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p3xFLAG-Myc-CMV™-23

Vector for expression and secretion of 3xFLAG- and Myc-tagged proteins in mammalian cells.

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p3xFLAG-Myc-CMV-23.dna
Map and Sequence File:    Download    Open   
Sequence Author:  MilliporeSigma
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DraIII (4576) NaeI (4470) NgoMIV (4468) ScaI (3904) AflIII - PciI (2531) BspQI - SapI (2415) BsrGI (276) SpeI (332) NdeI (567) SnaBI (673) Eco53kI (899) SacI (901) PstI (970) HindIII (1040) EagI - NotI (1047) EcoRI (1054) BglII (1066) EcoRV (1074) Acc65I (1078) KpnI (1082) SalI (1085) AccI (1086) XbaI (1091) BamHI (1097) TspMI - XmaI (1136) SmaI (1138) AleI (1196) BbsI (1319) EcoO109I (1326) BbvCI - Bpu10I (1422) BlpI (1466) BsmBI (1486) BstXI (1573) AgeI (1684) PflMI (1687) XcmI (1779) SexAI * (1871) SfiI (2057) StuI (2103) AvrII (2104) AbsI - PaeR7I - PspXI - XhoI (2126) p3xFLAG-Myc-CMV™-23 4798 bp
DraIII  (4576)
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  (4470)
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  (4468)
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  (3904)
1 site
A G T A C T T C A T G A
AflIII  (2531)
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  (2531)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2415)
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  (2415)
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.
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
PstI  (970)
1 site
C T G C A G G A C G T C
HindIII  (1040)
1 site
A A G C T T T T C G A A
EagI  (1047)
1 site
C G G C C G G C C G G C
NotI  (1047)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (1054)
1 site
G A A T T C C T T A A G
BglII  (1066)
1 site
A G A T C T T C T A G A
EcoRV  (1074)
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  (1078)
1 site
G G T A C C C C A T G G
KpnI  (1082)
1 site
G G T A C C C C A T G G
SalI  (1085)
1 site
G T C G A C C A G C T G
AccI  (1086)
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  (1091)
1 site
T C T A G A A G A T C T
BamHI  (1097)
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  (1136)
1 site
C C C G G G G G G C C C
XmaI  (1136)
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  (1138)
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  (1196)
1 site
C A C N N N N G T G G T G N N N N C A C
BbsI  (1319)
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  (1326)
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  (1422)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1422)
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  (1466)
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  (1486)
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  (1573)
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  (1684)
1 site
A C C G G T T G G C C A
PflMI  (1687)
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  (1779)
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  (1871)
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  (2057)
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  (2103)
1 site
A G G C C T T C C G G A
AvrII  (2104)
1 site
C C T A G G G G A T C C
AbsI  (2126)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (2126)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2126)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2126)
1 site
C T C G A G G A G C T C
AmpR
3351 .. 4211  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3351 .. 4142  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3351 .. 4211  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4143 .. 4211  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3351 .. 4211  =  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
1139 .. 1761  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1139 .. 1761  =  623 bp
human growth hormone polyadenylation signal
ori
2592 .. 3180  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2592 .. 3180  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4343 .. 4798  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4343 .. 4798  =  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
1970 .. 2105  =  136 bp
SV40 origin of replication
SV40 ori
1970 .. 2105  =  136 bp
SV40 origin of replication
AmpR promoter
4212 .. 4316  =  105 bp
AmpR promoter
4212 .. 4316  =  105 bp
3xFLAG
977 .. 1042  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG epitope tags, followed by an enterokinase cleavage site
3xFLAG
977 .. 1042  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG epitope tags, followed by an enterokinase cleavage site
preprotrypsin leader
932 .. 976  =  45 bp
15 amino acids  =  1.4 kDa
Product: leader sequence from mouse preprotrypsin
preprotrypsin leader
932 .. 976  =  45 bp
15 amino acids  =  1.4 kDa
Product: leader sequence from mouse preprotrypsin
Myc
1103 .. 1132  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
1103 .. 1132  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
MCS
1040 .. 1096  =  57 bp
multiple cloning site
MCS
1040 .. 1096  =  57 bp
multiple cloning site
ORF:  3481 .. 3747  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1503 .. 1727  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  3351 .. 4211  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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