The DNAmate is a Type IIS assembling systems, as Golden Gate or MoClo, that allows the single-pot directional assembly of up to 8 DNA fragments. Each fragment has a specific prefix and suffix that harbor a type IIS recognition sequence and a unique restriction site. This design allows to combine the best of both worlds since it offers the simultaneous cloning of multiple fragments of the Golden Gate and the possibility to manipulate at will the final construct using the unique restriction sites. The downside of DNAmate is that it leaves short undesired sequences (“scars”) between two adjacent fragments. This limitation is solved by carefully designing the scars so to not interfere with construct functions.