pCMV-(DYKDDDDK)-C

Vector for expressing a C-terminally FLAG-tagged protein in mammalian cells.

Sequence Author: Clontech (TaKaRa)

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PluTI (3610) SfoI (3608) NarI (3607) KasI (3606) BstAPI (3556) SspI (3188) XmnI (2983) ScaI (2864) NmeAIII (2532) BsaI (2445) AhdI (2384) AlwNI (1907) BfuAI - BspMI (12) SbfI (23) BtgZI (300) BsaAI - SnaBI (306) NcoI - StyI (326) Eco53kI (532) SacI (534) BbsI (600) BspEI (617) PpuMI (702) BseRI (730) StuI (769) PspOMI * (820) ApaI * (824) EcoRI (832) SalI (839) AccI (840) BglII (847) AvaI - BsoBI - PaeR7I - XhoI (853) Acc65I (858) KpnI (862) FLAG EagI - NotI (895) PsiI (1019) MfeI (1048) NheI (1130) BmtI (1134) M13 rev lac operator lac promoter BspQI - SapI (1375) AflIII - PciI (1491) BseYI (1795) PspFI (1799) pCMV-(DYKDDDDK)-C 3766 bp
PluTI  (3610)
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  (3608)
1 site
G G C G C C C C G C G G
NarI  (3607)
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  (3606)
1 site
G G C G C C C C G C G G
BstAPI  (3556)
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  (3188)
1 site
A A T A T T T T A T A A
XmnI  (2983)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (2864)
1 site
A G T A C T T C A T G A
NmeAIII  (2532)
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).
BsaI  (2445)
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  (2384)
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  (1907)
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
NcoI  (326)
1 site
C C A T G G G G T A C C
StyI  (326)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
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
* Blocked by Dcm methylation.
ApaI  (824)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
ApaI can be used between 25°C and 37°C.
EcoRI  (832)
1 site
G A A T T C C T T A A G
SalI  (839)
1 site
G T C G A C C A G C T G
AccI  (840)
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  (847)
1 site
A G A T C T T C T A G A
AvaI  (853)
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  (853)
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  (853)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (853)
1 site
C T C G A G G A G C T C
Acc65I  (858)
1 site
G G T A C C C C A T G G
KpnI  (862)
1 site
G G T A C C C C A T G G
EagI  (895)
1 site
C G G C C G G C C G G C
NotI  (895)
1 site
G C G G C C G C C G C C G G C G
PsiI  (1019)
1 site
T T A T A A A A T A T T
MfeI  (1048)
1 site
C A A T T G G T T A A C
NheI  (1130)
1 site
G C T A G C C G A T C G
BmtI  (1134)
1 site
G C T A G C C G A T C G
BspQI  (1375)
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  (1375)
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  (1491)
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  (1491)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseYI  (1795)
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  (1799)
1 site
C C C A G C G G G T C G
AmpR
2311 .. 3171  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   2311 .. 3102  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2311 .. 3171  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3103 .. 3171  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2311 .. 3171  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1552 .. 2140  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1552 .. 2140  =  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
905 .. 1039  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
905 .. 1039  =  135 bp
SV40 polyadenylation signal
AmpR promoter
3172 .. 3276  =  105 bp
AmpR promoter
3172 .. 3276  =  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
820 .. 863  =  44 bp
multiple cloning site
MCS
820 .. 863  =  44 bp
multiple cloning site
lac promoter
1198 .. 1228  =  31 bp
3 segments
   Segment 3:  -10  
   1198 .. 1204  =  7 bp
promoter for the E. coli lac operon
lac promoter
1198 .. 1228  =  31 bp
3 segments
   Segment 2:  
   1205 .. 1222  =  18 bp
promoter for the E. coli lac operon
lac promoter
1198 .. 1228  =  31 bp
3 segments
   Segment 1:  -35  
   1223 .. 1228  =  6 bp
promoter for the E. coli lac operon
lac promoter
1198 .. 1228  =  31 bp
3 segments
promoter for the E. coli lac operon
FLAG
867 .. 890  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an enterokinase cleavage site
FLAG
867 .. 890  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an enterokinase cleavage site
M13 rev
1150 .. 1166  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
1150 .. 1166  =  17 bp
common sequencing primer, one of multiple similar variants
lac operator
1174 .. 1190  =  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
1174 .. 1190  =  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).
ORF:  2441 .. 2707  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3557 .. 36  =  246 bp
ORF:  81 amino acids  =  9.5 kDa
ORF:  784 .. 1056  =  273 bp
ORF:  90 amino acids  =  10.2 kDa
ORF:  2311 .. 3171  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3517 .. 113  =  363 bp
ORF:  120 amino acids  =  13.4 kDa
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