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Plasmid Files

M-ST1cas

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

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M-ST1cas Sequence and MapM-ST1cas.dna
Map and Sequence File   
Sequence Author:  Church Lab / Addgene #48669
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 MfeI (8789) SgrDI (8626) SspI (8509) PvuI (8075) AhdI (7705) PciI (6812) BstZ17I (6433) BsmI (6381) RsrII (5975) PflFI - Tth111I (5577) PluTI (5462) SfoI (5460) NarI (5459) KasI (5458) SmaI (5272) TspMI - XmaI (5270) AvrII (5249) PmeI (4181) MluI (43) SpeI (64) NdeI (299) SnaBI (405) Eco53kI (631) SacI (633) BsmBI (654) BspEI (716) XbaI (723) BstEII (813) SgrAI (929) SacII (1095) PspOMI (1689) ApaI (1693) AarI (1950) BsgI (1996) BstXI (2823) SbfI (2843) AscI (3642) FspAI (4045) AgeI (4163) M-ST1cas 8813 bp
MfeI  (8789)
1 site
C A A T T G G T T A A C
SgrDI  (8626)
1 site
C G T C G A C G G C A G C T G C
SspI  (8509)
1 site
A A T A T T T T A T A A
PvuI  (8075)
1 site
C G A T C G G C T A G C
AhdI  (7705)
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.
PciI  (6812)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstZ17I  (6433)
1 site
G T A T A C C A T A T G
BsmI  (6381)
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.
RsrII  (5975)
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.
PflFI  (5577)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (5577)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
PluTI  (5462)
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  (5460)
1 site
G G C G C C C C G C G G
NarI  (5459)
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  (5458)
1 site
G G C G C C C C G C G G
SmaI  (5272)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (5270)
1 site
C C C G G G G G G C C C
XmaI  (5270)
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.
AvrII  (5249)
1 site
C C T A G G G G A T C C
PmeI  (4181)
1 site
G T T T A A A C C A A A T T T G
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
NdeI  (299)
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  (405)
1 site
T A C G T A A T G C A T
Eco53kI  (631)
1 site
G A G C T C C T C G A G
SacI  (633)
1 site
G A G C T C C T C G A G
BsmBI  (654)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BspEI  (716)
1 site
T C C G G A A G G C C T
XbaI  (723)
1 site
T C T A G A A G A T C T
BstEII  (813)
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.
SgrAI  (929)
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.
SacII  (1095)
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.
PspOMI  (1689)
1 site
G G G C C C C C C G G G
ApaI  (1693)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AarI  (1950)
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.
BsgI  (1996)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BstXI  (2823)
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.
SbfI  (2843)
1 site
C C T G C A G G G G A C G T C C
AscI  (3642)
1 site
G G C G C G C C C C G C G C G G
FspAI  (4045)
1 site
R T G C G C A Y Y A C G C G T R
AgeI  (4163)
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.
St1Cas9
748 .. 4110  =  3363 bp
1121 amino acids  =  129.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus thermophilus Type II CRISPR/Cas
system
generates RNA-guided double strand breaks in DNA
St1Cas9
748 .. 4110  =  3363 bp
1121 amino acids  =  129.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus thermophilus Type II CRISPR/Cas
system
generates RNA-guided double strand breaks in DNA
AmpR
7632 .. 8492  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7632 .. 8423  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7632 .. 8492  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   8424 .. 8492  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7632 .. 8492  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
5331 .. 6125  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
5331 .. 6125  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
6873 .. 7461  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6873 .. 7461  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
4493 .. 4921  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
4493 .. 4921  =  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
4935 .. 5264  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
4935 .. 5264  =  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
6301 .. 6422  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
6301 .. 6422  =  122 bp
SV40 polyadenylation signal
AmpR promoter
8493 .. 8597  =  105 bp
AmpR promoter
8493 .. 8597  =  105 bp
HSV TK poly(A) signal
4243 .. 4291  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
4243 .. 4291  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
SV40 NLS
4123 .. 4143  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4123 .. 4143  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 ori
5115 .. 5250  =  136 bp
SV40 origin of replication
SV40 ori
5115 .. 5250  =  136 bp
SV40 origin of replication
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