Abstract
Although gene synthesis efficiency has improved through the years, current methods offered by DNA synthesis vendors are limited when it comes to repetitive sequences. Here, we describe a method for the enzymatic assembly of repetitive small synthetic genes. The method involves an initial step where the gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden Gate-compatible four-base pair overhangs. Synthons are enzymatically synthesized by oligo extension and then assembled into the synthetic gene by Golden Gate Assembly. We construct eight different synthetic genes ranging from 133 to 456 base pairs encoding RNA structures with repetitive elements that are challenging to synthesize. We report assembly fidelities of up to 87.5 % that decrease for increasing number of synthons. This method is envisioned as an important addition to the molecular cloning toolbox and especially useful for construction of challenging and repetitive genes.
Competing Interest Statement
The authors have declared no competing interest.
