E coli strain nico21 de3

The CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to obtain intermediate constructs by direct PCR and restriction/ligation cloning of required genetic elements. The pRSET-EmGFP plasmid (Thermo Fisher Scientific) was used as an expression vector containing the GFP gene. High-fidelity restriction endonucleases BamHI-HF, BglII-HF, EcoRI-HF, NcoI-HF, and PstI-HF; T4-DNA-ligase; T4-polynucleotide-kinase; and shrimp alkaline phosphatase (rSAP) were purchased from New England Biolabs (Ipswich, MA, USA). The oligonucleotides were synthesised by the solid-phase method and purified by preparative polyacrylamide gel electrophoresis (PAGE) by Syntol LLC (Russia). Q5^® High-Fidelity DNA Polymerase (New England Biolabs) was used for all polymerase chain reaction (PCR). Ultrafree-DA Centrifugal Filter Units (Merck, Kenilworth, NJ, USA) were used for DNA extraction from agarose gel. The ZymoPURE™ Plasmid Miniprep Kit (Zymo Research, Irvine, CA, USA) was used for plasmid DNA purification. The authenticity of the plasmids was confirmed by Sanger sequencing performed by Eurogen CJSC (Russia). E. coli strain NiCo21(DE3) (#C2529H, New England Biolabs, Ipswich, MA, USA) was used for cloning and expression experiments according to the manufacturer’s protocol.