Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000583 | pT040 | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pT040
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5186 bp
- Type:
- Yeast Expression, CRISPR
- Replication origin:
- ori
- Selection Marker:
- URA3
- Copy Number:
- High Copy
- Promoter:
- SNR52
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- T3
pT040 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.
pT040 vector Sequence
LOCUS 40924_42039 5186 bp DNA circular SYN 13-MAY-2021
DEFINITION URA3 vector for cloning guide sequences using BclI-SwaI sites into
guide RNA expression cassette.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5186)
AUTHORS Laughery MF, Hunter T, Brown A, Hoopes J, Ostbye T, Shumaker T,
Wyrick JJ
TITLE New vectors for simple and streamlined CRISPR-Cas9 genome editing in
Saccharomyces cerevisiae.
JOURNAL Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep
21.
PUBMED 26305040
REFERENCE 2 (bases 1 to 5186)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5186)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1002/yea.3098";
journalName: "Yeast"; date: "2015-12"; volume: "32"; issue: "12";
pages: "711-20"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5186
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 27..131
/label=AmpR promoter
CDS 132..989
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1163..1751
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1905..1922
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 2039..2060
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2075..2105
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2113..2129
/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 2137..2153
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 2174..2192
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind 2225..2241
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2287..2303)
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
terminator complement(2378..2397)
/label=SUP4 terminator
/note="transcription terminator for the S. cerevisiae SUP4
tRNA gene"
promoter complement(2479..2747)
/label=SNR52 promoter
/note="promoter for the S. cerevisiae small nucleolar RNA
gene SNR52"
promoter complement(2774..2792)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2799..2815)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
misc_feature complement(3331..3623)
/label=CEN4
/note="S. cerevisiae CEN4 centromere"
rep_origin complement(3624..4077)
/direction=LEFT
/label=ARS1
/note="S. cerevisiae autonomously replicating sequence
ARS1"
promoter 4084..4304
/label=URA3 promoter
CDS 4305..5105
/codon_start=1
/label=URA3
/note="orotidine-5'-phosphate decarboxylase, required for
uracil biosynthesis"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"