Cloning of the pGEMM7 plasmid

All fragments for Gibson assembly were amplified using Phusion High-Fidelity DNA polymerase (New England Biolabs, USA) with the recommended standard reaction conditions from the supplier. Elongation times and primer annealing temperatures were varied according to primer length between 55 °C and 65 °C. Primers and remnants of the PCR reaction were removed using the Wizard PCR cleanup kit (Promega, USA). The concentration of the purified DNA was determined using an ND-2000 NanoDrop spectrophotometer. Purified PCR products were mixed following the pipetting scheme in Supplementary Table ³ plus 15 µL of prepared Gibson assembly mix containing 100 mM Tris-HCl, 50 mM MgCl₂, 0.2 mM each dNTP, 10 mM dithiothreitol (DTT), 5% w/v PEG-8000, 1 mM nicotinamide adenine dinucleotide (NAD), 5.33 U mL^–1 T5 Exonuclease, 33.3 U mL^–1 Phusion polymerase and 5.33 U mL^–1 Taq-ligase in a final volume of 20 µL. The Gibson assembly mixture was incubated at 50 °C for 1 h and 5 µL were subsequently used for transformation of 50 µL One Shot™ TOP10 Chemically Competent E. coli cells (ThermoFisher Scientific, USA, catalogue number C4040-10).

Transformed cells were recovered in 1 mL LB medium for 1 h and transferred on LB-Agar plates containing 50 µg mL^–1 ampicillin. After overnight incubation at 37 °C, ten colonies were selected for colony PCR using primers 91 and 397 which bind in the T7 terminator region and the RBS, respectively. Four of the tested colonies gave the expected pattern (Supplementary Fig. ^3a) and were subsequently grown overnight in LB medium. Their plasmid DNA was isolated using a PureYield miniprep kit (Promega, USA) and was further analysed with restriction digestion using the enzymes EcoRI-HF, SacI and DraI (New England Biolabs, USA). Supplementary Fig. ^3b shows that all four colonies gave the expected pattern consisting of digestion products of 4300 bp, 2836 bp, 1863 bp, 1395 bp, 692 bp, and 19 bp (indicated by black stars, only the 19-bp product was not visible), plus some side products attributed to incomplete DNA digestion. The correct DNA sequence was finally confirmed with Sanger sequencing (Macrogen, South-Korea).