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3xFLAG-dCas9/pMXs-puro

Retroviral vector with a puromycin resistance marker, encoding FLAG®-tagged dCas9 for engineered ChIP (enChIP) purification of specific genomic regions.

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3xFLAG-dCas9 pMXs-puro Sequence and Map3xFLAG-dCas9 pMXs-puro.dna
Map and Sequence File   
Sequence Author:  Fujii Lab / Addgene #51240
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 FseI (8984) BspQI - SapI (8101) SgrDI (7338) SacII (6896) RsrII (6798) BsiWI (6738) AvrII (6657) SfiI (6610) NotI (6311) PspXI (6305) NruI (5260) SpeI (752) PshAI (874) BsrGI (1390) AgeI (1690) PacI (1913) ATG BspDI - ClaI (1991) SbfI (2267) XcmI (2371) BmgBI (4530) FspAI (4970) 3xFLAG-dCas9/pMXs-puro 10,003 bp
FseI  (8984)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
BspQI  (8101)
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  (8101)
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.
SgrDI  (7338)
1 site
C G T C G A C G G C A G C T G C
SacII  (6896)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
RsrII  (6798)
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.
BsiWI  (6738)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
AvrII  (6657)
1 site
C C T A G G G G A T C C
SfiI  (6610)
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.
NotI  (6311)
1 site
G C G G C C G C C G C C G G C G
PspXI  (6305)
1 site
V C T C G A G B B G A G C T C V
NruI  (5260)
1 site
T C G C G A A G C G C T
SpeI  (752)
1 site
A C T A G T T G A T C A
PshAI  (874)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BsrGI  (1390)
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.
AgeI  (1690)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
PacI  (1913)
1 site
T T A A T T A A A A T T A A T T
BspDI  (1991)
1 site
A T C G A T T A G C T A
ClaI  (1991)
1 site
A T C G A T T A G C T A
SbfI  (2267)
1 site
C C T G C A G G G G A C G T C C
XcmI  (2371)
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.
BmgBI  (4530)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
FspAI  (4970)
1 site
R T G C G C A Y Y A C G C G T R
dCas9
2020 .. 6123  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   2020 .. 2046  =  27 bp
   9 amino acids  =  1.1 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
2020 .. 6123  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   2047 .. 2049  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
2020 .. 6123  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   2050 .. 4536  =  2487 bp
   829 amino acids  =  96.3 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
2020 .. 6123  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   4537 .. 4539  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
2020 .. 6123  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   4540 .. 6123  =  1584 bp
   528 amino acids  =  60.9 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
2020 .. 6123  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
AmpR
9030 .. 9890  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   9030 .. 9821  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9030 .. 9890  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   9822 .. 9890  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9030 .. 9890  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
6682 .. 7281  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
6682 .. 7281  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
LTR
22 .. 615  =  594 bp
long terminal repeat from Moloney murine leukemia
virus
LTR
22 .. 615  =  594 bp
long terminal repeat from Moloney murine leukemia
virus
ori
8278 .. 8866  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8278 .. 8866  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
LTR
7508 .. 7977  =  470 bp
long terminal repeat from Moloney murine leukemia
virus
LTR
7508 .. 7977  =  470 bp
long terminal repeat from Moloney murine leukemia
virus
gag (truncated)
1094 .. 1510  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
1094 .. 1510  =  417 bp
truncated MMLV gag gene lacking the start codon
pol region
1520 .. 1894  =  375 bp
Moloney murine leukemia virus (MMLV) pol region
containing the splice acceptor site
pol region
1520 .. 1894  =  375 bp
Moloney murine leukemia virus (MMLV) pol region
containing the splice acceptor site
MMLV Ψ
678 .. 1035  =  358 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
MMLV Ψ
678 .. 1035  =  358 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
SV40 promoter
6343 .. 6672  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
6343 .. 6672  =  330 bp
SV40 enhancer and early promoter
AmpR promoter
9891 .. 9995  =  105 bp
AmpR promoter
9891 .. 9995  =  105 bp
3xFLAG
1951 .. 2016  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
1951 .. 2016  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
SV40 NLS
6136 .. 6156  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6136 .. 6156  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
1948 .. 1950  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1948 .. 1950  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
6523 .. 6658  =  136 bp
SV40 origin of replication
SV40 ori
6523 .. 6658  =  136 bp
SV40 origin of replication
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