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Plasmid Files

eSpCas9(1.1)

Zhang lab vector for expressing a single guide RNA (sgRNA) together with an S. pyogenes Cas9 variant with improved targeting specificity.

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eSpCas9(1.1) Sequence and MapeSpCas9(1.1).dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #71814
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 BbsI (245) nucleoplasmin NLS BbsI (267) CMV enhancer chicken β- actin promoter ATG eSpCas9(1.1) 8506 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.
eSpCas9(1.1)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.1 kDa
   Segment 1:  
   1371 .. 3908  =  2538 bp
   846 amino acids  =  98.2 kDa
Product: Cas9 endonuclease from the Streptococcus
pyogenes
Type II CRISPR/Cas system, mutated to
improve targeting specificity (Slaymaker et al.,
2016)
carries the mutations K848A, K1003A, and R1060A
eSpCas9(1.1)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.1 kDa
   Segment 2:  
   3909 .. 3911  =  3 bp
   1 amino acid  =  89.1 Da
Product: Cas9 endonuclease from the Streptococcus
pyogenes
Type II CRISPR/Cas system, mutated to
improve targeting specificity (Slaymaker et al.,
2016)
carries the mutations K848A, K1003A, and R1060A
eSpCas9(1.1)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.1 kDa
   Segment 3:  
   3912 .. 4373  =  462 bp
   154 amino acids  =  18.0 kDa
Product: Cas9 endonuclease from the Streptococcus
pyogenes
Type II CRISPR/Cas system, mutated to
improve targeting specificity (Slaymaker et al.,
2016)
carries the mutations K848A, K1003A, and R1060A
eSpCas9(1.1)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.1 kDa
   Segment 4:  
   4374 .. 4376  =  3 bp
   1 amino acid  =  89.1 Da
Product: Cas9 endonuclease from the Streptococcus
pyogenes
Type II CRISPR/Cas system, mutated to
improve targeting specificity (Slaymaker et al.,
2016)
carries the mutations K848A, K1003A, and R1060A
eSpCas9(1.1)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.1 kDa
   Segment 5:  
   4377 .. 4544  =  168 bp
   56 amino acids  =  6.6 kDa
Product: Cas9 endonuclease from the Streptococcus
pyogenes
Type II CRISPR/Cas system, mutated to
improve targeting specificity (Slaymaker et al.,
2016)
carries the mutations K848A, K1003A, and R1060A
eSpCas9(1.1)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.1 kDa
   Segment 6:  
   4545 .. 4547  =  3 bp
   1 amino acid  =  89.1 Da
Product: Cas9 endonuclease from the Streptococcus
pyogenes
Type II CRISPR/Cas system, mutated to
improve targeting specificity (Slaymaker et al.,
2016)
carries the mutations K848A, K1003A, and R1060A
eSpCas9(1.1)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.1 kDa
   Segment 7:  
   4548 .. 5471  =  924 bp
   308 amino acids  =  35.1 kDa
Product: Cas9 endonuclease from the Streptococcus
pyogenes
Type II CRISPR/Cas system, mutated to
improve targeting specificity (Slaymaker et al.,
2016)
carries the mutations K848A, K1003A, and R1060A
eSpCas9(1.1)
1371 .. 5471  =  4101 bp
1367 amino acids  =  158.1 kDa
7 segments
Product: Cas9 endonuclease from the Streptococcus
pyogenes
Type II CRISPR/Cas system, mutated to
improve targeting specificity (Slaymaker et al.,
2016)
carries the mutations K848A, K1003A, and R1060A
AmpR
6826 .. 7686  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6826 .. 6894  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6826 .. 7686  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6895 .. 7686  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6826 .. 7686  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
7857 .. 8445  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7857 .. 8445  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5984 .. 6439  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5984 .. 6439  =  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
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
5553 .. 5760  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5553 .. 5760  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
5769 .. 5909  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
5769 .. 5909  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
6721 .. 6825  =  105 bp
AmpR promoter
6721 .. 6825  =  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
3xFLAG
1254 .. 1319  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
1254 .. 1319  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
nucleoplasmin NLS
5472 .. 5519  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
5472 .. 5519  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SV40 NLS
1326 .. 1346  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1326 .. 1346  =  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 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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