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Plasmid Files

pEF-DEST51

Gateway® destination vector for high-level constitutive expression of C-terminally V5- and 6xHis-tagged proteins in mammalian cells.

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pEF-DEST51 Sequence and MappEF-DEST51.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 ZraI (119) FspI (6885) AhdI (6663) BspQI - SapI (5654) PfoI (5194) BlpI (5098) PflFI - Tth111I (4977) PpuMI (4862) AvrII (4574) SexAI * (4341) PmeI (3525) SgrDI (120) AatII (121) AflII (854) FseI (1179) Eco53kI (1301) SacI (1303) KasI (1413) NarI (1414) SfoI (1415) PluTI (1417) PaeR7I - XhoI (1440) T7 promoter NotI (1846) BspEI (2162) EcoRI (2166) BsmBI (2390) PflMI * (2398) BssHII (2660) SrfI (3037) BmgBI (3071) BstXI (3154) XbaI * (3428) BstBI (3445) 6xHis pEF-DEST51 7464 bp
ZraI  (119)
1 site
G A C G T C C T G C A G
FspI  (6885)
1 site
T G C G C A A C G C G T
AhdI  (6663)
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.
BspQI  (5654)
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  (5654)
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.
PfoI  (5194)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
BlpI  (5098)
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.
PflFI  (4977)
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  (4977)
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  (4862)
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.
AvrII  (4574)
1 site
C C T A G G G G A T C C
SexAI  (4341)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
PmeI  (3525)
1 site
G T T T A A A C C A A A T T T G
SgrDI  (120)
1 site
C G T C G A C G G C A G C T G C
AatII  (121)
1 site
G A C G T C C T G C A G
AflII  (854)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
FseI  (1179)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
Eco53kI  (1301)
1 site
G A G C T C C T C G A G
SacI  (1303)
1 site
G A G C T C C T C G A G
KasI  (1413)
1 site
G G C G C C C C G C G G
NarI  (1414)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (1415)
1 site
G G C G C C C C G C G G
PluTI  (1417)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
PaeR7I  (1440)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1440)
1 site
C T C G A G G A G C T C
NotI  (1846)
1 site
G C G G C C G C C G C C G G C G
BspEI  (2162)
1 site
T C C G G A A G G C C T
EcoRI  (2166)
1 site
G A A T T C C T T A A G
BsmBI  (2390)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
PflMI  (2398)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
BssHII  (2660)
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.
SrfI  (3037)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (3071)
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.
BstXI  (3154)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
XbaI  (3428)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
BstBI  (3445)
1 site
T T C G A A A A G C T T
EF-1α promoter
475 .. 1653  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
EF-1α promoter
475 .. 1653  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
AmpR
6590 .. 7450  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6590 .. 7381  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6590 .. 7450  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7382 .. 7450  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6590 .. 7450  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
1953 .. 2612  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1953 .. 2612  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5831 .. 6419  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5831 .. 6419  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
3817 .. 4245  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3817 .. 4245  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
BSD
4703 .. 5101  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
4703 .. 5101  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
SV40 promoter
4259 .. 4589  =  331 bp
SV40 enhancer and early promoter
SV40 promoter
4259 .. 4589  =  331 bp
SV40 enhancer and early promoter
ccdB
2954 .. 3259  =  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
2954 .. 3259  =  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.
bGH poly(A) signal
3547 .. 3771  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
3547 .. 3771  =  225 bp
bovine growth hormone polyadenylation signal
attR1
1720 .. 1844  =  125 bp
recombination site for the Gateway® LR reaction
attR1
1720 .. 1844  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3300 .. 3424  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3300 .. 3424  =  125 bp
recombination site for the Gateway® LR reaction
SV40 poly(A) signal
5259 .. 5380  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5259 .. 5380  =  122 bp
SV40 polyadenylation signal
AmpR promoter
7451 .. 91  =  105 bp
AmpR promoter
7451 .. 91  =  105 bp
EM7 promoter
4637 .. 4684  =  48 bp
synthetic bacterial promoter
EM7 promoter
4637 .. 4684  =  48 bp
synthetic bacterial promoter
V5 tag
3450 .. 3491  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
3450 .. 3491  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
lac UV5 promoter
1869 .. 1899  =  31 bp
   Segment 1:  -35  
   1869 .. 1874  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1869 .. 1899  =  31 bp
   Segment 2:  
   1875 .. 1892  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1869 .. 1899  =  31 bp
   Segment 3:  -10  
   1893 .. 1899  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1869 .. 1899  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
T7 promoter
1670 .. 1688  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1670 .. 1688  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
3501 .. 3518  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
3501 .. 3518  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
EF-1α intron A
706 .. 1644  =  939 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
706 .. 1644  =  939 bp
intron upstream of the start codon of human EF-1α
SV40 ori
4440 .. 4575  =  136 bp
SV40 origin of replication
SV40 ori
4440 .. 4575  =  136 bp
SV40 origin of replication
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