Table 1. Primer Sequences and Cycling Conditions.

Construct and Primer Name	Nucleotide Position*	Primer Sequence (5′-3′)	Tm °C	Amplicon Size	PCR Cycling Conditions
C-4B a (1 to 255 aa)
4B-CT-Fb	6916–6934	acc acc atg g c ct AAC GAG ATG GGT TGG CTA G	68.3	777	95°C 5 min 1 cycle; 95°C 45 s, 64°C 45 s, 72°C 1 min, 30 cycles; 72°C 7 min 1 cycle
4B-CT-Rb	7680–7658	G TCT TTT TAG TCC TGG TTT TTC CA	55.2
C-174B a (−17 to 255 aa)
17-4B-CT-Fb	6865–6882	acc acc atg g c ct CTA GCC GTG TTC CTG ATT	65.9	826	95°C 5 min 1 cycle; 95°C 45 s, 60°C 45 s, 72°C 1 min, 30 cycles; 72°C 7 min 1 cycle
C-sig4B a (−44 to 255 aa)
sig-4B-CT-Fb	6784–6801	acc acc atg c GGA ATG TTG CTG CTC TCC	66.6	906	95°C 5 min 1 cycle; 95°C 45 s, 60°C 45 s, 72°C 1.5 min, 30 cycles; 72°C 7 min 1 cycle
C-4AB a (−149 to 255 aa)
4AB-CT-Fb	6469–6489	acc acc atg g c ct TCT CAG ATA GGG CTC ATT GAG	65	1224	95°C 5 min 1 cycle; 95°C 45 s, 60°C 45 s, 72°C 2 min, 30 cycles; 72°C 7 min 1 cycle
N-sig4B a (−44 to 255 aa)
si4B-NT-Fb	6784–6801	GGA ATG TTG CTG CTC TCC C	58.1	900	95°C 5 min 1 cycle; 95°C 45 s, 55°C 45 s, 72°C 1 min, 30 cycles; 72°C 7 min 1 cycle
4B-NT-Rb	7680–7658	TTC ATC TTT TTA GTC CTG GTT TTT CCA	53.5

^aConstruct name. The numbers in parentheses indicate the NS4B amino acid (aa) from 1–255 and the minus sign represent the number of aa to C-teminal ends of NS4A preceding NS4B protein.

^bPrimer name. The CT or NT in the primer name denotes C- or N-terminal end of NS4B with respect to GFP fusion protein whereas the F or R denotes the sense or antisense primer, respectively.

^cThe Kozak sequence is in bold, lowercase and italicize. The nucleotides in lower case only are codon filling nucleotides. The viral sequences are in uppercase.

^*GenBank Accession No. AF196835.