Fig. S.

Schematic representation of generation of full-length podhfr-ts using overlap extension PCR technique and analysis of amplicons. Four PCR reactions were required to generate the complete sequence: (1) pojr-ts (1134 bp) was PCR amplified from P. ovale DNA and ligated into pTZ57R/T; (2) podhfr-jr (863 bp) and pojr-ts (1134 bp) was acquired using pGEMT-podhfr and pTZ57R/T-pojr-ts as template respectively; (3) and (4) podhfr-ts (1920 bp) was obtained by 2-steps PCR overlap extension, in which dhfr-jr and jr-ts contain overlapping sequences that are complementary for extension shown by half arrow heads indicating the direction in which each fragment can act as primers for DNA polymerase. Dashed lines mark newly synthesized sequence or strand. Horizontal arrows represent primers used in PCR reactions: i, ii, iii, iv, and v are 5′Po-link, 3′PoTS, 5′PoDHFR, 3′Po-link, and 5′NhePo, respectively.