Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006923 | pSL0283 (pTnsABC) | 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:
- pSL0283 (pTnsABC)
- Antibiotic Resistance:
- Kanamycin
- Length:
- 6858 bp
- Type:
- Bacterial Expression, CRISPR ; Transposon
- Replication origin:
- ColA ori
- Copy Number:
- High Copy
- Promoter:
- T7
- Cloning Method:
- Restriction Enzyme
pSL0283 (pTnsABC) 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.
pSL0283 (pTnsABC) vector Sequence
LOCUS 40924_40607 6858 bp DNA circular SYN 13-MAY-2021
DEFINITION Expresses V. cholerae TnsA, TnsB, and TnsC from a T7 promoter..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6858)
AUTHORS Klompe SE, Vo PLH, Halpin-Healy TS, Sternberg SH
TITLE Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA
integration.
JOURNAL Nature. 2019 Jul;571(7764):219-225. doi: 10.1038/s41586-019-1323-z.
Epub 2019 Jun 12.
PUBMED 31189177
REFERENCE 2 (bases 1 to 6858)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6858)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/s41586-019-1323-z"; journalName: "Nature"; date: "2019-07";
volume: "571"; issue: "7764"; pages: "219-225"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6858
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind complement(164..185)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(201..1280)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(1281..1358)
/label=lacI promoter
promoter 1404..1422
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1423..1447
/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)."
RBS 1462..1484
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 3569..3580
/codon_start=1
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
/translation="IEGR"
terminator 5021..5068
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(5301..6113)
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
promoter complement(6114..6205)
/label=AmpR promoter
rep_origin complement(6223..6858)
/direction=LEFT
/label=ColA ori
/note="Plasmids containing the ColA origin of replication
can be propagated in E. coli cells that contain additional
plasmids with compatible origins."