Figure 2.

Synthesis of translation templates containing 3′ puromycin. (A) Puromycin⋅(HCl)₂ was protected and attached to a synthesis support to give CPG-puromycin, and the linker 30-P (dA₂₇dCdCP) was made by automated synthesis (see Materials and Methods). (B) Translation templates used were: 43-P (Met template), LP77 (short myc template), and LP154 (long myc template) containing ORFs of 1, 12, and 33 codons, respectively, and no stop codon. The 5′ untranslated regions (UTRs) of 43-P and LP77 contain a Shine–Dalgarno sequence complementary to five bases of 16S rRNA (33) and spaced similarly to ribosomal protein sequences (34). The 5′ UTR of LP154 contains a 22-nt sequence derived from the tobacco mosaic virus 5′ UTR encompassing two ACAAAUUAC direct repeats (26). LP154 codes for the peptide used to raise the mAb 9E10 and LP77 codes for the recognition epitope EQKLISEEDL (24).