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Plasmid Files

pcDNA™3.2/V5-DEST

Gateway® destination vector for high-level expression of C-terminally V5-tagged proteins in mammalian cells.

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pcDNA3.2 V5-DEST Sequence and MappcDNA3.2 V5-DEST.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 SgrDI (7709) PvuI (7158) PciI (5895) PfoI (5319) RsrII (5058) PflFI - Tth111I (4660) PluTI (4545) SfoI (4543) NarI (4542) KasI (4541) EagI (4448) BclI * (4383) AvrII (4332) StuI (4331) SexAI * (4099) MfeI (161) SpeI (249) CMV enhancer NdeI (484) SnaBI (590) Eco53kI (816) SacI (818) BstXI (1572) BmgBI (1651) SrfI (1685) BbvCI (1828) BamHI (2099) EcoRI (2552) BspEI (2556) XbaI * (3179) PspOMI (3185) ApaI (3189) SacII (3192) BstBI (3196) AgeI (3246) PmeI (3264) DraIII (3809) pcDNA™3.2/V5-DEST 7711 bp
SgrDI  (7709)
1 site
C G T C G A C G G C A G C T G C
PvuI  (7158)
1 site
C G A T C G G C T A G C
PciI  (5895)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PfoI  (5319)
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  (5058)
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.
PflFI  (4660)
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  (4660)
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.
PluTI  (4545)
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.
SfoI  (4543)
1 site
G G C G C C C C G C G G
NarI  (4542)
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.
KasI  (4541)
1 site
G G C G C C C C G C G G
EagI  (4448)
1 site
C G G C C G G C C G G C
BclI  (4383)
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.
AvrII  (4332)
1 site
C C T A G G G G A T C C
StuI  (4331)
1 site
A G G C C T T C C G G A
SexAI  (4099)
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.
MfeI  (161)
1 site
C A A T T G G T T A A C
SpeI  (249)
1 site
A C T A G T T G A T 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
Eco53kI  (816)
1 site
G A G C T C C T C G A G
SacI  (818)
1 site
G A G C T C C T C G A G
BstXI  (1572)
1 site
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.
BmgBI  (1651)
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.
SrfI  (1685)
1 site
G C C C G G G C C G G G C C C G
BbvCI  (1828)
1 site
C C T C A G C G G A G T C G
BamHI  (2099)
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  (2552)
1 site
G A A T T C C T T A A G
BspEI  (2556)
1 site
T C C G G A A G G C C T
XbaI  (3179)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
PspOMI  (3185)
1 site
G G G C C C C C C G G G
ApaI  (3189)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
SacII  (3192)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
BstBI  (3196)
1 site
T T C G A A A A G C T T
AgeI  (3246)
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  (3264)
1 site
G T T T A A A C C A A A T T T G
DraIII  (3809)
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.
AmpR
6715 .. 7575  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6715 .. 7506  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6715 .. 7575  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7507 .. 7575  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6715 .. 7575  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
4414 .. 5208  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
4414 .. 5208  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
CmR
2111 .. 2770  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2111 .. 2770  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5956 .. 6544  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5956 .. 6544  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
3576 .. 4004  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3576 .. 4004  =  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
4018 .. 4347  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
4018 .. 4347  =  330 bp
SV40 enhancer and early promoter
ccdB
1464 .. 1769  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
1464 .. 1769  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
attR1
911 .. 1035  =  125 bp
recombination site for the Gateway® LR reaction
attR1
911 .. 1035  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3051 .. 3175  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3051 .. 3175  =  125 bp
recombination site for the Gateway® LR reaction
SV40 poly(A) signal
5384 .. 5505  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5384 .. 5505  =  122 bp
SV40 polyadenylation signal
AmpR promoter
7576 .. 7680  =  105 bp
AmpR promoter
7576 .. 7680  =  105 bp
cat promoter
2771 .. 2873  =  103 bp
promoter of the E. coli cat gene
cat promoter
2771 .. 2873  =  103 bp
promoter of the E. coli cat gene
HSV TK poly(A) signal
3326 .. 3374  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
3326 .. 3374  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
V5 tag
3201 .. 3242  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
3201 .. 3242  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SV40 ori
4198 .. 4333  =  136 bp
SV40 origin of replication
SV40 ori
4198 .. 4333  =  136 bp
SV40 origin of replication
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