pcDNA3

Mammalian expression vector with the CMV promoter and a neomycin-resistance marker.

Sequence Author: Thermo Fisher (Invitrogen)

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SgrDI (5444) SspI (5327) ScaI (5003) PvuI (4893) AhdI (4523) PciI (3630) BstZ17I (3251) BsmI (3199) PfoI (3054) BstBI (2961) RsrII (2795) BglII (12) MfeI (161) Bpu10I (180) NruI (208) MluI (228) NdeI (484) SnaBI (590) HindIII (889) Acc65I (895) KpnI (899) BamHI (907) EcoRI (938) EcoRV (950) NotI (965) PaeR7I - PspXI - XhoI (971) XbaI (983) PspOMI (989) ApaI (993) BbsI (1231) DraIII (1545) CsiI - SexAI * (1836) BseRI (2065) StuI (2068) AvrII (2069) TspMI - XmaI (2090) SmaI (2092) KasI (2278) NarI (2279) SfoI (2280) PluTI (2282) MscI (2361) PflFI - Tth111I (2397) BssHII (2676) pcDNA3 5446 bp
SgrDI  (5444)
1 site
C G T C G A C G G C A G C T G C
SspI  (5327)
1 site
A A T A T T T T A T A A
ScaI  (5003)
1 site
A G T A C T T C A T G A
PvuI  (4893)
1 site
C G A T C G G C T A G C
AhdI  (4523)
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  (3630)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstZ17I  (3251)
1 site
G T A T A C C A T A T G
BsmI  (3199)
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  (3054)
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.
BstBI  (2961)
1 site
T T C G A A A A G C T T
RsrII  (2795)
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.
NruI  (208)
1 site
T C G C G A A G C G C T
MluI  (228)
1 site
A C G C G T T G C G C A
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  (889)
1 site
A A G C T T T T C G A A
Acc65I  (895)
1 site
G G T A C C C C A T G G
KpnI  (899)
1 site
G G T A C C C C A T G G
BamHI  (907)
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  (938)
1 site
G A A T T C C T T A A G
EcoRV  (950)
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  (965)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (971)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (971)
1 site
V C T C G A G B B G A G C T C V
XhoI  (971)
1 site
C T C G A G G A G C T C
XbaI  (983)
1 site
T C T A G A A G A T C T
PspOMI  (989)
1 site
G G G C C C C C C G G G
ApaI  (993)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BbsI  (1231)
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  (1545)
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  (1836)
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  (1836)
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.
BseRI  (2065)
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.
StuI  (2068)
1 site
A G G C C T T C C G G A
AvrII  (2069)
1 site
C C T A G G G G A T C C
TspMI  (2090)
1 site
C C C G G G G G G C C C
XmaI  (2090)
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  (2092)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
KasI  (2278)
1 site
G G C G C C C C G C G G
NarI  (2279)
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  (2280)
1 site
G G C G C C C C G C G G
PluTI  (2282)
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  (2361)
1 site
T G G C C A A C C G G T
PflFI  (2397)
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  (2397)
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  (2676)
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
4450 .. 5310  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   4450 .. 5241  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4450 .. 5310  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   5242 .. 5310  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4450 .. 5310  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
2151 .. 2945  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)