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Plasmid Files

pX334

Zhang lab vector for expressing a Cas9 nickase mutant, a tracrRNA, and a guide sequence. Also known as pX334-U6-DR-BB-DR-Cbh-NLS-hSpCas9n(D10A)-NLS-H1-shorttracr-PGK-puro.

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pX334 Sequence and MappX334.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #42333
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 DR polIII terminator tracrRNA BbsI (283) BbsI (305) DR CMV enhancer chicken β- actin promoter ATG HA SV40 NLS pX334 10,152 bp
BbsI  (283)
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  (305)
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)
1263 .. 5363  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 1:  
   1263 .. 1286  =  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)
1263 .. 5363  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 2:  
   1287 .. 1289  =  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)
1263 .. 5363  =  4101 bp
1367 amino acids  =  158.3 kDa
   Segment 3:  
   1290 .. 5363  =  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)
1263 .. 5363  =  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
8472 .. 9332  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   8472 .. 8540  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8472 .. 9332  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8541 .. 9332  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8472 .. 9332  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
6758 .. 7357  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
6758 .. 7357  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
9503 .. 10,091  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9503 .. 10,091  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
PGK promoter
6219 .. 6718  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
6219 .. 6718  =  500 bp
mouse phosphoglycerate kinase 1 promoter
f1 ori
7630 .. 8085  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
7630 .. 8085  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
390 .. 675  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
CMV enhancer
390 .. 675  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
chicken β-actin promoter
677 .. 954  =  278 bp
chicken β-actin promoter
677 .. 954  =  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
955 .. 1182  =  228 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
hybrid intron
955 .. 1182  =  228 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
H1 promoter
5651 .. 5865  =  215 bp
human H1 RNA promoter
H1 promoter
5651 .. 5865  =  215 bp
human H1 RNA promoter
bGH poly(A) signal
5430 .. 5637  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5430 .. 5637  =  208 bp
bovine growth hormone polyadenylation signal
AmpR promoter
8367 .. 8471  =  105 bp
AmpR promoter
8367 .. 8471  =  105 bp
tracrRNA
5876 .. 5954  =  79 bp
trans-activating CRISPR RNA for the Streptococcus
pyogenes
CRISPR/Cas9 system
tracrRNA
5876 .. 5954  =  79 bp
trans-activating CRISPR RNA for the Streptococcus
pyogenes
CRISPR/Cas9 system
DR
252 .. 287  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR system
DR
252 .. 287  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR system
DR
304 .. 339  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR system
DR
304 .. 339  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR system
HA
1203 .. 1229  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
1203 .. 1229  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
1233 .. 1253  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1233 .. 1253  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5367 .. 5387  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5367 .. 5387  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
polIII terminator
340 .. 344  =  5 bp
RNA polymerase III transcription terminator
polIII terminator
340 .. 344  =  5 bp
RNA polymerase III transcription terminator
polIII terminator
5955 .. 5959  =  5 bp
RNA polymerase III transcription terminator
polIII terminator
5955 .. 5959  =  5 bp
RNA polymerase III transcription terminator
ATG
1200 .. 1202  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1200 .. 1202  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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