Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007324 | pACT2 AD | 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
pACT2 generates a fusion of the GAL4 AD (amino acids 768–881), an HA epitope tag, and a protein of interest (or protein encoded by a cDNA in a fusion library) cloned into the MCS in the correct orientation and reading frame. pACT2, which is derived from pACT (1), contains a unique EcoR I site in the MCS. The hybrid protein is expressed at high levels in yeast host cells from the constitutive ADH1 promoter (P); transcription is terminated at the ADH1 transcription termination signal (T). The protein is targeted to the yeast nucleus by the nuclear localization sequence from SV40 T-antigen which has been cloned into the 5’ end of the GAL4 AD sequence (2). pACT2 is a shuttle vector that replicates autonomously in both E. coli and S. cerevisiae and carries the bla gene, which confers ampicillin resistance in E. coli. pACT2 also contains the LEU2 nutritional gene that allows yeast auxotrophs to grow on limiting synthetic media. Transformants with AD/library plasmids can be selected by complementation by the LEU2 gene by using an E. coli strain that carries a leuB mutation (e.g., HB101).
- Vector Name:
- pACT2 AD
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8117 bp
- Type:
- Yeast Two-Hybrid System
- Replication origin:
- ori
- Host:
- Yeast
- Promoter:
- LEU2
- Cloning Method:
- Enzyme digestion and ligation
pACT2 AD 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.
pACT2 AD vector Sequence
LOCUS 40924_3931 8117 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8117)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 8117)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8117
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 238..1580
/label=2u ori
/note="yeast 2u plasmid origin of replication"
promoter 2057..2461
/label=LEU2 promoter
CDS 2474..3565
/label=LEU2
/note="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
protein_bind 4186..4219
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
protein_bind complement(4263..4296)
/label=Cre recombinase binding site
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
protein_bind complement(4368..4401)
/label=Cre recombinase binding site
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
terminator complement(4419..4606)
/label=ADH1 terminator
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
CDS complement(5042..5068)
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
CDS complement(5081..5422)
/label=GAL4 activation domain
/note="activation domain of the GAL4 transcriptional
activator"
CDS complement(5438..5458)
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
promoter complement(5504..5902)
/label=ADH1 promoter
/note="promoter for alcohol dehydrogenase 1"
misc_feature 6042..6182
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(6368..6956)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7130..7987)
/label=AmpR
/note="beta-lactamase"
promoter complement(7988..8092)
/label=AmpR promoter