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Plasmid Files

pCas-Guide-EF1a-GFP

Mammalian vector for co-expressing a guide RNA (gRNA) together with Cas9 and TurboGFP, for genome editing using the S. pyogenes CRISPR/Cas9 system.

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pCas-Guide-EF1a-GFP Sequence and MappCas-Guide-EF1a-GFP.dna
Map and Sequence File   
Sequence Author:  OriGene
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 CF3 Primer (128 .. 149) FLAG SV40 NLS SV40 NLS BamHI (334) BsmBI (384) CMV enhancer pCas-Guide-EF1a-GFP 10,493 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
Cas9
3657 .. 7760  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
3657 .. 7760  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
EF-1α promoter
480 .. 1658  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
EF-1α promoter
480 .. 1658  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
AmpR
9454 .. 10,314  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   9454 .. 10,245  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9454 .. 10,314  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,246 .. 10,314  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9454 .. 10,314  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TurboGFP
1737 .. 2432  =  696 bp
232 amino acids  =  25.7 kDa
Product: improved green fluorescent protein from
Pontellina plumata (Evdokimov et al., 2006)
mammalian codon-optimized
TurboGFP
1737 .. 2432  =  696 bp
232 amino acids  =  25.7 kDa
Product: improved green fluorescent protein from
Pontellina plumata (Evdokimov et al., 2006)
mammalian codon-optimized
hGH poly(A) signal
7948 .. 8570  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
7948 .. 8570  =  623 bp
human growth hormone polyadenylation signal
ori
8691 .. 9279  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8691 .. 9279  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
3047 .. 3350  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
3047 .. 3350  =  304 bp
human cytomegalovirus immediate early enhancer
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
CMV promoter
3351 .. 3554  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
3351 .. 3554  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
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
Myc
7830 .. 7859  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
7830 .. 7859  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
FLAG
7878 .. 7901  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
7878 .. 7901  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
SV40 NLS
7761 .. 7781  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7761 .. 7781  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7788 .. 7808  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7788 .. 7808  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
3580 .. 3598  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3580 .. 3598  =  19 bp
promoter for bacteriophage T7 RNA polymerase
polIII terminator
460 .. 465  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
460 .. 465  =  6 bp
RNA polymerase III transcription terminator
EF-1α intron A
711 .. 1649  =  939 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
711 .. 1649  =  939 bp
intron upstream of the start codon of human EF-1α
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