Resources
Plasmid Files

pGuide

Mammalian vector for expressing a guide RNA (gRNA) under control of the U6 promoter, for genome editing using the S. pyogenes CRISPR/Cas9 system.

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pGuide.dna
Map and Sequence File:    Download    Open   
Sequence Author:  OriGene
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CF3 primer (128 .. 149) BamHI (334) BsmBI (384) pGuide 3152 bp
BamHI  (334)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
BsmBI  (384)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
CF3 primer
22-mer  /  45% GC
1 binding site
128 .. 149  =  22 annealed bases
Tm  =  56°C
primer for sequencing the inserted gRNA
AmpR
2113 .. 2973  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2113 .. 2904  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2113 .. 2973  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2905 .. 2973  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2113 .. 2973  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1361 .. 1949  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1361 .. 1949  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
U6 promoter
90 .. 330  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
90 .. 330  =  241 bp
RNA polymerase III promoter for human U6 snRNA
gRNA scaffold
384 .. 459  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
gRNA scaffold
384 .. 459  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
ORF:  1330 .. 1686  =  357 bp
ORF:  118 amino acids  =  13.2 kDa
ORF:  2243 .. 2509  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2113 .. 2973  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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