Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012312 | p426-SNR52p-gRNA.CAN1.Y-SUP4t | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- p426-SNR52p-gRNA.CAN1.Y-SUP4t
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6274 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM.
- Copy Number:
- High copy number
- Promoter:
- SNR52
p426-SNR52p-gRNA.CAN1.Y-SUP4t vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
p426-SNR52p-gRNA.CAN1.Y-SUP4t vector Sequence
LOCUS p426-SNR52p-gRNA 6274 bp DNA circular SYN 01-JAN-1980
DEFINITION Yeast high-copy plasmid for gRNA targeting of the negatively
selectable CAN1 gene.
ACCESSION .
VERSION .
KEYWORDS p426-SNR52p-gRNA.CAN1.Y-SUP4t
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6274)
AUTHORS DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM.
TITLE Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas
systems.
JOURNAL Nucleic Acids Res. 2013;41:4336-43.
PUBMED 23460208
REFERENCE 2 (bases 1 to 6274)
AUTHORS Church Lab / Addgene #43803
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6274)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic
Acids Res."; date: "2013"; volume: "41"; pages: "4336-43"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6274
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 69..1411
/label=2u ori
/note="yeast 2u plasmid origin of replication"
promoter 1438..1542
/label=AmpR promoter
CDS 1543..2400
/label=AmpR
/note="beta-lactamase"
rep_origin 2574..3162
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 3450..3471
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 3486..3516
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3524..3540
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 3548..3564
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 3585..3603
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 3621..3889
/label=SNR52 promoter
/note="promoter for the S. cerevisiae small nucleolar RNA
gene SNR52"
misc_feature 3890..3909
/label=CAN1.Y genomic target
/note="CAN1.Y genomic target"
/note="genomic target sequence located 207 bp downstream of
the start codon in the yeast CAN1 gene"
misc_RNA 3910..3984
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator 3989..4008
/label=SUP4 terminator
/note="transcription terminator for the S. cerevisiae SUP4
tRNA gene"
terminator 4014..4261
/label=CYC1 terminator
/note="transcription terminator for CYC1"
promoter complement(4280..4298)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4308..4324)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 4469..4924
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(5058..5858)
/label=URA3
/note="orotidine-5'-phosphate decarboxylase, required for
uracil biosynthesis"
promoter complement(5859..6074)
/label=URA3 promoter