Construction of PUb-RL and FerLCH-FFL Reporters

A gene cassette containing the Ae. aegypti polyubiquitin promoter-Renilla luciferase (PUb-RL) was excised from pSLfa-PUb-RL [33 (link)] using MluI and EcoRI (NEB) and treated with mung bean nuclease (NEB) to make the ends blunt. A separate plasmid containing the Ae. aegypti ferritin light chain (AAEL007383) promoter-firefly luciferase (FerLCH-FFL) [20 (link)] gene was linearized 3’ of the FFL ORF using PshAI and treated with shrimp alkaline phosphatase (NEB). The PUb-RL fragment was ligated into the linearized pGL3-LCH-FFL, and only tail-to-tail orientation constructs were sequenced to confirm the direction and integrity of the insert. A sequence-confirmed clone was purified using the endotoxin-free Midiprep (NucleoBond Xtra Midi EF, Machery-Nagel) for transfection.