Figure 5

Insertion of Gateway Destination cassette between rescued regulatory regions of PAP85. (A) Schematic diagram showing the location of primers and restriction enzyme sites used for analysis of integrity of candidate clones of pWY190 with the Rfa-cassette replacing the coding region of PAP85. Open-boxed region represents 5'- and 3'-regulatory region of PAP85, hatched-box region represents the Gateway Destination cassette, vertical striped-box region represents the 3'-regulatory region of PAP85 and the line represents vector sequence. Arrows indicate location of diagnostic PCR primers and BamHI sites used for evaluating the correctness of the assembled construct. (B) PCR-based evaluation of candidate clones of pWY190. The 5'-Test (primers Cm-5'-AS1 and PAP85-DEST-Integ-5'-Test) evaluates for correct fusion of attR1 with the 5' regulatory region of PAP85 with the presence of a 0.3 kb amplicon. Of 24 candidate clones assessed 16 are represented. The 3'-Test (primers C1 and PAP85-DEST-Integ-3'-Test) evaluates clones 9 and 13 for correct fusion of attR2 with the 3' regulatory region of PAP85 with the presence of a 1.1 kb amplicon. (C) Restriction enzyme analysis of candidate PAP85-Rfa fusion. pWY190 was digested with BamHI and exhibited the predicted restriction fragments of 0.7, 3.0 and 7.1 kb. M, DNA size marker (1 kb Plus ladder, Invitrogen) with representation from 0.1–1 kb (B) and 0.5–12 kb (C).