI (7585) BclI * (7103) ApaI (6914) PspOMI (6910) HpaI (6615) PshAI (6276) NruI (6018) FspAI (5536) PflFI - Tth111I (4771) BsaAI (4765) BspQI - SapI (4630) PciI (4513) SphI (0) BglII (189) T7 promoter XbaI (255) RBS NdeI (295) T7 tag (gene 10 leader) AleI (348) Xpress™ tag attR1 NotI (723) lac UV5 promoter EcoRI (1043) PasI (1275) NcoI (1344) AgeI (1543) BbvCI (1747) TspMI - XmaI (1891) SmaI - SrfI (1893) BmgBI (1927) SalI (2150) PacI (2290) BlpI (2351) BspDI - ClaI (2599) ZraI (2704) AatII (2706) AmpR promoter PvuI (3256) AhdI (3625) pET104.1-DEST 7650 bp
EcoNI  (7585)
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.
BclI  (7103)
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.
ApaI  (6914)
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  (6910)
1 site
G G G C C C C C C G G G
HpaI  (6615)
1 site
G T T A A C C A A T T G
PshAI  (6276)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
NruI  (6018)
1 site
T C G C G A A G C G C T
FspAI  (5536)
1 site
R T G C G C A Y Y A C G C G T R
PflFI  (4771)
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  (4771)
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.
BsaAI  (4765)
1 site
Y A C G T R R T G C A Y
BspQI  (4630)
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  (4630)
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.
PciI  (4513)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SphI  (0)
1 site
G C A T G C C G T A C G
BglII  (189)
1 site
A G A T C T T C T A G A
XbaI  (255)
1 site
T C T A G A A G A T C T
NdeI  (295)
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.
AleI  (348)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (723)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (1043)
1 site
G A A T T C C T T A A G
PasI  (1275)
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.
NcoI  (1344)
1 site
C C A T G G G G T A C C
AgeI  (1543)
1 site
A C C G G T T G G C C A
BbvCI  (1747)
1 site
C C T C A G C G G A G T C G
TspMI  (1891)
1 site
C C C G G G G G G C C C
XmaI  (1891)
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  (1893)
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  (1893)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (1927)
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.
SalI  (2150)
1 site
G T C G A C C A G C T G
PacI  (2290)
1 site
T T A A T T A A A A T T A A T T
BlpI  (2351)
1 site