Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001074 | pFC334 | 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:
- pFC334
- Antibiotic Resistance:
- Ampicillin
- Length:
- 17327 bp
- Type:
- Bacterial Expression, CRISPR ; Fungal expression
- Replication origin:
- ori
- Selection Marker:
- argB
- Promoter:
- A. nidulans gpdA promoter with Hammerhead ribozyme
- 5' Primer:
- CGGCATCTGTAGGGCGT
pFC334 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.
pFC334 vector Sequence
LOCUS V001074 17327 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V001074
VERSION V001074
KEYWORDS pFC334
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 17327)
AUTHORS Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH
TITLE A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi.
JOURNAL PLoS One. 2015 Jul 15;10(7):e0133085. doi:
10.1371/journal.pone.0133085. eCollection 2015.
PUBMED 26177455
REFERENCE 2 (bases 1 to 17327)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 17327)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
date: "2015-07-15"; pages: "
10.1371/journal.pone.0133085. eCollection 2015"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..17327
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(2..859)
/label="AmpR"
/note="beta-lactamase"
promoter complement(860..951)
/gene="bla"
/label="AmpR promoter"
primer_bind 993..1012
/label="Kan-R"
/note="Kanamycin resistance gene, reverse primer"
primer_bind 1018..1037
/label="pENTR-R"
/note="pENTR vectors, reverse primer"
protein_bind 1244..1277
/label="FRT (minimal)"
/note="supports FLP-mediated excision but not integration
(Turan and Bode, 2011)"
rep_origin complement(1590..6838)
/direction=LEFT
/label="AMA1"
/note="replication origin from Aspergillus nidulans
(Aleksenko and Clutterbuck, 1996)"
intron 7484..7599
/label="gpdA intron"
/note="intron from the Aspergillus nidulans
glyceraldehyde-3-phosphate dehydrogenase gene (Punt et al.,
1990)"
ncRNA 7615..7651
/label="hammerhead ribozyme"
misc_RNA 7672..7747
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
ncRNA 7752..7819
/label="HDV ribozyme"
CDS 8955..13058
/label="Cas9"
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 13062..13082
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 14297..15373
/gene="argB"
/label="Ornithine carbamoyltransferase, mitochondrial"
/note="Ornithine carbamoyltransferase, mitochondrial from
Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS
112.46 / NRRL 194 / M139). Accession#: P11803"
protein_bind complement(15827..15860)
/label="FRT (minimal)"
/note="supports FLP-mediated excision but not integration
(Turan and Bode, 2011)"
primer_bind complement(16330..16347)
/label="M13 Forward"
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(16330..16346)
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(16445..16462)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(16616..17204)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"