Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000736 | pCFD5 | 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:
- pCFD5
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6617 bp
- Type:
- Insect Expression, CRISPR
- Replication origin:
- ori
- Promoter:
- dU6-3
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- ACGTTTTATAACTTATGCCCCTAAG
- 3' Primer:
- GCCGAGCACAATTGTCTAGAATGC
pCFD5 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.
pCFD5 vector Sequence
LOCUS 40924_10496 6617 bp DNA circular SYN 13-MAY-2021
DEFINITION multiplex gRNA plasmid.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6617)
AUTHORS Port F, Bullock SL
TITLE Augmenting CRISPR applications in Drosophila with tRNA-flanked
sgRNAs.
JOURNAL Nat Methods. 2016 Oct;13(10):852-4. doi: 10.1038/nmeth.3972. Epub
2016 Sep 5.
PUBMED 27595403
REFERENCE 2 (bases 1 to 6617)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6617)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/nmeth.3972"; journalName: "Nat Methods"; date: "2016-10";
volume: "13"; issue: "10"; pages: "852-4"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6617
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(25..42)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(196..784)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(958..1815)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(1816..1920)
/label=AmpR promoter
rep_origin 1947..2403
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 2544..2560
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 2570..2588
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS complement(join(2780..2897,2974..3067,3159..3765,3820..3953,
4014..4174,4248..4273))
/codon_start=1
/product="tryptophan oxygenase"
/label=vermilion
/note="selectable marker, required to synthesize the brown
eye pigment in Drosophila"
/translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLSE
EDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRVVL
ILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYNQKY
SDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFLEAQV
QSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGAIMITF
YRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQYLRSTL
SDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV"
protein_bind 4588..4657
/label=attB
/note="attB site for the phi-C31 integrase (Groth et al.,
2000)"
protein_bind 4899..5284
/label=gypsy insulator
/note="chromatin insulator from Drosophila"
promoter 5298..5718
/label=dU6-3 promoter
/note="RNA polymerase III promoter for Drosophila U6-3
snRNA (Port et al., 2014)"
misc_RNA 5824..5899
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_RNA 5986..6061
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(6372..6390)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(6411..6427)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6435..6451)
/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(6459..6489)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(6504..6525)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."