Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000100 | pUC18-mini-Tn7T-Plac-dCas9 (Plac) | 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:
- pUC18-mini-Tn7T-Plac-dCas9 (Plac)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9404 bp
- Type:
- Bacterial Expression, CRISPR, Synthetic Biology
- Replication origin:
- ori
- Selection Marker:
- Gentamicin
- Copy Number:
- High Copy
pUC18-mini-Tn7T-Plac-dCas9 (Plac) 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.
pUC18-mini-Tn7T-Plac-dCas9 (Plac) vector Sequence
LOCUS 40924_44998 9404 bp DNA circular SYN 13-MAY-2021
DEFINITION Tn7 integrating plasmid with S. pasteurianus dCas9 expressed from
the Plac promoter for expression in P. aeruginosa.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9404)
AUTHORS Tan SZ, Reisch CR, Prather KLJ
TITLE A Robust CRISPR Interference Gene Repression System in Pseudomonas.
JOURNAL J Bacteriol. 2018 Mar 12;200(7). pii: JB.00575-17. doi:
10.1128/JB.00575-17. Print 2018 Apr 1.
PUBMED 29311279
REFERENCE 2 (bases 1 to 9404)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9404)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J
Bacteriol."; date: "2018-03-12"; pages: " 10.1128/JB.00575-17. Print
2018 Apr 1"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9404
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 100..119
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 534..550
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
terminator 578..672
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
terminator 775..861
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
protein_bind 907..954
/label=FRT
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
CDS complement(1087..1617)
/codon_start=1
/label=GmR
/note="gentamycin acetyltransferase"
/translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
EEVMHFDIDPSTAT"
promoter complement(1806..1834)
/label=Pc promoter
/note="class 1 integron promoter"
protein_bind 1875..1922
/label=FRT
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
CDS complement(1968..3047)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(3048..3125)
/label=lacIq promoter
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
promoter 3355..3385
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3393..3409
/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)."
CDS 3461..3487
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
terminator 6981..7007
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
mobile_element complement(7099..7264)
/label=Tn7L
/note="mini-Tn7 element (left end of the Tn7 transposon)"
rep_origin complement(7534..8122)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8296..9153)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(9154..9258)
/label=AmpR promoter
primer_bind 9326..9344
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(9382..9404)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"