|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||46012||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Backbone manufacturerLidstrom Lab (PMID 8021187)
- Backbone size (bp) 6000
Modifications to backboneTo insert loxP bounded antibiotic resistance gene: The 1.3-kb HincII fragment bearing the kanamycin resistance cassette from pUC4K (Vieira, 1982) was inserted into pLox1 (Palmeros, 2000), which had been cut with XbaI and blunted, to create pCM161. To introduce convenient multiple cloning sites, the loxP-bounded kanamycin cassette of pCM161 was amplified with following primer pair, CM-ufkMCS, 5′-TGACGTCTAGATCTGAATTCAGCTGTACAATTGGTACCATGGATGCATATGGCGGCCGCA-3′, and CM-dfkMCS, 5′-GACTAGTGAGCTCACCGGTTAACACGCGTACGTAGGGCCCGCGGTATCGATAAGCTGGATCC-3′. The resulting 1.4-kb PCR product was purified and cloned into pCR2.1 (Invitrogen, Carlsbad, CA, USA) to create pCM183. To preserve useful cloning sites, pAYC61 was cut with EcoRI and SmaI, blunted using T4 DNA polymerase, and selfligated to produce pCM182. Finally, the 1.4-kb AatII-SpeI fragment from pCM183 containing the loxP-flanked kanamycin cassette was ligated between the AatII and XbaI sites of pCM182 to create pCM184.
- Promoter na
Growth in Bacteria
Bacterial Resistance(s)Ampicillin and Kanamycin
- Cloning method Restriction Enzyme
- 5′ sequencing primer na (Common Sequencing Primers)
This allelic exchange vector is used with Cre expression vectors (Addgene plasmids 45863 or 45864) to generate unmarked mutants in a broad bacterial host range.
The full plasmid sequence is approximate and there may be small discrepancies present.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pCM184 was a gift from Mary Lidstrom (Addgene plasmid # 46012 ; http://n2t.net/addgene:46012 ; RRID:Addgene_46012)
For your References section:Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. Marx CJ, Lidstrom ME. Biotechniques. 2002 Nov;33(5):1062-7. 10.2144/02335rr01 PubMed 12449384