Overlap Extension PCR for DNA Assembly

In separate PCR steps (see Supplementary Fig. S1A), the assembly fragments (5′ untranslated region-AGT and 3′ untranslated region) were amplified with primers 5′-UTR fwd (5′-CCGCCGGTACCTCCCAAGCCTG-3′), 5′-UTR rev (5′-TGGCGAAAGGGGGATGTGC-3′), 3′ UTR fwd (5′-AGTGAGGATCCGGCTGCTAACAAAGCC-3′) and 3′ UTR rev (5′-TGCTAGCGCTATATGCGTTGATGC-3′), respectively, from the plasmid pIVEX-AGT-DHFR using Pfu Ultra II polymerase (Agilent Technologies). Identical thermo-cycling conditions were used as above apart from the annealing temperatures (60°C). After DpnI digestion and purification of the amplicons, the library was assembled by overlap extension PCR using the PCR fragments from the separate amplification steps described above using the primers LMB 2-6 (5′-ATGTGCTGCAAGGCGATTAAG-3′) and pIV-BG (5′-BG-­GCGTTGATGCAATTTCTATGC-3′). The final construct was subjected to PEG-MgCl₂ precipitation, as described in Stein and Hollfelder (2009) (link) to remove any remaining primer-dimers. Samples were run on an agarose gel to confirm DNA fragments with the correct size were amplified (see Supplementary Fig. S1B).