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p3xFLAG-CMV™-13

Vector with a neomycin resistance marker for expression and secretion of C-terminally 3xFLAG-tagged proteins in mammalian cells.

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p3xFLAG-CMV-13.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Sigma-Aldrich
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XmnI (5587) ScaI (5468) AflIII - PciI (4095) HpaI (3581) MfeI (3568) AfeI (3376) BsrGI (276) SpeI (332) NdeI (567) SnaBI (673) Eco53kI (899) SacI (901) HindIII (976) NotI (983) EcoRI (990) BspDI - ClaI (997) BglII (1002) EcoRV (1010) Acc65I (1014) KpnI (1018) XbaI (1027) BamHI (1033) AleI (1178) EcoO109I (1308) BbvCI - Bpu10I (1404) BsmBI (1468) BstXI (1555) AgeI (1666) PflMI (1669) XcmI (1761) SexAI * (1853) SfiI (2039) BseRI (2082) BssHII (2661) RsrII (2780) BstBI (2946) NruI (3119) p3xFLAG-CMV™-13 6362 bp
XmnI  (5587)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (5468)
1 site
A G T A C T T C A T G A
AflIII  (4095)
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  (4095)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
HpaI  (3581)
1 site
G T T A A C C A A T T G
MfeI  (3568)
1 site
C A A T T G G T T A A C
AfeI  (3376)
1 site
A G C G C T T C G C G A
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  (976)
1 site
A A G C T T T T C G A A
NotI  (983)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (990)
1 site
G A A T T C C T T A A G
BspDI  (997)
1 site
A T C G A T T A G C T A
ClaI  (997)
1 site
A T C G A T T A G C T A
BglII  (1002)
1 site
A G A T C T T C T A G A
EcoRV  (1010)
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  (1014)
1 site
G G T A C C C C A T G G
KpnI  (1018)
1 site
G G T A C C C C A T G G
XbaI  (1027)
1 site
T C T A G A A G A T C T
BamHI  (1033)
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.
AleI  (1178)
1 site
C A C N N N N G T G G T G N N N N C A C
EcoO109I  (1308)
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  (1404)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1404)
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.
BsmBI  (1468)
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  (1555)
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  (1666)
1 site
A C C G G T T G G C C A
PflMI  (1669)
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  (1761)
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  (1853)
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  (2039)
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.
BseRI  (2082)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
BssHII  (2661)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
RsrII  (2780)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BstBI  (2946)
1 site
T T C G A A A A G C T T
NruI  (3119)
1 site
T C G C G A A G C G C T
AmpR
4915 .. 5775  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4915 .. 5706  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4915 .. 5775  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5707 .. 5775  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4915 .. 5775  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
2136 .. 2930  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
NeoR/KanR
2136 .. 2930  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
hGH poly(A) signal
1121 .. 1743  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1121 .. 1743  =  623 bp
human growth hormone polyadenylation signal
ori
4156 .. 4744  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4156 .. 4744  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
5907 .. 6362  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
5907 .. 6362  =  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
SV40 promoter
1785 .. 2082  =  298 bp
SV40 enhancer and early promoter
SV40 promoter
1785 .. 2082  =  298 bp
SV40 enhancer and early promoter
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 poly(A) signal
3582 .. 3716  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3582 .. 3716  =  135 bp
SV40 polyadenylation signal
AmpR promoter
5776 .. 5880  =  105 bp
AmpR promoter
5776 .. 5880  =  105 bp
MCS
976 .. 1042  =  67 bp
multiple cloning site
MCS
976 .. 1042  =  67 bp
multiple cloning site
3xFLAG
1045 .. 1110  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG epitope tags, followed by an enterokinase cleavage site
3xFLAG
1045 .. 1110  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG epitope tags, followed by an enterokinase cleavage site
preprotrypsin leader
931 .. 975  =  45 bp
15 amino acids  =  1.4 kDa
Product: leader sequence from mouse preprotrypsin
preprotrypsin leader
931 .. 975  =  45 bp
15 amino acids  =  1.4 kDa
Product: leader sequence from mouse preprotrypsin
ORF:  2308 .. 2694  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  2951 .. 3331  =  381 bp
ORF:  126 amino acids  =  14.1 kDa
ORF:  5045 .. 5311  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2136 .. 2930  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  1485 .. 1709  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  2445 .. 2981  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
ORF:  3129 .. 3491  =  363 bp
ORF:  120 amino acids  =  13.1 kDa
ORF:  2981 .. 3226  =  246 bp
ORF:  81 amino acids  =  8.4 kDa
ORF:  994 .. 1221  =  228 bp
ORF:  75 amino acids  =  8.4 kDa
ORF:  3211 .. 3465  =  255 bp
ORF:  84 amino acids  =  9.3 kDa
ORF:  4915 .. 5775  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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