p3xFLAG-Myc-CMV-25

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

Sequence Author: MilliporeSigma

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ScaI (5495) AflIII - PciI (4122) HpaI (3608) MfeI (3595) AfeI (3403) BsrGI (276) SpeI (332) CMV enhancer NdeI (567) SnaBI (673) Eco53kI (899) SacI (901) HindIII (1050) NotI (1057) EcoRI (1064) BglII (1076) EcoRV (1084) Acc65I (1088) KpnI (1092) XbaI (1101) Myc BamHI (1140) AleI (1204) EcoO109I (1334) BbvCI - Bpu10I (1430) BlpI (1474) BsmBI - Esp3I (1494) BstXI (1581) AgeI (1692) PflMI (1695) XcmI (1787) CsiI - SexAI * (1879) SfiI (2066) BseRI (2109) PflFI - Tth111I (2409) BssHII (2688) RsrII (2807) BstBI (2973) NruI (3146) p3xFLAG-Myc-CMV™-25 6406 bp
ScaI  (5495)
1 site
A G T A C T T C A T G A
AflIII  (4122)
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  (4122)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
HpaI  (3608)
1 site
G T T A A C C A A T T G
MfeI  (3595)
1 site
C A A T T G G T T A A C
AfeI  (3403)
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  (1050)
1 site
A A G C T T T T C G A A
NotI  (1057)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (1064)
1 site
G A A T T C C T T A A G
BglII  (1076)
1 site
A G A T C T T C T A G A
EcoRV  (1084)
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  (1088)
1 site
G G T A C C C C A T G G
KpnI  (1092)
1 site
G G T A C C C C A T G G
XbaI  (1101)
1 site
T C T A G A A G A T C T
BamHI  (1140)
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  (1204)
1 site
C A C N N N N G T G G T G N N N N C A C
EcoO109I  (1334)
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  (1430)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1430)
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  (1474)
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  (1494)
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  (1494)
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  (1581)
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  (1692)
1 site
A C C G G T T G G C C A
PflMI  (1695)
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  (1787)
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  (1879)
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  (1879)
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  (2066)
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  (2109)
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.
PflFI  (2409)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (2409)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BssHII  (2688)
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  (2807)
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  (2973)
1 site
T T C G A A A A G C T T
NruI  (3146)
1 site
T C G C G A A G C G C T
AmpR
4942 .. 5802  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   4942 .. 5733  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4942 .. 5802  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   5734 .. 5802  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4942 .. 5802  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
2163 .. 2957  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
NeoR/KanR
2163 .. 2957  =  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
1147 .. 1769  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
1147 .. 1769  =  623 bp
human growth hormone polyadenylation signal
ori
4183 .. 4771  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4183 .. 4771  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
5934 .. 6406  =  473 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
5934 .. 6406  =  473 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
1811 .. 2109  =  299 bp
SV40 enhancer and early promoter
SV40 promoter
1811 .. 2109  =  299 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
preprotrypsin leader
942 .. 986  =  45 bp
15 amino acids  =  1.4 kDa
Product: leader sequence from mouse preprotrypsin
preprotrypsin leader
942 .. 986  =  45 bp
15 amino acids  =  1.4 kDa
Product: leader sequence from mouse preprotrypsin
3xFLAG
987 .. 1052  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG epitope tags, followed by an enterokinase cleavage site
3xFLAG
987 .. 1052  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG epitope tags, followed by an enterokinase cleavage site
Myc
1107 .. 1136  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
1107 .. 1136  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
SV40 poly(A) signal
3609 .. 3743  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3609 .. 3743  =  135 bp
SV40 polyadenylation signal
AmpR promoter
5803 .. 5907  =  105 bp
AmpR promoter
5803 .. 5907  =  105 bp
MCS
1050 .. 1106  =  57 bp
multiple cloning site
MCS
1050 .. 1106  =  57 bp
multiple cloning site
ORF:  2335 .. 2721  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  2978 .. 3358  =  381 bp
ORF:  126 amino acids  =  14.1 kDa
ORF:  5072 .. 5338  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2163 .. 2957  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  1511 .. 1735  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  3008 .. 3253  =  246 bp
ORF:  81 amino acids  =  8.4 kDa
ORF:  3238 .. 3492  =  255 bp
ORF:  84 amino acids  =  9.3 kDa
ORF:  4942 .. 5802  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  2472 .. 3008  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
ORF:  3156 .. 3518  =  363 bp
ORF:  120 amino acids  =  13.1 kDa
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