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Plasmid Files

pX330

Zhang lab plasmid for expressing a chimeric guide RNA (gRNA) together with human codon-optimized Cas9. Also known as pX330-U6-Chimeric_BB-CBh-hSpCas9.

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pX330 Sequence and MappX330.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #42230
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 pol III terminator BbsI (7954) BbsI (7932) nucleoplasmin NLS chicken β- actin promoter ATG pX330 8484 bp
BbsI  (7954)
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  (7932)
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
552 .. 4652  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
552 .. 4652  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
AmpR
6007 .. 6867  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6007 .. 6075  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6007 .. 6867  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6076 .. 6867  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6007 .. 6867  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
7038 .. 7626  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7038 .. 7626  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5165 .. 5620  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5165 .. 5620  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
8127 .. 8412  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
CMV enhancer
8127 .. 8412  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
chicken β-actin promoter
8414 .. 186  =  257 bp
contains a 19-bp deletion relative to the standard
β-actin promoter
chicken β-actin promoter
8414 .. 186  =  257 bp
contains a 19-bp deletion relative to the standard
β-actin promoter
U6 promoter
7688 .. 7928  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
7688 .. 7928  =  241 bp
RNA polymerase III promoter for human U6 snRNA
hybrid intron
187 .. 414  =  228 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
hybrid intron
187 .. 414  =  228 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
bGH poly(A) signal
4734 .. 4941  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
4734 .. 4941  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
4950 .. 5090  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
4950 .. 5090  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
5902 .. 6006  =  105 bp
AmpR promoter
5902 .. 6006  =  105 bp
gRNA scaffold
7955 .. 8030  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
7955 .. 8030  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
3xFLAG
435 .. 500  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
435 .. 500  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
nucleoplasmin NLS
4653 .. 4700  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
4653 .. 4700  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SV40 NLS
507 .. 527  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
507 .. 527  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
pol III terminator
8031 .. 8036  =  6 bp
RNA polymerase III transcription terminator
pol III terminator
8031 .. 8036  =  6 bp
RNA polymerase III transcription terminator
ATG
432 .. 434  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
432 .. 434  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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