Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V003382 | pSET5s | 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
pSET4s, pSET5s, and pSET6s are three thermosensitive (Ts) suicide vectors used for gene replacement in Streptococcus suis. These vectors could be propagated at 37°C in Escherichia coli, but their replication was blocked above 37°C in S. suis. orfC, replication regulatory protein; repATs, thermosensitive replication initiation–termination protein.
- Vector Name:
- pSET5s
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 4438 bp
- Type:
- Thermosensitive suicide vector
- Replication origin:
- ori
- Source/Author:
- Takamatsu D, Osaki M, Sekizaki T.
- Growth Strain(s):
- DH5alpha
- Growth Temperature:
- 37℃
pSET5s 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
- Ni J, Teng K, Liu G, Qiao C, Huan L, Zhong J. Autoregulation of lantibiotic bovicin HJ50 biosynthesis by the BovK-BovR two-component signal transduction system in Streptococcus bovis HJ50. Appl Environ Microbiol. 2011 Jan;77(2):407-15. doi: 10.1128/AEM.01278-10. Epub 2010 Nov 12. Retraction in: Appl Environ Microbiol. 2024 Mar 20;90(3):e0000624. doi: 10.1128/aem.00006-24. PMID: 21075878; PMCID: PMC3020555.
pSET5s vector Sequence
LOCUS V003382 4438 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V003382
VERSION V003382
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 4438)
AUTHORS Takamatsu D, Osaki M, Sekizaki T.
TITLE Thermosensitive suicide vectors for gene replacement in
Streptococcus suis
JOURNAL Plasmid 46 (2), 140-148 (2001)
PUBMED 11591139
REFERENCE 2 (bases 1 to 4438)
AUTHORS Takamatsu D, Osaki M, Sekizaki T.
TITLE Direct Submission
JOURNAL Submitted (08-FEB-2001) Daisuke Takamatsu, National Institute of
Animal Health, Laboratory of Molecular Bacteriology; Kannondai
3-1-1, Tsukuba, Ibaraki 305-0856, Japan
(E-mail:p1013dt@niah.affrc.go.jp, Tel:81-298-38-7743,
Fax:81-298-38-7743)
REFERENCE 3 (bases 1 to 4438)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 4438)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid";
date: "2001"; volume: "46"; issue: "2"; pages: "140-148"
SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(08-FEB-2001) Daisuke Takamatsu, National Institute of Animal
Health, Laboratory of Molecular Bacteriology"; volume: " Kannondai
3-1-1, Tsukuba, Ibaraki 305-0856, Japan
(E-mail:p1013dt@niah.affrc.go.jp, Tel:81-298-38-7743, Fax"; pages:
"81-298-38-7743"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4438
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(133..144)
/label="Factor Xa site"
/note="Factor Xa recognition and cleavage site"
CDS 528..689
/codon_start=1
/gene="orfC"
/product="OrfC"
/label="orfC"
/protein_id="BAB64884.1"
/translation="MVISESKKRVMISLTKEQDKKLTDMAKQKGFSKSAVAALAIEEYA
RKESEQKK"
gene 528..689
/gene="orfC"
/label="orfC"
CDS 756..1454
/codon_start=1
/gene="repAts"
/product="RepAts"
/label="repAts"
/protein_id="BAB64885.1"
/translation="MAIKNTKARNFGFLLYPDSIPNDWKEKLESLGVSMAVSPLHDMDE
KKDKDTWNNSNIIQNGKHYKKPHYHVIYIARNPVTIESVRNKIKRKLGNSSVAHVEILD
YIKGSYEYLTHESKDAIAKNKHIYDKKDILNINDFDIDRYITLDESQKRELKNLLLDIV
DDYNLVNTKDLMAFIRLRGAEFGILNTNDVKDIVSTNSSAFRLWFEGNYQCGYRASYAK
VLDAETGEIK"
gene 756..1454
/gene="repAts"
/label="repAts"
primer_bind 2036..2052
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 2053..2109
/label="MCS"
/note="pUC18/19 multiple cloning site"
primer_bind complement(2122..2138)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(2146..2162)
/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(2170..2200)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(2215..2236)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(2524..3112)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 3593..4240
/gene="cat"
/label="Chloramphenicol acetyltransferase"
/note="Chloramphenicol acetyltransferase from
Staphylococcus aureus. Accession#: P00485"