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Plasmid Files

pdCas9::BFP-humanized

Retroviral vector encoding human codon-optimized catalytically dead dCas9 fused to TagBFP, for visualizing genomic loci using a small guide RNA (sgRNA).

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pdCas9 BFP-humanized.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Qi Lab / Addgene #44247
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SgrAI (10,992) NdeI (10,620) BspDI - ClaI (7487) NsiI (7486) SacII (7047) RsrII (6949) BsiWI (6889) BfuAI - BspMI (6822) AgeI (6423) EcoRI (6303) PshAI (6190) BbsI (5858) BglII (1410) NcoI (1421) PflMI (1722) SgrDI (1789) BtgZI (1973) BsaBI * (2240) PsiI (2540) MluI (5256) pdCas9::BFP-humanized 11,170 bp
SgrAI  (10,992)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
NdeI  (10,620)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
BspDI  (7487)
1 site
A T C G A T T A G C T A
ClaI  (7487)
1 site
A T C G A T T A G C T A
NsiI  (7486)
1 site
A T G C A T T A C G T A
SacII  (7047)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
RsrII  (6949)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BsiWI  (6889)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
BfuAI  (6822)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (6822)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
AgeI  (6423)
1 site
A C C G G T T G G C C A
EcoRI  (6303)
1 site
G A A T T C C T T A A G
PshAI  (6190)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
BbsI  (5858)
1 site
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.
BglII  (1410)
1 site
A G A T C T T C T A G A
NcoI  (1421)
1 site
C C A T G G G G T A C C
PflMI  (1722)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
SgrDI  (1789)
1 site
C G T C G A C G G C A G C T G C
BtgZI  (1973)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
BsaBI  (2240)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
PsiI  (2540)
1 site
T T A T A A A A T A T T
MluI  (5256)
1 site
A C G C G T T G C G C A
dCas9
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   1423 .. 1449  =  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
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   1450 .. 1452  =  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
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   1453 .. 3939  =  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
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   3940 .. 3942  =  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
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   3943 .. 5526  =  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
1423 .. 5526  =  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
AmpR
9376 .. 10,236  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   9376 .. 10,167  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
9376 .. 10,236  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,168 .. 10,236  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
9376 .. 10,236  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TagBFP
5566 .. 6261  =  696 bp
232 amino acids  =  26.2 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
TagBFP
5566 .. 6261  =  696 bp
232 amino acids  =  26.2 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
PuroR
6833 .. 7432  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
6833 .. 7432  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
8617 .. 9205  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
8617 .. 9205  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
5' LTR
11,169 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem cell virus
5' LTR
11,169 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem cell virus
3' LTR
7532 .. 8046  =  515 bp
3' long terminal repeat from murine embryonic stem cell virus
3' LTR
7532 .. 8046  =  515 bp
3' long terminal repeat from murine embryonic stem cell virus
PGK promoter
6313 .. 6812  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
6313 .. 6812  =  500 bp
mouse phosphoglycerate kinase 1 promoter
gag (truncated)
987 .. 1403  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
987 .. 1403  =  417 bp
truncated MMLV gag gene lacking the start codon
MESV Ψ
579 .. 920  =  342 bp
packaging signal of murine embryonic stem cell virus
MESV Ψ
579 .. 920  =  342 bp
packaging signal of murine embryonic stem cell virus
AmpR promoter
10,237 .. 10,341  =  105 bp
AmpR promoter
10,237 .. 10,341  =  105 bp
SV40 NLS
5536 .. 5556  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5536 .. 5556  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
6271 .. 6291  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
6271 .. 6291  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
ORF:  1423 .. 6303  =  4881 bp
ORF:  1626 amino acids  =  187.2 kDa
ORF:  5273 .. 5557  =  285 bp
ORF:  94 amino acids  =  10.0 kDa
ORF:  6593 .. 7432  =  840 bp
ORF:  279 amino acids  =  30.1 kDa
ORF:  9506 .. 9772  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  10,622 .. 10,918  =  297 bp
ORF:  98 amino acids  =  11.7 kDa
ORF:  362 .. 637  =  276 bp
ORF:  91 amino acids  =  9.9 kDa
ORF:  674 .. 934  =  261 bp
ORF:  86 amino acids  =  9.5 kDa
ORF:  6758 .. 7483  =  726 bp
ORF:  241 amino acids  =  24.8 kDa
ORF:  3043 .. 3516  =  474 bp
ORF:  157 amino acids  =  17.4 kDa
ORF:  4828 .. 5214  =  387 bp
ORF:  128 amino acids  =  14.5 kDa
ORF:  5509 .. 6420  =  912 bp
ORF:  303 amino acids  =  31.7 kDa
ORF:  9376 .. 10,236  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3336 .. 3704  =  369 bp
ORF:  122 amino acids  =  14.1 kDa
ORF:  10,686 .. 10,916  =  231 bp
ORF:  76 amino acids  =  7.5 kDa
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