Cloning of A. queenslandica TNKS constructs

The cloning of human TNKS1 constructs was conducted as previously described for CFP-ARC5 [43 (link)] or SAM-ART and ART domains [44 (link)]. Cloning of the YFP-REAGDGEE construct was conducted as previously described [43 (link)]. The E. coli codon-optimized constructs for A. queenslandica TNKS (AqTNKS: XP_019848937.1) were obtained as gBlock from IDT. The construct ARC5 (AqTNKS Glu658-Met811) was cloned into the pNIC-CFP expression vector (Addgene #173074). The AqTNKS constructs SAM-ART (Pro889-Thr1181) and ART (Thr968-Thr1181) were cloned into the pNIC-MBP vector, which was prepared as previously described [43 (link)]. Cloning was conducted using the SLIC method [45 (link)]. Briefly, the vectors were linearized and mixed with insert and T4 DNA polymerase. The reaction mixture was used to transform chemicompetent E. coli NEB 5α cells (New England BioLabs). Colonies were grown at 37 °C on LB agar plates containing 5% sucrose and 50 μg/mL kanamycin; genes in the vector encoding kanamycin-resistance and SacB enzyme [46 (link),47 (link)] served as selection markers for successful transformation and vector linearization, respectively. All constructs were verified by sequencing of the insert regions.