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Plasmid Files

M-SPcas

Mammalian cell vector for expressing human codon-optimized Streptococcus pyogenes Cas9 with a G418-resistance marker.

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M-SPcas Sequence and MapM-SPcas.dna
Map and Sequence File   
Sequence Author:  Church Lab / Addgene #48668
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 MfeI (9529) SgrDI (9366) AhdI (8445) BstZ17I (7173) RsrII (6715) AvrII (5989) StuI (5988) SexAI * (5756) DraIII (5466) PmeI (4921) AgeI (4903) MluI (43) SpeI (64) SnaBI (405) XbaI (723) EcoRV (777) SbfI (995) XcmI (1099) BlpI (1696) Bsu36I (2014) BamHI (2753) BmgBI (3258) BstXI - PmlI (3537) FspAI (3698) AarI (4008) PaeR7I - XhoI (4291) EcoRI (4570) AscI (4703) M-SPcas 9553 bp
MfeI  (9529)
1 site
C A A T T G G T T A A C
SgrDI  (9366)
1 site
C G T C G A C G G C A G C T G C
AhdI  (8445)
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.
BstZ17I  (7173)
1 site
G T A T A C C A T A T G
RsrII  (6715)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
AvrII  (5989)
1 site
C C T A G G G G A T C C
StuI  (5988)
1 site
A G G C C T T C C G G A
SexAI  (5756)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
DraIII  (5466)
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.
PmeI  (4921)
1 site
G T T T A A A C C A A A T T T G
AgeI  (4903)
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.
MluI  (43)
1 site
A C G C G T T G C G C A
SpeI  (64)
1 site
A C T A G T T G A T C A
SnaBI  (405)
1 site
T A C G T A A T G C A T
XbaI  (723)
1 site
T C T A G A A G A T C T
EcoRV  (777)
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.
SbfI  (995)
1 site
C C T G C A G G G G A C G T C C
XcmI  (1099)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
BlpI  (1696)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
Bsu36I  (2014)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BamHI  (2753)
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.
BmgBI  (3258)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
BstXI  (3537)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
PmlI  (3537)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
FspAI  (3698)
1 site
R T G C G C A Y Y A C G C G T R
AarI  (4008)
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.
PaeR7I  (4291)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (4291)
1 site
C T C G A G G A G C T C
EcoRI  (4570)
1 site
G A A T T C C T T A A G
AscI  (4703)
1 site
G G C G C G C C C C G C G C G G
Cas9
748 .. 4851  =  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
748 .. 4851  =  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
AmpR
8372 .. 9232  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8372 .. 9163  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8372 .. 9232  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   9164 .. 9232  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8372 .. 9232  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
6071 .. 6865  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
6071 .. 6865  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
7613 .. 8201  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7613 .. 8201  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5233 .. 5661  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5233 .. 5661  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
50 .. 429  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
50 .. 429  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
5675 .. 6004  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
5675 .. 6004  =  330 bp
SV40 enhancer and early promoter
CMV promoter
430 .. 633  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
430 .. 633  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
7041 .. 7162  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
7041 .. 7162  =  122 bp
SV40 polyadenylation signal
AmpR promoter
9233 .. 9337  =  105 bp
AmpR promoter
9233 .. 9337  =  105 bp
HSV TK poly(A) signal
4983 .. 5031  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
4983 .. 5031  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
SV40 NLS
4864 .. 4884  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4864 .. 4884  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 ori
5855 .. 5990  =  136 bp
SV40 origin of replication
SV40 ori
5855 .. 5990  =  136 bp
SV40 origin of replication
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