TABLE 2.

Oligonucleotides used in this study

PrimerSequenceaFeature(s) and/or sequence amplified
rtnrfARI5′-ggaattcAGTTGGTGATTGCGCTG-3′EcoRI restriction site is underlined; primer binds bp −1357 to −1341 upstream of nrfA start codon
5nrfAexp5′-ctagatctatcctggcgcgcCATTCAAGCGTAAGATACCA-3′BssHII restriction site is underlined, and nrfA start codon position is in boldface
3nrfArevexp5′-cgccaggatagatctaggcctTAAATGAAAAATGCACCG-3′StuI restriction site is underlined, and nrfA stop codon is in boldface
lt1068nrfARI5′-ggaattcATAGTTTATCCTTTTCTTCG-3′EcoRI restriction site is underlined; primer binds bp +721 to +702 downstream of last base of nrfA stop codon
5′helORF5′-aaaatggcgcgctaagaacataatATGAAAACAACGTTAAAAATGAC-3′BssHII restriction site is underlined, and start codon of hel is in boldface
3′helORF5′-ttatgcataatctggcacatcatattgataTTTACCATCCCAAGCTTGTACTGC-3′Sequence complementary to HA-epitope coding sequence is underlined, and position of stop codon of hel-HA fusion is in boldface
5′nrfAsapI5′-ccctagctagctcttcTATGATTGAGGAATTGCGCGTAAG-3′SapI restriction site is underlined, and nrfA start codon is in boldface
3′nrfAsapI5′-ccacatcgtgctcttcCTTATTTTTGTGCTGGTGGATCAACTAATAAGC-3′SapI restriction site is underlined, and position of the nrfA stop codon is in boldface
5′FNRupstreamCK5′-GCGGCAAGAATGGCAGCGTTATCC-3′Primer binds bp −1663 to −1640 upstream of fnr start codon
3′FNRdownstreamCK5′-ACCTGTGCGTCCCACTGTGCC-3′Primer binds bp +954 to +934 downstream of last base of fnr stop codon
FNRMlu1FORWARD5′-gcgacgcgtAGGGGTTTATGGCTGATGTGAAC-3′MluI restriction site is underlined; primer binds bp −468 to −446 upstream of fnr start codon
FNRMlu1REVERSE5′-cgcacgcgtAAAAAGGCGTGATATTCAC-3′MluI restriction site is underlined; primer binds bp +27 to +9 downstream of last base of fnr stop codon
1kb5′16775′-ATTGGGCGATCTTTAACCCAAGCGA-3′Primer binds bp −1025 to −1001 upstream of ytfE start codon
HI1677-5′out5′-gctgctgcgtaaCATAAATCACTCCTATATTGATTAAGT-3′Complementary sequence directly upstream of ytfE start codon; ytfE start codon is in boldface
HI1677-3′out5′-agtaccgccaccTAATTTAATATTCGTCAAAAAATAACCG-3′Sequence directly downstream of ytfE stop codon; ytfE stop codon is in boldface
1kb3′16775′-ATATCTAGAACATAATCTGCATGG-3′Primer binds bp +1676 to +1699 upstream of ytfE start codon
aacC1-5ORF5′-ATGTTACGCAGCAGCAACGATGTTACGCAGCAGG-3′Start codon of aacC1 is in boldface
aacC1-3ORF5′-TTAGGTGGCGGTACTTGGGTCGAT-3′Stop codon for aacC1 is in boldface
1677upcomp5′-aaagatctgctcttcAATGGGCTTGAGCAAGATGGGTAATTTC-3′SapI site is underlined; primer binds bp −646 to −619 upstream of ytfE start codon
1677-3′sap5′-aaagatctgctcttcTTTAATTAAAGTGCGGTTATTTTTTGAC-3′SapI site is underlined, and position of ytfE stop codon is in boldface
3′HI16765′-GCCACCAATAGAAGCTAAATG-3′Primer binds bp −1455 to −1435 upstream of ytfE start codon
3′ytfe.down5′-GCGCAAGATTATTTGGACAGA-3′Primer binds bp +1065 to +1045 downstream of last base of ytfE stop codon
5′ytfEc5′-tttgctcttcTATGTCTTTTGCCCAACAAAAAC-3′Sequence directly downstream of the ytfE start codon, which is in boldface; SapI site is underlined
5′ytfE-RT5′-CATGGTGATCGTGATGACTGTCCT-3′Within ytfE coding sequence
3′ytfE-RT5′-CCAACTGAAACAAGCATCTGCTGG-3′Within ytfE coding sequence
5′napA-RT5′-GCACCCATTAGATGCACAAGCAC-3′Within napA coding sequence
3′napA-RT5′-ATTGGGTCTGTCGCATCTAGTGTTA-3′Within napA coding sequence
  • a Uppercase letters indicate sequences identical to the H. influenzae genome. Lowercase letters indicate sequences added to allow generation of recombinant DNA constructs.