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Plasmid Files

pcDNA™3.2/capTEV-NT/V5-DEST

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

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pcDNA3.2 capTEV-NT V5-DEST Sequence and MappcDNA3.2 capTEV-NT V5-DEST.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 SgrDI (7919) PvuI (7368) PciI (6105) PfoI (5529) NruI (5436) RsrII (5268) PflFI - Tth111I (4870) EagI (4658) BclI * (4593) AvrII (4542) StuI (4541) SexAI * (4309) DraIII (4019) MluI (215) SpeI (236) CMV enhancer NdeI (471) SnaBI (577) Eco53kI (803) SacI (805) AleI (875) BstEII (877) BmgBI (1945) SrfI (1979) BbvCI (2122) EcoRI (2848) BspEI (2852) EcoO109I (3737) pcDNA™3.2/capTEV-NT/V5-DEST 7921 bp
SgrDI  (7919)
1 site
C G T C G A C G G C A G C T G C
PvuI  (7368)
1 site
C G A T C G G C T A G C
PciI  (6105)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PfoI  (5529)
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.
NruI  (5436)
1 site
T C G C G A A G C G C T
RsrII  (5268)
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  (4870)
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  (4870)
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  (4658)
1 site
C G G C C G G C C G G C
BclI  (4593)
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  (4542)
1 site
C C T A G G G G A T C C
StuI  (4541)
1 site
A G G C C T T C C G G A
SexAI  (4309)
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  (4019)
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.
MluI  (215)
1 site
A C G C G T T G C G C A
SpeI  (236)
1 site
A C T A G T T G A T C A
NdeI  (471)
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  (577)
1 site
T A C G T A A T G C A T
Eco53kI  (803)
1 site
G A G C T C C T C G A G
SacI  (805)
1 site
G A G C T C C T C G A G
AleI  (875)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (877)
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.
BmgBI  (1945)
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  (1979)
1 site
G C C C G G G C C G G G C C C G
BbvCI  (2122)
1 site
C C T C A G C G G A G T C G
EcoRI  (2848)
1 site
G A A T T C C T T A A G
BspEI  (2852)
1 site
T C C G G A A G G C C T
EcoO109I  (3737)
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.
AmpR
6925 .. 7785  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6925 .. 7716  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6925 .. 7785  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7717 .. 7785  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6925 .. 7785  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
4624 .. 5418  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
4624 .. 5418  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
CmR
2413 .. 3066  =  654 bp
218 amino acids  =  25.6 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2413 .. 3066  =  654 bp
218 amino acids  =  25.6 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
6166 .. 6754  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6166 .. 6754  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
3786 .. 4214  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3786 .. 4214  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
222 .. 601  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
222 .. 601  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
4228 .. 4557  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
4228 .. 4557  =  330 bp
SV40 enhancer and early promoter
ccdB
1758 .. 2063  =  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
1758 .. 2063  =  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™
860 .. 1162  =  303 bp
101 amino acids  =  10.6 kDa
   Segment 1:  BioEase™ tag  
   860 .. 1078  =  219 bp
   73 amino acids  =  7.3 kDa
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
860 .. 1162  =  303 bp
101 amino acids  =  10.6 kDa
   Segment 2:  
   1079 .. 1090  =  12 bp
   4 amino acids  =  306.3 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
860 .. 1162  =  303 bp
101 amino acids  =  10.6 kDa
   Segment 3:  TEV site  
   1091 .. 1111  =  21 bp
   7 amino acids  =  870.0 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
860 .. 1162  =  303 bp
101 amino acids  =  10.6 kDa
   Segment 4:  
   1112 .. 1117  =  6 bp
   2 amino acids  =  259.3 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
860 .. 1162  =  303 bp
101 amino acids  =  10.6 kDa
   Segment 5:  TEV site  
   1118 .. 1138  =  21 bp
   7 amino acids  =  870.0 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
860 .. 1162  =  303 bp
101 amino acids  =  10.6 kDa
   Segment 6:  
   1139 .. 1144  =  6 bp
   2 amino acids  =  259.3 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
860 .. 1162  =  303 bp
101 amino acids  =  10.6 kDa
   Segment 7:  6xHis  
   1145 .. 1162  =  18 bp
   6 amino acids  =  840.9 Da
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
capTEV™
860 .. 1162  =  303 bp
101 amino acids  =  10.6 kDa
7 segments
Product: composite tag for in vivo biotinylation, TEV
protease cleavage, and 6xHis tagging
CMV promoter
602 .. 805  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
602 .. 805  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
attR1
1205 .. 1329  =  125 bp
recombination site for the Gateway® LR reaction
attR1
1205 .. 1329  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3347 .. 3471  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3347 .. 3471  =  125 bp
recombination site for the Gateway® LR reaction
SV40 poly(A) signal
5594 .. 5715  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5594 .. 5715  =  122 bp
SV40 polyadenylation signal
AmpR promoter
7786 .. 7890  =  105 bp
AmpR promoter
7786 .. 7890  =  105 bp
cat promoter
3067 .. 3169  =  103 bp
promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase
cat promoter
3067 .. 3169  =  103 bp
promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase
HSV TK poly(A) signal
3536 .. 3584  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
3536 .. 3584  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
V5 tag
1163 .. 1204  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
1163 .. 1204  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
SV40 ori
4408 .. 4543  =  136 bp
SV40 origin of replication
SV40 ori
4408 .. 4543  =  136 bp
SV40 origin of replication
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