plasmid construction - (Jul/24/2014 )
Hi. Im a newbie here.. Does anyone know if the order of the genes in the plasmid have an effect on its expression? or can I just insert genes on any restriction sites? like if its in a pathway if im going to put gene I, II, and III corresponding to step 1,2,3 in the pathway.. do i have to put these genes in order or is it okay to jumble it all up like gene III, I and II...?
In principle, the order should make little difference. But, you can have all sorts of effects, such as transcriptional read-though, poor transcriptional termination, or cryptic transcriptional and translational elements in your "gene" which make this untrue. Unfortunately, we're in an early stage of understanding and predicting all of the things that happen. My best advice is to do the easy thing, try it, and be prepared to be surprised.
Thank you... im still in the designing part of the experiment... so this really helps..^_^