pCas-Guide-CRISPRi

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CF3 Primer (128 .. 149) SV40 NLS SV40 NLS BamHI (334) BsmBI (384) polIII terminator pCas-Guide-CRISPRi 9198 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
1197 .. 5300  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 1:  
   1197 .. 1223  =  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
1197 .. 5300  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 2:  
   1224 .. 1226  =  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
1197 .. 5300  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 3:  
   1227 .. 3713  =  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
1197 .. 5300  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 4:  
   3714 .. 3716  =  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
1197 .. 5300  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 5:  
   3717 .. 5300  =  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
1197 .. 5300  =  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
5301 .. 5321  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 (simian virus 40) large T antigen
SV40 NLS
5301 .. 5321  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 (simian virus 40) large T antigen
SV40 NLS
5328 .. 5348  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 (simian virus 40) large T antigen
SV40 NLS
5328 .. 5348  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 (simian virus 40) large T antigen
KRAB
5409 .. 5603  =  195 bp
65 amino acids  =  7.8 kDa
Product: Krüppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)
KRAB
5409 .. 5603  =  195 bp
65 amino acids  =  7.8 kDa
Product: Krüppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)
SV40 NLS
5637 .. 5657  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 (simian virus 40) large T antigen
SV40 NLS
5637 .. 5657  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 (simian virus 40) large T antigen
MeCP2
5667 .. 6524  =  858 bp
286 amino acids  =  30.6 kDa
Product: transcriptional repression domain from rat methyl-CpG-binding protein 2 (Nan et al., 1997)
MeCP2
5667 .. 6524  =  858 bp
286 amino acids  =  30.6 kDa
Product: transcriptional repression domain from rat methyl-CpG-binding protein 2 (Nan et al., 1997)
AmpR
8159 .. 9019  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   8159 .. 8950  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
8159 .. 9019  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   8951 .. 9019  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
8159 .. 9019  =  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
6664 .. 7286  =  623 bp
human growth hormone polyadenylation signal
hGH poly(A) signal
6664 .. 7286  =  623 bp
human growth hormone polyadenylation signal
ori
7407 .. 7995  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
7407 .. 7995  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CMV enhancer
511 .. 890  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
511 .. 890  =  380 bp
human cytomegalovirus immediate early enhancer
U6 promoter
90 .. 330  =  241 bp
Truncated RNA polymerase III promoter for human U6 snRNA used in pSIREN vectors such as pSIREN-RetroQ-ZsGreen1. Expected transcription start site is 8bp downstream of this feature.
U6 promoter
90 .. 330  =  241 bp
Truncated RNA polymerase III promoter for human U6 snRNA used in pSIREN vectors such as pSIREN-RetroQ-ZsGreen1. Expected transcription start site is 8bp downstream of this feature.
CMV promoter
891 .. 1094  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
891 .. 1094  =  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
6546 .. 6575  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc proto-oncogene) epitope tag
Myc
6546 .. 6575  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc proto-oncogene) epitope tag
FLAG
6594 .. 6617  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
FLAG
6594 .. 6617  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an enterokinase cleavage site
polIII terminator
460 .. 465  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
460 .. 465  =  6 bp
RNA polymerase III transcription terminator
ORF:  7376 .. 7732  =  357 bp
ORF:  118 amino acids  =  13.2 kDa
ORF:  1050 .. 6527  =  5478 bp
ORF:  1825 amino acids  =  208.1 kDa
ORF:  8289 .. 8555  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1455 .. 1697  =  243 bp
ORF:  80 amino acids  =  9.6 kDa
ORF:  3249 .. 3593  =  345 bp
ORF:  114 amino acids  =  13.5 kDa
ORF:  3747 .. 4151  =  405 bp
ORF:  134 amino acids  =  15.6 kDa
ORF:  4620 .. 5243  =  624 bp
ORF:  207 amino acids  =  24.3 kDa
ORF:  5516 .. 5791  =  276 bp
ORF:  91 amino acids  =  10.1 kDa
ORF:  6257 .. 6559  =  303 bp
ORF:  100 amino acids  =  10.5 kDa
ORF:  7028 .. 7252  =  225 bp
ORF:  74 amino acids  =  8.1 kDa
ORF:  8159 .. 9019  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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