Figure 1

Construction of pSANG vectors. A . Sequence of the basic vector pSANG-10, which was created by inserting an oligo between a BglI site (GCCnnnnnGGC) and XhoI site (CTCGAG now destroyed) downstream of the promoter within the vector backbone derived from pET26 (+) plasmid (Novagen, San Diego, CA). The inserted sequence cloned between these restriction sites is shown. Translated sequences from the end of the signal peptide and hexa-histidine tag are indicated. The scFv encoding gene is sub-cloned at the NcoI/NotI site and the HindIII site is available for insertion of peptide tags and fusion partners. B. For comparison the sequence of pSANG10-3F is shown where a tri-FLAG tag has been inserted. An oligonucleotide encoding the tri-FLAG sequence was cloned into the HindIII site of pSANG10 destroying the site at 5' end and retaining it at 3' end for future use. C. Schematic view of the expression cassettes of the pSANG vectors. a-d: vectors used with BL21 (DE3) host strain for periplasmic expression. e-f: vectors used with Origami 2™ (DE3) host strain for cytoplasmic expression. MBP= Maltose Binding Protein; * = serine replaced by a cysteine.