pCas-Guide-CRISPRa

Mammalian gene activation vector for expressing a guide RNA (gRNA) containing MS2 aptamers plus a dCas9-VP64 fusion. Use together with pCRISPRa-Enhancer.

Sequence Author: OriGene

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CF3 Primer (128 .. 149) SV40 NLS SV40 NLS BamHI (334) BsmBI (384) CMV enhancer pCas-Guide-CRISPRa 8232 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.
BsmBI-v2 is an improved version of BsmBI.
CF3 Primer
22-mer  /  45% GC
1 binding site
128 .. 149  =  22 annealed bases
Tm  =  56°C
dCas9
1257 .. 5360  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 1:  
   1257 .. 1283  =  27 bp
   9 amino acids  =  1.1 kDa
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
dCas9
1257 .. 5360  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 2:  
   1284 .. 1286  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
dCas9
1257 .. 5360  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 3:  
   1287 .. 3773  =  2487 bp
   829 amino acids  =  96.3 kDa
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
dCas9
1257 .. 5360  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 4:  
   3774 .. 3776  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
dCas9
1257 .. 5360  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 5:  
   3777 .. 5360  =  1584 bp
   528 amino acids  =  60.9 kDa
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
dCas9
1257 .. 5360  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
SV40 NLS
5361 .. 5381  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 (simian virus 40) large T antigen
SV40 NLS
5361 .. 5381  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 (simian virus 40) large T antigen
SV40 NLS
5388 .. 5408  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 (simian virus 40) large T antigen
SV40 NLS
5388 .. 5408  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 (simian virus 40) large T antigen
VP64
5412 .. 5561  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)
VP64
5412 .. 5561  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)
Myc
5580 .. 5609  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc proto-oncogene) epitope tag
Myc
5580 .. 5609  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc proto-oncogene) epitope tag
FLAG
5628 .. 5651  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
FLAG
5628 .. 5651  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
AmpR
7193 .. 8053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   7193 .. 7984  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7193 .. 8053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   7985 .. 8053  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7193 .. 8053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
hGH poly(A) signal
5698 .. 6320  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
5698 .. 6320  =  623 bp
human growth hormone polyadenylation signal
ori
6441 .. 7029  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
6441 .. 7029  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CMV enhancer
571 .. 950  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
571 .. 950  =  380 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
951 .. 1154  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
951 .. 1154  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
gRNA scaffold
384 .. 519  =  136 bp
5 segments
   Segment 1:  
   384 .. 400  =  17 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system, modified to contain two MS2 RNA aptamers
gRNA scaffold
384 .. 519  =  136 bp
5 segments
   Segment 2:  MS2 aptamer  
   401 .. 419  =  19 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system, modified to contain two MS2 RNA aptamers
gRNA scaffold
384 .. 519  =  136 bp
5 segments
   Segment 3:  
   420 .. 470  =  51 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system, modified to contain two MS2 RNA aptamers
gRNA scaffold
384 .. 519  =  136 bp
5 segments
   Segment 4:  MS2 aptamer  
   471 .. 489  =  19 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system, modified to contain two MS2 RNA aptamers
gRNA scaffold
384 .. 519  =  136 bp
5 segments
   Segment 5:  
   490 .. 519  =  30 bp
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system, modified to contain two MS2 RNA aptamers
gRNA scaffold
384 .. 519  =  136 bp
5 segments
guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system, modified to contain two MS2 RNA aptamers
polIII terminator
520 .. 525  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
520 .. 525  =  6 bp
RNA polymerase III transcription terminator
ORF:  6410 .. 6766  =  357 bp
ORF:  118 amino acids  =  13.2 kDa
ORF:  1110 .. 5657  =  4548 bp
ORF:  1515 amino acids  =  175.2 kDa
ORF:  7323 .. 7589  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1515 .. 1757  =  243 bp
ORF:  80 amino acids  =  9.6 kDa
ORF:  3309 .. 3653  =  345 bp
ORF:  114 amino acids  =  13.5 kDa
ORF:  3807 .. 4211  =  405 bp
ORF:  134 amino acids  =  15.6 kDa
ORF:  4680 .. 5303  =  624 bp
ORF:  207 amino acids  =  24.3 kDa
ORF:  5345 .. 5593  =  249 bp
ORF:  82 amino acids  =  8.8 kDa
ORF:  6062 .. 6286  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  7193 .. 8053  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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