TABLE 2.

Oligonucleotide primers used for mutant construction^a

Gene target and primer name	PCR conditions	Oligonucleotide sequence (5′-3′)b	Product size (bp) (primers used)	Digestion enzyme
emrE	95°C for 2 min; 30 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1.7 min; 72°C for 10 min		1,588 (AD)
SOE-A		CCC CTG CAG AGA CCC TCG GCT TTG CGT CC	881 (AB)	PstI
SOE-B		GCA GGG GTT GTA GGC CTG AAC
SOE-C		GTT CAG GCC TAC AAC CCC TGC AAG TTC AAG TAC GAT AGC GAC	707 (CD)
SOE-D		CCC GGT ACC GAT GGC GTG AAA ACG GCG GC		KpnI
emrE-XF		GCC ACA AAA GGG CAG GTT
emrE-XR		TAA AGC TCT CCC GCA GTA CC
lmo1851	95°C for 2 min; 30 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 1.3 min; 72°C for 2 min		1,083 (AD)
SOE-A		CCC CTG CAG ATC CAT TAG AGC ATC AAT TTG	537 (AB)	PstI
SOE-B		TTA CTA AAA GAA ATC AGT TCT
SOE-C		AGA ACT GAT TTC TTT TAG TAA ATT AGC CAC TTC ATC TTC TAT	546 (CD)
SOE-D		CCC GGT ACC CAT TAT AGC AAC TTG ATT GTG		KpnI
sel1	95°C for 2 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1.5 min; 72°C for 10 min		3,603 (AD)
SOE-A		GG TCT AGA GCT GCT TGA TGA GGT ATG C	501 (AB)	XbaI
SOE-B		GCA TTC CAC ATT GAC CGC
SOE-C		GCG GTC AAT GTG GAA TGC CGG TAA CAG TAG CTT GCT ATC ATC	504 (CD)
SOE-D		GG GGT ACC ACA TGA GCC TAT CAG AAT TAA CCC		KpnI
sel1-XF		CAT CTA CAC CGA CAA ATA CCG CA
sel1-XR		GCA ATC TTG TGC GAG TCT TTC

^aAll primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).

^bEndonuclease restriction sites are underlined, and regions complementary to SOE-B primers are italicized.