Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments

Table 2 Oligonucleotide primers used for construction of rib5⁺ expression plasmid and for site-directed mutagenesis

Primer	Primer orientation	Amino acid replacement	Novel restriction site	Sequence (5'– 3')
SSP-RIB5-1	-	none	EcoRI	ATAATAGAATTC ATTAAAGAGGAGAAATTAACCATGTTTACCGGTCTTGTTGAAGCCATTGGTGTTG
SSP-RIB5-2	+	none	BamHI	TATTATGGATCC TCAAGCTTGGGTTTTCTTAATCCAGTC
1	-	none (flanking primer)		GGTCTAGATCACACAGAATTCATTAAAGAGG
2	+	none (flanking primer)		GACTGCAGTCAAGCTTGGGTTTTCTTAA
3	+	C48 → S	HpaI	TCTGTCACTGTAAGagA AGTACCGTTaAC GGCGATACT
4	+	C48 → A	HpaI	TCTGTCACTGTAAGagc AGTACCGTTaAC GGCGATACT
5	+	C48 → M	HpaI	TCTGTCACTGTAAGcat AGTACCGTTaAC GGCGATACT
5	-	S146 → C	KpnI	ATAGCCCTGGACGGTACc Tgt CTGACCATTACTCAT
6	-	S146 → A	KpnI	ATAGCCCTGGACGGTACc gCG CTGACCATTACTCAT

Oligonucleotides were designed to hybridize to the sense (+) and to the antisense (-) strand of the rib5⁺. Mutated bases are indicated in lower case. Codons specifying modified amino acid residues are shown in bold type. Novel restriction sites are underlined.

ISSN: 1471-2091