A ubiquitin promoter drives expression 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 P850A mutation that stabilizes the expressed protein under normoxia (by preventing hydroxylation of the protein). A mNeonGreen tag has been inserted between amino acids 725 and 726 in the oxygen-dependent degradation domain (ODD) and the coding sequence is tagged at the C-terminal end with Tag:V5.
