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pAbAi vector (V010329)

Price Information

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V010329 pAbAi In stock, instant shipping

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

To use pAbAi in a one-hybrid assay, clone one or more copies of a cis-acting element into the MCS. To facilitate recombination of the vector with the host’s ura3-52 locus, linearize the vector with either BbsI or BstBI, then introduce the linearized vector into competent yeast cells to integrate into the chromsome via homologous recombination between the vector’s URA3 gene and the ura3-52 locus of the yeast strain.

Vector Name:
pAbAi
Antibiotic Resistance:
Ampicillin
Length:
4894 bp
Type:
Yeast Plasmids
Replication origin:
ori
Host:
Yeast
Source/Author:
Clontech
Copy Number:
High copy number
Promoter:
URA3

pAbAi vector Map

Copy Sequence View Map
pAbAi4894 bp6001200180024003000360042004800MCSAurRM13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR promoterURA3URA3 promoter

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.

pAbAi vector Sequence

LOCUS       pAbAi.        4894 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Yeast integrating vector for cloning a DNA bait sequence to be used 
            in a one-hybrid screen.
ACCESSION   .
VERSION     .
KEYWORDS    pAbAi
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4894)
  AUTHORS   Clontech
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4894)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Linearize with BstBI or BbsI to integrate at the URA3 locus.
FEATURES             Location/Qualifiers
     source          1..4894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..45
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             185..1387
                     /label=AurR
                     /note="aureobasidin A-resistant mutant of S. cerevisiae
                     inositol phosphorylceramide synthase (Aeed et al., 2009)"
     primer_bind     complement(1645..1661)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1669..1685)
                     /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)."
     promoter        complement(1693..1723)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1738..1759)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(2047..2635)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2809..3666)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(3667..3771)
                     /label=AmpR promoter
     CDS             complement(3878..4678)
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
     promoter        complement(4679..4894)
                     /label=URA3 promoter