Gene fragments encoding PKAcs (H87Q/W196R mutant 31 ), HaloTag, and MBP were amplified by PCR (NEB Q5 polymerase), using PCR primers that added overlapping sequences to facilitate restriction enzyme-free cloning. PCR fragments were gel-isolated and combined with target vector (pNic28) in a Hot PFX reaction . The reactions were transformed into E. coli XL10-Gold Ultracompetent cells (Agilent), following manufacturer's instructions, and plated on selective media (LB-Agar with Kanamycin). Following overnight incubation (37 ˚C), individual colonies were selected, incubated overnight in a shaker incubator (37 °C, 250 RPM), followed by extraction of plasmid DNA by mini-prep (GeneJet (ThermoFisher)). DNA samples were then characterized by diagnostic restriction digest (HindIII, NdeI, NEB buffer 2.1) and subsequent electrophoresis on an agarose gel. Plasmids with inserts of the correct size were further characterized via Sanger sequencing. Correct clones were subsequently transformed into E. coli BL-21 (DE3) cells (NEB) for expression.