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Plasmid Files

pCas9D10A_GFP

Plasmid for co-expression in mammalian cells of enhanced GFP (EGFP) and human codon-optimized Cas9 with the D10A nickase mutation.

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pCas9D10A_GFP Sequence and MappCas9D10A_GFP.dna
Map and Sequence File   
Sequence Author:  Musunuru Lab / Addgene #44720
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 MfeI (9205) BspDI * - ClaI * (9055) SgrDI (9038) AhdI (8117) BspQI - SapI (7108) BstZ17I (6845) BsrGI (6660) AscI (5700) PaeR7I - XhoI (5288) FspAI (4695) MluI (1) SpeI (22) SnaBI (363) PspOMI (863) ApaI (867) SacII (1034) BsiWI (1190) MreI - SgrAI (1316) BstAPI (1350) NotI (1734) BsmBI (1907) SbfI (1992) XcmI (2096) Bsu36I (3011) TspMI - XmaI (3111) SmaI (3113) Eco53kI (3313) SacI (3315) BamHI (3750) BstXI - PmlI (4534) pCas9D10A_GFP 9271 bp
MfeI  (9205)
1 site
C A A T T G G T T A A C
BspDI  (9055)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (9055)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrDI  (9038)
1 site
C G T C G A C G G C A G C T G C
AhdI  (8117)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BspQI  (7108)
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  (7108)
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.
BstZ17I  (6845)
1 site
G T A T A C C A T A T G
BsrGI  (6660)
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.
AscI  (5700)
1 site
G G C G C G C C C C G C G C G G
PaeR7I  (5288)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (5288)
1 site
C T C G A G G A G C T C
FspAI  (4695)
1 site
R T G C G C A Y Y A C G C G T R
MluI  (1)
1 site
A C G C G T T G C G C A
SpeI  (22)
1 site
A C T A G T T G A T C A
SnaBI  (363)
1 site
T A C G T A A T G C A T
PspOMI  (863)
1 site
G G G C C C C C C G G G
ApaI  (867)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
SacII  (1034)
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.
BsiWI  (1190)
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.
MreI  (1316)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (1316)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
BstAPI  (1350)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
NotI  (1734)
1 site
G C G G C C G C C G C C G G C G
BsmBI  (1907)
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.
SbfI  (1992)
1 site
C C T G C A G G G G A C G T C C
XcmI  (2096)
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.
Bsu36I  (3011)
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.
TspMI  (3111)
1 site
C C C G G G G G G C C C
XmaI  (3111)
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  (3113)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
Eco53kI  (3313)
1 site
G A G C T C C T C G A G
SacI  (3315)
1 site
G A G C T C C T C G A G
BamHI  (3750)
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.
BstXI  (4534)
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.
PmlI  (4534)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
Cas9(D10A)
1745 .. 5848  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 1:  
   1745 .. 1771  =  27 bp
   9 amino acids  =  1.1 kDa
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
Cas9(D10A)
1745 .. 5848  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 2:  
   1772 .. 1774  =  3 bp
   1 amino acid  =  89.1 Da
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
Cas9(D10A)
1745 .. 5848  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 3:  
   1775 .. 5848  =  4074 bp
   1358 amino acids  =  157.3 kDa
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
Cas9(D10A)
1745 .. 5848  =  4104 bp
1368 amino acids  =  158.4 kDa
3 segments
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
chimeric intron
666 .. 1678  =  1013 bp
chimera between introns from chicken β-actin and
rabbit β-globin
chimeric intron
666 .. 1678  =  1013 bp
chimera between introns from chicken β-actin and
rabbit β-globin
AmpR
8044 .. 8904  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8044 .. 8835  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8044 .. 8904  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   8836 .. 8904  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8044 .. 8904  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
EGFP
5951 .. 6670  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 1:  
   5951 .. 5953  =  3 bp
   1 amino acid  =  149.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
5951 .. 6670  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 2:  1a  
   5954 .. 5956  =  3 bp
   1 amino acid  =  117.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
5951 .. 6670  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 3:  
   5957 .. 6670  =  714 bp
   237 amino acids  =  26.7 kDa
Product: enhanced GFP
mammalian codon-optimized
EGFP
5951 .. 6670  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
Product: enhanced GFP
mammalian codon-optimized
ori
7285 .. 7873  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7285 .. 7873  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
8 .. 387  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
8 .. 387  =  380 bp
human cytomegalovirus immediate early enhancer
chicken β-actin promoter
390 .. 665  =  276 bp
chicken β-actin promoter
390 .. 665  =  276 bp
SV40 poly(A) signal
6713 .. 6834  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
6713 .. 6834  =  122 bp
SV40 polyadenylation signal
AmpR promoter
8905 .. 9009  =  105 bp
AmpR promoter
8905 .. 9009  =  105 bp
T2A
5897 .. 5950  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
T2A
5897 .. 5950  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
SV40 NLS
5861 .. 5881  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5861 .. 5881  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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