Cloning and Sequencing of ACA1_384820 from A. castellanii

The ACA1_384820 coding sequence from A. castellanii was amplified by PCR from total cDNA with flanking NdeI and XhoI restriction sites, using the primers ACA1_384820_Fwd_NdeI and ACA1_384820_Rev_XhoI (Table 2). PCR fragments were cloned into the NdeI/XhoI sites of the expression plasmid pTBPF-eGFP [21 (link)]. For the pTBPF-empty, pTBPF-eGFP was digested by NdeI and XhoI (NEB), and the sticky 5’-overhangings ends of the vector were filled using DNA Polymerase I Large (Klenow) Fragment (Promega) and ligated (T4 DNA Ligase, Promega) following the manufacturer’s recommendations.
All plasmid constructs were transferred in chemically-competent Escherichia coli DH5α and validated by Sanger sequencing. DNA sequencing was completed with the ABI Prism BigDye™ terminator v3.1 sequencing kit (Applied Biosystems) and then analyzed using an automatic ABI Prism 3730 genetic analyzer (Applied Biosystems).