I'm making a large, complex vector containing an oncogene.
I want to limit expression to a specific subset of Cre-expressing cells, so I would like to add a loxP-stop-loxP to my vector.
After rummaging around on Addgene and in the literature, the only LoxP-stop sites I have found are very large (2.4kb and up).
Does anyone know of a short effective loxP-stop-loxP sequence? I don't need any RFP or other expressed proteins, I just need aborted transcription in non-Cre cells, and I would like it in as short a sequence as reasonably possible. (My vector is already 11kb)
Do you know any unique restriction sites on loxP-stop-loxP (Addgene), so you can re-engineer it to remove the original 'STOP' fragment and insert in a new shorter 'STOP' fragment?
I guess the more precise question would be: what is the minimum number of base pairs I need between a start and a stop codon after a CMV promoter? I can clone the loxp sites around any sequence I want.
Did you find an answer to this? I am having the same problem with trying to find a simple LoxP-STOP-LoxP plasmid. The below is the best I could find https://www.addgene.org/51269/
There is this paper with a 338 bp floxed stop cassette that is shown to work:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1919482/
Did you find a best answer? I am having the same question. I guess the answer for this question is important when we need to clone loxP-stop-LoxP in AAV vector. Does the above 338 bp fragment work?
As a final update for this -
Even with cloning lox-stop-lox I found some 'leaking' where the proteins were being expressed at a lower level even without Cre.
The solution that worked for me was to use a FLEx construct, where two pairs of loxP sites are used. I generated my FLEx sequence by purchasing paired oligos online and annealing them. (Its enormously difficult to use any sort of PCR method to subclone the palindromic LoxP sequences)
https://blog.addgene.org/plasmids-101-flex-vectors
Good luck, happy cloning!
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