Fig. 2

Schematic basis of Asis-Sal-Pac BioBrick strategy. To perform this assembly, a destination plasmid with two adjacent restriction sites of SalI and PacI is needed. The inserts with two restriction sites of SalI and PacI at the 5′-end, and an AsisI restriction site at the 3′-end will be used. Each assembly step results in the ligation of SalI sites from the plasmid and insert, re-creating a new SalI recognition site at the 5′-side of the insert. However, at the 3′-side of the insert the AsisI site after digestion will be compatible with PacI site in the destination plasmid, forming a scar sequence after ligation. The new plasmid with recreated SalI and the PacI site (sequence of the previous insert which is currently part of the vector sequence) will be ready to accept the next insert with SalI, PacI, and AsiSI sites in the next assembly step