Site-specificity of serine integrase demonstrated by the attB sequence preference of φBT1 integrase

Serine integrases mediate site-specific recombination and are extensively applied in genetic engineering and synthetic biology. However, which regions of the attachment sites determine site-specificity and how these regions function in recombination remain elusive. Here, we explored the sequence features of attB attachment sites recognized by phi BT1 integrase, a representative serine integrase. A 34-bp DNA motif is found that displays distinct base-specific preference for every position. Further investigation of mutations at different positions within the attB sequence shows different recombination efficiencies and binding affinities. We found four conserved regions within the attB motif that coincide with the results of recombination assays, and mutations in the attB sequence that hamper recombination almost all cause reduced binding affinity.