TABLE 1.

Primers for cloning and site-directed mutagenesis^a

Gene target or primer name	Restriction enzyme site	Primer sequence (5′ to 3′)
Primers for cloning
dzr	BamHI	ATAAGGATCCGATGCAGCACGCGAAATA
	NotI	ATATAGCGGCCGCTACACCATGCGCGCTAC
ifcA	NdeI	ATAATCATATGAAAGCTCAACTGAATC
	EcoRV	ATAAGATATCCTAGTGGTGATGATGGTGATGCTCAGCGTCCACGTC
ddr	BamHI	ATAAGGATCCGATGGCACAAGAGGTTAAG
	NotI	ATATAGCGGCCGCTTAGGCGATTTCGCCCTG
tdr	NdeI	ATAATCATATGGCAGAATTCGACGTTG
	KpnI	ATAAGGTACCTCAGTGGTGGTGGTGGTGGTGCATGTTTGCAATCGCGTG
Primers for site-directed mutagenesis of DHDR
P212A_F		CCGTGACCGCCGGCCTGGTGTG
P212A_B		CCAGGCCGGCGGTCACGGAATTG
H182Y_F		CAGGCATGCTACGCCGCCGCCAAG
H182Y_B		GGCGGCGGCGTAGCATGCCTGCGG

^adzr and ifcA were cloned into the pRSFDuet-1 vector at multicloning sites 1 and 2, respectively. ddr and tdr were cloned into the pCDFDuet-1 vector at multicloning sites 1 and 2, respectively. dzr and ddr had an N-terminal 6×His tag sequence that was originally positioned in the plasmids, whereas ifcA and tdr had a C-terminal 6×His tag sequence followed by a stop codon. In the case of sole cloning of ifcA, the pET24a vector was used, and the C-terminal cloning primer was modified by replacing the EcoRV restriction site with a HindIII restriction site. All genes were expressed under the control of the T7 promoter.