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Plasmid Files

SP-Cas9

Bacterial plasmid for regulated expression of bacterial codon-optimized S. pyogenes Cas9 with a C-terminal nuclear localization signal and 6xHis tag.

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SP-Cas9 Sequence and MapSP-Cas9.dna
Map and Sequence File   
Sequence Author:  Geijsen Lab / Addgene #62731
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 BamHI (9499) 6xHis BmtI (9035) NheI (9031) KflI (8694) KpnI (8300) Acc65I (8296) DraIII (7839) SalI (7828) BsiWI (7229) PmlI (6685) NdeI (5795) AarI (5735) NcoI (5315) RBS XbaI (5276) T7 promoter SgrAI (5169) EcoNI (4956) HindIII (9789) ScaI (515) BsaI (930) AhdI (996) BspQI - SapI (2001) BstZ17I (2117) Bpu10I (2779) FspAI (2907) MscI * (2917) AvaI - BsoBI (2934) BmeT110I (2935) PshAI (3647) PspOMI (4281) ApaI (4285) BstEII (4306) SP-Cas9 9822 bp
BamHI  (9499)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can
remain bound to DNA and alter its electrophoretic mobility.
BmtI  (9035)
1 site
G C T A G C C G A T C G
NheI  (9031)
1 site
G C T A G C C G A T C G
KflI  (8694)
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.
KpnI  (8300)
1 site
G G T A C C C C A T G G
Acc65I  (8296)
1 site
G G T A C C C C A T G G
DraIII  (7839)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
SalI  (7828)
1 site
G T C G A C C A G C T G
BsiWI  (7229)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
PmlI  (6685)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
NdeI  (5795)
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.
AarI  (5735)
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.
NcoI  (5315)
1 site
C C A T G G G G T A C C
XbaI  (5276)
1 site
T C T A G A A G A T C T
SgrAI  (5169)
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.
EcoNI  (4956)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
HindIII  (9789)
1 site
A A G C T T T T C G A A
ScaI  (515)
1 site
A G T A C T T C A T G A
BsaI  (930)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (996)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BspQI  (2001)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (2001)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
BstZ17I  (2117)
1 site
G T A T A C C A T A T G
Bpu10I  (2779)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
FspAI  (2907)
1 site
R T G C G C A Y Y A C G C G T R
MscI  (2917)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
AvaI  (2934)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (2934)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
BmeT110I  (2935)
1 site
C Y C G R G G R G C Y C
PshAI  (3647)
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.
PspOMI  (4281)
1 site
G G G C C C C C C G G G
ApaI  (4285)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BstEII  (4306)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
Cas9
5317 .. 9420  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
5317 .. 9420  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
lacI
3761 .. 4843  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
3761 .. 4843  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
AmpR
209 .. 1069  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   209 .. 277  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
209 .. 1069  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   278 .. 1069  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
209 .. 1069  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1240 .. 1828  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1240 .. 1828  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
2258 .. 2449  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2258 .. 2449  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
104 .. 208  =  105 bp
AmpR promoter
104 .. 208  =  105 bp
lacI promoter
4844 .. 4921  =  78 bp
lacI promoter
4844 .. 4921  =  78 bp
T7 terminator
9563 .. 9610  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
9563 .. 9610  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
5249 .. 5273  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
5249 .. 5273  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
RBS
5288 .. 5310  =  23 bp
efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)
RBS
5288 .. 5310  =  23 bp
efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)
SV40 NLS
9445 .. 9465  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
9445 .. 9465  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
5230 .. 5248  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5230 .. 5248  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
9478 .. 9495  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
9478 .. 9495  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
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