I am wondering what is the maximal distance the U6 promoter can be situated from its target gene and still drive expression? I am developing a model whereby U6 will be located approx. 160 bp away from the gene of interest (last base of U6 promoter to 1st base of target gene sequence). Should this still drive expression? Is there any literature that people are aware of on the topic? I was intending to test this in a plasmid model first but the cloning is being awkward. Any guidance appreciated.
Thanks in advance
Hi Dylan,
The U6 promoter is typically used for short RNA expression (gRNAs, shRNA etc) but I don't know of any example for protein coding genes. Depending on your application it might or might not be the right choice.
The transcription will start inevitably from the first "G" after the promoter, and it will stop at the first short stretch of poly-T (usually 5). 160 bp is quite a long gap for this promoter, and there's a risk of appending significant portions of RNA at the 5' end of your gene or, if a poly T occurs, stopping tanscription prematurely.
I would try to avoid such a big gap.
Hope it helps
Francesco
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