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p3xFLAG-CMV™-7.1

Vector for expression of N-terminally 3xFLAG-tagged proteins in mammalian cells. For an alternative MCS, use p3xFLAG‑CMV™-7.

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p3xFLAG-CMV-7.1.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Sigma-Aldrich
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DraIII (4495) NaeI (4389) NgoMIV (4387) XmnI (3942) ScaI (3823) AlwNI (2866) AflIII - PciI (2450) BsrGI (276) SpeI (332) NdeI (567) SnaBI (673) Eco53kI (899) SacI (901) HindIII (994) EagI - NotI (1001) EcoRI (1008) BglII (1020) EcoRV (1028) Acc65I (1032) KpnI (1036) SalI (1039) AccI (1040) XbaI (1045) BamHI (1051) TspMI - XmaI (1055) SmaI (1057) AleI (1115) BbsI (1238) EcoO109I (1245) BsgI (1299) BbvCI - Bpu10I (1341) BlpI (1385) BsmBI (1405) BstXI (1492) AgeI (1603) PflMI (1606) XcmI (1698) SexAI * (1790) SfiI (1976) StuI (2022) AvrII (2023) AbsI - PaeR7I - PspXI - XhoI (2045) BspQI - SapI (2334) p3xFLAG-CMV™-7.1 4717 bp
DraIII  (4495)
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  (4389)
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  (4387)
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  (3942)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3823)
1 site
A G T A C T T C A T G A
AlwNI  (2866)
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  (2450)
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  (2450)
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  (994)
1 site
A A G C T T T T C G A A
EagI  (1001)
1 site
C G G C C G G C C G G C
NotI  (1001)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (1008)
1 site
G A A T T C C T T A A G
BglII  (1020)
1 site
A G A T C T T C T A G A
EcoRV  (1028)
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  (1032)
1 site
G G T A C C C C A T G G
KpnI  (1036)
1 site
G G T A C C C C A T G G
SalI  (1039)
1 site
G T C G A C C A G C T G
AccI  (1040)
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  (1045)
1 site
T C T A G A A G A T C T
BamHI  (1051)
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  (1055)
1 site
C C C G G G G G G C C C
XmaI  (1055)
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  (1057)
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  (1115)
1 site
C A C N N N N G T G G T G N N N N C A C
BbsI  (1238)
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  (1245)
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  (1299)
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  (1341)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1341)
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  (1385)
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  (1405)
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  (1492)
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  (1603)
1 site
A C C G G T T G G C C A
PflMI  (1606)
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  (1698)
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  (1790)
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  (1976)
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  (2022)
1 site
A G G C C T T C C G G A
AvrII  (2023)
1 site
C C T A G G G G A T C C
AbsI  (2045)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (2045)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2045)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2045)
1 site
C T C G A G G A G C T C
BspQI  (2334)
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  (2334)
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
3270 .. 4130  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3270 .. 4061  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3270 .. 4130  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4062 .. 4130  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3270 .. 4130  =  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
1058 .. 1680  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1058 .. 1680  =  623 bp
human growth hormone polyadenylation signal
ori
2511 .. 3099  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2511 .. 3099  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4262 .. 4717  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4262 .. 4717  =  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
1889 .. 2024  =  136 bp
SV40 origin of replication
SV40 ori
1889 .. 2024  =  136 bp
SV40 origin of replication
AmpR promoter
4131 .. 4235  =  105 bp
AmpR promoter
4131 .. 4235  =  105 bp
3xFLAG
931 .. 996  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG epitope tags, followed by an enterokinase cleavage site
3xFLAG
931 .. 996  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG epitope tags, followed by an enterokinase cleavage site
ATG
928 .. 930  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
928 .. 930  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
MCS
994 .. 1060  =  67 bp
multiple cloning site
MCS
994 .. 1060  =  67 bp
multiple cloning site
ORF:  3400 .. 3666  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1422 .. 1646  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  3270 .. 4130  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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