TABLE 1.

Templates and primers used in the PCR walking technique to clone and sequence arfI

PCR	Template	PCR primers
		Namea	Sequenceb
1	C. xylanolytica genomic	F1	5′-GAR GCN GCN CAR TGG GT-3′ (forward)
	DNA	R1	5′-GCR TTR TTR TTR AAD ATR TT-3′ (reverse)
2	pUC19-EcoRI libraryc	F2	5′-CAG TCA CGA CGT TGT AAA ACG ACG GC-3′ (forward)
		R2d	5′-CCA AGT TTG ATG CCA CGA CTG TTC-3′ (reverse)
3	pUC19-KpnI libraryc	F2	5′-CAG TCA CGA CGT TGT AAA ACG ACG GC-3′ (forward)
		R3	5′-GAT ATC CCA GGG TTT ATC ACG ACC G-3′ (reverse)
4	pUC19-HindIII libraryc	F4	5′-CGC AGC ATC ACA AGT TCC TCA TGC-3′ (forward)
		R4	5′-TCA CAC AGG AAA CAG CTA TGA CCA TG-3′ (reverse)
5	C. xylanolytica genomic	F5	5′-GGT CGT TGG TGA AAT ACA GCG G-3′ (forward)
	DNA	R5	5′-GAC AAA TCG CTC CCA CCG AAC AC-3′ (reverse)

^aThe priming sites of F2 and R4 complemented regions within the multiple cloning site of pUC19. 

^bA, adenosine; T, thymidine; G, guanosine; C, cytosine; R, adenosine or guanosine; N, adenosine, thymidine, cytosine, or guanosine; D, adenosine, thymidine, or guanosine. 

^cLibrary of C. xylanolytica genomic DNA created by restriction with an enzyme and ligated into pUC19. 

^dThe second and fourth nucleotides of primer R2 (cytosine and adenosine, respectively) were identified incorrectly in the amplicon from PCR 1 and did not correspond to the homologous nucleotides in amplicons from PCR 4 and 5. However, their distances from the 3′ end did not compromise the efficacy of R2 as a primer for PCR 2.