Fig. 1. Schematic overview of recombinase-mediated 5′ UTR library screening strategy.

Naturally occurring 5′ UTRs were extracted, analyzed, and used as the training set to generate synthetic 5′ UTRs for screening. Oligos encoding the 5′ UTR library were synthesized and cloned into plasmids containing a recombinase-recognition site and a GFP reporter. The resulting plasmids were transfected into the HEK 293T-LP cell line with the corresponding recombinase recognition site, resulting in targeted genomic insertion. The cells were sorted into bins based on GFP intensities, and the 5′ UTR sequences of each bin were amplified, sequenced, counted, and compared. The 5′ UTR candidates that enhanced GFP expression were selected and validated experimentally. Finally, the top-ranked validated 5′ UTRs were combined to test for increased gene expression.