I have a gene of interest that has an important membrane binding domain at it's N-terminus. It was already designed with an 5' NcoI site, so I was just going to use the NcoI site in pET30b however there is a fairly large N-terminal affinity tag in PET30b. What I've done is cut the N-terminal tag coding sequence out with NdeI and KpnI, treated this with Klenow to blunt, and religated to generate what is essentially a pET30b vector lacking the N-terminal tag, and will now have the ATG start of the NcoI site.
This vector was confirmed through dna sequencing.
I was wondering if anyone done this before and it's worked, or if pET30b requires the NdeI site to properly express recombinant proteins. I have been having problems expressing protein in this modified vector. I should mention I haven't tried expression in the original pET30b yet simply because I had this modified vector on hand.