Table 2.

The primer sequence.The underlined sequence shown in 1 and 1’, 2 and 2’, 3 and 3’, 4 and 4’, 5 and 5’, 6 and 6’, 7 and 7’ are reverse complements. The boldface sequence gatcc, which was added to reserve a BamHI site in the overlapping region in B3-R, did not destroy the structure of the assembled sequence of the 4.98 kb DNA molecule in the final plasmid. The boldface sequence agc, which was added to introduce an Eco47III site in the overlapping region in W4-F and W5-F, did not destroy the structure of the assembled sequence of the 11.88 kb DNA molecule in the final plasmid.

The overlapping primers for assembly of the 4.98 kb DNA molecule

B1-F: 5′-cgactctagaggatcGGACAAGATCTTGAAAGTGCTCGACGTCAATT-3′

B1-R: 5′-agctcggtacccgggGATCCATGAGAGGAC 1AACAAGGAAGATAGCTAT-3′

B2-F: 5′-GTCCTCTCATGGATC 1′TTGGTCCACAAGTG-3′

B2-R: 5′-agctcggtacccgggGATCCGCGGCCCGGC 2CTGGAACG-3′

B3-F: 5′-GCCGGGCCGCGGATC 2′TGCCTCG-3′

B3-R: 5′-agctcggtacccggggatccACATCTCGCTCGGGTACCTGGTCGAAT-3′

The overlapping primers for assembly of the 7.09 kb DNA molecule

P1-F: 5′-ctctagaggatccccGGGTGGGGGAGGCAT 3CTGATATGCAGCAA-3′

P1-R: 5′-tcgagctcggtacccGCATGTGGACTGTGGAGGTGGCCAGTAATT-3′

P2-F: 5′-ctctagaggatccccGTCATGCATTCAGCCGTCAGAAAGGCTCAGA-3′

P2-R: 5′-ATGCCTCCCCCACCC 3′TACAACACCATGGCATCGATCGGATCAATCCTA-3′

The overlapping primers for assembly of the 11.88 kb DNA molecule

W1-F: 5′-ctctagaggatccccGGGAGCAAGAATTGT 4CGATGGAACCAGCT-3′

W1-R: 5′-tcgagctcggtacccCCTGTATAAGTTGAATTTGAATTTGCATGC-3′

W2-F: 5′-ctctagaggatccccGGGATAAATTAAACC 5ACGGTAATCTTCCAATAGAAC-3′

W2-R: 5′-ACAATTCTTGCTCCC 4′AACTTTGGGTATTTCAG-3′

W3-F: 5′-ctctagaggatccccGGGCGAGGCATCTTG 6GTAGCTGGCTT-3′

W3-R: 5′-GGTTTAATTTATCCC 5′AAGGTTACAGGTCTAGTAAT-3′

W4-F: 5′-ctctagaggatccccagc GCTGACGACGATTAA 7TTGTGTGTGCAACG-3′

W4-R: 5′-CAAGATGCCTCGCCC 6′CTGCCCGAGA-3′

W5-F: 5′-ctctagaggatccccagc GCTGCTTGCAAAACACTCTCCCTCTAAAAT-3′

W5-R: 5′-TTAATCGTCGTCAGC 7′CACGCTTTAATTTGTTC-3′