TABLE 2.

Primers used for constructing a signal peptide in the collagen α(IV) NC1 domain (pCI-neo/rSP-NC1)

Primer orientation/primer number	Primer, 5′–3′
Sense 1	GTGGTTAGCACAAGAATGAGAGGCTTCATCTTCAC
Sense 2	GCTCCTGGCTGCCCCGCCTGTGGTTAGCACAAGAAT
Sense 3	TTCTTGGCGCTCCTGCTGCCTATCCTGTTGCTGCTCCTGGCTGCCCCGC
Sense 4	CACTCGAGATGCACTCAGAGACTGCTCCAAGGTTCTTGGCGCTCCTGCT
Antisense	CAGCGGCCGCTTAGTGTCTTTTCTTCATGCACACCTG

The nucleotide sequence for the SP of rat collagen α3(IV) chain is: 5’- ATGCACTCAGAGACTGCTCCAAGGTTCTTGGCGCTCCTGCTGCCTATCCTGTTGCTGCTCCTGGCTGCCCCGCCTGTGGTTAGC-3’, which was constructed using the 4 sense primers and the antisense primer. Four overlapping PCRs were performed by using sense primer 1 and antisense primer in the first PCR (PCR-i), with Cola3(IV) NC1 cDNA (34) serving as the template, to be followed by sense primer 2 and antisense primer in the second PCR (PCR-ii) using PCR product from PCR-i as the template. Thereafter, sense primer 3 and antisense primer in the third PCR (PCR-iii) used PCR product from PCR-ii as the template. Finally, sense primer 4 and antisense primer in the fourth PCR (PCR-iv) using PCR product from PCR-iii as the template to clone the SP into Cola3(IV) NC1. The restriction sites are XhoI (italicized/dotted underlined in sense primer 4) at the 5ʹ end and NotI (italicized/dotted underlined in antisense primer) at the 3ʹ end. The start and stop codons of ATG and TTA, respectively, are double underlined. Nucleotide sequence was confirmed by direct nucleotide sequencing at Genewiz.