# Plant Synthetic Promoters
## 3 Objectives and General Methodology of Synthetic Promoter Creating
### Cis-Element Juggling and Domain Swapping
The construction of synthetic promoters uses novel cis-active elements such as the W-box sequence (GACTTTT), MYB-like (TGGTTT), bZIP-related (TACGTGACG), and TACGTCACG, from the genome of the fungal pathogen _Phytophthora sojae_. These cis-active motifs, assembled in tetrameric mode, were found to drive the expression of the CAMV35S core-based synthetic promoter in response to the elicitor peptide Pep25 (N-DVTAGAEVWNQPVRGFKVYEQTEM-C) from _P. sojae_[183]. The source of cis-active elements or larger promoter domains is complex and not fully characterized in plant genomes. The selection of cis-active motifs responsible for drought stress began with in silico analyses of 63 and 183 soybean drought-inducible genes, which were fused to the minimal 35S promoter as concatamers of 4-7 elements [184; 185]. Analogously, two cis-regulatory motifs M1.1 (TAAAATAAAGTTCTTTAATT) and M2.3 (ATAAATAAAGT) in the soybean (_G. max_) genome, reacting to the infection of cyst nematode (SCN), were used as fourfold repeats to develop synthetic promoters; these in turn were used to drive _GUS_ expression in roots infected by _Meloidogyne incognito_[189]. Moreover, the simultaneous response to fungal pathogens mediated by SA (salicylic acid) and MJ (methyl jasmonate) was achieved in synthetic promoters containing core 35S placed downstream to dimers of both D (31 bp) and F (39 bp) cis-active motifs from the _ArabidopsisAtCMPG1_ gene [190]. Since cis-elements in pathogen genes have evolved to utilize the plant's transcriptional machinery, such as TGACG-motif binding (TGA) of basic leucine zipper (bZIP) TFs, they are much stronger activators of synthetic promoters than other TFs found in plant constitutive promoters. However, the combination of three different plant cis-active elements recognized by weak and dissimilar activators induced considerably greater reporter gene expression; this may have been mediated by passive cooperativity and not the direct interaction between trans-factors, as the suggested result of extending the distance between the cis-active motifs [186]. Other properties of synthetic promoters not related to pathogen resistance could be engineered by the application of different cis-active elements. The deployment of a six-times repeated ABA responsive element (6 xABRE) upstream to a minimal CAMV35S promoter \(-90\) to \(-1\) produced a system that responded to ABA, salt and mannitol treatment in transformed _A. thaliana_ roots [233]. Combining a more complex set of seven cis-active elements from three photorespiratory genes in _A. thaliana_ (_AtPLGG1_, _AtBASS6_, _AtPLGP_) resulted in the formation of synthetic promoters that could respond to elevated temperature, low CO\({}_{2}\), and high irradiance stress. Among the analyzed cis-responsive elements, MYB and b-ZIP reduced transgene expression rate, measured by luciferase activity, by recruiting transcriptional repressors. However, bHLH increased luciferase activity in response to elevated temperatures, and AP2 in response to high irradiance [192]. Studies on the role of UTR regions in plants and viruses suggest that plant-derived UTRs outperform viral UTRs under ambient conditions. However, plant-derived UTRs are more sensitive to environmental stress conditions [192]. To construct synthetic promoters, both cis-elements and larger fragments could be assigned as enhancers or entire promoters of selected crop genes, extending the number of available plant synthetic promoters [128; 221]. To that end, the publicly available RNA-seq datasets were searched to find genes indicating a high dynamic range, activity in different plant species, and ubiquitous expression in diverse tissues. The obtained genes demonstrated a range of mean transcript abundances, suggesting differences in their strengths. A prepared set of 15 plant constitutive promoters driving these genes showed expression levels spanning nearly two orders of magnitude [221]. Seven of them were stronger than the NOS, and five are comparable with the CAMV35S promoter [221]. Moreover, [PERSON] et al.