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Plasmid Files

pcDNA™3.2/capTEV-CT/V5-DEST

Gateway® destination vector for mammalian cell expression of proteins C-terminally modified with V5 and 6xHis tags plus a cleavable biotinylation peptide.

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pcDNA3.2 capTEV-CT V5-DEST Sequence and MappcDNA3.2 capTEV-CT V5-DEST.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 SgrDI (7972) PvuI (7421) PciI (6158) PfoI (5582) RsrII (5321) PflFI - Tth111I (4923) EagI (4711) BclI * (4646) AvrII (4595) StuI (4594) SexAI * (4362) DraIII (4072) SpeI (213) CMV enhancer NdeI (448) SnaBI (554) Eco53kI (780) SacI (782) BmgBI (1615) SrfI (1649) BbvCI (1792) EcoRI (2518) BspEI (2522) XbaI * (3145) PspOMI (3151) ApaI (3155) SacII (3158) BstBI (3162) AleI (3299) BstEII (3301) PmeI (3527) pcDNA™3.2/capTEV-CT/V5-DEST 8010 bp
SgrDI  (7972)
1 site
C G T C G A C G G C A G C T G C
PvuI  (7421)
1 site
C G A T C G G C T A G C
PciI  (6158)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PfoI  (5582)
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  (5321)
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  (4923)
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  (4923)
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.
EagI  (4711)
1 site
C G G C C G G C C G G C
BclI  (4646)
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  (4595)
1 site
C C T A G G G G A T C C
StuI  (4594)
1 site
A G G C C T T C C G G A
SexAI  (4362)
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.
DraIII  (4072)
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.
SpeI  (213)
1 site
A C T A G T T G A T C A
NdeI  (448)
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  (554)
1 site
T A C G T A A T G C A T
Eco53kI  (780)
1 site
G A G C T C C T C G A G
SacI  (782)
1 site
G A G C T C C T C G A G
BmgBI  (1615)
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  (1649)
1 site
G C C C G G G C C G G G C C C G
BbvCI  (1792)
1 site
C C T C A G C G G A G T C G
EcoRI  (2518)
1 site
G A A T T C C T T A A G
BspEI  (2522)
1 site
T C C G G A A G G C C T
XbaI  (3145)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
PspOMI  (3151)
1 site
G G G C C C C C C G G G
ApaI  (3155)
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  (3158)
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  (3162)
1 site
T T C G A A A A G C T T
AleI  (3299)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (3301)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
PmeI  (3527)
1 site
G T T T A A A C C A A A T T T G
AmpR
6978 .. 7838  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6978 .. 7769  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6978 .. 7838  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7770 .. 7838  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6978 .. 7838  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
4677 .. 5471  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
4677 .. 5471  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
CmR
2083 .. 2736  =  654 bp
218 amino acids  =  25.6 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2083 .. 2736  =  654 bp
218 amino acids  =  25.6 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
6219 .. 6807  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6219 .. 6807  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
3839 .. 4267  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3839 .. 4267  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
199 .. 578  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
199 .. 578  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
4281 .. 4610  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
4281 .. 4610  =  330 bp
SV40 enhancer and early promoter
ccdB
1428 .. 1733  =  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
1428 .. 1733  =  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.
capTEV™
3218 .. 3502  =  285 bp
95 amino acids  =  10.0 kDa
   Segment 1:  6xHis  
   3218 .. 3235  =  18 bp
   6 amino acids  =  840.9 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
3218 .. 3502  =  285 bp
95 amino acids  =  10.0 kDa
   Segment 2:  
   3236 .. 3238  =  3 bp
   1 amino acid  =  75.1 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
3218 .. 3502  =  285 bp
95 amino acids  =  10.0 kDa
   Segment 3:  TEV site  
   3239 .. 3259  =  21 bp
   7 amino acids  =  870.0 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
3218 .. 3502  =  285 bp
95 amino acids  =  10.0 kDa
   Segment 4:  
   3260 .. 3265  =  6 bp
   2 amino acids  =  259.3 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
3218 .. 3502  =  285 bp
95 amino acids  =  10.0 kDa
   Segment 5:  TEV site  
   3266 .. 3286  =  21 bp
   7 amino acids  =  870.0 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
3218 .. 3502  =  285 bp
95 amino acids  =  10.0 kDa
   Segment 6:  BioEase™ tag  
   3287 .. 3502  =  216 bp
   72 amino acids  =  7.1 kDa
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
3218 .. 3502  =  285 bp
95 amino acids  =  10.0 kDa
6 segments
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
CMV promoter
579 .. 782  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
579 .. 782  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
attR1
875 .. 999  =  125 bp
recombination site for the Gateway® LR reaction
attR1
875 .. 999  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3017 .. 3141  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3017 .. 3141  =  125 bp
recombination site for the Gateway® LR reaction
SV40 poly(A) signal
5647 .. 5768  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5647 .. 5768  =  122 bp
SV40 polyadenylation signal
AmpR promoter
7839 .. 7943  =  105 bp
AmpR promoter
7839 .. 7943  =  105 bp
cat promoter
2737 .. 2839  =  103 bp
promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase
cat promoter
2737 .. 2839  =  103 bp
promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase
HSV TK poly(A) signal
3589 .. 3637  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
3589 .. 3637  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
V5 tag
3167 .. 3208  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
3167 .. 3208  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
T7 promoter
827 .. 845  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
827 .. 845  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SV40 ori
4461 .. 4596  =  136 bp
SV40 origin of replication
SV40 ori
4461 .. 4596  =  136 bp
SV40 origin of replication
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