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Table 2 Primer pairs used in this study a

From: Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii

Primer pair PCR Product/Description Primer sequences
P1: HVO_0665 preKO BamHI RV1 Primers anneal ~500 bp upstream and downstream of hvo_0665; used with H26 genomic DNA as template to generate pJAM2819 (pre-deletion) by ligation into HindIII to BamHI sites of pTA131 5’- ATGAAGCTT AACGCGAGTCTCCTGTGGGCGCTCGG-3'
P2: HVO_0665 HindIII preKO FW1 5’-ATTGGATCC GACGCGCGCACCTCGCCGTTC-3’
P4: HVO_0665 3’-inverse Used to generate plasmid pJAM2820 (Δhvo_0665) by inverse PCR using pJAM2819 as a template 5’-TCCCGCGCCGGCCGACGACTGA-3’
P3: HVO_0665 5’-inverse 5’-TCCGTCGCGTCGGTGAAGCCGTCGAACGACAT-3’
P7: HVO_0665 700 bp confirm FW Anneal ~700 bp upstream and downstream of hvo_0665; used to confirm Δhvo_0665 5’-GCTCGGCGGGGCGAACACG-3
P8: HVO_0665 700 bp confirm RV 5’-GTGACCCACGAGACGACCCACGCG-3’
P5: HVO_0665 NdeI Used to screen for Δhvo_0665 and generate pJAM2821 (in trans complement of Δhvo_0665) 5’-GGGCGGCATATGTCGTTCGACGGCTTCAC-3’
P6: HVO_0665 KpnI 5’-TTGGTACCGTCGTCGGCCGGCGC-3’
P7: C165A THI4 FW Used to generate pJAM2822 for synthesis of the site-directed variant HvThi4 C165A 5’- CGCGAACTCACGGCG GTCGACCCCATC-3’
P8: C165A THI4 RV 5’- GATGGGGTCGACCGC CGTGAGTTCGCG-3’
  1. aItalic in the sequences of primers P1/P2 and P7/P8 indicates the introduced BamHI and HindIII sites for ligation into pTA131 and mutation resulting to Cys to Ala conversion, respectively.