f1(+)origin138-444 f1 (+ori B-galactosidase a-fragment 463-816 multiple cloning site 653-760 lac promoter 817-938 ampicillin pUC origin 1158-1825 lacZ ampicillin resistance (bla)ORF 1976-2833 Kpn I pBluescript SK+ MCS 3.0kb -SacI TP lac pBS SK+ pUC ori pBluescript SK(+/-)Multiple Cloning Site Region (sequence shown 601-826) T7 Promoter Kpn I A8d1091 粉o1 TTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGT. M13-20 primer binding site T7 primer binding site KS primer binding site. 2tp1061 Hindll EcoRV EcoRI Pl Smal BamH I Spel Xbal .ATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCA. KS primer binding site SK primer binding site T3 Promoter B-gal a-fragment .GCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCC T3.prmer binding site M13 Reverse primer binding site 11 pBS SK+