pET104-DEST

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

Sequence Author: Thermo Fisher (Invitrogen)

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EcoNI (7553) BclI * (7071) ApaI (6882) PspOMI (6878) HpaI (6583) PshAI (6244) NruI (5986) FspAI (5504) PflFI - Tth111I (4739) BsaAI (4733) BspQI - SapI (4598) PciI (4481) SphI (0) BglII (189) T7 promoter XbaI (255) RBS NdeI (295) ATG AleI (312) Xpress™ tag attR1 NotI (687) lac UV5 promoter EcoRI (1007) PasI (1239) NcoI (1308) AgeI (1507) BbvCI (1711) TspMI - XmaI (1855) SmaI - SrfI (1857) BmgBI (1891) SalI (2114) PacI (2254) BlpI (2319) T7 terminator BspDI - ClaI (2567) ZraI (2672) AatII (2674) PvuI (3224) AhdI (3593) pET104-DEST 7618 bp
EcoNI  (7553)
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  (7071)
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  (6882)
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  (6878)
1 site
G G G C C C C C C G G G
HpaI  (6583)
1 site
G T T A A C C A A T T G
PshAI  (6244)
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  (5986)
1 site
T C G C G A A G C G C T
FspAI  (5504)
1 site
R T G C G C A Y Y A C G C G T R
PflFI  (4739)
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  (4739)
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  (4733)
1 site
Y A C G T R R T G C A Y
BspQI  (4598)
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  (4598)
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  (4481)
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  (312)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (687)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (1007)
1 site
G A A T T C C T T A A G
PasI  (1239)
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  (1308)
1 site
C C A T G G G G T A C C
AgeI  (1507)
1 site
A C C G G T T G G C C A
BbvCI  (1711)
1 site
C C T C A G C G G A G T C G
TspMI  (1855)
1 site
C C C G G G G G G C C C
XmaI  (1855)
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  (1857)
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  (1857)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (1891)
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  (2114)
1 site
G T C G A C C A G C T G
PacI  (2254)
1 site
T T A A T T A A A A T T A A T T
BlpI  (2319)
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.
BspDI  (2567)
1 site
A T C G A T T A G C T A
ClaI  (2567)
1 site
A T C G A T T A G C T A
ZraI  (2672)
1 site
G A C G T C C T G C A G
AatII  (2674)
1 site
G A C G T C C T G C A G
PvuI  (3224)
1 site
C G A T C G G C T A G C
AhdI  (3593)
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.
lacI
6358 .. 7440  =  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
6358 .. 7440  =  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
2806 .. 3666  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   2806 .. 2874  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2806 .. 3666  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   2875 .. 3666  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2806 .. 3666  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
794 .. 1453  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
794 .. 1453  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
3837 .. 4425  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3837 .. 4425  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ccdB
1774 .. 2079  =  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
1774 .. 2079  =  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.
ATG
297 .. 299  =  3 bp
1 amino acid  =  149.2 Da
Product: sta