Plasmid Construction Using USER and Gibson

Plasmids (listed in Supplementary Data 2) were constructed by PCR-based uracil-specific excision reagent (USER) cloning method^{63 (link),64 (link)} or using Gibson cloning^{65 (link)}. For USER cloning, one microliter of 5x HF buffer (Thermo Scientific) and 1U of USER™ enzyme mix (New England Biolabs, 1 U/µl) were added to 8 µl of the mixture of purified PCR products, plasmid-backbone or genes. All PCR products were amplified with oligonucleotides (Integrated DNA Technologies) having uracil incorporated, utilizing Phusion U polymerase (Thermo Fisher Scientific). The reaction mixture was incubated for 25 min at 37 °C, followed by 25 min of incubation at a temperature optimized for annealing of the fragments for 25 min. Eight microliters MilliQ was added to the reactions, reaching a final volume of 20 µl. In all, 2.5 µl of the diluted USER reaction was used to transform 50 µl competent E. coli. Gibson assemblies were carried out using Gibson Assembly Cloning Kit (NEB). Fragments (motif cassette) with ∼30 bp overlap regions were mixed together with the backbone fragment in 1:1 molar ratio together with an equal volume of Gibson master mix, incubated at 50 °C for 1 h and immediately transformed into E. coli.