"The minigene encoding the beta-(1-42) peptide was assembled in thress steps. The artificial signal peptide coding sequence of vector pPD50.52 was amplified by using primers SP-up (5'-CGGGATTGGCCAAAGGACCC) and SP-down (5'-CCCGGTACCTGCTGGTGCCAGAAAGAT), cleaved with Nhe I and Kpn I restriction endonucleases, and inserted between the unique Nhe I and Kpn I sites of vector pPD49.26, resulting in construct pCL2. This procedure results in a reengineering of the signal peptide, such that the signal-peptide cleavage site, as predicted by the consensus of von Heijne (1986), occurs immediately after the Gly-Thr dipeptide encoded by the Kpn I site. A 146-bp fragment encoding beta-(1-42) (including an artificial stop codon) was amplified from human beta-amyloid precursor protein cDNA clone p4
T4B (Ponte et al., 1988) by using primers Beta1-42-up (5'GGGGGTACCGATGCAGAATTCCGACATGA-3') and Beta1-42-down (5'CCCGAGCTCACGCTATGACAACACCGCCAA3'), cleaved with Kpn I and Sac I, and inserted between the unique Kpn I and Sac I sites of pCl2, generating pCL3. The signal peptide/Beta-(1-42) minigene fragment was removed from this plasmid by digestion with Nhe I and Sac I and inserted between the unique Nhe I and Sac I sites of pPD30.38 to construct pCL12. The sequence of the Beta-(1-42) minigene was confirmed by dideoxy DNA sequencing (coding strand only).Nhe I and Sac I sites of pPD30.38 to construct pCL12."