Resources
Plasmid Files

pSpCas9n(BB)-2A-Puro (PX462) V2.0

Zhang lab plasmid for expressing a chimeric guide RNA (gRNA) plus a puromycin resistance marker and human codon-optimized Cas9 with the D10A nickase mutation.

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

pSpCas9n(BB)-2A-Puro (PX462) V2.0 Sequence and MappSpCas9n(BB)-2A-Puro (PX462) V2.0.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #62987
Download Free Trial Get SnapGene Viewer

 BbsI (245) AAV2 ITR BbsI (267) polIII terminator chicken β- actin promoter ATG pSpCas9n(BB)-2A-Puro (PX462) V2.0 9175 bp
BbsI  (245)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BbsI  (267)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
Cas9(D10A)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 1:  
   1371 .. 1394  =  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)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 2:  
   1395 .. 1397  =  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)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 3:  
   1398 .. 5471  =  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)
1371 .. 5471  =  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
AmpR
7495 .. 8355  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7495 .. 7563  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7495 .. 8355  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7564 .. 8355  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7495 .. 8355  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
5589 .. 6188  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
5589 .. 6188  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
8526 .. 9114  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8526 .. 9114  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
6653 .. 7108  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6653 .. 7108  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
440 .. 725  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
CMV enhancer
440 .. 725  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
chicken β-actin promoter
727 .. 1004  =  278 bp
chicken β-actin promoter
727 .. 1004  =  278 bp
U6 promoter
1 .. 241  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
1 .. 241  =  241 bp
RNA polymerase III promoter for human U6 snRNA
hybrid intron
1005 .. 1233  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
hybrid intron
1005 .. 1233  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
bGH poly(A) signal
6222 .. 6429  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
6222 .. 6429  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
6438 .. 6578  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
6438 .. 6578  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
7390 .. 7494  =  105 bp
AmpR promoter
7390 .. 7494  =  105 bp
gRNA scaffold
268 .. 343  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
268 .. 343  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
3xFLAG
1254 .. 1319  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
1254 .. 1319  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
T2A
5535 .. 5588  =  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
5535 .. 5588  =  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.
nucleoplasmin NLS
5472 .. 5519  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
5472 .. 5519  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SV40 NLS
1326 .. 1346  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1326 .. 1346  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
polIII terminator
344 .. 349  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
344 .. 349  =  6 bp
RNA polymerase III transcription terminator
ATG
1251 .. 1253  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1251 .. 1253  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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