This protocol is used for performing Type IIS assembly by either BsaI or SapI-mediated restriction\/ligation using Loop assembly with either Loop or uLoop plasmids.Loop assembly comprises\u00a08\u00a0receiver plasmids in odd and even levels (4 per level), which contain\u00a0directional overhangs for higher level assembly. Odd and even level receiver plasmids are used in a recursive schema\u00a0for assembly in\u00a0iterative 'loops'. Any assembly (except for L0 part composition into a L1 transcriptional unit) requires the usage of 4 donor\u00a0plasmids (each in\u00a0a 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.\u00a0Loop odd receivers use the\u00a0Common Syntax standard for L0 part assembly into TUs (Patron,\u00a0et al.\u00a02015 [10.1111\/nph.13532]). Then, 4 L1 plasmids (positions 1-4) are assembled into an\u00a0even 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)\u00a0with an\u00a0odd receiver to yield a L3 assembly (16 TUs).The\u00a0Loop type IIS assembly protocol is based on:\u00a0\u201cPatron, NJ (2016) DNA Assembly for Plant Biology. Current Protocols in Plant Biology 1:1-13 [doi: 10.1002\/cppb.20038]\u201d, but with slight modifications for DNA concentrations:This\u00a0protocol uses a target amount of each donor DNA of 15 fmol and 7.5 fmol for\u00a0receiver plasmid DNA in a 10 \u00b5L reaction. In order to perform\u00a0accurate dispensing, the plasmid DNA is diluted to their corresponding\u00a0concentration of 15 fmol\/\u00b5L for donor parts and of 7.5 fmol\/\u00b5L for the receiver plasmid, and then 1 \u00b5L of each plasmid is added to the DNA mix.\u00a0The\u00a0plasmid part target concentration\u00a0(in ng\/uL) equals to each\u00a0donor plasmid length \/ 100, and of the receiver plasmid length \/ 200.\u00a0Volumes CAN be reduced by half, but expect more variability from aliquoting.