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pFLAG-CMV™-5.1

Vector for expression of C-terminally FLAG-tagged proteins in mammalian cells.

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pFLAG-CMV-5.1.dna
Map and Sequence File:    Download    Open   
Sequence Author:  MilliporeSigma
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DraIII (4460) NaeI (4354) NgoMIV (4352) XmnI (3907) ScaI (3788) AlwNI (2831) AflIII - PciI (2415) PsiI (4585) BsrGI (276) SpeI (332) NdeI (567) SnaBI (673) Eco53kI (899) SacI (901) HindIII (924) EagI - NotI (931) EcoRI (938) BspDI - ClaI (945) BglII (950) EcoRV (958) Acc65I (962) KpnI (966) SalI (969) AccI (970) XbaI (975) BamHI (981) TspMI - XmaI (985) SmaI (987) AleI (1080) BbsI (1203) EcoO109I (1210) BsgI (1264) BbvCI - Bpu10I (1306) BlpI (1350) BsmBI (1370) BstXI (1457) AgeI (1568) PflMI (1571) XcmI (1663) SexAI * (1755) SfiI (1941) StuI (1987) AvrII (1988) AbsI - PaeR7I - PspXI - XhoI (2010) BspQI - SapI (2299) pFLAG-CMV™-5.1 4682 bp
DraIII  (4460)
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.
NaeI  (4354)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
NgoMIV  (4352)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
XmnI  (3907)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3788)
1 site
A G T A C T T C A T G A
AlwNI  (2831)
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.
AflIII  (2415)
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  (2415)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PsiI  (4585)
1 site
T T A T A A A A T A T T
BsrGI  (276)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
SpeI  (332)
1 site
A C T A G T T G A T C A
NdeI  (567)
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  (673)
1 site
T A C G T A A T G C A T
Eco53kI  (899)
1 site
G A G C T C C T C G A G
SacI  (901)
1 site
G A G C T C C T C G A G
HindIII  (924)
1 site
A A G C T T T T C G A A
EagI  (931)
1 site
C G G C C G G C C G G C
NotI  (931)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (938)
1 site
G A A T T C C T T A A G
BspDI  (945)
1 site
A T C G A T T A G C T A
ClaI  (945)
1 site
A T C G A T T A G C T A
BglII  (950)
1 site
A G A T C T T C T A G A
EcoRV  (958)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
Acc65I  (962)
1 site
G G T A C C C C A T G G
KpnI  (966)
1 site
G G T A C C C C A T G G
SalI  (969)
1 site
G T C G A C C A G C T G
AccI  (970)
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.
XbaI  (975)
1 site
T C T A G A A G A T C T
BamHI  (981)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
TspMI  (985)
1 site
C C C G G G G G G C C C
XmaI  (985)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (987)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AleI  (1080)
1 site
C A C N N N N G T G G T G N N N N C A C
BbsI  (1203)
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.
EcoO109I  (1210)
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.
BsgI  (1264)
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).
BbvCI  (1306)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1306)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
BlpI  (1350)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
BsmBI  (1370)
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.
BstXI  (1457)
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.
AgeI  (1568)
1 site
A C C G G T T G G C C A
PflMI  (1571)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
XcmI  (1663)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
SexAI  (1755)
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.
SfiI  (1941)
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.
StuI  (1987)
1 site
A G G C C T T C C G G A
AvrII  (1988)
1 site
C C T A G G G G A T C C
AbsI  (2010)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (2010)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2010)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2010)
1 site
C T C G A G G A G C T C
BspQI  (2299)
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  (2299)
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.
AmpR
3235 .. 4095  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3235 .. 4026  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3235 .. 4095  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4027 .. 4095  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3235 .. 4095  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
hGH poly(A) signal
1023 .. 1645  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1023 .. 1645  =  623 bp
human growth hormone polyadenylation signal
ori
2476 .. 3064  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2476 .. 3064  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4227 .. 4682  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4227 .. 4682  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
CMV enhancer
318 .. 697  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
318 .. 697  =  380 bp
human cytomegalovirus immediate early enhancer
CMV promoter
698 .. 901  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
698 .. 901  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 ori
1854 .. 1989  =  136 bp
SV40 origin of replication
SV40 ori
1854 .. 1989  =  136 bp
SV40 origin of replication
AmpR promoter
4096 .. 4200  =  105 bp
AmpR promoter
4096 .. 4200  =  105 bp
MCS
924 .. 990  =  67 bp
multiple cloning site
MCS
924 .. 990  =  67 bp
multiple cloning site
FLAG
993 .. 1016  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an enterokinase cleavage site
FLAG
993 .. 1016  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an enterokinase cleavage site
ORF:  3365 .. 3631  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1387 .. 1611  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  3235 .. 4095  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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