Table 1. Mutants used for translation analysis of Cx41 5′ UTR.

Name	Template PCR	Primers PCRa	Mutationsb	Position	Effect
WT	–	5F1	CTATGG→	174	Introduction NcoI site
		5R1	CCATGG
Δ1	WT	5F2	uATG1→AAG	42	Annihilation of uAUG1
		5F1
		5R1
Δ23	WT	5F3	uATG2→AAG	98	Annihilation of uAUG2 and uAUG3
		5F1	uATG3→AAG	104	Mutation of two cysteine residues in uORF1 into serine
		5R1
#1	WT	5F1	TAG1→TTG	126	uORF1 connected to ORF GFP
		5R4	A insertion	164
Δ23#1	Δ23	5F1	uATG2→AAG	98	Annihilation of uAUG2 and uAUG3
		5R4	uATG3→AAG	104	uORF1 connected to ORF GFP
			TAG1→TTG	126
			A insertion	164
#23	WT	5F1	TAG2+3→AAG	121	uORF2+3 connected to ORF GFP
		5R5	TGA→AGA	139	uORF1 6 nt shorter
			TAA→AAA	160
CB–	WT	5F5	GCT→GCG	46	Creation of rare codons for ALPVc in uORF1
		5R1	CCA→CCG	52
			GTG→GTA	55
CB+	WT	5F6	TTA→CTG	47 + 49	Creation of common codons for ALPVc in uORF1
		5R1
#1CB–	#1	5F5	GCT→GCG	46	Creation of rare codons for ALPVc in uORF1
		5R4	CCA→CCG	52	uORF1 connected to ORF GFP
			GTG→GTA	55
			TAG1→TTG	126
			A insertion	164
#1CB+	#1	5F6	TTA→CTG	47 + 49	Creation of common codons for ALPVc in uORF1
		5R4	TAG1→TTG	126	uORF1 connected to ORF GFP
			A insertion	164
Δ23CB–	Δ23	5F5	GCT→GCG	46	Creation of rare codons for ALPVc in uORF1
		5R1	CCA→CCG	52	Annihilation of uAUG2 and uAUG3
			GTG→GTA	55
			uATG2→AAG	98
			uATG3→AAG	104
Δ23CB+	Δ23	5F6	TTA→CTG	47 + 49	Creation of common codons for ALPVc in uORF1
		5R1	uATG2→AAG	98	Annihilation of uAUG2 and uAUG3
			uATG3→AAG	104
Δ23#1CB–	Δ23#1	5F5	GCT→GCG	46	Creation of rare codons for ALPVc in uORF1
		5R4	CCA→CCG	52	uORF1 connected to ORF GFP
			GTG→GTA	55	Annihilation of uAUG2 and uAUG3
			uATG2→AAG	98
			uATG3→AAG	104
			TAG1→TTG	126
			A insertion	164
Δ23#1CB+	Δ23#1	5F6	TTA→CTG	47 + 49	Creation of common codons for ALPVc in uORF1
		5R4	uATG2→AAG	98	uORF1 connected to ORF GFP
			uATG3→AAG	104	Annihilation of uAUG2 and uAUG3
			TAG1→TTG	126
			A insertion	164

^aPrimers are listed in Table 2.

^bAll mutants are based on the WT Cx41 5′ UTR containing the NcoI restriction site.

^cALPV = alanine, leucine, proline, valine, amino acid residues 2–5 of uORF1.