Table 3. One step assembly of promoter, GOI and terminator by Hot Fusion reaction.

Hot Fusion	Work plan ID	Insert	Size	Conc. (ng/ul)	Total size (bp)	Blue colonies	White colonies	Colonies screened	Positive colonies
HF1161	CP58017	Promoter	2924	42.3	4615	12	608	8	8
		GOI	1113	42.69
		Terminator	578	13.92
HF1162	CP58833	Promoter	2622	20.01	5572	30	1,312	8	8
		GOI	2625	36.96
		Terminator	325	18.21
HF1163	CP58837	Promoter	2622	61.27	3978	22	2,576	8	8
		GOI	1031	30.33
		Terminator	325	15.34
HF1164	CP59426	Promoter	1940	48.56	5746	26	1,680	8	8
		GOI	3228	26.58
		Terminator	578	21.58
HF1165	CP59974	Promoter	941	24.88	2020	42	1,858	8	8
		GOI	579	36.62
		Terminator	500	33.58
HF1166	CP59975	Promoter	497	27.18	1657	38	2,976	8	8
		GOI	660	32.27
		Terminator	500	39.48
HF1167	CP60007	Promoter	2196	45.27	3874	44	1,940	8	8
		GOI	1428	25.35
		Terminator	250	25.67
HF1168	CP60348	Promoter	1583	63.67	4225	32	2,304	8	8
		GOI	2112	29.03
		Terminator	530	12.18
HF1169	CP60436	Promoter	2196	31.23	4288	27	1,648	8	8
		GOI	1842	39.75
		Terminator	250	24.87
HF1170	CP61067	Promoter	2001	95.99	5055	56	1,360	8	8
		GOI	2804	24.77
		Terminator	250	20.95
HF1192	CP61167	Promoter	2196	27.71	3895	22	768	8	8
		GOI	1449	20.79
		Terminator	250	8.58
HF1193	CP62253	Promoter	1931	14.45	4634	19	344	8	8
		GOI	2403	44.19
		Terminator	300	11.7
HF1171	Control				28	13	NA	NA	NA

A binary vector was digested with SfaAI to release the lacZ gene from the cloning site, heat-inactivated and used directly in this study. Blue colonies indicate the parental vector (background) and white colonies indicate potential positive clones. The last column indicates true positive clones based on PCR screening of 8 clones per construct.