Table S1.
Plasmids used in the study
| Plasmid* | Construction/comments† |
| pT7SecA | E. coli secA gene under T7 promoter control (44) |
| pT7SecA(Cys0) | pT7SecA derivative with all four cysteine codons changed to serine |
| pT7SecA(Cys321) | SecA codon 321 on pT7SecA(Cys0) was changed to a cysteine codon |
| pT7SecA(Amber721, Cys321) | SecA codon 721 on pT7SecA(Cys321) was changed to an amber codon |
| pT7SecA834 | Deletion of SecA codons 835–901 of pT7SecA |
| pT7SecA834–PhoA68 | Insertion of first 68 codons of PhoA between SecA codon 834 and his tag of pT7SecA834 in multiple stages: codons 1–7, 8–15, 16–21, 22–28, 29–34, 35–41, 42–48, 48–54, 55–61, and 62–68 |
| pT7SecA834(Cys0)–PhoA68 | SecA codon 98 on pT7SecA834–PhoA68 was changed to a serine codon |
| pT7SecA834(Cys0)–GS–PhoA68 | A linker sequence comprised of glycine and serine residues (SSGGSG) was inserted between SecA and PhoA on pT7SecA834(Cys0)–PhoA68 |
| pT7SecA834(Cys321)–GS–PhoA68 | SecA codon 321 on pT7SecA834(Cys0)–GS–PhoA68 was changed to a cysteine codon |
| pT7SecA834(Cys321)–GS–PhoA68(Amber2) | PhoA codon 2 on pT7SecA834(Cys321)–GS–PhoA68 was changed to an amber codon |
| pT7SecA834(Cys321)–GS–PhoA68(Amber22) | PhoA codon 22 on pT7SecA834(Cys321)–GS–PhoA68 was changed to an amber codon |
| pT7SecA834(Cys321)–GS–PhoA68(Amber37) | PhoA codon 37 on pT7SecA834(Cys321)–GS–PhoA68 was changed to an amber codon |
| pT7SecA834(Cys321)–GS–PhoA68(Amber45) | PhoA codon 45 on pT7SecA834(Cys321)–GS–PhoA68 was changed to an amber codon |
| pT7SecA834(Cys37)–GS–PhoA68 | SecA codon 37 on pT7SecA834(Cys0)–GS–PhoA68 was changed to a cysteine codon |
| pT7SecA834(Cys37)–GS–PhoA68(Amber2) | PhoA codon 2 on pT7SecA834(Cys37)–GS–PhoA68 was changed to an amber codon |
| pT7SecA834(Cys37)–GS–PhoA68(Amber 22) | PhoA codon 22 on pT7SecA834(Cys37)–GS–PhoA68 was changed to an amber codon |
| pT7SecA834(Cys37)–GS–PhoA68(Amber37) | PhoA codon 37 on pT7SecA834(Cys37)–GS–PhoA68 was changed to an amber codon |
| pT7SecA834(Cys37)–GS–PhoA68(Amber45) | PhoA codon 45 on pT7SecA834(Cys37)–GS–PhoA68 was changed to an amber codon |
| pBAD22 SecE–SecY(Cys0)–SecG | E. coli SecYEG lacking any cysteine under araBAD promoter control; courtesy of Tom Rapoport, Harvard Medical School, Boston |
| pBAD22 SecE–SecY(Cys292)–SecG | SecY codon 292 on pBAD22 SecE–SecY(Cys0)–SecG was changed to a cysteine codon; courtesy of Tom Rapoport |
| pEVOL–pAzF | Plasmid for incorporation of H-4-Azido-Phe-OH at amber codons (22) |
*
All SecA or SecA–PhoA chimeras contained a carboxyl-terminal hexahistidine tag, while the SecYEG-containing plasmids contained an amino-terminal hexahistidine tag on SecE.
†
QuikChange mutagenesis was used for all plasmid construction as described by the manufacturer. Plasmid DNA sequence was verified by the University of Pennsylvania DNA-Sequencing Facility.