Human Coronavirus NL63 Open Reading Frame 3 encodes a virion-incorporated N-glycosylated membrane protein

Table 2 Oligonucleotides^a used for cloning procedures

Primer	Sequence (5'-end to 3'-end)	+/-	NC_005831^b
pcDNA3.1/NT-GFP-TOPO
5'NL63-E-GFP	TTCCTTCGATTAATTGATGAC	+	25203-25223
5'NL63-M-GFP	TCTAATAGTAGTGTGCCTC	+	25445-25463
5'NL63-N-GFP	GCTAGTGTAAATTGGGCC	+	26136-26153
5'NL63-O3-GFP	CCTTTTGGTGGCCTATTTC	+	24545-24563
3'NL63-EpK R	TTAGACATTTAGTACTTCAGCTGG	-	25410-25433
3'NL63-MpK R	TTAGATTAAATGAAGCAACTTCTC	-	26099-26122
3'NL63-NpK R	TTAATGCAAAACCTCGTTGAC	-	27246-27266
3'NL63-O3	ACAAGGAGCCATAAAATG	-	25244-25261
pcDNA3.1/V5-His-TOPO
5'Leader-NL	GACTTTGTGTCTACTCTTC	+	45 - 63
3'NL63-O3s	ATTAATCGAAGGAACATC	-	25199-25216
NL63-O3mis-Asn16 F	CTTACTCTTGAAAGTACTATTCAGAAGAGTGTGGCTAATCTC	+	25567-25609
NL63-O3mis-Asn16 R	GAGATTAGCCACACTCTTCTGAATAGTACTTTCAAGAGTAAG	-	25567-25609
pCAGGS^c
5'Eco-FLAG_O3-63	GCAGCAGAATTC ATGGACTACAAGGACGACGATGACAAG CCTTTTGGTGGCCTATTTCAACTTAC	+	24544-24570
3'Not-O3-63	CCTCCTGCGGCCGC TCAATTAATCGAAGGAACATCTTCGTATAG	-	25190-25219
5'Eco-HA-E	GCAGCAGAATTC ATGTACCCATACGATGTTCCAGATTACGCT TTCCTTCGATTAATTGATGACAATG	+	25203-25227
3'Not-E	CCTCCTGCGGCCGC TTAGACATTTAGTACTTCAGCTG	-	25411-25433
5'Eco-HA-M	GCAGCAGAATTC ATGTACCCATACGATGTTCCAGATTACGCT TCTAATAGTAGTGTGCCTCTTTTAGAG	+	25446-25472
3'Not-M	CCTCCTGCGGCCGC TTAGATTAAATGAAGCAACTTCTCTC	-	26098-26123
5'Eco-HA-N	GCAGCAGAATTC ATGTACCCATACGATGTTCCAGATTACGCT GCTAGTGTAAATTGGGCCGATGACAG	+	26138-26163
3'Not-N	CCTCCTGCGGCCGC TTAATGCAAAACCTCGTTGACAATTTC	-	27242-27268

^aOligonucleotides were provided by TIBMolbiol, Berlin, Germany
^bAccession no. hCoV-NL63 strain Amsterdam I
^cUnderlined are additional nucleotides representing restriction sites and a FLAG or HA-tag (italics)

ISSN: 1743-422X