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pCMV-(DYKDDDDK)-N

Vector for expressing an N-terminally FLAG-tagged protein in mammalian cells.

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pCMV-(DYKDDDDK)-N.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Clontech (TaKaRa)
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PluTI (3623) SfoI (3621) NarI (3620) KasI (3619) BstAPI (3569) SspI (3201) XmnI (2996) ScaI (2877) NmeAIII (2545) BsrFI (2477) BsaI (2458) AhdI (2397) AlwNI (1920) BfuAI - BspMI (12) SbfI (23) BtgZI (300) BsaAI - SnaBI (306) Eco53kI (532) SacI (534) BbsI (600) BspEI (617) PpuMI (702) BseRI (730) StuI (769) PspOMI (820) ApaI (824) HindIII (853) MscI (862) SfiI (868) EcoRI (875) SalI (882) AccI (883) BglII (890) AvaI - BsoBI - PaeR7I - XhoI (896) BmeT110I (897) Acc65I (901) KpnI (905) EagI - NotI (908) PsiI (1032) MfeI (1061) NheI (1143) BmtI (1147) BspQI - SapI (1388) AflIII - PciI (1504) BseYI (1808) PspFI (1812) pCMV-(DYKDDDDK)-N 3779 bp
PluTI  (3623)
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  (3621)
1 site
G G C G C C C C G C G G
NarI  (3620)
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  (3619)
1 site
G G C G C C C C G C G G
BstAPI  (3569)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
SspI  (3201)
1 site
A A T A T T T T A T A A
XmnI  (2996)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (2877)
1 site
A G T A C T T C A T G A
NmeAIII  (2545)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BsrFI  (2477)
1 site
R C C G G Y Y G G C C R

Efficient cleavage requires at least two copies of the BsrFI recognition sequence.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
BsaI  (2458)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (2397)
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.
AlwNI  (1920)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
BfuAI  (12)
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  (12)
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.
SbfI  (23)
1 site
C C T G C A G G G G A C G T C C
BtgZI  (300)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
BsaAI  (306)
1 site
Y A C G T R R T G C A Y
SnaBI  (306)
1 site
T A C G T A A T G C A T
Eco53kI  (532)
1 site
G A G C T C C T C G A G
SacI  (534)
1 site
G A G C T C C T C G A G
BbsI  (600)
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  (617)
1 site
T C C G G A A G G C C T
PpuMI  (702)
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.
BseRI  (730)
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  (769)
1 site
A G G C C T T C C G G A
PspOMI  (820)
1 site
G G G C C C C C C G G G
ApaI  (824)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
HindIII  (853)
1 site
A A G C T T T T C G A A
MscI  (862)
1 site
T G G C C A A C C G G T
SfiI  (868)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
EcoRI  (875)
1 site
G A A T T C C T T A A G
SalI  (882)
1 site
G T C G A C C A G C T G
AccI  (883)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
BglII  (890)
1 site
A G A T C T T C T A G A
AvaI  (896)
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  (896)
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.
PaeR7I  (896)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (896)
1 site
C T C G A G G A G C T C
BmeT110I  (897)
1 site
C Y C G R G G R G C Y C
Acc65I  (901)
1 site
G G T A C C C C A T G G
KpnI  (905)
1 site
G G T A C C C C A T G G
EagI  (908)
1 site
C G G C C G G C C G G C
NotI  (908)
1 site
G C G G C C G C C G C C G G C G
PsiI  (1032)
1 site
T T A T A A A A T A T T
MfeI  (1061)
1 site
C A A T T G G T T A A C
NheI  (1143)
1 site
G C T A G C C G A T C G
BmtI  (1147)
1 site
G C T A G C C G A T C G
BspQI  (1388)
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  (1388)
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.
AflIII  (1504)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (1504)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseYI  (1808)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
PspFI  (1812)
1 site
C C C A G C G G G T C G
AmpR
2324 .. 3184  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2324 .. 3115  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2324 .. 3184  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3116 .. 3184  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2324 .. 3184  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1565 .. 2153  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1565 .. 2153  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CMV enhancer
27 .. 330  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
27 .. 330  =  304 bp
human cytomegalovirus immediate early enhancer
CMV promoter
331 .. 534  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
331 .. 534  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
918 .. 1052  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
918 .. 1052  =  135 bp
SV40 polyadenylation signal
AmpR promoter
3185 .. 3289  =  105 bp
AmpR promoter
3185 .. 3289  =  105 bp
SV40 intron
672 .. 768  =  97 bp
modified SV40 intron with splice donor and acceptor sites
SV40 intron
672 .. 768  =  97 bp
modified SV40 intron with splice donor and acceptor sites
MCS
860 .. 914  =  55 bp
multiple cloning site
MCS
860 .. 914  =  55 bp
multiple cloning site
lac promoter
1211 .. 1241  =  31 bp
   Segment 3:  -10  
   1211 .. 1217  =  7 bp
promoter for the E. coli lac operon
lac promoter
1211 .. 1241  =  31 bp
   Segment 2:  
   1218 .. 1235  =  18 bp
promoter for the E. coli lac operon
lac promoter
1211 .. 1241  =  31 bp
   Segment 1:  -35  
   1236 .. 1241  =  6 bp
promoter for the E. coli lac operon
lac promoter
1211 .. 1241  =  31 bp
3 segments
promoter for the E. coli lac operon
FLAG
832 .. 855  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an enterokinase cleavage site
FLAG
832 .. 855  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an enterokinase cleavage site
M13 rev
1163 .. 1179  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
1163 .. 1179  =  17 bp
common sequencing primer, one of multiple similar variants
lac operator
1187 .. 1203  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
1187 .. 1203  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
ATG
829 .. 831  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
829 .. 831  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  2454 .. 2720  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3570 .. 36  =  246 bp
ORF:  81 amino acids  =  9.5 kDa
ORF:  3530 .. 113  =  363 bp
ORF:  120 amino acids  =  13.4 kDa
ORF:  2324 .. 3184  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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