Fig. 2
From: High-throughput process development from gene cloning to protein production
Schematic diagram of the 3 A assembly process for assembling BioBrick parts. To perform 3 A assembly, the destination plasmid, the upstream part plasmid and the downstream part plasmid must have different antibiotic resistance markers from each other. Abbreviations are as follows: A = ampicillin resistance gene, K = kanamycin resistance gene, C = chloramphenicol resistance gene, S1, 2, 3 = sequence 1, 2, 3, E = EcoRI, X = XbaI, S = SpeI, P = PstI. 3 A assembly works as follows: Digest the upstream part plasmid with EcoRI and SpeI. Digest the downstream part plasmid with XbaI and PstI. Digest the destination vector with the EcoRI and PstI. Then, all digested plasmids are mixed, ligated, and transformed to a solid plate supplemented with antibiotic corresponding to the destination vector resistance marker for selection. The scar represents the mixed XbaI/ SpeI site