The plasmid backbone was generated by linearizing the pHKm vector via PCR. The primary genetic parts of the inducible plasmid, including an inducible promoter (ParaD or PabnE), the expression cassette of the repressor AraR (with codons optimized for A. thermocellus, see Table S3), and a 45-bp terminator (TaraT, the terminator region of araT), were synthesized by Tsingke Biotech. The reporter genes, lacZ (Thebr_0629) and gusB (SSO3036), which encode a thermophilic β-galactosidase (LacZ) and a hyperthermophilic β-glucuronidase (GusB) (Honarbakhsh et al., 2012), were amplified by PCR from the genomic DNA of Thermoanaerobacter brockii subsp. finnii Ako-1 and Sulfolobus solfataricus P2, respectively.
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