In this project, we are making efforts to convert ORF sequences to expression clones for further production of recombinant proteins using Flexi Vector Systems^2,3,4 , which contain Sgf I and Pme I sites where protein-coding regions could be cloned in the correct orientation and reading frame. Flexi Vector clones that contain a full-length protein-coding sequence have been constructed by the following three methods.

(1) Cloning using restriction enzymes
After addition of Sgf I and Pme I sites at the outer sides of ORF sequences of previously prepared clones ⁵, an Sgf I-Pme I fragment including the ORF is inserted into pF1K-based vectors, such that the resultant ORF sequence is flanked by BstB I and SnaB I sites.

(2) PCR cloning
ORF sequences flanked by Sgf I and Pme I sites are amplified by PCR, and cloned into a pF1K vector.

(3) ORF Trap cloning
Based on homologous recombination in Escherichia coli, ORF sequences are transferred into pF1K-based vectors without artificial introduction of mutations in ORF.

     As an evaluation of the Flexi clones, we digested them with Sgf I and Pme I , analyzed them by agarose gel electrophoresis and confirmed their insert sizes. In addition, we confirmed both the end sequences of the cloned ORF by single-pass sequencing. For the pF1K clones that were produced by PCR cloning, we determined the entire sequences of their inserts.

     The pF1K clones thus prepared can be easily transferred to other various types of Flexi Vectors following digestion with Sgf I and Pme I (Fig. c-1). The flanking sequences of ORF and vector types used here are shown in Fig. c-2 and Fig. c-3, respectively. 

Fig.c-1 Preparation of Flexi clones	Fig.c-2 Flanking sequences of ORF in Flexi clones	Fig.c-3 Type of vectors used for Flexi clones