Efficient Gibson Assembly of Ultra-Large cDNAs: Generation of Full-length Wild-Type and Mutated futsch transgenes in Drosophila

Research on ultra-large proteins (>5,000 amino acids) is often hindered by the lack of available full-length cDNA constructs and the technical challenges inherent with their cloning. Here, I present an efficient and reproducible method for cloning both wild-type and mutated full-length Drosophila futsch cDNA (16,488 bp), the homologue of mammalian MAP1B, using Gibson Assembly. These resulting cDNA were used to generate UAS-futsch transgenes. When expressed in neurons, the wild type transgenic Futsch associated with microtubule and rescued the synaptic morphological defects observed in futsch ^K68 mutants. This approach substantially reduces the time and complexity compared with traditional cloning techniques. Furthermore, I highlight common pitfalls encountered during the cloning process and provide practical solutions to enhance cloning efficiency. This protocol serves as a valuable reference for researchers aiming to clone other ultra-large cDNAs for functional and structural studies.