The AssemblX Toolkit for Reliable and User-Friendly Multigene Assemblies.

The implementation of complex cloning projects covering the assembly of entire biological pathways or large genetic circuits poses a major challenge in the field of biotechnology and synthetic biology, as such projects can be costly and time-consuming. To overcome these difficulties, we developed the software-assisted AssemblX toolkit, which allows even unexperienced users to design, build, and subsequently test large DNA constructs. Currently, AssemblX allows the assembly of up to 25 functional units (e.g., genes), from 75 or more subunits (e.g., promoters, coding sequences, terminators). At the first assembly level, AssemblX uses overlap-based, scar-free, and sequence-independent cloning methods. This allows the unrestricted design at the gene level without the need for laborious parts domestication. The standardized, polymerase chain reaction-free, and virtually sequence-independent assembly into multigene modules relies on rare cutting homing endonucleases and computationally optimized overlap sequences. Selection and marker switching strategies ensure an effective process, and the assembly product can be transferred to any desired expression host.