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Plasmid Files

Puro-Cas9 donor

Vector for targeting a tetracycline-inducible Cas9 cassette to the adeno-associated virus integration site (AAVS1) in the human PPP1R12C gene.

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Puro-Cas9 donor Sequence and MapPuro-Cas9 donor.dna
Map and Sequence File   
Sequence Author:  Huangfu Lab / Addgene #58409
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 SspI (11,489) NsiI (10,130) SnaBI (8906) attB1 AgeI (7467) ATG NcoI (7457) SV40 NLS PciI (667) SpeI (1054) SbfI (1065) PshAI (1456) TspMI - XmaI (1740) SmaI (1742) SV40 poly(A) signal PsiI (2938) HpaI (2958) MfeI (2967) SalI (3078) AccI (3079) BspDI - ClaI (3091) MluI (3110) attB2 AscI (3169) FseI (3214) Puro-Cas9 donor 11,573 bp
SspI  (11,489)
1 site
A A T A T T T T A T A A
NsiI  (10,130)
1 site
A T G C A T T A C G T A
SnaBI  (8906)
1 site
T A C G T A A T G C A T
AgeI  (7467)
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.
NcoI  (7457)
1 site
C C A T G G G G T A C C
PciI  (667)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SpeI  (1054)
1 site
A C T A G T T G A T C A
SbfI  (1065)
1 site
C C T G C A G G G G A C G T C C
PshAI  (1456)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
TspMI  (1740)
1 site
C C C G G G G G G C C C
XmaI  (1740)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (1742)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
PsiI  (2938)
1 site
T T A T A A A A T A T T
HpaI  (2958)
1 site
G T T A A C C A A T T G
MfeI  (2967)
1 site
C A A T T G G T T A A C
SalI  (3078)
1 site
G T C G A C C A G C T G
AccI  (3079)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the
recognition sequence.
Sticky ends from different AccI sites may not be compatible.
BspDI  (3091)
1 site
A T C G A T T A G C T A
ClaI  (3091)
1 site
A T C G A T T A G C T A
MluI  (3110)
1 site
A C G C G T T G C G C A
AscI  (3169)
1 site
G G C G C G C C C C G C G C G G
FseI  (3214)
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.
Cas9
3240 .. 7340  =  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
3240 .. 7340  =  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
10,612 .. 11,472  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   10,612 .. 11,403  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,612 .. 11,472  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   11,404 .. 11,472  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,612 .. 11,472  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
HA-R
7863 .. 8699  =  837 bp
right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the
human PPP1R12C gene
HA-R
7863 .. 8699  =  837 bp
right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the
human PPP1R12C gene
HA-L
1087 .. 1890  =  804 bp
left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the
human PPP1R12C gene
HA-L
1087 .. 1890  =  804 bp
left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the
human PPP1R12C gene
PuroR
2009 .. 2608  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
2009 .. 2608  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
23 .. 611  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
23 .. 611  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
tight TRE promoter
7532 .. 7846  =  315 bp
Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter
tight TRE promoter
7532 .. 7846  =  315 bp
Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter
ccdB
8913 .. 9215  =  303 bp
100 amino acids  =  11.6 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
8913 .. 9215  =  303 bp
100 amino acids  =  11.6 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
bGH poly(A) signal
2646 .. 2870  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
2646 .. 2870  =  225 bp
bovine growth hormone polyadenylation signal
SV40 poly(A) signal
2877 .. 2958  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2877 .. 2958  =  82 bp
SV40 polyadenylation signal
3xFLAG
7392 .. 7457  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
7392 .. 7457  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
T2A
1946 .. 1999  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
T2A
1946 .. 1999  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
nucleoplasmin NLS
3192 .. 3239  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
3192 .. 3239  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SA
1897 .. 1922  =  26 bp
splice acceptor site
SA
1897 .. 1922  =  26 bp
splice acceptor site
attB2
3142 .. 3166  =  25 bp
recombination site for the Gateway® BP reaction
attB2
3142 .. 3166  =  25 bp
recombination site for the Gateway® BP reaction
attB1
7491 .. 7515  =  25 bp
recombination site for the Gateway® BP reaction
attB1
7491 .. 7515  =  25 bp
recombination site for the Gateway® BP reaction
SV40 NLS
7365 .. 7385  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7365 .. 7385  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
7458 .. 7460  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
7458 .. 7460  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
tet operator
7606 .. 7624  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7606 .. 7624  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7642 .. 7660  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7642 .. 7660  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7677 .. 7695  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7677 .. 7695  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7713 .. 7731  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7713 .. 7731  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7749 .. 7767  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7749 .. 7767  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7784 .. 7802  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7784 .. 7802  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7820 .. 7838  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
7820 .. 7838  =  19 bp
bacterial operator O2 for the tetR and tetA genes
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