Recent progress in the field of synthetic biology has led to the creation of cells containing synthetic genomes. Although these first synthetic organisms contained copies of natural genomes, future work will be directed toward engineering of organisms with modified genomes and novel phenotypes. Much work, however, remains to be done to be able to routinely engineer novel biological functions. As a tool that will be useful for such purpose, we have recently developed a modular cloning system (MoClo) that allows high throughput assembly of multiple genetic elements. We present here new features of this cloning system that allow to increase the speed of assembly of multigene constructs. As an example, 68 DNA fragments encoding basic genetic elements were assembled using three one-pot cloning steps, resulting in a 50 kb construct containing 17 eukaryotic transcription units. This cloning system should be useful for generating the multiple construct variants that will be required for developing gene networks encoding novel functions, and fine-tuning the expression levels of the various genes involved.