pEF6 V5-His C

Mammalian vector for high-level constitutive expression of C-terminally V5- and 6xHis-tagged proteins. For other reading frames, use pEF6/V5-His A or pEF6/V5-His B.

Sequence Author: Thermo Fisher (Invitrogen)

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SspI (3) ScaI (5482) FspI (5224) AhdI (5002) BspQI - SapI (4004) BstZ17I (3741) BsmI (3689) PfoI (3544) BlpI (3448) BsaBI * (3351) PpuMI (3212) MscI (3056) SmaI (2947) TspMI - XmaI (2945) ZraI (119) SgrDI (120) AatII (121) AflII (852) BfuAI - BspMI (997) PspOMI (1086) ApaI (1090) FseI (1177) PasI (1221) BbvCI (1375) KasI (1411) NarI (1412) SfoI (1413) PluTI (1415) Acc65I (1713) KpnI (1717) BamHI (1725) SpeI (1731) EcoRI (1748) EcoRV (1760) NotI (1775) PspXI (1781) BstEII (1786) BstBI (1796) PmeI (1876) CsiI - SexAI * (2691) AvrII (2924) pEF6/V5-His C 5803 bp
SspI  (3)
1 site
A A T A T T T T A T A A
ScaI  (5482)
1 site
A G T A C T T C A T G A
FspI  (5224)
1 site
T G C G C A A C G C G T
AhdI  (5002)
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  (4004)
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  (4004)
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.
BstZ17I  (3741)
1 site
G T A T A C C A T A T G
BsmI  (3689)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
PfoI  (3544)
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  (3448)
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.
BsaBI  (3351)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
PpuMI  (3212)
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.
MscI  (3056)
1 site
T G G C C A A C C G G T
SmaI  (2947)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (2945)
1 site
C C C G G G G G G C C C
XmaI  (2945)
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.
ZraI  (119)
1 site
G A C G T C C T G C A 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  (852)
1 site
C T T A A G G A A T T C
BfuAI  (997)
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  (997)
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.
PspOMI  (1086)
1 site
G G G C C C C C C G G G
ApaI  (1090)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
FseI  (1177)
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.
PasI  (1221)
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.
BbvCI  (1375)
1 site
C C T C A G C G G A G T C G
KasI  (1411)
1 site
G G C G C C C C G C G G
NarI  (1412)
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  (1413)
1 site
G G C G C C C C G C G G
PluTI  (1415)
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.
Acc65I  (1713)
1 site
G G T A C C C C A T G G
KpnI  (1717)
1 site
G G T A C C C C A T G G
BamHI  (1725)
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.
SpeI  (1731)
1 site
A C T A G T T G A T C A
EcoRI  (1748)
1 site
G A A T T C C T T A A G
EcoRV  (1760)
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.
NotI  (1775)
1 site
G C G G C C G C C G C C G G C G
PspXI  (1781)
1 site
V C T C G A G B B G A G C T C V
BstEII  (1786)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
BstBI  (1796)
1 site
T T C G A A A A G C T T
PmeI  (1876)
1 site
G T T T A A A C C A A A T T T G
CsiI  (2691)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (2691)
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.
AvrII  (2924)
1 site
C C T A G G G G A T C C
EF-1α promoter
473 .. 1651  =  1179 bp
strong constitutive promoter for human elongation factor EF-1α
EF-1α promoter
473 .. 1651  =  1179 bp
strong constitutive promoter for human elongation factor EF-1α
AmpR
4929 .. 5789  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   4929 .. 5720  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4929 .. 5789  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   5721 .. 5789  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4929 .. 5789  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
4170 .. 4758  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4170 .. 4758  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2168 .. 2596  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2168 .. 2596  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
BSD
3053 .. 3451  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
3053 .. 3451  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
SV40 promoter
2610 .. 2939  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
2610 .. 2939  =  330 bp
SV40 enhancer and early promoter
bGH poly(A) signal
1898 .. 2122  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
1898 .. 2122  =  225 bp
bovine growth hormone polyadenylation signal
SV40 poly(A) signal
3609 .. 3730  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3609 .. 3730  =  122 bp
SV40 polyadenylation signal
AmpR promoter
5790 .. 91  =  105 bp
AmpR promoter
5790 .. 91  =  105 bp
MCS
1713 .. 1800  =  88 bp
multiple cloning site
MCS
1713 .. 1800  =  88 bp
multiple cloning site
V5 tag
1801 .. 1842  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
1801 .. 1842  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
6xHis
1852 .. 1869  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1852 .. 1869  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
EM7 promoter
2987 .. 3034  =  48 bp
synthetic bacterial promoter
EM7 promoter
2987 .. 3034  =  48 bp
synthetic bacterial promoter
T7 promoter
1668 .. 1686  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1668 .. 1686  =  19 bp
promoter for bacteriophage T7 RNA polymerase
EF-1α intron A
704 .. 1642  =  939 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
704 .. 1642  =  939 bp
intron upstream of the start codon of human EF-1α
SV40 ori
2790 .. 2925  =  136 bp
SV40 origin of replication
SV40 ori
2790 .. 2925  =  136 bp
SV40 origin of replication
ORF:  1108 .. 1341  =  234 bp
ORF:  77 amino acids  =  7.7 kDa