Construction and confirmation of purD gene deletion plasmid cassette “pUC19-Kanamycin-Up-Down”. (A) Illustration of map of purD gene deletion plasmid cassette pUC19-Kanamycin-Up-Down (A1) confirmation of pUC19-Kanamycin-up-down gene deletion plasmid cassette (B) illustration of map of pUC19 plasmid (B1) confirmation of pUC19 cloning in DH5α cells (C) illustration of Kanamycin fragment with restriction sites KpnI (GGTACC) and BamHI (GGATCC) (C1) amplification of Kanamycin fragment. (C2) Confirmation of Kanamycin cloning in pUC19 (D) Illustration of Up sequence fragment with restriction sites EcoRI (GAATTC) and KpnI (GGTACC) (D1) amplification of upstream fragment (D2) confirmation of up sequence cloning into pUC19-Kanamycin plasmid (E) Illustration of downstream fragment with restriction sites BamHI (GGATCC) and PstI (CTGCAG) (E1) amplification of downstream fragment (E2) confirmation of Downstream fragment into pUC19-Kanamycin-up.