Recombinant DNA molecule, composition, use of plant, seed, cell progeny or plant part, as well as methods for production of a basic product and for expression of a transcriptional DNA molecule

Description

[001] This application claims the benefit of the provisional application of United States 62 / 714,228, filed August 3, 2018, which is incorporated herein by reference in its entirety. INCORPORATION OF SEQUENCE LISTING

[002] The sequence listing contained in the file named 38-21-62691-0001_Seqlist_ST25.txt, is 31,060 bytes (as measured in Microsoft Windows®), was created on July 2, 2019, and is archived herewith by electronic submission and incorporated herein by reference. FIELD OF THE INVENTION

[003] The invention relates to the fields of plant molecular biology and plant genetic manipulation. More specifically, the invention relates to DNA molecules useful for modulating gene expression in plants. FUNDAMENTALS

[004] Regulatory elements are genetic elements that regulate gene activity by modulating the transcription of an operatively bound, transcribable DNA molecule. Such elements may include promoters, leaders, introns, and 3' untranslated regions and are useful in the fields of plant molecular biology and plant genetic engineering. SUMMARY OF THE INVENTION

[005] The invention provides new synthetic regulatory elements Petition 870260061195, dated 06 / 22 / 2026, page 8 / 178 2 / 65 of genes for use in plants. The invention also provides recombinant DNA molecule constructs comprising the regulatory elements. The present invention also provides transgenic plant cells, plants, and seeds comprising the regulatory elements. In one embodiment, the regulatory elements are operatively linked to a transcribable DNA molecule. In certain embodiments, the transcribable DNA molecule may be heterologous with respect to the regulatory sequence. Thus, a regulatory element sequence provided by the invention may, in particular embodiments, be defined as operatively linked to a heterologous transcribable DNA molecule.The present invention also provides methods for utilizing regulatory elements and for manufacturing and utilizing recombinant DNA molecules comprising the regulatory elements and transgenic plant cells, plants and seeds comprising the regulatory elements operatively linked to a transcribable DNA molecule.

[006] Thus, in one aspect, the invention provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least about 85 percent sequence identity with any of the SEQ ID NOs: 1-19 and SEQ ID NO: 26; (b) a sequence comprising any of the SEQ ID NOs: 1-19 and SEQ ID NO: 26; and (c) a fragment of any of the SEQ ID NOs: 1-19 and SEQ ID NO: 26, wherein the fragment has gene regulatory activity; wherein the sequence is operatively linked to a heterologous transcriptable DNA molecule. By “heterologous transcriptable DNA molecule” it is understood that the transcriptable DNA molecule is heterologous with respect to the polynucleotide sequence to which it is operatively linked. In specific embodiments, the recombinant DNA molecule comprises a DNA sequence with at least approximately 85 percent, at least Petition 870260061195, dated 06 / 22 / 2026, p. 9 / 178 3 / 65 less about 86 percent, at least about 87 percent, at least about 88 percent, at least about 89 percent, at least about 90 percent, at least 91 percent, at least 92 percent, at least 93 percent, at least 94 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent sequence identity with the DNA sequence of any of the SEQ ID NOs: 1-19 and SEQ ID NO: 26. In particular embodiments, the DNA sequence comprises a regulatory element. In some embodiments, the regulatory element comprises a promoter. In still other embodiments, the regulatory element comprises an intron. In still other embodiments, the regulatory element comprises a 3' UTR.In other embodiments, the heterologous transcriptable DNA molecule comprises a gene of agronomic interest, such as a gene capable of providing herbicide resistance in plants, or a gene capable of providing plant pest resistance in plants. In still other embodiments, the heterologous transcriptable DNA molecule comprises a sequence encoding a small RNA, such as a dsRNA, a miRNA, or a siRNA. In still other embodiments, the invention provides a construct comprising a recombinant DNA molecule as provided in this document.

[007] In another aspect, transgenic plant cells are provided herein comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least about 85 percent sequence identity with either SEQ ID NOs: 119 and SEQ ID NO: 26; (b) a sequence comprising either SEQ ID NOs: 1-19 and SEQ ID NO: 26; and (c) a fragment of either SEQ ID NOs: 1-19 and SEQ ID NO: 26, wherein the fragment has gene regulatory activity; wherein the sequence of Petition 870260061195, dated 06 / 22 / 2026, p. 10 / 178 4 / 65 DNA is operatively linked to a heterologous transcriptable DNA molecule. In certain embodiments, the transgenic plant cell is a monocotyledonous plant cell. In other embodiments, the transgenic plant cell is a dicotyledonous plant cell.

[008] In yet another aspect, a transgenic plant, or part thereof, comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85 percent sequence identity with any of the SEQ ID NOs: 1-19 and SEQ ID NO: 26; b) a sequence comprising any of the SEQ ID NOs: 1-19 and SEQ ID NO: 26; and c) a fragment of any of the SEQ ID NOs: 1-19 and SEQ ID NO: 26, wherein the fragment has gene regulatory activity; wherein the sequence is operatively linked to a heterologous transcriptable DNA molecule. In specific embodiments, the transgenic plant is a parent plant of any generation comprising the recombinant DNA molecule. A transgenic seed comprising the recombinant DNA molecule that produces a transgenic plant when grown is also provided in this document.

[009] In another aspect, the invention provides a method of producing a base product comprising obtaining a transgenic plant or a part thereof containing a recombinant DNA molecule of the invention and producing the base product therefrom. In one embodiment, the base product is seeds, processed seeds, protein concentrate, protein isolate, starch, grains, plant parts, seed oil, biomass, flour and meal.

[0010] In yet another aspect, the invention provides a method for producing a transgenic plant comprising a recombinant DNA molecule of the invention comprising transforming a Petition 870260061195, dated 06 / 22 / 2026, page 11 / 178 5 / 65 plant cell with the recombinant DNA molecule of the invention to produce a transformed plant cell and regenerate a transgenic plant from the transformed plant cell. BRIEF DESCRIPTION OF THE SEQUENCES

[0011] SEQ ID NO: 1 is a DNA sequence of a synthetic regulatory expression element (EXP) group, EXP-Zm.GSP850 comprising a synthetic promoter (P-Zm.GSP850.nno: 4), operatively linked in 5' to a synthetic leader (L-Zm.GSP850.nno:3).

[0012] SEQ ID NO: 2 is a DNA sequence of a synthetic promoter, P-Zm.GSP850.nno:4.

[0013] SEQ ID NO: 3 is a DNA sequence of a synthetic leader, L-Zm.GSP850.nno:3.

[0014] SEQ ID NO:4 is a DNA sequence of a synthetic EXP, EXP-Zm.GSP850.nno+ Zm.GSI153.nno:2 comprising a synthetic promoter (P-Zm.GSP850.nno:4), operatively linked in 5' to a synthetic leader (L-Zm.GSP850.nno:3), operatively linked in 5' to a synthetic intron (I-Zm.GSI153.nno:1).

[0015] SEQ ID NO:5 is a DNA sequence of a synthetic intron, I-Zm.GSI153.nno:1.

[0016] SEQ ID NO:6 is a DNA sequence of a synthetic EXP, EXP-Zm.GSP990 comprising a synthetic promoter (PZm.GSP990.nno:2), operatively linked in 5' to a synthetic leader (L-Zm.GSP990.nno:1).

[0017] SEQ ID NO: 7 is a DNA sequence of a synthetic promoter, P-Zm.GSP990.nno:2.

[0018] SEQ ID NO: 8 is a DNA sequence of a synthetic leader, L-Zm.GSP990.nno:1.

[0019] SEQ ID NO:9 is a DNA sequence of a synthetic EXP, EXP-Zm.GSP990.nno+ Zm.GSI197.nno:2 comprising a synthetic promoter (P-Zm.GSP990.nno:2), operatively linked at 5' Petition 870260061195, dated 06 / 22 / 2026, page 12 / 178 6 / 65 to a synthetic leader (L-Zm.GSP990.nno:1), operatively linked at 5' to a synthetic intron (I-Zm.GSI197.nno:1).

[0020] SEQ ID NO:10 is a DNA sequence of a synthetic intron, I-Zm.GSI197.nno:1.

[0021] SEQ ID NO:11 is a DNA sequence of a synthetic EXP, EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 comprising a synthetic promoter (P-Zm.GSP850.nno:4), operatively linked in 5' to a synthetic leader (L-Zm.GSP850.nno:3), operatively linked in 5' to a synthetic intron (I-Zm.GSI140.nno:1).

[0022] SEQ ID NO:12 is a DNA sequence of a synthetic intron, I-Zm.GSI140.nno:1.

[0023] SEQ ID NO: 13 is a DNA sequence of a synthetic 3' UTR, T-Zm.GST9.nno:2.

[0024] SEQ ID NO: 14 is a DNA sequence of a synthetic 3' UTR, T-Zm.GST18.nno:2.

[0025] SEQ ID NO:15 is a DNA sequence of a synthetic EXP, EXP-Zm.GSP850.nno+Zm.DnaK:1 comprising a synthetic promoter (P-Zm.GSP850.nno:4), operatively linked in 5' to a synthetic leader (L-Zm.GSP850.nno:3), operatively linked in 5' to an intron (I-Zm.DnaK:1).

[0026] SEQ ID NO:16 is a DNA sequence of a synthetic EXP, EXP-Zm.GSP990.nno+Zm.DnaK:1 comprising a synthetic promoter (P-Zm.GSP990.nno:2), operatively linked in 5' to a synthetic leader (L-Zm.GSP990.nno:1), operatively linked in 5' to an intron (I-Zm.DnaK:1).

[0027] SEQ ID NO:17 is a DNA sequence of a synthetic enhancer, E-Zm.GSP850, which is derived from the synthetic promoter, PZm.GSP850.nno:4.

[0028] SEQ ID NO:18 is a DNA sequence of a synthetic enhancer, E-Zm.GSP990, which is derived from the synthetic promoter, PPetition 870260061195, dated 06 / 22 / 2026, p. 13 / 178 7 / 65 Zm.GSP990.nno:2.

[0029] SEQ ID NO:19 is a DNA sequence of a 3' UTR, TSb.Nltp4-1:1:2 derived from the NLTP4 gene (non-specific lipid transfer protein 4) of Sorghum bicolor.

[0030] SEQ ID NO:20 is a synthetic coding sequence optimized for plant expression for β-glucuronidase (GUS) with a processable intron derived from the light-inducible tissue-specific ST-LS1 gene of potato (Genbank Accession: X04753).

[0031] SEQ ID NO:21 is a DNA sequence of EXP, EXPCaMV.35S comprising the 35S promoter and leader derived from Cauliflower Mosaic Virus.

[0032] SEQ ID NO:22 is a DNA sequence of the intron, IZm.DnaK:1 derived from the heat shock protein 70 (Hsp70) gene (DnaK) of Zea mays.

[0033] SEQ ID NO:23 is a DNA sequence of the UTR 3', TOs.LTP:1 derived from the lipid transfer protein (LTP)-like gene of Oryza sativa.

[0034] SEQ ID NO:24 is a coding sequence for plant expression of β-glucuronidase (GUS) with a processable intron derived from the light-inducible tissue-specific ST-LS1 gene of potato (Genbank Accession: X04753).

[0035] SEQ ID NO:25 is a coding sequence for the fluorescent luciferase protein NanoLuc® (Promega, Madison, WI 53711), Nluc, which was engineered by directed evolution from a deep-sea shrimp luciferase (Oplophorus gacilirostris).

[0036] SEQ ID NO: 26 is a DNA sequence of a synthetic 3' UTR, T-Zm.GST43.nno:1. DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention provides synthetic regulatory elements with gene regulatory activity in plants. The nucleotide sequences Petition 870260061195, dated 06 / 22 / 2026, p. 14 / 178 8 / 65 of these synthetic regulatory elements are provided as SEQ ID Nos: 1-18 and SEQ ID No: 26. These synthetic regulatory elements are capable of affecting the expression of an operatively linked transcribable DNA molecule in plant tissues and thus regulating the gene expression of an operatively linked transgene in transgenic plants. The invention also provides novel endogenous regulatory elements with gene regulatory activity in plants and provided as SEQ ID No: 19. The invention also provides methods for modifying, producing, and utilizing recombinant DNA molecules containing the provided synthetic and endogenous regulatory elements. The invention also provides compositions including transgenic plant cells, plants, plant parts, and seeds containing the recombinant DNA molecules of the invention, and methods for the preparation and utilization thereof.

[0038] The following definitions and methods are provided to better define the present invention and to guide those skilled in the art in practicing the present invention. Unless otherwise indicated, terms should be understood in accordance with conventional usage by those skilled in the relevant art. DNA molecules

[0039] As used in this document, the term DNA or DNA molecule refers to a double-stranded DNA molecule of genomic or synthetic origin, that is, a polymer of deoxyribonucleotide bases or a DNA molecule, read from the 5' (upstream) end to the 3' (downstream) end. As used in this document, the term DNA sequence refers to the nucleotide sequence of a DNA molecule. The nomenclature used in this document corresponds to that of Title 37 of the United States Code of Federal Regulations § 1.822, and presented in the tables of WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3. Petition 870260061195, dated 06 / 22 / 2026, p. 15 / 178 9 / 65

[0040] As used in this document, a “recombinant DNA molecule” is a DNA molecule comprising a combination of DNA molecules that would not naturally occur together without human intervention. For example, a recombinant DNA molecule may be a DNA molecule that is composed of at least two heterologous DNA molecules, a DNA molecule comprising a DNA sequence that deviates from DNA sequences existing in nature, a DNA molecule comprising a synthetic DNA sequence, or a DNA molecule that has been incorporated into the DNA of a host cell by genetic transformation or gene editing.

[0041] As used in this document, a synthetic nucleotide sequence or artificial nucleotide sequence is a nucleotide sequence that is not known to occur in nature or that does not occur naturally. The gene regulatory elements of the present invention comprise synthetic nucleotide sequences. Preferably, the synthetic nucleotide sequences share little or no extended homology with the natural sequences. Extended homology in this context generally refers to 100% sequence identity extending beyond about 25 contiguous sequence nucleotides.

[0042] The reference in this application to an “isolated DNA molecule,” or an equivalent term or phrase, is intended to mean that the DNA molecule is one that is present alone or in combination with other components, but not within its natural environment. For example, nucleic acid elements such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcriptional termination sequence, and the like, which are naturally found in the DNA of an organism’s genome, are not considered “isolated,” provided that the Petition 870260061195, dated 06 / 22 / 2026, p. 16 / 178 10 / 65 ment is within the organism's genome and at the site within the genome where it is naturally found. However, each of these elements, and subparts of these elements, would be "isolated" within the scope of this disclosure, provided that the element is not within the organism's genome and at the site within the genome where it is naturally found. Similarly, a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein would be an isolated nucleotide sequence provided that the nucleotide sequence is not in the DNA of the bacterium from which the protein-coding sequence is naturally found. A synthetic nucleotide sequence encoding the amino acid sequence of the naturally occurring insecticidal protein would be considered isolated for the purposes of this disclosure.For the purposes of this disclosure, any transgenic nucleotide sequence, that is, the nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell, or present in an extrachromosomal vector, would be considered an isolated nucleotide sequence, regardless of whether the transgenic nucleotide sequence is present within the plasmid or similar structure used to transform the cells, in the genome of the plant or bacterium, or present in detectable quantities in tissues, progeny, biological samples, or base products derived from the plant or bacterium.

[0043] As used in this document, the term “sequence identity” refers to the extent to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. An optimal sequence alignment is created by manually aligning two sequences, for example, a reference sequence and another sequence, to maximize the number of nucleotide matches in the alignment. Petition 870260061195, dated 06 / 22 / 2026, p. 17 / 178 11 / 65 sequences with appropriate internal nucleotide insertions, deletions, or gaps. As used in this document, the term reference sequence refers to a DNA sequence provided as SEQ ID NOs: 1-19 and SEQ ID NO: 26.

[0044] As used in this document, the term “percent sequence identity” or “percent identity” or “% identity” is the identity fraction multiplied by 100. The “identity fraction” for a sequence optimally aligned with a reference sequence is the number of nucleotide matches in the optimal alignment, divided by the total number of nucleotides in the reference sequence, for example, the total number of nucleotides in the entire length of the reference sequence.Thus, one embodiment of the invention provides a DNA molecule comprising a sequence that, when optimally aligned with a reference sequence, provided herein as SEQ ID Nos: 1-19 and SEQ ID No: 26, has at least about 85 percent identity, at least about 86 percent identity, at least about 87 percent identity, at least about 88 percent identity, at least 89 percent identity, at least 90 percent identity, at least 91 percent identity, at least 92 percent identity, at least 93 percent identity, at least about 94 percent identity, at least about 95 percent identity, at least about 96 percent identity, at least about 97 percent identity, at least about 98 percent identity, at least 99 percent identity, or at least 100 percent identity to the reference sequence.DNA molecules with a percent sequence identity to the reference molecule can exhibit the activity of the reference sequence. Regulatory elements Petition 870260061195, dated 06 / 22 / 2026, p. 18 / 178 12 / 65

[0045] Regulatory elements such as promoters, leaders (also known as 5' UTRs), enhancers, introns, and transcription termination regions (or 3' UTRs) play a fundamental role in the overall expression of genes in living cells. The term regulatory element, as used in this document, refers to a DNA molecule possessing gene regulatory activity. The term “gene regulatory activity,” as used in this document, refers to the ability to affect the expression of an operatively bound transcribable DNA molecule, for example, by affecting the transcription and / or translation of the operatively bound DNA molecule. Regulatory elements such as promoters, leaders, enhancers, introns, and 3' UTRs that function in plants are therefore useful for modifying plant phenotypes through genetic engineering.

[0046] As used in this document, a sequence group of regulatory expression elements or EXP may refer to a group of operatively linked regulatory elements, such as enhancers, promoters, leaders, and introns. For example, a group of regulatory expression elements may be understood, for instance, from a promoter operatively linked at 5' to a leader sequence. EXPs useful in the practice of the present invention include SEQ ID Nos: 1, 4, 6, 9, 11, 15, and 16.

[0047] Regulatory elements can be characterized by their gene expression pattern, for example, positive and / or negative effects, such as constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle and / or chemically responsive expression, and any combination thereof, as well as by quantitative or qualitative indications. As used in this document, a gene expression pattern is any transcription pattern of an open DNA molecule. Petition 870260061195, dated 06 / 22 / 2026, p. 19 / 178 13 / 65 actively bound to a transcribed RNA molecule. The transcribed RNA molecule can be translated to produce a protein molecule or it can provide an antisense RNA molecule or another regulatory RNA molecule, such as a double-stranded RNA (dsRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA), a microRNA (miRNA), a small interfering RNA (siRNA), and the like.

[0048] As used in this document, the term protein expression is any pattern of translation of a transcribed RNA molecule into a protein molecule. Protein expression can be characterized by its temporal, spatial, developmental, or morphological qualities, as well as by quantitative or qualitative indicators.

[0049] A promoter is useful as a regulatory element for modulating the expression of an operatively bound transcriptional DNA molecule. As used in this document, the term “promoter” generally refers to a DNA molecule that is involved in the recognition and binding of RNA polymerase II and other proteins, such as trans-activating transcription factors, to initiate transcription. A promoter may initially be isolated from the 5' untranslated region (5' UTR) of a genomic copy of a gene. Alternatively, promoters may be synthetically produced or manipulated DNA molecules. Promoters may also be chimeric. Chimeric promoters are produced by fusing two or more heterologous DNA molecules. Promoters useful in the practice of the present invention include promoter elements provided as SEQ ID NOs: 2 and 7, or comprised in any of the SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16, or fragments or variants thereof.In specific embodiments of the invention, the claimed DNA molecules and any variants or derivatives thereof. Petition 870260061195, dated 06 / 22 / 2026, page 20 / 178 14 / 65 as described in this document, are further defined as comprising promoter activity, i.e., they are capable of acting as a promoter in a host cell, such as in a transgenic plant. In yet other specific embodiments, a fragment may be defined as exhibiting promoter activity possessed by the starting promoter molecule from which it is derived, or a fragment may comprise a “minimal promoter” that provides a basal level of transcription and is comprised of a TATA box, or equivalent DNA sequence for recognition and binding of the RNA polymerase II complex for transcription initiation.

[0050] In one embodiment, fragments of an EXP sequence or a promoter sequence disclosed in this document are provided. The promoter fragments may comprise promoter activity, as described above, and may be useful alone or in combination with other promoters and promoter fragments, such as in the construction of chimeric promoters, or in combination with other expression elements and fragments of expression elements.In specific embodiments, fragments of a promoter are provided comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least 600, at least 700, at least 750, at least 800, at least 900, or at least about 1000 contiguous nucleotides, or more, of a DNA molecule possessing promoter activity as disclosed in this document. The methods for producing such fragments from a starting promoter molecule are well known in the art.

[0051] In other modalities, fragments of sequences of in Petition 870260061195, dated 06 / 22 / 2026, page 21 / 178 15 / 65 enhancer or intron fragments disclosed in this document are provided. Enhancer or intron fragments may comprise the activity of the base molecule from which they were derived and may be useful alone or in combination with other regulatory elements, including promoters, leaders, other enhancers, other introns, or fragments thereof.In specific embodiments, enhancer or intron fragments are provided comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 contiguous nucleotides, or more, of a DNA molecule with enhancer or intron activity as disclosed herein. The methods for producing such fragments from a starting molecule are well known in the art.

[0052] In other embodiments, fragments of UTR 3' sequences disclosed herein are provided. The UTR 3' fragments may comprise the activity of the base UTR 3' molecule from which they were derived and may be useful alone or in combination with other regulatory elements, including promoters, leaders, introns, or fragments thereof. In specific embodiments, fragments of an intron are provided comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least Petition 870260061195, dated 06 / 22 / 2026, page 22 / 178 16 / 65 approximately 750, at least approximately 800, at least approximately 900, or at least approximately 1000 contiguous nucleotides, or more, of a DNA molecule with 3' UTR activity as disclosed herein. The methods for producing such fragments from an initial 3' UTR molecule are well known in the art.

[0053] Compositions derived from any of the promoter elements provided as SEQ ID NOs: 2 and 7, or comprised within any of the SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16, such as internal or 5' deletions, for example, may be produced using methods known in the art to enhance or alter expression, including removing elements that have positive or negative effects on expression; duplicating elements that have positive or negative effects on expression; and / or duplicating or removing elements that have tissue- or cell-specific effects on expression. Compositions derived from any of the promoter elements provided as SEQ ID NOs: 2 and 7, or comprised within any of the SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16, comprised within 3' deletions in which the TATA box element or equivalent sequence thereof and the removed downstream sequence may be used, for example, to make enhancer elements.Other deletions can be made to remove any positive or negative elements; tissue-specific effects; cell-specific effects; or time-specific effects (such as, but not limited to, circadian rhythm) on expression. Any of the promoter elements provided as SEQ ID NOs: 2 and 7, or comprised in any of the SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16, and fragments or enhancers derived from them, can be used to make chimeric compositions of transcriptional regulatory elements.

[0054] According to the invention, a promoter or promoter fragment can be analyzed for the presence of promo elements. Petition 870260061195, dated 06 / 22 / 2026, page 23 / 178 17 / 65 known promoters, that is, features of the DNA sequence, such as a TATA box and other known transcription factor binding site motifs. The identification of such known promoter elements can be used by one skilled in the art to design promoter variants with an expression pattern similar to the original promoter.

[0055] As used in this document, the term leader refers to a DNA molecule isolated from the 5' untranslated region (UTR 5') of a gene and generally defined as a nucleotide segment between the transcription start site (TSS) and the protein-coding sequence start site. Alternatively, leaders may be synthetically produced or manipulated DNA elements. A leader may be used as a 5' regulatory element to modulate the expression of an operatively linked transcriptable DNA molecule. Leader molecules may be used with a heterologous promoter or with their native promoter. Useful leaders in the practice of the present invention include SEQ ID NOs: 3 and 8; or any of the leader elements comprised in any of the SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16 or fragments or variants thereof.In specific embodiments, such DNA sequences can be defined as being capable of acting as a leader in a host cell, including, for example, a transgenic plant cell. In one embodiment, these sequences are decoded as comprising leader activity.

[0056] The leader sequences (also referred to as 5' UTRs) presented as SEQ ID NOs: 3 and 8 or any of the leader elements comprised in any of the SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16 may be composed of regulatory elements, or may adopt secondary structures that may have an effect on the transcription or translation of an operationally transcribable DNA molecule. Petition 870260061195, dated 06 / 22 / 2026, p. 24 / 178 18 / 65 gada. The leader sequences presented as SEQ ID NOs: 3 and 8 or any of the leader elements comprised in any of the SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16 can be used according to the invention to make chimeric regulatory elements that affect the transcription or translation of an operatively linked transcribable DNA molecule.

[0057] As used in this document, the term intron refers to a DNA molecule that can be isolated or identified from a gene and can generally be defined as a region separated during messenger RNA (mRNA) processing before translation. Alternatively, an intron can be a synthetically produced or manipulated DNA element. An intron may contain enhancer elements that affect the transcription of operatively linked genes. An intron can be used as a regulatory element to modulate the expression of an operatively linked transcriptional DNA molecule. A construct may comprise an intron, and the intron may or may not be heterologous with respect to the transcriptional DNA molecule. Examples of introns in the art include the rice actin intron and the maize HSP70 intron.

[0058] In plants, the inclusion of certain introns in gene constructs leads to increased mRNA and protein accumulation compared to constructs without the intron. This effect has been termed “intron-mediated enhancement” (IME) of gene expression. Introns known to stimulate expression in plants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), rice genes (e.g., tpi), and genes in dicotyledonous plants such as petunia (e.g., rbcS) (e.g., st-ls1) and Arabidopsis thaliana (e.g., ubq3 and pat1). Deletions or mutations at intron splicing sites have been shown to reduce gene expression, indicating that splicing may be necessary for IME. Petition 870260061195, dated 06 / 22 / 2026, p. 25 / 178 19 / 65 Therefore, IME in dicotyledonous plants has been demonstrated by point mutations in the splicing sites of the pat1 gene of A. thaliana. Multiple uses of the same intron in a plant have been shown to exhibit disadvantages. In such cases, it is necessary to have a collection of basic control elements for the construction of appropriate recombinant DNA elements. The exemplary introns useful in the practice of the present invention are presented as SEQ ID NOs: 5, 10 and 12.

[0059] As used in this document, the terms 3' transcription termination molecule, 3' untranslated region, or 3' UTR refer to a DNA molecule that is used during transcription for the untranslated region of the 3' portion of an mRNA molecule. The 3' untranslated region of an mRNA molecule can be generated by specific cleavage and 3' polyadenylation, also known as a polyA tail. A 3' UTR may be operatively bound and located downstream of a transcristable DNA molecule and may include a polyadenylation signal and other regulatory signals capable of affecting transcription, mRNA processing, or gene expression. PolyA tails are believed to function in mRNA stability and translation initiation. Examples of 3' transcription termination molecules in the technique are the 3' region of nopalin synthase, the 3' region of wheat hsp17, the 3' region of pea rubisco small subunits, the 3' E6 region of cotton, and the 3' UTR of coixin.

[0060] 3' UTRs typically find beneficial use for the recombinant expression of specific DNA molecules. A weak 3' UTR has the potential to generate rereading, which can affect the expression of DNA molecules located in neighboring expression cassettes. Appropriate control of transcription termination can prevent rereading on downstream DNA sequences (e.g., other expression cassettes) and can even allow for efficient RNA polymerase recycling to enhance gene expression. Termination Petition 870260061195, dated 06 / 22 / 2026, page 26 / 178 Efficient 20 / 65 transcription (release of RNA Polymerase II from DNA) is a prerequisite for transcription restart and therefore directly affects the overall transcript level. After transcription termination, the mature mRNA is released from the synthesis site and the template is transported to the cytoplasm. Eukaryotic mRNAs accumulate as poly(A) forms in vivo, making it difficult to detect transcriptional termination sites by conventional methods. However, predicting functional and efficient 3' UTRs using bioinformatics methods is difficult because there are no conserved DNA sequences that allow for easy prediction of an effective 3' UTR.

[0061] From a practical standpoint, it is typically beneficial for a 3' UTR used in an expression cassette to possess the following characteristics. First, the 3' UTR must be able to efficiently and effectively terminate transgene transcription and prevent transcript reading on any neighboring DNA sequence, which may be composed of another expression cassette, as in the case of multiple expression cassettes residing on transfer DNA (T-DNA) or the neighboring chromosomal DNA into which the T-DNA has been inserted. Second, the 3' UTR must not cause a reduction in transcriptional activity transmitted by the promoter, leader, enhancers, and introns that are used to direct the expression of the DNA molecule.Finally, in plant biotechnology, the 3' UTR is frequently used to initiate amplification reactions of the reverse transcribed RNA extracted from the transformed plant and used to: (1) evaluate the transcriptional activity or expression of the expression cassette, once integrated into the plant chromosome; (2) evaluate the copy number of insertions in the plant DNA; and (3) evaluate the zygosity of the resulting seed after reproduction. The 3' UTR is also used in amplification reactions of DNA extracted from the transformed plant to characterize the integrity of the inserted cassette. 3' UTRs useful in the practice of the present invention. Petition 870260061195, dated 06 / 22 / 2026, page 27 / 178 21 / 65 are presented as SEQ ID Nos: 13, 14, 19 and 26.

[0062] As used in this document, the term 'enhancer' or 'enhancer element' refers to a cis-acting regulatory element also known as a cis element that confers an aspect of the overall expression pattern, but is generally insufficient on its own to direct the transcription of a bound transcriptional DNA molecule. Unlike promoters, enhancer elements generally do not include a transcription start site (TSS) or TATA box or equivalent DNA sequence. A promoter or promoter fragment may naturally comprise one or more enhancer elements that affect the transcription of an operatively bound DNA sequence. An enhancer element may also be fused to a promoter to produce a chimeric cis promoter element that confers an aspect of the overall modulation of gene expression.

[0063] Many promoter enhancer elements are believed to bind to DNA-binding proteins and / or affect DNA topology, producing local conformations that selectively allow or restrict RNA polymerase access to DNA or that facilitate selective opening of the double helix at the transcriptional initiation site. An enhancer element may function to bind transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and transcription factors may interact with different affinities with more than one enhancer domain.Enhancer elements can be identified by various techniques, including deletion analysis, i.e., the elimination of one or more nucleotides from the 5' end or internal to a promoter, DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility displacement assays, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), and other conventional assays. Petition 870260061195, dated 06 / 22 / 2026, page 28 / 178 22 / 65 nais, or by DNA sequence similarity analysis using cis-element motifs or enhancer elements as a target sequence or target motif with conventional DNA sequence comparison methods, such as BLAST. The fine structure of an enhancer domain can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods known in the art. Enhancer elements can be obtained by chemical synthesis or by isolation from regulatory elements that include these elements, and can be synthesized with additional flanking nucleotides containing restriction enzyme sites useful for facilitating subsequent manipulation. Thus, the design, construction, and use of enhancer elements according to the methods disclosed herein to modulate the expression of operatively linked transcriptional DNA molecules are covered by the invention.Useful illustrative intensifiers for practical application of this invention are presented as SEQ ID Nos: 17 and 18.

[0064] As used in this document, the term chimeric refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, where neither the first nor the second DNA molecule would normally be found in that configuration, i.e., fused with each other. The chimeric DNA molecule is therefore a novel DNA molecule not normally found in nature. As used in this document, the term chimeric promoter refers to a promoter produced through such manipulation of DNA molecules. A chimeric promoter can combine two or more DNA fragments; for example, the fusion of a promoter to an enhancer element. Thus, the design, construction, and use of chimeric promoters according to the methods disclosed herein to modulate the expression of mo Petition 870260061195, dated 06 / 22 / 2026, p. 29 / 178 23 / 65 operatively linked transcribable DNA molecules are covered by the present invention.

[0065] Chimeric regulatory elements can be designed to comprise several constituent elements that can be operatively linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation-independent cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of the regulatory element, as well as other methods known in the art. The resulting various chimeric regulatory elements may consist of the same, or variants of the same constituent elements, but differ in the DNA sequence or sequences comprising the linking DNA sequence or sequences that allow the constituent parts to be operatively linked.In the invention, the DNA sequences provided as SEQ ID NOs:1-19 and SEQ ID NO:26 can provide regulatory element reference sequences, wherein the constituent elements comprising the reference sequence can be joined by methods known in the art and may comprise substitutions, deletions, and / or insertions of one or more nucleotides or mutations that occur naturally in the transformation of bacterial and plant cells.

[0066] As used in this document, the term “variant” refers to a second DNA molecule, such as a regulatory element, that is similar in composition to, but not identical to, a first DNA molecule, and in which the second DNA molecule still retains the general functionality, i.e., the same or similar expression pattern, for example through more or less equivalent transcriptional activity, of the first DNA molecule. A variant may be a shorter or truncated version of the first DNA molecule or an altered version of the sequence of the first DNA molecule. Petition 870260061195, dated 06 / 22 / 2026, p. 30 / 178 24 / 65 DNA, such as one with different restriction enzyme sites and / or internal deletions, substitutions, or insertions. A “variant” may also encompass a regulatory element having a nucleotide sequence comprising a substitution, deletion, or insertion of one or more nucleotides from a reference sequence, wherein the derived regulatory element has more or less or equivalent transcriptional or translational activity than the corresponding parental regulatory molecule. “Variants” of the regulatory element will also encompass variants resulting from mutations that occur naturally in the transformation of bacterial and plant cells.In the present invention, the polynucleotide sequences provided as SEQ ID NOs:1-19 and SEQ ID NO:26 can be used to create variants that are similar in composition, but not identical, to the DNA sequence of the original regulatory element, while maintaining the overall functionality, i.e., the same or similar expression pattern, of the original regulatory element. The production of such variants of the invention is well within the common knowledge of the art in light of disclosure and is encompassed within the scope of the invention.

[0067] The effectiveness of the modifications, duplications or deletions described in this document on the desired expression aspects of a particular transgene can be empirically tested in sterile and transient plant assays, such as those described in the working examples presented in this document, in order to validate the results, which may vary depending on the changes made and the objective of the change in the initial DNA molecule. Constructs

[0068] As used in this document, the term construct means any recombinant DNA molecule, such as a plasmid, cosmid, virus, phage, or linear or circular DNA or RNA molecule, derived from any source, capable of genomic integration or Petition 870260061195, dated 06 / 22 / 2026, p. 31 / 178 25 / 65 autonomous replication, comprising a DNA molecule in which at least one DNA molecule has been linked to another DNA molecule in a functionally operative manner, i.e., operatively linked. As used in this document, the term “vector” means any construct that can be used for the purpose of transformation, i.e., the introduction of heterologous DNA or RNA into a host cell. A construct typically includes one or more expression cassettes. As used in this document, an “expression cassette” refers to a DNA molecule comprising at least one operatively linked transcribable DNA molecule to one or more regulatory elements, typically at least one promoter and a 3' UTR.

[0069] As used in this document, the term “operatively linked” refers to a first DNA molecule attached to a second DNA molecule, wherein the first and second DNA molecules are arranged in such a way that the first DNA molecule affects the function of the second DNA molecule. The two DNA molecules may or may not be part of a single contiguous DNA molecule and may or may not be adjacent. For example, a promoter is operatively linked to a transcribable DNA molecule if the promoter modulates the transcription of the transcribable DNA molecule of interest in a cell. A leader, for example, is operatively linked to the DNA sequence when it is capable of affecting the transcription or translation of the DNA sequence.

[0070] The constructs of the invention can be provided, in one embodiment, as tumor-inducing double plasmid border constructs (Ti) having right border (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of the Ti plasmid isolated from Agrobacterium tumefaciens comprising a T-DNA that, together with transfer molecules provided by the cells Petition 870260061195, dated 06 / 22 / 2026, p. 32 / 178 26 / 65 of A. tumefaciens allow the integration of T-DNA into the genome of a plant cell (see, for example, US Patent 6,603,061). The constructs may also contain the DNA segments of the plasmid backbone that provide replication function and antibiotic selection in bacterial cells, for example, an Escherichia coli origin of replication such as ori322, a wide range of host origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spec / Strp encoding the Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a selectable gentamicin marker gene (Gm, Gent). For plant transformation, the host bacterial strain is often ABI, C58, or LBA4404 of A. tumefaciens; However, other strains known to those skilled in plant transformation techniques may work in the invention.

[0071] Methods are known in the art for assembling and introducing constructs into a cell in such a way that the transcribable DNA molecule is transcribed into a functional mRNA molecule that is translated and expressed as a protein. For the practice of the invention, conventional compositions and methods for the preparation and use of constructs and host cells are well known to those skilled in the art. Typical vectors useful for the expression of nucleic acids in higher plants are well known in the art and include vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaMVCN transfer control vector.

[0072] Several regulatory elements may be included in a construct, including any of those provided in this document. Any of these regulatory elements may be provided in combination with other regulatory elements. Such combinations may be designed or modified to produce desirable regulatory features. In one embodiment, the constructs of the invention comprise Petition 870260061195, dated 06 / 22 / 2026, p. 33 / 178 27 / 65 dem at least one regulatory element operatively linked to a transcribable DNA molecule operatively linked to a 3' UTR.

[0073] The constructs of the invention may include any promoter or leader provided in this document or known in the art. For example, a promoter of the invention may be operatively linked to a heterologous untranslated 5' leader, such as one derived from a heat shock protein gene. Alternatively, a leader of the invention may be operatively linked to a heterologous promoter, such as the cauliflower virus 35S transcript promoter.

[0074] Expression cassettes may also include a transit peptide coding sequence that encodes a peptide that is useful for the subcellular targeting of an operatively bound protein, particularly to a chloroplast, leucoplast, or other plastidial organoplast; mitochondrion; peroxisome; vacuole; or an extracellular location. Many proteins located in chloroplasts are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, those associated with the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex protein I and protein II, thioredoxin F, and enolpyruvyl shikimate synthase phosphate (EPSPS). Chloroplast transit peptides are described, for example, in US Patent 7,193,133.It has been shown that non-chloroplastic proteins can be targeted to the chloroplast by the expression of a heterologous CTP operatively linked to the transgene encoding non-chloroplastic proteins. Transcribeable DNA molecules

[0075] As used in this document, the term molecule of Petition 870260061195, dated 06 / 22 / 2026, page 34 / 178 28 / 65 Transcribeable DNA refers to any DNA molecule capable of being transcribed into an RNA molecule, including, but not limited to, those that have protein-coding sequences and those that produce RNA molecules with sequences useful for gene deletion. The type of DNA molecule may include, but is not limited to, a DNA molecule from the same plant, a DNA molecule from another plant, a DNA molecule from a different organism, or a synthetic DNA molecule, such as an antisense message of a gene, or a DNA molecule that encodes an artificial, synthetic, or otherwise modified version of a transgene.Examples of transcribable DNA molecules for incorporation into constructs of the invention include, for example, DNA molecules or genes from a species different from the species in which the DNA molecule is incorporated, or genes that originate from or are present in the same species but are incorporated into recipient cells by genetic engineering methods rather than classical breeding techniques.

[0076] A transgene refers to a transcribable DNA molecule that is heterologous to a host cell at least with respect to its location in the host cell genome and / or a transcribable DNA molecule that has been artificially incorporated into the genome of a host cell in the current generation or any previous generation of the cell.

[0077] A regulatory element, such as a promoter of the invention, may be operatively linked to a transcribable DNA molecule that is heterologous with respect to the regulatory element. As used in this document, the term heterologous refers to the combination of two or more DNA molecules where such a combination is not normally found in nature. For example, the two DNA molecules may be derived from different species and / or the two DNA molecules may be derived from different genes, for example, different genes from the same species or the same genes from Petition 870260061195, dated 06 / 22 / 2026, p. 35 / 178 29 / 65 different species. A regulatory element is thus heterologous with respect to a transcribable DNA molecule, operationally linked, if this combination is not normally found in nature, that is, the transcribable DNA molecule does not occur naturally operationally linked to the regulatory element.

[0078] The transcribable DNA molecule can generally be any DNA molecule for which the expression of a transcript is desired. Such expression of a transcript can result in the translation of the resulting mRNA molecule, and thus protein expression. Alternatively, for example, a transcribable DNA molecule can be designed to ultimately cause decreased expression of a specific gene or protein. In one embodiment, this can be accomplished using a transcribable DNA molecule that is oriented in the antisense direction. One of common knowledge in the art is familiar with the use of such antisense technology. Any gene can be downregulated in this way, and, in one embodiment, a transcribable DNA molecule can be designed for the suppression of a specific gene through the expression of a dsRNA, siRNA, or miRNA molecule.

[0079] Thus, one embodiment of the invention is a molecule of Recombinant DNA comprising a regulatory element of the invention, such as those provided as SEQ ID Nos. 1-19 and SEQ ID No. 26, operatively linked to a heterologous transcriptable DNA molecule so as to modulate the transcription of the transcriptable DNA molecule at a desired level or in a desired pattern when the construct is integrated into the genome of a transgenic plant cell. In one embodiment, the transcriptable DNA molecule comprises a protein-coding region of a gene and in another embodiment the transcriptable DNA molecule comprises an antisense region of a gene. Genes of agronomic interest Petition 870260061195, dated 06 / 22 / 2026, page 36 / 178 30 / 65

[0080] A transcribable DNA molecule can be a gene of agronomic interest. As used in this document, the term gene of agronomic interest refers to a transcribable DNA molecule that, when expressed in a particular plant tissue, cell, or cell type, confers a desirable characteristic. The product of a gene of agronomic interest can act within the plant to cause an effect on plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, resistance to diseases or pests, and / or environmental or chemical tolerance, or act as a pesticidal agent in the diet of a pest that feeds on the plant. In one embodiment of the invention, a regulatory element of the invention is incorporated into a construct such that the regulatory element is operatively linked to a transcribable DNA molecule that is a gene of agronomic interest.In a transgenic plant containing such a construct, the expression of the gene of agronomic interest can confer a beneficial agronomic trait. A beneficial agronomic trait may include, for example, but is not limited to, herbicide tolerance, insect control, modified yield, disease resistance, pathogen resistance, modified plant growth and development, modified starch content, modified oil content, modified fatty acid content, modified protein content, modified fruit ripening, improved animal and human nutrition, biopolymer production, resistance to environmental stress, pharmaceutical peptides, improved processing qualities, improved flavor, hybrid seed production utility, improved fiber production, and desirable biofuel production.

[0081] Non-limiting examples of genes of agronomic interest known in the art include those for herbicide resistance (e.g., U.S. Patents 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and 5,463,175), Petition 870260061195, dated 06 / 22 / 2026, page 37 / 178 31 / 65 increased yield (e.g., US Patent USRE38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,098; and 5,716,837), insect control (US Patents No. 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756; 6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949; 6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013; 5,959,091; 5,942,664; 5,942,658; 5,880,275; 5,763,245; and 5,763,241), resistance to fungal disease (US Patents Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,506,962), virus resistance (US Patents Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematode resistance (US Patent No. 6,228,992), bacterial disease resistance (US Patent No. 5,516,671), plant growth and development (US Patents Nos. 6,723,897 and 6,518,488), starch production (US Patents Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oil production (US Patents (US Patents No. 6,444,876; 6,426,447; and 6,380,462), high oil production (US Patents No. 6,495,739; 5,608,149; 6,483,008; and 6,476,295), modified fatty acid content (US Patents No. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,461; and 6,459,018), high protein production (US Patent No. 6,380,466), fruit ripening (US Patent No. 5,512,466), enhanced animal and human nutrition (US Patents Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers (US Patent Nos. USRE37,543; 6,228,623; and 5,958,745 and 6,946,588), resistance to environmental stress (US Patent 6,072,103), pharmaceutical peptides and secretable peptides (US Patents 6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processing traces (US Patent 6,476,295), digestion. Petition 870260061195, dated 06 / 22 / 2026, page 38 / 178 32 / 65 tability (US Patent 6,531,648) low raffinose (US Patent 6,166,292), industrial enzyme production (US Patent 5,543,576), improved flavor (US Patent 6,011,199), nitrogen fixation (US Patent 5,229,114), hybrid seed production (US Patent 5,689,041), fiber production (US Patents 6,576,818; 6,271,443; 5,981,834; and 5,869,720) and biofuel production (US Patent 5,998,700).

[0082] Alternatively, a gene of agronomic interest may affect the aforementioned plant characteristics or phenotypes by encoding an RNA molecule that causes targeted modulation of gene expression of an endogenous gene, for example by antisense (see, for example, US Patent 5,107,065); inhibitory RNA (“RNAi”, including modulation of gene expression by mechanisms mediated by miRNA, siRNA, trans-regulatory siRNA, and in-phase sRNA, for example, as described in published applications US 2006 / 0200878 and US 2008 / 0066206, and in patent application US 11 / 974,469); or mechanisms mediated by co-suppression. RNA can also be a catalytic RNA molecule (e.g., a ribozyme or a riboswitch; see, for example, US 2006 / 0200878) modified to cleave a desired endogenous mRNA product.There are known methods in the field for constructing and introducing constructs into a cell in such a way that the transcribable DNA molecule is transcribed into a molecule capable of causing gene deletion. Selectable Markers

[0083] Selectable marker transgenes can also be used with the regulatory elements of the invention. As used in this document, the term selectable marker transgene refers to any DNA molecule that can be transcribed, the expression or lack thereof of which in a transgenic plant, tissue or cell can be screened or marked in some way. Marked genes Petition 870260061195, dated 06 / 22 / 2026, p. 39 / 178 33 / 65 selectable marker transgenes, and their associated selection and screening techniques, for use in the practice of the invention are known in the art and include, but are not limited to, transcribable DNA molecules encoding β-glucuronidase (GUS), green fluorescent protein (GFP), proteins conferring antibiotic resistance, and proteins conferring herbicide tolerance. Examples of selectable marker transgenes are provided as SEQ ID Nos: 20 and 24. Cellular Transformation

[0084] The invention also relates to a method of producing transformed cells and plants comprising one or more regulatory elements operatively linked to a transcribable DNA molecule.

[0085] The term transformation refers to the introduction of a DNA molecule into a recipient host. As used in this document, the term host refers to bacteria, fungi, or plants, including any cells, tissues, organs, or progeny of the bacteria, fungi, or plants. Plant tissues and cells of particular interest include protoplasts, calluses, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[0086] As used in this document, the term transformed refers to a cell, tissue, organ, or organism into which a foreign DNA molecule, such as a construct, has been introduced. The introduced DNA molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism, such that the introduced DNA molecule is inherited by subsequent offspring. A transgenic or transformed cell or organism may also include progeny of the cell or organism and progeny produced from a breeding program that employs a transgenic organism as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a DNA molecule. The DNA molecule Petition 870260061195, dated 06 / 22 / 2026, page 40 / 178 34 / 65 Introduced DNA can also be transiently introduced into the recipient cell, such that the introduced DNA molecule is not inherited by subsequent offspring. The term transgenic refers to a bacterium, fungus, or plant containing one or more heterologous DNA molecules.

[0087] There are many well-known methods among those skilled in the art for introducing DNA molecules into plant cells. The process generally comprises the steps of selecting a suitable host cell, transforming the host cell with a vector, and obtaining the transformed host cell. The methods and materials for transforming plant cells by introducing a plant construct into a plant genome in the practice of this invention may include any of the well-known and demonstrated methods. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct DNA delivery (e.g., by PEG-mediated transformation, desiccation / inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and acceleration of DNA-coated particles), gene editing (e.g., CRISPR-Cas systems), among others.

[0088] Host cells can be any cell or organism, such as a plant cell, algal cell, algae, fungal cell, fungus, bacterial cell, or insect cell. In specific embodiments, host cells and transformed cells may include cultured plant cells.

[0089] A transgenic plant can subsequently be regenerated from a transgenic plant cell of the invention. Using conventional breeding techniques or self-pollination, seeds can be produced from this transgenic plant. Such a seed, and the resulting progeny plant grown from that seed... Petition 870260061195, dated 06 / 22 / 2026, page 41 / 178 35 / 65 te will contain the recombinant DNA molecule of the invention and will therefore be transgenic.

[0090] Transgenic plants of the invention can be self-pollinated to provide seeds for homozygous transgenic plants of the invention (homozygous for the recombinant DNA molecule) or crossed with non-transgenic plants or different transgenic plants to provide seeds for heterozygous transgenic plants of the invention (heterozygous for the recombinant DNA molecule). Both homozygous and heterozygous transgenic plants are referred to in this document as progeny plants. Progeny plants are transgenic plants descended from the original transgenic plant and containing the recombinant DNA molecule of the invention. Seeds produced using a transgenic plant of the invention can be harvested and used to cultivate generations of transgenic plants, i.e., progeny plants of the invention, comprising the construct of this invention and expressing a gene of agronomic interest.Descriptions of breeding methods that are commonly used for different crops can be found in one of several reference books, see, for example, Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (1960); Simmonds, Principles of Crop Improvement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Perspectives on Plant Breeding, Wageningen (ed), Center for Agricultural Publication and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16: 249 (1987); Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).

[0091] The transformed plants can be analyzed for the presence of the gene or genes of interest and the level and / or expression profile conferred by the regulatory elements of the invention. The ver Petition 870260061195, dated 06 / 22 / 2026, p. 42 / 178 36 / 65 experts in the art are aware of the numerous methods available for the analysis of transformed plants. For example, methods for plant analysis include, but are not limited to, Southern blots or Northern blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, field assessments, and immunodiagnostic assays. The expression of a transcristable DNA molecule can be measured using TaqMan® reagents and methods (Applied Biosystems, Foster City, CA), as described by the manufacturer, and PCR cycle times determined using the TaqMan® test array. Alternatively, Invader® reagents and methods (Third Wave Technologies, Madison, WI) and the methods described by the manufacturer can be used to assess transgene expression.

[0092] The invention also provides plant parts of the invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. Plant parts may be viable, non-viable, regenerable, and / or non-regenerable. The invention also includes and provides transformed plant cells comprising a DNA molecule of the invention. The transformed or transgenic plant cells of the invention include regenerable and / or non-regenerable plant cells.

[0093] The invention also provides a base product that is produced from a transgenic plant or part thereof containing the recombinant DNA molecule of the invention. The base products of the invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs:1-19 and SEQ ID NO:26. As used in this document, a “base product” refers to any composition or product that consists of material derived from a transgenic plant, seed, plant cell or plant part containing the molecule of Petition 870260061195, dated 06 / 22 / 2026, p. 43 / 178 37 / 65 Recombinant DNA of the invention. Base products include, but are not limited to, processed seeds, grains, plant parts, and flours. A base product of the invention will contain a detectable amount of DNA corresponding to the recombinant DNA molecule of the invention. Detection of one or more of these DNA molecules in a sample can be used to determine the content or source of the base product. Any conventional method of detecting DNA molecules can be used, including detection methods disclosed in this document.

[0094] The invention can be more easily understood by reference to the following examples, which are provided for illustrative purposes only and are not intended to be limiting of the invention unless otherwise specified. It should be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the invention. However, those skilled in the art should, in the light of the present disclosure, appreciate that many alterations can be made to the specific embodiments that are disclosed and still obtain a similar or like result without departing from the spirit and scope of the invention; therefore, all matters set forth or shown in the accompanying figures should be interpreted as illustrative and not in a limiting sense. EXAMPLES Example 1 Design, synthesis and cloning of synthetic regulatory elements

[0095] Novel synthetic transcription regulatory elements are synthetic expression elements designed using algorithmic methods. These computationally designed regulatory elements were chemically synthesized and cloned to make clusters of synthetic regulatory expression elements (EXPs). Well over Petition 870260061195, dated 06 / 22 / 2026, page 44 / 178 38 / 65 One thousand synthetic regulatory elements were designed and tested in maize protoplasts and stably transformed maize plants to identify the synthetic regulatory elements that provided the desired characteristics, such as protein expression levels and expression patterns. The synthetic elements of the present invention provide several constitutive expression patterns useful in driving the expression of many different coding sequences and interfering RNAs of agronomic interest.

[0096] The engineered synthetic transcription regulatory elements have no extended homology with any known nucleic acid sequences that exist in nature, but affect the transcription of an operatively linked coding sequence in the same way as naturally occurring promoters, leaders, introns, and 3' UTRs. The synthetic EXPs and their corresponding synthetic promoters, leaders, and introns, as well as synthetic 3' UTRs, are presented in Table 1. The synthetic EXPs were cloned using methods known in the art into plant transformation binary vectors, operatively linked to a βglucuronidase (GUS) coding sequence, and expression levels and patterns in stably transformed maize plants were evaluated.

[0097] Analysis of the TSS regulatory element and intron / exon splice junctions can be performed using transformed plant tissue. Briefly, plants were transformed with plant expression vectors comprising operatively cloned DNA fragments ligated to a heterologous transcriptable DNA molecule. Subsequently, the RACE 5' System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen, Carlsbad, California 92008) was used to confirm the TSS regulatory element and intron / exon splice junctions by analyzing the DNA sequence of the produced mRNA transcripts. Synthetic 3' UTRs were characterized Petition 870260061195, dated 06 / 22 / 2026, page 45 / 178 39 / 65 rizadas por seu efeito na expressão de genes, como como pela terminar a próprio do transcripto.

[0098] In addition to synthetic elements of expression, a new An endogenous 3' UTR derived from the Sorghum bicolor non-specific lipid transfer protein 4 gene, T-Sb.Nltp4-1:1:2, is provided here and is presented as SEQ ID NO: 19. T-Sb.Nltp4-1:1:2 was characterized similarly to synthetic 3' UTRs. Table 1. Groups of regulatory expression elements of synthetic transcription, promoters, leaders, introns and 3' UTRs. Annotation SEQ ID NO: Size (bp) Description and / or regulatory elements of the EXP linked in the 5' ^ 3' direction (SEQ ID NOs): EXP-Zm.GSP850 1 500 EXP: P-Zm.GSP850.nno:4 (SEQ ID NO:2), L- Zm.GSP850.nno:3 (SEQ ID NO:3) P-Zm.GSP850.nno:4 2 450 Promoter L-Zm.GSP850.nno:3 3 50 Leader EXP- Zm.GSP850.nno+Zm.GSI153.nno: 2 4 1117 EXP: P-Zm.GSP850.nno:4 (SEQ ID NO:2), L- Zm.GSP850.nno:3 (SEQ ID NO:3), I- Zm.GSI153.nno:1 (SEQ ID NO:5) I-Zm.GSI153.nno: 1 5 610 Intron EXP-Zm.GSP990 6 500 EXP: P-Zm.GSP990.nno:2 (SEQ ID NO:7), L- Zm.GSP990.nno:1 (SEQ ID NO:8) P-Zm.GSP990.nno:2 7 450 Promoter L-Zm.GSP990.nno:1 8 50 Leader EXP- Zm.GSP990.nno+Zm.GSI197.nno: 2 9 1117 EXP: P-Zm.GSP990.nno:2 (SEQ ID NO:7), L- Zm.GSP990.nno:1 (SEQ ID NO:8), I- Zm.GSI197.nno:1 (SEQ ID NO:10) I-Zm.GSI197.nno:1 10 610 Intron EXP- Zm.GSP850.nno+Zm.GSI140.nno: 1 11 1117 EXP: P-Zm.GSP850.nno:4 (SEQ ID NO:2), L- Zm.GSP850.nno:3 (SEQ ID NO:3), I- Zm.GSI140.nno:1 (SEQ ID NO:12) I-Zm.GSI140.nno:1 12 610 Intron T-Zm.GST9.nno:2 13 300 UTR 3' T-Zm.GST18.nno:2 14 400 UTR 3'. Petition 870260061195, dated 06 / 22 / 2026, p. 46 / 178 40 / 65 Annotation SEQ ID NO: Size (bp) Description and / or regulatory elements EXP connected in the 5' ^ 3' direction (SEQ ID NOs): EXP-Zm.GSP850.nno+Zm.DnaK:1 15 1311 EXP: P-Zm.GSP850.nno:4 (SEQ ID NO:2), L- Zm.GSP850.nno:3 (SEQ ID NO:3), I- Zm.DnaK:1 (SEQ ID NO:22) EXP-Zm.GSP990.nno+Zm.DnaK:1 16 1311 EXP: P-Zm.GSP990.nno:2 (SEQ ID NO:7), L- Zm.GSP990.nno:1 (SEQ ID NO:8), I- Zm.DnaK:1 (SEQ ID NO:22) E-Zm.GSP850 17 418 E-Zm.GSP990 18 416 T-Zm.GST43.nno:1 26 300 UTR 3' Example 2 Analysis of synthetic regulatory elements that drive GUS in maize leaf protoplasts

[0099] Corn leaf protoplasts were transformed with vectors, specifically expression vectors containing a test regulatory element that directs the expression of the βglucuronidase (GUS) transgene. The resulting transformed corn leaf protoplasts were analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[00100] Corn protoplasts, derived from leaf tissue, were transformed with expression vectors comprising synthetic expression elements. The level and pattern of expression of these synthetic expression element vectors in corn protoplasts were compared to the level and pattern of expression of expression elements known in the art. Separate experiments were conducted to evaluate the activity of EXP, EXP-Zm.GSP850 (SEQ ID NO: 1) and EXP-Zm.GSP990 (SEQ ID NO: 6), the introns IZm.GSI153.nno: 1 (SEQ ID NO: 5) and I-Zm.GSI197.nno: 1 (SEQ ID NO: 10), and the 3' UTRs, T-Zm.GST9.nno:2 (SEQ ID NO: 13) and T-Zm .GST18.nno: 2 (SEQ ID NO: 14). The expression elements were cloned into expression vectors and operatively linked to one. Petition 870260061195, dated 06 / 22 / 2026, p. 47 / 178 41 / 65 GUS coding sequence, GOI-Ec.uidA+St.LS1:1:1 (SEQ ID NO:24), comprising a processable intron. The control expression vectors comprised different configurations of known expression elements that varied depending on the type of element to be evaluated (EXP, Intron, or 3' UTR). A plasmid used in protoplast cotransformation and data normalization was also constructed using methods known in the art. It comprised a transgene cassette composed of EXP, EXPCaMV.35S (SEQ ID NO: 21) operatively linked in 5' to a coding sequence encoding the fluorescent protein NanoLuc® luciferase (Promega, Madison, WI 53711), herein referred to as Nluc (SEQ ID NO: 25), which was operatively linked in 5' to a 3' UTR, T-Os.LTP:1 (SEQ ID NO: 23).

[00101] Corn leaf protoplasts were transformed using a PEG-based transformation method, similar to those known in the art. Protoplast cells were transformed into a ninety-six (96) well format. Twelve (12) micrograms of test vector DNA or control vector DNA and six (6) micrograms of NanoLuc® vector DNA were used to transform 3.2 x 105 protoplasts per well. After transformation, the protoplasts were incubated at 25°C in the dark for sixteen to twenty hours. After incubation, the protoplasts were lysed and the lysate was used to measure luciferase and GUS expression. To lyse the cells, the cells in the plate were pelleted by centrifugation, washed, resuspended in a smaller volume, and transferred to strip well tubes. The tubes were centrifuged again and the supernatant was aspirated, leaving behind the pellet of protoplastic cells.The cell pellet was resuspended in QB buffer (100 mM KPO4, pH 7.8; 1 mM EDTA; 1% Triton X-100; 10% Glycerol; 1 mM DTT). The cells were lysed by vigorously pipetting the cells several times, agi. Petition 870260061195, dated 06 / 22 / 2026, page 48 / 178 42 / 65 vortexing the tubes and allowing them to incubate on ice for five minutes. The lysate was then centrifuged to pelletize the cellular debris. The resulting lysate was then transferred to a clean plate.

[00102] Luciferase activity was tested using Nano-Glo® Luciferase Assay Substrate (Promega, Madison, WI 53711) in QB buffer. In summary, a small volume of lysate, QB buffer and the Nano-Glo® Luciferase / QB Assay Substrate solution were mixed in ninety-six (96) well blank plates. Fluorescence was then measured using a PHERAstar® plate reader (BMG LABTECH Inc., Cary, NC 27513).

[00103] GUS activity was evaluated using the fluorogenic substrate 4-methyleumbelliferyl-nD-glucuronide (MUG) in a total reaction volume of fifty (50) microliters. The reaction product, 4-methylumbelliferone (4-MU), is maximally fluorescent at high pH, ​​where the hydroxyl group is ionized. The addition of a basic sodium carbonate solution simultaneously stops the assay and adjusts the pH to quantify the fluorescent product. An aliquot of lysate was mixed with an aliquot of MUG dissolved in QB buffer and incubated at 37°C. A small aliquot of the lysate / MUG reaction mixture was removed and added to a stop buffer at three different time points: (1) immediately after mixing the lysate / MUG reaction as “Time zero minutes”; (2) twenty minutes; and (3) sixty minutes. Fluorescence was measured with excitation at 355 nm and emission at 460 nm using a PHERAstar® plate reader (BMG LABTECH Inc., Cary, NC 27513).The expression level is expressed as “nM MUG hydrolysate”, which is derived from the standard curve on the plate.

[00104] For each plate, each construct is transformed into four (4) to eight (8) wells. An aliquot was taken from each transformation for the MUG assay and “nM MUG hydrolysate” was derived from the pa curve. Petition 870260061195, dated 06 / 22 / 2026, page 49 / 178 43 / 65 plate standard. An aliquot was also taken from each transformation for NanoLuc® reading (NanoLuc® RLU). The average nM hydrolyzed MUG / NanoLuc® RLU for each construct was normalized relative to the EXP-CaMV.35S / I-Zm.DnaK:1 / T-Os.LTP:1 construct, which is set to 100%. Analysis of GUS expression in maize leaf protoplasts driven by synthetic EXP-Zm.GSP850.

[00105] Maize leaf protoplast cells were transformed with expression vectors that were constructed using methods known in the art, comprising expression elements that direct GUS expression. Two (2) test expression vectors comprised transgene cassettes comprising the EXP, synthetic EXP-Zm.GSP850 (SEQ ID NO: 1). The synthetic EXPZm.GSP850 is composed of a synthetic promoter, PZm.GSP850.nno:4 (SEQ ID NO: 2), operatively linked at 5' to a synthetic leader, L-Zm.GSP850.nno:3 (SEQ ID NO: 3). The first test vector comprised EXP-Zm.GSP850, operatively linked at 5' to a GUS encoding sequence (SEQ ID NO: 24) which comprised a processable intron, which was operatively linked at 5' to the UTR 3', T-Os.LTP:1 (SEQ ID NO: 23). The second transgene cassette comprised EXP-Zm.GSP850, operatively linked at 5' to the I-Zm intron.DnaK:1 (SEQ ID NO: 22), operationally connected in 5' to the GUS encoding sequence, which was operationally connected in 5' to UTR 3', T-Os.LTP:1.

[00106] Three (3) control expression vectors were also constructed and used to transform maize leaf protoplasts. The first control expression vector comprised a promoterless transgene cassette and was composed of the intron, I-Zm.DnaK:1, operatively linked at 5' to the GUS coding sequence, which was operatively linked at 5' to the UTR 3', T-Os.LTP:1. The second Petition 870260061195, dated 06 / 22 / 2026, p. 50 / 178 The 44 / 65 control vector comprised an intronless transgene cassette and was composed of EXP, EXP-CaMV.35S (SEQ ID NO:21), operatively linked 5' to the GUS coding sequence, which was operatively linked 5' to the 3' UTR, T-Os.LTP:1. The third control vector comprised a transgene cassette comprising EXP, EXP-CaMV.35S, operatively linked 5' to the IZm.DnaK:1 intron, operatively linked 5' to the GUS coding sequence, which was operatively linked 5' to the 3' UTR, T-Os.LTP:1.

[00107] Maize leaf protoplasts were transformed with all five (5) vectors. Transformation and lysis of protoplast cells were performed as described herein. Luciferase and GUS expression were assayed as described herein. Table 2 shows the average expression of GUS tested and is expressed as a percentage of expression relative to the third control expression vector comprising EXP-CaMV.35S and I-Zm.DnaK:1 driving GUS. Table 2. Average percentage expression of GUS from maize leaf protoplasts transformed with test and control vectors. Plate ID Promoter Intron Mean SD Reps 39 No Promoter I-Zm.DnaK:1 0.1 0.337 6 54 No Promoter I-Zm.DnaK:1 0.4 0.315 8 95 No Promoter I-Zm.DnaK:1 0.1 0.534 8 103 No Promoter I-Zm.DnaK:1 0.5 0.149 8 39 EXP-CaMV.35S No intron 48.3 2018 6 54 EXP-CaMV.35S No intron 46.5 3.949 8 95 EXP-CaMV.35S No intron 46.8 4.369 8 103 EXP-CaMV.35S No intron 40 3.333 8 39 EXP-CaMV.35S I-Zm.DnaK:1 100 5.117 6 54 EXP-CaMV.35S I-Zm.DnaK:1 100 6.465 8 95 EXP-CaMV.35S I-Zm.DnaK:1 100 18.603 8 103 EXP-CaMV.35S I-Zm.DnaK:1 100 5.164 8 95 EXP-Zm.GSP850 No intron 14 1.844 8 103 EXP-Zm.GSP850 No intron 8.4 0.336 8 39 EXP-Zm.GSP850 I-Zm.DnaK:1 19 0.732 4 54 EXP-Zm.GSP850 I-Zm.DnaK:1 22 1,954 8 Petition 870260061195, dated 06 / 22 / 2026, page 51 / 178 45 / 65

[00108] As can be seen in Table 2 above, EXP-Zm.GSP850 (SEQ ID NO:1) was able to drive the expression of the GUS transgene in maize leaf protoplasts when compared to maize leaf protoplast cells transformed with a promoter-less construct. Analysis of GUS expression in maize leaf protoplasts driven by synthetic EXP-Zm.GSP990.

[00109] Maize leaf protoplast cells were transformed with expression vectors that were constructed, comprising expression elements that direct the expression of GUS. A test expression vector comprised a transgene cassette comprising the synthetic EXP-Zm.GSP990 (SEQ ID NO: 6), operatively linked at 5' to the intron I-Zm.DnaK:1 (SEQ ID NO: 22), operatively linked at 5' to a coding sequence encoding GUS (SEQ ID NO: 20), which was operatively linked at 5' to the UTR 3', TOs.LTP:1. The synthetic EXP-Zm.GSP990 (SEQ ID NO: 6) is composed of a synthetic promoter, P-Zm.GSP990.nno:2 (SEQ ID NO: 7), operatively linked at 5' to a synthetic leader, L-Zm.GSP990.nno:1 (SEQ ID NO: 8). Three control expression vectors were also transformed into maize leaf protoplasts and constructed as described above. Table 3 shows the average percent expression relative to the third control expression vector comprising EXPCaMV.35S and I-Zm.DnaK:1 conducting GUS. Table 3. Average percentage expression of GUS from maize leaf protoplasts transformed with test and control vectors. Board ID Promoter Intron Average SD Reps 71 Without Promoter I-Zm.DnaK:1 0.3 0.125 6 108 Without Promoter I-Zm.DnaK:1 -0.6 1.211 8 71 EXP-CaMV.35S Without intron 45.1 7.791 6 108 EXP-CaMV.35S Without intron 39.9 2.636 8 Petition 870260061195, dated 06 / 22 / 2026, page 52 / 178 46 / 65 Plate ID Promoter Intron Mean SD Reps 71 EXP-CaMV.35S I-Zm.DnaK:1 100 13,591 6 108 EXP-CaMV.35S I-Zm.DnaK:1 100 7,425 8 71 EXP-Zm.GSP990 I-Zm.DnaK:1 52.3 13,658 4 108 EXP-Zm.GSP990 I-Zm.DnaK:1 45.2 5.468 8

[00110] As can be seen in Table 3, EXP-Zm.GSP990 (SEQ ID NO:6) was able to drive the expression of the GUS transgene in maize leaf protoplasts when compared to maize leaf protoplast cells transformed with a promoter-less construct. Analysis of the increase in GUS expression by the synthetic intron, IZm.GSI153.nno:1

[00111] Corn leaf protoplast cells were transformed with constructed expression vectors comprising expression elements that direct GUS expression. A test expression vector was used to test the enhancement of GUS expression from the synthetic intron, I-Zm.GSI153.nno:1 (SEQ ID NO: 5), driven by EXP-CaMV.35. The transgene cassette comprised EXP, EXP-CaMV.35 operatively linked at 5' to the synthetic intron, I-Zm.GSI153.nno:1 (SEQ ID NO: 5), operatively linked at 5' to a coding sequence encoding GUS (SEQ ID NO: 24), which was operatively linked at 5' to the 3' UTR, TOs.LTP:1. Two control expression vectors were also constructed and used to transform corn leaf protoplasts. The first control expression vector comprised an intron-free transgene cassette and was composed of EXP, EXP-CaMV.35S, operatively linked at 5' to the GUS coding sequence, which was operatively linked at 5' to the UTR 3', T-Os.LTP:1. The second control vector comprised a transgene cassette comprising EXP, EXP-CaMV.35S, operatively linked at 5' to the IZm.DnaK:1 intron, operatively linked at 5' to the coding sequence. Petition 870260061195, dated 06 / 22 / 2026, page 53 / 178 47 / 65 GUS, which was operatively connected at 5' to UTR 3', T-Os.LTP:1. Table 4 shows the average percentage expression in relation to the second control expression vector comprising both EXPCaMV.35S and I-Zm.DnaK:1 driving GUS. Table 4. Average percentage expression of GUS from maize leaf protoplasts transformed with test and control vectors. Plate ID Promoter Intron Mean SD Reps 10 EXP-CaMV.35S Intronless 52.8 8.428 6 13 EXP-CaMV.35S Intronless 44.4 4.586 8 10 EXP-CaMV.35S I-Zm.DnaK:1 100 16.646 6 13 EXP-CaMV.35S I-Zm.DnaK:1 100 13.123 8 10 EXP-CaMV.35S I-Zm.GSI153.nno: 1 83 5.601 4 13 EXP-CaMV.35S I-Zm.GSI153.nno: 1 83.6 7.596 8

[00112] As can be seen in Table 4, the synthetic intron, IZm.GSI153.nno:1 (SEQ ID NO: 5), increased the expression of the GUS transgene in maize leaf protoplasts driven by EXPCaMV.35S when compared to the control expression vector without an intron. Analysis of the increase in GUS expression by the synthetic intron, IZm.GSI197.nno:1

[00113] Corn leaf protoplast cells were transformed with expression vectors that were constructed, comprising expression elements that direct GUS expression. A test expression vector was used to test the increase in GUS expression from the synthetic intron, I-Zm.GSI197.nno:1 (SEQ ID NO: 10), driven by EXP-CaMV.35. The transgene cassette comprised EXP, EXP-CaMV.35 operatively linked at 5' to the synthetic intron, I-Zm.GSI197.nno:1, operatively linked at 5' to a coding sequence encoding GUS (SEQ ID NO: 24), which was operatively linked at 5' to the 3' UTR, T-Os.LTP:1. Three control expression vectors were also constructed and used to transform corn leaf protoplasts. The first vector of ex Petition 870260061195, dated 06 / 22 / 2026, page 54 / 178 The first control vector comprised a 48 / 65 control vector, a promoterless transgene cassette, and consisted of the intron, I-Zm.DnaK:1, operatively linked at 5' to the GUS coding sequence, which was operatively linked at 5' to the 3' UTR, T-Os.LTP:1. The second control vector comprised an intronless transgene cassette, consisting of EXP, EXP-CaMV.35S, operatively linked at 5' to the GUS coding sequence, which was operatively linked at 5' to the 3' UTR, T-Os.LTP:1. The third control vector comprised a transgene cassette comprising EXP, EXP-CaMV.35S, operatively linked at 5' to the intron I-Zm.DnaK:1, operatively linked at 5' to the GUS coding sequence, which was operatively linked at 5' to the 3' UTR, T-Os.LTP:1. Table 5 shows the average percentage expression in relation to the third control expression vector comprising both EXP-CaMV.35S and I-Zm.DnaK:1 driving GUS. Table 5. Average percentage expression of GUS from maize leaf protoplasts transformed with test and control vectors. Plate ID Promoter Intron Mean SD Reps 125 No Promoter I-Zm.DnaK:1 1.1 0.317 6 146 No Promoter I-Zm.DnaK:1 0.6 0.101 8 125 EXP-CaMV.35S No intron 43.8 4.081 6 146 EXP-CaMV.35S No intron 41 3,666 8 125 EXP-CaMV.35S I-Zm.DnaK:1 100 11,287 6 146 EXP-CaMV.35S I-Zm.DnaK:1 100 8,506 8 125 EXP-CaMV.35S I-Zm.GSI197.nno:1 150 12,451 4 146 EXP-CaMV.35S I-Zm.GSI197.nno:1 109.8 8.001 8

[00114] As can be seen in Table 5, the synthetic intron, IZm.GSI197.nno:1 (SEQ ID NO: 10), increased the expression of the GUS transgene in maize leaf protoplasts driven by EXPCaMV.35S when compared to the control expression vector without an intron. The increase in expression was greater than that conferred by the intron, I-Zm.DnaK:1, when compared to the third control expression vector comprising EXP, EXP-CaMV.35S, operation Petition 870260061195, dated 06 / 22 / 2026, page 55 / 178 49 / 65 actively connected in 5' to the intron I-Zm.DnaK:1. Analysis of the intensification of GUS expression by synthetic 3' UTRs, T-Zm.GST9.nno:2 and T-Zm.GST18.nno:2.

[00115] Corn leaf protoplast cells were transformed with expression vectors that were constructed, comprising expression elements that direct GUS expression. Two test vectors comprised a transgene cassette used for the analysis of the GUS expression increase conferred by the 3' UTRs, T-Zm.GST9.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14), and was composed of EXP-CaMV.35S operatively linked at 5' to the intron I-Zm.DnaK:1, operatively linked at 5' to a coding sequence encoding GUS (SEQ ID NO: 24), which was operatively linked at 5' to the 3' UTR, T-Zm.GST9.nno:2 (SEQ ID NO: 13) or to the 3' UTR, T-Zm.GST18.nno:2 (SEQ ID NO: 14). Three control expression vectors were also constructed, as described above, and used to transform maize leaf protoplasts. Table 6 shows the average percentage expression in relation to the third control expression vector, which includes both EXPCaMV.35S and I-Zm.DnaK:1 driving GUS. Table 6. Average percentage expression of GUS from maize leaf protoplasts transformed with test and control vectors. Plate ID Promoter Intron 3' UTR Mean SD Reps 119 No Promoter I-Zm.DnaK:1 T-Os.LTP: 1 3.5 2.163 8 184 No Promoter I-Zm.DnaK:1 T-Os.LTP: 1 0.1 0.205 6 185 No Promoter I-Zm.DnaK:1 T-Os.LTP: 1 0 0.331 8 328 No Promoter I-Zm.DnaK:1 T-Os.LTP: 1 10.2 3.017 6 119 EXP-CaMV.35S No intron T-Os.LTP: 1 45.5 3.729 8 184 EXP-CaMV.35S No intron T-Os.LTP: 1 33.7 2.719 6 185 EXP-CaMV.35S Intronless T-Os.LTP: 1 33.6 2.576 8 328 EXP-CaMV.35S Intronless T-Os.LTP: 1 48.8 3.69 6 119 EXP-CaMV.35S I-Zm.DnaK:1 T-Os.LTP: 1 100 7,305 8 184 EXP-CaMV.35S I-Zm.DnaK:1 T-Os.LTP: 1 100 6.91 6 Petition 870260061195, dated 06 / 22 / 2026, p. 56 / 178 50 / 65 Plate ID Promoter Intron 3' UTR Mean SD Reps 185 EXP-CaMV.35S I-Zm.DnaK:1 T-Os.LTP: 1 100 6.308 8 328 EXP-CaMV.35S I-Zm.DnaK:1 T-Os.LTP: 1 100 5.989 6 184 EXP-CaMV.35S I-Zm.DnaK:1 T-Zm.GST9.nno:2 202.5 17,582 4 119 EXP-CaMV.35S I-Zm.DnaK:1 T-Zm.GST18.nno:2 483.2 40,613 8 185 EXP-CaMV.35S I-Zm.DnaK:1 T-Zm.GST18.nno:2 254.5 18.347 8 328 EXP-CaMV.35S I-Zm.DnaK:1 T-Zm.GST18.nno:2 307.4 17.772 4

[00116] As can be seen in Table 6, the UTRs 3', TZm.GST9.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14) intensified GUS expression relative to the controls in maize leaf protoplasts. Example 3 Analysis of GUS expression driven by synthetic EXPs, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 and EXPZm.GSP850.nno+Zm.GSI140.nno:1 in LH244 maize plants with stable transformation.

[00117] Corn plants were transformed with a vector, specifically a plant expression vector containing a test regulatory element that drives the expression of the βglucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of the selected regulatory element on expression.

[00118] Corn plants were transformed with GUS plant expression constructs. The regulatory elements were cloned into a base plant expression vector using known standard methods in the art. The resulting plant expression vectors contained a left border region of Agrobacterium tumefaciens (B-AGRtu.leftborder), a first transgene selection cassette used for the selection of transformed plant cells conferring resistance to the herbicide glyphosate; a second transgene cassette to evaluate the activity of the synthetic regulatory elements. Petition 870260061195, dated 06 / 22 / 2026, page 57 / 178 51 / 65 tics, comprising the synthetic EXP, EXPZm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO:4) or EXPZm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO:11) operatively linked in 5' to a synthetic coding sequence designed for expression in a plant cell encoding β-glucuronidase (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20) containing a processable intron derived from the light-inducible tissue-specific ST-LS1 gene of potato (Genbank Accession: X04753), operatively linked in 5' to a 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO:19); and a right border region of Agrobacterium tumefaciens (BAGRtu. right border). The synthetic EXP, EXPZm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO: 4) is composed of a synthetic promoter, P-Zm.GSP850.nno:4 (SEQ ID NO: 2), operatively linked at 5' to a synthetic leader, L-Zm.GSP850.nno:3 (SEQ ID NO: 3), which is operatively linked at 5' to a synthetic intron, I-Zm.GSI153.nno:1 (SEQ ID NO: 5). The synthetic EXP, EXPZm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO: 11) consists of a synthetic promoter, P-Zm.GSP850.nno:4 (SEQ ID NO: 2), operatively linked at 5' to a synthetic leader, L-Zm.GSP850.nno:3 (SEQ ID NO: 3), which is operatively linked at 5' to a synthetic intron, IZm.GSI140.nno:1 (SEQ ID NO: 12).

[00119] Plant cells of maize of the LH244 variety were transformed using the binary transformation vector construct described above by Agrobacterium-mediated transformation, as is well known in the art. The resulting transformed plant cells were induced to form whole maize plants.

[00120] Qualitative and quantitative analysis of GUS was used to evaluate the expression element activity in selected plant organs and tissues in transformed plants. For qualitative analysis of GUS expression by histochemical staining, whole tissues or Petition 870260061195, dated 06 / 22 / 2026, page 58 / 178 52 / 65 sections were incubated with GUS staining solution containing 1 mg / mL of X-Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide) for 5 ha at 37°C and decolorized with 35% EtOH and 50% acetic acid. GUS expression was qualitatively determined by visual inspection of selected plant organs or tissues stained blue under a dissecting or compound microscope.

[00121] For quantitative analysis of GUS expression by enzymatic assays, total protein was extracted from selected tissues of transformed maize plants. One to two micrograms of total protein was incubated with the fluorogenic substrate, 4-methyleumbelliferyl-nnD-glucuronide (MUG) at a concentration of 1 mM in a total reaction volume of 50 microliters. After 1 h of incubation at 37°C, the reaction was stopped by the addition of 350 microliters of 200 mM sodium bicarbonate solution. The reaction product, 4-methylumbelliferone (4MU), is maximally fluorescent at high pH, ​​where the hydroxyl group is ionized. The addition of the basic sodium carbonate solution simultaneously stops the assay and adjusts the pH to quantify the fluorescent product 4-MU. The amount of 4-MU formed was estimated by measuring its fluorescence using a FLUOstar Omega Microplate Reader (BMG LABTECH) (excitation at 355 nm, emission at 460 nm).GUS activity values ​​are given in nmols of 4-MU / hour / mg of total protein.

[00122] The following tissues were sampled for GUS expression in the Ro generation: V4 stage leaf and root; V7 stage leaf and root; VT stage leaf, root, and flower / anther; R1 stage cob / silk; and R3 stage seed embryo and seed endosperm 21 days after pollination (DAP). Table 7 shows the mean values ​​of quantitative GUS expression for each of the synthetic EXPs. Table 7. Average quantitative GUS expression in corn plants. Petition 870260061195, dated 06 / 22 / 2026, page 59 / 178 53 / 65 varieties of stably transformed LH244 driven by synthetic EXPs, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 and EXPZm.GSP850.nno+Zm.GSI140.nno:1. Stage Organ EXP- Zm.GSP850.nno+Zm.GSI153. nno:2 (SEQ ID NO:4) EXP- Zm.GSP850.nno+Zm.GSI 140.nno:1 (SEQ ID NO:11) V4 Leaf 694 642 Root 870 116 V7 Leaf 1423 1265 Root 476 521 VT Leaf 2646 344 Root 424 70 Flower / Anther 5465 1267 R1 Cob / Silk 3618 1116 R3 Embryo Seed 21 DAP 260 224 Endosperm Seed 21 DAP 2648 1140

[00123] As can be seen in Table 7, the synthetic GSP850 promoter and leader (P-Zm.GSP850.nno:4 (SEQ ID NO: 2) and LZm.GSP850.nno:3 (SEQ ID NO: 3)) boosted constitutive GUS expression in stably transformed LH244 maize plants. Molecular analysis of the transcription start site demonstrated consistent TSS for the GSP850 promoter and leader. The synthetic introns, I-Zm.GSI153.nno:1 (SEQ ID NO: 5) and IZm.GSI140.nno:1 (SEQ ID NO: 12), affected expression differently in the different sampled tissues. Molecular analysis of the intron splicing sites demonstrated consistent processing of the synthetic introns. The overall increase in GUS expression was greater in most plant tissue samples comprising IZm.GSI153.nno: 1 (SEQ ID NO: 5), with the exception of the V4 stage leaf, V7 stage root, and R3 seed embryo where GUS expression levels were relatively similar. The increase in expression conferred by I-Zm.GSI153.nno:1 (SEQ ID NO: 5) in relation to IZm.GSI140.nno:1 (SEQ ID NO: 12) was approximately 7.5 times. Petition 870260061195, dated 06 / 22 / 2026, page 60 / 178 54 / 65 times greater in the V4 root, 7.7 times greater in the VT leaf, 6.0 times greater in the VT root, 4.3 times greater in the VT flower / anther, 3.2 times greater in the R1 ear / silk, and 2.3 times greater in the R3 seed endosperm. Example 4 Analysis of GUS expression driven by synthetic EXPs, EXP-Zm.GSP850.nno+Zm.DnaK:1, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 in 01DKD2 maize plants with stable transformation.

[00124] Corn plants were transformed with a vector, specifically a plant expression vector containing a test regulatory element that drives the expression of the βglucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of the selected regulatory element on expression.

[00125] Corn plants were transformed with GUS plant expression constructs. The regulatory elements were cloned into a base plant expression vector using standard methods known in the art. The resulting plant expression vectors contained a left-border region of Agrobacterium tumefaciens (B-AGRtu.leftborder), a first transgene selection cassette used for the selection of transformed plant cells that confer resistance to the herbicide glyphosate; a second transgene cassette to evaluate the activity of regulatory elements, comprising the synthetic EXP, EXP-Zm.GSP850.nno+Zm.DnaK:1 (SEQ ID NO:15), EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO:4) or EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO:11) operatively linked in 5' to a synthetic coding sequence designed for expression in a plant cell encoding βglucuronidase (GUS, GOI-Ec.uidA+St.LS1.nno: 1, SEQ ID NO: 20) containing a processable intron derived from the specific ST-LS1 gene. Petition 870260061195, dated 06 / 22 / 2026, p. 61 / 178 55 / 65 of light-inducible potato tissue (Genbank Accession: X04753), operatively linked at 5' to a 3' termination region, TSb.Nltp4-1:1:2 (SEQ ID NO:19); and a right-edge region of Agrobacterium tumefaciens (B-AGRtu. right edge). The synthetic EXP, EXP-Zm.GSP850.nno+Zm.DnaK:1 (SEQ ID NO:15) is composed of a synthetic promoter, P-Zm.GSP850.nno:4 (SEQ ID NO: 2), operatively linked at 5' to a synthetic leader, L-Zm.GSP850.nno:3 (SEQ ID NO: 3), which is operatively linked at 5' to an intron, IZm.DnaK:1 (SEQ ID NO: 22). Synthetic EXPs, EXPZm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO: 4) and EXPZm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO: 11) are described in Example 3.

[00126] Maize plant cells of the 01DKD2 variety were transformed using the binary transformation vector construct described above by Agrobacterium-mediated transformation, as is well known in the art. The resulting transformed plant cells were induced to form whole maize plants. The qualitative and quantitative expression of GUS was tested as described in Example 3. Table 8 shows the average values ​​of quantitative GUS expression for each of the synthetic EXPs. Table 8. Average quantitative expression of GUS in stably transformed maize plants of variety 01DKD2 conducted by the synthetic EXPs EXP-Zm.GSP850.nno+Zm.DnaK:1, EXPZm.GSP850.nno+Zm.GSI153.nno:2, and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1. Stage Organ EXP- Zm.GSP850.nno+Zm. DnaK:1 (SEQ ID NO: 15) EXPZm.GSP850.nno+Z m.GSI153.nno:2 (SEQ ID NO:4) EXP- Zm.GSP850.nno+Zm.GSI14 0.nno:1 (SEQ ID NO:11) V4 Sheet 208 374 519 Root 36 606 341 Petition 870260061195, dated 06 / 22 / 2026, page 62 / 178 56 / 65 Stage Organ EXP- Zm.GSP850.nno+Zm. DnaK:1 (SEQ ID NO: 15) EXPZm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO:4) EXP- Zm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO:11) V7 Leaf 314 539 619 Root 22 1158 303 VT Leaf 26 610 630 Root 32 770 797 Flower / Anther 104 797 1121 R1 Cob / Silk 160 1139 1312 R3 Embryo Seed 21 DAP 31 188 723 Endosperm Seed 21 DAP 98 1490 1635

[00127] As can be seen in Table 8, the synthetic GSP850 promoter and leader (P-Zm.GSP850.nno:4 (SEQ ID NO: 2) and LZm.GSP850.nno:3 (SEQ ID NO: 3)) boosted constitutive GUS expression in stably transformed LH244 maize plants. The synthetic introns, I-Zm.GSI153.nno:1 (SEQ ID NO: 5) and I-Zm.GSI140.nno:1 (SEQ ID NO: 12) intensified expression relative to the intron, I-Zm.DnaK:1 (SEQ ID NO: 22) in all tissues tested. Example 5 Analysis of GUS expression driven by synthetic EXPs EXP-Zm.GSP990.nno+Zm.DnaK:1 and EXP-Zm.GSP990.nno+Zm.G SI197.nno:2 in stably transformed maize plants of variety 01DKD2

[00128] Corn plants were transformed with a vector, specifically a plant expression vector containing a test regulatory element that drives the expression of the βglucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of the selected regulatory element on expression. Petition 870260061195, dated 06 / 22 / 2026, page 63 / 178 57 / 65

[00129] Corn plants were transformed with GUS plant expression constructs. The regulatory elements were cloned into a base plant expression vector using standard methods known in the art. The resulting plant expression vectors contained a left-border region of Agrobacterium tumefaciens (B-AGRtu.leftborder), a first transgene selection cassette used for the selection of transformed plant cells that confer resistance to the herbicide glyphosate; a second transgene cassette to evaluate the activity of regulatory elements, comprising the synthetic EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO:16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO:9) operatively linked in 5' to a synthetic coding sequence designed for expression in a plant cell encoding βglucuronidase (GUS, GOI-Ec.uidA+St.LS1.nno: 1, SEQ ID NO: 20) containing a processable intron derived from the light-inducible tissue-specific ST-LS1 gene of potato (Genbank Accession: X04753), operatively linked in 5' to a 3' termination region, TSb.Nltp4-1:1:2 (SEQ ID NO:19); and a right-border region of Agrobacterium tumefaciens (B-AGRtu. right-border). The synthetic EXP EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9) consists of a synthetic promoter, P-Zm.GSP990.nno:2 (SEQ ID NO: 7), operatively linked at 5' to a synthetic leader, L-Zm.GSP990.nno:1 (SEQ ID NO: 8), which is operatively linked at 5' to a synthetic intron, I-Zm.GSI197.nno:1 (SEQ ID NO: 10). The synthetic EXP, EXPZm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO:16) is composed of a synthetic promoter, P-Zm.GSP990.nno:2 (SEQ ID NO:7), operatively linked at 5' to a synthetic leader, L-Zm.GSP990.nno:1 (SEQ ID NO:8), which is operatively linked at 5' to an intron, I-Zm.DnaK:1 (SEQ ID NO: 22).

[00130] The plant cells of maize of variety 01DKD2 were Petition 870260061195, dated 06 / 22 / 2026, page 64 / 178 58 / 65 were transformed using the binary transformation vector construct described above by Agrobacterium-mediated transformation, as is well known in the art. The resulting transformed plant cells were induced to form whole maize plants. The qualitative and quantitative expression of GUS was tested as previously described in Example 3. Table 9 shows the average quantitative expression values ​​of GUS for each of the synthetic EXPs, where ND indicates not determined. Table 9. Average quantitative expression of GUS in stably transformed maize plants of variety 01DKD2 conducted by the synthetic EXPs EXP-Zm.GSP990.nno + Zm.DnaK:1 and EXP-Zm.GSP990.nno+Zm.GSI197.nno:2. Stage Organ EXP- Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO:16) EXP- Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO:9) V4 Leaf 99 75 Root 32 33 V7 Leaf 226 138 Root 29 40 VT Leaf 140 61 Root 55 96 Flower / Anther 87 231 R1 Cob / Silk 39 35 R3 Embryo Seed 21 DAP ND 21 Endosperm Seed 21 DAP ND 22

[00131] As can be seen, the synthetic GSP990 promoter and leader (P-Zm.GSP990.nno:2 (SEQ ID NO: 7) and L-Zm.GSP990.nno:1 (SEQ ID NO: 8)) directed GUS expression. Molecular analysis of the transcription start site demonstrated a consistent TSS for the GSP990 promoter and leader. The synthetic intron, I-Zm.GSI197.nno:1 (SEQ ID NO: 10), attenuated expression in some tissues, while intensifying it. Petition 870260061195, dated 06 / 22 / 2026, page 65 / 178 59 / 65 expression in other tissues, relative to the intron, I-Zm.DnaK:1 (SEQ ID NO: 22). For example, GUS expression was attenuated in the leaf at stages V4, V7, and VT. GUS expression was slightly intensified in the root at V7 and VT, relative to I-Zm.DnaK:1. Flower / anther expression was intensified approximately 2.7 times by IZm.GSI197.nno:1 (SEQ ID NO: 10), relative to I-Zm.DnaK:1. The differences in expression conferred by I-Zm.GSI197.nno:1 (SEQ ID NO: 10), relative to I-Zm.DnaK:1, can be very useful where lower leaf and upper flower / anther expression is desired. Molecular analysis of the intron splicing sites demonstrated consistent processing of the synthetic intron, I-Zm.GSI197.nno:1 (SEQ ID NO: 10). Example 6 Analysis of the effect on GUS expression by the synthetic 3' UTRs T-Zm.GST9.nno:2, T-Zm.GST18.nno:2, and T-Zm.GST43.nno:1, and the native TSb.Ntlp4-1:1:2 in a variety of stably transformed maize plants 01DKD2

[00132] Corn plants were transformed with a vector, specifically a plant expression vector containing a test regulatory element that drives the expression of the βglucuronidase (GUS) transgene. The resulting plants were analyzed for GUS protein expression to assess the effect of the selected regulatory element on expression.

[00133] Corn plants were transformed with GUS plant expression constructs. The regulatory elements were cloned into a base plant expression vector using known standard methods in the art. The resulting plant expression vectors contained a left-border region of Agrobacterium tumefaciens (B-AGRtu. left-border), a first transgene selection cassette used for selection of transformed plant cells conferring resistance to the herbicide glyphosate; a second cassette of Petition 870260061195, dated 06 / 22 / 2026, page 66 / 178 60 / 65 transgene to evaluate the activity of UTR 3' regulatory elements, which comprised EXP EXP-CaMV.35S (SEQ ID NO: 21), operatively linked 5' to the intron, I-Zm.DnaK:1 (SEQ ID NO: 22), operatively linked 5' to a synthetic coding sequence designed for expression in a plant cell encoding β glucuronidase (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20) containing a processable intron derived from the light-inducible tissue-specific ST-LS1 gene of potato (Genbank Accession: X04753), operatively linked 5' to a 3' termination region; and a right-border region of Agrobacterium tumefaciens (B-AGRtu. right-border). Three test expression vectors comprised the UTRs 3' TZm.GST9.nno:2 (SEQ ID NO: 13), T-Zm.GST18.nno:2 (SEQ ID NO: 14) or T-Zm.GST43.nno:1 (SEQ ID NO: 26) operatively linked to the GUS encoding sequence. An additional test expression vector comprised the UTR 3' T-Sb.Nltp4-1:1:2 (SEQ ID NO:19) is operatively linked to the GUS encoding sequence and was used to compare the expression between native and synthetic 3' UTRs.

[00134] Maize plant cells of the 01DKD2 variety were transformed using the binary transformation vector construct described above by Agrobacterium-mediated transformation, as is well known in the art. The resulting transformed plant cells were induced to form whole maize plants.

[00135] Qualitative and quantitative analysis of GUS was used to evaluate the expression element activity in the V4 leaf and root tissues of transformed plants and was performed as described above in Example 3. The effect of synthetic 3' UTRs was evaluated by comparison with the effect of expression of the 3' UTR TSb.Nltp4-1:1:2 (SEQ ID NO:19). The resulting transcripts were analyzed to determine if proper termination occurred and if there was no transcript readout. One method to assess whether readout occurred was Petition 870260061195, dated 06 / 22 / 2026, page 67 / 178 61 / 65 through the use of cDNA amplification transcribed using an amplification primer corresponding to a portion of the T-DNA boundary sequence that is 3' to the 3' UTR. The average GUS expression of plants transformed with the four constructs comprising T-Zm.GST9.nno:2 (SEQ ID NO: 13), T-Zm.GST18.nno:2 (SEQ ID NO: 14), T-Zm.GST43.nno:1 (SEQ ID NO: 26) and T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19) is provided in Table 10. Table 10. Average quantitative expression of GUS in maize plants variety 01DKD2 stably transformed from constructs comprising three different UTRs 3'. Organ Stage T-Sb.Nltp41:1:2 (SEQ ID NO:19) T-Zm.GST9.nno:2 (SEQ ID NO:13) T-Zm.GST18.nno:2 (SEQ ID NO:14) T-Zm.GST43.nno:1 (SEQ ID NO:26) V4 Leaf 350 684 423 113 Root 778 924 810 883

[00136] As can be seen in Table 10, both T-Zm.GST9.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14) intensified the expression of GUS driven by EXP-CaMV.35S operatively linked to I-Zm.DnaK:1, relative to UTR 3', T-Sb.Nltp4-1:1:2. GUS expression in plants comprising T-Zm.GST9.nno:2 (SEQ ID NO: 13) was higher than in plants comprising TZm.GST18.nno:2. T-Zm.GST43.nno:1 (SEQ ID NO: 26) increased GUS expression in the V4 root relative to T-Sb.Nltp4-1:1:2, but attenuated expression in the V4 leaf. Analysis of the GUS transcripts of all four constructs demonstrated proper transcription termination and no evidence of reading in the resulting GUS transcripts. The 3' UTRs T-Zm.GST9.nno:2 (SEQ ID NO: 13), T-Zm.GST18.nno:2 (SEQ ID NO: 14), and T-Zm.GST43.nno:1 (SEQ ID NO: 26) operate similarly to native 3' UTRs and demonstrated modulation of GUS expression, relative to the native 3' UTR T-Sb.Nltp4-1:1:2.All four synthetic 3' UTRs and the additional native T-Sb.Nltp4-1:1:2 3' UTRs provide a range of expression values ​​that are... Petition 870260061195, dated 06 / 22 / 2026, page 68 / 178 62 / 65 useful in fine-tuning expression in stably transformed corn plants. Example 7 Enhancer elements derived from regulatory elements.

[00137] Enhancers are derived from promoter elements presented as SEQ ID NOs: 2 and 7. Enhancer elements may consist of one or more cis regulatory elements that, when operatively linked 5' or 3' to a promoter element, or operatively linked 5' or 3' to additional enhancer elements that are operatively linked to a promoter, may enhance or modulate the expression levels of a transcribable DNA molecule, or provide the expression of a transcribable DNA molecule in a specific cell or plant organ type or at a specific developmental time point or circadian rhythm. Enhancers are made by removing the TATA box or functionally similar elements and any downstream sequences from the promoters that allow transcription to be initiated from the promoter presented as SEQ ID NOs: 2 and 7 or fragments thereof.

[00138] The TATA box in plant promoters is not as highly conserved as in some other eukaryotic organisms. Therefore, in order to define a fragment as an enhancer, one must first identify the transcription start site (TSS) of the gene, where the 5' UTR is first transcribed. An enhancer derived from the synthetic promoter, P-Zm.GSP850.nno:4 (SEQ ID NO: 2) could comprise nucleotides 1 to 418 of SEQ ID NO: 2, resulting in the synthetic enhancer, E-Zm.GSP850 (SEQ ID NO: 17). An enhancer derived from the synthetic promoter, P-Zm.GSP990.nno:2 (SEQ ID NO: 7) could comprise nucleotides 1 to 416 of SEQ ID NO: 7, Petition 870260061195, dated 06 / 22 / 2026, page 69 / 178 63 / 65 resulting in the synthetic enhancer, E-Zm.GSP990 (SEQ ID NO: 18). Enhancers derived from the promoters may comprise fragments of SEQ ID NOs: 17 and 18, or duplications of SEQ ID NOs: 17 and 18 or their respective fragments. The efficacy of synthetic enhancers derived from synthetic promoters is empirically determined by constructing a chimeric transcriptional regulatory element comprising fragments derived from the synthetic promoters P-Zm.GSP850.nno:4 (SEQ ID NO: 2) or P-Zm.GSP990.nno:2 (SEQ ID NO: 7), which is operatively linked to a promoter and leader and used to drive the expression of a transcribable DNA molecule, such as GUS, in a stable or transient plant assay.

[00139] Further refinement of the enhancer element may be required and is empirically validated. Furthermore, the position of the enhancer element relative to other elements within a chimeric transcription regulatory element is also empirically determined, since the order of each element within the chimeric transcription regulatory element can impart different effects depending on the relative positions of each element. Some promoter elements will have multiple TATA box or TATA box-like elements and possibly multiple transcription start sites. In these circumstances, it may be necessary to first identify where the first TSS is located and then begin designing enhancers using the first TSS to prevent potential transcription start from occurring within a putative enhancer element.

[00140] Enhancer elements, derived from the promoter elements presented as SEQ ID NOs: 2 and 7, are cloned using methods known in the art as being operatively linked at 5' or within a promoter element, or operatively linked at 5' or 3' to additional enhancer elements that are operatively Petition 870260061195, dated 06 / 22 / 2026, page 70 / 178 64 / 65 linked to a promoter. Alternatively, enhancer elements can be cloned, using methods known in the art, to provide a larger enhancer element consisting of two or more copies of the enhancer and cloned using methods known in the art as being operatively linked in 5' or 3' to a promoter element, or operatively linked in 5' or 3' to additional enhancer elements that are operatively linked to a promoter that produces a chimeric transcriptional regulatory element. Enhancer elements derived from promoters derived from genes of multiple organisms of the genus can be operatively linked to enhancers derived from synthetic promoters.

[00141] A plant expression transformation vector for GUS can be constructed using methods known in the art similar to that described in Example 3, wherein the resulting plant expression vectors contain a left-border region of Agrobacterium tumefaciens (left-border B-AGRtu), a first transgene selection cassette used for selection of transformed plant cells conferring resistance to the herbicide glyphosate; and a second transgene cassette to test the enhancer element composed of the enhancer element operatively linked in 5' or 3' to a promoter element or operatively linked in 5' or 3' to additional enhancer elements which in turn are operatively linked to a promoter which is operatively linked in 5' to a leader element, operatively linked in 5' to an intron element, operatively linked to a coding sequence for β-glucuronidase (GOI-Ec.uidA+St.LS1).nno:1, SEQ ID NO: 20) containing a processable intron derived from the light-inducible tissue-specific ST-LS1 gene of potato (Genbank accession: X04753), operatively linked to a 3' termination region of the Oryza sativa Lipid-like Protein Transfer gene (T-Os.LTP:1, SEQ. Petition 870260061195, dated 06 / 22 / 2026, page 71 / 178 65 / 65 ID NO:23); and a region of the right border of A. tumefaciens (right border B-AGRtu). The resulting plasmids are used to transform maize plants or other monocotyledonous plants using the methods described above. Alternatively, protoplast cells derived from maize or other monocotyledonous plants are transformed using methods known in the art to perform transient assays.

[00142] The expression of GUS driven by a regulatory element comprising one or more enhancers is evaluated in stable or transient plant assays to determine the effects of the enhancer element on the expression of a transcribable DNA molecule. Modifications to one or more enhancer elements or duplication of one or more enhancer elements can be performed based on empirical experimentation and the resulting regulation of gene expression observed using each regulatory element composition. Altering the relative positions of one or more enhancers in the resulting regulatory or chimeric elements can affect the transcriptional activity or specificity of the regulatory or chimeric regulatory element and is empirically determined to identify the best enhancers for the desired transgene expression profile in maize or other plant genus.

[00143] Having illustrated and described the principles of the present invention, it should be evident to those skilled in the art that the invention can be modified in arrangement and in detail without departing from such principles. All modifications that are within the spirit and scope of the claims are claimed. All publications and patent documents cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

1. Recombinant DNA molecule, characterized in that it comprises a DNA sequence selected from the group consisting of: (a) a sequence having at least 85 percent sequence identity with any of the SEQ ID NOs: 9, 7 and 10; (b) a sequence comprising any of the SEQ ID NOs: 9, 7 and 10; and (c) a fragment of any of the SEQ ID NOs: 9, 7 and 10, wherein the fragment has gene regulatory activity; wherein said DNA sequence is operatively linked to a heterologous transcriptable DNA molecule.

2. Recombinant DNA molecule, according to claim 1, characterized in that said sequence comprises at least 90 percent sequence identity with the DNA sequence of any of the SEQ ID Nos: 9, 7 and 10.

3. Recombinant DNA molecule, according to claim 1, characterized in that said sequence comprises at least 95 percent sequence identity with the DNA sequence of any of the SEQ ID Nos: 9, 7 and 10.

4. Recombinant DNA molecule, according to claim 1, characterized in that the DNA sequence comprises gene regulatory activity.

5. Recombinant DNA molecule, according to claim 1, characterized in that the heterologous transcribable DNA molecule comprises a gene of agronomic interest.

6. Recombinant DNA molecule, according to claim 5, characterized in that the gene of agronomic interest confers herbicide tolerance in plants.

7. Recombinant DNA molecule, according to claim 5, characterized in that the gene of agronomic interest confers resistance to pests in plants.

8. Recombinant DNA molecule, according to claim 1, characterized in that the heterologous transcriptable DNA molecule encodes a dsRNA, a miRNA or an siRNA.

9. Transgenic plant cell, characterized in that it comprises a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least 85 percent sequence identity with any of the SEQ ID NOs: 9, 7 and 10; (b) a sequence comprising any of the SEQ ID NOs: 9, 7 and 10; and (c) a fragment of any of the SEQ ID NOs: 9, 7 and 10, wherein the fragment has gene regulatory activity; wherein said DNA sequence is operatively linked to a heterologous transcriptable DNA molecule.

10. Transgenic plant cell, according to claim 9, characterized in that said transgenic plant cell is a monocotyledonous plant cell.

11. Transgenic plant cell, according to claim 9, characterized in that said transgenic plant cell is a dicotyledonous plant cell.

12. Transgenic plant, or part thereof, characterized in that it comprises the recombinant DNA molecule, as defined in claim 1.

13. A plant progeny of the transgenic plant, as defined in claim 12, or a part thereof, characterized in that the plant progeny or part thereof comprises said recombinant DNA molecule.

14. Transgenic seed, characterized in that the seed comprises the recombinant DNA molecule, as defined in claim 1.

15. Method for producing a basic product, characterized in that it comprises obtaining a transgenic plant or part thereof, as defined in claim 12, and producing the basic product from the same.

16. Method according to claim 15, characterized in that the base product is seeds, processed seeds, protein concentrate, protein isolate, starch, grains, plant parts, seed oil, biomass, flour or bran.

17. Method for expressing a transcription-capable DNA molecule, characterized in that it comprises obtaining a transgenic plant, as defined in claim 12, and cultivating the plant in which the transcription-capable DNA is expressed.

18. Use of a plant, seed, cell progeny or part of a plant comprising recombinant DNA molecule, as defined in any one of claims 1 to 8, characterized in that it is for crossing with a second plant, regeneration of a transgenic plant, planting or growing a field of transgenic plants, producing plant products or producing a base product.

19. Composition, characterized in that it includes transgenic plant cells, plants, plant parts and / or seeds containing the recombinant DNA molecule, as defined in one of claims 1 to 8.

20. Invention, characterized by any of its modalities or categories of claims encompassed by the object initially disclosed in the patent application or in its examples presented herein.