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Plasmid Files

3xFLAG-dCas9/pCMV-7.1

Plasmid for expression in mammalian cells of FLAG®-tagged catalytically inactive dCas9, for engineered ChIP (enChIP) purification of specific genomic regions.

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3xFLAG-dCas9 pCMV-7.1 Sequence and Map3xFLAG-dCas9 pCMV-7.1.dna
Map and Sequence File   
Sequence Author:  Fujii Lab / Addgene #47948
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 DraIII (8626) BspQI - SapI (6465) AbsI - PspXI (6176) AvrII (6154) StuI (6153) SfiI (6107) SexAI * (5921) AgeI (5734) BbvCI (5472) AleI (5246) XbaI (5176) KpnI (5167) Acc65I (5163) AscI - BssHII (4955) EcoRI (4822) AarI (4260) BsrGI (276) SpeI (332) CMV enhancer SnaBI (673) ATG BspDI - ClaI (971) SbfI (1247) Bsu36I (2266) BmgBI (3510) PmlI (3789) FspAI (3950) NruI (4240) 3xFLAG-dCas9/pCMV-7.1 8848 bp
DraIII  (8626)
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.
BspQI  (6465)
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  (6465)
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.
AbsI  (6176)
1 site
C C T C G A G G G G A G C T C C
PspXI  (6176)
1 site
V C T C G A G B B G A G C T C V
AvrII  (6154)
1 site
C C T A G G G G A T C C
StuI  (6153)
1 site
A G G C C T T C C G G A
SfiI  (6107)
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.
SexAI  (5921)
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.
AgeI  (5734)
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.
BbvCI  (5472)
1 site
C C T C A G C G G A G T C G
AleI  (5246)
1 site
C A C N N N N G T G G T G N N N N C A C
XbaI  (5176)
1 site
T C T A G A A G A T C T
KpnI  (5167)
1 site
G G T A C C C C A T G G
Acc65I  (5163)
1 site
G G T A C C C C A T G G
AscI  (4955)
1 site
G G C G C G C C C C G C G C G G
BssHII  (4955)
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.
EcoRI  (4822)
1 site
G A A T T C C T T A A G
AarI  (4260)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
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
SnaBI  (673)
1 site
T A C G T A A T G C A T
BspDI  (971)
1 site
A T C G A T T A G C T A
ClaI  (971)
1 site
A T C G A T T A G C T A
SbfI  (1247)
1 site
C C T G C A G G G G A C G T C C
Bsu36I  (2266)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BmgBI  (3510)
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.
PmlI  (3789)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
FspAI  (3950)
1 site
R T G C G C A Y Y A C G C G T R
NruI  (4240)
1 site
T C G C G A A G C G C T
dCas9
1000 .. 5103  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   1000 .. 1026  =  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
1000 .. 5103  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   1027 .. 1029  =  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
1000 .. 5103  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   1030 .. 3516  =  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
1000 .. 5103  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   3517 .. 3519  =  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
1000 .. 5103  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   3520 .. 5103  =  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
1000 .. 5103  =  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
7401 .. 8261  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7401 .. 8192  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7401 .. 8261  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   8193 .. 8261  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7401 .. 8261  =  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
5189 .. 5811  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
5189 .. 5811  =  623 bp
human growth hormone polyadenylation signal
ori
6642 .. 7230  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6642 .. 7230  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
8393 .. 8848  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
8393 .. 8848  =  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
5840 .. 6169  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
5840 .. 6169  =  330 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
AmpR promoter
8262 .. 8366  =  105 bp
AmpR promoter
8262 .. 8366  =  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
SV40 NLS
5116 .. 5136  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5116 .. 5136  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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
SV40 ori
6020 .. 6155  =  136 bp
SV40 origin of replication
SV40 ori
6020 .. 6155  =  136 bp
SV40 origin of replication
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