The engineering of fusion proteins for structural biology and protein nanotechnology often requires seamless DNA assembly with slight variations in the domain boundaries. To improve the molecular biology workflow for such projects, we evaluated the use of sub-terminal homologous sequences (HS) for co-transformation cloning and for T5 exonuclease / Phusion DNA polymerase mediated in vitro assembly. To quantify the effects of different HS-to-ends distances on cloning efficiency, we designed a blue-white-pink screening system that allowed us to easily identify positive clones (blue colonies), negative clones resulting from circular template plasmid (pink colonies) and negative colonies originating from linearized plasmids that have recircularized without an insert (white colonies). Our experiments show that both methods are feasible with HS-to-ends distances up to at least 10 base pairs. Using a combination of co-transformation cloning at sub-terminal HS and nucleotide insertions in non-annealing primer 5'-overhangs, we integrated a fusion protein into the third intracellular loop (ICL) of a G-protein-coupled receptor (GPCR) with nine different linker boundaries, using only a single plasmid linearization reaction. This molecular cloning approach is an invaluable tool for protein engineering, protein nanotechnology and synthetic biology that extends the range of applications of DNA assembly strategies.