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Plasmid Files

pSpCas9(BB)-2A-GFP (PX458)

Zhang lab plasmid for expressing a chimeric guide RNA (gRNA) plus EGFP and human codon-optimized Cas9.

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pSpCas9(BB)-2A-GFP (PX458) Sequence and MappSpCas9(BB)-2A-GFP (PX458).dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #48138
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 BbsI (245) AAV2 ITR EcoRI (6302) EcoRI (5519) BbsI (267) polIII terminator chicken β- actin promoter ATG pSpCas9(BB)-2A-GFP (PX458) 9288 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.
EcoRI  (6302)
2 sites
G A A T T C C T T A A G
EcoRI  (5519)
2 sites
G A A T T C C T T A A G
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
1370 .. 5470  =  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
1370 .. 5470  =  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
7608 .. 8468  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7608 .. 7676  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7608 .. 8468  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7677 .. 8468  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7608 .. 8468  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
EGFP
5588 .. 6301  =  714 bp
238 amino acids  =  26.8 kDa
   Segment 1:  1a  
   5588 .. 5590  =  3 bp
   1 amino acid  =  117.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
5588 .. 6301  =  714 bp
238 amino acids  =  26.8 kDa
   Segment 2:  
   5591 .. 6301  =  711 bp
   237 amino acids  =  26.7 kDa
Product: enhanced GFP
mammalian codon-optimized
EGFP
5588 .. 6301  =  714 bp
238 amino acids  =  26.8 kDa
2 segments
Product: enhanced GFP
mammalian codon-optimized
ori
8639 .. 9227  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8639 .. 9227  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
6766 .. 7221  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6766 .. 7221  =  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
6335 .. 6542  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
6335 .. 6542  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
6551 .. 6691  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
6551 .. 6691  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
7503 .. 7607  =  105 bp
AmpR promoter
7503 .. 7607  =  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
3xFLAG
1253 .. 1318  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
1253 .. 1318  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
T2A
5534 .. 5587  =  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
5534 .. 5587  =  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.
nucleoplasmin NLS
5471 .. 5518  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
5471 .. 5518  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SV40 NLS
1325 .. 1345  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1325 .. 1345  =  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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