Table 1.

Cloning of the proposed epivancosamine biosynthetic genes

Gene	5′ primer (5′-3′)	3′ primer (5′-3′)	Vector	Restriction sites	Predicted mass, kDa
orf14	GAATTCCATATGTCGGTCACGTCCCA	TGATGTCCTCGAGGCTCACGTGCACGTT	pET22b(+)	NdeI/XhoI	45
orf23	GGGAATTTCCATATGTCGTCCTTCGTCGTTCCATCA	AAACCGCTCGAGTCATGCACCTCCCCGAGGCTGGGC	pET24b(+)	NdeI/XhoI	53
orf24	GGTCGATCATATGAAGCTGATCACCG	ATACACCTCGAGTCATGCGCGAGC	pET16b(+)	NdeI/XhoI	34
orf25	GGAATTCATATGACCACGCGTGTATGGGAT	GGGTAACTCGAGGGTCGACAGCACTTCGCG	pET16b(+)	NdeI/XhoI	41
orf26	GGAGCACATATGCAAGCACGCAAACTCG	CAGAAGCTTGGTCGAGACGGGGACCG	pET22b(+)	NdeI/HindIII	23

The proposed epivancosamine biosynthetic genes were amplified by PCR from Amycolatopsis orientalis genomic DNA with listed primer pairs. The restriction site introduced by each primer is shown in boldface. The PCR products were gel purified, digested with the appropriate restriction enzymes, and ligated into the designated vector with matching restriction sites.