Deletions of pJL22/FoMV that led to the construction of the FECT vector series. Fragments containing various lengths of TGB1 subgenomic promoter were created by PCR between an upstream primer binding in the FoMV replicase region (nt. 3044) and a downstream primer which mutated the TGB1 AUG start codon to AUC and also added a PacI and AvrII site downstream of the AUC. This fragment was digested with BamHI and AvrII and inserted into pJL22/FoMV to take advantage of the native BamHI site in the replicase (3081) and the AvrII site 93 bases upstream of the CP ORF translational stop (stop at 6018) to create FECT0. FECT0 retains a subgenomic promoter consisting of the replicase 3' end and the TGB1 RNA leader but has no TGB1 ORF codons; it also retains 93 bases of the 3' end of the CP ORF. A PacI/AvrII cloning site is present after the TGB1 leader in FECT0 and subsequent FECT versions. FECT22 and FECT40 extend the potential TGB1 subgenomic promoter by an additional 22 and 40 bases, respectively, of TGB1 upstream ORF sequence. These were created from FECT0 with the upstream primer at 3044 and primers downstream of the AUC in FECT0. This fragment was digested with native BamHI and added PacI (contained in the sequence of the downstream primers) and inserted into these sites in FECT0.
Liu and Kearney BMC Biotechnology 2010 10:88 doi:10.1186/1472-6750-10-88