The DNA fragments
MMV FLt (-193 to +63 bp),
MMV Sgt (-306 to -125 bp), and
FMV Sgt (-270 to -63 bp) were chemically synthesized (Gene Universal, Inc., Newark, USA) (
Supplemental Table 1). The FM′M promoter was first synthesized as a single DNA fragment. Subsequently, each domain was amplified by PCR using the specific primers MMFg-F, MMSg-F, FsMf-F, FsMf-R, M′FM-R, M′FM-F, and FM′M-R (
Supplemental Table 2). To insert the UAS×4 motif and/or the single or double zinc finger binding motifs into the FM′M promoter, the primers FM′M-U-F and FM′M-R or FM′M-US-F and FM′M-US-R or FM′M-UD-F and FM′M-UD-R were used for overlapping PCR (
Supplemental Table 2). All promoter fragments were digested with
PstI and
XbaI, and ligated into pCAMBIA1300, digested with the same restriction endonucleases. The
BiP : GFP:HDEL recombinant construct (Islam et al., 2020 (
link)) was ligated to each of the hybrid promoters following digestion with the restriction endonucleases
XbaI and
XhoI. The reporter gene encoding BiP : RBD:SD1:6×His : HDEL (Bangaru et al., 2020 (
link)) was digested with the restriction endonucleases
XbaI and
XhoI, and ligated to the FM′M-UD or CaMV 35S promoters that had previously been digested with the same restriction endonucleases. The recombinant construct,
BiP : MP:CBM3:bdSUMO:hIL6:HDEL (Islam et al., 2019 (
link)), was ligated to the FM′M-UD or CaMV 35S promoters following digestion with
XbaI and
XhoI restriction endonucleases. All these constructs contained the RD29B terminator from
Arabidopsis thaliana RD29B (D.13044.1) or the recombinant 3PR terminator (
Supplemental Table 1). The 3PR terminator sequence was chemically synthesized (Gene Universal, Inc., Newark, United States). These terminators were ligated into the constructs after digestion with restriction endonucleases
XhoI and
EcoRI. The DNA fragments encoding the transcription factors GAL4:VP16, GAL4:TAC3d2, and ZinC7:TAC3d2 were chemically synthesized (Gene Universal, Inc., Newark, United States), and contained
XbaI and
XhoI restriction endonuclease sites. The MacT promoter and RD29B terminator were used for the expression of these transcription factors. All the primer sequences used in this study are listed in
Supplemental Table 2.
Yun A., Kang J., Lee J., Song S.J, & Hwang I. (2023). Design of an artificial transcriptional system for production of high levels of recombinant proteins in tobacco (Nicotiana benthamiana). Frontiers in Plant Science, 14, 1138089.