TABLE 1.

Plasmids used for in vitro transcription and PCR primers used for their construction

Mutagenesis methods used are: o, overlap extension PCR method; i, inverse PCR.

Plasmid	Mutation	Forward primer (5′-3′)	Reverse primer (5′-3′)	Mutagenesis
M8:Ex1(WT)-based synonymous codon substitutions
pYY23	Ile-74(AUA)	TGAGCATAAAAGCCCGTAGAA	GGCTTTTATGCTCAGCTGAC	o
pYY22	Lys-75(AAG)	GCATTAAGGCCCGTAGAAACT	ACGGGCCTTAATGCTCAGCT	o
pYY24	Ala-76(GCG)	TAAAGCGCGTAGAAACTGTAGC	TCTACGCGCTTTAATGCTCAG	o
pYY25	Ala-76(GCA)	TAAAGCACGTAGAAACTGTAGC	TCTACGTGCTTTAATGCTCAG	o
pYY26	Arg-77(CGG)	TAAAGCCCGGAGAAACTGTAGC	GTTTCTCCGGGCTTTAATGC	o
pYY28	Arg-77(AGG)	TAAAGCCAGGAGAAACTGTAGC	GTTTCTCCUGGCTTTAATGCTCAG	o
pYY27	Arg-77(AGA)	TAAAGCCAGAAGAAACTGTAGC	GTTTCTUCUGGCTTTAATGCTCAG	o
pYY21	Ile-83(AUA)	ACATAGGTGTTGCACAGATCG	GCAACACCTATGTTGCTACAG	o
pYY20	Gly-84(GGG)	ATCGGGGTTGCACAGATCG	GCAACCCCGATGTTGCTAC	o
pYY19	Gly-84(GGA)	ATCGGAGTTGCACAGATCG	GCAACTCCGATGTTGCTAC	o
pYY17	Val-85(GUG)	CGGTGTGGCACAGATCG	TGTGCCACACCGATGTTGC	o
pYY18	Val-85(GUA)	CGGTGTAGCACAGATCG	TGTGCCACACCGATGTTGC	o
pYY43	Ser-94(UCG)	CTAAGTGGTCGAACAACCCATC	GGTTGTTCGACCACTTAGC	o
pYY44	Ser-94(AGC)	CTAAGTGGAGCAACAACCCATC	GGTTGTTGCTCCACTTAGC	o
M8:ND5(C80A)-based cysteine substitutions
pYY37	K75C, C80A	TTTAATGCTCAGCTGACGGAC	TGTCGTAGAAACGCTAGCAACATC	i
pYY38	A76C, C80A	TGTGCCCGTAGAAACGCTAGCAA	AATGCTCAGCTGACGGACGA	i
pYY39	R77C, C80A	TGAGCATTAAAGCCTGTAGAAAC	CAGTTTCTACAGGCTTTAATGCTC	o
pYY40	R78C, C80A	AAGCCCGTTGTAACGCTAG	TGCTAGCGTTACAACGGGCT	o
pYY41	N79C, C80A	AAGCCCGTAGATGTGCTAGCAA	TTGCTAGCACATCTACGGGCTT	o
pYY42	K92C, C80A	GCGGCTTGTTGGTCCAACAAC	GGACCAACAAGCCGCCACGAT	o