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Plasmid Files

PX856

Zhang lab split-Cas9 plasmid for expressing FKBP fused to a C-terminal fragment of catalytically inactive dCas9 and the VP64 transcriptional activation domain.

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PX856 Sequence and MapPX856.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #62888
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 BbsI (245) BbsI (267) CMV enhancer hybrid intron ATG PX856 7351 bp
BbsI  (245)
2 sites
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.
BbsI  (267)
2 sites
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.
dCas9(C)
1668 .. 4166  =  2499 bp
833 amino acids  =  96.4 kDa
   Segment 1:  
   1668 .. 2648  =  981 bp
   327 amino acids  =  38.1 kDa
Product: C-terminal portion of catalytically inactive
Streptococcus pyogenes dCas9 carrying the N863A
mutation (Zetsche et al., 2015)
dCas9(C)
1668 .. 4166  =  2499 bp
833 amino acids  =  96.4 kDa
   Segment 2:  
   2649 .. 2651  =  3 bp
   1 amino acid  =  89.1 Da
Product: C-terminal portion of catalytically inactive
Streptococcus pyogenes dCas9 carrying the N863A
mutation (Zetsche et al., 2015)
dCas9(C)
1668 .. 4166  =  2499 bp
833 amino acids  =  96.4 kDa
   Segment 3:  
   2652 .. 4166  =  1515 bp
   505 amino acids  =  58.3 kDa
Product: C-terminal portion of catalytically inactive
Streptococcus pyogenes dCas9 carrying the N863A
mutation (Zetsche et al., 2015)
dCas9(C)
1668 .. 4166  =  2499 bp
833 amino acids  =  96.4 kDa
3 segments
Product: C-terminal portion of catalytically inactive
Streptococcus pyogenes dCas9 carrying the N863A
mutation (Zetsche et al., 2015)
AmpR
5671 .. 6531  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5671 .. 5739  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5671 .. 6531  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5740 .. 6531  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5671 .. 6531  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
6702 .. 7290  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6702 .. 7290  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
4829 .. 5284  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
4829 .. 5284  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
FKBP
1302 .. 1622  =  321 bp
107 amino acids  =  11.8 kDa
Product: human FK506-binding protein FKBP12
synthetic gene with alternative codons
FKBP
1302 .. 1622  =  321 bp
107 amino acids  =  11.8 kDa
Product: human FK506-binding protein FKBP12
synthetic gene with alternative codons
CMV enhancer
440 .. 725  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
CMV enhancer
440 .. 725  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
chicken β-actin promoter
727 .. 1004  =  278 bp
chicken β-actin promoter
727 .. 1004  =  278 bp
U6 promoter
1 .. 241  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
1 .. 241  =  241 bp
RNA polymerase III promoter for human U6 snRNA
hybrid intron
1005 .. 1233  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
hybrid intron
1005 .. 1233  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
bGH poly(A) signal
4398 .. 4605  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
4398 .. 4605  =  208 bp
bovine growth hormone polyadenylation signal
VP64
4209 .. 4358  =  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
4209 .. 4358  =  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)
AAV2 ITR
4614 .. 4754  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
4614 .. 4754  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
5566 .. 5670  =  105 bp
AmpR promoter
5566 .. 5670  =  105 bp
gRNA scaffold
268 .. 343  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
268 .. 343  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
SV40 NLS
1257 .. 1277  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1257 .. 1277  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4170 .. 4190  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4170 .. 4190  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
polIII terminator
344 .. 349  =  6 bp
RNA polymerase transcription terminator
polIII terminator
344 .. 349  =  6 bp
RNA polymerase transcription terminator
ATG
1251 .. 1253  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1251 .. 1253  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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