A modular cloning system (MoClo) was developed to enable the standardized assembly of complex eukaryotic multigene constructs. This system allows the creation of any desired multigene construct from pre-made standardized genetic modules, including promoters, 5' untranslated regions, signal peptides, coding sequences, and terminators. The MoClo system is based on Golden Gate cloning technology, which enables the efficient directional assembly of multiple DNA fragments in a single reaction. The system uses type IIS restriction enzymes to cleave DNA outside their recognition sites, allowing for seamless ligation of DNA fragments with compatible overhangs. The MoClo system consists of hierarchical modules: level 0 modules are basic genetic elements, level 1 modules are combinations of level 0 modules, and level 2 modules are assembled from level 1 modules. The system allows for the assembly of constructs in three steps, starting with the creation of level 0 modules, followed by the assembly of level 1 modules, and finally the assembly of level 2 constructs. This approach enables the efficient and standardized assembly of complex constructs, which is particularly useful for applications such as gene stacking and metabolic engineering. The system was tested by assembling a 33 kb construct encoding 11 transcription units, made from 44 individual basic modules, in only three successive cloning steps. This demonstrates the high efficiency and flexibility of the MoClo system in assembling complex genetic constructs. The system also allows for the addition of more transcription units by incorporating new type IIS restriction sites into the constructs. The MoClo system is highly automated and can be used to generate multiple variants of constructs for optimization of desired phenotypes. The system is based on the use of standardized modules, which can be reused across different applications, facilitating the development of standardized construct assembly strategies. The MoClo system represents a significant advancement in synthetic biology, enabling the efficient and standardized assembly of complex genetic constructs.A modular cloning system (MoClo) was developed to enable the standardized assembly of complex eukaryotic multigene constructs. This system allows the creation of any desired multigene construct from pre-made standardized genetic modules, including promoters, 5' untranslated regions, signal peptides, coding sequences, and terminators. The MoClo system is based on Golden Gate cloning technology, which enables the efficient directional assembly of multiple DNA fragments in a single reaction. The system uses type IIS restriction enzymes to cleave DNA outside their recognition sites, allowing for seamless ligation of DNA fragments with compatible overhangs. The MoClo system consists of hierarchical modules: level 0 modules are basic genetic elements, level 1 modules are combinations of level 0 modules, and level 2 modules are assembled from level 1 modules. The system allows for the assembly of constructs in three steps, starting with the creation of level 0 modules, followed by the assembly of level 1 modules, and finally the assembly of level 2 constructs. This approach enables the efficient and standardized assembly of complex constructs, which is particularly useful for applications such as gene stacking and metabolic engineering. The system was tested by assembling a 33 kb construct encoding 11 transcription units, made from 44 individual basic modules, in only three successive cloning steps. This demonstrates the high efficiency and flexibility of the MoClo system in assembling complex genetic constructs. The system also allows for the addition of more transcription units by incorporating new type IIS restriction sites into the constructs. The MoClo system is highly automated and can be used to generate multiple variants of constructs for optimization of desired phenotypes. The system is based on the use of standardized modules, which can be reused across different applications, facilitating the development of standardized construct assembly strategies. The MoClo system represents a significant advancement in synthetic biology, enabling the efficient and standardized assembly of complex genetic constructs.