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Plasmid Files

pSpCas9 (PX165)

Zhang lab vector for expressing human codon-optimized Cas9. sgRNA sequences can be co-transfected as PCR products.

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pSpCas9 (PX165) Sequence and MappSpCas9 (PX165).dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #48137
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 XbaI (148) AflIII - PciI (8221) SacII (7268) FspI (7110) PvuI (6964) PsiI (6062) PluTI (5634) SfoI (5632) NarI (5631) KasI (5630) NotI (5482) EcoRI (5243) FseI (5220) nucleoplasmin NLS BsmI (4728) BsaBI * (4586) KflI (4465) Acc65I (154) KpnI (158) NdeI (315) SnaBI (421) chicken β- actin promoter BseRI (832) AarI (937) AgeI (959) ATG BglII (1338) PspOMI (2443) ApaI (2447) PasI (2857) EcoRV (2941) PmlI (3877) pSpCas9 (PX165) 8226 bp
XbaI  (148)
1 site
T C T A G A A G A T C T
AflIII  (8221)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (8221)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SacII  (7268)
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.
FspI  (7110)
1 site
T G C G C A A C G C G T
PvuI  (6964)
1 site
C G A T C G G C T A G C
PsiI  (6062)
1 site
T T A T A A A A T A T T
PluTI  (5634)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
SfoI  (5632)
1 site
G G C G C C C C G C G G
NarI  (5631)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
KasI  (5630)
1 site
G G C G C C C C G C G G
NotI  (5482)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (5243)
1 site
G A A T T C C T T A A G
FseI  (5220)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
BsmI  (4728)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
BsaBI  (4586)
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.
KflI  (4465)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
Acc65I  (154)
1 site
G G T A C C C C A T G G
KpnI  (158)
1 site
G G T A C C C C A T G G
NdeI  (315)
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.
SnaBI  (421)
1 site
T A C G T A A T G C A T
BseRI  (832)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
AarI  (937)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
AgeI  (959)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BglII  (1338)
1 site
A G A T C T T C T A G A
PspOMI  (2443)
1 site
G G G C C C C C C G G G
ApaI  (2447)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PasI  (2857)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
EcoRV  (2941)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
PmlI  (3877)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
Cas9
1091 .. 5191  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
1091 .. 5191  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
AmpR
6546 .. 7406  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6546 .. 6614  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6546 .. 7406  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6615 .. 7406  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6546 .. 7406  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
7577 .. 8165  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7577 .. 8165  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5704 .. 6159  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5704 .. 6159  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
160 .. 445  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
CMV enhancer
160 .. 445  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
chicken β-actin promoter
447 .. 724  =  278 bp
chicken β-actin promoter
447 .. 724  =  278 bp
hybrid intron
725 .. 953  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
hybrid intron
725 .. 953  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
bGH poly(A) signal
5273 .. 5480  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5273 .. 5480  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
1 .. 141  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
1 .. 141  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
5489 .. 5629  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
5489 .. 5629  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
6441 .. 6545  =  105 bp
AmpR promoter
6441 .. 6545  =  105 bp
3xFLAG
974 .. 1039  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
974 .. 1039  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
nucleoplasmin NLS
5192 .. 5239  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
5192 .. 5239  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SV40 NLS
1046 .. 1066  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1046 .. 1066  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
971 .. 973  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
971 .. 973  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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