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URL: http://www.addgene.org/91567
Proper Citation: RRID:Addgene_91567
Bacterial Resistance: Other
Defining Citation: PMID:28614382
Vector Backbone Description: Backbone Marker:N/A; Backbone Size:10690; Vector Backbone:pRK415; Vector Types:Bacterial Expression; Bacterial Resistance:Other
Comments: Please note- 2 mutations in tse2 (H30R and K33Q) were identified during Addgene's quality control process. The depositing lab has noted that these mutations have no affect on plasmid function, and are likely a strain variant. The Pseudomonas aeruginosa PAO1 Tse2 toxin on the backbone acts as a counter selection marker for allelic exchange. Expression of Tse2 is highly toxic to a huge variety of cells, and can result in less background than traditional markers like SacB. See: Khetrapal, Varnica et al. “A Set of Powerful Negative Selection Systems for Unmodified Enterobacteriaceae.” Nucleic Acids Research 43.13 (2015): e83. PMC. Web. 11 Apr. 2017. Toxin expression is tightly repressed by TetR, until induction by anhydrotetracycline. Anhydrotetracycline is non-toxic to most bacteria, and freely crosses membrane barriers without need of specific transporters, potentially enabling this vector to work in a wide variety of bacteria. The ampicillin resistance gene is flanked by two BsaI sites. It can be cleanly removed and replaced with another marker via Golden Gate cloning. If to be used for mating, this plasmid or derivatives can be transformed into commonly used strains such as S17-1λpir, SM10, DH5a(pRK2013), because these strains provide the RK2/RP4 TrfA protein in trans. Important: This suicide plasmid IS NOT based on the R6K gamma ori like most suicide plasmids, so it won't replicate in strains like DH5aλpir.
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Source: Addgene
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