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Plasmid Files

pEF6/V5-His-TOPO® (linearized)

Linearized vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products and expression of tagged proteins in mammalian cells.

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pEF6 V5-His-TOPO (linearized) Sequence and MappEF6 V5-His-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 SspI (4084) ScaI (3760) FspI (3502) AhdI (3280) BspQI - SapI (2271) BstZ17I (2008) BsmI (1956) PfoI (1811) BlpI (1715) BsaBI * (1618) PflFI - Tth111I (1594) PpuMI (1479) MscI (1323) SmaI (1214) TspMI - XmaI (1212) AvrII (1191) ZraI (4200) SgrDI (4201) AatII (4202) AflII (4935) BfuAI - BspMI (5080) FseI (5260) PasI (5304) BbvCI (5458) KasI (5494) NarI (5495) SfoI (5496) PluTI (5498) Acc65I (5796) KpnI (5800) BamHI (5808) SpeI (5814) BstXI (5827) End (5841) Start (1) EcoRV (18) BstXI (28) NotI (33) PspXI (39) XbaI (45) BstBI (62) 6xHis PmeI (142) SexAI * (958) pEF6/V5-His-TOPO® 5840 bp
SspI  (4084)
1 site
A A T A T T T T A T A A
ScaI  (3760)
1 site
A G T A C T T C A T G A
FspI  (3502)
1 site
T G C G C A A C G C G T
AhdI  (3280)
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  (2271)
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  (2271)
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  (2008)
1 site
G T A T A C C A T A T G
BsmI  (1956)
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  (1811)
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  (1715)
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  (1618)
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.
PflFI  (1594)
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  (1594)
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  (1479)
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  (1323)
1 site
T G G C C A A C C G G T
SmaI  (1214)
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  (1212)
1 site
C C C G G G G G G C C C
XmaI  (1212)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
AvrII  (1191)
1 site
C C T A G G G G A T C C
ZraI  (4200)
1 site
G A C G T C C T G C A G
SgrDI  (4201)
1 site
C G T C G A C G G C A G C T G C
AatII  (4202)
1 site
G A C G T C C T G C A G
AflII  (4935)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
BfuAI  (5080)
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  (5080)
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.
FseI  (5260)
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  (5304)
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  (5458)
1 site
C C T C A G C G G A G T C G
KasI  (5494)
1 site
G G C G C C C C G C G G
NarI  (5495)
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  (5496)
1 site
G G C G C C C C G C G G
PluTI  (5498)
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  (5796)
1 site
G G T A C C C C A T G G
KpnI  (5800)
1 site
G G T A C C C C A T G G
BamHI  (5808)
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  (5814)
1 site
A C T A G T T G A T C A
BstXI  (5827)
2 sites
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.
End  (5841)
0 sites
Start  (1)
0 sites
EcoRV  (18)
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.
BstXI  (28)
2 sites
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.
NotI  (33)
1 site
G C G G C C G C C G C C G G C G
PspXI  (39)
1 site
V C T C G A G B B G A G C T C V
XbaI  (45)
1 site
T C T A G A A G A T C T
BstBI  (62)
1 site
T T C G A A A A G C T T
PmeI  (142)
1 site
G T T T A A A C C A A A T T T G
SexAI  (958)
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.
EF-1α promoter
4556 .. 5734  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
EF-1α promoter
4556 .. 5734  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
AmpR
3207 .. 4067  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3207 .. 3998  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3207 .. 4067  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3999 .. 4067  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3207 .. 4067  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2448 .. 3036  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2448 .. 3036  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
434 .. 862  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
434 .. 862  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
BSD
1320 .. 1718  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
1320 .. 1718  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
SV40 promoter
876 .. 1206  =  331 bp
SV40 enhancer and early promoter
SV40 promoter
876 .. 1206  =  331 bp
SV40 enhancer and early promoter
bGH poly(A) signal
164 .. 388  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
164 .. 388  =  225 bp
bovine growth hormone polyadenylation signal
SV40 poly(A) signal
1876 .. 1997  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1876 .. 1997  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4068 .. 4172  =  105 bp
AmpR promoter
4068 .. 4172  =  105 bp
EM7 promoter
1254 .. 1301  =  48 bp
synthetic bacterial promoter
EM7 promoter
1254 .. 1301  =  48 bp
synthetic bacterial promoter
V5 tag
67 .. 108  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
67 .. 108  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
T7 promoter
5751 .. 5769  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5751 .. 5769  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
118 .. 135  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
118 .. 135  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
EF-1α intron A
4787 .. 5725  =  939 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
4787 .. 5725  =  939 bp
intron upstream of the start codon of human EF-1α
SV40 ori
1057 .. 1192  =  136 bp
SV40 origin of replication
SV40 ori
1057 .. 1192  =  136 bp
SV40 origin of replication
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