WO2022046957A2 - Cannabis dna constructs and methods of regulating gene expression in plants - Google Patents

Abstract

The present application relates to Cannabis DNA constructs and methods of regulating gene expression in plants. In particular, regulatory sequence elements from Cannabis are disclosed, including Cannabis Ubiquitin genes and functional fragments thereof, and their use in the expression of heterologous nucleotides in plant cells.

Description

CANNABIS DNA CONSTRUCTS AND METHODS OF REGULATING GENE EXPRESSION IN PLANTS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63/070,642, filed August 26, 2020, which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present application relates to Cannabis DNA constructs and methods of regulating gene expression in plants.

BACKGROUND

[0003] Cannabis refers to a genus of flowering plants in the family Cannabaceae. and includes at least three recognized species: Cannabis saliva. Cannabis indica. and Cannabis ruderalis. Various types of cannabis plants can exist within the same species, including narrow leaf and broad leaf types, as well as medicinal and non-medicinal types. Cannabis is a versatile plant producing products ranging from fiber extracted from stems for paper and textiles, seeds used for food and oil, and flowers producing secondary metabolites called cannabinoids. Cannabinoids include a range of compounds, including delta-9 tetrahydrocannabinol (“THC”) used for its psychoactive properties and cannabidiol (“CBD”), which is used for therapeutic purposes such as the treatment of certain types of epilepsy, nausea, and for pain and inflammation.

[0004] Cannabis varieties can be classified into five classes of cannabinoid content referred to as chemotypes or chemovars. Chemotype 1 (marijuana) has high THC and low CBD content. Chemotype 2 has approximately equal amounts of THC and CBD. Chemotype 3 (hemp) has high CBD and low THC. Chemotype 4 has high cannabigerol (“CBG”), a precursor of THC and CBD. Chemotype 5 does not produce cannabinoids. Hemp is defined by the Agricultural Improvement Act of 2018 as any Cannabis plant, or derivative thereof, that contains not more than 0.3% THC on a dry-weight basis.

[0005] In Cannabis and other plants, there is a need for novel DNA constructs with regulatory elements that allow the application of technologies to control gene expression for the development of new traits. DNA constructs can provide transient expression of a gene of interest, or they can be used to introduce transgenes that are stably integrated into the genome of a plant species using transformation technologies. Through transient or stable transformation of DNA constructs, plant species can be developed or modified to have novel traits that are of agronomic interest. [0006] Control and regulation of gene expression can occur through numerous mechanisms. Although some sequence elements such as promoters, 3′ terminators, introns, and enhancers from plants and plant viruses that affect gene expression have been discovered and characterized, there is still a need for novel sequences. Heterologous expression of DNA is often most efficient when promoter sequences from the host organism are used to control expression. In addition, use of different promoter, 3′ terminator and other sequence elements in a vector help to stabilize the DNA construct, prevent homologous recombination and secondary structure formation, and reduce potential competition for regulatory factors between similar or identical sequences used more than once in a DNA construct. Repeated use of promoter and other sequence elements can also lead to transcriptional or post-transcriptional gene silencing. Therefore, a need exists for new regulatory elements that can control the desired levels of expression of heterologous sequences in plants and plant cells. [0007] The present application is directed to overcoming these and other deficiencies in the art. SUMMARY [0008] One aspect of the present application relates to a DNA construct comprising a first nucleotide sequence comprising a Cannabis Ubiquitin promoter with at least 70% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof. The DNA construct also has a second nucleotide sequence heterologous to the first nucleotide sequence, where the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence is capable of controlling expression of the second nucleotide sequence. [0009] Another aspect of the present application relates to a method of expressing a coding sequence in a transformed plant cell. This method involves transforming a plant cell with a DNA construct comprising a Cannabis Ubiquitin promoter with at least 70% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof. The DNA construct also has a second nucleotide sequence heterologous to the first nucleotide sequence, where the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence is capable of controlling expression of the second nucleotide sequence. Transforming the plant cell with the DNA construct expresses the coding sequence in the transformed plant cell. [0010] A further aspect of the present application relates to a DNA construct comprising a first nucleotide sequence comprising a Cannabis Ubiquitin 3′ terminator sequence with at least 70% sequence identity to any one of SEQ ID NOs:34-53 or a functional fragment thereof. The DNA construct also has a second nucleotide sequence heterologous to the first nucleotide sequence, where the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence contributes to transcriptional termination of the second nucleotide sequence. [0011] Another aspect of the present application relates to a method of expressing a coding sequence in a transformed plant cell. This method involves transforming a plant cell with a DNA construct comprising a first nucleotide sequence comprising a Cannabis Ubiquitin 3′ terminator sequence with at least 70% sequence identity to any one of SEQ ID NOs:34-53 or a functional fragment thereof. The DNA construct also has a second nucleotide sequence heterologous to the first nucleotide sequence, where the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence contributes to transcriptional termination of the second nucleotide sequence. Transforming the plant cell with the DNA construct expresses the coding sequence in the transformed plant cell. [0012] Yet another aspect of the present application relates to a DNA construct comprising a first nucleotide sequence comprising a Cannabis Ubiquitin intron sequence with at least 70% sequence identity to any one of SEQ ID NOs:6, 17, 28, or a functional fragment thereof. The DNA construct also has a second nucleotide sequence heterologous to the first nucleotide sequence, where the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence contributes to transcriptional regulation of the second nucleotide sequence. [0013] Other aspects of the present application relate to vectors; cells; and transgenic plants, seeds, pollen, floral buds, or clones comprising the DNA constructs described herein. BRIEF DESCRIPTION OF THE DRAWINGS [0014] FIGs.1A-C show a nucleotide sequence alignment of promoter regions of UbiChr1.1P4 from multiple varieties of Cannabis. This promoter sequence comprises an upstream element, a 5′ UTR and an intron. Vertical lines indicate where UbiChr1.1P4 (“P4”) (SEQ ID NO:1), UbiChr1.1P3 (“P3”) (SEQ ID NO:2), UbiChr1.1P2 (“P2”) (SEQ ID NO:3) and UbiChr1.1P1 (“P1”) (SEQ ID NO:4) begin. Brackets show the position of the putative 5′ UTR (SEQ ID NO:5) and intron (SEQ ID NO:6). The nucleotide sequence alignment ends at the translational start codon (ATG) of the endogenous polyubiquitin coding sequence. Boxed elements indicate putative TATA boxes and CAAT sequences. Shown are sequences from Cannabis variety CBDRx (GenBank GCA_900626175.1; “U1P4_Cs10”; SEQ ID NO:54), variety BaOx (“U1P4_BaOX”; SEQ ID NO:1), variety Z2 from a female plant (“U1P4_Z2-F”; SEQ ID NO:55), and variety Z2 from a male plant (“U1P4_Z2-M”; SEQ ID NO:56). [0015] FIGs.2A-C show a nucleotide sequence alignment of promoter regions of UbiChr3P4 from multiple varieties of Cannabis. This promoter comprises an upstream element and a 5’ UTR. Vertical lines indicate where UbiChr3P4 (“P4”) (SEQ ID NO:7), UbiChr3P3 (“P3”) (SEQ ID NO:8), UbiChr3P2 (“P2”) (SEQ ID NO:9), and UbiChr3P1 (“P1”) (SEQ ID NO:10) begin. Brackets show the position of the putative 5’ UTR (SEQ ID NO:11).The nucleotide sequence alignment ends at the translational start codon (ATG) of the endogenous polyubiquitin coding sequence. A putative TATA box is shown in a box. Shown are sequences from Cannabis variety CBDRx (GenBank GCA_900626175.1; “U3P4_Cs10”; SEQ ID NO:57), variety BaOx (“U3P4_BaOX”; SEQ ID NO:7), variety Z2 from a female plant (“U3P4_Z2-F”; SEQ ID NO:58), and variety Z2 from a male plant (“U3P4_Z2-M”; SEQ ID NO:59). [0016] FIGs.3A-C show a nucleotide sequence alignment of promoter regions of UbiChrXP4 from multiple varieties of Cannabis. This promoter comprises an upstream element, a 5’ UTR, and an intron. Vertical lines indicate where UbiChrXP4 (“P4”) (SEQ ID NO:12), UbiChrXP3 (“P3”) (SEQ ID NO:13), UbiChrXP2 (“P2”) (SEQ ID NO:14), and UbiChrXP1 (“P1”) (SEQ ID NO:15) begin. Brackets show the position of the putative 5’ UTR (SEQ ID NO:16) and intron (SEQ ID NO:17). The sequence alignment ends at the translational start codon (ATG) of the endogenous polyubiquitin coding sequence. Boxed elements indicate a putative TATA box and a CAAT sequence. Shown are sequences from Cannabis variety CBDRx (GenBank GCA_900626175.1; “UXP4_Cs10”; SEQ ID NO:60), variety BaOx (“UXP4_BaOX”; SEQ ID NO:12), variety Z2 from a female plant (“UXP4_Z2-F”; SEQ ID NO:61), and variety Z2 from a male plant (“UXP4_Z2-M”; SEQ ID NO:62). [0017] FIGs.4A-C show a nucleotide sequence alignment of promoter regions of UbilikeChr9P4 from multiple varieties of Cannabis. This promoter comprises an upstream element and a 5’ UTR. Vertical lines indicate where UbilikeChr9P4 (“P4”) (SEQ ID NO:18), UbilikeChr9P3 (“P3”) (SEQ ID NO:19), UbilikeChr9P2 (“P2”) (SEQ ID NO:20), and UbilikeChr9P1 (“P1”) (SEQ ID NO:21) begin. Brackets show the position of a putative 5’ UTR (SEQ ID NO:22). The sequence alignment ends at the translational start codon (ATG) of the endogenous ubiquitin-like coding sequence. A box denotes the position of an upstream gene’s stop codon. Shown are sequences from Cannabis variety CBDRx (GenBank GCA_900626175.1; “UL9P4_Cs10”; SEQ ID NO:63), variety BaOx (“UL9P4_BaOX”; SEQ ID NO:18), variety Z2 from a female plant (“UL9P4_Z2-F”; SEQ ID NO:64), and variety Z2 from a male plant (“UL9P4_Z2-M”; SEQ ID NO:65). [0018] FIGs.5A-C show a nucleotide sequence alignment of promoter regions of UbiChr1.2P4 from multiple varieties of Cannabis. This promoter comprises an upstream element, a 5’ UTR, and an intron. Vertical lines indicate where UbiChr1.2P4 (“P4”) (SEQ ID NO:23), UbiChr1.2P3 (“P3”) (SEQ ID NO:24), UbiChr1.2P2 (“P2”) (SEQ ID NO:25), and UbiChr1.2P1 (“P1”) (SEQ ID NO:26) begin. Brackets show the position of the putative 5’ UTR (SEQ ID NO:27) and intron (SEQ ID NO:28). The sequence alignment ends at the translational start codon (ATG) of the endogenous ubiquitin coding sequence. A box denotes the position of an upstream gene’s start codon. Shown are sequences from Cannabis variety CBDRx (GenBank GCA_900626175.1; “U2P4_Cs10”; SEQ ID NO:66), variety BaOx (“U2P4_BaOX”; SEQ ID NO:23), variety Z2 from a female plant (“U2P4_Z2-F”; SEQ ID NO:67), and variety Z2 from a male plant (“U2P4_Z2-M”; SEQ ID NO:68). [0019] FIG.6 shows a nucleotide sequence alignment of transcriptional termination regions of UbiChr3T4, including a 3’ terminator sequences from multiple varieties of Cannabis. The sequence alignment begins at the translational stop codon (TGA) of the endogenous polyubiquitin coding sequence. A box denotes the position of a putative polyadenylation signal. Shown are sequences from Cannabis variety CBDRx (GenBank GCA_900626175.1; “U3T4_Cs10”; SEQ ID NO:72), variety BaOx (“U3T4_BaOX”; SEQ ID NO:38), variety Z2 from a female plant (“U3T4_Z2F”; SEQ ID NO:73), and variety Z2 from a male plant (“U3T4_Z2M”; SEQ ID NO:74). [0020] FIG.7 shows a nucleotide sequence alignment of transcriptional termination regions of UbiChr1.1T4, including a 3’ terminator sequences from multiple varieties of Cannabis. The sequence alignment begins at the translational stop codon (TGA) of the endogenous polyubiquitin coding sequence. A box denotes the position of a putative polyadenylation signal. Shown are sequences from Cannabis variety CBDRx (GenBank GCA_900626175.1; “U1T4_Cs10”; SEQ ID NO:69), variety BaOx (“U1T4_BaOX”; SEQ ID NO:34), variety Z2 from a female plant (“U1T4_Z2F”; SEQ ID NO:70), and variety Z2 from a male plant (“U1T4_Z2M”; SEQ ID NO:71). [0021] FIG.8 shows a nucleotide sequence alignment of transcriptional termination regions of UbilikeChr9T4, including a 3’ terminator sequence from multiple varieties of Cannabis. The sequence alignment begins at the translational stop codon (TAA) of the endogenous polyubiquitin coding sequence. Shown are sequences from Cannabis variety CBDRx (GenBank GCA_900626175.1; “Ul9T4_Cs10”; SEQ ID NO:78), variety BaOx (“Ul9T4_BaOX”; SEQ ID NO:46), variety Z2 from a female plant (“Ul9T4_Z2F”; SEQ ID NO:79), and variety Z2 from a male plant (“Ul9T4_Z2M”; SEQ ID NO:80). [0022] FIG.9 shows a nucleotide sequence alignment of transcriptional termination regions of UbiChrXT4, including a 3’ terminator sequence from multiple varieties of Cannabis. The sequence alignment begins at the boxed in translational stop codon (TAA) of the endogenous ubiquitin-like coding sequence. Shown are sequences from Cannabis variety CBDRx (GenBank GCA_900626175.1; “UXT4_Cs10”; SEQ ID NO:75), variety BaOx (“UXT4_BaOX”; SEQ ID NO:42), variety Z2 from a female plant (“UXT4_Z2F”; SEQ ID NO:76), and variety Z2 from a male plant (“UXT4_Z2M”; SEQ ID NO:77). [0023] FIG.10 shows a nucleotide sequence alignment of transcriptional termination regions of UbiChr1.2T4, including 3’ terminator sequence from multiple varieties of Cannabis. The sequence alignment begins at the boxed in translational stop codon (TGA) of the endogenous ubiquitin coding sequence. Shown are sequences from Cannabis variety CBDRx (GenBank GCA_900626175.1; “U2T4_Cs10”; SEQ ID NO:81), variety BaOx (“U2T4_BaOX”; SEQ ID NO:50), variety Z2 from a female plant (“U2T4_Z2F”; SEQ ID NO:82), and variety Z2 from a male plant (“U2T4_Z2M”; SEQ ID NO:83). [0024] FIG.11 is a schematic illustration of vector pARC1295, which has EcoRI, SacI, and KpnI restriction sites for cloning Ubiquitin promoter sequences 5’ to the AtCAS9 gene sequence and NOS 3’ terminator for expression analysis. The vector also contains a double 35S (“d35S”) promoter driving a plant selectable marker, NPTII, with a CaMV 3′ UTR. [0025] FIG.12 is a schematic illustration of vector pARC1297, which has AscI and PacI restriction sites for cloning Ubiquitin 3’ terminator sequences 3’ to the AtCAS9 sequences for expression analysis. The vector also contains a d35S promoter driving a plant selectable marker, NPTII, with a CaMV 3′ UTR. [0026] FIG.13 shows exemplary nucleotide sequences comprising the Parsley Ubiquitin promoter and 5’UTR (SEQ ID NO:88) with the CsUbiChr1.1 intron (SEQ ID NO:6), and the beginning of the AtCAS9 sequence (SEQ ID NO:85) for testing the effect of the Cannabis Ubiquitin introns on transcription. [0027] FIGs.14A-B show bar graphs of relative expression of Cas9 as calculated using the transgene, EcNPTII, as standard. These bar graphs compare the difference in expression of Cas9 between Cannabis Ubi promoters, the parsley Ubi (PcUbi), and PcUbi with the intron replaced by Cannabis Ubi introns for CsUbi-1.1, CsUbi-X, and CsUbi-1.2. FIG 14A shows the initial screen of promoters. Vectors are ordered by fragment size (0.7 kb, 1.2 kb, 1.5 kb, and 2.0 kb) and grouped by gene. The horizontal box shows the level of expression of Cas9 by PcUbi. In FIG.14B, the number after the dash represents the qPCR plate the vector was analyzed on and the number in parenthesis indicates the promoter fragment size. The vertical rectangles in FIG. 14A show the promoters and promoter lengths that were chosen for additional analysis. FIG. 4B shows a replicate analysis of expression of selected CsUbi promoter constructs using qPCR ssays repeated with technical duplicates. DETAILED DESCRIPTION 0028] The present application relates to DNA constructs comprising Cannabis Ubiquitin gulatory elements and methods of their use in plants. 0029] One aspect of the present application relates to a DNA construct comprising a rst nucleotide sequence comprising a Cannabis Ubiquitin promoter with at least 70% sequence entity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment hereof. The DNA construct also has a second nucleotide sequence heterologous to the first ucleotide sequence, where the second nucleotide sequence is operably linked to the first ucleotide sequence, such that the first nucleotide sequence is capable of controlling expression f the second nucleotide sequence. 0030] The nucleotide sequences of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, and 23-26 are et forth in Table 1 (infra). 0031] Plant Ubiquitin is a small, highly conserved protein of 76 amino acids that has a ole in a wide range of cellular functions from protein turnover to chromosome structure. biquitin is covalently linked to proteins to target them for degradation by the proteasome. argeted degradation of proteins in plants controls growth and plant hormone signaling, among her functions. 0032] In accordance with this and all aspects of the present application, the terms ucleotide, nucleic acid, and polynucleotide are used interchangeably, unless indicated herwise by context. In some embodiments, a nucleotide of the present application is isolated. he term isolated refers to a synthesized, cloned, and/or partial sequence from the naturally- ccurring sequence. 0033] As used herein, a regulatory element is a nucleotide sequence that contributes to he regulation of expression of a gene with which it is associated. Regulatory elements include, without limitation, promoters, enhancers, silencers, RNA polymerase binding sites, 5’ untranslated regions, introns, 3’ terminators, polyadenylation sites, and 3’ untranslated regions, among others. [0034] A promoter refers to a nucleic acid molecule capable of controlling transcription of another nucleic acid molecule. A promoter is a non-coding genomic DNA sequence, usually upstream (5′) to the relevant coding sequence, and its primary function is to act as a binding site for RNA polymerase to initiate transcription by the RNA polymerase. Thus, a promoter is capable of controlling expression, capable of initiating transcription, or driving expression of a DNA sequence when it is able to carry out this primary function of a promoter. Promoter function includes expression of RNA, including functional RNA, or the expression of a polypeptide for operably linked encoding nucleotide sequences, as the transcribed RNA ultimately may be translated into the corresponding polypeptide. Promoters vary in their strength (i.e., their ability to promote transcription). The nucleotide sequence of the promoter determines the nature of the RNA polymerase binding and other related protein factors that attach to the RNA polymerase and/or promoter, and the rate of RNA synthesis. [0035] Promoter sequences in general include proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an enhancer is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. Different promoters may direct the expression of a gene or functional RNA in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Since, in many cases, the exact boundaries of regulatory sequences have not been completely defined, a functional fragment of some variation of the promoter may also have promoter activity. [0036] As used herein, the terms Cannabis Ubiquitin promoter, Ubiquitin promoter, or Ubi promoter are used interchangeably, and refer to the promoter of a Cannabis Ubiquitin gene, or a functional fragment thereof. The term Cannabis Ubi promoter includes both a native Cannabis Ubiquitin promoter (or a functional fragment thereof) and an engineered sequence comprising at least a fragment of the native Cannabis Ubiquitin promoter with, for example, a DNA linker attached to facilitate cloning. A DNA linker may comprise a restriction enzyme site. [0037] In some embodiments of the methods and compositions described herein, Cannabis Ubiquitin promoter sequences include, without limitation, sequences disclosed in SEQ ID NOs:1-4, 7-10, 12-15, 18-21, and 23-26. In some embodiments, the promoter may include the nucleotide sequence of any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, or 23-26. In some embodiments, the promoter is the nucleotide sequence of any one of SEQ ID NOs:1-4, 7-10, 12- 15, 18-21, or 23-26. In some embodiments, the promoter has a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, or 23-26, or a functional fragment thereof. For example, in some embodiments, the Cannabis Ubiquitin promoter as the first nucleotide sequence comprises at least 80% sequence identity to any one of SEQ ID NOs:1-4, 7- 10, 12-15, 18-21, or 23-26, or a functional fragment thereof. In some embodiments of the DNA constructs taught herein, the Cannabis Ubiquitin promoter as the first nucleotide sequence comprises at least 90% sequence identity to any one of in SEQ ID NOs:1-4, 7-10, 12-15, 18-21, or 23-26, or a functional fragment thereof. In some embodiments of the DNA constructs taught herein, the Cannabis Ubiquitin promoter as the first nucleotide sequence comprises at least 95% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, or 23-26, or a functional fragment thereof. And in some embodiments of the DNA constructs taught herein, the Cannabis Ubiquitin promoter as the first nucleotide sequence comprises any one of SEQ ID NOs:1-4, 7- 10, 12-15, 18-21, or 23-26, or a functional fragment thereof. In some embodiments of the DNA constructs taught herein, the Cannabis Ubiquitin promoter sequence comprises any of SEQ ID NOs:54-68, or a functional fragment thereof. The nucleotide sequences of SEQ ID NOs:54-68 are set forth in Table 6 (infra). [0038] In other embodiments, the promoters disclosed herein can be modified relative to their sequences disclosed herein. Those skilled in the art can create promoters that have variations in the nucleotide sequence. The nucleotide sequence of the promoters of the present application as shown in SEQ ID NOs:1-4, 7-10, 12-15, 18-21, and 23-26 may be modified or altered to enhance their control characteristics. Modification or alteration of the promoter sequence can also be made without substantially affecting the promoter function. This can be accomplished using methods that are well known in the art. For example, nucleotide sequences can be modified by insertion, deletion, or replacement of template sequences in a PCR-based DNA modification approach or by chemically synthesizing the variant promoter or a portion thereof, or by altering the sequence by site-directed mutagenesis. [0039] As used herein, the term “functional fragment” refers to a portion or subsequence of the Cannabis Ubiquitin regulatory sequence elements described herein, which still has the ability to function substantially similarly to the full length regulatory sequence. For example, a functional fragment of the Cannabis Ubiquitin promoter is a portion or subsequence of the full- length promoter that still has the ability to initiate, control, or regulate transcription of a heterologous nucleotide sequence. In one embodiment, the functional fragment of a promoter includes a TATA box. TATA boxes are present in some, but not all promoters and, when present, are located approximately 25-35 bp upstream of a transcriptional start site. In another embodiment, the functional fragment of a promoter includes a CAAT box. CAAT boxes are binding sites for transcription factors and are located in some, but not all promoters and, when present, are located approximately 60-100 bp upstream of the start of transcription. In some embodiments, the functional fragment of a promoter optionally includes a 5’ UTR. In some embodiments, the functional fragment of a promoter optionally includes an intron. In some embodiments, a functional fragment of an intron includes splice sites to allow splicing of the intron out of pre-mRNA. [0040] In some embodiments, the DNA construct comprising a Cannabis Ubiquitin promoter further comprises an intron. As used herein, an intron is an intervening sequence in a gene that is transcribed into RNA but is then excised in the process of generating the mature mRNA. The term intron is also used for the excised RNA sequences. Introns may be used in combination with a promoter sequence to enhance transcription, translation, and/or mRNA stability. Some introns have been identified that can enhance gene expression when included in DNA constructs. In some cases, introns can influence tissue specificity of expression by a promoter (Jeon et al., “Tissue-Preferential Expression of a Rice α-Tubulin Gene, OsTubA1, Mediated by the First Intron,” Plant Phys.123:1005-1014 (2000), which is hereby incorporated by reference in its entirety). An intron may comprise sequences from a Ubiquitin gene disclosed herein, or an intron may be derived from a heterologous sequence. One such heterologous intron is the first intron of gene II of the histone H3 variant of Arabidopsis (Chaubet-Gigot et al., “Tissue-Dependent Enhancement of Transgene Expression by Introns of Replacement Histone H3 Genes of Arabidopsis,” Plant Mol Biol 45:17-30 (2001), which is hereby incorporated by reference in its entirety), or any other commonly known intron sequence. In some embodiments, the intron comprises SEQ ID NOs:6, 17, or 28. In some embodiments, the intron is SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:28, or a functional fragment thereof. [0041] An exon is a portion of the nucleotide sequence of a gene that is transcribed and is found in the mature messenger RNA derived from the gene, but is not necessarily a part of the sequence that encodes the final gene product. [0042] In some embodiments, the DNA construct comprising a Cannabis Ubiquitin promoter further comprises a 5’ UTR. A 5′ UTR is an untranslated segment at the 5′ end of pre- mRNAs or mature mRNAs. For example, on mature mRNAs, a 5′ UTR typically harbors on its 5′ end a 7-methylguanosine cap and is involved in many processes such as splicing, polyadenylation, mRNA export towards the cytoplasm, identification of the 5′ end of the mRNA by the translational machinery, and protection of the mRNAs against degradation. The putative transcriptional start sites of the Cannabis Ubiquitin genes is predicted to be near the 5’UTR. In some embodiments the 5’ UTR comprises any one of SEQ ID NOs:5, 11, 16, 22 or 27. In some embodiments, the 5’ UTR is SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:27, or a functional fragment thereof. [0043] In some embodiments, the promoter includes an enhancer regulatory element (such as the 35S enhancer exemplified in SEQ ID NO:84) added to the promoter sequence. An enhancer is a regulatory element is a binding site for transcriptional activators that increases transcription. [0044] In some embodiments of the DNA constructs of the present application, the second nucleotide sequence is operably linked to a 3’ terminator sequence. A 3’ terminator sequence is an untranslated regulatory element at the 3′ end of the coding region of a gene, which comprises sequences capable of affecting transcription or mRNA processing. The 3’ terminator may also comprise signals for polyadenylation of the mRNA transcript. In some embodiments, non-limiting examples of a suitable 3’ terminator sequence is any one of SEQ ID NOs:34-53. In some embodiments, the 3' terminator is the nopaline synthase 3' region (nos 3') (Fraley et al, “Expression of Bacterial Genes in Plant Cells,” Proc. Natl. Acad. Sci.80:4803- 4807 (1983), which is hereby incorporated by reference in its entirety). An example of the use of different 3' non-translated regions is provided in Ingelbrecht et al., “Different 3’ End Regions Strongly Influence the Level of Gene Expression in Plant Cells,” Plant Cell 1:671-680 (1989), which is hereby incorporated by reference in its entirety). Non-limiting examples of polyadenylation signals include one from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al., “Tissue-Specific and Light-Regulated Expression of a Pea Nuclear Gene Encoding the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase,” EMBO J.3:1671-1679 (1984), which is hereby incorporated by reference in its entirety) and AGRtu.nos (GenBank Accession No. E01312, which is hereby incorporated by reference in its entirety). [0045] A functional fragment of the Cannabis Ubiquitin 3’ terminator is a portion or subsequence with the ability to terminate transcription. In one embodiment, a functional fragment of a 3’terminator includes a polyadenylation signal. [0046] There is natural variation in the genetic sequence of the Cannabis Ubiquitin regulatory elements of different Cannabis varieties. For example, FIGs.1-5 show sequence variation between Cannabis Ubiquitin promoter sequences from three different Cannabis varieties. FIGs.6-10 show sequence variation between Cannabis Ubiquitin 3’ terminator sequences from three different Cannabis varieties. The present application encompasses more than the specific exemplary sequences. [0047] As used herein, sequence identity means that sequences (e.g., nucleotide sequences or protein sequences) are identical (i.e., on a nucleotide-by-nucleotide basis for nucleic acids or amino acid-by-amino acid basis for polypeptides) over a window of comparison. Methods of calculating sequence identity are known in the art. For example, sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Multalin program (Corpet, “Multiple Sequence Alignment with Hierarchical Clustering,” Nucleic Acids Res.16:10881-90 (1988), which is hereby incorporated by reference in its entirety), or the Megalign^® program of the LASERGENE^® bioinformatics computing suite (DNASTAR^® Inc., Madison, Wis.). Sequences may also be aligned using algorithms known in the art including, but not limited to, CLUSTAL V algorithm or the BLASTN or BLAST 2 sequence programs. [0048] The DNA construct of the present application may comprise a second heterologous nucleotide sequence operably linked to the Cannabis Ubiquitin promoter. A promoter is capable of controlling the expression of an operably linked sequence. By “operably linked” it is meant an association in which the function of one is affected by the other. For example, a nucleotide promoter sequence may be operably linked to a nucleotide sequence encoding a polypeptide or a functional RNA if the nucleotide promoter sequence affects the transcription of the sequence encoding a polypeptide or a functional RNA. Other elements of the DNA constructs described herein may also be operably linked. For example the sequence encoding a polypeptide or a functional RNA may be operably linked to a 3’ terminator sequence such that the 3’ terminator sequence contributes to termination of transcription of the sequence encoding a polypeptide or a functional RNA. [0049] In the DNA constructs described herein, the second nucleotide sequence is heterologous to the first nucleotide sequence. A heterologous nucleotide sequence refers to a nucleotide sequence that is not naturally occurring with the Cannabis Ubiquitin promoter, 3’ terminator sequence, 5’UTR, intron, or other regulatory element described herein. While this nucleotide sequence is heterologous to the promoter, 3’ terminator, 5’UTR, or intron sequence, it may be homologous, native, heterologous, or foreign, to the plant host. For example, a promoter operably linked to a heterologous nucleotide sequence refers to a nucleotide sequence from an organism or species different from that from which the promoter was derived or, if from the same organism or species, a nucleotide sequence which is not naturally associated with the promoter. [0050] In some embodiments, the second nucleotide sequence encodes a polypeptide or a functional RNA. In some embodiments, the second nucleotide sequence of the DNA construct is selected from the group consisting of a functional RNA molecule; a molecule that confers increased yield, improved fiber content, increased oil content, increased protein content, disease resistance, insecticidal resistance, herbicide tolerance, nitrogen use efficiency, water use efficiency, nutritional quality; a DNA binding protein; a cannabinoid content gene; a terpene content gene; a reporter gene; a genome editing nuclease; a selectable marker; or combinations thereof. [0051] In some embodiments, heterologous sequences of interest include genes conferring traits to improve a variety of characteristics of a plant. These include, without limitation, genes involved in transferring information, such as transcription factors and kinases. Also included are genes involved in sterility, seed characteristics, oil, starch, carbohydrate, terpene, cannabinoids, increasing protein content or other nutritional value quality, increasing yield, conferring insect or disease resistance, increasing drought and stress tolerance, improving horticultural qualities (e.g., pigmentation and growth), altering growth habit, imparting herbicide tolerance, enabling the production of industrially useful compounds and/or materials from the plant, and/or enabling the production of pharmaceuticals. [0052] The second nucleotide sequence of the DNA construct may encode a functional RNA molecule, which refers to an RNA molecule that may not be translated but has a biological role. Functional RNA molecules include, but are not limited to, guide RNA (“gRNA”), short hairpin RNA (“shRNA”), microRNA (“miRNA”), artificial micro RNA (“amiRNA”), RNA interference (“RNAi”), small interfering RNA (“siRNA”), CRISPR RNA (“crRNA”), trans- activating CRISPR RNA (“tracrRNA”), antisense RNA, transfer RNA (“tRNA”), and ribosomal RNA (“rRNA”). [0053] The second nucleotide sequence of the DNA constructs described herein may encode a genome editing endonuclease. In some embodiments, the genome editing nuclease is a zinc finger nuclease (“ZFN”), a transcriptional activator-like effector nuclease (“TALEN”), a meganuclease, or a Cre recombinase. In other embodiments, the genome editing endonuclease refers to a protein such as a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated nuclease. Non-limiting examples of CRISPR associated nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, Mad7, homologs thereof, or modified versions, and endonuclease inactive versions thereof. CRISPR/Cas systems can be a type I, a type II, or a type III system. Use of such systems for gene editing has been widely described. For example, the use of CRISPR guide RNA in conjunction with CRISPR-Cas9 technology to target RNA is described in Wiedenheft et al., “RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,” Nature 482:331-338 (2012); Zhang et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems,” Science 339(6121): 819-23 (2013); and Gaj et al., “ZFN, TALEN, and CRISPR/Cas-based Methods for Genome Engineering,” Cell 31(7):397-405 (2013), which are hereby incorporated by reference in their entirety. When a guide RNA and a CRISPR endonuclease or “Cas” endonuclease are expressed in the cell, the genomic target sequence can be modified or permanently disrupted. The guide RNA/genome editing endonuclease complex is recruited to the target sequence by the base-pairing between the guide RNA sequence and the complementarity to the target sequence in the genomic DNA. [0054] In some embodiments, the genome editing endonuclease is Cas9, but can also be an endonuclease from one of many related CRISPR systems that have been described. “Cas9” refers to a Cas endonuclease of a type II CRISPR system that forms a complex with a crRNA and a tracrRNA, or with a guide RNA, for specifically recognizing and cleaving all or part of a DNA target sequence. For example, a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In another example, a Cas9 can be in complex with a guide RNA. Thus, crRNA, tracrRNA, and guide RNA are non-limiting examples of RNA components described herein. [0055] In another embodiment, the Cas endonuclease gene is a plant optimized Cas9 endonuclease gene. As used herein, the term “plant codon-optimized” means that the coding sequence has been optimized to improve expression such as by reducing the use of rarely used codons in plants. In another embodiment, the Cas endonuclease gene is a Cpf1 (also known as Cas12a) or a Mad7 endonuclease. [0056] In some embodiments, the recognition and cutting of a target sequence by a genome editing endonuclease occurs if the correct protospacer-adjacent motif (“PAM”) is located at or adjacent to the 3′ end of the DNA target sequence. For example, Cas9 will make a double strand break (“DSB”) 3-4 nucleotides upstream of the PAM sequence. A double strand break in DNA can be repaired through one of two general repair pathways: (1) non-homologous end joining (“NHEJ”) DNA repair pathway or (2) the homologous directed repair (“HDR”) pathway. The NHEJ repair pathway often results in insertions/deletions (“InDels”) at the DSB site that can lead to frameshifts and/or premature stop codons, effectively disrupting the open reading frame (“ORF”) of the targeted gene. The HDR pathway requires the presence of a repair template, which is used to fix the DSB. HDR faithfully copies the sequence of the repair template to the cut target sequence. Specific nucleotide changes can be introduced into a targeted gene by the use of HDR with a repair template. A prerequisite for cleavage for Cas is the presence of a conserved PAM downstream of the target DNA. The sequence and length of a PAM can differ depending on the Cas protein or Cas protein complex used, but are typically 2, 3, 4, 5, 6, 7, or 8 nucleotides long. For example, a PAM has the sequence 5'-NGG-3' but less frequently NAG. Specificity is provided by the sequence approximately 12 bases upstream of the PAM, which matches between the RNA and target DNA. Cpf1 acts in a similar manner to Cas9, but Cpf1 does not require a tracrRNA and it recognizes a T-rich PAM sequence adjacent to the 5′ end of the DNA target sequence. Specificity of the CRISPR/Cas system is based on the guide RNA that uses complementary base pairing to recognize target DNA sequences. [0057] Other aspects of the present application are directed to a vector comprising the DNA constructs described herein. As used herein, a “vector” means any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, T-DNA vector, etc., which is capable of replication when associated with the proper control elements, and/or which is capable of transferring gene sequences into cells. Thus, the term includes cloning and expression vectors, as well as viral vectors. [0058] In some embodiments, the vector includes left and right Agrobacterium T-DNA border sequences (Peralta and Ream, “T-DNA Border Sequences Required for Crown Gall Tumorigenesis,” Proc. Natl. Acad. Sci.82:5112-5116 (1985), which is hereby incorporated by reference in its entirety). These border sequences allow the introduction of heterologous DNA located between the left and right T-DNA border sequences into a host cell when using Agrobacterium-mediated DNA transformation. [0059] Standard cloning procedures known in the art can be used to prepare the DNA construct and/or the vector, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, New York (2001), which is hereby incorporated by reference in its entirety. [0060] In some embodiments, the vector comprising the DNA construct is introduced into a host cell. “Introduced” includes the incorporation of a nucleotide into a eukaryotic or prokaryotic cell, where the nucleotide may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleotide or protein to the cell. The term also may include a reference to stable or transient transformation methods, as well as sexually crossing. Thus, “introduced” in the context of inserting a nucleotide fragment (e.g., a DNA construct/expression construct) into a cell, means “transfection”, “transformation”, or “transduction” and includes the incorporation of a nucleotide fragment into a eukaryotic or prokaryotic cell where the nucleotide fragment is incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed. [0061] DNA constructs and vectors can be introduced into cells via transformation. Transformation refers to both stable transformation and transient transformation. A transient transformation refers to the introduction of the DNA construct into the plant cell of a host organism resulting in gene expression without genetically stable inheritance. [0062] A stable transformation refers to the introduction of the DNA construct into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleotide fragment is stably integrated in the genome of the host organism and any subsequent generation. [0063] Selectable markers may be used to select for plants or plant cells that comprise a DNA construct. Selection of transformed cells comprising the DNA construct utilizes an antibiotic or other compound useful for selective growth as a supplement to the media. The compound to be used will be dictated by the selectable marker element present in the vector with which the host cell was transformed. The marker may encode biocide resistance, antibiotic resistance (e.g., kanamycin, Geneticin (G418), bleomycin, hygromycin, etc.), or herbicide resistance (e.g., glyphosate, glufosinate, etc.). Examples of selectable markers include, but are not limited to, a neo gene which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc.; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene which encodes glyphosate resistance; a nitrilase gene, which confers resistance to bromoxynil; a mutant acetolactate synthase gene (ALS), which confers imidazolinone or sulfonylurea resistance; and a methotrexate resistant DHFR gene. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, spectinomycin, rifampicin, streptomycin and tetracycline, etc. Examples of selectable markers are described in, e.g., U.S. Patent Nos.5,550,318; 5,633,435; 5,780,708; and 6,118,047, which are each hereby incorporated by reference in their entirety. [0064] In some embodiments, reporter genes that encode enzymes providing for production of an identifiable compound, or other markers that indicate relevant information regarding the outcome of transformation are used for selection. The most widely used reporter gene for gene fusion experiments has been uidA, a gene from Escherichia coli that encodes the β-glucuronidase protein, also known as GUS (Jefferson et al., “GUS Fusions: β-Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher Plants,” EMBO J.6:3901-3907 (1987), which is hereby incorporated by reference in its entirety); and GUSPlus, (Vickers et al., “pGFPGUSPlus, a New Binary Vector for Gene Expression Studies and Optimizing Transformation Systems in Plants,” Biotechnol Lett.11:1793-1796 (2007), which is hereby incorporated by reference in its entirety). Other exemplary reporter genes include a GFP gene from A. victoria which encodes for green fluorescent light emission under UV light (Chalfie et al., “Green Fluorescent Protein as a Marker For Gene Expression,” Science 263:802-805 (1994), which is hereby incorporated by reference in its entirety), other fluorescent protein markers (Shaner et al., “Improved Monomeric Red, Orange and Yellow Fluorescent Proteins Derived from Discosoma sp. Red Fluorescent Protein,” Nat Biotech 22:1567-1572 (2004), which is hereby incorporated by reference in its entirety); an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., “Molecular Cloning of the Maize R-nj Allele by Transposon Tagging with Ac.” In 18 Stadler Genetics Symposium, P. Gustafson and R. Appels, eds. (New York: Plenum), pp.263-82 (1988), which is hereby incorporated by reference in its entirety); a β-lactamase gene (Sutcliffe et al., “Nucleotide Sequence of the Ampicillin Resistance Gene of Escherichia coli Plasmid pBR322,” Proc. Natl. Acad. Sci. USA 75:3737-41 (1978), which is hereby incorporated by reference in its entirety); a gene which encodes an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin); a luciferase gene (Ow et al., “Transient and Stable Expression of the Firefly Luciferase Gene in Plant Cells and Transgenic Plants,” Science 234:856-9 (1986), which is hereby incorporated by reference in its entirety); a lE gene that encodes a catechol dioxygenase that can convert chromogenic catechols (Zukowski et al., “Chromogenic Identification of Genetic Regulatory Signals in Bacillus subtilis Based on Expression of a Cloned Pseudomonas Gene,” Proc. Natl. Acad. Sci. USA 80:1101-1105 (1983), which is hereby incorporated by reference in its entirety); an amylase gene (Ikatu et al., “The α- Amylase Gene as a Marker for Gene Cloning: Direct Screening of Recombinant Clones,” Bio/Technol.8:241-2 (1990), which is hereby incorporated by reference in its entirety); a tyrosinase gene that encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to melanin (Katz et al., “Cloning and Expression of the Tyrosinase Gene from Streptomyces antibioticus in Streptomyces lividans,” J. Gen. Microbiol. 129:2703-14 (1983), which is hereby incorporated by reference in its entirety); and an α- galactosidase. [0065] Transient or stable transformation of DNA constructs described herein may be performed using particle bombardment (also known as biolistic transformation). In some embodiments, particle bombardment involves propelling inert or biologically active particles at cells. This technique is disclosed, for example, in Klein et al., “High-Velocity Microprojectiles for Delivering Nucleic Acids Into Living Cells,” Nature 327:70-73 (1987), which is hereby incorporated by reference in its entirety, and is also known as biolistic transformation of the host cell, as disclosed in U.S. Patent Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., and in Emerschad et al., “Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera),” Plant Cell Reports 14:6-12 (1995), which are hereby incorporated by reference in their entirety. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. In other embodiments, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into cells. [0066] In some embodiments, biolistic methods use gold or tungsten particles typically of 0.5 to 2 micrometers in size and coated with DNA, RNA, or ribonucleotide particles that has been precipitated onto the particles; the particles are discharged using a “gene gun” powered by a gas at high pressure (typically hundreds to thousands pounds per square inch) onto a plant held in an evacuated chamber. More recent biolistic methods using equipment such as the Helios^® gene gun (Bio-Rad Laboratories, Inc.) use lower pressures (in the hundreds pounds per square inch). Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells. Other variations of particle bombardment, now known or hereafter developed, can also be used. [0067] Delivery of DNA constructs described herein for modification of a plant genome can be accomplished by plant transformation, including, for example, infection with a microbe, such as Rhizobia or Agrobacterium infection. The Ti (or Ri) plasmid of Agrobacterium enables the highly successful transfer of a foreign nucleotide molecule into plant cells. A variation of Agrobacterium transformation uses vacuum infiltration in which whole plants are used (Senior, “Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998), which is hereby incorporated by reference in its entirety). In some embodiments, transformation involves fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies (Fraley et al., “Liposome-Mediated Delivery of Tobacco Mosaic Virus RNA into Tobacco Protoplasts: A Sensitive Assay for Monitoring Liposome-Protoplast Interactions,” Proc. Natl. Acad. Sci. USA 79:1859-63 (1982), which is hereby incorporated by reference in its entirety). [0068] In some embodiments, transformation can be accomplished by electroporation (Fromm et al., “Expression of Genes Transferred into Monocot and Dicot Plant Cells by Electroporation,” Proc. Natl. Acad. Sci. USA 82:5824 (1985), which is hereby incorporated by reference in its entirety). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate. [0069] In other embodiments, transformation can be accomplished through PEG- mediated DNA transfer, microinjection, or vacuum infiltration to provide for stable or transient expression of the DNA construct. Other methods of transformation include polyethylene- mediated plant transformation, micro-injection, physical abrasives, and laser beams (Senior, “Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998), which is hereby incorporated by reference in its entirety). In any of these methods, transformation can be enhanced by the use of a suitable microbe, such as a Rhizobia microbe, or an Agrobacterium, to facilitate DNA uptake by plant cells. [0070] Wounding of a target plant tissue prior to or during DNA delivery, for example using Agrobacterium or a Rhizobia species, such as Ensifer adhaerens, may also be employed to cause transformation. Various methods of wounding are employed in plant transformation methods, including for example, microprojectile bombardment; treatment with glass beads; cutting, scratching or slicing; sonication; or silicon carbide fibers or whiskers. [0071] Any method of transformation that results in efficient transformation of the host cell of choice is appropriate for practicing the present application. [0072] In the present application, a cell may comprise the DNA construct. In some embodiments, the cell comprising the DNA construct is a plant cell. A plant cell includes, without limitation, cells from plant tissue, including seeds, suspension cultures, embryos, meristematic regions, cotyledons, hypocotyls, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, microspores, cotyledons, zygotic and somatic embryos, protoplasts, pollen, embryos, anthers, and the like. [0073] The means of transformation chosen in practicing the methods described herein include those most suited to the tissue to be transformed. In some embodiments, the DNA construct is introduced into a plant, meaning a whole plant, plant organ, plant tissue, seed, cutting, clone, or progeny. In some embodiments, the DNA construct is not incorporated into a plant’s genome. [0074] A plant cell comprising a DNA construct described herein may be a monocot plant cell. Monocot plants include, without limitation, major plant crops such as rice, maize, sorghum, wheat, barley, and oats. [0075] A plant cell comprising a DNA construct described herein may also be a dicot plant cell. Dicot plants include, without limitation, the family Cannabaceae among many other families. Examples of dicot plants include, without limitation, Cannabis, hops, soybean, alfalfa, sunflower, cotton, canola, and sugar beet, to name a few. In some embodiments, the plant cell is from a Cannabis plant. Cannabis refers to a genus of flowering plants in the family Cannabaceae, and includes at least three recognized species: Cannabis sativa, Cannabis indica, and Cannabis ruderalis. In some embodiments, the plant cell comprising the DNA construct is a Cannabis plant cell. [0076] After transformation, transformed plant cells can be regenerated. Means for regeneration vary from species to species of plant, but generally a petri plate containing explants or a suspension of transformed protoplasts is first provided. Callus tissue is formed and transformation of callus tissue can be performed. Shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Efficient regeneration will depend on the medium, genotype, and history of the culture. [0077] Transformed cells may first be identified using a selection marker simultaneously introduced into the host cells along with the DNA construct or vector of the present application. Suitable selection markers are described above. Cells or tissues are grown on a selection medium containing the appropriate antibiotic, whereby generally only those transformants expressing the antibiotic resistance marker continue to grow. Other types of markers are also suitable for inclusion in the vector of the present application, such as reporter genes as described above. The selection employed will depend on the target species; for certain target species, different antibiotics, herbicide, or biosynthesis selection markers may be preferred. [0078] Plant cells and tissues selected by means of an inhibitory agent or other selection marker are then tested for the acquisition of the DNA construct. In some embodiments, a transgenic cell comprises the DNA construct. In some embodiments, the transgenic cell is a transgenic plant cell. As used herein, a transgenic cell or plant cell comprises within its genome a heterologous nucleotide introduced by a transformation step. The heterologous nucleotide is stably integrated within the genome such that the nucleotide is passed on to successive generations. As used herein, genome, as it applies to plant cells, encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell. The heterologous nucleotide may be integrated into the genome alone or as part of a DNA construct. In some embodiments, a transgenic plant comprises the transgenic plant cell. [0079] A transgenic plant can also comprise more than one heterologous nucleotide sequence within its genome. Each heterologous nucleotide may confer a different trait to the transgenic plant. A heterologous nucleotide can include a sequence that originates from a foreign species, or, if from the same species, can be substantially modified from its native form. Transgenic can include any cell, cell line, callus, tissue, plant part or plant, or clone, the genotype of which has been altered by the presence of a heterologous nucleotide including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic plant. In some embodiments, the transgenic plant is a monocot plant. In some embodiments, the transgenic monocot plant is a rice plant. In some embodiments, the transgenic plant is a dicot plant. In other embodiments the transgenic dicot plant is a Cannabis plant. In another embodiment, transgenic seeds, clone, pollen, or floral buds of the transgenic plant comprise the DNA construct. [0080] The alterations of the genome (chromosomal or extra-chromosomal) by genome editing procedures that do not result in an insertion of a foreign polynucleotide, or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non- recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation are not intended to be regarded as transgenic. [0081] Another aspect of the present application relates to a method of expressing a coding sequence in a transformed plant cell. This method involves transforming a plant cell with a DNA construct comprising a first nucleotide sequence comprising a Cannabis Ubiquitin promoter with at least 70% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof. The DNA construct also has a second nucleotide sequence heterologous to the first nucleotide sequence, where the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence is capable of controlling expression of the second nucleotide sequence. Transforming expresses the coding sequence in the transformed plant cell. [0082] This aspect of the present application can be carried out with any of the embodiments described above. [0083] A further aspect of the present application relates to a DNA construct comprising a first nucleotide sequence comprising a Cannabis Ubiquitin 3’ terminator sequence with at least 70% sequence identity to any one of SEQ ID NOs:34-53 or a functional fragment thereof. The DNA construct also has a second nucleotide sequence heterologous to the first nucleotide equence, where the second nucleotide sequence is operably linked to the first nucleotide equence, such that the first nucleotide sequence contributes to transcriptional termination of the econd nucleotide sequence. 0084] The nucleotide sequences of SEQ ID NOs:34-53 are set forth in Table 5 (infra). 0085] In some embodiments of the methods and compositions described herein, Cannabis Ubiquitin 3’ terminator sequences include, without limitation, sequences disclosed in SEQ ID NOs:34-53. In some embodiments, the 3’ terminator comprises the nucleotide sequence of any one of SEQ ID NOs:34-35. In some embodiments, the 3’ terminator is the nucleotide equence of any one of SEQ ID NOs:34-53. Also encompassed are 3’ terminators with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% equence identity to any one of SEQ ID NOs:34-53, or a functional fragment thereof. For example, in some embodiments, the Cannabis Ubiquitin 3’ terminator as the first nucleotide equence comprises at least 80% sequence identity to any one of SEQ ID NOs:34-53, or a unctional fragment thereof. In some embodiments, the Cannabis Ubiquitin 3’ terminator as the irst nucleotide sequence comprises at least 90% sequence identity to any one of in SEQ ID NOs:34-53, or a functional fragment thereof. In some embodiments, the Cannabis Ubiquitin 3’ erminator as the first nucleotide sequence comprises at least 95% sequence identity to any one of SEQ ID NOs:34-53, or a functional fragment thereof. And in some embodiments, the Cannabis Ubiquitin 3’ terminator as the first nucleotide sequence comprises any one of SEQ ID NOs: 34-53, or a functional fragment thereof. In some embodiments, the Cannabis Ubiquitin 3’ erminator sequences include any of SEQ ID NOs:69-83, or a functional fragment thereof. The nucleotide sequences of SEQ ID NOs:69-83 are set forth in Table 6 (infra). 0086] In some embodiments, the DNA construct comprising the 3’ terminator, further comprises a promoter sequence. In some embodiments, the promoter sequence comprises at least 95% identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof. In some embodiments, the promoter sequence comprises any one of SEQ ID NOs:1-4, 7- 10, 12-15, 18-21, 23-26, or a functional fragment thereof. Other promoters not described herein may also be used in the DNA construct comprising the 3′ terminator described herein. [0087] This aspect of the present application can also be carried out with any of the embodiments described above. [0088] Another aspect of the present application relates to a method of expressing a coding sequence in a transformed plant cell. This method involves transforming a plant cell with a DNA construct comprising a first nucleotide sequence comprising a Cannabis Ubiquitin 3’ terminator sequence with at least 70% sequence identity to any one of SEQ ID NOs:34-53 or a functional fragment thereof. The DNA construct also has a second nucleotide sequence heterologous to the first nucleotide sequence, where the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence contributes to transcriptional termination of the second nucleotide sequence. Transforming expresses the coding sequence in the plant cell. [0089] This aspect of the present application can also be carried out with any of the embodiments described above. [0090] Yet another aspect of the present application relates to a DNA construct comprising a first nucleotide sequence comprising a Cannabis Ubiquitin intron sequence with at least 70% sequence identity to any one of SEQ ID NOs:6, 17, 28, or a functional fragment thereof. The DNA construct also has a second nucleotide sequence heterologous to the first nucleotide sequence, where the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence contributes to transcriptional regulation of the second nucleotide sequence. [0091] This aspect of the present application can also be carried out with any of the embodiments described above. [0092] The following examples are provided to illustrate embodiments of the present application but are by no means intended to limit its scope. EXAMPLES Example 1 – Identification of Cannabis Ubiquitin Genes [0093] Ubiquitin is a highly conserved protein of 76 amino acids. The promoters of maize, Arabidopsis, and parsley Ubiquitin genes have been characterized as driving high to moderate expression (Christensen et al., “Maize Polyubiquitin Genes: Structure, Thermal Perturbation of Expression and Transcript Splicing, and Promoter Activity Following Transfer to Protoplasts by Electroporation,” Plant Mol. Biol.18:675-689 (1992); Kawalleck et al., “Polyubiquitin Gene Expression and Structural Properties of the ubi4-2 Gene in Petroselinum crispum,” Plant Mol. Biol.21:673-684 (1993), and Norris et al., “The Intron of Arabidopsis thaliana Polyubiquitin Genes is Conserved in Location and is a Quantitative Determinant of Chimeric Gene Expression,” Plant Mol. Biol.21:895-906 (1993), which are hereby incorporated by reference in their entirety). Five Arabidopsis Ubiquitin genes, AtUbi3 (GenBank Accession No. NM_120402), AtUbi4 (GenBank Accession No. NM_001343676), AtUbi10 (GenBank Accession No. NM_178968), AtUBI11 (NM_001036508), and AtUBI14 (NM_001125450) (each of which is hereby incorporated by reference in its entirety) were used to BLAST search the genome sequence of Cannabis sativa genome sequence of variety CBDRx (“Cs10”; GenBank GCA_900626175.1, which is hereby incorporated by reference in its entirety). [0094] Three Cannabis polyubiquitin genes CsUbi-1.1 (LOC115707819; GenBank Accession No. XM_030635894, which is hereby incorporated by reference in its entirety), CsUbi-3 (LOC115709395; GenBank Accession No. XM_030637486, which is hereby incorporated by reference in its entirety), and CsUbi-X (LOC115702720; GenBank Accession No. XM_030630147, which is hereby incorporated by reference in its entirety) were identified in the Cannabis genome. One Ubiquitin-like gene with less sequence identity was also identified and called CsUbilike-9 (LOC115723255; GenBank Accession No. XM_030652698, which is hereby incorporated by reference in its entirety). A final gene that encodes a single Ubiquitin called CsUbi-1.2 (LOC115705966; GenBank Accession No. XM_030633466, hereby incorporated by reference in its entirety) was identified by its annotation in the Cs10 genome as a ubiquitin gene. Example 2 – Novel Cannabis Ubiquitin Promoter Elements [0095] Nucleotide sequences of each Ubiquitin LOCUS from Cannabis variety BaOX was obtained by identifying approximately 3000 bp of the upstream regions of the five Ubiquitin or Ubiquitin-like genes of interest in the Cannabis genome. These regions were then used to BLAST search an in-house genome assembly of Cannabis sativa variety BaOX for that varietal’s specific sequence. Various lengths of the predicted promoter sequences were isolated from each gene as represented in Table 1. Promoter fragments of various sizes from BaOX CsUbi-1.1 represented in Table 1 are called UbiChr1.1P4, SEQ ID NO:1; UbiChr1.1P3, SEQ ID NO:2; UbiChr1.1P2, SEQ ID NO:3; and UbiChr1.1P1, SEQ ID NO:4 (FIGs.1A-C). These promoters comprise a 5’ UTR element of CsUbi-1.1 represented as SEQ ID NO:5, and an intron element represented as SEQ ID NO:6. [0096] Promoter fragments of various sizes from BaOX CsUbi-3 represented in Table 1 are called UbiChr3P4, SEQ ID NO:7; UbiChr3P3, SEQ ID NO:8; UbiChr3P2, SEQ ID NO:9; and UbiChr3P1, SEQ ID NO:10 (FIGs.2A-C). These promoters comprise a 5’ UTR element of CsUbi-3 represented as SEQ ID NO:11. CsUbi-3 has no intron. [0097] Promoter fragments of various sizes from BaOX CsUbi-X represented in Table 1 are called UbiChrXP4, SEQ ID NO:12; UbiChrXP3, SEQ ID NO:13; UbiChrXP2, SEQ ID NO:14; and UbiChrXP1, SEQ ID NO:15 (FIGs.3A-C). These promoters comprise a 5’ UTR element of CsUbi-X represented by SEQ ID NO:16 and an intron element represented by SEQ ID NO:17. [0098] Promoter fragments of various sizes from BaOX CsUbilike-9 represented in Table 1 are called UbilikeChr9P4, SEQ ID NO:18; UbilikeChr9P3, SEQ ID NO:19; UbilikeChr9P2, SEQ ID NO:20; and UbilikeChr9P1, SEQ ID NO:21 (FIGs.4A-C). These promoters comprise a 5’ UTR element of CsUbilike-9 represented as SEQ ID NO:22. CsUbilike-9 has no intron. [0099] Promoter fragments of various sizes from BaOX CsUbi-1.2 represented in Table 1 are called UbiChr1.2P4, SEQ ID NO:23; UbiChr1.2P3, SEQ ID NO:24; UbiChr1.2P2, SEQ ID NO:25; and UbiChr1.2P1, SEQ ID NO:26 (FIGs.5A-C). These promoters comprise a 5’ UTR element of CsUbi-1.2 represented as SEQ ID NO:27, and an intron element represented by SEQ ID NO:28. T l 1 i i i i P El

TCTTGGAAAGGCCAACTCGACAAAACTAATATTCCAAACTTTGCGTGTAAGCGGAGCGTAAGA

GTCGTATCTTTGAATTTTCTTTTCTGTTTATATTTGAAGGAACTGTATCAATTATATTTGGTT

TTGTTTTAATTCTGTATTTTTAG

GGGTTGCCGTGTGGTCGCCGTGGAGTCGCCCTCCGATTGCCTAGGCTGGCTGCAGAGAAATAT

UbiChr3P2 (SEQ ID NO:9)

ACAAGAACGATTTTCTTTTTCTCTGCGATTTTCAATTGTTGAAT

AATTACGGGTTGAAAAATTACAAAATCAGCATATGCAGTTTGGTTTGAAAAAATAGTCTAAAC

CTTGTATCGTATCTTTGAATTTTTTAGAGTATTTAATTCTCGACCTGTTTGTATTTAATTTTT

CCCAGGTCTTAACCAGCCCCTTTCAACTACTTTGAATGGTGCTCTTGGAAATGATCTATTTGC

TCTCCTGAACTTGGACCTCACTTGGGTTCTACACCTTTCATTACACTTAGAAGAAGACTTTAG

AACAATATTTTTTTCCTTTGTGATGAATCAATAGCTATTTGATGATGAAAAACCTCCCAAAAC

GAATGATTGGTGAATTTTTGGGTGATTGAATTTGAATTACATGTTGCTTGCTAACCACATTTG

TGGGCCTGGGCCAGGCCGATATGAATGTGGAAAAGTGCATAGTGTTTGCTTTTGAACACTCCA

CTAAGCAATTTGCCTATGACAATATGTGTATGGGTTTTTTGAGTTGTACTTGTAGTGGGTTGA

[0100] The CsUbi-1.1 gene encodes a protein with 7 Ubiquitin repeats, listed as SEQ ID NO:29 (GenBank Accession No. XP_030491754.1, which is hereby incorporated by reference in its entirety). The CsUbi-3 gene encodes a protein with 6 Ubiquitin repeats, listed as SEQ ID NO:30 (GenBank Accession No. XP_0300493346.1, which is hereby incorporated by reference in its entirety). The CsUbi-X gene encodes a protein with 5 Ubiquitin repeats listed as SEQ ID NO:31 (GenBank Accession No. XP_030486007.1, which is hereby incorporated by reference in its entirety). The CsUbilike-9 gene encodes a protein listed as SEQ ID NO:32 (GenBank Accession No. XP_030508558.1, which is hereby incorporated by reference in its entirety). The CsUbi-1.2 gene encodes a single Ubiquitin protein listed as SEQ ID NO:33 (GenBank Accession No. XP_030489326.1, which is hereby incorporated by reference in its entirety). Table 2. Protein Sequences of Ubiquitin Genes

QLEDGRTLADYNIQKESTLHLVLRLRGGI

NDSVVALTLRKDDNEFEDVNIARPTDFYQSRDPEGGSW Example 3 – Cloning of Cannabis Ubiquitin Promoters [0101] To evaluate the Ubiquitin promoters, proximate regions from Cannabis Ubiquitin genes (SEQ ID NOs: 1-4, 7-10, 12-15, 18-21, and 23-26) were synthesized for promoter cloning5 and evaluation using standard gene synthesis service (PriorityGene) into DNA vector pUC57- Kan by Genewiz (South Plainfield, NJ). KpnI restriction sites were added to flank the promoter sequences. Promoters were cloned into pARC1295 (FIG.11) using the KpnI restriction site 5’ of the Arabidopsis codon optimized CAS9 gene (SEQ ID NO:85, Table 4) (based on Fauser et al., “Both CRISPR/Cas-Based Nucleases and Nickases can be Used Efficiently for Genome Engineering in Arabidopsis thaliana,” Plant J.79:348-359 (2014), which is hereby incorporated by reference in its entirety) with an alternate N-terminal NLS and a NOS 3’ terminator already present in the vector. pARC1295 also has a double 35S promoter driving NPTII plant selectable marker d35S:NPTII:3’CaMV. Constructs are shown in Table 3. Table 3. DNA Constructs with Ubi Promoters

pARC1319 (2.0 kb) pARC1295 CsUbi-1.2 CsUbi-1.2 26 [0102] Optionally, a 35S enhancer (SEQ ID NO:84, Table 4) can be added to the DNA construct using the SacI restriction site 5’ of the KpnI restriction site. In many cases, the enhancer is added upstream of (5’ to) the Ubiquitin promoter to increase expression. Table 4. Other Exemplary Sequences

GACCTCTCGCTAGAGGAAACTCAAGATTCGCTTGGATGACCAGAAAGTCTGAGGAAACCATCAC

CGAGAAGTTGAAGGGATCTCCAGAAGATAACGAGCAGAAGCAACTTTTCGTTGAGCAGCACAAG

GAATTCGAATCCAAAAATTACGGATATGAATATAGGCATATCCGTATCCGAATTATCCGTTTGA

AGAGCAGCCGATTGTCTGTTG Example 4 – Evaluation of Ubiquitin Promoter Expression [0103] Promoter strength of Cannabis Ubiquitin promoter elements was evaluated after transformation of the DNA construct into Cannabis callus tissue or leaf tissue. To induce Cannabis callus tissue, seeds of hemp variety BaOX were surface sterilized then germinated in water with 5 mL/L PPM (plant preservative mixture) for 2-3 days at room temperature in the dark. Germinated seedlings were then transferred to Germination media (MS with B5 vitamins (4.43 g/L), sucrose (20 g/L), phytagar (3 g/L), pH 5.8) and incubated at 24°C under light (16 hours light and 8 hours dark) for 2-3 days. Once the seedlings became dark green-purple, the shoot tissue (hypocotyl, cotyledons, and emerging true leaves) were cut into 0.5 cm pieces and placed on Resting media (MS with B5 vitamins (4.43 g/L), sucrose (30 g/L), casein (0.5 g/L), glutamine (0.5 g/L), MES (0.5 g/L), gelzan (3 g/L), pH 5.8, TDZ (2 mg/L), NAA (0.5 mg/L)) in the dark for 2 weeks. Calli were sub-cultured by transferring 1 mm² or larger size calli to fresh media and incubated for another 4 weeks. [0104] Vectors comprising the DNA constructs from Examples 3, 5, or 8 were prepared for transformation and particle bombardment. On the day of particle bombardment, 5 µL of the prepared DNA (0.1-0.5 µg/µl), 10% sterilized gold particles (size 1.0 µm, re-suspended in nuclease-free, sterile water), 0.1 M spermidine (Sigma-Aldrich), and 2.5 M CaCl₂ (Sigma- Aldrich) were vortexed at medium speed for 15 minutes. The particles were then washed with absolute ethanol on ice three times and eluted in 70 µL absolute ethanol. [0105] Approximately 30-50 calli were placed in a 2.5 cm diameter circle, centered on Osmotic media (MS with vitamins (4.73 g/L), sucrose (30 g/L), sorbitol (30 g/L), mannitol (30 g/L), Phytagel (3.5 g/L), pH 5.8, Timentin (2 mg/L), NAA(0.5 mg/L)) in a 100 x 15 mm Petri dish and incubated at 24°C for 4 to 24 hours prior to bombardment. Gold particles were coated as described previously then spread evenly across 4 macro carriers and allowed to dry for 1 minute. The helium tank was set to a pressure of at least 1100 psi. The DNA-coated gold was loaded on a biolistic particle delivery system (Biorad PDS-1000/He™, 100/120V System) and delivered to the prepared plate of calli, set 6 cm from the particles, at 28 Hg vacuum and 1100 to 1300 psi, according to manufacturer’s instructions. Each plate was shot once, rotated 180°, then shot again. Two plates, or approximately 100 calli, were shot per vector. Two to 24 hours following bombardment, calli were transferred to Selection media (Resting media, described above, with 75 mg/L selectable marker, G418 for NPTII or Hygromycin for HPTII) and incubated at 24°C under 16 hours light and 8 hours dark for 1-2 weeks. After 1-2 weeks, the promoter expression of the heterologous functional RNA was assessed by grinding all transiently transformed calli tissue for a single bombardment plate in liquid nitrogen then extracting RNA from 100-250 mg of the crushed tissue in duplicate, per pool of tissue. Individual calli or smaller pools of calli can also be extracted for analysis. [0106] Cannabis Ubiquitin promoter activities were represented by the expression levels of Cas9 in Cannabis callus or leaf tissue. The expression levels were determined by reverse transcriptase quantitative PCR (RT-qPCR). Total RNA was extracted from individual or pooled transformed callus samples using MagMax Plant RNA isolation kit (Invitrogen), including a DNase I (Invitrogen) treatment step to remove any DNA contamination according to manufacturer’s instructions. [0107] Four-fifths to 1 microgram of total RNA was reverse-transcribed to first-strand cDNA using SuperScript^TM IV VILO cDNA synthesis kit then diluted to 8-10 ng/uL with DEPC- treated, nuclease-free water for the following qPCR. Specific primers designed for CAS9 include forward SEQ ID NOs:89 and 90 (Table 4) to measure CAS9 expression. A transgene from E.coli, NPTII (EcNPTII) SEQ ID NOs: 91 and 92 (Table 4), was used as a transformation calibrator for gene expression in both Cannabis and rice. [0108] Each qPCR reaction mixture contained 2 µl of diluted cDNA, 200 µM of MgCl₂, 200 nM of each primer, 0.75x passive reference dye ROX, and 1x DyNAmo™ HS SYBR^® Green qPCR master mix in a total volume of 20 µl. The reactions were initially denatured at 50°C for 2 min and 94°C for 10 min, followed by 40 cycles of amplification as follows: 10 sec at 94°C, 1 min at 60°C. Fluorescence of bound SYBR-Green was detected with the QuantStudio 5 real-time PCR system at 60°C followed by 60-95°C dissociation curve to confirm amplicon specificity. Cas9 transcription levels were measured in triplicate and normalized against the expression of EcNPTII. Primary data analysis for Ct values and amplicon melting temperatures was performed using the QuantStudio systems Design and Analysis Software v1.5.1. Relative Quantification (RQ) values were determined using the GeoMean method (Vandesompele et al, “Accurate Normalization of Real-Time Quantitative RT-PCR Data by Geometric Averaging of Multiple Internal Control Genes,” Genome Biol.3(7):research0034.1-research0034.11 (2002), which is hereby incorporated by reference in its entirety), yielding relative expression difference as the fold gene expression of subjects over calibrators as shown in FIG 14A. The promoters of CsUbi genes expressing Cas9 the most and the next smallest size of the same gene were repeated with technical repeats to verify results as shown in FIG 14B. Of all promoters tested, only the two larger fragment sizes of CsUbi-1.1, 1.5 kb and 2.0 kb, doubled expression of Cas9. There was no expression in rice. Example 5 – Novel Cannabis Ubiquitin 3’ Terminator Elements [0109] Nucleotide sequences of each Ubiquitin LOCUS described in Example 1 were obtained from Cannabis variety BaOX by identifying the coding region and putative 3’ terminator region of the five Ubiquitin or Ubiquitin-like genes of interest in the Cannabis genome. These regions were then used to BLAST search an in-house genome assembly of Cannabis sativa variety BaOX for that varietal’s specific sequence (FIGs.6-10). Various lengths of the predicted Ubiquitin 3’ terminator sequence elements are shown in Table 5. Ubiquitin 3’ terminator fragments of various sizes from CsUbi-1.1 represented in Table 5 are called UbiChr1.1T4, SEQ ID NO:34; UbiChr1.1T3, SEQ ID NO:35; UbiChr1.1T2, SEQ ID NO:36; and UbiChr1.1T1, SEQ ID NO:37. [0110] 3’ terminator fragments of various sizes from CsUbi-3 represented in Table 5 are called UbiChr3T4, SEQ ID NO:38; UbiChr3T3, SEQ ID NO:39; UbiChr3T2, SEQ ID NO:40; and UbiChr3T1, SEQ ID NO:41. [0111] 3’ terminator fragments of various sizes from CsUbi-X represented in Table 5 are called UbiChrXT4, SEQ ID NO:42; UbiChrXT3, SEQ ID NO:43; UbiChrXT2, SEQ ID NO:44; and UbiChrXT1, SEQ ID NO:45. [0112] 3’ terminator fragments of various sizes from CsUbilike-9 represented in Table 5 are called UbilikeChr9T4, SEQ ID NO:46; UbilikeChr9T3, SEQ ID NO:47; UbilikeChr9T2, SEQ ID NO:48; and UbilikeChr9T1, SEQ ID NO:49. [0113] 3’ terminator fragments of various sizes from CsUbi-1.2 represented in Table 5 are called UbiChr1.2T4, SEQ ID NO:50; UbiChr1.2T3, SEQ ID NO:51; UbiChr1.2T2, SEQ ID NO:52; and UbiChr1.2T1, SEQ ID NO:53. Table 5. Cannabis Ubiquitin 3’ Terminator Element Sequences

TTTGCTGAACAAAGTGTTCTTTATTTTCAGTTTTAA

UbiChr1.2T4 SEQ ID NO:50

GAGACCATTGCCTCAGCTTGTAAAGCCATGGACTATAATACATATATATG Prophetic Example 6 – Cloning of Cannabis Ubiquitin 3’ terminators [0114] Four sizes of each of the 3’ terminator regions downstream of the Cannabis Ubiquitin gene translational stop signal are synthesized for 3’ terminator cloning and evaluation (SEQ ID NOs:34-37, 38-41, 42-45, 46-49, and 50-53, Table 5) using standard gene synthesis service (PriorityGene) with the addition of AscI and PacI restriction enzyme sites to the 5’ and 3’ ends of the terminator sequences, respectively, to ease cloning in the next step. The Ubiquitin 3’ terminator fragments are cloned into a DNA vector pARC1297 (FIG.12) comprising a d35S:NPTII:3’CaMV plant selectable marker and a parsley Ubiquitin promoter (“PcUbi”) (SEQ ID NO:86, Table 4) driving CAS9 PcUBI:CAS9:3’CaMV. The 3’CaMV terminator of the Cas9 gene is replaced using AscI and PacI restriction sites situated 3’ to the CAS9 gene sequence in the vector according to standard protocols. As a result, each Cannabis Ubiquitin 3’ UTR is operably linked 3’ to the Arabidopsis codon optimized Cas9 gene PcUBI:CAS9:Ubi 3’ terminator. Prophetic Example 7 – Effect of Ubiquitin 3’ Terminator on Expression [0115] Vectors comprising the DNA constructs with each Ubiquitin 3’ terminator cloned into pARC1297 as described in Example 6 (PcUbi:CAS9:Ubi 3’ terminators and d35S:NPTII:3’CaMV selectable marker) are prepared for transformation and particle bombardment. Cannabis callus tissue is induced and transformed using Particle bombardment as described in Example 4. [0116] The effect of Cannabis Ubiquitin 3’ terminator elements on transcription are reflected by the expression levels of CAS9 in Cannabis callus tissue. The expression levels are determined by quantitative PCR (“qPCR”) as described in Example 4. Example 8 – Cloning of Cannabis Ubiquitin Introns [0117] Sequence of each of the introns from SEQ ID NOs:6, 17, and 28 were combined with the promoter and 5’UTR of PcUbi (SEQ ID NO:88, Table 4), synthesized, then cloned into pARC1295 (FIG.11) via the KpnI restriction site 5’ to Cas9 using a standard gene synthesis service via BioBasic Canada Inc. In these vectors, the original intron sequence of the PcUbi promoter (SEQ ID NO:87, Table 4) was replaced by one of the Cannabis Ubiquitin introns (SEQ

ID NOs:6, 17 and 28) and operably linked between the Arabidopsis codon optimized CAS9 gene and the PcUBI promoter and 5’UTR (SEQ ID NO:88) as shown in FIG.13. Example 9 – Effect of Cannabis Ubiquitin Introns on Gene Expression [0118] The effect of Cannabis Ubiquitin introns on gene expression was evaluated after transformation of the DNA constructs from Example 8 into Cannabis callus tissue and qPCR analysis of CAS9 expression as described in Example 4. All introns, when combined with the PcUbi promoter, including that for CsUbi-1.1, decreased expression of Cas9, as shown in FIG 14A. The appropriate intron, therefore, is important to normal expression of the transgene. Example 10 – Sequence Heterogeneity of Ubiquitin Regulatory Elements in Cannabis Varieties [0119] Alignments of Cannabis Ubiquitin regulatory sequences illustrates the sequence variability between different varieties of Cannabis. For example, an alignment of Cannabis Ubiquitin promoter UbiChrXP4 sequences from BaOX, SEQ ID NO:12; Cs10, SEQ ID NO:60; Z2 Female, SEQ ID NO:61; and Z2 male, SEQ ID NO:62; using Multalin, is shown in FIGs.3A- C. Sequence identities were calculated using the NCBI BLASTN suite (Zheng et al., “A Greedy Algorithm for Aligning DNA Sequences,” J. Comput. Biol.7(1-2):203-214 (2000), which is hereby incorporated by reference in its entirety). Sequence identity between UbiChrXP4 (BaOX) and UbiChrXP4 (Cs10) was 96.86%. Sequence identity between UbiChrXP4 (BaOX) and UbiChrXP4 (Z2F) was 96.30%. Sequence identity between UbiChrXP4 (BaOX) and UbiChrXP4 (Z2M) was 97.34%. [0120] An alignment of Cannabis Ubiquitin 3’ terminator UbiChrXT4 sequences UXT4_BaOX, SEQ ID NO:12; UXT4_Cs10, SEQ ID NO:75; UXT4_Z2F, SEQ ID NO:76; and UXT4_Z2M, SEQ ID NO:77 using Multalin, is shown in FIG.8. Sequence identities were calculated using the NCBI BLASTN suite (Zheng et al., “A Greedy Algorithm for Aligning DNA Sequences,” J. Comput. Biol.7(1-2):203-214 (2000), which is hereby incorporated by reference in its entirety). Sequence identity between UbiChrXT4 (BaOX) and UbiChrXT4 (Cs10) was 99.33%. Sequence identity between UbiChrXT4 (BaOX) and UbiChrXT4 (Z2F) was 99.67%. Sequence identity between UbiChrXT4 (BaOX) and UbiChrXT4 (Z2M) was 99.67%. Table 6. Cannabis Regulatory Elements from Different Varieties

ATTGAACGAACTGTATCAATGATCTTGGGTTTCATTTAATTTTAGGTTTTTTAATTTACCGAATA

UbiChr1.1P4 (Z2M) SEQ ID NO:56

AATAACTCTTTTTTTTTTTTTTTTAAAGAAATAAAAAACTCAAATTTCTACTAAAATAACATACA

TCCTCTGTTGCTTGCTAGCTACAGTATCTTAGTCGATAATGATCTTTATWAGGTACGAACGATCG

TGTCCAATCACAAGTCAAAAAATTTTAATAGTATATATATCTTTATAAAACATAAATAGCAAATT

AGGCCACACGCTATCTTGGGCTGCTCATTGACGCTGTCAGTAACGGTTCGATTAACGTTTAACAA

TGCTTCCTTAGTCAAAATTGCTTCTGATCATAGAACACTCGTATATTTGAATTTTTTTTGTTGGA

AATAATATTGGGTTTCATACAATTTTAGGGTTTTATTATTTAATCAGGCAAGATTGTTTTTTTTT

GCAGCAACAAGAACTTGAGATGCTGAGGCGGCGGTTGGAGGAAATTGAGTTGGAATTGCGATCGA

CCATCGCTAATGTGAGTCCAACTCCCTTGACATTTTCACACCAGATTTGTCAAGAGACTCCAGGG

AGTGTTTGCTTTTGAACACTCCAAAGCAACAACTTCCAAGGAAACAAATTCACTTTCCTCCATTC

GCTTACTGAGAAACCCATTAAAGGAATTTGAGACCATAATCAAACAAAATTATTTTCTGGACTGG

AATGAAATGCTAACATTATCTATTTGTTCTCAATTCTTTAATTCAATCAAAATTCCTTGGCTGTT

UbiChr3T4 (Z2M) SEQ ID NO:74

UbilikeChr9T4 (Z2M) SEQ ID NO:80

AGTCTCAAACTGCATATGTATACATGCTTTTTCTCCACTT [0121] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the present application and these are therefore considered to be within the scope of the present application.

Claims

WHAT IS CLAIMED: 1. A DNA construct comprising: a first nucleotide sequence comprising a Cannabis Ubiquitin promoter, wherein the first nucleotide sequence comprises a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof and a second nucleotide sequence heterologous to the first nucleotide sequence, wherein the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence is capable of controlling expression of the second nucleotide sequence.

2. The DNA construct of claim 1, wherein the first nucleotide comprises any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof.

3. The DNA construct of claim 1, wherein the first nucleotide sequence has at least 80% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof.

4. The DNA construct of claim 1, wherein the first nucleotide sequence has at least 90% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof.

5. The DNA construct of claim 1, wherein the first nucleotide sequence has at least 95% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof.

6. The DNA construct of any one of claims 1-5, wherein the first nucleotide sequence further comprises an intron.

7. The DNA construct of claim 6, wherein the intron comprises SEQ ID NOs:6, 17, or 28.

8. The DNA construct of any one of claims 1-5, wherein the first nucleotide sequence further comprises a 5′ UTR.

9. The DNA construct of claim 8, wherein the 5′ UTR comprises any one of SEQ ID NOs:5, 11, 16, 22, or 27.

10. The DNA construct of any one of claims 1-5, wherein the second nucleotide sequence is operably linked to a 3′ terminator sequence.

11. The DNA construct of claim 10, wherein the 3′ terminator sequence is any one of SEQ ID NOs:34-53.

12. The DNA construct of any one of claims 1-5, wherein the second nucleotide sequence encodes a polypeptide or a functional RNA.

13. The DNA construct of any one of claims 1-5, wherein the second nucleotide sequence encodes a functional RNA molecule; a molecule that confers increased yield, improved fiber content, increased oil content, increased protein content, disease resistance, insecticidal resistance, herbicide tolerance, nitrogen use efficiency, water use efficiency, nutritional quality; a DNA binding protein; a cannabinoid content gene; a terpene content gene; a reporter gene; a genome editing nuclease; a selectable marker; or combinations thereof.

14. A vector comprising the DNA construct of any one of claims 1-13.

15. A cell comprising the DNA construct of any one of claims 1-13.

16. The cell of claim 15, wherein the cell is a plant cell.

17. The cell of claim 16, wherein the DNA construct is not incorporated into the plant’s genome.

18. The cell of claim 16, wherein the plant cell is from a monocot plant.

19. The cell of claim 16, wherein the plant cell is from a dicot plant.

20. The cell of claim 19, wherein the plant cell is from a Cannabis plant.

21. A transgenic cell comprising the DNA construct of any one of claims 1-13 stably incorporated into the genome.

22. The transgenic cell of claim 21, wherein the transgenic cell is a transgenic plant cell.

23. A transgenic plant comprising the transgenic plant cell of claim 22.

24. The transgenic plant of claim 23, wherein said plant is a monocot plant.

25. The transgenic plant of claim 23, wherein said plant is a dicot plant.

26. The transgenic plant of claim 25, wherein the plant is a Cannabis plant.

27. A transgenic seed, clone, pollen, or floral bud from the transgenic plant of claim 26, wherein the seed, clone, pollen, or floral bud comprises the DNA construct.

28. A method of expressing a coding sequence in a transformed plant cell, said method comprising: transforming a plant cell with a DNA construct comprising: a first nucleotide sequence comprising a Cannabis Ubiquitin promoter, wherein the first nucleotide sequence comprises a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26 or a functional fragment thereof, and a second nucleotide sequence heterologous to the first nucleotide sequence, wherein the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence is capable of controlling expression of the second nucleotide sequence; wherein said transforming expresses the coding sequence in the transformed plant cell.

29. The method of claim 28, wherein the first nucleotide comprises any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof.

30. The method of claim 28, wherein the first nucleotide sequence has at least 80% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof.

31. The method of claim 28, wherein the first nucleotide sequence has at least 90% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26 or a functional fragment thereof.

32. The method of claim 28, wherein the first nucleotide sequence has at least 95% sequence identity to any one of SEQ ID NOs:1-4, 7-10, 12-15, 18-21, 23-26 or a functional fragment thereof.

33. A DNA construct comprising: a first nucleotide sequence comprising a Cannabis Ubiquitin 3′ terminator sequence having at least 70% sequence identity to any one of SEQ ID NOs:34-53 or a functional fragment thereof and a second nucleotide sequence heterologous to the first nucleotide sequence, wherein the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence contributes to transcriptional termination of the second nucleotide sequence.

34. The DNA construct of claim 33, wherein the first nucleotide sequence comprises any one of SEQ ID NOs:34-53 or a functional fragment thereof.

35. The DNA construct of claim 33, wherein the first nucleotide sequence has at least 80% sequence identity to any one of SEQ ID NOs:34-53 or a functional fragment thereof.

36. The DNA construct of claim 33, wherein the first nucleotide sequence has at least 90% sequence identity to any one of SEQ ID NOs:34-53 or a functional fragment thereof.

37. The DNA construct of claim 33, wherein the first nucleotide sequence has at least 95% sequence identity to any one of SEQ ID NOs:34-53 or a functional fragment thereof.

38. The DNA construct of any one of claims 33-37 further comprising: a promoter sequence.

39. The DNA construct of claim 38, wherein the promoter sequence comprises at least 95% identity to any one of SEQ ID NOs: 1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof.

40. The DNA construct of claim 38, wherein the promoter sequence comprises any one of SEQ ID NOs: 1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof.

41. The DNA construct of any one of claims 33-37, wherein the second nucleotide sequence encodes a polypeptide or a functional RNA.

42. The DNA construct of any one of claims 33-37, wherein the second nucleotide sequence encodes a functional RNA molecule; a molecule that confers increased yield, improved fiber content, increased oil content, increased protein content, disease resistance, insecticidal resistance, herbicide tolerance, nitrogen use efficiency, water use efficiency, nutritional quality; a DNA binding protein; a cannabinoid content gene; a terpene content gene; a reporter gene; a genome editing nuclease; a selectable marker; or combinations thereof.

43. A vector comprising the DNA construct of any one of claims 33-37.

44. A cell comprising the DNA construct of any one of claims 33-37.

45. The cell of claim 44, wherein the cell is a plant cell.

46. The cell of claim 45, wherein the DNA construct is not incorporated into the plant’s genome.

47. The cell of claim 45, wherein the plant cell is from a monocot plant.

48. The cell of claim 45, wherein the plant cell is from a dicot plant.

49. The cell of claim 48, wherein the plant cell is from a Cannabis plant.

50. A transgenic cell comprising the DNA construct of any one of claims 33- 37 stably incorporated into the genome.

51. The transgenic cell of claim 50, wherein the transgenic cell is a transgenic plant cell.

52. A transgenic plant comprising the transgenic plant cell of claim 51.

53. The transgenic plant of claim 52, wherein said plant is a monocot plant.

54. The transgenic plant of claim 52, wherein said plant is a dicot plant.

55. The transgenic plant of claim 54 wherein the plant is a Cannabis plant.

56. A transgenic seed, clone, pollen or floral bud from the transgenic plant of claim 55, wherein the seed, clone, pollen, or floral bud comprises the DNA construct.

57. A method of expressing a coding sequence in a transformed plant cell, said method comprising: transforming a plant cell with a DNA construct comprising: a first nucleotide sequence comprising a Cannabis Ubiquitin 3′ terminator sequence having at least 70% sequence identity to any one of SEQ ID NOs:34-53, and a second nucleotide sequence heterologous to the first nucleotide sequence, wherein the second nucleotide sequence is operably linked to the first nucleotide sequence such that the first nucleotide sequence contributes to transcriptional termination of the second nucleotide sequence; wherein said transforming expresses the coding sequence in the plant cell.

58. A DNA construct comprising: a first nucleotide sequence comprising a Cannabis Ubiquitin intron sequence having at least 70% sequence identity to any one of SEQ ID NOs:6, 17, 28, or a functional fragment thereof and a second nucleotide sequence heterologous to the first nucleotide sequence, wherein the second nucleotide sequence is operably linked to the first nucleotide sequence, such that the first nucleotide sequence contributes to transcription regulation of the second nucleotide sequence.

59. The DNA construct of claim 58, wherein the first nucleotide comprises any one of SEQ ID NO’s: 6, 17 or 28 or a functional fragment thereof.

60. The DNA construct of claim 58, wherein the first nucleotide sequence has at least 80% sequence identity to any one of SEQ ID NO’s: 6, 17 or 28 or a functional fragment thereof.

61. The DNA construct of claim 58, wherein the first nucleotide sequence has at least 90% sequence identity to any one of SEQ ID NO’s: 6, 17 or 28 or a functional fragment thereof.

62. The DNA construct of claim 58, wherein the first nucleotide sequence has at least 95% sequence identity to any one of SEQ ID NO’s: 6, 17 or 28 or a functional fragment thereof.

63. The DNA construct of any one of claims 58-62 further comprising: a promoter sequence.

64. The DNA construct of claim 63, wherein the promoter sequence comprises at least 95% identity to any one of SEQ ID NOs: 1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof.

65. The DNA construct of claim 63, wherein the promoter sequence comprises any one of SEQ ID NOs: 1-4, 7-10, 12-15, 18-21, 23-26, or a functional fragment thereof.

66. The DNA construct of any one of claims 58-62, wherein the first nucleotide sequence further comprises a 5′ UTR.

67. The DNA construct of claim 66, wherein the 5′ UTR comprises any one of SEQ ID NOs:5, 11, 16, 22, or 27.

68. The DNA construct of any one of claims 58-62, wherein the second nucleotide sequence is operably linked to a 3′ terminator sequence.

69. The DNA construct of claim 68, wherein the 3’ terminator sequence is any one of SEQ ID NOs:34-53.

70. The DNA construct of any one of claims 58-62, wherein the second nucleotide sequence encodes a polypeptide or a functional RNA.

71. The DNA construct of any one of claims 58-62, wherein the second nucleotide sequence encodes a functional RNA molecule; a molecule that confers increased yield, improved fiber content, increased oil content, increased protein content, disease resistance, insecticidal resistance, herbicide tolerance, nitrogen use efficiency, water use efficiency, nutritional quality; a DNA binding protein; a cannabinoid content gene; a terpene content gene; a reporter gene; a

enome editing nuclease; a selectable marker; or combinations thereof.

72. A vector comprising the DNA construct of any one of claims 58-62.

73. A cell comprising the DNA construct of any one of claims 58-62.

74. The cell of claim 73, wherein the cell is a plant cell.

75. The cell of claim 74, wherein the DNA construct is not incorporated into the plant’s genome.

76. The cell of claim 74, wherein the plant cell is from a monocot plant.

77. The cell of claim 74, wherein the plant cell is from a dicot plant. 78. The cell of claim 77, wherein the plant cell is from a Cannabis plant. 79. A transgenic cell comprising the DNA construct of any one of claims 58- 62 stably incorporated into the genome. 80. The transgenic cell of claim 79, wherein the transgenic cell is a transgenic plant cell. 81. A transgenic plant comprising the transgenic plant cell of claim 80. 82. The transgenic plant of claim 81, wherein said plant is a monocot plant. 83. The transgenic plant of claim 81, wherein said plant is a dicot plant. 84. The transgenic plant of claim 83 wherein the plant is a Cannabis plant. 85. A transgenic seed, clone, pollen or floral bud from the transgenic plant of claim 84, wherein the seed, clone, pollen, or floral bud comprises the DNA construct.