The present disclosure provides a
protein having
luciferase activity comprising a secondary structural arrangement H1-L1-H2-L2-B1-L3-B2-L4-H3-L5-B3-L6-L4-L7-L5-L8-B6 wherein (i) "H" is a
helical domain, "L" is a loop domain, and "B" is a beta chain domain wherein the length of the B5 domain is at least 11, 12, 13, or 14 amino acids, and wherein "L" is a cyclic domain, and "B" is a beta chain domain. And residue 11 of the B5 domain is F, Y or L; and the length of the B4 domain is at least 12 amino acids, and the residue 10 of the B4 domain is F, Y, L, I, K or M; and / or residue 12 of the B4 domain is F, L, R, D, M, Q or V; (ii) the H1 domain is at least 18 or 19 amino acids in length; the residue 9 of the H1 domain is T, S, H, R, C, L, D, V, A, Q, G, E, K, I, N or M; (iii) the length of the B3 domain is at least 6, 7, 8, 9 or 10 amino acids, and the residue 1 of the B3 domain is W or H; (iv) the length of the B4 domain is at least 12 amino acids, and the residue 10 of the B4 domain is F, Y, L, I, K or M; or (v) the length of the B4 domain is at least 12 amino acids, and the residue 12 of the B4 domain is L, R, D, M, Q, or V. Additional proteins disclosed herein are illustrated in the claims. Also provided herein are
luciferase substrates,
assay buffers, and kits comprising one or more of a
protein having
luciferase activity or a
nucleic acid encoding the
protein, an
assay buffer, and a luciferase substrate.