TABLE 2.

Oligonucleotides used in this study

PrimerOligonucleotide sequencea
CFP up SD PstI5′-AAACTGCAGAGGAGGACAGCTATGGTGAGCAAGGGCGAGGAG
C/YFP up SD XbaI5′-TCTAGAAGGAGGACAAGCTATGGTGAGCAAGGGCGAGGAG
YFP up SD BamHI (for YFP-PilT and YFP-PilU truncations)5′-AAGGATCCAGGAGGACAGCTATGGTGAGCAAGGGCGAGGAG
YFP up SD EcoRI (for YFP-PilB)5′-AAAAGAATTCAGGAGGACAGCTATGGTGAGCAAGGGCGAGGAG
YFP dn EcoRI (for YFP-PilB)5′-AAAAGAATTCGAGTCCGGACTTGTACAGCTCGTCCATGCC
C/YFP dn SalI (for C/YFP and YFP-PilU)5′-AAAGTCGACTGCAGAATTCGAAGC
C/YFP up PstI (for PilT-CFP)5′-CGAATTCTGCAGTCGACGGTA
C/YFP dn HindIII (for PilT-CFP)5′-ACAAAGCTTGTATGGCTGATTATGA
C/YFP dn XbaI (for YFP-PilT)5′-ATCTAGATTGAGCTCGAGATCTGAGTC
CFP dn PstI (for CFP-PilT)5′-AAAGTCGACTGCAGAATTCGAAGC
pilB XbaI up (for YFP-PilB)5′-AATCTAGAATGAACGACAGCATCCAACT
pilB XbaI dn (for YFP-PilB)5′-AATCTAGATTAATCCTTGGTCACGCGGT
pilT up BamHI (for PilT-CFP)5′-GGGATCCGCTCATCCGGTGTTTTCCTT
pilT dn PstI (for PilT-CFP)5′-ATTCTGCAGGAAGTTTTCCGGGATCTTCGCC
pilT up PstI (for CFP-PilT)5′-AAACTGCAGGGATGGATATTACCGAGCTGCTCGCT
pilT dn HindIII (for CFP-PilT and YFP-PilT)5′-GGGAAGCTTCTTGAATCCTAGACGCAGTT
pilT up XbaI (for YFP-PilT)5′-ATCTAGAATGGATATTACCGAGCTGCT
pilU up SalI (for YFP-PilU)5′-AAAGTCGACATGGAATTCGAAAAGCTGCTG
pilU dn HindIII (for YFP-PilU)5′-AAAAAGCTTCCCGAATCCCGGAACTGCGT
pilU117 dn XbaI5′-AATCTAGATCACTTCTTGAGGATTTCCGGGA
pilU118 up SalI5′-AAAGTCGACGCTGGCGCTGACCAAGCGCGGCCTG
pilU156 dn XbaI5′-AAAATCTAGATCAGGAGATGATGTGCCCGGTGGAGTTC
pilU168 dn XbaI5′-ATCTAGATCAGTGCTGGTGGATGTACTCGATCGGG
pilU197 dn XbaI5′-AAAATCTAGATCACGGCGCCTGGCGCAGGGTGTTCTTC
pilU221 dn XbaI5′-AAAATCTAGATCAGCCGGTCTCGGCGAAGGCCACGGCG
pilU244 dn XbaI5′-AATCTAGATCAGAAGAAGTGGATGATCCGCT
pilT156 dn BamHI5′-TTTTGGATCCTCAGAGGATGTGGTGGTACTTGG
pilT168 dn BamHI5′-TTTTGGATCTTCACGATTCGTGGACGAATTCGATCGGG
pilT197 dn BamHI5′-TTTTGGATCCTCACGGGTCCTCCCGCAGCGCCG
pilT221 dn BamHI5′-TTTTGGATCCTCAGCCGGTCTCCGCCGCGGTCA
NpilT dn + pilU (junction for YFP-N′PilT-C′PilU chimera)5′-CTCTTCCAGGGTCGGGATGTTGGAGGGAATGGTCCGGAATACCGCGCCGGCGCCACGGTTC
NpilU dn + pilT (junction for YFP-N′PilU-C′PilT chimera)5′-CTCCTCCATGGTCAGTACCTTGGTCTCGATCCGGCGCAATACCATGCCCACCAGGTTGCGC-3′
  • a Restriction endonuclease sequences are indicated by boldface type. The underlined sequences are Shine-Delgarno and spacer sequences for ribosomal binding sites. Stop codons used in the truncation primers are indicated by italics.