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Plasmid Files

pNW3

Plasmid for mammalian cell expression of the Csy4 CRISPR-associated endoribonuclease and a D10A nickase mutant of Cas9.

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pNW3 Sequence and MappNW3.dna
Map and Sequence File   
Sequence Author:  Joung Lab / Addgene #53372
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 SexAI * (9650) ScaI (9216) PvuI (9106) BsaI (8797) AhdI (8736) SV40 poly(A) signal AvrII (7510) SfiI (7463) HindIII (7139) BstXI (6652) NotI (6592) PaeR7I - XhoI (6514) SV40 NLS BmtI (6030) NheI (6026) FspAI (5334) SpeI (18) BlpI (1154) MreI - SgrAI (1311) XbaI (1623) Acc65I (1735) KpnI (1739) TspMI - XmaI (1746) SmaI (1748) AgeI (1756) BsmBI (2240) HpaI (2349) BstZ17I (2598) BmgBI (2603) BstEII (3098) BsiWI (3192) BsrGI (3942) EcoRV (4225) PshAI (4366) PvuII (4468) pNW3 9654 bp
SexAI  (9650)
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.
ScaI  (9216)
1 site
A G T A C T T C A T G A
PvuI  (9106)
1 site
C G A T C G G C T A G C
BsaI  (8797)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (8736)
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.
AvrII  (7510)
1 site
C C T A G G G G A T C C
SfiI  (7463)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
HindIII  (7139)
1 site
A A G C T T T T C G A A
BstXI  (6652)
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.
NotI  (6592)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (6514)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (6514)
1 site
C T C G A G G A G C T C
BmtI  (6030)
1 site
G C T A G C C G A T C G
NheI  (6026)
1 site
G C T A G C C G A T C G
FspAI  (5334)
1 site
R T G C G C A Y Y A C G C G T R
SpeI  (18)
1 site
A C T A G T T G A T C A
BlpI  (1154)
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.
MreI  (1311)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (1311)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
XbaI  (1623)
1 site
T C T A G A A G A T C T
Acc65I  (1735)
1 site
G G T A C C C C A T G G
KpnI  (1739)
1 site
G G T A C C C C A T G G
TspMI  (1746)
1 site
C C C G G G G G G C C C
XmaI  (1746)
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  (1748)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AgeI  (1756)
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.
BsmBI  (2240)
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.
HpaI  (2349)
1 site
G T T A A C C A A T T G
BstZ17I  (2598)
1 site
G T A T A C C A T A T G
BmgBI  (2603)
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.
BstEII  (3098)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
BsiWI  (3192)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
BsrGI  (3942)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
EcoRV  (4225)
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.
PshAI  (4366)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
PvuII  (4468)
1 site
C A G C T G G T C G A C
Cas9(D10A)
2384 .. 6487  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 1:  
   2384 .. 2410  =  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)
2384 .. 6487  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 2:  
   2411 .. 2413  =  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)
2384 .. 6487  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 3:  
   2414 .. 6487  =  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)
2384 .. 6487  =  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
chimeric intron
662 .. 1670  =  1009 bp
chimera between introns from chicken β-actin and
rabbit β-globin
chimeric intron
662 .. 1670  =  1009 bp
chimera between introns from chicken β-actin and
rabbit β-globin
AmpR
8663 .. 9523  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8663 .. 9454  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8663 .. 9523  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   9455 .. 9523  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8663 .. 9523  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
7904 .. 8492  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7904 .. 8492  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
Csy4
1769 .. 2329  =  561 bp
187 amino acids  =  21.4 kDa
Product: CRISPR-associated endoribonuclease
responsible for pre-crRNA processing in
Pseudomonas aeruginosa (Haurwitz et al., 2010)
Csy4
1769 .. 2329  =  561 bp
187 amino acids  =  21.4 kDa
Product: CRISPR-associated endoribonuclease
responsible for pre-crRNA processing in
Pseudomonas aeruginosa (Haurwitz et al., 2010)
CMV enhancer
4 .. 383  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4 .. 383  =  380 bp
human cytomegalovirus immediate early enhancer
chicken β-actin promoter
386 .. 661  =  276 bp
chicken β-actin promoter
386 .. 661  =  276 bp
SV40 promoter
7330 .. 7525  =  196 bp
SV40 early promoter
SV40 promoter
7330 .. 7525  =  196 bp
SV40 early promoter
SV40 poly(A) signal
7531 .. 7665  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
7531 .. 7665  =  135 bp
SV40 polyadenylation signal
AmpR promoter
9524 .. 9628  =  105 bp
AmpR promoter
9524 .. 9628  =  105 bp
3xFLAG
6521 .. 6586  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
6521 .. 6586  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
β-globin poly(A) signal
6741 .. 6796  =  56 bp
rabbit β-globin polyadenylation signal
β-globin poly(A) signal
6741 .. 6796  =  56 bp
rabbit β-globin polyadenylation signal
T2A
2330 .. 2383  =  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
2330 .. 2383  =  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.
SV40 NLS
6494 .. 6514  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6494 .. 6514  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 ori
7376 .. 7511  =  136 bp
SV40 origin of replication
SV40 ori
7376 .. 7511  =  136 bp
SV40 origin of replication
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