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Plasmid Files

PX551

Zhang lab AAV vector, also known as AAV-SpCas9 or pAAV-SpCas9, for expressing Cas9 in neurons. Use together with PX552.

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PX551 Sequence and MapPX551.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #60957
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 AflIII - PciI (7395) SacII (6442) BpmI (6438) AseI (6332) FspI (6284) PvuI (6138) PsiI (5236) BtgZI (5103) PluTI (4808) SfoI (4806) NarI (4805) KasI (4804) NotI (4656) EcoRI (4601) BsmI (4101) BsaBI * (3959) SphI (3727) XbaI (148) HindIII (154) MfeI (212) AarI (300) AgeI - SgrAI (389) ATG HA SV40 NLS PspOMI (1816) ApaI (1820) PasI (2230) EcoRV (2314) Eco53kI (2698) SacI (2700) PmlI (3250) PX551 7400 bp
AflIII  (7395)
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  (7395)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SacII  (6442)
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.
BpmI  (6438)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI
recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its
electrophoretic mobility.
BpmI quickly loses activity at 37°C.
AseI  (6332)
1 site
A T T A A T T A A T T A
FspI  (6284)
1 site
T G C G C A A C G C G T
PvuI  (6138)
1 site
C G A T C G G C T A G C
PsiI  (5236)
1 site
T T A T A A A A T A T T
BtgZI  (5103)
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.
PluTI  (4808)
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  (4806)
1 site
G G C G C C C C G C G G
NarI  (4805)
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  (4804)
1 site
G G C G C C C C G C G G
NotI  (4656)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (4601)
1 site
G A A T T C C T T A A G
BsmI  (4101)
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  (3959)
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.
SphI  (3727)
1 site
G C A T G C C G T A C G
XbaI  (148)
1 site
T C T A G A A G A T C T
HindIII  (154)
1 site
A A G C T T T T C G A A
MfeI  (212)
1 site
C A A T T G G T T A A C
AarI  (300)
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  (389)
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.
SgrAI  (389)
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.
PspOMI  (1816)
1 site
G G G C C C C C C G G G
ApaI  (1820)
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  (2230)
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  (2314)
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.
Eco53kI  (2698)
1 site
G A G C T C C T C G A G
SacI  (2700)
1 site
G A G C T C C T C G A G
PmlI  (3250)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
Cas9
464 .. 4564  =  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
464 .. 4564  =  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
5720 .. 6580  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5720 .. 5788  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5720 .. 6580  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5789 .. 6580  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5720 .. 6580  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
6751 .. 7339  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6751 .. 7339  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
4878 .. 5333  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
4878 .. 5333  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
Mecp2 promoter
160 .. 388  =  229 bp
promoter for selective expression in neurons (Adachi
et al., 2005; Gray et al., 2011)
Mecp2 promoter
160 .. 388  =  229 bp
promoter for selective expression in neurons (Adachi
et al., 2005; Gray et al., 2011)
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
4663 .. 4803  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
4663 .. 4803  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
5615 .. 5719  =  105 bp
AmpR promoter
5615 .. 5719  =  105 bp
poly(A) signal
4607 .. 4655  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4607 .. 4655  =  49 bp
synthetic polyadenylation signal
HA
404 .. 430  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
404 .. 430  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
434 .. 454  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
434 .. 454  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4568 .. 4588  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4568 .. 4588  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
401 .. 403  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
401 .. 403  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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