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Plasmid Files

pCAG-T3-hCasD10A-pA

Plasmid for mammalian cell expression or in vitro transcription of human codon-optimized Cas9 with the D10A nickase mutation.

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pCAG-T3-hCasD10A-pA Sequence and MappCAG-T3-hCasD10A-pA.dna
Map and Sequence File   
Sequence Author:  Fujii & Naito Labs / Addgene #51638
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 SV40 poly(A) signal BspQI - SapI (9212) AhdI (8207) SpeI (7265) CMV enhancer SnaBI (6928) ApaI (6445) PspOMI (6441) BsiWI (6113) MreI - SgrAI (5987) XbaI (5672) AleI (5531) NotI (5520) SrfI (5510) ATG SV40 NLS BsmBI (5252) SbfI (5175) XcmI (5068) AvrII (156) StuI (161) SfiI (210) SphI (1092) PspXI (1097) Acc65I (1268) KpnI (1272) BspDI - ClaI (1275) AscI (1459) NruI (2178) FspAI (2468) PmlI (2629) BmgBI (2908) pCAG-T3-hCasD10A-pA 9272 bp
BspQI  (9212)
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  (9212)
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.
AhdI  (8207)
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.
SpeI  (7265)
1 site
A C T A G T T G A T C A
SnaBI  (6928)
1 site
T A C G T A A T G C A T
ApaI  (6445)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (6441)
1 site
G G G C C C C C C G G G
BsiWI  (6113)
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  (5987)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (5987)
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.
XbaI  (5672)
1 site
T C T A G A A G A T C T
AleI  (5531)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (5520)
1 site
G C G G C C G C C G C C G G C G
SrfI  (5510)
1 site
G C C C G G G C C G G G C C C G
BsmBI  (5252)
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  (5175)
1 site
C C T G C A G G G G A C G T C C
XcmI  (5068)
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.
AvrII  (156)
1 site
C C T A G G G G A T C C
StuI  (161)
1 site
A G G C C T T C C G G A
SfiI  (210)
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.
SphI  (1092)
1 site
G C A T G C C G T A C G
PspXI  (1097)
1 site
V C T C G A G B B G A G C T C V
Acc65I  (1268)
1 site
G G T A C C C C A T G G
KpnI  (1272)
1 site
G G T A C C C C A T G G
BspDI  (1275)
1 site
A T C G A T T A G C T A
ClaI  (1275)
1 site
A T C G A T T A G C T A
AscI  (1459)
1 site
G G C G C G C C C C G C G C G G
NruI  (2178)
1 site
T C G C G A A G C G C T
FspAI  (2468)
1 site
R T G C G C A Y Y A C G C G T R
PmlI  (2629)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BmgBI  (2908)
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.
Cas9(D10A)
1316 .. 5416  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 3:  
   1316 .. 5389  =  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)
1316 .. 5416  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 2:  
   5390 .. 5392  =  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)
1316 .. 5416  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 1:  
   5393 .. 5416  =  24 bp
   8 amino acids  =  923.1 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)
1316 .. 5416  =  4101 bp
1367 amino acids  =  158.3 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
5630 .. 6646  =  1017 bp
chimera between introns from chicken β-actin and
rabbit β-globin
chimeric intron
5630 .. 6646  =  1017 bp
chimera between introns from chicken β-actin and
rabbit β-globin
AmpR
7420 .. 8280  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7420 .. 7488  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7420 .. 8280  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7489 .. 8280  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7420 .. 8280  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
8451 .. 9039  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8451 .. 9039  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
6905 .. 7284  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
6905 .. 7284  =  380 bp
human cytomegalovirus immediate early enhancer
chicken β-actin promoter
6647 .. 6903  =  257 bp
contains a 21-bp deletion relative to the standard
chicken β-actin promoter
chicken β-actin promoter
6647 .. 6903  =  257 bp
contains a 21-bp deletion relative to the standard
chicken β-actin promoter
SV40 promoter
146 .. 341  =  196 bp
SV40 early promoter
SV40 promoter
146 .. 341  =  196 bp
SV40 early promoter
SV40 poly(A) signal
6 .. 140  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
6 .. 140  =  135 bp
SV40 polyadenylation signal
AmpR promoter
7315 .. 7419  =  105 bp
AmpR promoter
7315 .. 7419  =  105 bp
poly(A)
1103 .. 1197  =  95 bp
enables microinjection of poly(A)-tailed mRNA after
in vitro transcription
poly(A)
1103 .. 1197  =  95 bp
enables microinjection of poly(A)-tailed mRNA after
in vitro transcription
3' UTR
1198 .. 1267  =  70 bp
portion of the 3' UTR from the human TBPL1 gene
3' UTR
1198 .. 1267  =  70 bp
portion of the 3' UTR from the human TBPL1 gene
β-globin poly(A) signal
875 .. 930  =  56 bp
rabbit β-globin polyadenylation signal
β-globin poly(A) signal
875 .. 930  =  56 bp
rabbit β-globin polyadenylation signal
FLAG
5471 .. 5494  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
5471 .. 5494  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
SV40 NLS
1283 .. 1303  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1283 .. 1303  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5441 .. 5461  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5441 .. 5461  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T3 promoter
5555 .. 5573  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T3 promoter
5555 .. 5573  =  19 bp
promoter for bacteriophage T3 RNA polymerase
ATG
5495 .. 5497  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
5495 .. 5497  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
160 .. 295  =  136 bp
SV40 origin of replication
SV40 ori
160 .. 295  =  136 bp
SV40 origin of replication
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