pFA6a-kanMX6-PGAL1-GFP vector (V010295)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V010295	pFA6a-kanMX6-PGAL1-GFP	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:: pFA6a-kanMX6-PGAL1-GFP

Antibiotic Resistance:: Ampicillin

Length:: 5404 bp

Type:: Yeast Plasmids

Replication origin:: ori

Source/Author:: Longtine MS, McKenzie A, Demarini DJ, Shah NG, Wach A, Brachat A,

Copy Number:: High copy number

Promoter:: GAL1

pFA6a-kanMX6-PGAL1-GFP vector Map

Copy Sequence View 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.

pFA6a-kanMX6-PGAL1-GFP vector Sequence

LOCUS       pFA6a-kanMX6-PGA        5404 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid with a kanMX marker for swapping in the GAL1 promoter and 
            adding a GFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    pFA6a-kanMX6-PGAL1-GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5404)
  AUTHORS   Longtine MS, McKenzie A, Demarini DJ, Shah NG, Wach A, Brachat A, 
            Philippsen P, Pringle JR.
  TITLE     Additional modules for versatile and economical PCR-based gene 
            deletion and modification in Saccharomyces cerevisiae.
  JOURNAL   Yeast 1998;14:953-61.
  PUBMED    9717241
REFERENCE   2  (bases 1 to 5404)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5404)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Yeast"; 
            date: "1998"; volume: "14"; pages: "953-61"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(50..237)
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae
                     alcohol dehydrogenase 1 (ADH1) gene"
     CDS             complement(261..971)
                     /label=GFP (S65T)
                     /note="S65T variant of Aequorea victoria green fluorescent 
                     protein (Heim et al., 1995)"
     CDS             complement(984..986)
                     /codon_start=1
                     /label=start codon
                     /note="start codon"
                     /translation="M"
     promoter        complement(1006..1447)
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     gene            1581..2937
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin
                     resistance (Wach et al., 1994)"
     promoter        complement(3042..3060)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(3318..3906)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4080..4937)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4938..5042)
                     /label=AmpR promoter
     promoter        5388..5404
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"