Psc a amp kan

Stenotrophomonas maltophilia Oak Ridge strain 02 (ATCC #53510) was purchased from the American Type Culture Collection (Manassas, VA, USA), Enterobacter sp. YSU was described previously [29 (link)] and StrataClone SoloPack Competent Cells and the plasmid, pSC-A-amp/kan, were purchased from Agilent (Santa Clara, CA, USA) as components of the StrataClone PCR (Polymerase Chain Reaction) Cloning Kit. TransforMax™ EC100D™ pir-116 Escherichia coli (E. coli) was purchased from Lucigen (Middleton, WI, USA) [30 (link)].
Lennox LB medium was purchased from Amresco (Solon, OH, USA), and R3A-tris medium was described previously [29 (link)]. When required, LB or R3A-tris medium was supplemented with 100 µg/mL ampicillin (Amresco), 50 µg/mL kanamycin (Amresco) and varying concentrations of HgCl₂, ZnCl₂, CdCl₂, CuSO₄ (Fisher Scientific, Fair Lawn, NJ, USA) and HAuCl₄·3H₂O (Amresco).

The Irx3 alleles of the mouse strains B6JHanZtm, B6NCrl and B6J were amplified with Q5 high-fidelity Taq polymerase (New England Biolabs, Ipswich, MA, USA) using the primer pair 5'-GACGACAGGAGGAGAGTGTAAACTAG-3' and 5'-GGCAGACCTGCCGGTTATAGTCAAAA-3', producing a fragment that covered 3136 bp of the B6J allele (ACCESSION No: NC_000074.6). The thermocycling conditions were as follows: (i) an initial denaturation step of 30 s at 94 °C; (ii) 35 cycles of 30 s at 98 °C, 30 s at 60 °C (annealing temperature) and 210 s at 72 °C; and (iii) a final elongation step of 600 s at 72 °C. The PCR products were directly cloned into the TOPO TA-cloning vector pSC-A amp/kan (Agilent, Waldbronn, Germany). Two independent clones from each strain were sequenced (Eurofins Genomics, Ebersberg, Germany) with vector-specific T3 and T7 oligonucleotides and with five Irx3-specific primers (Irx3_1: 5′-TCTGGGTCCCTATCCAATGTG-3′, Irx3_2: 5′-AGGAGAACAAGATGACGTGG-3′, Irx3_3: 5′-AGAAGCCCAAGATCTGGTCA-3′, IRX3_4: 5′-TCCTACAGATCGCTGTAGTG-3′, Irx3_5: 5′-CTCTGGTCTTATCAGCTCT-3'). Sequence analysis and alignment were performed with ApE-A Plasmid Editor v2.0.47 software.