pDEST10

Gateway® destination vector for expressing N-terminally 6xHis-tagged proteins in insect cells using the Bac-to-Bac® baculovirus system.

Sequence Author: Thermo Fisher (Invitrogen)

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BbsI (6677) Pc promoter PflFI - Tth111I (6148) BseRI (5881) SacII (5688) BspQI - SapI (5301) PspFI (4884) BseYI (4880) FspI (4073) PvuI (3927) BsaHI (3756) NaeI (3049) SnaBI (93) RsrII (251) 6xHis TEV Site PvuII (824) MluI (1371) BssHII (1418) BbvCI (1649) TspMI - XmaI (1793) SmaI - SrfI (1795) BmgBI (1829) BfuAI - BspMI (2039) StuI (2208) SgrDI (2214) Eco53kI (2222) SacI (2224) SpeI (2227) BstBI (2243) XbaI (2248) PaeR7I - XhoI (2263) SphI (2273) Acc65I (2275) KpnI (2279) HindIII (2281) MfeI (2396) HpaI (2409) BclI * (2545) AvrII (2560) NgoMIV (3047) pDEST™10 6708 bp
BbsI  (6677)
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  (6148)
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  (6148)
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  (5881)
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.
SacII  (5688)
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  (5301)
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  (5301)
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  (4884)
1 site
C C C A G C G G G T C G
BseYI  (4880)
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  (4073)
1 site
T G C G C A A C G C G T
PvuI  (3927)
1 site
C G A T C G G C T A G C
BsaHI  (3756)
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.
NaeI  (3049)
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.
SnaBI  (93)
1 site
T A C G T A A T G C A T
RsrII  (251)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
PvuII  (824)
1 site
C A G C T G G T C G A C
MluI  (1371)
1 site
A C G C G T T G C G C A
BssHII  (1418)
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  (1649)
1 site
C C T C A G C G G A G T C G
TspMI  (1793)
1 site
C C C G G G G G G C C C
XmaI  (1793)
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  (1795)
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  (1795)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (1829)
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  (2039)
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  (2039)
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.
StuI  (2208)
1 site
A G G C C T T C C G G A
SgrDI  (2214)
1 site
C G T C G A C G G C A G C T G C
Eco53kI  (2222)
1 site
G A G C T C C T C G A G
SacI  (2224)
1 site
G A G C T C C T C G A G
SpeI  (2227)
1 site
A C T A G T T G A T C A
BstBI  (2243)
1 site
T T C G A A A A G C T T
XbaI  (2248)
1 site
T C T A G A A G A T C T
PaeR7I  (2263)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (2263)
1 site
C T C G A G G A G C T C
SphI  (2273)
1 site
G C A T G C C G T A C G
Acc65I  (2275)
1 site
G G T A C C C C A T G G
KpnI  (2279)
1 site
G G T A C C C C A T G G
HindIII  (2281)
1 site
A A G C T T T T C G A A
MfeI  (2396)
1 site
C A A T T G G T T A A C
HpaI  (2409)
1 site
G T T A A C C A A T T G
BclI  (2545)
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  (2560)
1 site
C C T A G G G G A T C C
NgoMIV  (3047)
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.
AmpR
3509 .. 4369  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3509 .. 3577  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3509 .. 4369  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3578 .. 4369  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3509 .. 4369  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
711 .. 1370  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
711 .. 1370  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
4540 .. 5128  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4540 .. 5128  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
GmR
5722 .. 6255  =  534 bp
177 amino acids  =  19.4 kDa
Product: gentamycin acetyltransferase
confers resistance to gentamycin
GmR
5722 .. 6255  =  534 bp
177 amino acids  =  19.4 kDa
Product: gentamycin acetyltransferase
confers resistance to gentamycin
f1 ori
2922 .. 3377  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2922 .. 3377  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
ccdB
1712 .. 2017  =  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
1712 .. 2017  =  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
5431 .. 5655  =  225 bp
mini-Tn7 element (right end of the Tn7 transposon)
Tn7R
5431 .. 5655  =  225 bp
mini-Tn7 element (right end of the Tn7 transposon)
Tn7L
2573 .. 2738  =  166 bp
mini-Tn7 element (left end of the Tn7 transposon)
Tn7L
2573 .. 2738  =  166 bp
mini-Tn7 element (left end of the Tn7 transposon)
SV40 poly(A) signal
2410 .. 2544  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2410 .. 2544  =  135 bp
SV40 polyadenylation signal
attR1
337 .. 461  =  125 bp
recombination site for the Gateway® LR reaction
attR1
337 .. 461  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2058 .. 2182  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2058 .. 2182  =  125 bp
recombination site for the Gateway® LR reaction
AmpR promoter
3404 .. 3508  =  105 bp
AmpR promoter
3404 .. 3508  =  105 bp
cat promoter
608 .. 710  =  103 bp
promoter of the E. coli cat gene
cat promoter
608 .. 710  =  103 bp
promoter of the E. coli cat gene
polyhedrin promoter
116 .. 207  =  92 bp
promoter for the baculovirus polyhedrin gene
polyhedrin promoter
116 .. 207  =  92 bp
promoter for the baculovirus polyhedrin gene
ATG
262 .. 264  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
262 .. 264  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
274 .. 291  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
274 .. 291  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TEV Site
313 .. 333  =  21 bp
7 amino acids  =  869.9 Da
Product: tobacco etch virus (TEV) recognition and cleavage site
TEV Site
313 .. 333  =  21 bp
7 amino acids  =  869.9 Da
Product: tobacco etch virus (TEV) recognition and cleavage site
Pc promoter
6444 .. 6472  =  29 bp
3 segments
   Segment 3:  -10  
   6444 .. 6449  =  6 bp
class 1 integron promoter
Pc promoter
6444 .. 6472  =  29 bp
3 segments
   Segment 2:  
   6450 .. 6466  =  17 bp
class 1 integron promoter
Pc promoter
6444 .. 6472  =  29 bp
3 segments
   Segment 1:  -35  
   6467 .. 6472  =  6 bp
class 1 integron promoter
Pc promoter
6444 .. 6472  =  29 bp
3 segments
class 1 integron promoter
ORF:  5860 .. 6192  =  333 bp
ORF:  110 amino acids  =  11.9 kDa
ORF:  6466 .. 6708  =  243 bp
ORF:  80 amino acids  =  9.1 kDa
ORF:  1712 .. 2017  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  3509 .. 4369  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  711 .. 1370  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  5565 .. 5867  =  303 bp
ORF:  100 amino acids  =  11.8 kDa
ORF:  6357 .. 128  =  480 bp
ORF:  159 amino acids  =  18.5 kDa
ORF:  3973 .. 4239  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  5722 .. 6255  =  534 bp
ORF:  177 amino acids  =  19.4 kDa
ORF:  6447 .. 47  =  309 bp
ORF:  102 amino acids  =  11.4 kDa
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