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Plasmid Files

p426-SNR52p-gRNA.CAN1.Y-SUP4t

Yeast high-copy plasmid for gRNA targeting of the negatively selectable CAN1 gene.

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p426-SNR52p-gRNA.CAN1.Y-SUP4t Sequence and Mapp426-SNR52p-gRNA.CAN1.Y-SUP4t.dna
Map and Sequence File   
Sequence Author:  Church Lab / Addgene #43803
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 PfoI (6223) NdeI (5942) BsgI (5913) PstI - SbfI (5879) BfuAI - BspMI (5868) EcoRV (5669) NcoI (5648) BsmI (5497) ApaI * (5483) PspOMI * (5479) StuI (5421) Bpu10I (5124) BtgZI (4694) NaeI (4596) NgoMIV (4594) KpnI (4270) Acc65I (4266) EagI (4261) MluI (4186) BsrGI (4179) SUP4 terminator CAN1.Y genomic target BspDI * - ClaI * (3888) BmtI (3758) NheI (3754) MfeI (115) BmgBI (331) SnaBI (769) AvaI - BsoBI (1115) BmeT110I (1116) BsaHI (1790) NmeAIII (2183) AhdI (2330) p426-SNR52p-gRNA.CAN1.Y-SUP4t 6274 bp
PfoI  (6223)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
NdeI  (5942)
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.
BsgI  (5913)
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).
PstI  (5879)
1 site
C T G C A G G A C G T C
SbfI  (5879)
1 site
C C T G C A G G G G A C G T C C
BfuAI  (5868)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (5868)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
EcoRV  (5669)
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.
NcoI  (5648)
1 site
C C A T G G G G T A C C
BsmI  (5497)
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.
ApaI  (5483)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
ApaI can be used between 25°C and 37°C.
PspOMI  (5479)
1 site
G G G C C C C C C G G G
* Blocked by Dcm methylation.
StuI  (5421)
1 site
A G G C C T T C C G G A
Bpu10I  (5124)
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.
BtgZI  (4694)
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.
NaeI  (4596)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
NgoMIV  (4594)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
KpnI  (4270)
1 site
G G T A C C C C A T G G
Acc65I  (4266)
1 site
G G T A C C C C A T G G
EagI  (4261)
1 site
C G G C C G G C C G G C
MluI  (4186)
1 site
A C G C G T T G C G C A
BsrGI  (4179)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
BspDI  (3888)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (3888)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
BmtI  (3758)
1 site
G C T A G C C G A T C G
NheI  (3754)
1 site
G C T A G C C G A T C G
MfeI  (115)
1 site
C A A T T G G T T A A C
BmgBI  (331)
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.
SnaBI  (769)
1 site
T A C G T A A T G C A T
AvaI  (1115)
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  (1115)
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  (1116)
1 site
C Y C G R G G R G C Y C
BsaHI  (1790)
1 site
G R C G Y C C Y G C R G

BsaHI is typically used at 37°C, but is even more active at 60°C.
NmeAIII  (2183)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AhdI  (2330)
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.
2μ ori
69 .. 1411  =  1343 bp
yeast 2μ plasmid origin of replication
2μ ori
69 .. 1411  =  1343 bp
yeast 2μ plasmid origin of replication
AmpR
1543 .. 2403  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1543 .. 1611  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1543 .. 2403  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1612 .. 2403  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1543 .. 2403  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
URA3
5055 .. 5858  =  804 bp
267 amino acids  =  29.3 kDa
Product: orotidine-5'-phosphate decarboxylase,
required for uracil biosynthesis
yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)
URA3
5055 .. 5858  =  804 bp
267 amino acids  =  29.3 kDa
Product: orotidine-5'-phosphate decarboxylase,
required for uracil biosynthesis
yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)
ori
2574 .. 3162  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2574 .. 3162  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
4469 .. 4924  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
4469 .. 4924  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SNR52 promoter
3621 .. 3889  =  269 bp
promoter for the S. cerevisiae small nucleolar RNA
gene SNR52
SNR52 promoter
3621 .. 3889  =  269 bp
promoter for the S. cerevisiae small nucleolar RNA
gene SNR52
CYC1 terminator
4014 .. 4261  =  248 bp
transcription terminator for CYC1
CYC1 terminator
4014 .. 4261  =  248 bp
transcription terminator for CYC1
URA3 promoter
5859 .. 6074  =  216 bp
URA3 promoter
5859 .. 6074  =  216 bp
AmpR promoter
1438 .. 1542  =  105 bp
AmpR promoter
1438 .. 1542  =  105 bp
gRNA scaffold
3910 .. 3984  =  75 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
3910 .. 3984  =  75 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
CAN1.Y genomic target
3890 .. 3909  =  20 bp
genomic target sequence located 207 bp
downstream of the start codon in the yeast CAN1
gene
CAN1.Y genomic target
3890 .. 3909  =  20 bp
genomic target sequence located 207 bp
downstream of the start codon in the yeast CAN1
gene
SUP4 terminator
3989 .. 4008  =  20 bp
transcription terminator for the S. cerevisiae SUP4
tRNA gene
SUP4 terminator
3989 .. 4008  =  20 bp
transcription terminator for the S. cerevisiae SUP4
tRNA gene
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