Synthetic dna

cDNA sequences coding for potential ancestors and the corresponding mutants were constructed as synthetic DNA (Thermofisher). KnRAV and CaARF (full-length, fragments (CaARF-DBD (residues 1–421), CaARF-PB1 (residues 644–750), KnRAV-DBD (residues 256–523), KnRAV-PB1 (residues 724–798)) or mutants) coding sequences were cloned into pETM40 plasmid (EMBL) that contains a MBP-tag in the N-terminal region except for PB1 domains from both proteins that were cloned into pETM11 (EMBL) that confers a N-terminal His-tag.
KnRAV and CaARF specific domains were isolated by PCR from synthetic cDNA sequences (S6 Table). Full-length ARF2, ARF5 and ARF10 were cloned into pHMGWA vectors (Addgene) containing N-terminal His-MBP-His tags.

The pH-LAMP mix for HPV DNA test contained the following: 2.4  $\mathrm{\mu }$ L of Betaine (stock at 5 M), 1.5  $\mathrm{\mu }$ L of customised isothermal buffer (pH 8.5–9), 0.9  $\mathrm{\mu }$ L of MgSO4 (stock at 100 mmol/L), 0.9  $\mathrm{\mu }$ L of bovine serum albumin (20 mg/mL), 0.84  $\mathrm{\mu }$ L of dNTPs (stock at 25 mmol/L), 0.375  $\mathrm{\mu }$ L of Syto9 (20 mmol/L stock), 0.375  $\mathrm{\mu }$ L of sodium hydroxide (0.2 M), 0.04  $\mathrm{\mu }$ L of Bst 2.0 DNApolymerase (120,000 U/mL) (New England Biolabs, Hitchin, UK), 1.5  $\mathrm{\mu }$ L of $10\times$ primer mixture (20 mmol/L BIP/FIP, 10 mmol/L LB/LF, and 2.5 mmol/L B3/F3), 3 µL of synthetic DNA (Integrated DNA Technologies) template solution, and enough nuclease-free water to bring the volume to 15  $\mathrm{\mu }$ L. The pH-LAMP mix for hTERT and GAPDH mRNA tests contained the same reagents as the HPV DNA test, in addition to 0.375  $\mathrm{\mu }$ L of SuperScript $\circledR$ III RT/Platinum ${}^{\circledR }$ Taq (Thermo FisherScientific, Waltham, USA), 0.15  $\mathrm{\mu }$ L of ${\text{RNaseOUT}}^{\text{TM}}$ Recombinant Ribonuclease Inhibitor (Thermo FisherScientific, Waltham, USA) and enough nuclease-free water to bring the volume to 15  $\mathrm{\mu }$ L.

The pBBRLIC-lacZ vector was used for ligation independent cloning of promoter-lacZ reporter gene fusions (Ebert et al., 2017 (link)). A 329 bp fragment of the bchF promoter region, encoding sequences from −289 to +41 with respect to the translation start of bchF was PCR amplified from D. shibae DFL12^T genomic DNA using the primer pair EH677 (5′-CCGCGGGCTTTCCCAGCATGGGATCTTGCAGGTT-3′) and EH678 (5′-GTTCCTCCTTCCCACCAGTCCGACCCTGTTTTCT-3′). Subsequently, the bchF promoter fragment was cloned into pBBRLIC-lacZ, resulting in the plasmid pBBR1LIC_bchF-lacZ. For the generation of bchF(mut)-lacZ reporter gene fusion, the bchF promoter fragment containing mutations of two potential PpsR binding sites at positions −79/−77 from TGT to CCA and −30/−28 from ACA to TGG with respect to the translational start site was ordered as synthetic DNA (Thermo Fisher Scientific Inc., Waltham, United States) and subsequently cloned into the pBBRLIC-lacZ vector via ligation independent cloning. This resulted in the plasmid pBBR1LIC_bchF(mut)-lacZ.