Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012314 | p414-TEF1p-Cas9-CYC1t | 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:
- p414-TEF1p-Cas9-CYC1t
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9524 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Host:
- Yeast
- Source/Author:
- DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM.
- Copy Number:
- High copy number
- Promoter:
- TEF1
p414-TEF1p-Cas9-CYC1t 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.
p414-TEF1p-Cas9-CYC1t vector Sequence
LOCUS p414-TEF1p-Cas9- 9524 bp DNA circular SYN 01-JAN-1980
DEFINITION Yeast centromeric plasmid for expressing human codon-optimized Cas9
with the constitutive TEF1 promoter.
ACCESSION .
VERSION .
KEYWORDS p414-TEF1p-Cas9-CYC1t
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9524)
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 9524)
AUTHORS Church Lab / Addgene #43802
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9524)
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..9524
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature complement(69..572)
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
promoter 609..713
/label=AmpR promoter
CDS 714..1571
/label=AmpR
/note="beta-lactamase"
rep_origin 1745..2333
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 2621..2642
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2657..2687
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2695..2711
/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 2719..2735
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 2756..2774
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 2791..3190
/label=TEF1 promoter
/note="promoter for EF-1-alpha"
primer_bind 3194..3210
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
CDS 3222..7325
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 7338..7358
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
terminator 7374..7621
/label=CYC1 terminator
/note="transcription terminator for CYC1"
promoter complement(7640..7658)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(7668..7684)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 7829..8284
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(8386..9057)
/label=TRP1
/note="phosphoribosylanthranilate isomerase, required for
tryptophan biosynthesis"
promoter complement(9058..9338)
/label=TRP1 promoter