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Plasmid Files

pCas-Guide-Nickase

Mammalian vector for co-expressing a guide RNA (gRNA) together with the D10A nickase mutant of Cas9, for genome editing using the CRISPR/Cas9 system.

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pCas-Guide-Nickase Sequence and MappCas-Guide-Nickase.dna
Map and Sequence File   
Sequence Author:  OriGene
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 CF3 Primer (128 .. 149) FLAG SV40 NLS SV40 NLS BamHI (334) BsmBI (384) polIII terminator CMV enhancer pCas-Guide-Nickase 8033 bp
BamHI  (334)
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.
BsmBI  (384)
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.
CF3 Primer
22-mer  /  45% GC
1 binding site
128 .. 149  =  22 annealed bases
Tm  =  56°C
Cas9(D10A)
1197 .. 5300  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 1:  
   1197 .. 1223  =  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)
1197 .. 5300  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 2:  
   1224 .. 1226  =  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)
1197 .. 5300  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 3:  
   1227 .. 5300  =  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)
1197 .. 5300  =  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
6994 .. 7854  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6994 .. 7785  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6994 .. 7854  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7786 .. 7854  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6994 .. 7854  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
hGH poly(A) signal
5488 .. 6110  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
5488 .. 6110  =  623 bp
human growth hormone polyadenylation signal
ori
6231 .. 6819  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6231 .. 6819  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
511 .. 890  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
511 .. 890  =  380 bp
human cytomegalovirus immediate early enhancer
U6 promoter
90 .. 330  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
90 .. 330  =  241 bp
RNA polymerase III promoter for human U6 snRNA
CMV promoter
891 .. 1094  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
891 .. 1094  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
gRNA scaffold
384 .. 459  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
384 .. 459  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
Myc
5370 .. 5399  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
5370 .. 5399  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
FLAG
5418 .. 5441  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
5418 .. 5441  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
SV40 NLS
5301 .. 5321  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5301 .. 5321  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5328 .. 5348  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5328 .. 5348  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
1120 .. 1138  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1120 .. 1138  =  19 bp
promoter for bacteriophage T7 RNA polymerase
polIII terminator
460 .. 465  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
460 .. 465  =  6 bp
RNA polymerase III transcription terminator
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