NcoI (10 U/µL)

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	The synthetic gene, provided in a standard cloning vector, was subcloned into a pET28 plasmid (Novagen, Merck Millipore, Burlington, Massachusetts, USA) by restriction cloning using NcoI and XhoI (Fermentas, Waltham, Massachusetts, USA) as restriction enzymes.

Protocol tips
The synthetic gene, provided in a standard cloning vector, was subcloned into a pET28 plasmid (Novagen, Merck Millipore, Burlington, Massachusetts, USA) by restriction cloning using NcoI and XhoI (Fermentas, Waltham, Massachusetts, USA) as restriction enzymes.

Publication protocol

"In order to produce the enzyme in E. coli, the amino acid sequence of MtFae1a (Uniprot ID: G2QND5) was analyzed using SignalP 4.0 (Petersen et al. 2011). Te frst 18 amino acids were identifed as a signal peptide. Te signal peptide part was removed from the DNA sequence,
which was then codon optimized for expression in E. coli and ordered as a synthetic gene from Eurofns (Luxembourg, Luxembourg) (GenBank ID: MK955161). Tesynthetic gene, provided in a standard cloning vector,was subcloned into a pET28 plasmid (Novagen, Merck Millipore, Burlington, Massachusetts, USA) by restriction cloning using NcoI and XhoI (Fermentas, Waltham,Massachusetts, USA) as restriction enzymes. The newly
constructed plasmid, pET28-MtFae1a, encoded the same amino acid sequence as MtFae1a, minus the signal peptide, and with the addition of a C-terminal His6-tag. pET28-MtFae1a was transformed into an E. coli BL21(DE3) strain (Novagen) that had already been transformed with a plasmid coding for chaperones, pGro7
plasmid (Takara, Kusatsu, Shiga prefecture, Japan). pGro7 encodes the groES–groEL chaperones, is inducible by l-Arabinose and carries a chloramphenicol resistance marker. "

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