Targeted Lys acylation via insertion of the KL-tag N-terminally, internally and/or C-terminally into sfGFP. Reactions were run in 100 mM HEPES buffer, pH 8, containing 5 µM sfGFP, 100 µM biotin-R NGL H, 400 µM NiSO 4 and 0.5 µM Oa AEP1 for 2 h at 25 °C. Each attached biotin-RN label culminated in a mass shift of +497 Da (calc. +497 Da). Further reaction time points are shown in Extended Data Fig. 5.
