Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V005433 | pIMAY | 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
The pIMAY is a E. coli/staphylococcal temperature-sensitive plasmid for allelic exchange. E.coli bearing pIMAY MUST be cultured at 37℃ in Chloramphenicol (10µg/ml).
- Vector Name:
- pIMAY
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 5743 bp
- Type:
- Staphylococcal allelic exchange vector
- Replication origin:
- p15A ori
- Source/Author:
- Monk IR, Shah IM, Xu M, Tan MW, Foster TJ.
- Copy Number:
- Low copy number
- Promoter:
- T3
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pIMAY vector 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
- Monk IR, Shah IM, Xu M, Tan MW, Foster TJ. Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. mBio. 2012;3(2):e00277-11. Published 2012 Mar 20. doi:10.1128/mBio.00277-11
pIMAY vector Sequence
LOCUS V005433 5743 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V005433
VERSION V005433
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 5743)
AUTHORS Monk IR, Shah IM, Xu M, Tan MW, Foster TJ.
TITLE Transforming the Untransformable: Application of Direct
Transformation To Manipulate Genetically Staphylococcus aureus and
Staphylococcus epidermidis
JOURNAL MBio 3 (2), e00277-11 (2012)
PUBMED 22434850
REFERENCE 2 (bases 1 to 5743)
AUTHORS Monk IR.
TITLE Direct Submission
JOURNAL Submitted (03-FEB-2012) Department of Microbiology, Trinity College
Dublin, Nassau St, Dublin, Dublin 2, Ireland
REFERENCE 3 (bases 1 to 5743)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 5743)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "MBio";
date: "2012"; volume: "3"; issue: "2"; pages: "e00277-11"
SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(03-FEB-2012) Department of Microbiology, Trinity College Dublin,
Nassau St, Dublin, Dublin 2, Ireland"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5743
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(157..168)
/label="Factor Xa site"
/note="Factor Xa recognition and cleavage site"
CDS 302..511
/codon_start=1
/gene="repB"
/product="RepB"
/label="repB"
/protein_id="AFG28167.1"
/translation="MGGKEANFASVLRPPIKCRVPIFVPKTLYPNWLKGLRGFSIANES
PTFSPTFFINLYLSSFIVVFMITK"
gene 302..511
/gene="repB"
/label="repB"
/note="derived from pVE6007"
CDS 552..713
/codon_start=1
/gene="repC"
/product="RepC"
/label="repC"
/protein_id="AFG28168.1"
/translation="MVISESKKRVMISLTKEQDKKLTDMAKQKGFSKSAVAALAIEEYA
RKESEQKK"
gene 552..713
/gene="repC"
/label="repC"
/note="derived from pVE6007"
CDS 780..1478
/codon_start=1
/gene="repA(ts)"
/product="RepA(ts)"
/label="repA(ts)"
/protein_id="AFG28172.1"
/translation="MAIKNTKARNFGFLLYPDSIPNDWKEKLESLGVSMAVSPLHDMDE
KKDKDTWNNSNIIQNGKHYKKPHYHVIYIARNPVTIESVRNKIKRKLGNSSVAHVEILD
YIKGSYEYLTHESKDAIAKNKHIYDKKDILNINDFDIDRYITLDESQKRELKNLLLDIV
DDYNLVNTKDLMAFIRLRGAEFGILNTNDVKDIVSTNSSAFRLWFEGNYQCGYRASYAK
VLDAETGEIK"
gene 780..1478
/gene="repA(ts)"
/label="repA(ts)"
/note="derived from pVE6007"
CDS 1475..1624
/codon_start=1
/gene="repD"
/product="RepD"
/label="repD"
/protein_id="AFG28169.1"
/translation="MTNKEKELFAENEELKKEIKDLKERIERYREMEVELSTTIDLLRG
GIIE"
gene 1475..1624
/gene="repD"
/label="repD"
/note="derived from pVE6007"
CDS complement(1827..2474)
/gene="cat"
/label="Chloramphenicol acetyltransferase"
/note="Chloramphenicol acetyltransferase from
Staphylococcus aureus. Accession#: P00485"
regulatory complement(2475..2674)
/regulatory_class="promoter"
promoter 2675..2693
/label="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
misc_feature complement(2706..2813)
/label="MCS"
/note="pBluescript multiple cloning site"
promoter complement(2822..2840)
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin complement(3347..3892)
/direction=LEFT
/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."
oriT complement(4034..4143)
/direction=LEFT
/label="oriT"
/note="incP origin of transfer"
ncRNA 4373..4933
/product="secY"
/label="antisense_RNA"
/note="derived from pKOR1"
/ncRNA_class="antisense_RNA"
protein_bind 4957..4975
/label="tet operator"
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O1 for the tetR and tetA genes"
protein_bind complement(5042..5060)
/label="tet operator"
/note="bacterial operator O1 for the tetR and tetA genes"
CDS 5082..5705
/label="TetR"
/note="tetracycline repressor TetR"