Table 1. Primers used for site-specific mutagenesis.

Name a	Sequence b	Annealing Temperature (°C) c
Q38R_F	TCTTTGACGCTGGATCGGTCCGTC	60
Q38R_R	CTGCAAACCTTTGTCTTTATGGTCGAGCGG	60
E92K_F	ATTACCTTGAAGAGTGGAGAGTTCCAAACAA	65
E92K_R	GAGCTGCCCGTCCACTTCGATTTG	65
I114T_F	CAGACCGAGCAAACACAAGATTCGGAGCATTCC	70
I114T_R	AAAGGCGGTTAAGGCGGAATGGCT	70
R130G_F	AAACGCCAGTTCGGAATCGGCGAC	70
R130G_R	CGCAACCATCTTCCCGGAATGCTC	70
I134V_F	ATCGGCGACGTAGCGGGCGAACATACA	70
I134V_R	TCTGAACTGGCGTTTCGCAACCATCTTCCC	70
H138L_F	GCGGGCGAACTTACATCTTTTGACAAG	60
H138L_R	TATGTCGCCGATTCTGAACTGGCGTTT	60
S211T_F	ATCAGCGGTACCGTCCTTTACAACCAA	70
S211T_R	GACGGCATGGCGTTTTCCATCC	70
G236C_F	GAAGTTGCCTGCAGCGCGGAAGTG	70
G236C_R	CTGGGCTTTTCCGCCAAAGATACCGAG	70
H248L_F	ATACGCCTTATCGGCCTTGCC	65
H248L_R	GCCGTTTACGGTTTTCACTTCCG	65
S223R_F	AGTTACCGCCTCGGTATCTTTGGCG	60
S223R_R	GCCTTTCTCGGCTTGGTTGTAA	60
K199E_F	GCCGATATCGAGCCGGATGGAAAA	65
K199E_R	GGCGGCCAGGTCGACATTG	65

^a Name of primer for each mutant based on numbering of mature FHbp ID 1 sequence, beginning with lipidated cysteine residue; F, forward; R, reverse

^b Sequence shown from 5’ to 3’; nucleotide changes relative to wild-type sequence in forward primer are underlined

^c Annealing temperature used to amplify mutant FHbp gene