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Plasmid Files

pFLAG-CMV™-1

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

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pFLAG-CMV-1.dna
Map and Sequence File:    Download    Open   
Sequence Author:  MilliporeSigma
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PsiI (4617) DraIII (4492) NaeI (4386) NgoMIV (4384) ScaI (3820) AflIII - PciI (2447) BsrGI (269) SpeI (325) NdeI (560) SnaBI (666) Eco53kI (892) SacI (894) PstI (963) HindIII (991) EagI - NotI (998) EcoRI (1005) BspDI - ClaI (1012) BglII (1017) EcoRV (1025) Acc65I (1029) KpnI (1033) SalI (1036) AccI (1037) XbaI (1042) BamHI (1048) TspMI - XmaI (1052) SmaI (1054) AleI (1112) BbsI (1235) EcoO109I (1242) BbvCI - Bpu10I (1338) BlpI (1382) BsmBI (1402) BstXI (1489) AgeI (1600) PflMI (1603) XcmI (1695) SexAI * (1787) SfiI (1973) StuI (2019) AvrII (2020) AbsI - PaeR7I - PspXI - XhoI (2042) BspQI - SapI (2331) pFLAG-CMV™-1 4732 bp
PsiI  (4617)
1 site
T T A T A A A A T A T T
DraIII  (4492)
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  (4386)
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  (4384)
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.
ScaI  (3820)
1 site
A G T A C T T C A T G A
AflIII  (2447)
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  (2447)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BsrGI  (269)
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  (325)
1 site
A C T A G T T G A T C A
NdeI  (560)
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  (666)
1 site
T A C G T A A T G C A T
Eco53kI  (892)
1 site
G A G C T C C T C G A G
SacI  (894)
1 site
G A G C T C C T C G A G
PstI  (963)
1 site
C T G C A G G A C G T C
HindIII  (991)
1 site
A A G C T T T T C G A A
EagI  (998)
1 site
C G G C C G G C C G G C
NotI  (998)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (1005)
1 site
G A A T T C C T T A A G
BspDI  (1012)
1 site
A T C G A T T A G C T A
ClaI  (1012)
1 site
A T C G A T T A G C T A
BglII  (1017)
1 site
A G A T C T T C T A G A
EcoRV  (1025)
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  (1029)
1 site
G G T A C C C C A T G G
KpnI  (1033)
1 site
G G T A C C C C A T G G
SalI  (1036)
1 site
G T C G A C C A G C T G
AccI  (1037)
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  (1042)
1 site
T C T A G A A G A T C T
BamHI  (1048)
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  (1052)
1 site
C C C G G G G G G C C C
XmaI  (1052)
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  (1054)
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  (1112)
1 site
C A C N N N N G T G G T G N N N N C A C
BbsI  (1235)
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  (1242)
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.
BbvCI  (1338)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1338)
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  (1382)
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  (1402)
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  (1489)
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  (1600)
1 site
A C C G G T T G G C C A
PflMI  (1603)
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  (1695)
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  (1787)
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  (1973)
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  (2019)
1 site
A G G C C T T C C G G A
AvrII  (2020)
1 site
C C T A G G G G A T C C
AbsI  (2042)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (2042)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2042)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2042)
1 site
C T C G A G G A G C T C
BspQI  (2331)
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  (2331)
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
3267 .. 4127  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3267 .. 4058  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3267 .. 4127  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4059 .. 4127  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3267 .. 4127  =  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
1055 .. 1677  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1055 .. 1677  =  623 bp
human growth hormone polyadenylation signal
ori
2508 .. 3096  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2508 .. 3096  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4259 .. 4714  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4259 .. 4714  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
CMV enhancer
311 .. 690  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
311 .. 690  =  380 bp
human cytomegalovirus immediate early enhancer
CMV promoter
691 .. 894  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
691 .. 894  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 ori
1886 .. 2021  =  136 bp
SV40 origin of replication
SV40 ori
1886 .. 2021  =  136 bp
SV40 origin of replication
AmpR promoter
4128 .. 4232  =  105 bp
AmpR promoter
4128 .. 4232  =  105 bp
preprotrypsin leader
925 .. 969  =  45 bp
15 amino acids  =  1.4 kDa
Product: leader sequence from mouse preprotrypsin
preprotrypsin leader
925 .. 969  =  45 bp
15 amino acids  =  1.4 kDa
Product: leader sequence from mouse preprotrypsin
FLAG
970 .. 993  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an enterokinase cleavage site
FLAG
970 .. 993  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG epitope tag, followed by an enterokinase cleavage site
MCS
991 .. 1057  =  67 bp
multiple cloning site
MCS
991 .. 1057  =  67 bp
multiple cloning site
ORF:  3397 .. 3663  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1419 .. 1643  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  3267 .. 4127  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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