Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V006369 | pFA6a-5FLAG-natMX6 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pFA6a-5FLAG-natMX6 is a yeast integration vector used for C-terminal gene tagging. It enables targeted genomic insertion to fuse a 5×FLAG epitope tag to a protein of interest, with selection in yeast via the nourseothricin resistance marker natMX6.
- Vector Name:
- pFA6a-5FLAG-natMX6
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4075 bp
- Type:
- Tagging vector
- Replication origin:
- ori
- Host:
- Yeast
- Source/Author:
- Noguchi C, Garabedian MV, Malik M, Noguchi E.
- Copy Number:
- High copy number
- Promoter:
- TEF
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pFA6a-5FLAG-natMX6 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
- Noguchi C, Garabedian MV, Malik M, Noguchi E. A vector system for genomic FLAG epitope-tagging in Schizosaccharomyces pombe. Biotechnol J. 2008 Oct;3(9-10):1280-5. doi: 10.1002/biot.200800140. PMID: 18729046.
pFA6a-5FLAG-natMX6 vector Sequence
LOCUS Exported 4075 bp DNA circular SYN 03-NOV-2025
DEFINITION Plasmid for yeast gene deletion using the natMX6 selectable marker
conferring nourseothricin resistance.
ACCESSION .
VERSION .
KEYWORDS pFA6a-5FLAG-natMX6
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4075)
AUTHORS Hentges P, Van Driessche B, Tafforeau L, Vandenhaute J, Carr AM.
TITLE Three novel antibiotic marker cassettes for gene disruption and
marker switching in Schizosaccharomyces pombe.
JOURNAL Yeast 2005;22:1013-9.
PUBMED 16200533
REFERENCE 2 (bases 1 to 4075)
AUTHORS EUROSCARF
TITLE Direct Submission
REFERENCE 3 (bases 1 to 4075)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 4075)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 4075)
TITLE Direct Submission
REFERENCE 6 (bases 1 to 4075)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Yeast";
date: "2005"; volume: "22"; pages: "1013-9"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT SGRef: number: 4; type: "Journal Article"
COMMENT SGRef: number: 5; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4075
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 67..198
/codon_start=1
/label=5xFLAG
/note="five tandem FLAG(R) epitope tags"
/translation="DYKDDDDKDYKDDDDKLMDYKDDDDKDYKDDDDKLMDYKDDDDK"
terminator 225..412
/label=ADH1 terminator
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
gene 487..1606
/label=natMX6
/note="yeast selectable marker conferring nourseothricin
resistance"
promoter 487..830
/label=TEF promoter
/note="Ashbya gossypii TEF promoter"
CDS 831..1403
/codon_start=1
/gene="Streptomyces noursei nat1"
/product="nourseothricin acetyltransferase"
/label=NrsR
/note="confers resistance to nourseothricin"
/translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
LCGLDTALYDGTASDGEQALYMSMPCP"
terminator 1409..1606
/label=TEF terminator
/note="Ashbya gossypii TEF terminator"
promoter complement(1711..1729)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin complement(1987..2575)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2746..3606)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(3607..3711)
/label=AmpR promoter
promoter 4057..4075
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"