pDEST8

Gateway® destination vector for expressing proteins in insect cells using the Bac-to-Bac® baculovirus system.

Sequence Author: Thermo Fisher (Invitrogen)

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BbsI (6403) PflFI - Tth111I (5874) BseRI (5607) EcoRV (5471) SacII (5414) BspQI - SapI (5027) PspFI (4610) BseYI (4606) FspI (3799) PvuI (3653) BsaHI (3482) SnaBI (1) NotI (286) PvuII (647) EcoRI (747) NcoI (1048) MluI (1194) BamHI (1200) BssHII (1241) BbvCI (1472) TspMI - XmaI (1616) SmaI - SrfI (1618) BmgBI (1652) BfuAI - BspMI (1862) PstI (1873) SalI (1875) MfeI (2123) HpaI (2136) BclI * (2272) AvrII (2287) NgoMIV (2773) NaeI (2775) BanII (2807) pDEST™8 6526 bp
BbsI  (6403)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
PflFI  (5874)
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  (5874)
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.
BseRI  (5607)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
EcoRV  (5471)
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.
SacII  (5414)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
BspQI  (5027)
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  (5027)
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.
PspFI  (4610)
1 site
C C C A G C G G G T C G
BseYI  (4606)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
FspI  (3799)
1 site
T G C G C A A C G C G T
PvuI  (3653)
1 site
C G A T C G G C T A G C
BsaHI  (3482)
1 site
G R C G Y C C Y G C R G

BsaHI is typically used at 37°C, but is even more active at 60°C.
SnaBI  (1)
1 site
T A C G T A A T G C A T
NotI  (286)
1 site
G C G G C C G C C G C C G G C G
PvuII  (647)
1 site
C A G C T G G T C G A C
EcoRI  (747)
1 site
G A A T T C C T T A A G
NcoI  (1048)
1 site
C C A T G G G G T A C C
MluI  (1194)
1 site
A C G C G T T G C G C A
BamHI  (1200)
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.
BssHII  (1241)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
BbvCI  (1472)
1 site
C C T C A G C G G A G T C G
TspMI  (1616)
1 site
C C C G G G G G G C C C
XmaI  (1616)
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  (1618)
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  (1618)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (1652)
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.
BfuAI  (1862)
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  (1862)
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.
PstI  (1873)
1 site
C T G C A G G A C G T C
SalI  (1875)
1 site
G T C G A C C A G C T G
MfeI  (2123)
1 site
C A A T T G G T T A A C
HpaI  (2136)
1 site
G T T A A C C A A T T G
BclI  (2272)
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.
AvrII  (2287)
1 site
C C T A G G G G A T C C
NgoMIV  (2773)
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  (2775)
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.
BanII  (2807)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
AmpR
3235 .. 4095  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3235 .. 3303  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3235 .. 4095  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3304 .. 4095  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3235 .. 4095  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
534 .. 1193  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
534 .. 1193  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
4266 .. 4854  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4266 .. 4854  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
GmR
5448 .. 5981  =  534 bp
177 amino acids  =  19.4 kDa
Product: gentamycin acetyltransferase
confers resistance to gentamycin
GmR
5448 .. 5981  =  534 bp
177 amino acids  =  19.4 kDa
Product: gentamycin acetyltransferase
confers resistance to gentamycin
f1 ori
2648 .. 3103  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2648 .. 3103  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
ccdB
1535 .. 1840  =  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
1535 .. 1840  =  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.
Tn7R
5157 .. 5381  =  225 bp
mini-Tn7 element (right end of the Tn7 transposon)
Tn7R
5157 .. 5381  =  225 bp
mini-Tn7 element (right end of the Tn7 transposon)
Tn7L
2300 .. 2465  =  166 bp
mini-Tn7 element (left end of the Tn7 transposon)
Tn7L
2300 .. 2465  =  166 bp
mini-Tn7 element (left end of the Tn7 transposon)
SV40 poly(A) signal
2137 .. 2271  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2137 .. 2271  =  135 bp
SV40 polyadenylation signal
attR1
160 .. 284  =  125 bp
recombination site for the Gateway® LR reaction
attR1
160 .. 284  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1881 .. 2005  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1881 .. 2005  =  125 bp
recombination site for the Gateway® LR reaction
AmpR promoter
3130 .. 3234  =  105 bp
AmpR promoter
3130 .. 3234  =  105 bp
cat promoter
431 .. 533  =  103 bp
promoter of the E. coli cat gene
cat promoter
431 .. 533  =  103 bp
promoter of the E. coli cat gene
polyhedrin promoter
24 .. 115  =  92 bp
promoter for the baculovirus polyhedrin gene
polyhedrin promoter
24 .. 115  =  92 bp
promoter for the baculovirus polyhedrin gene
Pc promoter
6170 .. 6198  =  29 bp
3 segments
   Segment 3:  -10  
   6170 .. 6175  =  6 bp
class 1 integron promoter
Pc promoter
6170 .. 6198  =  29 bp
3 segments
   Segment 2:  
   6176 .. 6192  =  17 bp
class 1 integron promoter
Pc promoter
6170 .. 6198  =  29 bp
3 segments
   Segment 1:  -35  
   6193 .. 6198  =  6 bp
class 1 integron promoter
Pc promoter
6170 .. 6198  =  29 bp
3 segments
class 1 integron promoter
ORF:  3235 .. 4095  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1535 .. 1840  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  5291 .. 5593  =  303 bp
ORF:  100 amino acids  =  11.8 kDa
ORF:  6083 .. 36  =  480 bp
ORF:  159 amino acids  =  18.5 kDa
ORF:  534 .. 1193  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  5586 .. 5918  =  333 bp
ORF:  110 amino acids  =  11.9 kDa
ORF:  6192 .. 6434  =  243 bp
ORF:  80 amino acids  =  9.1 kDa
ORF:  6173 .. 6481  =  309 bp
ORF:  102 amino acids  =  11.4 kDa
ORF:  3699 .. 3965  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  5448 .. 5981  =  534 bp
ORF:  177 amino acids  =  19.4 kDa
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