Figure 3

Representative maps of parallel ccdB vectors for protein expression in E. coli , Baculovirus and HEK293E. For each of these hosts, one example of the pET (E. coli) (A), pFastBac (Baculovirus) (E) and pTT (HEK293E) (D) ccdB vector series is shown. All three backbones share common LP1 (3C) and LP2 (ccdB) primer binding sites for parallel cloning. (B) Primer design is illustrated for the universal PreScission 3C-ccdB primer pair of the pCoofy vector series. Gene specific primer sequences are fused to sequence overhangs of 20–24 nucleotides which are complementary to the corresponding vector amplification primer (see Table 2). (C) 2^ndgeneration ccdB cassette including C-tags The ccdB cassette as shown in Figure 2A starting at TTGACA (−35 region) was fused to a row of C-terminal tags each separated by a stop codon. LP1 (3C) sequence was added upstream of the ccdB coding sequence. Restriction sites at both ends were also added for subsequent cloning into different vector backbones. The complete cassette was synthesized by GeneArt (now part of Life Technologies) with concomitant codon optimization of the tags for eukaryotic expression.