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Fusion protein questions - Botched deletion primer (Nov/08/2006 )


I am trying to delete a portion of a protein's cytoplasmic tail. I designed my primers and I thought life was good, until the sequence came back. I was trying to delete a portion and keep it in frame then use the vector's 6XHis tag and stop codon. Well I botched the primer design and rather than delete the unwanted section, I just inserted the 6xHis tag and stop codon, then I have the rest of the sequence which then goes into the vector's 6xHis and stop.

Basically what I have is,

xxxxxxxxxxHHHHHHstopyyyyyyyyyyyyHHHHHHstop...(remainder of vector)

Where X is what I want. Y is what I wanted to delete.

What I wanted was,
xxxxxxxxxxHHHHHHstop....(remainder fo vector)

I haven't worked with fusion protein's before, did I just make one?

the first codon after the first stop codon is an ATG, will that start translation again or do I need some kind of kozak to start translation?

What about if there is an inframe ATG further down in the "y" section? Will that start translation again?

I am hoping not to have to re-purchase primers after this dumb mistake, but with my limited knowledge of protein translation I am at a loss of what effects this mistake will have.

Thanks for any help!


my guess would be that the ribosome will stop mainly at the frist stop codon and fall off the mRNA... unless you have some amber or orche mutation...

So things should be okay...


Thanks for the reply! I think that is right, I just received emails from two biotech companies tech support and they said the same thing!