pcDNA3.1 V5-His A

Mammalian vector for expressing C-terminally V5- and 6xHis-tagged proteins. For other reading frames, use pcDNA™3.1/V5-His B or pcDNA™3.1/V5-His C.

Sequence Author: Thermo Fisher (Invitrogen)

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SgrDI (5500) SspI (5383) ScaI (5059) PvuI (4949) AhdI (4579) PciI (3686) BstZ17I (3307) BsmI (3255) PfoI (3110) RsrII (2849) BglII (12) MfeI (161) Bpu10I (180) NdeI (484) SnaBI (590) HindIII (902) Acc65I (908) KpnI (912) BamHI (920) EcoRI (943) EcoRV (955) NotI (970) PaeR7I - PspXI - XhoI (976) XbaI (982) PspOMI (988) ApaI (992) BstBI (995) AgeI (1045) PmeI (1075) BbsI (1286) DraIII (1600) CsiI - SexAI * (1890) StuI (2122) AvrII (2123) TspMI - XmaI (2144) SmaI (2146) BsaBI * (2192) KasI (2332) NarI (2333) SfoI (2334) PluTI (2336) MscI (2415) PflFI - Tth111I (2451) BssHII (2730) pcDNA™3.1/V5-His A 5502 bp
SgrDI  (5500)
1 site
C G T C G A C G G C A G C T G C
SspI  (5383)
1 site
A A T A T T T T A T A A
ScaI  (5059)
1 site
A G T A C T T C A T G A
PvuI  (4949)
1 site
C G A T C G G C T A G C
AhdI  (4579)
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.
PciI  (3686)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstZ17I  (3307)
1 site
G T A T A C C A T A T G
BsmI  (3255)
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  (3110)
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.
RsrII  (2849)
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.
BglII  (12)
1 site
A G A T C T T C T A G A
MfeI  (161)
1 site
C A A T T G G T T A A C
Bpu10I  (180)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
NdeI  (484)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
SnaBI  (590)
1 site
T A C G T A A T G C A T
HindIII  (902)
1 site
A A G C T T T T C G A A
Acc65I  (908)
1 site
G G T A C C C C A T G G
KpnI  (912)
1 site
G G T A C C C C A T G G
BamHI  (920)
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.
EcoRI  (943)
1 site
G A A T T C C T T A A G
EcoRV  (955)
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  (970)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (976)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (976)
1 site
V C T C G A G B B G A G C T C V
XhoI  (976)
1 site
C T C G A G G A G C T C
XbaI  (982)
1 site
T C T A G A A G A T C T
PspOMI  (988)
1 site
G G G C C C C C C G G G
ApaI  (992)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BstBI  (995)
1 site
T T C G A A A A G C T T
AgeI  (1045)
1 site
A C C G G T T G G C C A
PmeI  (1075)
1 site
G T T T A A A C C A A A T T T G
BbsI  (1286)
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.
DraIII  (1600)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
CsiI  (1890)
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  (1890)
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.
StuI  (2122)
1 site
A G G C C T T C C G G A
AvrII  (2123)
1 site
C C T A G G G G A T C C
TspMI  (2144)
1 site
C C C G G G G G G C C C
XmaI  (2144)
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  (2146)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BsaBI  (2192)
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.
KasI  (2332)
1 site
G G C G C C C C G C G G
NarI  (2333)
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  (2334)
1 site
G G C G C C C C G C G G
PluTI  (2336)
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.
MscI  (2415)
1 site
T G G C C A A C C G G T
PflFI  (2451)
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  (2451)
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.
BssHII  (2730)
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.
AmpR
4506 .. 5366  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   4506 .. 5297  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4506 .. 5366  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   5298 .. 5366  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4506 .. 5366  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
2205 .. 2999  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
NeoR/KanR
2205 .. 2999  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
ori
3747 .. 4335  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3747 .. 4335  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
1367 .. 1795  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
1367 .. 1795  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
1809 .. 2138  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
1809 .. 2138  =  330 bp
SV40 enhancer and early promoter
bGH poly(A) signal
1097 .. 1321  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
1097 .. 1321  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
3175 .. 3296  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3175 .. 3296  =  122 bp
SV40 polyadenylation signal
AmpR promoter
5367 .. 5471  =  105 bp
AmpR promoter
5367 .. 5471  =  105 bp
MCS
902 .. 999  =  98 bp
multiple cloning site
MCS
902 .. 999  =  98 bp
multiple cloning site
V5 tag
1000 .. 1041  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
1000 .. 1041  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
6xHis
1051 .. 1068  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag