pcDNA4 HisMax-TOPO (linearized)

Linearized mammalian vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products and expression of N-terminally 6xHis-tagged proteins.

Sequence Author: Thermo Fisher (Invitrogen)

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SgrDI (4088) SspI (3971) ScaI (3647) PvuI (3537) FspI (3389) AhdI (3167) PciI (2277) BspQI - SapI (2161) BstZ17I (1898) BsmI (1846) FseI (1538) BmgBI (1396) SgrAI (1376) BglII (4102) MfeI (4251) Bpu10I (4270) NruI (4298) MluI (4318) SpeI (4339) CMV enhancer NdeI (4574) SnaBI (4680) Eco53kI (4906) SacI (4908) AflII (4998) HindIII (5001) BlpI (5004) SrfI (5051) ATG 6xHis T7 tag (gene 10 leader) Xpress™ tag End (5274) Start (1) BamHI (16) EcoRI (31) PstI (40) EcoRV (43) NotI (58) PaeR7I - PspXI - XhoI (64) XbaI (70) PspOMI (76) ApaI (80) BbsI (296) StuI (1132) MscI (1265) MauBI (1298) pcDNA™4/HisMax-TOPO® 5273 bp
SgrDI  (4088)
1 site
C G T C G A C G G C A G C T G C
SspI  (3971)
1 site
A A T A T T T T A T A A
ScaI  (3647)
1 site
A G T A C T T C A T G A
PvuI  (3537)
1 site
C G A T C G G C T A G C
FspI  (3389)
1 site
T G C G C A A C G C G T
AhdI  (3167)
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  (2277)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2161)
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  (2161)
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  (1898)
1 site
G T A T A C C A T A T G
BsmI  (1846)
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.
FseI  (1538)
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.
BmgBI  (1396)
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.
SgrAI  (1376)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
BglII  (4102)
1 site
A G A T C T T C T A G A
MfeI  (4251)
1 site
C A A T T G G T T A A C
Bpu10I  (4270)
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  (4298)
1 site
T C G C G A A G C G C T
MluI  (4318)
1 site
A C G C G T T G C G C A
SpeI  (4339)
1 site
A C T A G T T G A T C A
NdeI  (4574)
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  (4680)
1 site
T A C G T A A T G C A T
Eco53kI  (4906)
1 site
G A G C T C C T C G A G
SacI  (4908)
1 site
G A G C T C C T C G A G
AflII  (4998)
1 site
C T T A A G G A A T T C
HindIII  (5001)
1 site
A A G C T T T T C G A A
BlpI  (5004)
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.
SrfI  (5051)
1 site
G C C C G G G C C G G G C C C G
End  (5274)
0 sites
Start  (1)
0 sites
BamHI  (16)
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  (31)
1 site
G A A T T C C T T A A G
PstI  (40)
1 site
C T G C A G G A C G T C
EcoRV  (43)
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  (58)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (64)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (64)
1 site
V C T C G A G B B G A G C T C V
XhoI  (64)
1 site
C T C G A G G A G C T C
XbaI  (70)
1 site
T C T A G A A G A T C T
PspOMI  (76)
1 site
G G G C C C C C C G G G
ApaI  (80)
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  (296)
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.
StuI  (1132)
1 site
A G G C C T T C C G G A
MscI  (1265)
1 site
T G G C C A A C C G G T
MauBI  (1298)
1 site
C G C G C G C G G C G C G C G C
AmpR
3094 .. 3954  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3094 .. 3885  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3094 .. 3954  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3886 .. 3954  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3094 .. 3954  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2338 .. 2923  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2338 .. 2923  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
377 .. 805  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
377 .. 805  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
CMV enhancer
4325 .. 4704  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4325 .. 4704  =  380 bp
human cytomegalovirus immediate early enhancer
BleoR
1262 .. 1636  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
BleoR
1262 .. 1636  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
SV40 promoter
819 .. 1148  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
819 .. 1148  =  330 bp
SV40 enhancer and early promoter