Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V004246 | pNW33N | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
AmpR fragment is non-functional - Chloramphenicol should be used as the resistance marker.
- Vector Name:
- pNW33N
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 4217 bp
- Type:
- Geobacillus stearothermophilus Expression
- Replication origin:
- ori
- Source/Author:
- Mee EK, Welker NE.
- Growth Strain(s):
- Top10
- Growth Temperature:
- 37℃
pNW33N 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
- Nilasari D, Dover N, Rech S, Komives C. Expression of recombinant green fluorescent protein in Bacillus methanolicus. Biotechnol Prog. 2012 May-Jun;28(3):662-8.
- Olson DG, Lynd LR. Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles. J Biol Eng. 2012 May 11;6(1):5. doi: 10.1186/1754-1611-6-5. PMID: 22578246; PMCID: PMC3464808.
- Zarschler K, Janesch B, Zayni S, Schäffer C, Messner P. Construction of a gene knockout system for application in Paenibacillus alvei CCM 2051T, exemplified by the S-layer glycan biosynthesis initiation enzyme WsfP. Appl Environ Microbiol. 2009 May;75(10):3077-85. doi: 10.1128/AEM.00087-09. Epub 2009 Mar 20. PMID: 19304819; PMCID: PMC2681630.
pNW33N vector Sequence
LOCUS V004246 4217 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V004246
VERSION V004246
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 4217)
AUTHORS Mee EK, Welker NE.
TITLE Development of Genetic Tools to Analyze and Characterize Genes of
the Thermophile Geobacillus stearothermophilus
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 4217)
AUTHORS Mee EK, Welker NE.
TITLE Direct Submission
JOURNAL Submitted (14-FEB-2003) Biochemistry, Molecular Biology, and
Cellular Biology, Northwestern University, 2205 Tech Drive,
Evanston, IL 60208, USA
REFERENCE 3 (bases 1 to 4217)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 4217)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(14-FEB-2003) Biochemistry, Molecular Biology, and Cellular Biology,
Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4217
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 1..51
/label="multiple cloning site from plasmid pUC19"
/note="multiple cloning site from plasmid pUC19"
primer_bind complement(69..85)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(93..109)
/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(117..147)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(162..183)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(316..963)
/gene="cat"
/label="Chloramphenicol acetyltransferase"
/note="Chloramphenicol acetyltransferase from
Staphylococcus aureus. Accession#: P00485"
CDS complement(1073..2074)
/label="repB"
/note="RepB replication protein"
rep_origin complement(2766..3354)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 4217
/label="MCS"
/note="pUC18/19 multiple cloning site"