A ubiquitin promoter drives expression of a reporter of hypoxia. The reporter consists of the full-length sima coding sequence, carrying three amino acid substitutions (Q339E, V413E and Y417T) to prevent dimerization with tgo protein (this prevents the activation of hypoxia signalling by the reporter) and with a mNeonGreen tag inserted between amino acids 725 and 726 in the oxygen-dependent degradation domain (ODD). The coding sequence is tagged at the C-terminal end with Tag:V5. Under normoxia, the expressed protein undergoes oxygen-modulated degradation (mediated by hydroxylation of the P850 residue). Under hypoxia, the expressed protein is stabilized, resulting in an increase in both Tag:V5 immunoreactivity and mNeonGreen fluorescence (the increase is more apparent with Tag:V5 immunoreactivity).
