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Plasmid Files

pcDNA™3.3-TOPO® (linearized)

Linearized mammalian vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products and expression with the CMV promoter.

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pcDNA3.3-TOPO (linearized) Sequence and MappcDNA3.3-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 Bpu10I (4661) MfeI (4642) SgrDI (4479) SspI (4362) ScaI (4038) PvuI (3928) AhdI (3558) PciI (2665) BstZ17I (2286) BsmI (2234) PfoI (2089) RsrII (1828) MluI (4709) SpeI (4730) NdeI (4965) SnaBI (5071) Eco53kI (5297) SacI (5299) BsmBI (5320) BbsI (5365) BspEI (5382) XbaI (5389) End (5407) Start (1) AgeI (16) PmeI (34) EcoO109I (297) DraIII (579) SexAI * (869) BseRI (1098) StuI (1101) AvrII (1102) AvaI - BsoBI - TspMI - XmaI (1123) BmeT110I (1124) SmaI (1125) BclI * (1153) BsaBI * (1171) EagI (1218) KasI (1311) NarI (1312) SfoI (1313) PluTI (1315) PstI (1365) MscI (1394) PflFI - Tth111I (1430) BssHII (1709) pcDNA™3.3-TOPO® 5406 bp
Bpu10I  (4661)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
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.
MfeI  (4642)
1 site
C A A T T G G T T A A C
SgrDI  (4479)
1 site
C G T C G A C G G C A G C T G C
SspI  (4362)
1 site
A A T A T T T T A T A A
ScaI  (4038)
1 site
A G T A C T T C A T G A
PvuI  (3928)
1 site
C G A T C G G C T A G C
AhdI  (3558)
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  (2665)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstZ17I  (2286)
1 site
G T A T A C C A T A T G
BsmI  (2234)
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  (2089)
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  (1828)
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.
MluI  (4709)
1 site
A C G C G T T G C G C A
SpeI  (4730)
1 site
A C T A G T T G A T C A
NdeI  (4965)
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  (5071)
1 site
T A C G T A A T G C A T
Eco53kI  (5297)
1 site
G A G C T C C T C G A G
SacI  (5299)
1 site
G A G C T C C T C G A G
BsmBI  (5320)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BbsI  (5365)
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.
BspEI  (5382)
1 site
T C C G G A A G G C C T
XbaI  (5389)
1 site
T C T A G A A G A T C T
End  (5407)
0 sites
Start  (1)
0 sites
AgeI  (16)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
PmeI  (34)
1 site
G T T T A A A C C A A A T T T G
EcoO109I  (297)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
DraIII  (579)
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.
SexAI  (869)
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  (1098)
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  (1101)
1 site
A G G C C T T C C G G A
AvrII  (1102)
1 site
C C T A G G G G A T C C
AvaI  (1123)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (1123)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
TspMI  (1123)
1 site
C C C G G G G G G C C C
XmaI  (1123)
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.
BmeT110I  (1124)
1 site
C Y C G R G G R G C Y C
SmaI  (1125)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BclI  (1153)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BsaBI  (1171)
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.
EagI  (1218)
1 site
C G G C C G G C C G G C
KasI  (1311)
1 site
G G C G C C C C G C G G
NarI  (1312)
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  (1313)
1 site
G G C G C C C C G C G G
PluTI  (1315)
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.
PstI  (1365)
1 site
C T G C A G G A C G T C
MscI  (1394)
1 site
T G G C C A A C C G G T
PflFI  (1430)
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  (1430)
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  (1709)
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
3485 .. 4345  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3485 .. 4276  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3485 .. 4345  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4277 .. 4345  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3485 .. 4345  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
1184 .. 1978  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
1184 .. 1978  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
2726 .. 3314  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2726 .. 3314  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
346 .. 774  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
346 .. 774  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
4716 .. 5095  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4716 .. 5095  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
788 .. 1117  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
788 .. 1117  =  330 bp
SV40 enhancer and early promoter
CMV promoter
5096 .. 5299  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
5096 .. 5299  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
2154 .. 2275  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2154 .. 2275  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4346 .. 4450  =  105 bp
AmpR promoter
4346 .. 4450  =  105 bp
HSV TK poly(A) signal
96 .. 144  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
96 .. 144  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
SV40 ori
968 .. 1103  =  136 bp
SV40 origin of replication
SV40 ori
968 .. 1103  =  136 bp
SV40 origin of replication
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