Linear DNA library construction
Linear DNA libraries were constructed by annealing two single stranded DNA (ssDNA) oligos and extension by Klenow polymerase. Oligos were ordered through FASMAC Inc., (Supplementary Table 1). A final concentration of 3 μM of Display-F and Display-FLAG-random-R oligos were used for the synthesis of FLAG-random library, as well as Display-F and Display-10aa-random-R oligos for the synthesis of 10aa-random library and Display-F and FLAG-control-R oligo for the synthesis of fixed FLAG (DYKDDDDK) sequence for western blot analysis. Oligo pairs were mixed with final 200 μM dNTPs in Klenow buffer (Takara Bio) and annealed by heating at 92 ℃ for 30 seconds followed by cooling to room temperature. Then 10U of Klenow Fragment (Large Fragment E. coli DNA Polymerase I;l Takara Bio) was added to the reaction mix and an extension step was performed at 37 ℃ for 1 hour, followed by inactivation of the enzyme at 50 ℃ for 15 min. The Linear DNA libraries (FLAG-random and 10aa-random) were further column purified using the Wizard SV Gel and PCR Clean-Up System (Promega) and quantified by Nanodrop2000c (Thermo Fisher Scientific).