Construction of plasmid pKT0/

Copyright information:

Taken from "Characterization of the frameshift signal of , a mammalian example of programmed −1 ribosomal frameshifting"

Nucleic Acids Research 2005;33(5):1553-1563.

Published online 14 Mar 2005

PMCID:PMC1065257.

© The Author 2005. Published by Oxford University Press. All rights reserved

A 1230 bp DNA fragment (631–1861) encompassing the frameshift region was amplified by PCR from plasmid pSP64T/ () and cloned into NcoI/HindIII digested plasmid pKT0 (). The 5′ and 3′ portions of the cloned segment are shown in lower case, numbered according to the mRNA sequence (A of natural AUG start site is base 452; accession no. AJ006464). The AUG for the expression of sequences in pKT0/ is derived from the vector (upper case, underlined). In ribosomal frameshifting assays, capped mRNAs were prepared by SP6 transcription of NdeI-linearized templates (unless otherwise stated). The predicted size of the non-frameshifted (stop) and frameshifted (fs) products generated from the translation of this mRNA in RRL is 29 and 32 kDa, respectively. For structural analysis of the stimulatory RNA, a T3 promoter was introduced into the sequence (at position 1357) to generate plasmid pKT0//T3. Linearization of this plasmid with NdeI and subsequent transcription with T3 RNA polymerase yields a transcript of 152 nt.