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Plasmid Files

PX855

Zhang lab split-Cas9 plasmid for expressing an N-terminal fragment of catalytically inactive dCas9 (residues 2-535) fused to FRB.

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PX855 Sequence and MapPX855.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #62887
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 U6 promoter BbsI (245) BbsI (267) polIII terminator PX855 6205 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(N)
1278 .. 2879  =  1602 bp
534 amino acids  =  61.8 kDa
   Segment 1:  
   1278 .. 1301  =  24 bp
   8 amino acids  =  923.1 Da
Product: N-terminal portion of catalytically inactive
Streptococcus pyogenes dCas9 carrying the D10A
mutation (Zetsche et al., 2015)
dCas9(N)
1278 .. 2879  =  1602 bp
534 amino acids  =  61.8 kDa
   Segment 2:  
   1302 .. 1304  =  3 bp
   1 amino acid  =  89.1 Da
Product: N-terminal portion of catalytically inactive
Streptococcus pyogenes dCas9 carrying the D10A
mutation (Zetsche et al., 2015)
dCas9(N)
1278 .. 2879  =  1602 bp
534 amino acids  =  61.8 kDa
   Segment 3:  
   1305 .. 2879  =  1575 bp
   525 amino acids  =  60.8 kDa
Product: N-terminal portion of catalytically inactive
Streptococcus pyogenes dCas9 carrying the D10A
mutation (Zetsche et al., 2015)
dCas9(N)
1278 .. 2879  =  1602 bp
534 amino acids  =  61.8 kDa
3 segments
Product: N-terminal portion of catalytically inactive
Streptococcus pyogenes dCas9 carrying the D10A
mutation (Zetsche et al., 2015)
AmpR
4525 .. 5385  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4525 .. 4593  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4525 .. 5385  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4594 .. 5385  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4525 .. 5385  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
5556 .. 6144  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5556 .. 6144  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
3683 .. 4138  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3683 .. 4138  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
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
FRB
2925 .. 3197  =  273 bp
91 amino acids  =  11.0 kDa
Product: FKBP-rapamycin binding domain of human
FRAP
FRB
2925 .. 3197  =  273 bp
91 amino acids  =  11.0 kDa
Product: FKBP-rapamycin binding domain of human
FRAP
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
3252 .. 3459  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
3252 .. 3459  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
3468 .. 3608  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
3468 .. 3608  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
4420 .. 4524  =  105 bp
AmpR promoter
4420 .. 4524  =  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
polIII terminator
344 .. 349  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
344 .. 349  =  6 bp
RNA polymerase III 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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