Resources
Plasmid Files

pST1374-N-NLS-flag-linker-Cas9-D10A

Plasmid for mammalian cell expression of Cas9 with the D10A nickase mutation and with an N-terminal nuclear localization signal followed by a FLAG® tag.

To see this sequence with restriction sites, features, and translations, please download
 SnapGene or the free  SnapGene Viewer.

pST1374-N-NLS-flag-linker-Cas9-D10A Sequence and MappST1374-N-NLS-flag-linker-Cas9-D10A.dna
Map and Sequence File   
Sequence Author:  Huang Lab / Addgene #51130
Download Free Trial Get SnapGene Viewer

 AhdI (8304) FspI (8081) SgrDI (7378) MfeI (7215) SpeI (7127) CMV enhancer SnaBI (6790) SacI (6566) Eco53kI (6564) BmtI (6485) NheI (6481) ATG SV40 NLS NotI (6444) XbaI (6412) PflMI (6377) RsrII (6118) Bsu36I (5864) PaeR7I - XhoI (5176) BbvCI (5120) BamHI (4974) BstZ17I (258) MscI (943) TspMI - XmaI (1050) SmaI (1052) AvrII (1071) StuI (1076) SexAI * (1303) PmeI (2123) AgeI (2149) PspOMI (2206) ApaI (2210) KflI (2937) BstXI (3532) BmgBI (3807) EcoRV (4464) pST1374-N-NLS-flag-linker-Cas9-D10A 9317 bp
AhdI  (8304)
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.
FspI  (8081)
1 site
T G C G C A A C G C G T
SgrDI  (7378)
1 site
C G T C G A C G G C A G C T G C
MfeI  (7215)
1 site
C A A T T G G T T A A C
SpeI  (7127)
1 site
A C T A G T T G A T C A
SnaBI  (6790)
1 site
T A C G T A A T G C A T
SacI  (6566)
1 site
G A G C T C C T C G A G
Eco53kI  (6564)
1 site
G A G C T C C T C G A G
BmtI  (6485)
1 site
G C T A G C C G A T C G
NheI  (6481)
1 site
G C T A G C C G A T C G
NotI  (6444)
1 site
G C G G C C G C C G C C G G C G
XbaI  (6412)
1 site
T C T A G A A G A T C T
PflMI  (6377)
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  (6118)
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  (5864)
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  (5176)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (5176)
1 site
C T C G A G G A G C T C
BbvCI  (5120)
1 site
C C T C A G C G G A G T C G
BamHI  (4974)
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.
BstZ17I  (258)
1 site
G T A T A C C A T A T G
MscI  (943)
1 site
T G G C C A A C C G G T
TspMI  (1050)
1 site
C C C G G G G G G C C C
XmaI  (1050)
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  (1052)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AvrII  (1071)
1 site
C C T A G G G G A T C C
StuI  (1076)
1 site
A G G C C T T C C G G A
SexAI  (1303)
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  (2123)
1 site
G T T T A A A C C A A A T T T G
AgeI  (2149)
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.
PspOMI  (2206)
1 site
G G G C C C C C C G G G
ApaI  (2210)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
KflI  (2937)
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.
BstXI  (3532)
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.
BmgBI  (3807)
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.
EcoRV  (4464)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
Cas9(D10A)
2212 .. 6315  =  4104 bp
1367 amino acids  =  158.3 kDa
   Segment 3:  
   2212 .. 6288  =  4077 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)
2212 .. 6315  =  4104 bp
1367 amino acids  =  158.3 kDa
   Segment 2:  
   6289 .. 6291  =  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)
2212 .. 6315  =  4104 bp
1367 amino acids  =  158.3 kDa
   Segment 1:  
   6292 .. 6315  =  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)
2212 .. 6315  =  4104 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
AmpR
7517 .. 8377  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7517 .. 7585  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7517 .. 8377  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7586 .. 8377  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7517 .. 8377  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
8548 .. 9136  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8548 .. 9136  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1404 .. 1832  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1404 .. 1832  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
BSD
549 .. 947  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
549 .. 947  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
CMV enhancer
6767 .. 7146  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
6767 .. 7146  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
1061 .. 1390  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
1061 .. 1390  =  330 bp
SV40 enhancer and early promoter
bGH poly(A) signal
1878 .. 2102  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
1878 .. 2102  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
6563 .. 6766  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
6563 .. 6766  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
270 .. 391  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
270 .. 391  =  122 bp
SV40 polyadenylation signal
AmpR promoter
7412 .. 7516  =  105 bp
AmpR promoter
7412 .. 7516  =  105 bp
EM7 promoter
966 .. 1013  =  48 bp
synthetic bacterial promoter
EM7 promoter
966 .. 1013  =  48 bp
synthetic bacterial promoter
FLAG
6418 .. 6441  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
6418 .. 6441  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
SV40 NLS
6451 .. 6471  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6451 .. 6471  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
6500 .. 6518  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
6500 .. 6518  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ATG
6475 .. 6477  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
6475 .. 6477  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
1075 .. 1210  =  136 bp
SV40 origin of replication
SV40 ori
1075 .. 1210  =  136 bp
SV40 origin of replication
Try SnapGene and create your own beautiful maps

Individual Sequences & Maps

SnapGene offers the fastest and easiest way to plan, visualize, and document your molecular biology procedures.

Priced accessibly so that everyone in your lab can have a license.

Learn More...

SnapGene Viewer is a versatile tool for creating and sharing richly annotated sequence files. It opens many common file formats.

Free! Because there should be no barriers to seeing your data.

Learn More...

The map, notes, and annotations on this page and in the sequence/map file are copyrighted material. This material may be used without restriction by academic, nonprofit, and governmental entities, except that the source must be cited as "www.snapgene.com/resources". Commercial entities must contact GSL Biotech LLC for permission and terms of use.

Copyright © 2016 GSL Biotech LLC | Site Map | Privacy | Legal Disclaimers   Subscribe to Our Newsletter