Oligonucleotides were purchased from Qiagen with 'salt-free' purification. Sequence 261-1020 of pGFPuv (GenBank accession no. U62636 with T357C, T811A and C812G base substitutions) was assembled using 40mer (37) and 20mer (2) oligonucleotides with 20 bp overlap (Supplementary Table 1 ). Assembly reactions contained the following components: 64 nM each oligonucleotide, 200 mM dNTPs, 1 mM MgSO 4 , 1· buffer and 0.02 U/ml KOD Hot Start DNA Polymerase. Assembly was carried out using 25 cycles of 94 C for 30 s, 52 C for 30 s and 72 C for 2 min. PCR amplification of assembly products contained the following components: 10-fold dilution of assembly reaction, 25 mM of 20 bp outside primers, 200 mM dNTPs, 1 mM MgSO 4 , 1· buffer and 0.02 U/ml KOD Hot Start DNA Polymerase. PCR was carried out using 35 cycles of 94 C for 30 s, 55 C for 30 s and 72 C for 1 min followed by a final extension at 72 C for 10 min. PCR products were purified using the Qiagen QIAquick PCR purification kit with elution in dH 2 O followed by speed-vac concentration. Assuming an error rate of 1 · 10 À6 /bp/duplication for KOD DNA polymerase (24) , 35 cycles of PCR would be expected to introduce $0.053 mutations per assembled GFPuv molecule.