This protocol is used for performing Type IIS assembly by either BsaI or SapI-mediated restriction/ligation using the Loop assembly system.Loop assembly comprises 8 receiver plasmids in odd and even levels (4 per level), which contain directional overhangs for higher level assembly. Odd and even level receiver plasmids are used in a recursive schema for assembly in iterative 'loops'. Any assembly (except for L0 part composition into a L1 transcriptional unit) requires the usage of 4 donor plasmids (each in a specific position) and one receiver plasmid. BsaI is used for assembly into odd levels using kanamycin selection, and SapI is used in even levels using spectynomycin selection. Loop odd receivers use the Common Syntax standard for L0 part assembly into TUs (Patron, et al. 2015 [10.1111/nph.13532]). Then, 4 L1 plasmids (positions 1-4) are assembled into an even receiver plasmid to provide a L2 assembly (4 TUs). For higher level assemblies the same assembly structure is followed, 4 L2 plasmids (positions 1-4) with an odd receiver to yield a L3 assembly (16 TUs).The Loop type IIS assembly protocol is based on: “Patron, NJ (2016) DNA Assembly for Plant Biology. Current Protocols in Plant Biology 1:1-13 [doi: 10.1002/cppb.20038]”, but with slight modifications for DNA concentrations:This protocol uses a target amount of each donor DNA of 15 fmol and 7.5 fmol for receiver plasmid DNA in a 10 µL reaction. In order to perform accurate dispensing, the plasmid DNA is diluted to their corresponding concentration of 15 fmol/µL for donor parts and of 7.5 fmol/µL for the receiver plasmid, and then 1 µL of each plasmid is added to the DNA mix. The plasmid part target concentration (in ng/uL) equals to each donor plasmid length / 100, and of the receiver plasmid length / 200.