Huseyin Kucuktas <kucukhu at mail.auburn.edu> writes:
>> I am trying to express a protein which is supposed to be a product
> of a 5406 bp DNA fragment. However the actual ORF is small and
> the gene reads on the complementary strand.
> I HAVE SOME QUESTIONS!
> 1- Maximum how many extra bases I can have before the ATG
> codon of my "gene"?
You should avoid having any other ATG, regions with strong secondary
structure, and long stretches of G/C upstream from the initiating
ATG. Other than these features there is no clear correlation between
length of untranslated region and expression level - make it as
short as is conveniently possible.
> 2- It appears that I do not have a lot of desirable restriction
> sites around the beginning of the ORF, how am I going to get rid
> the extra bases?
Use whatever site is closest to the ATG. Since your ORF is small you
may even find a 4-base cutter that does not cut within the ORF. If
you can cut within 50 bp (or even 100 bp) of the ATG, I would try