This paper presents a hierarchical modular cloning system, called Modular Cloning (MoClo), designed to efficiently assemble complex eukaryotic multigene constructs. The system leverages the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. Key components include: 1. **Basic Modules**: These are standardized genetic elements such as promoters, 5' untranslated regions, signal peptides, coding sequences, and terminators. 2. **Golden Gate Cloning Technology**: This method allows directional assembly of multiple DNA fragments in a single reaction, with high efficiency. 3. **Destination Vectors**: These vectors are designed to facilitate the assembly of level 0 modules into higher-level constructs through a series of Golden Gate reactions. 4. **End-Linkers**: These are used to connect assembled modules and enable further assembly steps. The system was demonstrated by constructing a 33 kb DNA molecule containing 11 transcription units from 44 individual basic modules in just three successive cloning steps. The efficiency and flexibility of the MoClo system are highlighted, making it suitable for applications such as gene stacking and metabolic engineering. The modular nature of the system allows for the creation of construct libraries, facilitating the optimization of complex pathways in biotechnology.This paper presents a hierarchical modular cloning system, called Modular Cloning (MoClo), designed to efficiently assemble complex eukaryotic multigene constructs. The system leverages the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. Key components include: 1. **Basic Modules**: These are standardized genetic elements such as promoters, 5' untranslated regions, signal peptides, coding sequences, and terminators. 2. **Golden Gate Cloning Technology**: This method allows directional assembly of multiple DNA fragments in a single reaction, with high efficiency. 3. **Destination Vectors**: These vectors are designed to facilitate the assembly of level 0 modules into higher-level constructs through a series of Golden Gate reactions. 4. **End-Linkers**: These are used to connect assembled modules and enable further assembly steps. The system was demonstrated by constructing a 33 kb DNA molecule containing 11 transcription units from 44 individual basic modules in just three successive cloning steps. The efficiency and flexibility of the MoClo system are highlighted, making it suitable for applications such as gene stacking and metabolic engineering. The modular nature of the system allows for the creation of construct libraries, facilitating the optimization of complex pathways in biotechnology.