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Plasmid Files

hCas9_D10A

Mammalian vector with a G418 resistance marker for expressing human codon-optimized Cas9 with the D10A nickase mutation.

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hCas9_D10A.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Church Lab / Addgene #41816
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MfeI (9529) SgrDI (9366) AhdI (8445) BstZ17I (7173) RsrII (6715) AvrII (5989) StuI (5988) SexAI * (5756) DraIII (5466) PmeI (4921) AgeI (4903) MluI (43) SpeI (64) SnaBI (405) XbaI (723) SbfI (995) XcmI (1099) BlpI (1696) Bsu36I (2014) BamHI (2753) BmgBI (3258) BstXI - PmlI (3537) FspAI (3698) AarI (4008) PaeR7I - XhoI (4291) EcoRI (4570) AscI (4703) hCas9_D10A 9553 bp
MfeI  (9529)
1 site
C A A T T G G T T A A C
SgrDI  (9366)
1 site
C G T C G A C G G C A G C T G C
AhdI  (8445)
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.
BstZ17I  (7173)
1 site
G T A T A C C A T A T G
RsrII  (6715)
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.
AvrII  (5989)
1 site
C C T A G G G G A T C C
StuI  (5988)
1 site
A G G C C T T C C G G A
SexAI  (5756)
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.
DraIII  (5466)
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.
PmeI  (4921)
1 site
G T T T A A A C C A A A T T T G
AgeI  (4903)
1 site
A C C G G T T G G C C A
MluI  (43)
1 site
A C G C G T T G C G C A
SpeI  (64)
1 site
A C T A G T T G A T C A
SnaBI  (405)
1 site
T A C G T A A T G C A T
XbaI  (723)
1 site
T C T A G A A G A T C T
SbfI  (995)
1 site
C C T G C A G G G G A C G T C C
XcmI  (1099)
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.
BlpI  (1696)
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.
Bsu36I  (2014)
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.
BamHI  (2753)
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.
BmgBI  (3258)
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.
BstXI  (3537)
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  (3537)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
FspAI  (3698)
1 site
R T G C G C A Y Y A C G C G T R
AarI  (4008)
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.
PaeR7I  (4291)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (4291)
1 site
C T C G A G G A G C T C
EcoRI  (4570)
1 site
G A A T T C C T T A A G
AscI  (4703)
1 site
G G C G C G C C C C G C G C G G
Cas9(D10A)
748 .. 4851  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 1:  
   748 .. 774  =  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)
748 .. 4851  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 2:  
   775 .. 777  =  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)
748 .. 4851  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 3:  
   778 .. 4851  =  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)
748 .. 4851  =  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
AmpR
8372 .. 9232  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8372 .. 9163  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
8372 .. 9232  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   9164 .. 9232  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
8372 .. 9232  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
6071 .. 6865  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
6071 .. 6865  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
ori
7613 .. 8201  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
7613 .. 8201  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
5233 .. 5661  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
5233 .. 5661  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
CMV enhancer
50 .. 429  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
50 .. 429  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
5675 .. 6004  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
5675 .. 6004  =  330 bp
SV40 enhancer and early promoter
CMV promoter
430 .. 633  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
430 .. 633  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
7041 .. 7162  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
7041 .. 7162  =  122 bp
SV40 polyadenylation signal
AmpR promoter
9233 .. 9337  =  105 bp
AmpR promoter
9233 .. 9337  =  105 bp
HSV TK poly(A) signal
4983 .. 5031  =  49 bp
herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
4983 .. 5031  =  49 bp
herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)
SV40 NLS
4864 .. 4884  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
4864 .. 4884  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 ori
5855 .. 5990  =  136 bp
SV40 origin of replication
SV40 ori
5855 .. 5990  =  136 bp
SV40 origin of replication
ORF:  589 .. 4887  =  4299 bp
ORF:  1432 amino acids  =  166.3 kDa
ORF:  6071 .. 6865  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  6243 .. 6629  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  8502 .. 8768  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  6380 .. 6634  =  255 bp
ORF:  84 amino acids  =  9.6 kDa
ORF:  8372 .. 9232  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  724 .. 1227  =  504 bp
ORF:  167 amino acids  =  18.5 kDa
ORF:  2152 .. 2670  =  519 bp
ORF:  172 amino acids  =  20.1 kDa
ORF:  2734 .. 3267  =  534 bp
ORF:  177 amino acids  =  19.9 kDa
ORF:  4261 .. 4794  =  534 bp
ORF:  177 amino acids  =  20.0 kDa
ORF:  648 .. 890  =  243 bp
ORF:  80 amino acids  =  9.6 kDa
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