Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V004242 | pNZ5319 | 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
For L. plantarum, 10 μg/ml chloramphenicol and 10 μg/ml or (for replica plating) 30 μg/ml erythromycin were used. For E. coli, 10 μg/ml chloramphenicol (coud be as high as 37.5 μg/ml) and 250 μg/ml erythromycin were used.
- Vector Name:
- pNZ5319
- Antibiotic Resistance:
- Erythromycin
- Length:
- 3814 bp
- Type:
- Mutagenesis vector
- Replication origin:
- p15A ori
- Source/Author:
- Lambert JM, Bongers RS, Kleerebezem M.
- Copy Number:
- Medium copy number
- Growth Strain(s):
- DH5alpha
- Growth Temperature:
- 37℃
pNZ5319 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
- Lambert JM, Bongers RS, Kleerebezem M. Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum. Appl Environ Microbiol. 2007 Feb;73(4):1126-35. doi: 10.1128/AEM.01473-06. Epub 2006 Dec 1. PMID: 17142375; PMCID: PMC1828656.
pNZ5319 vector Sequence
LOCUS Exported 3814 bp DNA circular SYN 05-AUG-2024
DEFINITION Mutagenesis vector pNZ5319, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3814)
AUTHORS Lambert JM, Bongers RS, Kleerebezem M.
TITLE Cre-lox-based system for multiple gene deletions and
selectable-marker removal in Lactobacillus plantarum
JOURNAL Appl. Environ. Microbiol. 73 (4), 1126-1135 (2007)
PUBMED 17142375
REFERENCE 2 (bases 1 to 3814)
AUTHORS Lambert JM, Bongers RS, Kleerebezem M.
TITLE Direct Submission
JOURNAL Submitted (27-JUN-2005) Health and Safety Department, NIZO Food
Research, Kernhemseweg 2, Ede, Gld 6718ZB, The Netherlands
REFERENCE 3 (bases 1 to 3814)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 3814)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 3814)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Appl.
Environ. Microbiol."; date: "2007"; volume: "73"; issue: "4"; pages:
"1126-1135"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(27-JUN-2005) Health and Safety Department, NIZO Food Research,
Kernhemseweg 2, Ede, Gld 6718ZB, The Netherlands"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..3814
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 1..648
/gene="cat"
/label=cat
/note="Chloramphenicol acetyltransferase from
Staphylococcus aureus. Accession#: P00485"
protein_bind 772..805
/label=lox71
/note="Left element (LE) mutant of loxP (Araki et al.,
2010). Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
regulatory complement(949..1050)
/label=Tlas
/note="Tlas"
/regulatory_class="terminator"
CDS complement(1165..1899)
/gene="ermBP"
/label=ermBP
/note="rRNA adenine N-6-methyltransferase from Enterococcus
faecalis. Accession#: P0A4D5"
rep_origin 2328..2873
/direction=RIGHT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin."
regulatory 3285..3311
/label=TpepN
/note="TpepN"
/regulatory_class="terminator"
protein_bind complement(3505..3538)
/label=lox66
/note="Right element (RE) mutant of loxP (Araki et al.,
2010). Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
regulatory 3613..3798
/label=P32
/note="P32"
/regulatory_class="promoter"