. 2014 Sep 8;289(44):30343–30354. doi: 10.1074/jbc.M114.598656

TABLE 2.

Primers and templates for site-directed mutagenesis of pilF

Primer	Sequence 5′-3′	Template plasmids for PCR	Resulting plasmids
pilF_pDM12_for	`ATTACATATGCACCATCATCACCACCATCATAGCGTGCTCACCATAGGGGACAAAAGG`	pET28a-pilF wild type and variants	pDM12-pilF wild type and variants
pilF_pDM12_rev	`ACTTAGATGCGGCCGCTTACTCAATGGTACGCGCCAG`	pET28a-pilF wild type and variants	pDM12-pilF wild type and variants
MutA781_for	`GGTGGAGGTCAAGCCGGACCCC`	pET28a-pilFwt	pDM12-pilFC781A
MutA781_rev	`TTGCAGTGCTCGGCCACCCTGCGC`	pET28a-pilFwt	pDM12-pilFC781A
MutA784_for	`AGCCGGACCCCGAGACCCTCAG`	pET28a-pilFwt	pDM12-pilFC784A
MutA784_rev	`TGACCTCCACCTTGGCGTGCTCGCACAC`	pET28a-pilFwt	pDM12-pilFC784A
MutA784_for	`AGCCGGACCCCGAGACCCTCAG`	pET28a-pilFC784A	pDM12-pilFH783A/C784A
MutA783/784_rev	`TGACCTCCACCTTGGCGGCCTCGCACAC`	pET28a-pilFC784A	pDM12-pilFH783A/C784A
MutA816_for	`TGCGGCGGCACCGGGTACAAGGG`	pET28a-pilFwt	pDM12-pilFC816A
MutA816_rev	`CCGCTCGGCCCCCATGCCCTTGTAG`	pET28a-pilFwt	pDM12-pilFC816A
MutA819_for	`GGGCATGGGGTGCGAGCGGGCCGGCGGCACC`	pET28a-pilFwt	pDM12-pilFC819A
MutA819_rev	`GGATGGCGTAGCGGCCCTTGTACCC`	pET28a-pilFwt	pDM12-pilFC819A
Mut2Cys_for	`GCGCAGGGTGGCCGAGCACGCCAAGGTGGAG`	pET28a-pilFwt	pDM12-pilFC781A/H783A/C784A
Mut2Cys_rev	`CCTGAGGGTCTCGGGGTCCGGCTTGAC`	pET28a-pilFwt	pDM12-pilFC781A/H783A/C784A
Mut3Cys_for	`GGCATGGGGGCCGAGCGGTG`	pET28a-pilFC781A/H783AC784A	pDM12-pilFC781A/H783A/C784A/C816A
Mut3Cys_rev	`CCTTGTACCCGGTGCCGCCG`	pET28a-pilFC781A/H783AC784A	pDM12-pilFC781A/H783A/C784A/C816A
Mut4Cys_for	`CGAGCGGGCGGGCGGCACCG`	pET28apilFC781A/H783A/C784A/C816A/C819A	pDM12pilFC781A/H783AC784A/C816A/C819A
Mut4Cys_rev	`GGCGTAGCGGCCCTTGTACC`	pET28apilFC781A/H783A/C784A/C816A/C819A	pDM12pilFC781A/H783AC784A/C816A/C819A