Hi, I'm planing on integrating into the genome one fragment containing three genes (one of which is a resistance gene) in this manner:
Gene1 -> Resistance Gene -> Gene2 ->
The arrows indicate the reading frame direction.
Every gene has it's own promoter and terminator.
I was wondering if someone has experience with such cassettes, is there a problem with having the genes close and in the same direction? I was thinking it may cause a promoter to work in the opposite direction and therefore create an antisense for one of the genes and silence it.
Thanks
if the upstream promoter of the gene1 or resistance marker (most likely agTEF promoter which is strong) it would case an increase in the transcription in the downstream gene in this case Resistance Gene and Gene2 respectively if the termination efficiency of the terminators is not high. If this does not mess with your experiment then it is fine. However, I would suggest if you integrate such a large fragment is to use a larger regions of homology as opposed to the commonly used 50bp especially if the genes you are inserting originate from cerevisiae itself (since the genes themselves will provide a large homology fragment which might cause mis-targeting)
This should not cause problems. I have integrated even bigger constructs with more genes in different yeasts and never had a problem with antisense RNA. Just make sure you use good terminator regions.
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