Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V005894 | pGRB2.3 | 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
It is a Candida glabrata expression plasmid. pGRB2.3 contains yEGFP under the control of the PGK1 promoter.
- Vector Name:
- pGRB2.3
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6534 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Host:
- Yeast
- Source/Author:
- Zordan RE, Ren Y, Pan SJ, Rotondo G, Penas Ade L, Iluore J, Cormack BP.
- Promoter:
- URA3
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pGRB2.3 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.
References
- Zordan RE, Ren Y, Pan SJ, et al. Expression plasmids for use in Candida glabrata [published correction appears in G3 (Bethesda). 2014 Jul;4(7):1361]. G3 (Bethesda). 2013;3(10):1675-1686. Published 2013 Oct 3. doi:10.1534/g3.113.006908
pGRB2.3 vector Sequence
LOCUS 40924_22563 6534 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pGRB2.3, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6534)
AUTHORS Zordan RE, Ren Y, Pan SJ, Rotondo G, Penas Ade L, Iluore J, Cormack
BP.
TITLE Expression Plasmids for Use in Candida glabrata
JOURNAL G3 (Bethesda) 3 (10), 1675-1686 (2013)
PUBMED 23934995
REFERENCE 2 (bases 1 to 6534)
AUTHORS Zordan RE, Cormack BP.
TITLE Direct Submission
JOURNAL Submitted (14-MAY-2013) Molecular Biology and Genetics, Johns
Hopkins School of Medicine, 725 N Wolfe St., Hunterian 609,
Baltimore, MD 21205, USA
REFERENCE 3 (bases 1 to 6534)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 6534)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "G3
(Bethesda)"; date: "2013"; volume: "3"; issue: "10"; pages:
"1675-1686"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(14-MAY-2013) Molecular Biology and Genetics, Johns Hopkins School
of Medicine, 725 N Wolfe St., Hunterian 609, Baltimore, MD 21205,
USA"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6534
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature complement(66..664)
/label=Candida glabrata CEN/ARS
/note="Candida glabrata CEN/ARS"
promoter 666..737
/label=AmpR promoter
CDS 738..1595
/label=AmpR
/note="beta-lactamase"
rep_origin 1769..2357
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 2645..2666
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2681..2711
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2719..2735
/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 2743..2759
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 2780..2798
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
regulatory 2817..3367
/label=Saccharomyces cerevisiae PGK1 promoter
/note="Saccharomyces cerevisiae PGK1 promoter"
/regulatory_class="promoter"
primer_bind 3368..3384
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
CDS 3404..4117
/label=yeGFP
/note="yeast-enhanced green fluorescent protein"
regulatory 4139..4536
/label=region downstream of HIS3 in Candida glabrata BG2
/note="region downstream of HIS3 in Candida glabrata BG2"
/regulatory_class="terminator"
promoter complement(4551..4569)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4579..4595)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 4736..5191
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(5325..6125)
/label=URA3
/note="orotidine-5'-phosphate decarboxylase, required for
uracil biosynthesis"
promoter complement(6126..6341)
/label=URA3 promoter