pFLAG-CMV-2

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

Sequence Author: MilliporeSigma

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PsiI (4582) DraIII (4457) NaeI (4351) NgoMIV (4349) XmnI (3904) ScaI (3785) AlwNI (2828) AflIII - PciI (2412) BsrGI (276) SpeI (332) NdeI (567) SnaBI (673) Eco53kI (899) SacI (901) ATG HindIII (956) EagI - NotI (963) EcoRI (970) BspDI - ClaI (977) BglII (982) EcoRV (990) Acc65I (994) KpnI (998) SalI (1001) AccI (1002) XbaI (1007) BamHI (1013) TspMI - XmaI (1017) SmaI (1019) AleI (1077) BbsI (1200) EcoO109I (1207) BsgI (1261) BbvCI - Bpu10I (1303) BlpI (1347) BsmBI - Esp3I (1367) BstXI (1454) AgeI (1565) PflMI (1568) XcmI (1660) CsiI - SexAI * (1752) SfiI (1938) StuI (1984) AvrII (1985) AbsI - PaeR7I - PspXI - XhoI (2007) BspQI - SapI (2296) pFLAG-CMV™-2 4679 bp
PsiI  (4582)
1 site
T T A T A A A A T A T T
DraIII  (4457)
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  (4351)
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  (4349)
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  (3904)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3785)
1 site
A G T A C T T C A T G A
AlwNI  (2828)
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  (2412)
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  (2412)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
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  (956)
1 site
A A G C T T T T C G A A
EagI  (963)
1 site
C G G C C G G C C G G C
NotI  (963)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (970)
1 site
G A A T T C C T T A A G
BspDI  (977)
1 site
A T C G A T T A G C T A
ClaI  (977)
1 site
A T C G A T T A G C T A
BglII  (982)
1 site
A G A T C T T C T A G A
EcoRV  (990)
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  (994)
1 site
G G T A C C C C A T G G
KpnI  (998)
1 site
G G T A C C C C A T G G
SalI  (1001)
1 site
G T C G A C C A G C T G
AccI  (1002)
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  (1007)
1 site
T C T A G A A G A T C T
BamHI  (1013)
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  (1017)
1 site
C C C G G G G G G C C C
XmaI  (1017)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (1019)
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  (1077)
1 site
C A C N N N N G T G G T G N N N N C A C
BbsI  (1200)
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  (1207)
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  (1261)
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  (1303)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1303)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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  (1347)
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  (1367)
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.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (1367)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
BstXI  (1454)
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  (1565)
1 site
A C C G G T T G G C C A
PflMI  (1568)
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  (1660)
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.
CsiI  (1752)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (1752)
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  (1938)
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  (1984)
1 site
A G G C C T T C C G G A
AvrII  (1985)
1 site
C C T A G G G G A T C C
AbsI  (2007)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (2007)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2007)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2007)
1 site
C T C G A G G A G C T C
BspQI  (2296)
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  (2296)
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
3232 .. 4092  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3232 .. 4023  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3232 .. 4092  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   4024 .. 4092  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3232 .. 4092  =  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
1020 .. 1642  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1020 .. 1642  =  623 bp
human growth hormone polyadenylation signal
ori
2473 .. 3061  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2473 .. 3061  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4224 .. 4679  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4224 .. 4679  =  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
1851 .. 1986  =  136 bp
SV40 origin of replication
SV40 ori
1851 .. 1986  =  136 bp
SV40 origin of replication
AmpR promoter
4093 .. 4197  =  105 bp
AmpR promoter
4093 .. 4197  =  105 bp
ATG
932 .. 934  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
932 .. 934  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
FLAG
935 .. 958  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an enterokinase cleavage site
FLAG
935 .. 958  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an enterokinase cleavage site
MCS
956 .. 1022  =  67 bp
multiple cloning site
MCS
956 .. 1022  =  67 bp
multiple cloning site
ORF:  3362 .. 3628  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1384 .. 1608  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  3232 .. 4092  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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