Figure 3
From: Amplified insert assembly: an optimized approach to standard assembly of BioBrickTM genetic circuits
Three Antibiotic (3A) Assembly. Using this method of assembly, the promoter is digested with EcoRI and SpeI and the coding sequence is digested with XbaI and PstI. A PCR linearized "construction plasmid" with a different antibiotic resistance than the others is also digested with EcoRI and PstI and may or may not be treated with DpnI and phosphatase to remove some of the background plasmids. These three digests are then ligated together and the circular product of that ligation is transformed into cells.