Extended Data Fig. 8: The modular cloning or MoClo principle.
From: OPENPichia: licence-free Komagataella phaffii chassis strains and toolkit for protein expression
In Level -1, source DNA, such as PCR fragments or synthetic DNA are flanked with the proper Type IIS restriction sites and 4 nt overhangs, which are then accommodated in a Level 0 Entry vector through BsmBI digest and T4 DNA ligation. Then, selected Level 0 vectors are assembled into a Level 1 Expression vector by means of a BsaI digest and T4 DNA ligation. Finally, the system allows the assembly of multiple transcription units (promoter, CDS, terminator) from the individual Level 1 vectors, into a higher order Level 2 vector, in case the assembly connector sequences were properly selected during the assembly of the Level 1 vectors. Note that the Part 3 Coding Sequence can be split up in a Part 3a and Part 3b, to allow additional modularity. Likewise, the Part 4 Terminator can be split up in a Part 4a and a Part 4b.
