Hi, I did not get this part: "I've order the gene to be synthesized in a codon optimized form onto our expression plasmid but will need to remove two sections for a secondary truncated construct "

If the problem in PCR then you could try adding betaine, it makes the difference! 

Typically, I generate new plasmids by PCR adding a golden-gate compatible overhangs followed by golden gate cloning.  Gibson assembly is also a very good alternative especially is you could not pcr a part of the vector. 

An alternative may be inverse PCR (https://www.ncbi.nlm.nih.gov/pubmed/12904664, schematics: http://www.idtdna.com/pages/images/decoded/figure-3.png?sfvrsn=0) to introduce large deletions. If the plasmid itself is not too large, you can consider this option. If the problem was not due to GC-content, but poor primers for CDS amplification - of course. We use it routinely on small bacterial plasmids, and we have found that Phusion (Thermo) fidelity is adequate when used for few cycles (

Clarification: I've outsourced the initial cloning of the gene and requested codon optimization for E. coli which will remove the GC bias. This construct will be used for the full length protein but we also want to try expressing a single domain of the protein which would require the removal of two sections of the gene and are planning on using the optimized plasmid as a template.

Hi Gareth,

It sounds like good cloning fun. As an alternative to betaine if it does not work, give a go to DMSO. DMSO (in a dilution series, you need to get the best concentration). will be good for high-GC content PCR.

If the PCR approach does not work, I think recombineering (https://redrecombineering.ncifcrf.gov/) would be a great way to go. I don't suggest it as a first option since I assume you don't have it set up in the lab. However, for big (or huge!) DNA fragments and those not PCR-friendly it's excellent.

Best,

Ruben