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Plasmid Files

pST1374-N-NLS-flag-linker-Cas9-H840A

Plasmid for mammalian cell expression of Cas9 with the H840A nickase mutation and with an N-terminal nuclear localization signal followed by a FLAG® tag.

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pST1374-N-NLS-flag-linker-Cas9-H840A Sequence and MappST1374-N-NLS-flag-linker-Cas9-H840A.dna
Map and Sequence File   
Sequence Author:  Huang Lab / Addgene #51129
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 AmpR promoter FspI (8617) AhdI (8395) BstZ17I (7123) PflFI - Tth111I (6709) MscI (6438) EM7 promoter SmaI (6329) TspMI - XmaI (6327) AvrII (6306) StuI (6305) SexAI * (6073) PmeI (5257) AgeI (5227) ApaI (5174) PspOMI (5170) SgrDI (9316) MfeI (161) SpeI (249) SnaBI (590) Eco53kI (816) SacI (818) NheI (895) BmtI (899) ATG SV40 NLS NotI (932) XbaI (964) PflMI (1006) RsrII (1259) Bsu36I (1513) PaeR7I - XhoI (2200) BbvCI (2257) BamHI (2402) BmgBI (3573) BstXI (3852) KflI (4440) pST1374-N-NLS-flag-linker-Cas9-H840A 9318 bp
FspI  (8617)
1 site
T G C G C A A C G C G T
AhdI  (8395)
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  (7123)
1 site
G T A T A C C A T A T G
PflFI  (6709)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (6709)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
MscI  (6438)
1 site
T G G C C A A C C G G T
SmaI  (6329)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (6327)
1 site
C C C G G G G G G C C C
XmaI  (6327)
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.
AvrII  (6306)
1 site
C C T A G G G G A T C C
StuI  (6305)
1 site
A G G C C T T C C G G A
SexAI  (6073)
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.
PmeI  (5257)
1 site
G T T T A A A C C A A A T T T G
AgeI  (5227)
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.
ApaI  (5174)
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  (5170)
1 site
G G G C C C C C C G G G
SgrDI  (9316)
1 site
C G T C G A C G G C A G C T G C
MfeI  (161)
1 site
C A A T T G G T T A A C
SpeI  (249)
1 site
A C T A G T T G A T C A
SnaBI  (590)
1 site
T A C G T A A T G C A T
Eco53kI  (816)
1 site
G A G C T C C T C G A G
SacI  (818)
1 site
G A G C T C C T C G A G
NheI  (895)
1 site
G C T A G C C G A T C G
BmtI  (899)
1 site
G C T A G C C G A T C G
NotI  (932)
1 site
G C G G C C G C C G C C G G C G
XbaI  (964)
1 site
T C T A G A A G A T C T
PflMI  (1006)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
RsrII  (1259)
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.
Bsu36I  (1513)
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.
PaeR7I  (2200)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (2200)
1 site
C T C G A G G A G C T C
BbvCI  (2257)
1 site
C C T C A G C G G A G T C G
BamHI  (2402)
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  (3573)
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  (3852)
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.
KflI  (4440)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
Cas9(H840A)
1066 .. 5169  =  4104 bp
1367 amino acids  =  158.2 kDa
   Segment 1:  
   1066 .. 3579  =  2514 bp
   838 amino acids  =  97.3 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 H840A mutation in the HNH catalytic
domain
Cas9(H840A)
1066 .. 5169  =  4104 bp
1367 amino acids  =  158.2 kDa
   Segment 2:  
   3580 .. 3582  =  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 H840A mutation in the HNH catalytic
domain
Cas9(H840A)
1066 .. 5169  =  4104 bp
1367 amino acids  =  158.2 kDa
   Segment 3:  
   3583 .. 5169  =  1587 bp
   528 amino acids  =  60.9 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 H840A mutation in the HNH catalytic
domain
Cas9(H840A)
1066 .. 5169  =  4104 bp
1367 amino acids  =  158.2 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 H840A mutation in the HNH catalytic
domain
AmpR
8322 .. 9182  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8322 .. 9113  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8322 .. 9182  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   9114 .. 9182  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8322 .. 9182  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
7563 .. 8151  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7563 .. 8151  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5549 .. 5977  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5549 .. 5977  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
BSD
6435 .. 6833  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
6435 .. 6833  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
5991 .. 6321  =  331 bp
SV40 enhancer and early promoter
SV40 promoter
5991 .. 6321  =  331 bp
SV40 enhancer and early promoter
bGH poly(A) signal
5279 .. 5503  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5279 .. 5503  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
6991 .. 7112  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
6991 .. 7112  =  122 bp
SV40 polyadenylation signal
AmpR promoter
9183 .. 9287  =  105 bp
AmpR promoter
9183 .. 9287  =  105 bp
EM7 promoter
6369 .. 6416  =  48 bp
synthetic bacterial promoter
EM7 promoter
6369 .. 6416  =  48 bp
synthetic bacterial promoter
FLAG
940 .. 963  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
940 .. 963  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
SV40 NLS
910 .. 930  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
910 .. 930  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ATG
904 .. 906  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
904 .. 906  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
6172 .. 6307  =  136 bp
SV40 origin of replication
SV40 ori
6172 .. 6307  =  136 bp
SV40 origin of replication
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