Level 0 modules are the base for MoClo system, where they contain genetic elements like a promoter, a 5' untranslated region (UTR), a coding sequence, and a terminator. For the purpose of Golden Gate Cloning, the internal sequences of level 0 modules should not contain type IIS restriction enzymes sites for BsaI, BpiI, and Esp3I while surrounded by two BsaI restriction sites in inverted orientation. Level 0 modules without type IIS restriction sites flanking can add the BsaI sites during the process of Golden Gate Cloning.
If the level 0 modules contains any unwanted restriction site, they can be mutated in silico by removing one nucleotide from the Type IIS restriction site. In this process, one needs to make sure that the introduced mutation will not affect the genetic function encoded by the sequence of interest. A silent mutation in the coding sequence is preferred, for it neither changes the protein sequence nor the function of the gene of interest.