pSpCas9(BB)-2A-Puro (PX459) V2.0

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

Sequence Author: Zhang Lab / Addgene #62988

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BbsI (245) AAV2 ITR BbsI (267) polIII terminator chicken β-actin promoter ATG pSpCas9(BB)-2A-Puro (PX459) V2.0 9175 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.
ATG
1251 .. 1253  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1251 .. 1253  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
3xFLAG
1254 .. 1319  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags, followed by an enterokinase cleavage site
3xFLAG
1254 .. 1319  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags, followed by an enterokinase cleavage site
SV40 NLS
1326 .. 1346  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
1326 .. 1346  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
Cas9
1371 .. 5471  =  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
1371 .. 5471  =  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
nucleoplasmin NLS
5472 .. 5519  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from nucleoplasmin
nucleoplasmin NLS
5472 .. 5519  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from nucleoplasmin
T2A
5535 .. 5588  =  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
5535 .. 5588  =  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.
PuroR
5589 .. 6188  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
5589 .. 6188  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
AmpR
7495 .. 8355  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   7495 .. 7563  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7495 .. 8355  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   7564 .. 8355  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7495 .. 8355  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
8526 .. 9114  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
8526 .. 9114  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
6653 .. 7108  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
6653 .. 7108  =  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 .. 1233  =  229 bp
hybrid between chicken β-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)
hybrid intron
1005 .. 1233  =  229 bp
hybrid between chicken β-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)
bGH poly(A) signal
6222 .. 6429  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
6222 .. 6429  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
6438 .. 6578  =  141 bp
inverted terminal repeat of adeno-associated virus serotype 2
AAV2 ITR
6438 .. 6578  =  141 bp
inverted terminal repeat of adeno-associated virus serotype 2
AmpR promoter
7390 .. 7494  =  105 bp
AmpR promoter
7390 .. 7494  =  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
polIII terminator
344 .. 349  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
344 .. 349  =  6 bp
RNA polymerase III transcription terminator
ORF:  1189 .. 1428  =  240 bp
ORF:  79 amino acids  =  9.1 kDa
ORF:  7495 .. 8355  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  635 .. 982  =  348 bp
ORF:  115 amino acids  =  12.1 kDa
ORF:  6452 .. 6676  =  225 bp
ORF:  74 amino acids  =  8.3 kDa
ORF:  825 .. 1136  =  312 bp
ORF:  103 amino acids  =  10.7 kDa
ORF:  1251 .. 6188  =  4938 bp
ORF:  1645 amino acids  =  188.4 kDa
ORF:  2717 .. 3010  =  294 bp
ORF:  97 amino acids  =  10.8 kDa
ORF:  3281 .. 3730  =  450 bp
ORF:  149 amino acids  =  17.3 kDa
ORF:  6352 .. 6633  =  282 bp
ORF:  93 amino acids  =  10.4 kDa
ORF:  1230 .. 3887  =  2658 bp
ORF:  885 amino acids  =  93.1 kDa
ORF:  7959 .. 8225  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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