Fig. 3.

The integrated gene synthesis workflow and direct functional assay in E. coli. (A) EGFP was synthesised using two intermediate block fragments of 12 oligonucleotides which were then subjected to Surveyor digestion for 120 min. Full-length EGFP was then assembled efficiently from the digest products by OE-PCR. (B) Expression of the synthetic gene for EGFP in E. coli produced 85% colonies with green fluorescence under blue light. DNA sequencing confirmed that all fluorescent colonies contained the correct EGFP sequence. (C) The D5 variant of MAO-N (1518 bp) was synthesised using four intermediate fragments (labelled 1–4) using the same method. Expression of this construct is shown in Fig. 2C.