Hello! I am trying to generate a construct in this format:

seqA - seqB - linker - seqC - seqB into pSF-CMV-Ub-Puro-Ascl plasmid

0.5kb 2kb 66 0.25kb 2kb

I have tried many different ways but none of them work. The last attempt was to try cloning (seqA-seqB-linker) into a plasmid already containing (seqC-seqB). In other words by splitting the ligation process into two steps, because fusing the segments by PCR or Gibson and inserting them at once didn't work. I was not even able to get a correct fused product.

Does anyone have experience in cloning the same gene twice into the same plasmid?

I was wondering if that could cause a problem.

The ligated clones I got so far have large portions of seqB (the repeating gene) deleted.

(I have checked the presence of RE sites in the final product, those should not be an issue)

I would greatly appreciate any help or advice! Thank you in advance!