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pFA-LIFEACT-GFP-URA3 vector (V006382)

Price Information

Cat No. Plasmid Name Availability Add to cart
V006382 pFA-LIFEACT-GFP-URA3 In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
pFA-LIFEACT-GFP-URA3
Antibiotic Resistance:
Ampicillin
Length:
5074 bp
Type:
Cloning vector
Replication origin:
ori
Source/Author:
Epp E, Nazarova E, Douglas LM, Konopka JB, Vogel J, Whiteway M.

pFA-LIFEACT-GFP-URA3 vector Map

Copy Sequence View Map
pFA-LIFEACT-GFP-URA35074 bp6001200180024003000360042004800LifeactGFPSaccharomyces cerevisiae URA3 terminatorOrotidine 5'-phosphate decarboxylaseT7 promoteroriAmpRAmpR promoterSP6 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.

pFA-LIFEACT-GFP-URA3 vector Sequence

LOCUS       V006382                 5074 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V006382
VERSION     V006382
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 5074)
  AUTHORS   Epp E, Nazarova E, Douglas LM, Konopka JB, Vogel J, Whiteway M.
  TITLE     Actin dynamics and endocytosis in the absence of Arp2/3 in Candida
            albicans
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5074)
  AUTHORS   Epp E, Nazarova E, Douglas LM, Konopka JB, Vogel J, Whiteway M.
  TITLE     Direct Submission
  JOURNAL   Submitted (24-MAY-2011) Biology, McGill University, 1205 Docteur
            Penfield, Montreal, Quebec H3A 1B1, Canada
REFERENCE   3  (bases 1 to 5074)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5074)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName:
            "Unpublished"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (24-MAY-2011) Biology, McGill University, 1205 Docteur Penfield,
            Montreal, Quebec H3A 1B1, Canada"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             151..201
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et
                     al., 2008)"
                     /label="Lifeact"
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             226..936
                     /label="GFP"
                     /note="green fluorescent protein"
     regulatory      940..1195
                     /label="Saccharomyces cerevisiae URA3 terminator"
                     /note="Saccharomyces cerevisiae URA3 terminator"
                     /regulatory_class="terminator"
     CDS             1598..2407
                     /gene="URA3"
                     /label="Orotidine 5'-phosphate decarboxylase"
                     /note="Orotidine 5'-phosphate decarboxylase from Candida
                     albicans (strain SC5314 / ATCC MYA-2876). Accession#:
                     P13649"
     promoter        complement(2712..2730)
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(2988..3576)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(3750..4607)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(4608..4712)
                     /label="AmpR promoter"
     promoter        5058..5074
                     /label="SP6 promoter"
                     /note="promoter for bacteriophage SP6 RNA polymerase"