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Plasmid Files

pcDNA™6.2/nGeneBLAzer™-DEST

Gateway® destination vector for in vivo or in vitro detection of mammalian gene expression using an N-terminal β-lactamase reporter.

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pcDNA6.2 nGeneBLAzer-DEST Sequence and MappcDNA6.2 nGeneBLAzer-DEST.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 SgrDI (7562) PciI (5751) BspQI - SapI (5635) PfoI (5175) BsgI (4871) PpuMI (4843) BbsI (4769) BclI * (4606) AvrII (4555) StuI (4554) SexAI * (4322) MfeI (161) SpeI (249) CMV enhancer NdeI (484) SnaBI (590) Eco53kI (816) SacI (818) NotI (1838) BspEI (2154) EcoRI (2158) BsmBI (2382) PflMI * (2390) BssHII (2652) BbvCI (2883) SrfI (3029) BmgBI (3063) BstXI (3146) BfuAI - BspMI (3273) PstI (3284) AgeI (3469) PmeI (3487) pcDNA™6.2/nGeneBLAzer™-DEST 7564 bp
SgrDI  (7562)
1 site
C G T C G A C G G C A G C T G C
PciI  (5751)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5635)
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  (5635)
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.
PfoI  (5175)
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.
BsgI  (4871)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
PpuMI  (4843)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
BbsI  (4769)
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.
BclI  (4606)
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  (4555)
1 site
C C T A G G G G A T C C
StuI  (4554)
1 site
A G G C C T T C C G G A
SexAI  (4322)
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
NotI  (1838)
1 site
G C G G C C G C C G C C G G C G
BspEI  (2154)
1 site
T C C G G A A G G C C T
EcoRI  (2158)
1 site
G A A T T C C T T A A G
BsmBI  (2382)
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.
PflMI  (2390)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
BssHII  (2652)
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.
BbvCI  (2883)
1 site
C C T C A G C G G A G T C G
SrfI  (3029)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (3063)
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.
BstXI  (3146)
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.
BfuAI  (3273)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (3273)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
PstI  (3284)
1 site
C T G C A G G A C G T C
AgeI  (3469)
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  (3487)
1 site
G T T T A A A C C A A A T T T G
AmpR
6568 .. 7428  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6568 .. 7359  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6568 .. 7428  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7360 .. 7428  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6568 .. 7428  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
bla(M)
911 .. 1702  =  792 bp
264 amino acids  =  29.1 kDa
Product: β-lactamase lacking the signal sequence
allows cytosolic expression of β-lactamase
bla(M)
911 .. 1702  =  792 bp
264 amino acids  =  29.1 kDa
Product: β-lactamase lacking the signal sequence
allows cytosolic expression of β-lactamase
CmR
1945 .. 2604  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1945 .. 2604  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
5812 .. 6397  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5812 .. 6397  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
3799 .. 4227  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3799 .. 4227  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
BSD
4684 .. 5082  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
4684 .. 5082  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
4241 .. 4570  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
4241 .. 4570  =  330 bp
SV40 enhancer and early promoter
ccdB
2946 .. 3251  =  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
2946 .. 3251  =  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
1712 .. 1836  =  125 bp
recombination site for the Gateway® LR reaction
attR1
1712 .. 1836  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3292 .. 3416  =  125 bp
recombination site for the Gateway® LR reaction
attR2
3292 .. 3416  =  125 bp
recombination site for the Gateway® LR reaction
SV40 poly(A) signal
5240 .. 5361  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5240 .. 5361  =  122 bp
SV40 polyadenylation signal
AmpR promoter
7429 .. 7533  =  105 bp
AmpR promoter
7429 .. 7533  =  105 bp
HSV TK poly(A) signal
3549 .. 3597  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
3549 .. 3597  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
EM7 promoter
4618 .. 4665  =  48 bp
synthetic bacterial promoter
EM7 promoter
4618 .. 4665  =  48 bp
synthetic bacterial promoter
V5 tag
3424 .. 3465  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
3424 .. 3465  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
lac UV5 promoter
1861 .. 1891  =  31 bp
   Segment 1:  -35  
   1861 .. 1866  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1861 .. 1891  =  31 bp
   Segment 2:  
   1867 .. 1884  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1861 .. 1891  =  31 bp
   Segment 3:  -10  
   1885 .. 1891  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1861 .. 1891  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
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
4421 .. 4556  =  136 bp
SV40 origin of replication
SV40 ori
4421 .. 4556  =  136 bp
SV40 origin of replication
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