Why does 3A Assembly need three different antibiotic resistance plasmids? Why wouldn't two be enough? Let's say that I have two part samples in pSB1A3. I would cut one of the part samples using EcoRI and XbaI and the other sample with SpeI and PstI. Then I would cut my plasmid backbone pSB1K3 with EcoRI and PstI, ligate the products of all three digests, and transform into E.coli and grow it on LB plate with kanamycin. Wouldn't that just require two antibiotic resistance plasmids?