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Plasmid Files

piCRg Entry

Entry vector encoding human codon-optimized S. pyogenes Cas9 together with a chimeric guide RNA (gRNA) expression cassette.

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piCRg Entry Sequence and MappiCRg Entry.dna
Map and Sequence File   
Sequence Author:  Huangfu Lab / Addgene #58904
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 polIII terminator BbsI (7772) BbsI (7750) bGH poly(A) signal ATG piCRg Entry 8302 bp
BbsI  (7772)
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  (7750)
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
3108 .. 7208  =  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
3108 .. 7208  =  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
NeoR/KanR
1296 .. 2090  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
1296 .. 2090  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
481 .. 1069  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
481 .. 1069  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hPGK promoter
2337 .. 2847  =  511 bp
human phosphoglycerate kinase 1 promoter
hPGK promoter
2337 .. 2847  =  511 bp
human phosphoglycerate kinase 1 promoter
U6 promoter
7506 .. 7746  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
7506 .. 7746  =  241 bp
RNA polymerase III promoter for human U6 snRNA
bGH poly(A) signal
7290 .. 7497  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
7290 .. 7497  =  208 bp
bovine growth hormone polyadenylation signal
attL1
2858 .. 2957  =  100 bp
recombination site for the Gateway® LR reaction
attL1
2858 .. 2957  =  100 bp
recombination site for the Gateway® LR reaction
attL2
7966 .. 8065  =  100 bp
recombination site for the Gateway® LR reaction
attL2
7966 .. 8065  =  100 bp
recombination site for the Gateway® LR reaction
gRNA scaffold
7773 .. 7848  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
7773 .. 7848  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
3xFLAG
2991 .. 3056  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
2991 .. 3056  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
nucleoplasmin NLS
7209 .. 7256  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
7209 .. 7256  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SV40 NLS
3063 .. 3083  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
3063 .. 3083  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
polIII terminator
7849 .. 7854  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
7849 .. 7854  =  6 bp
RNA polymerase III transcription terminator
ATG
2988 .. 2990  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
2988 .. 2990  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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