Hi,
I was wondering if anyone here has some insight into what should be the distance between the promoter and the translation start site / initiator-ATG when making a DNA construct? In particular, I`m interested in this in making an S. cerevisiae expression plasmid. And I`m for example wondering how much of a multi-cloning-site I could put between the promoter and ATG.
One of the critical issues seems to be how "promoter" is defined. Should I look for specific sequences here (e.g. TATA box; does anyone know the sequence or a regular expression to look for them?)? The same issue for the translation start: Do I just look for the first ATG?
Thank you!
if this is just a standard construct with no specific optimization, you might be overthinking this a little bit. Most people just clone in MCS according to the enzymes that are compatible with their insert. This will lead to a non optimal kozak,however, in most cases the expression is fine. If you want to be absolutely sure you can add a yeast kozak (AAAAA) preceding your ATG then add the restriction enzyme site before the kozak.
Thanks you. I`ll check out if I`ll have anything looking like a Kozak sequence in the plasmids.
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