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Plasmid Files

pX335

Zhang lab plasmid for expressing a chimeric guide RNA (gRNA) plus Cas9 with the D10A nickase mutation. Also known as pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A).

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pX335 Sequence and MappX335.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #42335
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 BbsI (245) SV40 NLS BbsI (267) CMV enhancer chicken β- actin promoter ATG SV40 NLS pX335 8433 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)
1313 .. 5413  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 1:  
   1313 .. 1336  =  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)
1313 .. 5413  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 2:  
   1337 .. 1339  =  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)
1313 .. 5413  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 3:  
   1340 .. 5413  =  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)
1313 .. 5413  =  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
6753 .. 7613  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6753 .. 6821  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6753 .. 7613  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6822 .. 7613  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6753 .. 7613  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
7784 .. 8372  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7784 .. 8372  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5911 .. 6366  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5911 .. 6366  =  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 .. 1232  =  228 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
hybrid intron
1005 .. 1232  =  228 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
bGH poly(A) signal
5480 .. 5687  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5480 .. 5687  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
5696 .. 5836  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
5696 .. 5836  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
6648 .. 6752  =  105 bp
AmpR promoter
6648 .. 6752  =  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
HA
1253 .. 1279  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
1253 .. 1279  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
1283 .. 1303  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1283 .. 1303  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5417 .. 5437  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5417 .. 5437  =  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
1250 .. 1252  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1250 .. 1252  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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