I'm interested in generating a conditional knockout in S. cerevisiae. To do this, I'm thinking of using homologous recombination to insert loxP sites up and downstream of the ORF. To track the efficiency of the knockout, I will also have an epitope tag, a terminator sequence, and a selection cassette included in the genetic material to be flanked by loxP sites. As a whole, this will be just under 3 kb. Will Cre still effectively recombine this stretch of DNA, or will I need to rethink my strategy and place the LoxP sites closer together?
The loxP system can be used to generate translocations between mammalian chromosomes (http://www.pnas.org/content/108/26/10632.full), so essentially any distance is possible. The further away though, the less efficient.
3 kb can be considered a very small distance. This should be no problem at all.
Dr. Daneshvar, can you provide a reference where 4kb was floxed with high efficiency? I want to flox a 4kb construct and cannot find examples in the literature. thanks.
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