Patent application title: Gene Expression or Activity Enhancing Elements
Inventors:
IPC8 Class: AC12N1582FI
USPC Class:
1 1
Class name:
Publication date: 2020-11-19
Patent application number: 20200362358
Abstract:
The present invention relates to transgenic nucleic acids, expression
cassettes, vectors, plant cells, plant organs and plants. The invention
also relates to methods for increasing expression or activity of a target
gene, particularly in a plant cell or plant organ, and also to uses of
recombinant nucleic acids and expression cassettes to increase expression
or activity of a target gene or for manufacturing of a vector, plant
cell, plant organ or plant. Incidentally, the invention relates to
enhancers for achieving increased expression or activity of a target
gene, particularly in a plant cell or plant organ, when operably linked
to a promoter functional in such plant cell, plant organ or plant. The
invention is described herein with reference to the technical field of
production of polyunsaturated fatty acids (PUFAs), without being limited
to this technical field. For the production of desired molecules in plant
cells, e.g. PUFAs, it is frequently required to express a target gene
heterologous to the plant cell, or to overexpress a target gene naturally
found in said plant cell.Claims:
1. A recombinant nucleic acid, comprising a target gene and an
untranslated region adjacent to the target gene, wherein the untranslated
region comprises an enhancer of at least 18 consecutive nucleotides,
wherein at least 14 nucleotides are adenosine or cytidine.
2. A recombinant nucleic acid of claim 1, wherein the enhancer comprises any sequence according to SEQ ID NOS. 84, 85, 86, 87, 88 or 89.
3. A recombinant nucleic acid of claim 1, wherein the enhancer comprises or consists of i) 18 consecutive nucleotides, of which at least 15 nucleotides are adenosine or cytidine, or ii) 21 consecutive nucleotides, of which at least 15 nucleotides are adenosine or cytidine, or iii) 22 consecutive nucleotides, of which at least 16 nucleotides are adenosine or cytidine or iv) 24 consecutive nucleotides, of which at least 18nucleotides are adenosine or cytidine, or v) 36 consecutive nucleotides, of which at least 27 nucleotides are adenosine or cytidine or vi) 57 consecutive nucleotides, of which at least 42 nucleotides are adenosine or cytidine, or vii) 83 consecutive nucleotides, of which at least 62 nucleotides are adenosine or cytidine.
4. A recombinant nucleic acid of claim 1, wherein the enhancer comprises any sequence according to a) any of SEQ ID NOS. 20 to 45, or any sequence according to b) the last 18, 21, 22, 24, 36 or 57 nucleotides of any of SEQ ID NOS.46-83, 161-170, 221-230, 276-285 or 301-310, or c) any of SEQ ID NOS. 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 or 137, or d) a sequence according to b) or c) with 1 additional base inserted therein.
5. A recombinant nucleic acid, comprising a plant promoter and an untranslated region adjacent to the promoter, wherein the untranslated region comprises an enhancer of at least 18 consecutive nucleotides, wherein at least 14 nucleotides are adenosine or cytidine.
6. A recombinant nucleic acid according to claim 5, wherein the enhancer comprises or consists of i) 18 consecutive nucleotides, of which at least 15 nucleotides are adenosine or cytidine, or ii) 21 consecutive nucleotides, of which at least 15 nucleotides are adenosine or cytidine, or iii) 22 consecutive nucleotides, of which at least 16 nucleotides are adenosine or cytidine, or iv) 24 consecutive nucleotides, of which at least 18 nucleotides are adenosine or cytidine, or v) 36 consecutive nucleotides, of which at least 27 nucleotides are adenosine or cytidine, or vi) 57 consecutive nucleotides, of which at least 42 nucleotides are adenosine or cytidine, or vii) 83 consecutive nucleotides, of which at least 62 nucleotides are adenosine or cytidine.
7. A recombinant nucleic acid of claim 5, wherein the enhancer comprises any sequence according to a) any of SEQ ID NOS. 20 to 45, or any sequence according to b) the last 18, 21, 22, 24, 36 or 57 nucleotides of any of SEQ ID NOS. 46 to 83, or c) any of SEQ ID NOS. 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 or 137, or d) a sequence according to b) or c) with 1 additional base inserted therein.
8. A recombinant nucleic acid of claim 1, wherein the enhancer comprises a) a CCAAT-Box comprising SEQ ID NO. 100, and/or b) a Dof1/MNB1a binding site comprising SEQ ID NO. 102.
9. A recombinant nucleic acid of claim 1, wherein the target gene is a fatty acid desaturase or elongase gene.
10. An expression cassette, comprising a recombinant nucleic acid of claim 1 and a plant promoter, wherein the promoter comprises a TATA-box, and a CPRF factor binding site, and a TCP class I transcription factor binding site, and a bZIP protein G-Box binding factor 1 binding site.
11. An expression cassette of claim 10, wherein the promoter further comprises one or more of the following sequences: a Ry motif, a prolamin box, a Cis-element as in GAPDH promoters conferring light inducibility, a SBF-1 binding site, a Sunflower homeodomain leucine-zipper protein Hahb-4 binding site.
12. An expression cassette of claim 10 , wherein the promoter comprises or consists of a) a nucleic acid according to any of SEQ ID NOS. 141, 142, 144, 145, 147, 148, 150, 151, 153, 154, 156, 157, 159 to 310, or b) a nucleic acid having at least 70% sequence identity to any of the nucleic acid sequences according to a).
13. An expression cassette of claim 10, comprising or consisting of the sequence according to SEQ ID NO. 1 or of a sequence having at least 70% sequence identity to the sequence of SEQ ID NO. 1.
14. An expression cassette of claim 10, wherein the target gene does not consist of a sequence according to SEQ ID NO. 311.
15. A vector comprising an expression cassette of claim 10.
16. A plant, plant organ or plant cell comprising a recombinant nucleic acid of claim 1, operably linked to a promotor.
17. Method of increasing expression or activity of a target gene, comprising the steps of i) providing, upstream of the target gene, an untranslated region and a plant promoter to obtain an expression cassette of claim 9, and ii) introducing the expression cassette into a plant cell.
18. A plant, plant organ or plant cell comprising an expression cassette comprising a recombinant nucleic acid comprising a target gene and an untranslated region adjacent to the target gene, wherein the untranslated region comprises an enhancer of at least 18 consecutive nucleotides, wherein at least 14 nucleotides are adenosine or cytidine and a plant promoter, wherein the promoter comprises a TATA-box, and a CPRF factor binding site, and a TCP class I transcription factor binding site, and a bZIP protein G-Box binding factor 1 binding site.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent application Ser. No. 14/902,715, which is a National Stage application of International Application No. PCT/IB2014/062816 filed Jul. 3, 2014, which claims priority under 35 U.S.C. .sctn. 119 to European Patent Application No. 13175398.0, filed Jul. 5, 2013; all of the aforementioned applications are hereby incorporated herein by reference in their entirety.
[0002] The present invention relates to transgenic nucleic acids, expression cassettes, vectors, plant cells, plant organs and plants. The invention also relates to methods for increasing expression or activity of a target gene, particularly in a plant cell or plant organ, and also to uses of recombinant nucleic acids and expression cassettes to increase expression or activity of a target gene or for manufacturing of a vector, plant cell, plant organ or plant. Incidentally, the invention relates to enhancers for achieving increased expression or activity of a target gene, particularly in a plant cell or plant organ, when operably linked to a promoter functional in such plant cell, plant organ or plant. The invention is described herein with reference to the technical field of production of polyunsaturated fatty acids (PUFAs), without being limited to this technical field.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0003] This application was filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled "75088A_Seqlisting.TXT" created on Jul. 13, 2020, and is 203,784 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
[0004] WO 02/102970 discloses two Conlinin genes (Conlinin 1 and 2) and their respective promoter regions obtained from flax which can be utilized to improve seed traits, modify the fatty acid composition of seed oil and amino acid composition of seed storage protein, and produce bioactive compounds in plant seeds. The document also mentions methods based on using theses promoters to direct seed-specific expression of a gene of interest, which for example might be involved in lipid biosynthesis like e.g. acyl carrier protein, saturases, desaturases, and elongases.
[0005] WO 01/16340 discloses methods allowing the seed-specific expression of heterologous genes in flax and other plants. Of particular interest were promoters associated with fatty acid metabolism, such as acyl carrier protein, saturases, desaturases, elongases and the like.
[0006] Promoters function to initiate transcription of DNA into mRNA. Generally, transcribed mRNA comprises a translated region, also called a gene sequence, and upstream thereof an untranslated region. This untranslated region is generally believed not to have any profound influence on the translation of the gene sequence or the stability of the mRNA. Thus, the region between a promoter TATA box and a start codon is normally treated as being unimportant. For example, WO 01/16340 discloses a putative conlinin promotor, but the only GUS expression construct disclosed (herein reproduced as SEQ ID NO. 311) in this document is shortened on the 3' side of the putative promoter sequence.
[0007] Finding enhancer genetic elements which can improve the expression or activity of a target gene in a cell of interest is an ongoing demand for the development of improved agronomic traits. Specifically, oilseed crops producing modified fatty acid composition of the seed oil is a demand which makes the identification of further enhancing elements necessary; preferably, promoters are needed further improving the expression of genes of the fatty acid biosynthesis.
[0008] It has now been unexpectedly found that certain nucleic acids can improve expression or activity of a target or reporter gene when the gene is operably linked to a promoter.
[0009] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, plant species or genera, constructs, and reagents described as such. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.
[0010] It must be noted that as used herein and in the appended claims, the singular forms "a", "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art, and so forth. The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent, preferably 10 percent up or down (higher or lower). As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.
[0011] To overcome, reduce or mitigate the aforementioned disadvantages and/or to further the aforementioned goals and/or improve the aforementioned advantages, the invention provides a recombinant nucleic acid comprising a target gene and an untranslated region adjacent to the target gene, wherein the untranslated region comprises an enhancer of at least 18 consecutive nucleotides, wherein at least 14 nucleotides are adenosine or cytidine.
[0012] Describing the invention from another perspective, the invention provides a recombinant nucleic acid comprising a plant promoter and an untranslated region adjacent to the promoter, wherein the untranslated region comprises an enhancer of at least 18 consecutive nucleotides, wherein at least 14 nucleotides are adenosine or cytidine.
[0013] According to the invention is also provided an enhancer comprising
[0014] a) a CCAAT-Box comprising SEQ ID NO. 100, preferably comprising a sequence having at least 90% identity to SEQ ID NO. 101 and comprising SEQ ID NO. 100, and more preferably SEQ ID NO. 101, and/or
[0015] b) a Dof1/MNB1a binding site comprising SEQ ID NO. 102, preferably comprising a sequence having at least 90% identity to SEQ ID NO. 103 and comprising SEQ ID NO. 102, and more preferably SEQ ID NO. 103.
[0016] Further according to the invention is provided an expression cassette comprising a recombinant nucleic acid according to the invention, and, if not already comprised in the recombinant nucleic acid, a plant promoter, wherein the promoter comprises
[0017] a TATA-box, preferably comprising SEQ ID NO. 108, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 107 and comprising SEQ ID NO. 108, and more preferably comprising SEQ ID NO. 107, and
[0018] a CPRF factor binding site, preferably comprising SEQ ID NO. 114, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 113 and comprising SEQ ID NO. 114, and more preferably comprising SEQ ID NO. 113, and
[0019] a TCP class I transcription factor binding site, preferably comprising SEQ ID NO. 116, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 115 and comprising SEQ ID NO. 116, and more preferably comprising SEQ ID NO. 115, and 1
[0020] a bZIP protein G-Box binding factor 1 binding site, preferably comprising SEQ ID NO. 118, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 117 and comprising SEQ ID NO. 118, and more preferably comprising SEQ ID NO. 117.
[0021] The invention also provides a vector comprising the expression cassette of the present invention.
[0022] Further, the invention provides plants, plant organs or plant cells comprising a recombinant nucleic acid according to the present invention or an expression cassette according to the present invention.
[0023] The invention also teaches a method of increasing expression or activity of a target gene, comprising the steps of
[0024] i) providing, upstream of the target gene, an untranslated region and a plant promoter to obtain an expression cassette according to any of claims 9 to 11, and
[0025] ii) introducing the expression cassette into a plant cell to allow expression of the target gene.
[0026] According to the invention, an enhancer as described herein according to the invention or an expression cassette according to the invention can be used for
[0027] increasing expression or activity of a target gene,
[0028] producing a vector according to claim 15, or for
[0029] producing a plant, plant organ or plant cell according to claim 16.
[0030] The invention is hereinafter described in more detail. Unless specifically stated otherwise, the definitions of the chapter "definitions" apply throughout all of this text.
DETAILED DESCRIPTION OF THE INVENTION
[0031] One way to look at the invention is to understand that according to the present invention a recombinant nucleic acid is provided, said nucleic acid comprising a target gene and an untranslated region adjacent to the target gene. According to the invention, the untranslated region comprises an enhancer of at least 18 consecutive nucleotides, wherein at least 14 nucleotides are adenosine or cytidine.
[0032] The inventors have found that a particular section of a nucleic acid--preferred embodiments of which will be described hereinafter--functions as an enhancer in host cells, particularly plant cells, that is, activity of a target or reporter gene product can be increased by operably linking the reporter gene with the enhancer. The enhancer and the target gene are thus functionally linked, i.e. the enhancer influences or modifies transcription or translation of the target gene. Increase of activity by the enhancer of the present invention can be achieved by an increase in production of mRNA by the host cell, or can be achieved by an increased rate of translation of the mRNA, e.g. by improving the binding of ribosomes to the mRNA, or by protection of the mRNA against degradation. The invention, however, is not limited by any of these mechanisms.
[0033] The enhancer is preferably heterologous to the target gene and/or a promoter driving expression of the target gene. Thus, the following sequences are not part of the present invention: SEQ ID NO. 311 to 323 which are particularly comprised in WO0116340, WO2002102970, WO2009130291
[0034] The target gene can be any gene whose activity in a plant is desired to be increased. Increase is determined by comparison of the activity of the target gene being expressed in the same type of cell, e.g. seed cells, root cells and so on, without being functionally linked to the enhancer. Examples of useful target genes are fatty acid desaturase and fatty acid elongase genes, particularly d12d15Des(Ac_GA) (cf. WO 2007042510), d12Des(Ce_GA) (cf. US 2003172398), d12Des(Co_GA2) (cf. WO 200185968), d12Des(Fg) (cf. WO 2007133425), d12Des(Ps_GA) (cf. WO 2006100241), d12Des(Tp_GA) (cf. WO 2006069710), d6Des(Ol_febit) (cf. WO 2008040787), d6Des(Ol_febit)2 (cf. WO 2008040787), d6Des(Ot_febit) (cf. WO 2008040787), d6Des(Ot_GA) (cf. WO 2005083093), d6Des(Ot_GA2) (cf. WO 2005083093), d6Des(Pir) (cf. WO 2002026946), d6Des(Pir_GAI) (cf. WO 2002026946), d6Des(Plu) (cf. WO 2007051577), d6Elo(Pp_GA) (cf. WO 2001059128), d6Elo(Pp_GA2) (cf. WO 2001059128), d6Elo(Pp_GA3) (cf. WO 2001059128), d6Elo(Tp_GA) (cf. WO 2005012316) and d6Elo(Tp_GA2) (cf. WO 2005012316).
[0035] The enhancer is comprised in or forms an untranslated region adjacent to the target gene. For the present invention, the untranslated region is considered adjacent to the target gene if no translated region other than a region belonging to the target gene is located between the untranslated region comprising or consisting of the enhancer and the target gene. Thus, for example, in cases where the target gene comprises several exons, the untranslated region is considered to be located adjacent to the target gene when the untranslated region us located upstream of the first exon such that no translated region is located between the untranslated region comprising or being the enhancer and the first exon. For the sake of the present invention, exons are counted in 5' to 3' direction, so that the first exon is the one comprising the start codon of the target gene.
[0036] It is to be noted that the untranslated region may comprise, in addition to the enhancer of the present invention, further functional units including transcription or translation enhancing sequences. Regardless of whether the untranslated region comprises such further functional units the untranslated region is, for the purposes of the present invention, located adjacent to the target gene under the aforementioned conditions, that is, no translated region is located between the untranslated region and the target gene. It is preferred but not required that the enhancer of the present invention as such is adjacent to the translation start codon of the target gene.
[0037] The untranslated region may or may not be transcribed in a cell. In particular, the untranslated region may comprise translation enhancing sequences or mRNA stability enhancing sequences which are transcribed but not translated.
[0038] The untranslated region and the enhancer are preferably located upstream of the target gene. If the nucleobases of the target gene were numbered starting from 1 for the 5'-most nucleobase of the first translated codon (normally "A" of the codon "ATG" of a DNA sequence corresponding to "AUG" of the target gene mRNA) and incrementing the number in 3' direction, then nucleobases of the enhancer would be designated by negative numbers, as the untranslated region is preferably located in 5' direction of the target gene.
[0039] It is particularly preferred that the untranslated region comprises, in 5' to 3' direction, the enhancer of the present invention and one or more further functional units, particularly one or more NEENAs or RENAs as described for example in WO2013038294.
[0040] At the junction of the untranslated region and the target gene preferably a Kozak sequence is located. Preferably a Kozak sequence comprises the nucleotide sequence "ATGG", wherein the "ATG" is the start codon of the target gene. Kozak sequences facilitate the translation of the target gene. The skilled person can adapt the exact nucleotide sequence of the Kozak sequence according to the cell he would like to use, and also according to the expression needs of the target gene. For example, the skilled person could create all 256 variants of the sequence "NNATGNN", where "N" designates any nucleobase "A", "C", "G" or "T", and clone these variants in the cell he intends to use. By analyzing the activity of the target gene he will find the Kozak sequence optimal for his needs. The number of variants can be significantly reduced if the second amino acid is important for the functioning of the target gene, because in such cases at least the first nucleotide after (that is, in 3' direction of) the "ATG" start codon is limited to one or two alternatives. A preferred Kozak sequence is "CCATGG", as this sequence is also recognized by the restriction enzymes NcoI or Bsp19I, thus facilitating cloning of the target gene adjacent to the untranslated region. For the purposes of the present invention the leading "CC" nucleobases are considered to belong to the untranslated region.
[0041] The enhancer of the present invention comprises or consists of at least 18 consecutive nucleotides. The enhancer is thus not interrupted by any other element, be it a functional element or a non-functional element. As is described below, the enhancer and also the untranslated region can be substantially longer than 18 nucleotides, and preferably the enhancer consists of 57 or 58 consecutive nucleotides as described below.
[0042] The enhancer of the present invention has several beneficial features. For one, the enhancer sequence is short compared to other expression inducing sequences like NEENAs as described for example in WO2011023537, WO 2011023539, WO2011023800 or WO2013/005152. The enhancer thus can be incorporated with ease also in such constructs which are under severe size limitation, e.g. due to the number and/or size of genes to be incorporated in the respective construct.
[0043] Also, the enhancer has been shown to be active for a huge number of different target genes, particularly for desaturase and elongase genes of highly disparate sequence. Thus, the present invention provides a universally applicable enhacer for use in plants.
[0044] The enhancer of the present invention also is functional not only in Arabidopsis but also in other plants, particularly crop plants as described below, particularly in plant cells of the Brassicaceae family, even more in plant cells of genus Brassica and even more in particular in cells of Brassica napus, Brassica oleracea, Brassica carinata, Brassica nigra, Brassica juncea and Brassica rapa.
[0045] Another advantage of the enhancer of the present invention is that it is useful for increasing expression or activity of a target gene expressed under the control of a seed-specific promoter. Particularly the enhancer of the present invention can be combined with a Conlinin-type promoter to achieve seed-specific expression as described in WO2002102970. However, the enhancer of the present invention can also be combined with other promoters to increase expression or activity of a target gene.
[0046] The untranslated region or enhancer of the present invention preferably comprises any nucleotide sequence according to SEQ ID NO. 84, 85, 86, 87, 88 or 89. It is particularly preferred that the untranslated region or enhancer comprises two copies of one or more of the aforementioned sequences. The nucleotide sequences according to SEQ ID NO. 84, 85, 86, 87, 88 or 89 can be present in the untranslated region or enhancer in an overlapping form. For example, a nucleotide sequence of two cytidines followed by five adenosine nucleobases would simultaneously embody the sequences according to SEQ ID NO. 85, 86 and 87.
[0047] It is particularly preferred that the enhancer comprises the nucleotide sequence according to SEQ ID NO. 84. This sequence comprises the core motif SEQ ID NO. 100 of the plant CCAAT-box found in plant promoters. Thus, the presence of the sequence according to SEQ ID NO. 84 is particularly suitable for achieving the effects of transcription factor binding to this sequence. It is particularly preferred that the enhancer or untranslated region comprises two copies of SEQ ID NO. 84 separated by approximately 5 turns in a DNA helix, that is, a DNA sequence comprising the enhancer or untranslated region preferably comprises two instances of SEQ ID NO. 84 separated by 52, 53, 54, 55, 56, 57 or 58 nucleotides counting from the 1st nucleotide of the first (i.e. 5'-most) instance of SEQ ID NO. 84 to the last nucleotide in 5' direction in front of the second instance of SEQ ID NO. 84, preferably they are separated by 54, 55 or 56 nucleotides and most preferably by 55 nucleotides. For example, in the nucleotide sequence according to SEQ ID NO. 143 the instances of SEQ ID NO. 84 of the untranslated region are separated by 55 nucleotides.
[0048] It is also particularly preferred that the enhancer is functionally linked to a promoter such that the enhancer can be transcribed in a cell. This aspect of the invention is described in greater detail below. In such cases it is particularly preferred that the enhancer comprises at least one copy or instance of SEQ ID NO. 84.
[0049] According to the present invention, the enhancer preferably comprises or consists of 18 consecutive nucleotides, of which at least 15, preferably at least 17 nucleotides are adenosine or cytidine, and most preferably at most 1 nucleotide is neither adenosine nor cytidine. Preferred embodiments of such enhancers are described in SEQ ID NO. 20 to SEQ ID NO. 45. Of these, the sequences according to SEQ ID NO. 20, 22, 25, 28, 34, 35 and 36 are preferred as they comprise an instance of SEQ ID NO. 84. Particularly preferred is the sequence according to SEQ ID NO. 25, as this sequence comprises all of SEQ ID NO. 84, 100 and 101 and thus closely resembles a plant CCAAT box.
[0050] It is also preferred that the enhancer comprises or consists of 21 consecutive nucleotides, of which at least 15, preferably at least 16 nucleotides are adenosine or cytidine, and most preferably at most 2 nucleotides are neither adenosine nor cytidine. A correspondingly preferred sequence is given by SEQ ID NO. 95 and by the last 21 nucleotides of any of SEQ ID NO. 46, 96 161-170, 221-230, 276-285 and 301-310.
[0051] It is also preferred that the enhancer comprises or consists of 22 consecutive nucleotides, of which at least 16, preferably at least 17 nucleotides are adenosine or cytidine, and most preferably at most 2 nucleotides are neither adenosine nor cytidine. A preferred instance of such sequence is given by the last 22 nucleotides of any of SEQ ID NO. 46, 96 161-170, 221-230, 276-285 and 301-310.
[0052] It is also preferred that the enhancer comprises or consists of 24 consecutive nucleotides, of which at least 18, preferably at least 19 nucleotides are adenosine or cytidine, and most preferably at most 3 nucleotides are neither adenosine nor cytidine. A preferred instance of such sequence is given by the last 24 nucleotides of any of SEQ ID NO. 46, 96 161-170, 221-230, 276-285 and 301-310.
[0053] It is also preferred that the enhancer comprises or consists of 36 consecutive nucleotides, of which at least 27, preferably at least 28 nucleotides are adenosine or cytidine, and most preferably at most 6 nucleotides are neither adenosine nor cytidine. A preferred instance of such sequence is given by SEQ ID NO. 96 and by the last 36 nucleotides of any of SEQ ID NO. 46, 161-170, 221-230, 276-285 and 301-310.
[0054] It is also preferred that the enhancer comprises or consists of 57 consecutive nucleotides, of which at least 42, preferably at least 45 nucleotides are adenosine or cytidine, and most preferably at most 8 nucleotides are neither adenosine nor cytidine. Preferred examples of such enhancer are given by any of SEQ ID NO. 46 to 83, or the last 57 nucleotides of any of SEQ ID NO. 46, 96 161-170, 221-230, 276-285 and 301-310.
[0055] It is also preferred that the enhancer comprises or consists of 83 consecutive nucleotides, of which at least 62, preferably at least 65 nucleotides are adenosine or cytidine, and most preferably at most 8 nucleotides are neither adenosine nor cytidine. A preferred instance of such sequence is given by the last 83 nucleotides of any of SEQ ID NO. 161-170, 221-230, 276-285 and 301-310. Further preferred instances of such sequence are given by the last (i.e. counting from the 3' end) 83 nucleotides of a combination of SEQ ID NO. 140 and any of SEQ ID NO. 46 to 83, wherein the sequence of SEQ ID NO. 140 is fused immediately to the 5' end of any of SEQ ID NO. 46 to 83.
[0056] The sequences of SEQ ID NO. 161-170, 221-230, 276-285 and 301-310 are, for each group, sorted in descending order of preference. For example SEQ ID NO. 161 is more preferred than SEQ ID NO. 162, and SEQ ID NO. 221 is more preferred than SEQ ID NO. 230. The groups, however, are sorted in ascending order of preference, such that for example SEQ ID NO. 221 is more preferred than SEQ ID NO. 161 or SEQ ID NO. 170.
[0057] Among the sequences disclosed in the present application, the sequences of SEQ ID NO. 161-170, 221-230, 276-285 and 301-310 are special, because these sequences were checked not to affect major known or predicted cis-regulatory elements of the sequence according to SEQ ID NO. 1. The cis-regulatory elements that were checked comprise those mentioned below in greater detail, i.e. TATA-box, CPRF factor binding site, TCP class I transcription factor binding site, bZIP protein G-Box binding factor 1 binding site, Ry motif, prolamin box, Cis-element as in GAPDH promoters conferring light inducibility, SBF-1 binding site and Sunflower homeodomain leucine-zipper protein Hahb-4 binding site. This approach has been demonstrated to provide functional variants of the seed-specific p-PvARC5, the p-VfSBP and the p-BnNapin promoters in a GUS reporter gene assay and is described in more details in WO2012077020, which is incorporated herein by reference.
[0058] According to the present invention it is thus preferred if the enhancer comprises any sequence according to
[0059] a) any of SEQ ID NO. 20 to SEQ ID NO. 45, or any sequence according to
[0060] b) the last 18, 21, 22, 24, 36 or 57 nucleotides of any of SEQ ID NO.46-83, 161-170, 221-230, 276-285 or 301-310, or
[0061] c) any of SEQ ID NO. 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 or 137, or
[0062] d) a sequence according to b) or c) with 1 additional base inserted therein.
[0063] It is to be understood that preferably regardless of the number of A, C, G and T nucleotides an enhancer is considered an enhancer of the present invention if it consists of or comprises SEQ ID NO. 137.
[0064] Another way to look at the invention is to understand that according to the present invention a recombinant nucleic acid is provided, said nucleic acid comprising a plant promoter and an untranslated region adjacent to the promoter, wherein the untranslated region comprises an enhancer of the present invention. As described above, the enhancer consists of or comprises at least 18 consecutive nucleotides, wherein at least 14 nucleotides are adenosine or cytidine. Preferred enhancers and untranslated regions are described particularly above in greater detail. As described above, the enhancer is preferably heterologous to the promoter.
[0065] According to the present invention the untranslated region or enhancer is adjacent to a promoter as long as no translated region is present between the 3'-most TATA box of the promoter and the 5' end of the untranslated region or enhancer. Preferably, the enhancer is located immediately contiguous to the promoter 3' end, or is preferably separated from the promoter 3' end by at most 56 nucleotides, even more preferably by at most 39 nucleotides and even more preferably by at most 17 nucleotides. A preferred spacer sequence of 17 nucleotides length is given in SEQ ID NO. 140.
[0066] The promoter of the present invention preferably is a minimal promoter, and thus preferably consists only of the minimum length and nucleotide sequence required to achieve expression of a target gene functionally linked to said promoter and enhancer or untranslated region. This way the advantages described above due to the short length of the enhancer of the present invention are preserved. Particularly, a combination of a minimal promoter and the enhancer of the present invention allows to provide an expression cassette as described below that is not much longer than the target gene. Thus, the combination of a minimal promoter and the enhancer of the present invention allows cloning of long nucleotide sequences also in vectors and using transformation means which are restricted in size. This is particularly important when trying to establish, in plants, new metabolic pathways which require the introduction of multiple genes. Such pathways are for example described in WO2005083093, WO2007017419, WO2007042510 and WO2007096387.
[0067] The promoter can also be minimal in the sense that it consists only of the minimum length an nucleotide sequence required to function as a promoter under specific circumstances, e.g. driving expression of a gene functionally linked to said promoter only in specific plant tissues, developmental stages or under specific environmental conditions like heat stress or attempted pathogen infection. The promoter can, according to the present invention, also be longer or comprise more transcription influencing elements (e.g. transcription factor binding sites) than a minimal promoter. Suitable promoters are described e.g. in WO2002102970, WO2009077478, WO2010000708 and WO2012077020, the contents of which are incorporated herein by reference.
[0068] A preferred promoter comprises a TATA-box, preferably comprising SEQ ID NO. 108, more preferably comprising a sequence having at least 89% identity to SEQ ID NO. 107 and comprising SEQ ID NO. 108, and more preferably comprising SEQ ID NO. 107. Such TATA box facilitates onset of transcription particularly in plant cells. As the TATA box at least comprises the core motif SEQ ID NO. 108 of plant TATA boxes, at least a minimal activity of the promoter in plant can be achieved. If the promoter does not comprise the exact sequence SEQ ID NO. 107, then the promoter preferably comprises at least a sequence similar thereto. Such similar sequence contains the exact sequence SEQ ID NO. 108 and has a minimum of 89% identity to SEQ ID NO. 107 and thus preferably differs by at most two nucleotides from the sequence of SEQ ID NO. 107, and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 107.
[0069] A preferred promoter comprises a CPRF factor binding site, preferably comprising SEQ ID NO. 114, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 113 and comprising SEQ ID NO. 114, and more preferably comprising SEQ ID NO. 113. Where such promoter does not comprise the exact sequence SEQ ID NO. 113, it comprises a sequence differing from SEQ ID NO. 113 by at most one nucleotide and contains in this sequence the exact sequence SEQ ID NO. 114.
[0070] A preferred promoter comprises a TCP class I transcription factor binding site, preferably comprising SEQ ID NO. 116, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 115 and comprising SEQ ID NO. 116, and more preferably comprising SEQ ID NO. 115. Where such promoter does not comprise the exact sequence SEQ ID NO. 115, it comprises a sequence differing from SEQ ID NO. 115 by at most one nucleotide and contains in this sequence the exact sequence SEQ ID NO. 116.
[0071] A preferred promoter comprises a bZIP protein G-Box binding factor 1 binding site, preferably comprising SEQ ID NO. 118, more preferably comprising a sequence having at least 85% identity to SEQ ID NO. 117 and comprising SEQ ID NO. 118, and more preferably comprising SEQ ID NO. 117. If the promoter does not comprise the exact sequence SEQ ID NO. 117, then the promoter preferably comprises at least a sequence similar thereto. Such similar sequence contains the exact sequence SEQ ID NO. 118 and has a minimum of 85% identity to SEQ ID NO. 117 and thus preferably differs by at most three nucleotides from the sequence of SEQ ID NO. 117, more preferably differs by at most two nucleotides from the sequence of SEQ ID NO 117 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 117.
[0072] A preferred promoter comprises a Ry motif, preferably comprising SEQ ID NO. 110, more preferably comprising a sequence having at least 88% identity to SEQ ID NO. 109 and comprising SEQ ID NO. 110, and more preferably comprising SEQ ID NO. 109. Where such promoter does not comprise the exact sequence SEQ ID NO. 109, contains the exact sequence SEQ ID NO. 110 and has a minimum of 88% identity to SEQ ID NO. 109 and thus preferably differs by at most three nucleotides from the sequence of SEQ ID NO. 109, more preferably differs by at most two nucleotides from the sequence of SEQ ID NO 109 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 109.
[0073] A preferred promoter comprises a prolamin box, preferably comprising SEQ ID NO. 112, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 111 and comprising SEQ ID NO. 112, and more preferably comprising SEQ ID NO. 111. Where such promoter does not comprise the exact sequence SEQ ID NO. 111, contains the exact sequence SEQ ID NO. 112 and has a minimum of 90% identity to SEQ ID NO. 111 and thus preferably differs by at most two nucleotides from the sequence of SEQ ID NO 111 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 111.
[0074] A preferred promoter comprises a Cis-element as in GAPDH promoters conferring light inducibility, preferably comprising SEQ ID NO. 120, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 119 and comprising SEQ ID NO. 120, and more preferably comprising SEQ ID NO. 119. Where such promoter does not comprise the exact sequence SEQ ID NO. 119, contains the exact sequence SEQ ID NO. 120 and has a minimum of 90% identity to SEQ ID NO. 119 and thus preferably differs by at most two nucleotides from the sequence of SEQ ID NO 119 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 119.
[0075] A preferred promoter comprises a SBF-1 binding site, preferably comprising SEQ ID NO. 122, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 121 and comprising SEQ ID NO. 122, and more preferably comprising SEQ ID NO. 121. Where such promoter does not comprise the exact sequence SEQ ID NO. 121, contains the exact sequence SEQ ID NO. 122 and has a minimum of 90% identity to SEQ ID NO. 121 and thus preferably differs by at most two nucleotides from the sequence of SEQ ID NO 121 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 121.
[0076] A preferred promoter comprises a Sunflower homeodomain leucine-zipper protein Hahb-4 binding site, preferably comprising SEQ ID NO. 124, more preferably comprising a sequence having at least 80% identity to SEQ ID NO. 123 and comprising SEQ ID NO. 124, and more preferably comprising SEQ ID NO. 123. Where such promoter does not comprise the exact sequence SEQ ID NO. 123, contains the exact sequence SEQ ID NO. 124 and has a minimum of 80% identity to SEQ ID NO. 123 and thus preferably differs by at most two nucleotides from the sequence of SEQ ID NO 123 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 123.
[0077] A preferred promoter comprises a Transcriptional repressor BELLRINGER, preferably comprising SEQ ID NO. 126, more preferably comprising a sequence having at least 80% identity to SEQ ID NO. 125 and comprising SEQ ID NO. 126, and more preferably comprising SEQ ID NO. 125. Where such promoter does not comprise the exact sequence SEQ ID NO. 125, contains the exact sequence SEQ ID NO. 126 and has a minimum of 80% identity to SEQ ID NO. 125 and thus preferably differs by at most two nucleotides from the sequence of SEQ ID NO 125 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 125.
[0078] A preferred promoter comprises a Floral homeotic protein APETALA1, preferably comprising SEQ ID NO. 128, more preferably comprising a sequence having at least 85% identity to SEQ ID NO. 127 and comprising SEQ ID NO. 128, and more preferably comprising SEQ ID NO. 127. Where such promoter does not comprise the exact sequence SEQ ID NO. 127, contains the exact sequence SEQ ID NO. 128 and has a minimum of 85% identity to SEQ ID NO. 127 and thus preferably differs by at most three nucleotides from the sequence of SEQ ID NO 127, more preferably by at most two nucleotides from the sequence of SEQ ID NO 127 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 127.
[0079] A preferred promoter comprises an inducer of CBF expression 1, also called AtMYC2 (rd22BP1), preferably comprising SEQ ID NO. 130, more preferably comprising a sequence having at least 85% identity to SEQ ID NO. 129 and comprising SEQ ID NO. 130, and more preferably comprising SEQ ID NO. 129. Where such promoter does not comprise the exact sequence SEQ ID NO. 129, contains the exact sequence SEQ ID NO. 130 and has a minimum of 85% identity to SEQ ID NO. 129 and thus preferably differs by at most two nucleotides from the sequence of SEQ ID NO 129 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 129.
[0080] A preferred promoter comprises a binding site for bZIP factors DPBF-1 and/or 2, preferably comprising SEQ ID NO. 132, more preferably comprising a sequence having at least 81% identity to SEQ ID NO. 131 and comprising SEQ ID NO. 132, and more preferably comprising SEQ ID NO. 131. Where such promoter does not comprise the exact sequence SEQ ID NO. 131, contains the exact sequence SEQ ID NO. 132 and has a minimum of 81% identity to SEQ ID NO. 131 and thus preferably differs by at most two nucleotides from the sequence of SEQ ID NO 131 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 131.
[0081] A preferred promoter comprises a binding site for Class I GATA factors, preferably comprising SEQ ID NO. 134, more preferably comprising a sequence having at least 88% identity to SEQ ID NO. 133 and comprising SEQ ID NO. 134, and more preferably comprising SEQ ID NO. 133. Where such promoter does not comprise the exact sequence SEQ ID NO. 133, contains the exact sequence SEQ ID NO. 134 and has a minimum of 88% identity to SEQ ID NO. 133 and thus preferably differs by at most two nucleotides from the sequence of SEQ ID NO 133 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 133.
[0082] A preferred promoter comprises a binding site for Dof2 single zinc finger transcription factor, preferably comprising SEQ ID NO. 136, more preferably comprising a sequence having at least 88% identity to SEQ ID NO. 135 and comprising SEQ ID NO. 136, and more preferably comprising SEQ ID NO. 135. Where such promoter does not comprise the exact sequence SEQ ID NO. 135, contains the exact sequence SEQ ID NO. 136 and has a minimum of 88% identity to SEQ ID NO. 135 and thus preferably differs by at most two nucleotides from the sequence of SEQ ID NO 135 and even more preferably differs by at most one nucleotide from the sequence of SEQ ID NO 135.
[0083] A preferred promoter comprises a combination of two or more of the aforementioned transcription factor binding sites or cis-active elements. Preferably, the promoter comprises a TATA-box, a CPRF binding site, a TCP class I transcription factor binding site and a bZIP protein G-Box binding factor 1 binding site, each as defined above. Particularly preferred is a promoter comprising the sequence SEQ ID NO. 138 and/or SEQ ID NO. 139, or a sequence being at least 70% identical, preferably 80% identical, more preferably at least 90% identical to any of these sequences, and even more preferably differing from any of these sequences by at most 10 nucleotides, even more preferably by at most 9 nucleotides, even more preferably by at most 8 nucleotides, even more preferably by at most 7 nucleotides, even more preferably by at most 6 nucleotides, even more preferably by at most 5 nucleotides, even more preferably by at most 4 nucleotides, even more preferably by at most 3 nucleotides, even more preferably by at most 2 nucleotides, even more preferably by at most 1 nucleotide. Where such promoter comprises a sequence being at least 70% identical to SEQ ID NO. 138, the promoter preferably comprises at least one binding site for each of the transcription factors CPRF, TCP class I transcription factor and bZIP protein G-Box binding factor 1 as defined above, and preferably comprises at least each sequence according to SEQ ID NO. 114, 118 and 120. Where such promoter comprises a sequence being at least 70% identical to SEQ ID NO. 139, the promoter preferably comprises at least one binding site for each of the transcription factors BELLRINGER, APETALA1, CBF expression inducer 1, DPBF-1 and 2, Class I GATA factors and Dof2 as defined above, and preferably comprises at least each sequence according to SEQ ID NO. 126, 128, 130, 132, 134 and 136.
[0084] The function of the transcription factors referred herein are known to the skilled person. By providing the corresponding transcription factor binding sites ,e.g. as defined above, the skilled person achieves the benefits inherent in the action of these transcription factors. Particularly, the skilled person can combine two or more and preferably all of the aforementioned transcription factor binding sites.
[0085] With respect to the present invention the difference between nucleic acid sequences is calculated as the minimum number of substitutions, insertions or deletions required to transform one sequence into the other. Thus, for example, a sequence "ACGT" and "ATGT" differ by one nucleotide and have 75% sequence identity relative to the first sequence, and the sequences "AACCGGTT" and "AACTGTT" differ by two nucleotides, i.e one deletion and one substitution, and have 87.5% sequence identity relative to the first sequence. For the purposes of the present invention, sequences are given in the form of DNA sequences, the corresponding RNA sequences being considered identical, such that a substitution of "T" by "U" and vice versa is disregarded.
[0086] The promoter preferably has a length of at least or exactly 98 nucleotides, even more preferably at least or exactly 142 nucleotides, even more preferably at least or exactly 160 nucleotides, even more preferably at least or exactly 197 nucleotides, even more preferably at least or exactly 235 nucleotides and even more preferably at least or exactly 1063 nucleotides. A promoter having a length of not more than 98 nucleotides is particularly suitable for cloning of target genes under severe size limitation, a promoter of not more than 142 nucleotides is also still useful for cloning of target genes under severe size limitation, a promoter of not more than 160 nucleotides is also still useful for cloning of target genes under severe size limitation but is less preferred due to its larger size, a promoter of not more than 197 nucleotides is also still useful for cloning of target genes under severe size limitation but is less preferred due to its larger size, a promoter of not more than 235 nucleotides is also still useful for cloning of target genes under severe size limitation but is less preferred due to its larger size. Suitable promoters are selected preferably among those given in any of SEQ ID NO. 141, 144, 147, 150, 153, 156 and 159, and also in any of SEQ ID NO. 171-220, 231-275 and 286-300. Suitable promoters are also selected preferably among those having at least 70%, more preferably at least 80% and more preferably at least 90% sequence identity to any of SEQ ID NO. 141, 144, 147, 150, 153, 156, 159, 171-220, 231-275 and 286-300, and preferably comprise two or more transcription factor binding sites as described above.
[0087] Preferred nucleic acid sequences comprising a combination of a promoter and an enhancer of the present invention are selected from those of SEQ ID NO. 143, 146, 149, 152, 155, 158 and 1, and also from those of SEQ ID NO. 161-170, 221-230, 276-285 and 301-310. The order of preference for the sequences of SEQ ID NO. 161-170, 221-230, 276-285 and 301-310 and the reasons therefore are given above.
[0088] The invention also provides an expression cassette, comprising or consisting of a recombinant nucleic acid as described above. Where such recombinant nucleic acid does not already comprise a promoter, the expression cassette additionally comprises a promoter, preferably a plant promoter as described above. Thus, an expression cassette according to the present invention comprises, in 5' to 3' direction, a promoter, an untranslated region being or comprising the enhancer of the present invention, a target gene and optionally a terminator or other elements. The expression cassette of the present invention preferably comprises a promoter as defined above and an untranslated region or enhancer as described above. This way, the advantages attributed supra to the promoter and enhancer can be achieved using the expression cassette of the present invention. The expression cassette allows an easy transfer of a target gene into an organism, preferably a cell and preferably a plant cell.
[0089] Thus, the expression cassette of the present invention preferably comprises a promoter which in turn comprises
[0090] a TATA-box, preferably comprising SEQ ID NO. 108, more preferably comprising a sequence having at least 89% identity to SEQ ID NO. 107 and comprising SEQ ID NO. 108, and more preferably comprising SEQ ID NO. 107, and
[0091] a CPRF factor binding site, preferably comprising SEQ ID NO. 114, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 113 and comprising SEQ ID NO. 114, and more preferably comprising SEQ ID NO. 113, and
[0092] a TCP class I transcription factor binding site, preferably comprising SEQ ID NO. 116, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 115 and comprising SEQ ID NO. 116, and more preferably comprising SEQ ID NO. 115, and
[0093] a bZIP protein G-Box binding factor 1 binding site, preferably comprising SEQ ID NO. 118, more preferably comprising a sequence having at least 85% identity to SEQ ID NO. 117 and comprising SEQ ID NO. 118, and more preferably comprising SEQ ID NO. 117, and preferably also comprises at least one, preferably at least two, more preferably at least three and most preferably all of the following elements:
[0094] a Ry motif, preferably comprising SEQ ID NO. 110, more preferably comprising a sequence having at least 88% identity to SEQ ID NO. 109 and comprising SEQ ID NO. 110, and more preferably comprising SEQ ID NO. 109,
[0095] a prolamin box, preferably comprising SEQ ID NO. 112, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 111 and comprising SEQ ID NO. 112, and more preferably comprising SEQ ID NO. 111,
[0096] a Cis-element as in GAPDH promoters conferring light inducibility, preferably comprising SEQ ID NO. 120, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 119 and comprising SEQ ID NO. 120, and more preferably comprising SEQ ID NO. 119,
[0097] a SBF-1 binding site, preferably comprising SEQ ID NO. 122, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 121 and comprising SEQ ID NO. 122, and more preferably comprising SEQ ID NO. 121, and
[0098] a Sunflower homeodomain leucine-zipper protein Hahb-4 binding site, preferably comprising SEQ ID NO. 124, more preferably comprising a sequence having at least 90% identity to SEQ ID NO. 123 and comprising SEQ ID NO. 124, and more preferably comprising SEQ ID NO. 123.
[0099] Also preferably the promotor comprises or consists of
[0100] a) a nucleic acid according to any of SEQ ID NO. 141, 142, 144, 145, 147, 148, 150, 151, 153, 154, 156, 157, 159 to 310, or
[0101] b) a nucleic acid having at least 70% sequence identity to any of the nucleic acid sequences according to a).
[0102] The advantages conferred with such promoters are described above.
[0103] Most preferred is a promoter-enhancer combination comprising or consisting of the sequence according to SEQ ID NO 1 or of a sequence having at least 70% sequence identity to the sequence of SEQ ID NO. 1. Such sequence is found flax (Linum usitatissimum) and allows for seed specific and highly active expression of more or less any target gene expressible in plant seeds. Interestingly the advantages conferred by the combination of a promoter, particularly the promoter found in SEQ ID NO. 1, and the enhancer of the present invention had not been noticed despite attempts in the prior art to analyze the characteristics of the promoter. For example, in WO0116340 a construct is created comprising the promoter found in SEQ ID NO. 1, but the enhancer region had been deleted. Thus, only a sequence as given in SEQ ID NO. 311 has been fused in this document to a GUS reporter gene. However, it has now been found that by including the enhancer of the present invention a substantial increase of reporter gene activity can be achieved.
[0104] The expression cassette of the present invention is preferably comprised in a vector. Thus, the vector of the present invention allows to transform a cell, preferably a plant cell, with a long target gene or a combination of multiple genes while achieving a high expression or activity of the target gene functionally linked to the enhancer of the present invention.
[0105] Correspondingly the invention provides a plant, plant organ or plant cell comprising an expression cassette according to the present invention or a recombinant nucleic acid according to the present invention. Of course the recombinant nucleic acid should also comprise a promoter such as to allow for the expression of the target gene, because an increase of expression or activity of the target gene by the enhancer of the present invention obviously cannot be effected if the target gene is not expressed at all due to lack of a promoter. The plant, plant organ or plant cell makes use of the advantages conferred by the enhancer, recombinant nucleic acid or expression cassette of the present invention such that expression or activity of the target gene is increased compared to a plant, plant organ or plant cell comprising the same promoter and target gene combination without the enhancer of the present invention.
[0106] From what is given above it is clear that the invention also provides a method of increasing expression or activity of a target gene, comprising the steps of
[0107] i) providing, upstream of the target gene, an untranslated region and a plant promotor to obtain an expression cassette according to the present invention, and
[0108] ii) introducing the expression cassette into a plant cell.
[0109] The enhancer is, corresponding to the indications given above, preferably heterologous to the promoter and/or to the target gene. The expression cassette is introduced into the plant cell to allow for expression of the target gene in the plant cell or in plant cells derived from the exact plant cell that was subjected to introduction of the expression cassette. Thus, the above method of the invention encompasses the introduction of the expression cassette into a first plant cell and growth of further cells from the first cells, wherein the further cells can form for example a full plant or a plant organ, preferably a seed. Depending on the promoter of the expression cassette, the target gene is expressed in one or more of the further cells or during a selected stage of growth, for example during seed formation, or under selected environmental conditions, for example heat or drought stress or pathogen infection.
[0110] Also as described above the enhancer or expression cassette of the present invention is used for
[0111] increasing expression or activity of a target gene,
[0112] producing a vector according to the present invention, and/or for
[0113] producing a plant, plant organ or plant cell according to the present invention.
[0114] The advantages conferred by the above uses have been described supra in detail.
[0115] Unless indicated otherwise, the following definitions apply for the current invention:
[0116] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof ("polynucleotides") in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base, which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
[0117] A "codon" is a nucleotide sequence of three nucleotides which encodes a specific amino acid.
[0118] One nucleotide sequence can be "complementary" to another sequence, meaning that they have the base on each position is the complementary (i.e. A to T, C to G) and in the reverse order. If one strand of the double-stranded DNA is considered the "sense" strand, then the other strand, considered the "antisense" strand, will have the complementary sequence to the sense strand. This distinction is due to "sense" sequences which code for proteins, and the complementary "antisense" sequences which are by nature non-functional.
[0119] A "nucleic acid fragment" is a fragment of a given nucleic acid molecule.
[0120] "Genetic elements" are nucleic acid fragments of solitary building blocks like genes, introns, promoters etc.
[0121] In higher plants, deoxyribonucleic acid (DNA) is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
[0122] The terms "nucleic acid" or "nucleic acid sequence" or "polynucleotide sequence" are used interchangeably.
[0123] The "degeneracy code" is reflecting the redundancy of the genetic code characterized by its non-ambiguity. For example, although codons GAA and GAG both specify glutamic acid (redundancy), neither of them specifies any other amino acid (no ambiguity). Degeneracy results because there are more codons than amino acids to be encoded. Degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer 1991; Ohtsuka 1985; Rossolini 1994).
[0124] The term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. For example, gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences. Genes also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
[0125] The terms "genome" or "genomic DNA" is referring to the heritable genetic information of a host organism. Said genomic DNA comprises the DNA of the nucleus (also referred to as chromosomal DNA) but also the DNA of the plastids (e.g. chloroplasts) and other cellular organelles (e.g. mitochondria). Preferably the terms genome or genomic DNA is referring to the chromosomal DNA of the nucleus.
[0126] The term "chromosomal DNA" or "chromosomal DNA-sequence" is to be understood as the genomic DNA of the cellular nucleus independent from the cell cycle status. Chromosomal DNA might therefore be organized in chromosomes or chromatids, they might be condensed or uncoiled. An insertion into the chromosomal DNA can be demonstrated and analyzed by various methods known in the art like e.g., polymerase chain reaction (PCR) analysis, Southern blot analysis, fluorescence in situ hybridization (FISH), and in situ PCR.
[0127] "Coding sequence" refers to a DNA or RNA molecule that codes for a specific amino acid molecule and excludes the "non-coding sequences". It may constitute an "uninterrupted coding sequence", i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions. An "intron" is a molecule of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.
[0128] A "regulatory sequence" refers to nucleotide molecules influencing the transcription, RNA processing or stability, or translation of the associated (or functionally linked) nucleotide molecules to be transcribed. The transcription regulating nucleotide molecule may have various localizations with respect to the nucleotide molecules to be transcribed. The transcription regulating nucleotide molecule may be located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of the molecule to be transcribed (e.g., a coding sequence). The transcription regulating nucleotide molecule may be selected from the group comprising enhancers, promoters, translation leader sequences, introns, 5'-untranslated sequences, 3'-untranslated sequences, and polyadenylation signal sequences. They include natural and synthetic molecules as well as molecules, which may be a combination of synthetic and natural molecules. The term "transcription regulating nucleotide molecule" is not limited to promoters. However, preferably a transcription regulating nucleotide molecule of the invention comprises at least one promoter molecule (e.g., a molecule localized upstream of the transcription start of a gene capable to induce transcription of the downstream molecules). In one preferred embodiment the transcription regulating nucleotide molecule of the invention comprises the promoter molecule of the corresponding gene and--optionally and preferably--the native 5'-untranslated region of said gene. Furthermore, the 3'-untranslated region and/or the polyadenylation region of said gene may also be employed. As used herein, the term "cis-element" or "promoter motif" refers to a cis-acting transcriptional regulatory element that confers an aspect of the overall control of gene expression. A cis-element may function to bind transcription factors, transacting protein factors that regulate transcription. Some cis-elements bind more than one transcription factor, and transcription factors may interact with different affinities with more than one cis-element.
[0129] A "functional RNA" refers to an antisense RNA, microRNA, siRNA, ribozyme, or other RNA that is not translated.
[0130] "Transcription" takes place when RNA polymerase makes a copy from the DNA to mRNA. "mRNA" conveys genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression. Non-eukaryotic mRNA is, in essence, mature upon transcription and normally requires no processing. Eukaryotic pre-mRNA, requires "processing", meaning that the pre-mRNA is modified post-transcriptionally. Processing includes e.g. 5' cap addition, splicing, polyadenylation.
[0131] The term "RNA transcript" refers to the product resulting from RNA polymerase catalyzed transcription of a DNA molecule. When the RNA transcript is a perfect complementary copy of the DNA molecule, it is referred to as the primary transcript or it may be a RNA molecule derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.
[0132] "Messenger RNA" (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell.
[0133] "cDNA" refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA.
[0134] The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. "Translation" proceeds in four phases: initiation, elongation, translocation and termination. The terms "initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides ("codon") in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation). Initiation involves the small subunit of the ribosome binding to the 5' end of mRNA with the help of initiation factors (IF). The start codon is the first codon of a mRNA transcript translated by a ribosome. The start codon always codes for methionine in eukaryotes and a modified Met (fMet) in prokaryotes. The most common start codon is AUG. Termination of the polypeptide happens when the A site of the ribosome faces a stop codon (UAA, UAG, or UGA).
[0135] "5' non-coding sequence" or "5'-untranslated sequence" or "-region" refers to a sequence of a nucleotide molecule located 5' (upstream) to the codikeine ahnungng sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
[0136] "3' non-coding sequence" or "3'-untranslated sequence" or "-region" refers to a sequence of a nucleotide molecule located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al., 1989.
[0137] "Promoter" refers to a nucleotide molecule, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. "Promoter" also refers to a nucleotide molecule that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter molecule consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, such an "enhancer" is a DNA molecule which 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. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. 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 be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors, which control the effectiveness of transcription initiation in response to physiological or developmental conditions. A person skilled in the art is aware of methods for rendering a unidirectional to a bidirectional promoter and of methods to use the complement or reverse complement of a promoter sequence for creating a promoter having the same promoter specificity as the original sequence. Such methods are for example described for constitutive as well as inducible promoters by Xie et al. (2001) "Bidirectionalization of polar promoters in plants" nature biotechnology 19 pages 677-679. The authors describe that it is sufficient to add a minimal promoter to the 5' prime end of any given promoter to receive a promoter controlling expression in both directions with same promoter specificity. The promoters of the present invention desirably contain cis-elements that can confer or modulate gene expression, also called transcription factor binding sites. Cis-elements can be identified by a number of techniques, including deletion analysis, i.e., deleting one or more nucleotides from the 5' end or internal to a promoter; DNA binding protein analysis using DNase I footprinting, methylation interference, electrophoresis mobility-shift assays, in vivo genomic footprinting by ligation-mediated PCR, and other conventional assays; or by DNA sequence similarity analysis with known cis-element motifs by conventional DNA sequence comparison methods. The fine structure of a cis-element can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods. Cis-elements can be obtained by chemical synthesis or by isolation from promoters that include such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequent manipulation.
[0138] The "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.
[0139] Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters." In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A "minimal or core promoter" thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.
[0140] "Constitutive promoter" refers to a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant. Each of the transcdankmachs gut, and fuhl dich gedrucktription-activating elements do not exhibit an absolute tissue-specificity, but mediate transcriptional activation in most plant parts at a level of at least 1% of the level reached in the part of the plant in which transcription is most active.
[0141] "Regulated promoter" refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and includes both tissue-specific and inducible promoters. It includes natural and synthetic molecules as well as molecules which may be a combination of synthetic and natural molecules. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al. (1989). Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysone-inducible systems.
[0142] "Tissue-specific promoter" refers to regulated promoters that are not expressed in all plant cells but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as epidermis, green tissue, embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporally regulated, such as in early or late embryogenesis, during leaf expansion fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence.
[0143] "Tissue-specific transcription" in the context of this invention means the transcription of a nucleic acid molecule by a transcription regulating nucleic acid molecule in a way that transcription of said nucleic acid molecule in said tissue contribute to more than 90%, preferably more than 95%, more preferably more than 99% of the entire quantity of the RNA transcribed from said nucleic acid molecule in the entire plant during any of its developmental stage. The transcription regulating nucleotide molecules specifically disclosed herein are considered to be tissue-specific transcription regulating nucleotide molecules.
[0144] "Tissue-preferential transcription" in the context of this invention means the transcription of a nucleic acid molecule by a transcription regulating nucleic acid molecule in a way that transcription of said nucleic acid sequence in the said tissue contribute to more than 50%, preferably more than 70%, more preferably more than 80% of the entire quantity of the RNA transcribed from said nucleic acid sequence in the entire plant during any of its developmental stage.
[0145] "Inducible promoter" refers to those regulated promoters that can be turned on in one or more cell types or that cause increased expression upon an external stimulus, such as a chemical, light, hormone, stress, or a pathogen.
[0146] A terminator, or transcription terminator is a section of genetic sequence that marks the end of gene or operon on genomic DNA for transcription.
[0147] The term "translation leader sequence" refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
[0148] As part of gene expression, "translation" is the process through which cellular ribosomes manufacture proteins. In translation, messenger RNA (mRNA) produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein. In bacteria, translation occurs in the cell's cytoplasm, where the large and small subunits of the ribosome are located, and bind to the mRNA. In eukaryotes, translation occurs across the membrane of the endoplasmic reticulum in a process called vectorial synthesis. The ribosome facilitates decoding by inducing the binding of transfer RNAs (tRNA) with complementary anticodon sequences to that of the mRNA.
[0149] The Kozak sequence on an mRNA molecule is recognized by the ribosome as the translational start site, from which a protein is coded by that mRNA molecule. The ribosome requires this sequence, or a possible variation to initiate translation. The sequence is identified by the notation (gcc)gccRccAUGG,
[0150] which summarizes data analysed by Kozak from a wide variety of sources (about 699 in all; Kozak M (October 1987). "An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs". Nucleic Acids Res. 15 (20): 8125-8148.) as follows: a lower case letter denotes the most common base at a position where the base can nevertheless vary; upper case letters indicate highly-conserved bases, i.e. the `AUGG` sequence is constant or rarely changes, `R` which indicates that a purine (adenine or guanine) is always observed at this position (with adenine being claimed by Kozak to be more frequent); and the sequence in brackets ((gcc)) is of uncertain significance. Preferably, the Kozak consensus sequence it that of Arabidopsis thaliana AAA-AUG-GC.
[0151] A transfer RNA (tRNA) is an adaptor molecule composed of RNA that serves as the physical link between the nucleotide sequence of nucleic acids (DNA and RNA) and the amino acid sequence of proteins. It does this by carrying an amino acid to the protein synthetic machinery of a cell (i.e. the ribosome) as directed by a codon in the mRNA.
[0152] "Expression" refers to the transcription and/or translation of an endogenous gene, ORF or portion thereof, or a transgene in plants. For example, in the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. In addition, expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein.
[0153] The "expression pattern" of a promoter (with or without enhancer) is the pattern of expression levels, which shows where in the plant and in what developmental stage transcription is initiated by said promoter. Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter. The level of expression of a promoter can be determined by measuring the "steady state" concentration of a standard transcribed reporter mRNA. This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded. Therefore, the steady state level is the product of synthesis rates and degradation rates. The rate of degradation can however be considered to proceed at a fixed rate when the transcribed molecules are identical, and thus this value can serve as a measure of synthesis rates. When promoters are compared in this way, techniques available to those skilled in the art are hybridization S1-RNAse analysis, northern blots and competitive RT-PCR. This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA. The analysis of transcription start points in practically all promoters has revealed that there is usually no single base at which transcription starts, but rather a more or less clustered set of initiation sites, each of which accounts for some start points of the mRNA. Since this distribution varies from promoter to promoter the sequences of the reporter mRNA in each of the populations would differ from each other. Since each mRNA species is more or less prone to degradation, no single degradation rate can be expected for different reporter mRNAs. It has been shown for various eukaryotic promoter molecules that the sequence surrounding the initiation site ("initiator") plays an important role in determining the level of RNA expression directed by that specific promoter. This includes also part of the transcribed sequences. The direct fusion of promoter to reporter molecules would therefore lead to suboptimal levels of transcription. A commonly used procedure to analyze expression patterns and levels is through determination of the "steady state" level of protein accumulation in a cell. Commonly used candidates for the reporter gene, known to those skilled in the art are beta-glucuronidase (GUS), chloramphenicol acetyl transferase (CAT) and proteins with fluorescent properties, such as green fluorescent protein (GFP) from Aequora victoria. In principle, however, many more proteins are suitable for this purpose, provided the protein does not interfere with essential plant functions. For quantification and determination of localization a number of tools are suited. Detection systems can readily be created or are available which are based on, e.g., immunochemical, enzymatic, fluorescent detection and quantification. Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression. Generally, individual transformed lines with one chimeric promoter reporter construct will vary in their levels of expression of the reporter gene. Also frequently observed is the phenomenon that such transformants do not express any detectable product (RNA or protein). The variability in expression is commonly ascribed to `position effects`, although the molecular mechanisms underlying this inactivity are usually not clear.
[0154] Preferably, the level of expression of a promoter of the current invention is analyzed on the basis of the target gene activity (conversion efficiency) as calculated by the sum of target gene products (in the examples below: ARA and EPA) divided by the total of target gene substrates and products (in the examples below: 20:3n-6, 20:4n-3, ARA and EPA).
[0155] "Constitutive expression" refers to expression using a constitutive or regulated promoter. "Conditional" and "regulated expression" refer to expression controlled by a regulated promoter.
[0156] "Specific expression" is the expression of gene products, which is limited to one or a few tissues (spatial limitation) and/or to one or a few developmental stages (temporal limitation) e.g. of a plant. It is acknowledged that hardly a true specificity exists: promoters seem to be preferably switch on in some tissues, while in other tissues there can be no or only little activity. This phenomenon is known as leaky expression. However, with specific expression in this invention is meant preferable expression in one or a few plant tissues.
[0157] The terms "polypeptide", "peptide", "oligopeptide", "gene product", "expression product" and "protein" are used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues. As used herein, the term "amino acid sequence" or a "polypeptide sequence" refers to a list of abbreviations, letters, characters or words representing amino acid residues. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used herein are conventional one letter codes for the amino acids: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine ; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid (see L. Stryer, Biochemistry, 1988, W. H. Freeman and Company, New York. The letter "x" as used herein within an amino acid sequence can stand for any amino acid residue.
[0158] The term "wild-type", "natural" or "natural origin" means with respect to an organism, polypeptide, or nucleic acid sequence that said organism is naturally occurring or available in at least one naturally occurring organism which is not changed, mutated, or otherwise manipulated by man.
[0159] "Recombinant DNA molecule" is a combination of DNA sequences that are joined together using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook et al., 1989.
[0160] "Genetic modification" is the result of recombinant DNA modification, meaning an organism is recombinantly modified resulting in modified characteristics compared to the wild-type organism, which has not been genetically modified.
[0161] A "transgene" refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.
[0162] The terms "heterologous DNA molecule", or "heterologous nucleic acid," as used herein, each refer to a molecule that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA molecule. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous DNA molecule" is a DNA molecule that is naturally associated with a host cell into which it is introduced.
[0163] The heterologous nucleotide molecule to be expressed in e.g. a plant tissue, plant organ, plant, seed or plant cell is preferably operably linked to one or more introns having expression enhancing effects, NEENAs (WO2 011023537, WO 2011023539), 5' and or 3'-untranslated regions, transcription termination and/or polyadenylation signals. 3'-untranslated regions are suitable to stabilize mRNA expression and structure. This can result in prolonged presence of the mRNA and thus enhanced expression levels. Termination and polyadenylation signals are suitable to stabilize mRNA expression (e.g., by stabilization of the RNA transcript and thereby the RNA level) to ensure constant mRNA transcript length and to prevent read-through transcription. Especially in multigene expression constructs this is an important feature. Furthermore correct termination of transcription is linked to re-initiation of transcription from the regulatory 5'nucleotide sequence resulting in enhanced expression levels. The above-mentioned signals can be any signal functional in plants and can for example be isolated from plant genes, plant virus genes or other plant pathogens. However, in a preferred embodiment the 3'-untranslated regions, transcription termination and polyadenylation signals are from the genes employed as the source for the promoters of this invention.
[0164] "Target gene" refers to a gene on the replicon that expresses the desired target coding sequence, functional RNA, or protein. The target gene is not essential for replicon replication. Additionally, target genes may comprise native non-viral genes inserted into a non-native organism, or chimeric genes, and will be under the control of suitable regulatory sequences. Thus, the regulatory sequences in the target gene may come from any source, including the virus. Target genes may include coding sequences that are either heterologous or homologous to the genes of a particular plant to be transformed. However, target genes do not include native viral genes. Typical target genes include, but are not limited to genes encoding a structural protein, a seed storage protein, a protein that conveys herbicide resistance, and a protein that conveys insect resistance. Proteins encoded by target genes are known as "foreign proteins". The expression of a target gene in a plant will typically produce an altered plant trait.
[0165] A "reporter gene" is a special target gene. Meaning that such reporter genes are often attached to regulatory sequences because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism. A "marker gene" encodes a selectable trait to be screened for.
[0166] The term "chimeric gene" refers to any gene that contains
[0167] DNA sequences, including regulatory and coding sequences, that are not functionally linked together in nature, or
[0168] sequences encoding parts of proteins not naturally adjoined, or
[0169] parts of promoters that are not naturally adjoined.
[0170] Accordingly, a chimeric gene may comprise regulatory molecules and coding sequences that are derived from different sources, or comprise regulatory molecules, and coding sequences derived from the same source, but arranged in a manner different from that found in nature.
[0171] "Chimeric transacting replication gene" refers either to a replication gene in which the coding sequence of a replication protein is under the control of a regulated plant promoter other than that in the native viral replication gene, or a modified native viral replication gene, for example, in which a site specific sequence(s) is inserted in the 5' transcribed but untranslated region. Such chimeric genes also include insertion of the known sites of replication protein binding between the promoter and the transcription start site that attenuate transcription of viral replication protein gene.
[0172] "Replication gene" refers to a gene encoding a viral replication protein. In addition to the ORF of the replication protein, the replication gene may also contain other overlapping or non-overlapping ORF(s), as are found in viral sequences in nature. While not essential for replication, these additional ORFs may enhance replication and/or viral DNA accumulation. Examples of such additional ORFs are AC3 and AL3 in ACMV and TGMV geminiviruses, respectively.
[0173] An "oligonucleotide" corresponding to a nucleotide sequence of the invention, e.g., for use in probing or amplification reactions, may be about 30 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21, 22, 23, or 24, or any number between 9 and 30). Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16 to 24 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100's or even 1000's of nucleotides in length.
[0174] An "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
[0175] A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein of interest chemicals. The nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant (variant) forms. Such variants will continue to possess the desired activity, i.e., either promoter activity or the activity of the product encoded by the open reading frame of the non-variant nucleotide sequence.
[0176] "Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to a nucleotide sequence of interest, which is--optionally--operably linked to termination signals and/or other regulatory elements. An expression cassette may also comprise sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a non-translated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one, which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. An expression cassette may be assembled entirely extracellularly (e.g., by recombinant cloning techniques). However, an expression cassette may also be assembled using in part endogenous components. For example, an expression cassette may be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, which thereby becomes functionally linked and controlled by said promoter sequences. Likewise, a nucleic acid sequence to be expressed may be placed (or inserted) downstream of an endogenous promoter sequence thereby forming an expression cassette. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development. In a preferred embodiment, such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is preferably provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes. The cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants. The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such, as the octopine synthase and nopaline synthase termination regions and others described below (see also, Guerineau 1991; Proudfoot 1991; Sanfacon 1991; Mogen 1990; Munroe 1990; Ballas 1989; Joshi 1987).
[0177] "Vector" is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).
[0178] Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast or fungal cells).
[0179] Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
[0180] "Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid molecules on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA molecule is said to be "operably linked to" or "associated with" a DNA molecule that codes for an RNA or a polypeptide if the two molecules are situated such that the regulatory DNA molecule affects expression of the coding DNA molecule (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory molecules in sense or antisense orientation.
[0181] "Cloning vectors" typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance.
[0182] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic cells", and organisms comprising transgenic cells are referred to as "transgenic organisms". Examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation (De Blaere 1987) and particle bombardment technology (U.S. Pat. No. 4,945,050). Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan (see, for example, Fromm 1990).
[0183] "Transformed", "transgenic", and "recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. For example, "transformed", "transformant", and "transgenic" plants or calli have been through the transformation process and contain a foreign gene integrated into their chromosome. The term "untransformed" refers to normal plants that have not been through the transformation process.
[0184] "Transiently transformed" refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium-mediated transformation or biolistic bombardment), but not selected for stable maintenance. "Stably transformed" refers to cells that have been selected and regenerated on a selection media following transformation.
[0185] "Genetically stable" and "heritable" refer to chromosomally-integrated genetic elements that are stably maintained in the plant and stably inherited by progeny through successive generations.
[0186] "Chromosomally-integrated" refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes are not "chromosomally integrated", they may be "transiently expressed." Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus. "Transient expression" refers to expression in cells in which a virus or a transgene is introduced by viral infection or by such methods as Agrobacterium-mediated transformation, electroporation, or biolistic bombardment, but not selected for its stable maintenance.
[0187] "Overexpression" refers to the level of expression in transgenic cells or organisms that exceeds levels of expression in normal or untransformed (non-transgenic) cells or organisms.
[0188] "Signal peptide" refers to the amino terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into the secretory pathway. The term "signal sequence" refers to a nucleotide sequence that encodes the signal peptide. The term "transit peptide" as used herein refers part of an expressed polypeptide (preferably to the amino terminal extension of a polypeptide), which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into a cell organelle (such as the plastids (e.g., chloroplasts) or mitochondria). The term "transit sequence" refers to a nucleotide sequence that encodes the transit peptide.
[0189] The activity of a transcription regulating nucleotide molecule is considered equivalent if transcription is initiated in the same tissues as is by the reference molecule. Such expression profile is preferably demonstrated using reporter genes operably linked to said transcription regulating nucleotide sequence. Preferred reporter genes (Schenborn 1999) in this context are green fluorescence protein (GFP) (Chuff 1996; Leffel 1997), chloramphenicol transferase, luciferase (Millar 1992), .beta.-glucuronidase or .beta.-galactosidase. Especially preferred is .beta.-glucuronidase (Jefferson 1987).
[0190] Beside this the transcription regulating activity of a functional equivalent homolog or fragment of the transcription regulating nucleotide molecule may vary from the activity of its parent sequence, especially with respect to expression level. The expression level may be higher or lower than the expression level of the parent sequence. Both derivations may be advantageous depending on the nucleic acid sequence of interest to be expressed. Preferred are such functional equivalent sequences, which--in comparison with its parent sequence--does, not derivate from the expression level of said parent sequence by more than 50%, preferably 25%, more preferably 10% (as to be preferably judged by either mRNA expression or protein (e.g., reporter gene) expression). Furthermore preferred are equivalent sequences which demonstrate an increased expression in comparison to its parent sequence, preferably an increase by at least 50%, more preferably by at least 100%, most preferably by at least 500%.
[0191] What is meant by "substantially the same activity" or "the same activity" when used in reference to a polynucleotide fragment or a homolog is that the fragment or homolog has at least 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to at least 99% of the expression regulating activity of the full length polynucleotide.
[0192] "Significant increase" is an increase that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater.
[0193] The word "plant" refers to any plant, particularly to agronomically useful plants (e.g., seed plants), and "plant cell" is a structural and physiological unit of the plant, which comprises a cell wall but may also refer to a protoplast. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ differentiated into a structure that is present at any stage of a plant's development. Such structures include one or more plant organs including, but are not limited to, fruit, shoot, stem, leaf, flower petal, etc. Preferably, the term "plant" includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seeds (including embryo, endosperm, and seed coat) and fruits (the mature ovary), plant tissues (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous. Included within the scope of the invention are all genera and species of higher and lower plants of the plant kingdom. Included are furthermore the mature plants, seed, shoots and seedlings, and parts, propagation material (for example seeds and fruit) and cultures, for example cell cultures, derived therefrom. Preferred are plants and plant materials of the following plant families: Amaranthaceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Tetragoniaceae. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred host organisms for the generation of transgenic plants. The use of the recombination system, or method according to the invention is furthermore advantageous in all ornamental plants, forestry, fruit, or ornamental trees, flowers, cut flowers, shrubs or turf. Said plant may include--but shall not be limited to--bryophytes such as, for example, Hepaticae (hepaticas) and Musci (mosses); pteridophytes such as ferns, horsetail and clubmosses; gymnosperms such as conifers, cycads, ginkgo and Gnetaeae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms) and Euglenophyceae. Plants for the purposes of the invention may comprise the families of the Rosaceae such as rose, Ericaceae such as rhododendrons and azaleas, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such as pinks, Solanaceae such as petunias, Gesneriaceae such as African violet, Balsaminaceae such as touch-me-not, Orchidaceae such as orchids, Iridaceae such as gladioli, iris, freesia and crocus, Compositae such as marigold, Geraniaceae such as geraniums, Liliaceae such as Drachaena, Moraceae such as ficus, Araceae such as philodendron and many others. The transgenic plants according to the invention are furthermore selected in particular from among dicotyledonous crop plants such as, for example, from the families of the Leguminosae such as pea, alfalfa and soybean; the family of the Umbelliferae, particularly the genus Daucus (very particularly the species carota (carrot)) and Apium (very particularly the species graveolens var. dulce (celery)) and many others; the family of the Solanaceae, particularly the genus Lycopersicon, very particularly the species esculentum (tomato) and the genus Solanum, very particularly the species tuberosum (potato) and melongena (aubergine), tobacco and many others; and the genus Capsicum, very particularly the species annum (pepper) and many others; the family of the Leguminosae, particularly the genus Glycine, very particularly the species max (soybean) and many others; and the family of the Cruciferae, particularly the genus Brassica, very particularly the species napus (oilseed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli); and the genus Arabidopsis, very particularly the species thaliana and many others; the family of the Compositae, particularly the genus Lactuca, very particularly the species sativa (lettuce) and many others. The transgenic plants according to the invention may be selected among monocotyledonous crop plants, such as, for example, cereals such as wheat, barley, sorghum and millet, rye, triticale, maize, rice or oats, and sugarcane. Further preferred are trees such as apple, pear, quince, plum, cherry, peach, nectarine, apricot, papaya, mango, and other woody species including coniferous and deciduous trees such as poplar, pine, sequoia, cedar, oak, etc. Especially preferred are Arabidopsis thaliana, Nicotiana tabacum, oilseed rape, soybean, corn (maize), wheat, Linum usitatissimum (linseed and flax), Camelina sativa, Brassica juncea, potato and tagetes. Brassica napus is used synonymously with rapeseed and canola herein.
[0194] "Plant tissue" includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture such as single cells, protoplast, embryos, and callus tissue. The plant tissue may be in plants or in organ, tissue or cell culture.
[0195] "Mature seed" is a seed that has fully developed and has undergone all the stages of its development successfully. Such a seed can germinate into a seedling if provided with the necessary physical conditions. What are harvested are usually mature seeds.
[0196] The term "altered plant trait" means any phenotypic or genotypic change in a transgenic plant relative to the wild-type or non-transgenic plant host.
[0197] A "transgenic plant" is a plant having one or more plant cells that contain an expression vector or recombinant expression construct.
[0198] "Primary transformant" and "T0 generation" refer to transgenic plants that are of the same genetic generation as the tissue which was initially transformed (i.e., not having gone through meiosis and fertilization since transformation).
[0199] "Secondary transformants" and the "T1, T2, T3, etc. generations" refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants.
[0200] The term variant or "homolog" with respect to a sequence (e.g., a polypeptide or nucleic acid sequence such as--for example--a transcription regulating nucleotide molecule of the invention) is intended to mean substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein. Generally, nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide sequence identity to the native (wild type or endogenous) nucleotide sequence.
[0201] Sequence comparisons maybe carried out using a Smith-Waterman sequence alignment algorithm (see e.g., Waterman (1995)). The localS program, version 1.16, is preferably used with following parameters: match: 1, mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2.
[0202] The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity".
[0203] As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence or isolated nucleic acid sequence capable of regulating expression in plants, preferably the complete cDNA or gene sequence or isolated nucleic acid sequence capable of regulating expression in plants is the reference sequence.
[0204] As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. In a preferred embodiment the comparison window defining the homology of sequence consists of the entire query sequence. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
[0205] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Preferred, non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, 1988; the local homology algorithm of Smith et al. 1981; the homology alignment algorithm of Needleman and Wunsch 1970; the search-for-similarity-method of Pearson and Lipman 1988; the algorithm of Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993.
[0206] Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described (Higgins 1988, 1989; Corpet 1988; Huang 1992; Pearson 1994). The ALIGN program is based on the algorithm of Myers and Miller, supra. The BLAST programs of Altschul et al., 1990, are based on the algorithm of Karlin and Altschul, supra. Multiple aligments (i.e. of more than 2 sequences) are preferably performed using the Clustal W algorithm (Thompson 1994; e.g., in the software VectorNTI.TM., version 9; Invitrogen Inc.) with the scoring matrix BLOSUM62MT2 with the default settings (gap opening penalty 15/19, gap extension penalty 6.66/0.05; gap separation penalty range 8; % identity for alignment delay 40; using residue specific gaps and hydrophilic residue gaps).
[0207] Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
[0208] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
[0209] To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. 1997. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al., supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g. BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
[0210] For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein is preferably made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program.
[0211] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
[0212] As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, preferably the complete query or reference sequence as defined by SEQ ID NO: x, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
[0213] The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 90%, 91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 90%, 95%, and most preferably at least 98%.
[0214] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1.degree. C. to about 20.degree. C., depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
[0215] The term "substantial identity" in the context of a polypeptide indicates that a peptide comprises a sequence with at least 90%, 91%, 92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
[0216] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. The reference sequences of the invention is defined by SEQ ID NO: x.
[0217] An indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
[0218] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence dependent, and are different under different environmental parameters. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T.sub.m can be approximated from the equation of Meinkoth and Wahl, 1984:
T.sub.m=81.5.degree. C.+16.6(log.sub.10 M)+0.41 (% GC)-0.61 (% form)-500/L
where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T.sub.m is reduced by about 1.degree. C. for each 1% of mismatching; thus, T.sub.m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point I for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting point I; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting point I; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C. lower than the thermal melting point I. Using the equation, hybridization and wash compositions, and desired T, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, 1993. Generally, highly stringent hybridization and wash conditions are selected to be about 5.degree. C. lower than the thermal melting point T.sub.m for the specific sequence at a defined ionic strength and pH.
[0219] An example of highly stringent wash conditions is 0.15 M NaCl at 72.degree. C. for about 15 minutes. An example of stringent wash conditions is a 0.2.times.SSC wash at 65.degree. C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4 to 6.times.SSC at 40.degree. C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30.degree. C. and at least about 60.degree. C. for long robes (e.g., >50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2.times. (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
[0220] Very stringent conditions are selected to be equal to the T.sub.m for a particular probe. An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. C. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree. C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times. SSC at 55 to 60.degree. C.
[0221] The following are examples of sets of hybridization/wash conditions that may be used to clone orthologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree. C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
[0222] The term "fatty acid" refers to long chain aliphatic acids (alkanoic acids) of varying chain lengths, from about C.sub.12 to C.sub.22 (although both longer and shorter chain-length acids are known). The predominant chain lengths are between C.sub.16 and C.sub.22. Additional details concerning the differentiation between "saturated fatty acids" versus "unsaturated fatty acids", "monounsaturated fatty acids" versus "polyunsaturated fatty acids" (or "PUFAs"), and "omega-6 fatty acids" (.omega.-6 or n-6) versus "omega-3 fatty acids" (.omega.-3 or n-3) are provided in WO 2004/101757.
[0223] Fatty acids are described herein by a simple notation system of "X:Y", wherein the number before the colon indicates the number of carbon atoms in the fatty acid and the number after the colon is the number of double bonds that are present. The number following the fatty acid designation indicates the position of the double bond from the carboxyl end of the fatty acid with the "c" affix for the cis configuration of the double bond [e.g., palmitic acid (16:0), stearic acid (18:0), oleic acid (18: 1, 9c), petroselinic acid (18: 1, 6c), LA (18:2, 9c, 12c), GLA (18:3, 6c,9c, 12c) and ALA (18:3, 9c,12c,15c)]. Unless otherwise specified 18:1, 18:2 and 18:3 refer to oleic, LA and linolenic fatty acids. If not specifically written as otherwise, double bonds are assumed to be of the cis configuration. For instance, the double bonds in 18:2 (9, 12) would be assumed to be in the cis configuration.
[0224] Nomenclature of polyunsaturated fatty acids (PUFAs):
TABLE-US-00001 Common name Chemical name linoleic acid LA cis-9,12-octadecadienoic acid 18:2 .omega.-6 gamma-linoleic acid GLA cis-6,9,12-octadecatrienoic acid 18:3 .omega.-6 alpha-linoleic acid ALA cis-9,12,15-octadecatrienoic acid 18:3 .omega.-3 stearidonic acid STA cis-6,9,12,15-octadecatetraenoic acid 18:4 .omega.-3 eicosadienoic acid EDA cis-11,14-eicosadienoic acid 20:2 .omega.-6 dihomo-gamma linoleic acid DGLA cis-8,11,14-eicosatrienoic acid 20:3 .omega.-6 eicosatrienoic acid ETra cis-11,14,17-eicosatrienoic acid 20:3 .omega.-3 arachidonic acid AA cis-5,8,11,14-eicosatetraenoic acid 20:4 .omega.-6 eicosatetraenoic acid ETA cis-8,11,14,17-eicosatetraenoic acid 20:4 .omega.-3 eicosapentaenoic acid ETA cis-5,8,11,14,17-eicosapentaenoic acid 20:5 .omega.-3 docosapentaenoic acid DPA cis-7,10,13,16,19-docosapentaenoic acid 22:5 .omega.-3 docosahexaenoic acid DHA cis-4,7,10,13,16,19-docosapentaenoic acid 22:6 .omega.-3
[0225] The term "fat" refers to a lipid substance that is solid at 25.degree. C. and usually saturated.
[0226] The term "oil" refers to a lipid substance that is liquid at 25.degree. C. and usually polyunsaturated. PUFAs are found in the oils of some algae, oleaginous yeasts and filamentous fungi. "Microbial oils" or "single cell oils" are those oils naturally produced by microorganisms during their lifespan. Such oils can contain long chain PUFAs.
[0227] The term "PUFA biosynthetic pathway" refers to a metabolic process that converts oleic acid to LA, EDA, GLA, DGLA, ARA, ALA, STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in the literature (e.g., see WO 2005/003322). Simplistically, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special desaturation and elongation enzymes (i.e., "PUFA biosynthetic pathway enzymes") present in the endoplasmic reticulim membrane. More specifically, "PUFA biosynthetic pathway enzymes" refer to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: a delta-4 desaturase, a delta-S desaturase, a delta-6 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-8 desaturase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20 elongase and/or a C.sub.20/22 elongase.
[0228] "Desaturase" is a polypeptide which can desaturate one or more fatty acids to produce a mono- or poly-unsaturated fatty acid or precursor which is of interest. Of particular interest herein are delta-8 desaturases that will desaturate a fatty acid between the 8.sup.th and 9.sup.th carbon atom numbered from the carboxyl-terminal end of the molecule and that can, for example, catalyze the conversion of EDA to DGLA and/or ETrA to ETA Other useful fatty acid desaturases include, for example:
[0229] a. delta-5 desaturases that catalyze the conversion of DGLA to ARA and/or ETA to EPA;
[0230] b. delta-6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA;
[0231] c. delta-4 desaturases that catalyze the conversion of DPA to DHA;
[0232] d. delta-12 desaturases that catalyze the conversion of oleic acid to LA;
[0233] e. delta-15 desaturases that catalyze the conversion of LA to ALA and/or GLA to STA;
[0234] f. delta-17 desaturases that catalyze the conversion of ARA to EPA and/or DGLA to ETA; and
[0235] g. delta-9 desaturases that catalyze the conversion of palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid (18:1).
[0236] The term "elongase system" refers to a suite of four enzymes that are responsible for elongation of a fatty acid carbon chain to produce a fatty acid that is two carbons longer than the fatty acid substrate that the elongase system acts upon. More specifically, the process of elongation occurs in association with fatty acid synthase, whereby CoA is the acyl carrier (Lassner et al., The Plant Cell 8:281-292 (1996)). In the first step, which has been found to be both substrate-specific and also rate-limiting, malonyl-GoA is condensed with a long-chain acyl-CoA to yield CO.sub.2 and a beta-ketoacyl-CoA (where the acyl moiety has been elongated by two carbon atoms). Subsequent reactions include reduction to beta-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a second reduction to yield the elongated acyl-CoA. Examples of reactions catalyzed by elongase systems are the conversion of GLA to DGLA, STA to ETA and EPA to DPA. For the purposes herein, an enzyme catalyzing the first condensation reaction (i.e., conversion of malonyl-GoA to beta-ketoacyl-CoA) will be referred to generically as an "elongase". In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree of unsaturation. Accordingly, elongases can have different specificities. For example, a C.sub.16/18 elongase will utilize a C.sub.16 substrate (e.g., palmitate), a C.sub.18/20 elongase will utilize a C.sub.18 substrate (e.g., GLA, STA) and a C.sub.20/22 elongase will utilize a C.sub.20 substrate (e.g., EPA). In like manner, a delta-9 elongase is able to catalyze the conversion of LA and ALA to EDA and ETrA, respectively (see WO 2002/077213). It is important to note that some elongases have broad specificity and thus a single enzyme may be capable of catalyzing several elongase reactions (e.g., thereby acting as both a C.sub.16/18 elongase and a C.sub.18/20 elongase).
[0237] The following figures and examples describe the invention in further detail. The figures and examples are not meant to limit the scope of the invention or of the claims in any way.
[0238] FIG. 1 depicts the general pathways for polyunsaturated fatty acid synthesis up to arachidonic acid and eicosapentaenoic acid.
[0239] FIG. 2 depicts the general cloning strategy applied in the examples.
[0240] FIG. 3 depicts an alignment of sequences according to the present invention and a prior art sequence(SEQ-001-plus_A (SEQ ID NO: 327); SEQ-001-Kozak_ATG (SEQ ID NO: 328); SEQ-159-promoter (SEQ ID NO: 159); SEQ-020-minimal_enhancer (SEQ ID NO: 20); SEQ-046-enhancer (SEQ ID NO: 46); SEQ-137-enhancer_TFB (SEQ ID NO: 137); SEQ-138-promoter_TFB1 (SEQ ID NO: 138); SEQ-139-promoter_TFB2 (SEQ ID NO: 139); SEQ-140-spacer (SEQ ID NO: 140); SEQ-141-promoter_98bp (SEQ ID NO: 141); SEQ-147-promoter_160bp (SEQ ID NO: 147); SEQ-156-promoter_240bp (SEQ ID NO: 156); SEQ-003-1039bp+2 (SEQ ID NO: 3); SEQ-326-p1039_2UTR (SEQ ID NO: 326); SEQ-002-1039bp+38 (SEQ ID NO: 2); SEQ-324_p1039_38UTR (SEQ ID NO: 324); SEQ-325-p1039_38_differing_part (SEQ ID NO: 325); and WO0116340 GUS data (SEQ ID NO: 311).
[0241] FIG. 4 depicts the sequences referred to in the present application.
EXAMPLES
[0242] With regards to the present invention, the terms "binary vector, "T-DNA containing plasmid" and "T-plasmid" are used interchangeably. An overview of binary vectors and their usage is given by Hellens et al, Trends in Plant Science (2000) 5: 446-451.
Example 1
General Cloning Methods
[0243] Cloning methods, e.g. use of restriction endonucleases to cut double stranded DNA at specific sites, agarose gel electrophoreses, purification of DNA fragments, transfer of nucleic acids onto nitrocellulose and nylon membranes, joining of DNA-fragments, transformation of E. coli cells and culture of bacteria, were performed as described in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87965-309-6). Polymerase chain reaction was performed using Phusion.TM. High-Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufacturer's instructions. In general, primers used in PCR were designed such that at least 20 nucleotides of the 3' end of the primer anneal perfectly with the template to amplify. Restriction sites were added by attaching the corresponding nucleotides of the recognition sites to the 5' end of the primer. Fusion PCR, for example described by K. Heckman and L. R. Pease, Nature Protocols (2007) 2, 924-932, was used as an alternative method to join two fragments of interest, e.g. a promoter to a gene or a gene to a terminator.
Example 2
Assembly of Genes Required for EPA and DHA Synthesis Within Binary Vectors
[0244] The general cloning strategy is depicted in FIG. 2.
[0245] Following the modular cloning scheme depicted in FIG. 2, genes were either synthesized by GeneArt (Regensburg) or PCR-amplified using Phusion.TM. High-Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufacturer's instructions from cDNA. In both cases a Nco I and/or Asc I restriction site at the 5' terminus, and a Pac I restriction site at the 3' terminus (FIG. 2A) were introduced to enable cloning of these genes between functional elements such as promoters and terminators using these restriction sites such that the genes are functionally linked to both the respective promoter and terminator (see below in this example).
[0246] Promoter-terminator modules were created by complete synthesis by GeneArt (Regensburg) or by joining the corresponding expression elements using fusion PCR as described in example 1 and cloning the PCR-product into the TOPO-vector pCR2.1 (Invitrogen) according to the manufacturer's instructions (FIG. 2B). While joining terminator sequences to promoter sequences, recognition sequences for the restriction endonucleases Xma I, Sbf I, Fse I, Kas I, Fso I, Not I were added to either side of the modules in FIG. 2B, and the recognition sites for the restriction endonucleases Nco I, Asc I and Pac I were introduced between promoter and terminator (see FIG. 2B).
[0247] To obtain the final expression modules, PCR-amplified genes were cloned between promoter and terminator or intron and terminator via Nco I and/or Pac I restriction sites (FIG. 2C)
[0248] Employing the custom multiple cloning site (MCS) containing the recognition sequences for the restriction endonucleases Xma I, Sbf I, Fse I, Kas I, Fso I, Not I, up to three of expression modules were combined as desired to yield expression cassettes harbored by either one of pENTR/A, pENTR/B or pENTR/C constructs(FIG. 2D).
[0249] Finally, the Multisite Gateway.TM. System (Invitrogen) was used to combine three expression cassette harbored by pENTR/A, pENTR/B and pENTR/C (FIG. 2E) to obtain the final binary T-plasmids for plant transformation. Besides features for maintenance of the binary plasmid in E. coli and agrobacteria, the binary T-plasmid contains an acetohydroxyacid synthase (AHAS) gene to allow selection of transgenic plants.
[0250] To demonstrate the effectiveness of the enhancer of the invention, particularly of SEQ ID NO. 20 and SEQ ID NO. 46, three different promoter-enhancer combinations (SEQ ID NO. 1-3) based on the Conlinin-1 promotor as described in WO02102970 (FIG. 8) were prepared as described above.
[0251] The nucleic acid sequence A comprises a conlinin-1 promoter of SEQ ID NO. 159, a delta-5-desaturase as target gene coding for the amino acid sequence SEQ ID NO. 11 and between the promoter and the target gene an untranslated region of the sequence of SEQ ID NO. 140 fused with the enhancer of SEQ ID NO. 46. SEQ ID NO. 1 thus comprises the promotor and UTR up to the start codon.
[0252] The nucleic acid sequence B comprises the conlinin-1 promotor of SEQ ID NO. 159 and the delta-5 desaturase target gene coding for the polypeptide of SEQ ID NO. 11, and between the promoter and the target gene an untranslated region according to SEQ ID NO. 324. This sequence lacks the last 24 nucleotides of the enhancer of the invention according to SEQ ID NO. 46 and completely lacks the enhancer sequence SEQ ID NO. 20. Instead, the last 24 nucleotides of SEQ ID NO. 46 have been replaced by the 38 nucleotides of SEQ ID NO. 325. Even though the sequences A and B are of similar length, the latter sequence has the enhancer of the invention replaced by a sequence of significantly different number of G and T nucleotides. SEQ ID NO. 2 thus comprises the promotor and UTR up to the start codon.
[0253] The nucleic acid sequence C comprises the conlinin-1 promotor of SEQ ID NO. 159 and the delta-5 desaturase target gene coding for the polypeptide of SEQ ID NO. 11, and between the promoter and the target gene an untranslated region according to SEQ ID NO. 326. This sequence has the last 24 nucleotides of the enhancer of the invention according to SEQ ID NO. 46 replaced by the sequence "CC". SEQ ID NO. 3 thus comprises the promotor and UTR up to the start codon.
[0254] The delta-5 desaturase target gene converts the fatty acid 20:3n-6 to 20:4n-6 (arachidonic acid, ARA) and 20:4n-3 to 20:5n-3 (eicosapentaenoic acid, EPA). The reaction scheme is given in FIG. 1. In order to provide the substrates 20:3n-6 and 20:4n-3 for the delta-5-desaturase reporter gene in Brassica napus seeds, the constructs comprised in addition to the sequences A, B or C, respectively, further desaturase and elongase genes driven by other seed specific promoters in various combinations. This way it is assured that the activity and expression of the target gene is not dependent on any interaction with the desaturase and elongase genes or enzymes necessary for providing the substrates of the target gene. Among the desaturase and elongase genes used where d12d15Des(Ac_GA) (cf. WO 2007042510), d12Des(Ce_GA) (cf. US 2003172398), d12Des(Co_GA2) (cf. WO 200185968), d12Des(Fg) (cf. WO 2007133425), d12Des(Ps_GA) (cf. WO 2006100241), d12Des(Tp_GA) (cf. WO 2006069710), d6Des(Ol_febit) (cf. WO 2008040787), d6Des(Ol_febit)2 (cf. WO 2008040787), d6Des(Ot_febit) (cf. WO 2008040787), d6Des(Ot_GA) (cf. WO 2005083093), d6Des(Ot_GA2) (cf. WO 2005083093), d6Des(Pir) (cf. WO 2002026946), d6Des(Pir_GAI) (cf. WO 2002026946), d6Des(Plu) (cf. WO 2007051577), d6Elo(Pp_GA) (cf. WO 2001059128), d6Elo(Pp_GA2) (cf. WO 2001059128), d6Elo(Pp_GA3) (cf. WO 2001059128), d6Elo(Tp_GA) (cf. WO 2005012316) and d6Elo(Tp_GA2) (cf. WO 2005012316).
[0255] Activity of the delta-5 desaturase was analyzed by measuring fatty acid concentrations in seeds as described in example 4 and calculating the sum of desaturated products (ARA and EPA) divided by the total of desaturase substrates and products (20:3n-6, 20:4n-3, ARA and EPA) to obtain the conversion efficiency. The constructs frequently comprised genes for omega-3 desaturases. Presence of omega-3-desaturase genes was not motivated by the invention; the respective genes were present to answer questions unrelated to the present invention. Omega-3 desaturase only shift the ratio of the substrates between each other, as well as the ratios of the products between each other; their mode of action is depicted in FIG. 1. Thus, the presence of omega-3 desaturases does not influence the analysis of conversion efficiency nor does it perceptibly influence the activity of the target gene or any other fatty acid desaturase activity.
[0256] An alignment of the sequences found in the constructs of the present invention is shown in FIG. 3. An overview of genetic elements employed in the constructs is given in Table 1. The delta-5 desaturase gene sequences SEQ ID NO. 10 and SEQ ID NO. 12 code for the identical polypeptide sequence. The activity of the desaturase is not dependent on either gene sequence. Instead, the sequences can be arbitrarily exchanged without altering the outcome of the comparison experiments.
TABLE-US-00002 TABLE 1 Overview of genetic elements. "p- . . . ": Promoter; "d5Des": delta-5 desaturase; "o3Des": omega-3 desaturase Genetic element SEQ ID NO. DNA SEQ ID NO. Prot p-(1064 bp) 1 p-(1039 bp + 38) 2 p-(1039 bp + 2) 3 p-BnNapin 4 p-LuPXR 5 p-PvArc 6 p-VfSBP 7 p-BnFAE1 8 p-VfUSP 9 d5Des(Tc_GA) 10 11 d5Des(Tc_GA2) 12 11 o3Des(Cp_GA) 13 14 o3Des(Cp_GA2)_V282L 15 16 o3Des(Pi_GA2) 17 18 o3Des(Pi_GA) 19 18
Example 3
General Procedure for Production of Transgenic Plants
[0257] In general, the transgenic rapeseed plants were generated by a modified protocol according to Moloney et al. 1992, Plant Cell Reports, 8:238-242). For the generation rapeseed plants, the binary vectors described in example 2 were transformed into Agrobacterium tumefaciens C58C1:pGV2260 (Deblaere et al. 1984, Nucl. Acids. Res. 13: 4777-4788).
[0258] Overnight cultures of agrobacteria harbouring the binary vectors described in example 2 were grown in Murashige-Skoog Medium (Murashige and Skoog 1962 Physiol. Plant. 15, 473) supplemented by 3% saccharose (3MS-Medium). Hypocotyls of sterile rapeseed plants were incubated in a petri dish in a 1:50 diluted agrobacterial suspension obtained from the overnight cultures for 5-10 minutes. This was followed by a three day co-incubation in darkness at 25.degree. C. on 3MS-Medium with 0.8% bacto-agar. After three days the culture was transferred on MS-medium containing 500 mg/l Claforan (Cefotaxime-Natrium), 100 nM Imazethapyr, 20 microM Benzylaminopurin (BAP) and 1,6 g/l Glucose where they were cultivated for 7 days at 25.degree. C. under 16 hours light/8 hours darkness conditions. Growing sprouts--indicating the presence of the T-DNA harboring the AHAS selectable marker, were transferred to MS-Medium containing 2% saccharose, 250 mg/l Claforan and 0.8% Bacto-Agar. Rooting could be stimulated by adding a growth hormone, for example 2-indolbutyl acid.
[0259] Regenerated sprouts have been obtained on 2MS-Medium with Imazetapyr and Claforan and were transferred to the greenhouse for further development. After flowering, the mature seeds were harvested and analysed for expression of the genes listed in example 2 via lipid analysis as described in example 4.
Example 4
Lipid Extraction and Lipid Analysis of Plant Oils
[0260] Total lipids were extracted from fresh or freeze-dried homogenized plant material (seed or cotyledons) by liquid/liquid extraction using tert-butyl methyl ether.
[0261] The fatty acid composition of the extracted lipids was subsequently determined by the means of gas chromatography with flame-ionization detection or mass-selective detection after derivatization of the extracted lipids with trimethylsulfonium hydroxide.
[0262] Gas chromatographic separation of the so generated fatty acid methyl esters was performed on a suitable capillary column (50%-Cyanopropylphenyl)-dimethylpolysiloxane as stationary phase).
[0263] Identification and quantification of the separated chromatographic signals is accomplished by comparison of the respective retention times and signal intensities to chromatograms of standard solutions with known composition and content of fatty acid methyl esters.
[0264] To generate transgenic plants containing the genetic element described in example 2 for production of ARA and EPA in seeds, Canola (Brassica napus) was transformed as described in example 3. Selected plants containing the genetic elements described in example 2 where grown until development of mature seeds (Day/night cycle: 16 h at 200 mE and 21.degree. C., 8 h at darkness and 19.degree. C.). Fatty acids from harvested seeds were extracted and analyzed using gas chromatography.
Example 5
Comparison of Construct Containing the Promoter According to the Invention with Construct Containing Promoter Not According to the Invention
[0265] Two constructs (LJB950=comprising SEQ ID NO. 2 without any omega-3 desaturase and LJB997=comprising SEQ ID NO. 1 without any omega-3 desaturase) were evaluated which were identical in all genetic elements and their arrangement in the construct, apart from the promoter-untranslated region combination driving the reporter gene expression. Table 2 shows the result of the three independent transgenic plants (events) obtained for each of the two constructs.
TABLE-US-00003 TABLE 2 Comparison of conversion efficiency resulting from the use of two different versions of the Conlinin promoter. 20:3n-6 + Convertion 20:4n- ARA + Efficiency n (# of N = (# of 3 EPA (%) Events) constructs) B) LJB950 (Conlinin 1039 bp + 38) 6.9 6.9 50 3 1 A) LJB997 (Conlinin 1064 bp) 3.6 8.7 71 3 1
[0266] Surprisingly, use of sequence SEQ ID NO: 1 resulted in significantly higher conversion efficiency compared to SEQ ID NO: 2.
Example 6
Comparison of Constructs Containing the Promoter According to the Invention with Constructs Containing Promoters Not According to the Invention
[0267] A total of 69 constructs were evaluated which all express the delta-5-desaturase protein as shown in SEQ ID NO: 11 as a reporter gene. The reporter gene was functionally linked to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The differences between these three promoter versions are depicted in FIG. 3. In order to provide the substrates 20:3n-6 and 20:4n-3 for the delta-5-desaturase reporter gene in Brassica napus seeds, the constructs further contained desaturase and elongase genes driven by other seed specific promoters in various combinations as described in example 2 and table 1.
[0268] Table 3 shows that constructs using the SEQ ID NO: 1 constantly display a significantly higher conversion efficiency compared to constructs using SEQ ID NO: 2 or SEQ ID NO:3. This was particularly unexpected as WO 0116340 taught the uses of the promoter similar to SEQ ID NO: 3 using a reporter gene.
TABLE-US-00004 TABLE 3 Comparison of conversion efficiency resulting from the use of different constructs; delta-5 desaturase according to SEQ ID NO. 11 was the target gene for all constructs 20:3n-6 + Convertion 20:4n- ARA + Efficiency n (# of N = (# of 3 EPA (%) Events) constructs) constructs comprising SEQ ID 2 2.6 57 425 16 NO. 2 and an omega-3 desaturase constructs comprising SEQ ID 4.1 3.3 45 363 10 NO. 2 without an omega-3 desaturase constructs comprising SEQ ID 1.8 2 53 448 16 NO. 3 and an omega-3 desaturase constructs comprising SEQ ID 1.3 3.1 70 143 7 NO. 1 and an omega-3 desaturase constructs comprising SEQ ID 1.8 4 69 797 20 NO. 1 without an omega-3 desaturase
Sequence CWU
1
1
32811063DNAArtificial Sequencepromoter plus untranslated region incl
enhancer 1ttagcagata tttggtgtct aaatgtttat tttgtgatat gttcatgttt
gaaatggtgg 60tttcgaaacc agggacaacg ttgggatctg atagggtgtc aaagagtatt
atggattggg 120acaatttcgg tcatgagttg caaattcaag tatatcgttc gattatgaaa
attttcgaag 180aatatcccat ttgagagagt ctttacctca ttaatgtttt tagattatga
aattttatca 240tagttcatcg tagtcttttt ggtgtaaagg ctgtaaaaag aaattgttca
cttttgtttt 300cgtttatgtg aaggctgtaa aagattgtaa aagactattt tggtgttttg
gataaaatga 360tagtttttat agattctttt gcttttagaa gaaatacatt tgaaattttt
tccatgttga 420gtataaaata ccgaaatcga ttgaagatca tagaaatatt ttaactgaaa
acaaatttat 480aactgattca attctctcca tttttatacc tatttaaccg taatcgattc
taatagatga 540tcgatttttt atataatcct aattaaccaa cggcatgtat tggataatta
accgatcaac 600tctcacccct aatagaatca gtattttcct tcgacgttaa ttgatcctac
actatgtagg 660tcatatccat cgttttaatt tttggccacc attcaattct gtcttgcctt
tagggatgtg 720aatatgaacg gccaaggtaa gagaataaaa ataatccaaa ttaaagcaag
agaggccaag 780taagataatc caaatgtaca cttgtcattg ccaaaattag taaaatactc
ggcatattgt 840attcccacac attattaaaa taccgtatat gtattggctg catttgcatg
aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc ccatgcaaag ttaacactca
cgaccccatt 960cctcagtctc cactatataa acccaccatc cccaatctca ccaaacccac
cacacaactc 1020acaactcact ctcacacctt aaagaaccaa tcaccaccaa aaa
106321077DNAArtificial Sequencepromoter 1039 plus 38pb without
enhancer 2ttagcagata tttggtgtct aaatgtttat tttgtgatat gttcatgttt
gaaatggtgg 60tttcgaaacc agggacaacg ttgggatctg atagggtgtc aaagagtatt
atggattggg 120acaatttcgg tcatgagttg caaattcaag tatatcgttc gattatgaaa
attttcgaag 180aatatcccat ttgagagagt ctttacctca ttaatgtttt tagattatga
aattttatca 240tagttcatcg tagtcttttt ggtgtaaagg ctgtaaaaag aaattgttca
cttttgtttt 300cgtttatgtg aaggctgtaa aagattgtaa aagactattt tggtgttttg
gataaaatga 360tagtttttat agattctttt gcttttagaa gaaatacatt tgaaattttt
tccatgttga 420gtataaaata ccgaaatcga ttgaagatca tagaaatatt ttaactgaaa
acaaatttat 480aactgattca attctctcca tttttatacc tatttaaccg taatcgattc
taatagatga 540tcgatttttt atataatcct aattaaccaa cggcatgtat tggataatta
accgatcaac 600tctcacccct aatagaatca gtattttcct tcgacgttaa ttgatcctac
actatgtagg 660tcatatccat cgttttaatt tttggccacc attcaattct gtcttgcctt
tagggatgtg 720aatatgaacg gccaaggtaa gagaataaaa ataatccaaa ttaaagcaag
agaggccaag 780taagataatc caaatgtaca cttgtcattg ccaaaattag taaaatactc
ggcatattgt 840attcccacac attattaaaa taccgtatat gtattggctg catttgcatg
aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc ccatgcaaag ttaacactca
cgaccccatt 960cctcagtctc cactatataa acccaccatc cccaatctca ccaaacccac
cacacaactc 1020acaactcact ctcacacctt ctagaggatc tgatatctgc ggccgcggcg
cgccacc 107731041DNAArtificial Sequencepromoter 1039 without
enhancer 3ttagcagata tttggtgtct aaatgtttat tttgtgatat gttcatgttt
gaaatggtgg 60tttcgaaacc agggacaacg ttgggatctg atagggtgtc aaagagtatt
atggattggg 120acaatttcgg tcatgagttg caaattcaag tatatcgttc gattatgaaa
attttcgaag 180aatatcccat ttgagagagt ctttacctca ttaatgtttt tagattatga
aattttatca 240tagttcatcg tagtcttttt ggtgtaaagg ctgtaaaaag aaattgttca
cttttgtttt 300cgtttatgtg aaggctgtaa aagattgtaa aagactattt tggtgttttg
gataaaatga 360tagtttttat agattctttt gcttttagaa gaaatacatt tgaaattttt
tccatgttga 420gtataaaata ccgaaatcga ttgaagatca tagaaatatt ttaactgaaa
acaaatttat 480aactgattca attctctcca tttttatacc tatttaaccg taatcgattc
taatagatga 540tcgatttttt atataatcct aattaaccaa cggcatgtat tggataatta
accgatcaac 600tctcacccct aatagaatca gtattttcct tcgacgttaa ttgatcctac
actatgtagg 660tcatatccat cgttttaatt tttggccacc attcaattct gtcttgcctt
tagggatgtg 720aatatgaacg gccaaggtaa gagaataaaa ataatccaaa ttaaagcaag
agaggccaag 780taagataatc caaatgtaca cttgtcattg ccaaaattag taaaatactc
ggcatattgt 840attcccacac attattaaaa taccgtatat gtattggctg catttgcatg
aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc ccatgcaaag ttaacactca
cgaccccatt 960cctcagtctc cactatataa acccaccatc cccaatctca ccaaacccac
cacacaactc 1020acaactcact ctcacacctc c
10414664DNAArtificial Sequencepromotor p-BnNapin 4taaggatgac
ctacccattc ttgagacaaa tgttacattt tagtatcaga gtaaaatgtg 60tacctataac
tcaaattcga ttgacatgta tccattcaac ataaaattaa accagcctgc 120acctgcatcc
acatttcaag tattttcaaa ccgttcggct cctatccacc gggtgtaaca 180agacggattc
cgaatttgga agattttgac tcaaattccc aatttatatt gaccgtgact 240aaatcaactt
taacttctat aattctgatt aagctcccaa tttatattcc caacggcact 300acctccaaaa
tttatagact ctcatcccct tttaaaccaa cttagtaaac gttttttttt 360taattttatg
aagttaagtt tttaccttgt ttttaaaaag aatcgttcat aagatgccat 420gccagaacat
tagctacacg ttacacatag catgcagccg cggagaattg tttttcttcg 480ccacttgtca
ctcccttcaa acacctaaga gcttctctct cacagcacac acatacaatc 540acatgcgtgc
atgcattatt acacgtgatc gccatgcaaa tctcctttat agcctataaa 600ttaactcatc
ggcttcactc tttactcaaa ccaaaactca tcaatacaaa caagattaaa 660aaca
66451727DNAArtificial Sequencepromotor p-LuPXR 5cacgggcagg acatagggac
tactacaagc atagtatgct tcagacaaag agctaggaaa 60gaactcttga tggaggttaa
gagaaaaaag tgctagaggg gcatagtaat caaacttgtc 120aaaaccgtca tcatgatgag
ggatgacata atataaaaag ttgactaagg tcttggtagt 180actctttgat tagtattata
tattggtgag aacatgagtc aagaggagac aagaaaccga 240ggaaccatag tttagcaaca
agatggaagt tgcaaagttg agctagccgc tcgattagtt 300acatctccta agcagtacta
caaggaatgg tctctatact ttcatgttta gcacatggta 360gtgcggattg acaagttaga
aacagtgctt aggagacaaa gagtcagtaa aggtattgaa 420agagtgaagt tgatgctcga
caggtcagga gaagtccctc cgccagatgg tgactaccaa 480ggggttggta tcagctgaga
cccaaataag attcttcggt tgaaccagtg gttcgaccga 540gactcttagg gtgggatttc
actgtaagat ttgtgcattt tgttgaatat aaattgacaa 600ttttttttat ttaattatag
attatttaga atgaattaca tatttagttt ctaacaagga 660tagcaatgga tgggtatggg
tacaggttaa acatatctat tacccaccca tctagtcgtc 720gggttttaca cgtacccacc
cgtttacata aaccagaccg gaattttaaa ccgtacccgt 780ccgttagcgg gtttcagatt
tacccgttta atcgggtaaa acctgattac taaatatata 840ttttttattt gataaacaaa
acaaaaatgt taatattttc atattggatg caattttaag 900aaacacatat tcataaattt
ccatatttgt aggaaaataa aaagaaaaat atattcaaga 960acacaaattt caccgacatg
acttttatta cagagttgga attagatcta acaattgaaa 1020aattaaaatt aagatagaat
atgttgagga acatgacata gtataatgct gggttacccg 1080tcgggtaggt atcgaggcgg
atactactaa atccatccca ctcgctatcc gataatcact 1140ggtttcgggt atacccattc
ccgtcaacag gcctttttaa ccggataatt tcaacttata 1200gtgaatgaat tttgaataaa
tagttagaat accaaaatcc tggattgcat ttgcaatcaa 1260attttgtgaa ccgttaaatt
ttgcatgtac ttgggataga tataatagaa ccgaattttc 1320attagtttaa tttataactt
actttgttca aagaaaaaaa atatctatcc aatttactta 1380taataaaaaa taatctatcc
aagttactta ttataatcaa cttgtaaaaa ggtaagaata 1440caaatgtggt agcgtacgtg
tgattatatg tgacgaaatg ttatatctaa caaaagtcca 1500aattcccatg gtaaaaaaaa
tcaaaatgca tggcaggctg tttgtaacct tggaataaga 1560tgttggccaa ttctggagcc
gccacgtacg caagactcag ggccacgttc tcttcatgca 1620aggatagtag aacaccactc
cacccacctc ctatattaga cctttgccca accctcccca 1680actttcccat cccatccaca
aagaaaccga catttttatc ataaatc 172761151DNAArtificial
Sequencepromotor p-PvArc 6tactatagaa aatgtgttat atcgacatga ccagacaaag
gggcaacagt taacaaaaca 60attaattctt tcatttgaga ttaaggaagg taaggtacta
aaaagattaa aaaaaatgag 120cttatctctt tgtttctgta ataataatat aagtgtgata
aacttttaat ataataattg 180taattaggtt ttctacagat gagcaccact cagagacaag
ataagaagaa aacaattttg 240ttaaacatga ttatagaaac ttttagttaa gtcttgaagt
atcaatataa caaaaaaaag 300tacacacgac tatgacaata aacccactac cgtcaggtta
tcatttcgat gaaatgtttt 360gatatcatta aatataacag tcacaaaaaa tcatctaatt
ataacaatat aacttataca 420tatatttaac taaaaactta gagtttttgt aatgattcta
attgatgatt agagtttata 480gaaatacaat taaataaaaa atataatttt aaaaaaacat
agtaaagtca atgagatcct 540ctctgacctc agtgatcatt tagtcatgta tgtacaacaa
tcattgttca tcacatgact 600gtaaaataaa taaggataaa cttgggaata tatataatat
attgtattaa ataaaaaagg 660gaaatacaaa tatcaatttt agattcccga gttgacacaa
ctcaccatgc acgctgccac 720ctcagctccc agctctcgtc acatgtctca tgtcagttag
gtctttggtt tttagtcttt 780gacacaactc gccatgcatg ttgccacgtg agctcgttcc
tcttcccatg atctcaccac 840tgggcatgca tgctgccacc tcagctggca cctcttctct
atatgtccct agaggccatg 900cacagtgcca cctcagcact cctctcagaa cccatacgta
cctgccaatc ggcttctctc 960cataaatatc tatttaaatt ataactaatt atttcatata
cttaattgat gacgtggatg 1020cattgccatc gttgtttaat aattgttaat tacgacatga
taaataaaat gaaagtaaaa 1080agtacgaaag attttccatt tgttgttgta taaatagaga
agtgagtgat gcataatgca 1140tgaatgcatg a
115171799DNAArtificial Sequencepromotor 7tcgacggccc
ggactgtatc caacttctga tctttgaatc tctctgttcc aacatgttct 60gaaggagttc
taagactttt cagaaagctt gtaacatgct ttgtagactt tctttgaatt 120actcttgcaa
actctgattg aacctacgtg aaaactgctc cagaagttct aaccaaattc 180cgtcttggga
aggcccaaaa tttattgagt acttcagttt catggacgtg tcttcaaaga 240tttataactt
gaaatcccat catttttaag agaagttctg ttccgcaatg tcttagatct 300cattgaaatc
tacaactctt gtgtcagaag ttcttccaga atcaacttgc atcatggtga 360aaatctggcc
agaagttctg aacttgtcat atttcttaac agttagaaaa atttctaagt 420gtttagaatt
ttgacttttc caaagcaaac ttgacttttg actttcttaa taaaacaaac 480ttcatattct
aacatgtctt gatgaaatgt gattcttgaa atttgatgtt gatgcaaaag 540tcaaagtttg
acttttcagt gtgcaattga ccattttgct cttgtgccaa ttccaaacct 600aaattgatgt
atcagtgctg caaacttgat gtcatggaag atcttatgag aaaattcttg 660aagactgaga
ggaaaaattt tgtagtacaa cacaaagaat cctgtttttc atagtcggac 720tagacacatt
aacataaaac accacttcat tcgaagagtg attgaagaag gaaatgtgca 780gttacctttc
tgcagttcat aagagcaact tacagacact tttactaaaa tactacaaag 840aggaagattt
taacaactta gagaagtaat gggagttaaa gagcaacaca ttaaggggga 900gtgttaaaat
taatgtgttg taaccaccac tacctttagt aagtattata agaaaattgt 960aatcatcaca
ttataattat tgtccttatt taaaattatg ataaagttgt atcattaaga 1020ttgagaaaac
caaatagtcc tcgtcttgat ttttgaatta ttgttttcta tgttactttt 1080cttcaagcct
atataaaaac tttgtaatgc taaattgtat gctggaaaaa aatgtgtaat 1140gaattgaata
gaaattatgg tatttcaaag tccaaaatcc atcaatagaa atttagtaca 1200aaacgtaact
caaaaatatt ctcttatttt aaattttaca acaatataaa aatattctct 1260tattttaaat
tttacaataa tataatttat cacctgtcac ctttagaata ccaccaacaa 1320tattaatact
tagatatttt attcttaata attttgagat ctctcaatat atctgatatt 1380tattttatat
ttgtgtcata ttttcttatg ttttagagtt aacccttata tcttggtcaa 1440actagtaatt
caatatatga gtttgtgaag gacacattga catcttgaaa cattggtttt 1500aaccttgttg
gaatgttaaa ggtaataaaa cattcagaat tatgaccatc tattaatata 1560cttcctttgt
cttttaaaaa agtgtgcatg aaaatgctct atggtaagct agagtgtctt 1620gctggcctgt
gtatatcaat tccatttcca gatggtagaa actgccacta cgaataatta 1680gtcataagac
acgtatgtta acacacgtcc ccttgcatgt tttttgccat atattccgtc 1740tctttctttt
tcttcacgta taaaacaatg aactaattaa tagagcgatc aagctgaac
179981430DNAArtificial Sequencepromotor p-BnFAE1 8aagctttaca acgctacaca
aaacttataa ccgtaatcac cattcattaa cttaactact 60atcacatgca ttcatgaatt
gaaacgagaa ggatgtaaat agttgggaag ttatctccac 120gttgaagaga tcgttagcga
gagctgaaag accgagggag gagacgccgt caacacggac 180agagtcgtcg accctcacat
gaagtaggag gaatctccgt gaggagccag agagacgtct 240ttggtcttcg gtttcgatcc
ttgatctgac ggagaagacg agagaagtgc gactggactc 300cgtgaggacc aacagagtcg
tcctcggttt cgatcgtcgg tattggtgga gaaggcggag 360gaatctccgt gacgagccag
agagatgtcg tcggtcttcg gtttcgatcc ttgatctgac 420ggagaagacg agagaagtgc
gacgagactc cgtgaggacc aacagagttg tcctcggttt 480cgatcgtcgg tttcggcgga
gaaggcggag gaatctccgt gaggagccag agagacgtcg 540ttggtcttcg gtttcgatcc
ttgatctgtt ggagaagacg agacaagtgg gacgagactc 600aacgacggag tcagagacgt
cgtcggtctt cggtttcggc cgagaaggcg gagtcggtct 660tcggtttcgg ccgagaaggc
ggaggagacg tcttcgattt gggtctctcc tcttgacgaa 720gaaaacaaag aacacgagaa
ataatgagaa agagaacaaa agaaaaaaaa ataaaaataa 780aaataaaatt tggtcctctt
atgtggtgac acgtggtttg aaacccacca aataatcgat 840cacaaaaaac ctaagttaag
gatcggtaat aacctttcta attaattttg atttatatta 900aatcactctt tttatttata
aaccccacta aattatgcga tattgattgt ctaagtacaa 960aaattctctc gaattcaata
cacatgtttc atatatttag ccctgttcat ttaatattac 1020tagcgcattt ttaatttaaa
attttgtaaa cttttttggt caaagaacat ttttttaatt 1080agagacagaa atctagactc
tttatttgga ataatagtaa taaagatata ttaggcaatg 1140agtttatgat gttatgttta
tatagtttat ttcattttaa attgaaaagc attattttta 1200tcgaaatgaa tctagtatac
aatcaatatt tatgtttttt catcagatac tttcctattt 1260tttggcacct ttcatcggac
tactgattta tttcaatgtg tatgcatgca tgagcatgag 1320tatacacatg tcttttaaaa
tgcatgtaaa gcgtaacgga ccacaaaaga ggatccatac 1380aaatacatct catcgcttcc
tctactattc tccgacacac acactgagca 14309684DNAArtificial
Sequencepromotor p-VfUSP 9ctgcagcaaa tttacacatt gccactaaac gtctaaaccc
ttgtaatttg tttttgtttt 60actatgtgtg ttatgtattt gatttgcgat aaatttttat
atttggtact aaatttataa 120caccttttat gctaacgttt gccaacactt agcaatttgc
aagttgatta attgattcta 180aattattttt gtcttctaaa tacatatact aatcaactgg
aaatgtaaat atttgctaat 240atttctacta taggagaatt aaagtgagtg aatatggtac
cacaaggttt ggagatttaa 300ttgttgcaat gctgcatgga tggcatatac accaaacatt
caataattct tgaggataat 360aatggtacca cacaagattt gaggtgcatg aacgtcacgt
ggacaaaagg tttagtaatt 420tttcaagaca acaatgttac cacacacaag ttttgaggtg
catgcatgga tgccctgtgg 480aaagtttaaa aatattttgg aaatgatttg catggaagcc
atgtgtaaaa ccatgacatc 540cacttggagg atgcaataat gaagaaaact acaaatttac
atgcaactag ttatgcatgt 600agtctatata atgaggattt tgcaatactt tcattcatac
acactcacta agttttacac 660gattataatt tcttcatagc cagt
684101320DNAArtificial Sequenced5Des Tc GA delta 5
desaturase of Thraustochytrium, codon optimized 10atgggaaaag
gatctgaggg aagatctgct gctagagaga tgactgctga ggctaacgga 60gataagagaa
agaccatcct cattgaggga gtgttgtacg atgctaccaa cttcaaacac 120ccaggaggtt
ccattattaa cttcctcacc gagggagaag ctggagttga tgctacccaa 180gcttacagag
agttccatca gagatccgga aaggctgata agtacctcaa gtccctccca 240aagttggatg
cttctaaggt ggagtctagg ttctctgcta aggagcaggc tagaagggac 300gctatgacca
gggattacgc tgctttcaga gaggagttgg ttgctgaggg atacttcgat 360ccatctatcc
cacacatgat ctacagagtg gtggagattg tggctttgtt cgctttgtct 420ttctggttga
tgtctaaggc ttctccaacc tctttggttt tgggagtggt gatgaacgga 480atcgctcaag
gaagatgcgg atgggttatg catgagatgg gacacggatc tttcactgga 540gttatctggc
tcgatgatag gatgtgcgag ttcttctacg gagttggatg tggaatgtct 600ggacactact
ggaagaacca gcattctaag caccatgctg ctccaaacag attggagcac 660gatgtggatt
tgaacacctt gccactcgtt gctttcaacg agagagttgt gaggaaggtt 720aagccaggat
ctttgttggc tttgtggctc agagttcagg cttatttgtt cgctccagtg 780tcttgcttgt
tgatcggatt gggatggacc ttgtacttgc acccaagata tatgctcagg 840accaagagac
atatggagtt tgtgtggatc ttcgctagat atatcggatg gttctccttg 900atgggagctt
tgggatattc tcctggaact tctgtgggaa tgtacctctg ctctttcgga 960cttggatgca
tctacatctt cctccaattc gctgtgtctc atacccattt gccagttacc 1020aacccagagg
atcaattgca ttggcttgag tacgctgctg atcataccgt gaacatctct 1080accaagtctt
ggttggttac ctggtggatg tctaacctca acttccaaat cgagcatcat 1140ttgttcccaa
ccgctccaca attcaggttc aaggagatct ctccaagagt tgaggctctc 1200ttcaagagac
ataacctccc ttactacgat ttgccataca cctctgctgt ttctactacc 1260ttcgctaacc
tctactctgt tggacattct gttggagctg ataccaagaa gcaggattga
132011439PRTArtificial Sequenced5Des Tc GA delta 5 desaturase of
Thraustochytrium, codon optimized 11Met Gly Lys Gly Ser Glu Gly Arg Ser
Ala Ala Arg Glu Met Thr Ala1 5 10
15Glu Ala Asn Gly Asp Lys Arg Lys Thr Ile Leu Ile Glu Gly Val
Leu 20 25 30Tyr Asp Ala Thr
Asn Phe Lys His Pro Gly Gly Ser Ile Ile Asn Phe 35
40 45Leu Thr Glu Gly Glu Ala Gly Val Asp Ala Thr Gln
Ala Tyr Arg Glu 50 55 60Phe His Gln
Arg Ser Gly Lys Ala Asp Lys Tyr Leu Lys Ser Leu Pro65 70
75 80Lys Leu Asp Ala Ser Lys Val Glu
Ser Arg Phe Ser Ala Lys Glu Gln 85 90
95Ala Arg Arg Asp Ala Met Thr Arg Asp Tyr Ala Ala Phe Arg
Glu Glu 100 105 110Leu Val Ala
Glu Gly Tyr Phe Asp Pro Ser Ile Pro His Met Ile Tyr 115
120 125Arg Val Val Glu Ile Val Ala Leu Phe Ala Leu
Ser Phe Trp Leu Met 130 135 140Ser Lys
Ala Ser Pro Thr Ser Leu Val Leu Gly Val Val Met Asn Gly145
150 155 160Ile Ala Gln Gly Arg Cys Gly
Trp Val Met His Glu Met Gly His Gly 165
170 175Ser Phe Thr Gly Val Ile Trp Leu Asp Asp Arg Met
Cys Glu Phe Phe 180 185 190Tyr
Gly Val Gly Cys Gly Met Ser Gly His Tyr Trp Lys Asn Gln His 195
200 205Ser Lys His His Ala Ala Pro Asn Arg
Leu Glu His Asp Val Asp Leu 210 215
220Asn Thr Leu Pro Leu Val Ala Phe Asn Glu Arg Val Val Arg Lys Val225
230 235 240Lys Pro Gly Ser
Leu Leu Ala Leu Trp Leu Arg Val Gln Ala Tyr Leu 245
250 255Phe Ala Pro Val Ser Cys Leu Leu Ile Gly
Leu Gly Trp Thr Leu Tyr 260 265
270Leu His Pro Arg Tyr Met Leu Arg Thr Lys Arg His Met Glu Phe Val
275 280 285Trp Ile Phe Ala Arg Tyr Ile
Gly Trp Phe Ser Leu Met Gly Ala Leu 290 295
300Gly Tyr Ser Pro Gly Thr Ser Val Gly Met Tyr Leu Cys Ser Phe
Gly305 310 315 320Leu Gly
Cys Ile Tyr Ile Phe Leu Gln Phe Ala Val Ser His Thr His
325 330 335Leu Pro Val Thr Asn Pro Glu
Asp Gln Leu His Trp Leu Glu Tyr Ala 340 345
350Ala Asp His Thr Val Asn Ile Ser Thr Lys Ser Trp Leu Val
Thr Trp 355 360 365Trp Met Ser Asn
Leu Asn Phe Gln Ile Glu His His Leu Phe Pro Thr 370
375 380Ala Pro Gln Phe Arg Phe Lys Glu Ile Ser Pro Arg
Val Glu Ala Leu385 390 395
400Phe Lys Arg His Asn Leu Pro Tyr Tyr Asp Leu Pro Tyr Thr Ser Ala
405 410 415Val Ser Thr Thr Phe
Ala Asn Leu Tyr Ser Val Gly His Ser Val Gly 420
425 430Ala Asp Thr Lys Lys Gln Asp
435121320DNAArtificial Sequenced5Des Tc GA delta 5 desaturase of
Thraustochytrium, codon optimized 2nd version 12atgggaaaag gatctgaggg
aagatctgct gctagagaga tgactgctga ggctaacgga 60gataagagaa agaccatcct
cattgaggga gtgttgtacg atgctaccaa cttcaaacac 120ccaggaggtt ccattattaa
cttcctcacc gagggagaag ctggagttga tgctacccaa 180gcttacagag agttccatca
gagatccgga aaggctgata agtacctcaa gtccctccca 240aagttggatg cttctaaggt
ggagtctagg ttctctgcta aggagcaggc tagaagggac 300gctatgacca gggattacgc
tgctttcaga gaggagttgg ttgctgaggg atacttcgat 360ccatctatcc cacacatgat
ctacagagtg gtggagattg tggctttgtt cgctttgtct 420ttctggttga tgtctaaggc
ttctccaacc tctttggttt tgggagtggt gatgaacgga 480atcgctcaag gaagatgcgg
atgggttatg cacgagatgg gacacggatc tttcactgga 540gttatctggc tcgatgatag
gatgtgcgag ttcttctacg gagttggatg tggaatgtct 600ggacactact ggaagaacca
gcactctaag caccacgctg ctccaaacag attggagcac 660gatgtggatt tgaacacctt
gccactcgtt gctttcaacg agagagttgt gaggaaggtt 720aagccaggat ctttgttggc
tttgtggctc agagttcagg cttatttgtt cgctccagtg 780tcttgcttgt tgatcggatt
gggatggacc ttgtacttgc acccaagata tatgctcagg 840accaagagac acatggagtt
tgtgtggatc ttcgctagat atatcggatg gttctccttg 900atgggagctt tgggatattc
tcctggaact tctgtgggaa tgtacctctg ctctttcgga 960cttggatgca tctacatctt
cctccaattc gctgtgtctc acacccactt gccagttacc 1020aacccagagg atcaattgca
ctggcttgag tacgctgctg atcacaccgt gaacatctct 1080accaagtctt ggttggttac
ctggtggatg tctaacctca acttccaaat cgagcaccac 1140ttgttcccaa ccgctccaca
attcaggttc aaggagatct ctccaagagt tgaggctctc 1200ttcaagagac acaacctccc
ttactacgat ttgccataca cctctgctgt ttctactacc 1260ttcgctaacc tctactctgt
tggacactct gttggagctg ataccaagaa gcaggattga 1320131434DNAArtificial
Sequenceo3Des Cp GA omega 3 desaturase Claviceps purpurea; codon
optimized 13atggctgcta ctacctctgc tatgagcaag gatgctgttc ttagaagaac
tgctgctgct 60actactgcta tcgatcacga aagctctacc tctgcttctc cagctgattc
tcctagactc 120tctgcttctt ctacctctct ctcttctctc agctctctcg acgctaagga
taaggatgat 180gagtacgctg gacttcttga tacttacgga aacgctttca cccctcctga
tttcactatc 240aaggatatca gagatgctat ccctaagcac tgcttcgagc gttctgctat
caagggatac 300gcttatatcc tcagagatgt ggcttgcctt tctaccactt tctacctctt
ccacaacttc 360gttacccctg agaacgttcc ttacacccct cttagagttt tcctctgggg
agtttacact 420gctcttcagg gacttttcgg aactggactc tggattatcg ctcacgagtg
tggacacggt 480gctttctctc cttctaccct cactaacgat cttactggat gggttctcca
ctctgctctt 540ctcgtgcctt acttctcttg gaagttctct cactctgctc accacaaggg
aaccggaaat 600atggaaaggg atatggcttt cctccctaga actagggctc aatacgctac
cagattcgga 660agagctatgg atcagcttgg agatctttgc gaggaaaccc ctatctacac
tgctggattc 720cttgttttcc agcagcttct tggatggcct tcttacttga tcgctaacgt
tactggacac 780gatcttcacg agagacagag agagggaaga ggaaagggaa agaagaacgg
attcggagga 840actgttaacc acttcgaccc tcgttctcct atcttcgatg acaagcacgc
taagtttatc 900gttctcagcg atatcggact tggacttgct atcgctgctc ttgtttacct
cggaaacaga 960ttcggatggg ctaacgttgc tgtttggtac ttcgttcctt acctctgggt
taaccactgg 1020atcgttgcta tcactttcct tcagcacact gatcctactc ttcctcacta
cactgctgag 1080gaatggaact tcgttcgtgg agctgctgct acaatcgata gagagatggg
atttatcggt 1140agacacctct tccacggaat cgttgagact cacgtgcttc accactacgt
ttcttcaatc 1200cctttctaca acgctgatga ggcttctgag gctatcaagc ctgttatggg
aaagcactac 1260cgttctgaga ctaaggatgg acctatgggt tttatcaggg ctttgtggaa
aactgctaga 1320tggtgtcaat gggttgagcc ttctgctgat gctcaaggtg ctggtgaagg
tgttctcttc 1380ttcaggaaca gaaacggact tggaactaag cctatctcta tgaggaccca
gtga 143414477PRTArtificial Sequenceo3Des Cp GA omega 3
desaturase Claviceps purpurea; codon optimized 14Met Ala Ala Thr Thr
Ser Ala Met Ser Lys Asp Ala Val Leu Arg Arg1 5
10 15Thr Ala Ala Ala Thr Thr Ala Ile Asp His Glu
Ser Ser Thr Ser Ala 20 25
30Ser Pro Ala Asp Ser Pro Arg Leu Ser Ala Ser Ser Thr Ser Leu Ser
35 40 45Ser Leu Ser Ser Leu Asp Ala Lys
Asp Lys Asp Asp Glu Tyr Ala Gly 50 55
60Leu Leu Asp Thr Tyr Gly Asn Ala Phe Thr Pro Pro Asp Phe Thr Ile65
70 75 80Lys Asp Ile Arg Asp
Ala Ile Pro Lys His Cys Phe Glu Arg Ser Ala 85
90 95Ile Lys Gly Tyr Ala Tyr Ile Leu Arg Asp Val
Ala Cys Leu Ser Thr 100 105
110Thr Phe Tyr Leu Phe His Asn Phe Val Thr Pro Glu Asn Val Pro Tyr
115 120 125Thr Pro Leu Arg Val Phe Leu
Trp Gly Val Tyr Thr Ala Leu Gln Gly 130 135
140Leu Phe Gly Thr Gly Leu Trp Ile Ile Ala His Glu Cys Gly His
Gly145 150 155 160Ala Phe
Ser Pro Ser Thr Leu Thr Asn Asp Leu Thr Gly Trp Val Leu
165 170 175His Ser Ala Leu Leu Val Pro
Tyr Phe Ser Trp Lys Phe Ser His Ser 180 185
190Ala His His Lys Gly Thr Gly Asn Met Glu Arg Asp Met Ala
Phe Leu 195 200 205Pro Arg Thr Arg
Ala Gln Tyr Ala Thr Arg Phe Gly Arg Ala Met Asp 210
215 220Gln Leu Gly Asp Leu Cys Glu Glu Thr Pro Ile Tyr
Thr Ala Gly Phe225 230 235
240Leu Val Phe Gln Gln Leu Leu Gly Trp Pro Ser Tyr Leu Ile Ala Asn
245 250 255Val Thr Gly His Asp
Leu His Glu Arg Gln Arg Glu Gly Arg Gly Lys 260
265 270Gly Lys Lys Asn Gly Phe Gly Gly Thr Val Asn His
Phe Asp Pro Arg 275 280 285Ser Pro
Ile Phe Asp Asp Lys His Ala Lys Phe Ile Val Leu Ser Asp 290
295 300Ile Gly Leu Gly Leu Ala Ile Ala Ala Leu Val
Tyr Leu Gly Asn Arg305 310 315
320Phe Gly Trp Ala Asn Val Ala Val Trp Tyr Phe Val Pro Tyr Leu Trp
325 330 335Val Asn His Trp
Ile Val Ala Ile Thr Phe Leu Gln His Thr Asp Pro 340
345 350Thr Leu Pro His Tyr Thr Ala Glu Glu Trp Asn
Phe Val Arg Gly Ala 355 360 365Ala
Ala Thr Ile Asp Arg Glu Met Gly Phe Ile Gly Arg His Leu Phe 370
375 380His Gly Ile Val Glu Thr His Val Leu His
His Tyr Val Ser Ser Ile385 390 395
400Pro Phe Tyr Asn Ala Asp Glu Ala Ser Glu Ala Ile Lys Pro Val
Met 405 410 415Gly Lys His
Tyr Arg Ser Glu Thr Lys Asp Gly Pro Met Gly Phe Ile 420
425 430Arg Ala Leu Trp Lys Thr Ala Arg Trp Cys
Gln Trp Val Glu Pro Ser 435 440
445Ala Asp Ala Gln Gly Ala Gly Glu Gly Val Leu Phe Phe Arg Asn Arg 450
455 460Asn Gly Leu Gly Thr Lys Pro Ile
Ser Met Arg Thr Gln465 470
475151434DNAArtificial Sequenceo3Des Cp GA2 V282L; omega 3 desaturase
Claviceps purpurea; mutation V282L; codon optimized 15atggctgcta
ccacttctgc aatgtctaag gacgctgttc tgcggcgcac tgctgccgca 60acgactgcca
tcgatcacga gtcgtcgacc tctgccagtc cagccgactc gcctagactc 120tcagcctcgt
ccacgtcgct ttcgtcgctt tcttctctcg atgcgaagga caaggacgac 180gagtatgccg
gccttcttga cacatacgga aacgccttca caccccccga cttcactatc 240aaggacatcc
gtgatgccat acccaagcat tgcttcgaac gctctgccat caagggatac 300gcatatattc
ttcgcgacgt cgcctgtctt tctactacgt tctacctgtt ccacaacttc 360gtgacgcccg
agaacgtccc ctacactccc cttcgtgtct ttctctgggg tgtttacact 420gccctgcaag
gtctatttgg aactggactc tggattattg cccacgaatg tggccacgga 480gccttctctc
cttcaacctt gaccaacgac cttaccggct gggtccttca ctcagctctc 540cttgttccct
atttcagctg gaagttttcc cacagtgcgc atcacaaagg aactggaaac 600atggagcgcg
acatggcttt ccttccccgc acacgtgcgc agtatgccac tcgatttgga 660cgtgcgatgg
atcaacttgg tgacctttgc gaagagacac ccatttacac ggctgggttc 720ttggttttcc
agcagctcct aggctggcct agctatctta tagcgaacgt cacaggtcac 780gacctccacg
aacgccagcg tgagggtcga ggtaagggca agaagaacgg tttcgggggc 840accttaaatc
actttgatcc ccgcagccct attttcgatg acaaacacgc caagttcatt 900gttctctctg
acatcggcct gggtcttgct atcgctgctc tggtgtatct tggcaaccgt 960ttcggctggg
ctaacgtggc tgtttggtat ttcgtgccct atctttgggt gaatcactgg 1020atcgttgcca
tcacgttcct ccagcatacg gatccaactc tgccgcatta caccgccgaa 1080gagtggaact
ttgttcgcgg tgccgctgct accattgatc gcgagatggg cttcattggc 1140cgccaccttt
tccacggcat tgtcgagacc catgtcctcc atcactatgt cagctctata 1200ccgttctaca
acgcggacga agcctccgag gccataaaac cggttatggg caagcactat 1260cgatctgaaa
ccaaagacgg acctatggga tttatccgcg ctctttggaa gactgctcgc 1320tggtgccagt
gggtagagcc tagtgccgat gcgcaaggtg ctggagaggg cgtgttgttc 1380ttccgcaacc
gaaatggtct cggcacgaaa cccatttcga tgagaactca gtag
143416477PRTArtificial Sequenceo3Des Cp GA2 V282L; omega 3 desaturase
Claviceps purpurea; mutation V282L; codon optimized 16Met Ala Ala Thr
Thr Ser Ala Met Ser Lys Asp Ala Val Leu Arg Arg1 5
10 15Thr Ala Ala Ala Thr Thr Ala Ile Asp His
Glu Ser Ser Thr Ser Ala 20 25
30Ser Pro Ala Asp Ser Pro Arg Leu Ser Ala Ser Ser Thr Ser Leu Ser
35 40 45Ser Leu Ser Ser Leu Asp Ala Lys
Asp Lys Asp Asp Glu Tyr Ala Gly 50 55
60Leu Leu Asp Thr Tyr Gly Asn Ala Phe Thr Pro Pro Asp Phe Thr Ile65
70 75 80Lys Asp Ile Arg Asp
Ala Ile Pro Lys His Cys Phe Glu Arg Ser Ala 85
90 95Ile Lys Gly Tyr Ala Tyr Ile Leu Arg Asp Val
Ala Cys Leu Ser Thr 100 105
110Thr Phe Tyr Leu Phe His Asn Phe Val Thr Pro Glu Asn Val Pro Tyr
115 120 125Thr Pro Leu Arg Val Phe Leu
Trp Gly Val Tyr Thr Ala Leu Gln Gly 130 135
140Leu Phe Gly Thr Gly Leu Trp Ile Ile Ala His Glu Cys Gly His
Gly145 150 155 160Ala Phe
Ser Pro Ser Thr Leu Thr Asn Asp Leu Thr Gly Trp Val Leu
165 170 175His Ser Ala Leu Leu Val Pro
Tyr Phe Ser Trp Lys Phe Ser His Ser 180 185
190Ala His His Lys Gly Thr Gly Asn Met Glu Arg Asp Met Ala
Phe Leu 195 200 205Pro Arg Thr Arg
Ala Gln Tyr Ala Thr Arg Phe Gly Arg Ala Met Asp 210
215 220Gln Leu Gly Asp Leu Cys Glu Glu Thr Pro Ile Tyr
Thr Ala Gly Phe225 230 235
240Leu Val Phe Gln Gln Leu Leu Gly Trp Pro Ser Tyr Leu Ile Ala Asn
245 250 255Val Thr Gly His Asp
Leu His Glu Arg Gln Arg Glu Gly Arg Gly Lys 260
265 270Gly Lys Lys Asn Gly Phe Gly Gly Thr Leu Asn His
Phe Asp Pro Arg 275 280 285Ser Pro
Ile Phe Asp Asp Lys His Ala Lys Phe Ile Val Leu Ser Asp 290
295 300Ile Gly Leu Gly Leu Ala Ile Ala Ala Leu Val
Tyr Leu Gly Asn Arg305 310 315
320Phe Gly Trp Ala Asn Val Ala Val Trp Tyr Phe Val Pro Tyr Leu Trp
325 330 335Val Asn His Trp
Ile Val Ala Ile Thr Phe Leu Gln His Thr Asp Pro 340
345 350Thr Leu Pro His Tyr Thr Ala Glu Glu Trp Asn
Phe Val Arg Gly Ala 355 360 365Ala
Ala Thr Ile Asp Arg Glu Met Gly Phe Ile Gly Arg His Leu Phe 370
375 380His Gly Ile Val Glu Thr His Val Leu His
His Tyr Val Ser Ser Ile385 390 395
400Pro Phe Tyr Asn Ala Asp Glu Ala Ser Glu Ala Ile Lys Pro Val
Met 405 410 415Gly Lys His
Tyr Arg Ser Glu Thr Lys Asp Gly Pro Met Gly Phe Ile 420
425 430Arg Ala Leu Trp Lys Thr Ala Arg Trp Cys
Gln Trp Val Glu Pro Ser 435 440
445Ala Asp Ala Gln Gly Ala Gly Glu Gly Val Leu Phe Phe Arg Asn Arg 450
455 460Asn Gly Leu Gly Thr Lys Pro Ile
Ser Met Arg Thr Gln465 470
475171086DNAArtificial Sequenceo3Des(Pi GA2); Phytophthora infestans;
codon optimized, 2nd version 17atggctacaa aggaggctta cgttttccca
actctcaccg agatcaagag atctctccca 60aaggattgct tcgaggcttc tgtgcctttg
tctctctact acactgtgag atgcttggtt 120attgctgtgg ctttgacctt cggattgaac
tacgctagag ctttgccaga ggttgagtct 180ttctgggctt tggatgctgc tttgtgcact
ggatatatcc tcctccaggg aattgtgttc 240tggggattct tcactgttgg acacgatgct
ggacacggag ctttctctag ataccacctc 300ttgaacttcg ttgtgggaac cttcatgcac
tctctcatct tgaccccatt cgagtcttgg 360aagttgaccc acagacacca ccacaagaac
accggaaaca tcgatagaga tgaggtgttc 420tacccacaga gaaaggctga tgatcaccca
ttgtccagga acttgatctt ggctttggga 480gctgcttggc ttgcttattt ggtggaggga
ttcccaccaa gaaaggtgaa ccacttcaac 540ccattcgagc cactttttgt gagacaagtg
tccgctgtgg ttatctcttt gctcgctcac 600ttcttcgttg ctggactctc tatctacttg
tctctccagt tgggacttaa gaccatggct 660atctactact acggaccagt tttcgtgttc
ggatctatgt tggtgattac caccttcttg 720caccacaacg atgaggagac tccatggtat
gctgattctg agtggactta cgtgaaggga 780aacttgtcct ctgtggatag atcttacggt
gctctcatcg ataacctctc ccacaacatc 840ggaactcacc agatccacca cctcttccca
attatcccac actacaagct caagaaggct 900actgctgctt tccaccaagc tttcccagag
cttgtgagaa agtccgatga gccaatcatc 960aaggctttct tcagagtggg aaggttgtat
gctaactacg gagtggttga tcaagaggct 1020aagctcttca ctttgaagga ggctaaggct
gctactgaag ctgctgctaa gaccaagtct 1080acctga
108618361PRTArtificial Sequenceo3Des(Pi
GA2); omega 3 desaturase; Phytophthora infestans; codon optimized,
2nd version 18Met Ala Thr Lys Glu Ala Tyr Val Phe Pro Thr Leu Thr Glu Ile
Lys1 5 10 15Arg Ser Leu
Pro Lys Asp Cys Phe Glu Ala Ser Val Pro Leu Ser Leu 20
25 30Tyr Tyr Thr Val Arg Cys Leu Val Ile Ala
Val Ala Leu Thr Phe Gly 35 40
45Leu Asn Tyr Ala Arg Ala Leu Pro Glu Val Glu Ser Phe Trp Ala Leu 50
55 60Asp Ala Ala Leu Cys Thr Gly Tyr Ile
Leu Leu Gln Gly Ile Val Phe65 70 75
80Trp Gly Phe Phe Thr Val Gly His Asp Ala Gly His Gly Ala
Phe Ser 85 90 95Arg Tyr
His Leu Leu Asn Phe Val Val Gly Thr Phe Met His Ser Leu 100
105 110Ile Leu Thr Pro Phe Glu Ser Trp Lys
Leu Thr His Arg His His His 115 120
125Lys Asn Thr Gly Asn Ile Asp Arg Asp Glu Val Phe Tyr Pro Gln Arg
130 135 140Lys Ala Asp Asp His Pro Leu
Ser Arg Asn Leu Ile Leu Ala Leu Gly145 150
155 160Ala Ala Trp Leu Ala Tyr Leu Val Glu Gly Phe Pro
Pro Arg Lys Val 165 170
175Asn His Phe Asn Pro Phe Glu Pro Leu Phe Val Arg Gln Val Ser Ala
180 185 190Val Val Ile Ser Leu Leu
Ala His Phe Phe Val Ala Gly Leu Ser Ile 195 200
205Tyr Leu Ser Leu Gln Leu Gly Leu Lys Thr Met Ala Ile Tyr
Tyr Tyr 210 215 220Gly Pro Val Phe Val
Phe Gly Ser Met Leu Val Ile Thr Thr Phe Leu225 230
235 240His His Asn Asp Glu Glu Thr Pro Trp Tyr
Ala Asp Ser Glu Trp Thr 245 250
255Tyr Val Lys Gly Asn Leu Ser Ser Val Asp Arg Ser Tyr Gly Ala Leu
260 265 270Ile Asp Asn Leu Ser
His Asn Ile Gly Thr His Gln Ile His His Leu 275
280 285Phe Pro Ile Ile Pro His Tyr Lys Leu Lys Lys Ala
Thr Ala Ala Phe 290 295 300His Gln Ala
Phe Pro Glu Leu Val Arg Lys Ser Asp Glu Pro Ile Ile305
310 315 320Lys Ala Phe Phe Arg Val Gly
Arg Leu Tyr Ala Asn Tyr Gly Val Val 325
330 335Asp Gln Glu Ala Lys Leu Phe Thr Leu Lys Glu Ala
Lys Ala Ala Thr 340 345 350Glu
Ala Ala Ala Lys Thr Lys Ser Thr 355
360191086DNAArtificial Sequenceo3Des(Pi GA); omega 3 desaturase;
Phytophthora infestans; codon optimized 19atggctacaa aggaggctta
cgttttccca actctcaccg agatcaagag atctctccca 60aaggattgct tcgaggcttc
tgtgcctttg tctctctact acactgtgag atgcttggtt 120attgctgtgg ctttgacctt
cggattgaac tacgctagag ctttgccaga ggttgagtct 180ttctgggctt tggatgctgc
tttgtgcact ggatatatcc tcctccaggg aattgtgttc 240tggggattct tcactgttgg
acacgatgct ggacatggag ctttctctag ataccacctc 300ttgaacttcg ttgtgggaac
cttcatgcat tctctcatct tgaccccatt cgagtcttgg 360aagttgaccc atagacacca
tcataagaac accggaaaca tcgatagaga tgaggtgttc 420tacccacaga gaaaggctga
tgatcatcca ttgtccagga acttgatctt ggctttggga 480gctgcttggc ttgcttattt
ggtggaggga ttcccaccaa gaaaggtgaa ccacttcaac 540ccattcgagc cactttttgt
gagacaagtg tccgctgtgg ttatctcttt gctcgctcac 600ttcttcgttg ctggactctc
tatctacttg tctctccagt tgggacttaa gaccatggct 660atctactact acggaccagt
tttcgtgttc ggatctatgt tggtgattac caccttcttg 720caccataacg atgaggagac
tccatggtat gctgattctg agtggactta cgtgaaggga 780aacttgtcct ctgtggatag
atcttacggt gctctcatcg ataacctctc ccataacatc 840ggaactcatc agatccatca
cctcttccca attatcccac actacaagct caagaaggct 900actgctgctt tccatcaagc
tttcccagag cttgtgagaa agtccgatga gccaatcatc 960aaggctttct tcagagtggg
aaggttgtat gctaactacg gagtggttga tcaagaggct 1020aagctcttca ctttgaagga
ggctaaggct gctactgaag ctgctgctaa gaccaagtct 1080acctga
10862018DNAArtificial
Sequenceenhancer end sequence according to invention 20accaatcacc
accaaaaa
182118DNAArtificial Sequence5' untranslated region based on 6-tuples
21aatcaccacc accaaaaa
182218DNAArtificial Sequence5' untranslated region based on 6-tuples
22accaccaatc accaaaaa
182318DNAArtificial Sequence5' untranslated region based on 6-tuples
23caccaccacc accaaaaa
182418DNAArtificial Sequence5' untranslated region based on 5-tuples
24aaagaaccac caccaaaa
182518DNAArtificial Sequence5' untranslated region based on 5-tuples
25aaccaatcac accaaaaa
182618DNAArtificial Sequence5' untranslated region based on 5-tuples
26aactcaccac caccaaaa
182718DNAArtificial Sequence5' untranslated region based on 5-tuples
27aatcaccacc accaaaaa
182818DNAArtificial Sequence5' untranslated region based on 5-tuples
28accaatcaca caccaaaa
182918DNAArtificial Sequence5' untranslated region based on 5-tuples
29actcacacac caccaaaa
183018DNAArtificial Sequence5' untranslated region based on 5-tuples
30caactcacca caccaaaa
183118DNAArtificial Sequence5' untranslated region based on 5-tuples
31caatcacacc accaaaaa
183218DNAArtificial Sequence5' untranslated region based on 5-tuples
32cacaactcac accaaaaa
183318DNAArtificial Sequence5' untranslated region based on 5-tuples
33cacaactcac caccaaaa
183418DNAArtificial Sequence5' untranslated region based on 5-tuples
34caccaccaat caccaaaa
183518DNAArtificial Sequence5' untranslated region based on 5-tuples
35ccaatcacca ccaaaaaa
183618DNAArtificial Sequence5' untranslated region based on 5-tuples
36ccaccaatca caccaaaa
183718DNAArtificial Sequence5' untranslated region based on 4-tuples
37accacactca caaccaaa
183818DNAArtificial Sequence5' untranslated region based on 4-tuples
38actcaccaca accaaaaa
183918DNAArtificial Sequence5' untranslated region based on 4-tuples
39cacaaccact caccaaaa
184018DNAArtificial Sequence5' untranslated region based on 4-tuples
40cacaactcac caccaaaa
184118DNAArtificial Sequence5' untranslated region based on 4-tuples
41caccacacaa tcaccaaa
184218DNAArtificial Sequence5' untranslated region based on 4-tuples
42caccaccaca caaccaaa
184318DNAArtificial Sequence5' untranslated region based on 4-tuples
43ccaactcaca ccacaaaa
184418DNAArtificial Sequence5' untranslated region based on 4-tuples
44ccaccaactc acaccaaa
184518DNAArtificial Sequence5' untranslated region based on 4-tuples
45ctcacaccac accacaaa
184657DNAArtificial Sequencelonger enhancer 46ccaccacaca actcacaact
cactctcaca ccttaaagaa ccaatcacca ccaaaaa 574757DNAArtificial
Sequence5' untranslated region based on 6-tuples 47acaactcaca actcacacct
taaagaacca atcaccaatc accaccacca ccaaaaa 574857DNAArtificial
Sequence5' untranslated region based on 6-tuples 48acaactcaca actcactctc
acaactcaca ccttaaagaa ccaatcacca ccaaaaa 574957DNAArtificial
Sequence5' untranslated region based on 6-tuples 49acaactcaca actcactctc
acaccttaaa gaaccaatca ccaccaatca ccaaaaa 575057DNAArtificial
Sequence5' untranslated region based on 6-tuples 50actctcacac cttaaagaac
caatcaccaa tcaccaatca ccaatcacca ccaaaaa 575157DNAArtificial
Sequence5' untranslated region based on 6-tuples 51atcaccacac aactcactct
cacaccttaa agaaccaatc accaccacca ccaaaaa 575257DNAArtificial
Sequence5' untranslated region based on 6-tuples 52caccttaaag aaccaatcac
caccacacct taaagaacca atcaccacca ccaaaaa 575357DNAArtificial
Sequence5' untranslated region based on 6-tuples 53ccaatcacca cacaactcac
tctcacaact cactctcaca ccttaaagaa ccaaaaa 575457DNAArtificial
Sequence5' untranslated region based on 6-tuples 54tcacacaact cacaccttaa
agaaccaatc accaccacca ccaccaatca ccaaaaa 575557DNAArtificial
Sequence5' untranslated region based on 5-tuples 55aactcactct cacaactctc
acacaactca caccaatcac caccaatcac accaaaa 575657DNAArtificial
Sequence5' untranslated region based on 5-tuples 56acacaactct cactcacaac
tcacaactca caactcactc acaactcacc accaaaa 575757DNAArtificial
Sequence5' untranslated region based on 5-tuples 57actcacaact ctcaccacca
caactctcac accttaaaga accaatcaca ccaaaaa 575857DNAArtificial
Sequence5' untranslated region based on 5-tuples 58actcactcac aactcacaac
tctcacacac caccacacca atcaccacca ccaaaaa 575957DNAArtificial
Sequence5' untranslated region based on 5-tuples 59agaaccaatc actcaccaat
caccttaaag aaccaccacc aatcacacca ccaaaaa 576057DNAArtificial
Sequence5' untranslated region based on 5-tuples 60atcacaactc tcactcacca
ccaatcactc acaccaatca ccaatcacac accaaaa 576157DNAArtificial
Sequence5' untranslated region based on 5-tuples 61caatcaccac acacaactca
ctcactcaca actctcacac caatcaccac caaaaaa 576257DNAArtificial
Sequence5' untranslated region based on 5-tuples 62cacacacaca actcacacac
caatcactct caccaatcac aactcaccac accaaaa 576357DNAArtificial
Sequence5' untranslated region based on 5-tuples 63ccacaactct caccacaact
caccacctta aagaaccaat cacaactctc accaaaa 576457DNAArtificial
Sequence5' untranslated region based on 5-tuples 64ccaccaatca ctcaccacac
cacaactctc acaactcaca accaccaatc accaaaa 576557DNAArtificial
Sequence5' untranslated region based on 5-tuples 65ccttaaagaa ccaatcacaa
ctcacacacc aatcactctc acaactcaca ccaaaaa 576657DNAArtificial
Sequence5' untranslated region based on 5-tuples 66cttaaagaac caatcacacc
aatcactctc acaactcaca actcaccacc accaaaa 576757DNAArtificial
Sequence5' untranslated region based on 5-tuples 67caaccacctt aaagaaccac
aactcacacc ttaaagaacc accaccaatc accaaaa 576857DNAArtificial
Sequence5' untranslated region based on 5-tuples 68tcacaactct cactcactca
ctctcacaac tcacacctta aagaaccacc accaaaa 576957DNAArtificial
Sequence5' untranslated region based on 5-tuples 69tctcacaact cacaccacaa
ctcactctca ccaatcacaa ctcacacacc accaaaa 577057DNAArtificial
Sequence5' untranslated region based on 4-tuples 70aactcacaac caactcacac
tcaccacaac cacaaccaca ccacactcac aaccaaa 577157DNAArtificial
Sequence5' untranslated region based on 4-tuples 71aagaactcac acaatcacca
caatcaccac cacacaactc acaaccactc accaaaa 577257DNAArtificial
Sequence5' untranslated region based on 4-tuples 72aagaactctc tcacacaaag
aactcaccac tctcaccacc acaactcacc accaaaa 577357DNAArtificial
Sequence5' untranslated region based on 4-tuples 73acacaactca cacaccacca
aagaactcac caactctcac accacacaat caccaaa 577457DNAArtificial
Sequence5' untranslated region based on 4-tuples 74accaccaaag aaccaatcac
caccaccaac tctcaccaac caactcacac cacaaaa 577557DNAArtificial
Sequence5' untranslated region based on 4-tuples 75cacaactcac cacaactctc
tctcactcac accacaccac caccaactca caccaaa 577657DNAArtificial
Sequence5' untranslated region based on 4-tuples 76caccaatcac caatcacaat
cactcaccac aatcacaatc accaccacac aaccaaa 577757DNAArtificial
Sequence5' untranslated region based on 4-tuples 77caccacaacc aatcacctta
aagaactcac cttaaaagaa ctcaccacaa ccaaaaa 577857DNAArtificial
Sequence5' untranslated region based on 4-tuples 78caccacactc tcaccaccaa
ctcacactca ccacaccaac tcacaccaca ccacaaa 577957DNAArtificial
Sequence5' untranslated region based on 4-tuples 79ccacaactca ctcacacacc
accaaaaaga actcaccaat cacacaatca caaaaaa 578057DNAArtificial
Sequence5' untranslated region based on 4-tuples 80ccaatcacct taaagaactc
accttaaaga actcacaatc acaccaccaa ccacaaa 578157DNAArtificial
Sequence5' untranslated region based on 4-tuples 81ccacaccaca actcaccaca
caatcacaac tcaccaagaa ctctctcacc accaaaa 578257DNAArtificial
Sequence5' untranslated region based on 4-tuples 82ccactcacaa ctcacaacac
caccaactca ccacaactca caccaactca ccttaaa 578357DNAArtificial
Sequence5' untranslated region based on 4-tuples 83ctcaccacca caatcaccac
tcacaactct ctcacacacc aaccaatcac caccaaa 57845DNAArtificial
Sequencetuples at end of 5' untranslated region 84ccaat
5855DNAArtificial
Sequencetuples at end of 5' untranslated region 85ccaaa
5865DNAArtificial
Sequencetuples at end of 5' untranslated region 86caaaa
5875DNAArtificial
Sequencetuples at end of 5' untranslated region 87aaaaa
5885DNAArtificial
Sequencetuples at end of 5' untranslated region 88accaa
5895DNAArtificial
Sequencetuples at end of 5' untranslated region 89waaag
5904DNAArtificial
Sequenceartificial tuples in 5' untranslated region 90actc
4916DNAArtificial
Sequencetuples in 5' untranslated region 91caactc
6927DNAArtificial Sequencetuples
in 5' untranslated region 92acaactc
7937DNAArtificial Sequencetuples in 5'
untranslated region 93cacaact
79420DNAArtificial Sequence5' untranslated region
94gaaccaatca ccaccaaaaa
209521DNAArtificial Sequence5' untranslated region 95agaaccaatc
accaccaaaa a
219636DNAArtificial Sequencelonger 5' untranslated region 96actctcacac
cttaaagaac caatcaccac caaaaa
369715DNAArtificial Sequencelonger 5' untranslated region 97actctcacac
cttaa
159816DNAArtificial Sequencelonger 5' untranslated region 98actctcacac
cttaaa
169919DNAArtificial Sequencelonger 5' untranslated region 99actctcacac
cttaaagaa
191004DNAArtificial SequenceCCAAT-Box core 100ccaa
41019DNAArtificial
SequenceCCAAT-Box 101aaccaatca
91024DNAArtificial SequenceDof1 core 102aaag
410317DNAArtificial SequenceDof1 103cacaccttaa agaacca
1710415DNAArtificial SequenceSEF3,
Soybean embryo factor 3 104tataaaccca ccatc
1510515DNAArtificial SequenceSEF3, Soybean embryo
factor 3 105accaaaccca ccaca
151064DNAArtificial SequenceSEF3, Soybean embryo factor 3 core
106accc
410719DNAArtificial SequencePlant TATA box 107cactatataa acccaccat
191085DNAArtificial
SequencePlant TATA box core 108tataa
510927DNAArtificial SequenceRY and Sph motifs
conserved in seed-specific promoters 109gtgtagccca tgcaaagtta
acactca 271104DNAArtificial
SequenceRY and Sph motifs conserved in seed-specific promoters core
110catg
411117DNAArtificial SequenceProlamin box, conserved in cereal seed
storage protein gene promoters 111gcccatgcaa agttaac
171124DNAArtificial SequenceProlamin
box, conserved in cereal seed storage protein gene promoters core
112aaag
411313DNAArtificial SequenceCommon plant regulatory factor (CPRF) from
parsley 113aagaacagcc cat
131144DNAArtificial SequenceCommon plant regulatory factor
(CPRF) from parsley core 114acgt
411513DNAArtificial SequenceTCP class I
transcription factor (Arabidopsis) 115gtaagcccaa aag
131164DNAArtificial SequenceTCP class I
transcription factor (Arabidopsis) core 116gccc
411721DNAArtificial
SequencebZIP protein G-Box binding factor 1 117ataatactac gtgtaagccc a
211184DNAArtificial
SequencebZIP protein G-Box binding factor 1 core 118acgt
411915DNAArtificial
SequenceCis-element in the GAPDH promoters conferring light
inducibility 119ttgcatgaat aatac
151204DNAArtificial SequenceCis-element in the GAPDH
promoters conferring light inducibility core 120atga
412117DNAArtificial
SequenceSBF-1 121cacattatta aaatacc
171224DNAArtificial SequenceSBF-1 core 122ttaa
412311DNAArtificial
SequenceSunflower homeodomain leucine-zipper protein Hahb-4
123cacattatta a
111244DNAArtificial SequenceSunflower homeodomain leucine-zipper protein
Hahb-4 core 124atta
412511DNAArtificial SequenceTranscriptional repressor
BELLRINGER 125aaaattagta a
111264DNAArtificial SequenceTranscriptional repressor
BELLRINGER core 126atta
412721DNAArtificial SequenceFloral homeotic protein
APETALA1 127cattgccaaa attagtaaaa t
211284DNAArtificial SequenceFloral homeotic protein APETALA1 core
128caaa
412915DNAArtificial SequenceICE (inducer of CBF expression 1), AtMYC2
(rd22BP1) 129aatgtacact tgtca
151304DNAArtificial SequenceICE (inducer of CBF expression 1),
AtMYC2 (rd22BP1) core 130acac
413111DNAArtificial SequencebZIP factors
DPBF-1 and 2 (Dc3 promoter binding factor-1 and 2) 131tacacttgtc a
111324DNAArtificial SequencebZIP factors DPBF-1 and 2 (Dc3 promoter
binding factor-1 and 2) core 132acac
413317DNAArtificial SequenceClass I GATA
factors 133agtaagataa tccaaat
171344DNAArtificial SequenceClass I GATA factors core 134gata
413517DNAArtificial SequenceDof2 - single zinc finger transcription
factor 135tccaaattaa agcaaga
171364DNAArtificial SequenceDof2 - single zinc finger transcription
factor core 136aaag
413721DNAArtificial SequenceTranscription factor binding
site box enhancer 137cacaccttaa agaaccaatc a
2113843DNAArtificial SequenceTranscription factor
binding site box short promoter 138ataatactac gtgtaagccc aaaagaaccc
acgtgtagcc cat 4313969DNAArtificial
SequenceTranscription factor binding site box long promoter
139tccaaattaa agcaagagag gccaagtaag ataatccaaa tgtacacttg tcattgccaa
60aattagtaa
6914017DNAArtificial Sequencespacer 140ccccaatctc accaaac
1714198DNAArtificial Sequence98nt
promoter 141ataatactac gtgtaagccc aaaagaaccc acgtgtagcc catgcaaagt
taacactcac 60gaccccattc ctcagtctcc actatataaa cccaccat
98142115DNAArtificial Sequence98nt promoter with spacer
142ataatactac gtgtaagccc aaaagaaccc acgtgtagcc catgcaaagt taacactcac
60gaccccattc ctcagtctcc actatataaa cccaccatcc ccaatctcac caaac
115143172DNAArtificial Sequence98nt promoter with spacer and enhancer
143ataatactac gtgtaagccc aaaagaaccc acgtgtagcc catgcaaagt taacactcac
60gaccccattc ctcagtctcc actatataaa cccaccatcc ccaatctcac caaacccacc
120acacaactca caactcactc tcacacctta aagaaccaat caccaccaaa aa
172144142DNAArtificial Sequence142nt promoter 144cacattatta aaataccgta
tatgtattgg ctgcatttgc atgaataata ctacgtgtaa 60gcccaaaaga acccacgtgt
agcccatgca aagttaacac tcacgacccc attcctcagt 120ctccactata taaacccacc
at 142145159DNAArtificial
Sequence142nt promoter with spacer 145cacattatta aaataccgta tatgtattgg
ctgcatttgc atgaataata ctacgtgtaa 60gcccaaaaga acccacgtgt agcccatgca
aagttaacac tcacgacccc attcctcagt 120ctccactata taaacccacc atccccaatc
tcaccaaac 159146216DNAArtificial Sequence142nt
promoter with spacer and enhancer 146cacattatta aaataccgta tatgtattgg
ctgcatttgc atgaataata ctacgtgtaa 60gcccaaaaga acccacgtgt agcccatgca
aagttaacac tcacgacccc attcctcagt 120ctccactata taaacccacc atccccaatc
tcaccaaacc caccacacaa ctcacaactc 180actctcacac cttaaagaac caatcaccac
caaaaa 216147160DNAArtificial Sequence160nt
promoter 147cggcatattg tattcccaca cattattaaa ataccgtata tgtattggct
gcatttgcat 60gaataatact acgtgtaagc ccaaaagaac ccacgtgtag cccatgcaaa
gttaacactc 120acgaccccat tcctcagtct ccactatata aacccaccat
160148177DNAArtificial Sequence160nt promoter with spacer
148cggcatattg tattcccaca cattattaaa ataccgtata tgtattggct gcatttgcat
60gaataatact acgtgtaagc ccaaaagaac ccacgtgtag cccatgcaaa gttaacactc
120acgaccccat tcctcagtct ccactatata aacccaccat ccccaatctc accaaac
177149234DNAArtificial Sequence160nt promoter with spacer and enhancer
149cggcatattg tattcccaca cattattaaa ataccgtata tgtattggct gcatttgcat
60gaataatact acgtgtaagc ccaaaagaac ccacgtgtag cccatgcaaa gttaacactc
120acgaccccat tcctcagtct ccactatata aacccaccat ccccaatctc accaaaccca
180ccacacaact cacaactcac tctcacacct taaagaacca atcaccacca aaaa
234150197DNAArtificial Sequence197nt promoter 150aatgtacact tgtcattgcc
aaaattagta aaatactcgg catattgtat tcccacacat 60tattaaaata ccgtatatgt
attggctgca tttgcatgaa taatactacg tgtaagccca 120aaagaaccca cgtgtagccc
atgcaaagtt aacactcacg accccattcc tcagtctcca 180ctatataaac ccaccat
197151214DNAArtificial
Sequence197nt promoter with spacer 151aatgtacact tgtcattgcc aaaattagta
aaatactcgg catattgtat tcccacacat 60tattaaaata ccgtatatgt attggctgca
tttgcatgaa taatactacg tgtaagccca 120aaagaaccca cgtgtagccc atgcaaagtt
aacactcacg accccattcc tcagtctcca 180ctatataaac ccaccatccc caatctcacc
aaac 214152271DNAArtificial Sequence197nt
promoter with spacer and enhancer 152aatgtacact tgtcattgcc aaaattagta
aaatactcgg catattgtat tcccacacat 60tattaaaata ccgtatatgt attggctgca
tttgcatgaa taatactacg tgtaagccca 120aaagaaccca cgtgtagccc atgcaaagtt
aacactcacg accccattcc tcagtctcca 180ctatataaac ccaccatccc caatctcacc
aaacccacca cacaactcac aactcactct 240cacaccttaa agaaccaatc accaccaaaa a
271153235DNAArtificial Sequence235nt
promoter 153tccaaattaa agcaagagag gccaagtaag ataatccaaa tgtacacttg
tcattgccaa 60aattagtaaa atactcggca tattgtattc ccacacatta ttaaaatacc
gtatatgtat 120tggctgcatt tgcatgaata atactacgtg taagcccaaa agaacccacg
tgtagcccat 180gcaaagttaa cactcacgac cccattcctc agtctccact atataaaccc
accat 235154252DNAArtificial Sequence235nt promoter with spacer
154tccaaattaa agcaagagag gccaagtaag ataatccaaa tgtacacttg tcattgccaa
60aattagtaaa atactcggca tattgtattc ccacacatta ttaaaatacc gtatatgtat
120tggctgcatt tgcatgaata atactacgtg taagcccaaa agaacccacg tgtagcccat
180gcaaagttaa cactcacgac cccattcctc agtctccact atataaaccc accatcccca
240atctcaccaa ac
252155309DNAArtificial Sequence235nt promoter with spacer and enhancer
155tccaaattaa agcaagagag gccaagtaag ataatccaaa tgtacacttg tcattgccaa
60aattagtaaa atactcggca tattgtattc ccacacatta ttaaaatacc gtatatgtat
120tggctgcatt tgcatgaata atactacgtg taagcccaaa agaacccacg tgtagcccat
180gcaaagttaa cactcacgac cccattcctc agtctccact atataaaccc accatcccca
240atctcaccaa acccaccaca caactcacaa ctcactctca caccttaaag aaccaatcac
300caccaaaaa
309156240DNAArtificial Sequence240nt promoter 156aataatccaa attaaagcaa
gagaggccaa gtaagataat ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact
cggcatattg tattcccaca cattattaaa ataccgtata 120tgtattggct gcatttgcat
gaataatact acgtgtaagc ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc
acgaccccat tcctcagtct ccactatata aacccaccat 240157257DNAArtificial
Sequence240nt promoter with spacer 157aataatccaa attaaagcaa gagaggccaa
gtaagataat ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg
tattcccaca cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact
acgtgtaagc ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc acgaccccat
tcctcagtct ccactatata aacccaccat 240ccccaatctc accaaac
257158314DNAArtificial Sequence240nt
promoter with spacer and enhancer 158aataatccaa attaaagcaa gagaggccaa
gtaagataat ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg
tattcccaca cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact
acgtgtaagc ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc acgaccccat
tcctcagtct ccactatata aacccaccat 240ccccaatctc accaaaccca ccacacaact
cacaactcac tctcacacct taaagaacca 300atcaccacca aaaa
314159989DNAArtificial Sequence1064nt
promoter 159ttagcagata tttggtgtct aaatgtttat tttgtgatat gttcatgttt
gaaatggtgg 60tttcgaaacc agggacaacg ttgggatctg atagggtgtc aaagagtatt
atggattggg 120acaatttcgg tcatgagttg caaattcaag tatatcgttc gattatgaaa
attttcgaag 180aatatcccat ttgagagagt ctttacctca ttaatgtttt tagattatga
aattttatca 240tagttcatcg tagtcttttt ggtgtaaagg ctgtaaaaag aaattgttca
cttttgtttt 300cgtttatgtg aaggctgtaa aagattgtaa aagactattt tggtgttttg
gataaaatga 360tagtttttat agattctttt gcttttagaa gaaatacatt tgaaattttt
tccatgttga 420gtataaaata ccgaaatcga ttgaagatca tagaaatatt ttaactgaaa
acaaatttat 480aactgattca attctctcca tttttatacc tatttaaccg taatcgattc
taatagatga 540tcgatttttt atataatcct aattaaccaa cggcatgtat tggataatta
accgatcaac 600tctcacccct aatagaatca gtattttcct tcgacgttaa ttgatcctac
actatgtagg 660tcatatccat cgttttaatt tttggccacc attcaattct gtcttgcctt
tagggatgtg 720aatatgaacg gccaaggtaa gagaataaaa ataatccaaa ttaaagcaag
agaggccaag 780taagataatc caaatgtaca cttgtcattg ccaaaattag taaaatactc
ggcatattgt 840attcccacac attattaaaa taccgtatat gtattggctg catttgcatg
aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc ccatgcaaag ttaacactca
cgaccccatt 960cctcagtctc cactatataa acccaccat
9891601006DNAArtificial Sequence1064nt promoter with spacer
but without enhancer 160ttagcagata tttggtgtct aaatgtttat tttgtgatat
gttcatgttt gaaatggtgg 60tttcgaaacc agggacaacg ttgggatctg atagggtgtc
aaagagtatt atggattggg 120acaatttcgg tcatgagttg caaattcaag tatatcgttc
gattatgaaa attttcgaag 180aatatcccat ttgagagagt ctttacctca ttaatgtttt
tagattatga aattttatca 240tagttcatcg tagtcttttt ggtgtaaagg ctgtaaaaag
aaattgttca cttttgtttt 300cgtttatgtg aaggctgtaa aagattgtaa aagactattt
tggtgttttg gataaaatga 360tagtttttat agattctttt gcttttagaa gaaatacatt
tgaaattttt tccatgttga 420gtataaaata ccgaaatcga ttgaagatca tagaaatatt
ttaactgaaa acaaatttat 480aactgattca attctctcca tttttatacc tatttaaccg
taatcgattc taatagatga 540tcgatttttt atataatcct aattaaccaa cggcatgtat
tggataatta accgatcaac 600tctcacccct aatagaatca gtattttcct tcgacgttaa
ttgatcctac actatgtagg 660tcatatccat cgttttaatt tttggccacc attcaattct
gtcttgcctt tagggatgtg 720aatatgaacg gccaaggtaa gagaataaaa ataatccaaa
ttaaagcaag agaggccaag 780taagataatc caaatgtaca cttgtcattg ccaaaattag
taaaatactc ggcatattgt 840attcccacac attattaaaa taccgtatat gtattggctg
catttgcatg aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc ccatgcaaag
ttaacactca cgaccccatt 960cctcagtctc cactatataa acccaccatc cccaatctca
ccaaac 1006161172DNAArtificial SequenceProPer 98nt
promoter with spacer and enhancer 161ataatactac gtgtaagccc aaaagaaccc
acgtgtagcc catgcaaagt taacactcat 60gaccccattc ctcagtctcc actatataaa
cccaccatcc cctatctcac caaacccacc 120acacaactca caactcactc tcacacctta
aagaaccaat caccaccaaa aa 172162172DNAArtificial SequenceProPer
98nt promoter with spacer and enhancer 162ataatactac gtgtaagccc
aaaagaaccc acgtgtagcc catgcaaagt taacactcac 60aaccccattc ctcagtctcc
actatataaa cccaccatct tacttctcac caaacccacc 120acacaactca caactcactc
tcacacctta aagaaccaat caccaccaaa aa 172163172DNAArtificial
SequenceProPer 98nt promoter with spacer and enhancer 163ataatactac
gtgtaagccc aaaagaaccc acgtgtagcc catgcaaagt taacactcaa 60gaccccattc
ctcagtctcc actatataaa cccaccatcc atcgtctcac caaacccacc 120acacaactca
caactcactc tcacacctta aagaaccaat caccaccaaa aa
172164172DNAArtificial SequenceProPer 98nt promoter with spacer and
enhancer 164ataatactac gtgtaagccc aaaagaaccc acgtgtagcc catgcaaagt
taacactcat 60aaccccattc ctcagtctcc actatataaa cccaccatca tctctctcac
caaacccacc 120acacaactca caactcactc tcacacctta aagaaccaat caccaccaaa
aa 172165172DNAArtificial SequenceProPer 98nt promoter with
spacer and enhancer 165ataatactac gtgtaagccc aaaagaaccc acgtgtagcc
catgcaaagt taacactcat 60taccccattc ctcagtctcc actatataaa cccaccatcg
aatttctcac caaacccacc 120acacaactca caactcactc tcacacctta aagaaccaat
caccaccaaa aa 172166172DNAArtificial SequenceProPer 98nt
promoter with spacer and enhancer 166ataatactac gtgtaagccc aaaagaaccc
acgtgtagcc catgcaaagt taacactcag 60caccccattc ctcagtctcc actatataaa
cccaccatcc tcgctctcac caaacccacc 120acacaactca caactcactc tcacacctta
aagaaccaat caccaccaaa aa 172167172DNAArtificial SequenceProPer
98nt promoter with spacer and enhancer 167ataatactac gtgtaagccc
aaaagaaccc acgtgtagcc catgcaaagt taacactcag 60gaccccattc ctcagtctcc
actatataaa cccaccatcg gatgtctcac caaacccacc 120acacaactca caactcactc
tcacacctta aagaaccaat caccaccaaa aa 172168172DNAArtificial
SequenceProPer 98nt promoter with spacer and enhancer 168ataatactac
gtgtaagccc aaaagaaccc acgtgtagcc catgcaaagt taacactcac 60caccccattc
ctcagtctcc actatataaa cccaccatca catctctcac caaacccacc 120acacaactca
caactcactc tcacacctta aagaaccaat caccaccaaa aa
172169172DNAArtificial SequenceProPer 98nt promoter with spacer and
enhancer 169ataatactac gtgtaagccc aaaagaaccc acgtgtagcc catgcaaagt
taacactcag 60gaccccattc ctcagtctcc actatataaa cccaccatca acaatctcac
caaacccacc 120acacaactca caactcactc tcacacctta aagaaccaat caccaccaaa
aa 172170172DNAArtificial SequenceProPer 98nt promoter with
spacer and enhancer 170ataatactac gtgtaagccc aaaagaaccc acgtgtagcc
catgcaaagt taacactcac 60taccccattc ctcagtctcc actatataaa cccaccatcc
aacatctcac caaacccacc 120acacaactca caactcactc tcacacctta aagaaccaat
caccaccaaa aa 172171177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 171attaaaatac cgtatataaa cccacgtgta gcccatgcaa
agttaacact cacgacccca 60ttcccacaca ttattaaaat actacgtgta agcccatgca
aagttaacac tcaccatccc 120cattcccaca cattattaaa ataccgtata taaacccacc
aaaagaaccc accaaac 177172177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 172acgtgtaagc ccaatctcac catccccatt cctcagtctc
cactatataa acccacgtgt 60agcccatgca aagttaacac tcacgacccc atgcaaagtt
aacactcacg accccaatct 120caccatcccc attcctcagt ctccactata taaacccacc
aaaagaaccc accaaac 177173177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 173ctacgtgtaa gcccaatctc acgaccccat gcaaagttaa
cactcaccaa aagaacccac 60caaaagaacc cacgtgtagc ccaatctcac gaccccattc
ctcagtctcc actatataaa 120cccacacatt attaaaatac cgtatataaa cccacgtgta
gcccaatctc accaaac 177174177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 174tcctcagtct ccactatatg tattgtattc ctcagtctcc
actatataaa cccacgtgta 60gcccattccc acacattatt aaaatactac gtgtagccca
tgcaaagtta acactcacca 120tccccattcc cacacattat taaaataccg tatataaacc
cacgtgtaag cccaaac 177175177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 175tgtaagccca tgcaaagtta acactcacga ccccattccc
acacattatt aaaataccgt 60atataaaccc accatcccca aaagaaccca cgtgtagccc
atgcaaagtt aacactcacg 120accccattcc cacacattat taaaataccg tatataaacc
cacgtgtaag cccaaac 177176177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 176acattattaa aataccgtat atgtattggc tgcatgaata
atactacgtg tagcccatgc 60aaagttaaca ctcaccatcc ccattcccac gtgtagccca
tgcaaagtta acactcacca 120tccccattcc tcagtctcca ctatataaac ccacgtgtaa
gcccaatctc accaaac 177177177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 177cgaccccatg caaagttaac actcacgacc ccaatctcac
caaaagaacc caccatcccc 60atgcaaagtt aacactcacc atccccatgc aaagttaaca
ctcaccatcc ccaaaagaac 120ccacgtgtaa gcccattcct cagtctccac tatataaacc
cacgtgtaag cccaaac 177178177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 178aacactcacg accccaatct cacgacccca aaagaaccca
cgtgtagccc aatctcacga 60ccccatgcaa agttaacact caccatcccc attcctcagt
ctccactata tgtattgtat 120tcccacgtgt agcccattcc tcagtctcca ctatataaac
ccacgtgtag cccaaac 177179177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 179aaaagaaccc acacattatt aaaataccgt atataaaccc
acgtgtaagc ccaaaagaac 60ccacacatta ttaaaatact acgtgtaagc ccatgcaaag
ttaacactca ccaaaagaac 120ccacacatta ttaaaatacc gtatataaac ccacgtgtaa
gcccaatctc accaaac 177180177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 180cccacgtgta agcccaatct cacgacccca ttcccacaca
ttattaaaat actacgtgta 60agcccattcc cacgtgtagc ccatgcaaag ttaacactca
cgaccccatt cctcagtctc 120cactatatgt attcctcagt ctccactata taaacccacc
aaaagaaccc accaaac 177181177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 181aacccacaca ttattaaaat accgtatatg tattcccacc
atccccatgc aaagttaaca 60ctcaccatcc ccaaaagaac ccacacatta ttaaaatacc
gtatatgtat tcccacgtgt 120agcccattcc tcagtctcca ctatataaac ccacgtgtaa
gcccattccc accaaac 177182177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 182cccatgcaaa gttaacactc acgaccccaa aagaacccac
acattattaa aataccgtat 60atgtattcct cagtctccac tatataaacc cacgtgtaag
cccattccca cacattatta 120aaataccgta tatgtattcc tcagtctcca ctatataaac
ccacgtgtag cccaaac 177183177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 183cgtatatgta ttgtattccc acgtgtaagc ccaatctcac
gaccccatgc aaagttaaca 60ctcacgaccc catgcaaagt taacactcac caaaagaacc
cacacattat taaaatacta 120cgtgtagccc attcctcagt ctccactata taaacccacc
aaaagaaccc accaaac 177184177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 184acacattatt aaaataccgt atataaaccc acgtgtagcc
catgcaaagt taacactcac 60gaccccaaaa gaacccacac attattaaaa tactacgtgt
aagcccattc ctcagtctcc 120actatatgta ttgtattcct cagtctccac tatataaacc
cacgtgtaag cccaaac 177185177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 185tatatgtatt cccacacatt attaaaatac tacgtgtagc
ccattcccac acattattaa 60aatactacgt gtaagcccat tcctcagtct ccactatata
aacccacgtg tagcccattc 120ccacacatta ttaaaatacc gtatataaac ccacgtgtaa
gcccattccc accaaac 177186177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 186taaacccacc atccccattc ctcagtctcc actatataaa
cccacgtgta gcccatgcaa 60agttaacact caccatcccc aatctcacca tccccaatct
cacgacccca tgcaaagtta 120acactcacga ccccattcct cagtctccac tatataaacc
cacgtgtaag cccaaac 177187177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 187ctacgtgtag cccattccca cgtgtaagcc caatctcacg
accccattcc cacgtgtaag 60cccattccca cgtgtagccc atgcaaagtt aacactcacc
atccccaatc tcaccatccc 120caatctcacg accccattcc tcagtctcca ctatataaac
ccacgtgtag cccaaac 177188177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 188atgcaaagtt aacactcacg accccatgca aagttaacac
tcaccaaaag aacccacaca 60ttattaaaat actacgtgta gcccattccc acgtgtaagc
ccattcctca gtctccacta 120tatgtattcc tcagtctcca ctatataaac ccacgtgtaa
gcccaatctc accaaac 177189177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 189gaacccacac attattaaaa tactacgtgt agcccaatct
caccatcccc aaaagaaccc 60acacattatt aaaataccgt atatgtattg tattgtattg
gctgcatttg catgaataat 120actacgtgta agcccattcc tcagtctcca ctatataaac
ccacgtgtag cccaaac 177190177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 190cctcagtctc cactatataa acccacgtgt agcccaatct
caccatcccc atgcaaagtt 60aacactcacc atccccaaaa gaacccacac attattaaaa
taccgtatat gtattgtatt 120cccacacatt attaaaatac cgtatataaa cccacgtgta
gcccaatctc accaaac 177191177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 191agttaacact cacgacccca atctcaccat ccccattccc
acgtgtagcc caatctcacg 60accccaatct cacgacccca atctcaccaa aagaacccac
gtgtaagccc atgcaaagtt 120aacactcacg accccattcc tcagtctcca ctatataaac
ccacgtgtag cccaaac 177192177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 192gcccatgcaa agttaacact caccatcccc attcctcagt
ctccactata tgtattccca 60cacattatta aaataccgta tataaaccca cgtgtagccc
atgcaaagtt aacactcacg 120accccattcc tcagtctcca ctatataaac ccacgtgtaa
gcccaatctc accaaac 177193177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 193cattattaaa ataccgtata tgtattccca cgtgtaagcc
caatctcacg accccatgca 60aagttaacac tcaccatccc caatctcacg accccattcc
tcagtctcca ctatatgtat 120tgtattgtat tcctcagtct ccactatata aacccaccat
ccccaatctc accaaac 177194177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 194cccattccca cgtgtaagcc catgcaaagt taacactcac
gaccccaatc tcacgacccc 60attcccacca aaagaaccca cacattatta aaataccgta
tatgtattgg ctgcatgaat 120aataccgtat atgtattcct cagtctccac tatataaacc
cacgtgtaag cccaaac 177195177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 195atgtattggc tgcatgaata ataccgtata tgtattcctc
agtctccact atatgtattc 60ctcagtctcc actatatgta ttggctgcat gaataatact
acgtgtaagc ccaatctcac 120gaccccattc ctcagtctcc actatataaa cccacgtgta
gcccaatctc accaaac 177196177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 196ccatccccat tcctcagtct ccactatata aacccacaca
ttattaaaat actacgtgta 60gcccattccc acgtgtagcc catgcaaagt taacactcac
gaccccaatc tcaccaaaag 120aacccaccat ccccattcct cagtctccac tatataaacc
cacgtgtaag cccaaac 177197177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 197accccatgca aagttaacac tcacgacccc attcccacac
attattaaaa taccgtatat 60gtattcctca gtctccacta tataaaccca ccaaaagaac
ccacacatta ttaaaatacc 120gtatatgtat tggctgcatg aataataccg tatataaacc
cacgtgtaag cccaaac 177198177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 198cactatatgt attggctgca tttgcatttg catgaataat
actacgtgta agcccaatct 60cacgacccca aaagaaccca cacattatta aaataccgta
tataaaccca cacattatta 120aaataccgta tatgtattcc tcagtctcca ctatataaac
ccacgtgtag cccaaac 177199177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 199cacgacccca aaagaaccca cgtgtaagcc caatctcacg
accccatgca aagttaacac 60tcaccatccc catgcaaagt taacactcac caaaagaacc
cacacattat taaaatacta 120cgtgtagccc attcctcagt ctccactata taaacccacc
aaaagaaccc accaaac 177200177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 200actacgtgta gcccattcct cagtctccac tatatgtatt
gtattcccac gtgtaagccc 60atgcaaagtt aacactcacg accccaaaag aacccacaca
ttattaaaat actacgtgta 120agcccattcc cacacattat taaaataccg tatataaacc
cacgtgtaag cccaaac 177201177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 201aataataccg tatatgtatt ggctgcattt gcatgaataa
tactacgtgt aagcccaatc 60tcaccatccc cattcctcag tctccactat ataaacccac
acattattaa aataccgtat 120atgtattggc tgcatgaata ataccgtata taaacccacc
aaaagaaccc accaaac 177202177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 202accgtatatg tattggctgc atttgcatga ataataccgt
atataaaccc acgtgtaagc 60ccatgcaaag ttaacactca ccatccccat tcctcagtct
ccactatatg tattcctcag 120tctccactat atgtattcct cagtctccac tatataaacc
cacgtgtaag cccaaac 177203177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 203ccaaaagaac ccacgtgtaa gcccatgcaa agttaacact
caccatcccc aatctcacca 60tccccattcc cacacattat taaaataccg tatataaacc
cacacattat taaaatacta 120cgtgtagccc attcctcagt ctccactata taaacccacc
aaaagaaccc accaaac 177204177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 204atgcaaagtt aacactcacg accccatgca aagttaacac
tcaccatccc catgcaaagt 60taacactcac caaaagaacc caccatcccc atgcaaagtt
aacactcacc atccccattc 120ccacacatta ttaaaatacc gtatataaac ccacgtgtaa
gcccaatctc accaaac 177205177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 205atactacgtg taagcccatt cctcagtctc cactatatgt
attcccacgt gtaagcccaa 60aagaacccac gtgtaagccc atgcaaagtt aacactcacg
accccaatct caccatcccc 120aatctcacga ccccattcct cagtctccac tatataaacc
cacgtgtaag cccaaac 177206177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 206cactcacgac cccatgcaaa gttaacactc acgaccccat
gcaaagttaa cactcacgac 60cccaatctca cgaccccatt cccacacatt attaaaatac
cgtatataaa cccaccaaaa 120gaacccacca tccccattcc tcagtctcca ctatataaac
ccacgtgtag cccaaac 177207177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 207acattattaa aataccgtat ataaacccac acattattaa
aatactacgt gtaagcccaa 60aagaacccac catccccaat ctcacgaccc caatctcacg
accccattcc tcagtctcca 120ctatataaac ccacacatta ttaaaatacc gtatataaac
ccacgtgtag cccaaac 177208177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 208aaaataccgt atataaaccc acgtgtaagc ccaatctcac
catccccatg caaagttaac 60actcacgacc ccaatctcac gaccccatgc aaagttaaca
ctcacgaccc cattcctcag 120tctccactat atgtattcct cagtctccac tatataaacc
cacgtgtaag cccaaac 177209177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 209taagcccaat ctcaccatcc ccattcctca gtctccacta
tatgtattcc cacgtgtaag 60cccaatctca cgaccccatg caaagttaac actcacgacc
ccaaaagaac ccaccatccc 120caaaagaacc cacacattat taaaataccg tatataaacc
cacgtgtaag cccaaac 177210177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 210acattattaa aatactacgt gtaagcccat gcaaagttaa
cactcaccat ccccaatctc 60accaaaagaa cccacgtgta agcccaatct caccatcccc
atgcaaagtt aacactcacg 120accccattcc tcagtctcca ctatataaac ccacgtgtaa
gcccattccc accaaac 177211177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 211cactcaccaa aagaacccac catccccaaa agaacccacg
tgtagcccaa tctcacgacc 60ccattcccac catccccatg caaagttaac actcacgacc
ccattcccac acattattaa 120aataccgtat atgtattcct cagtctccac tatataaacc
cacgtgtaag cccaaac 177212177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 212acccacgtgt agcccatgca aagttaacac tcaccatccc
caaaagaacc cacgtgtaag 60cccattccca cacattatta aaataccgta tatgtattcc
tcagtctcca ctatataaac 120ccacacatta ttaaaatacc gtatataaac ccacgtgtaa
gcccaatctc accaaac 177213177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 213atgtattcct cagtctccac tatatgtatt cccacgtgta
agcccaatct caccatcccc 60aaaagaaccc accaaaagaa cccacacatt attaaaatac
tacgtgtagc ccaatctcac 120gaccccattc ctcagtctcc actatataaa cccacgtgta
gcccaatctc accaaac 177214177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 214attaaaatac cgtatatgta ttggctgcat gaataatacc
gtatataaac ccacgtgtaa 60gcccaaaaga acccacacat tattaaaata ccgtatataa
acccacgtgt agcccaaaag 120aacccacaca ttattaaaat accgtatata aacccaccat
ccccaatctc accaaac 177215177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 215cactatatgt attcctcagt ctccactata taaacccaca
cattattaaa ataccgtata 60tgtattggct gcatgaataa tactacgtgt agcccatgca
aagttaacac tcaccatccc 120caaaagaacc cacacattat taaaataccg tatataaacc
cacgtgtaag cccaaac 177216177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 216actacgtgta agcccatgca aagttaacac tcacgacccc
atgcaaagtt aacactcacg 60accccaatct cacgacccca ttcctcagtc tccactatat
aaacccacgt gtaagcccat 120tcccacgtgt agcccattcc tcagtctcca ctatataaac
ccacgtgtag cccaaac 177217177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 217aagaacccac acattattaa aataccgtat ataaacccac
acattattaa aataccgtat 60atgtattcct cagtctccac tatataaacc caccatcccc
atgcaaagtt aacactcacg 120accccattcc tcagtctcca ctatataaac ccacgtgtaa
gcccaatctc accaaac 177218177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 218acattattaa aatactacgt gtaagcccaa tctcaccaaa
agaacccacg tgtaagccca 60atctcacgac cccatgcaaa gttaacactc accaaaagaa
cccacgtgta agcccaaaag 120aacccacaca ttattaaaat accgtatata aacccaccat
ccccattccc accaaac 177219177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 219ctcacgaccc cattcccacg tgtagcccat gcaaagttaa
cactcaccat ccccaatctc 60accaaaagaa cccaccatcc ccaaaagaac ccacacatta
ttaaaatact acgtgtaagc 120ccaaaagaac ccacacatta ttaaaatacc gtatataaac
ccacgtgtag cccaaac 177220177DNAArtificial Sequencesynthetic 160nt
promoter with spacer 220tccccatgca aagttaacac tcaccatccc caatctcacc
aaaagaaccc acacattatt 60aaaataccgt atataaaccc acacattatt aaaatactac
gtgtaagccc atgcaaagtt 120aacactcacg accccattcc tcagtctcca ctatataaac
ccacgtgtag cccaaac 177221234DNAArtificial SequenceProPer 160nt
promoter with spacer and enhancer 221aaggcatcgc tgaaagagca cattattaaa
ataccgtata tgtattggct gcatttgcat 60gaataatact acgtgtaagc ccaaaagaac
ccacgtgtag cccatgcaaa gttaacactc 120aggaccccat tcctcagtct ccactatata
aacccaccat cggggttctc accaaaccca 180ccacacaact cacaactcac tctcacacct
taaagaacca atcaccacca aaaa 234222234DNAArtificial SequenceProPer
160nt promoter with spacer and enhancer 222gaggcgaggg tctgtcgaca
cattattaaa ataccgtata tgtattggct gcatttgcat 60gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag cccatgcaaa gttaacactc 120actaccccat tcctcagtct
ccactatata aacccaccat cctctctctc accaaaccca 180ccacacaact cacaactcac
tctcacacct taaagaacca atcaccacca aaaa 234223234DNAArtificial
SequenceProPer 160nt promoter with spacer and enhancer 223ctggtgatgc
cagggttgca cattattaaa ataccgtata tgtattggct gcatttgcat 60gaataatact
acgtgtaagc ccaaaagaac ccacgtgtag cccatgcaaa gttaacactc 120aaaaccccat
tcctcagtct ccactatata aacccaccat caacattctc accaaaccca 180ccacacaact
cacaactcac tctcacacct taaagaacca atcaccacca aaaa
234224234DNAArtificial SequenceProPer 160nt promoter with spacer and
enhancer 224cgagtgggaa ccgttcctca cattattaaa ataccgtata tgtattggct
gcatttgcat 60gaataatact acgtgtaagc ccaaaagaac ccacgtgtag cccatgcaaa
gttaacactc 120ataaccccat tcctcagtct ccactatata aacccaccat cagccctctc
accaaaccca 180ccacacaact cacaactcac tctcacacct taaagaacca atcaccacca
aaaa 234225234DNAArtificial SequenceProPer 160nt promoter with
spacer and enhancer 225attctacggt tccctcaaca cattattaaa ataccgtata
tgtattggct gcatttgcat 60gaataatact acgtgtaagc ccaaaagaac ccacgtgtag
cccatgcaaa gttaacactc 120accaccccat tcctcagtct ccactatata aacccaccat
ctctcttctc accaaaccca 180ccacacaact cacaactcac tctcacacct taaagaacca
atcaccacca aaaa 234226234DNAArtificial SequenceProPer 160nt
promoter with spacer and enhancer 226agatccgcgg cacaaagcca cattattaaa
ataccgtata tgtattggct gcatttgcat 60gaataatact acgtgtaagc ccaaaagaac
ccacgtgtag cccatgcaaa gttaacactc 120agcaccccat tcctcagtct ccactatata
aacccaccat cggcaatctc accaaaccca 180ccacacaact cacaactcac tctcacacct
taaagaacca atcaccacca aaaa 234227234DNAArtificial SequenceProPer
160nt promoter with spacer and enhancer 227agatacgcac ccaccctaca
cattattaaa ataccgtata tgtattggct gcatttgcat 60gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag cccatgcaaa gttaacactc 120attaccccat tcctcagtct
ccactatata aacccaccat ctgacttctc accaaaccca 180ccacacaact cacaactcac
tctcacacct taaagaacca atcaccacca aaaa 234228234DNAArtificial
SequenceProPer 160nt promoter with spacer and enhancer 228gacggtgccc
tgcgtggcca cattattaaa ataccgtata tgtattggct gcatttgcat 60gaataatact
acgtgtaagc ccaaaagaac ccacgtgtag cccatgcaaa gttaacactc 120aacaccccat
tcctcagtct ccactatata aacccaccat cctagatctc accaaaccca 180ccacacaact
cacaactcac tctcacacct taaagaacca atcaccacca aaaa
234229234DNAArtificial SequenceProPer 160nt promoter with spacer and
enhancer 229cctgggagag gtctcttaca cattattaaa ataccgtata tgtattggct
gcatttgcat 60gaataatact acgtgtaagc ccaaaagaac ccacgtgtag cccatgcaaa
gttaacactc 120aaaaccccat tcctcagtct ccactatata aacccaccat ctgtaatctc
accaaaccca 180ccacacaact cacaactcac tctcacacct taaagaacca atcaccacca
aaaa 234230234DNAArtificial SequenceProPer 160nt promoter with
spacer and enhancer 230gcagctttcc gttacgggca cattattaaa ataccgtata
tgtattggct gcatttgcat 60gaataatact acgtgtaagc ccaaaagaac ccacgtgtag
cccatgcaaa gttaacactc 120accaccccat tcctcagtct ccactatata aacccaccat
ccttcctctc accaaaccca 180ccacacaact cacaactcac tctcacacct taaagaacca
atcaccacca aaaa 234231220DNAArtificial Sequencesynthetic 240nt
promoter with spacer 231ccaaattaaa gcaagagagg ccaagtaaaa ttagtaaaat
actacgtgta gcccattccc 60accatcccca atctcaccat ccccattcct cagtctccac
tatatgtatt gtattcccac 120gtgtagccca tgcaaagtta acactcacga ccccaaatta
aaatactcgg catattgtat 180tcctcagtct ccactatata aacccacgtg tagcccaaac
220232248DNAArtificial Sequencesynthetic 240nt
promoter with spacer 232gtgtaagccc aaattaaaat accgtatata aacccacaca
ttattaaagc aagagaggcc 60aaaatactac gtgtagccca aaattaaaat accgtatata
aacccacgtg tagcccaatc 120tcacgacccc atgcaaagtt aacactcacg accccaaaag
aacccacaca ttattaaagc 180aagagaggcc aaatgtattg tattcctcag tctccactat
ataaacccac gtgtaagata 240atccaaac
248233275DNAArtificial Sequencesynthetic 240nt
promoter with spacer 233gcatttgcat gaataatcca aaataccgta tatgtacact
tgtcattgcc aagtaaaatt 60aaaattaaag caagagaggc caaattaaaa tactacgtgt
aagcccattc ccacgtgtag 120cccatgcaaa gttaacactt gtcattgcca aattaaaata
ctcggcatat tggctgcatg 180aataatacta cgtgtagccc aatctcacga ccccaaaata
ctacgtgtaa gataataccg 240tatataaacc cacgtgtagc ccaatctcac caaac
275234282DNAArtificial Sequencesynthetic 240nt
promoter with spacer 234cacacattat taaagcaaga gaggccaaaa gaacccacac
attattaaag caagagaggc 60caagtaagat aatccaaatt aaaatactac gtgtagccca
ttcccacgtg taagcccaat 120ctcacgaccc cattcctcag tctccactat ataaacccac
catccccatg caaagttaac 180acttgtcatt gccaagtaaa attagtaaaa tactacgtgt
aagcccattc ccacacatta 240ttaaaatacc gtatataaac ccacgtgtaa gataatccaa
ac 282235302DNAArtificial Sequencesynthetic 240nt
promoter with spacer 235accaaaagaa cccacgtgta agcccaaaag aacccacgtg
taagcccaaa tgtattccca 60cacattatta aagcaagaga ggccaagtaa aatactacgt
gtagcccatg caaagttaac 120actcaccaaa tgtattccca ccaaattagt aaaatactac
gtgtaagccc attcccacca 180aaagaaccca cgtgtagccc atgcaaagtt aacacttgtc
attgccaagt aaaatactcg 240gcatattggc tgcatgaata ataccgtata taaacccacg
tgtaagccca atctcaccaa 300ac
302236324DNAArtificial Sequencesynthetic 240nt
promoter with spacer 236tcagtctcca ctatataaac ccacacatta ttaaagcaag
agaggccaaa atactacgtg 60taagcccatt cccacgtgta gcccaaatta gtaagcccat
gcaaagttaa cactcacgac 120cccattccca cgtgtaagat aatccaaatt agtaaaatac
tcggcatatt gtattggctg 180catgaataat ccaaaatact cggcatattg tattgtattc
ccaccaaaat actcggcata 240ttgtattgta ttcctcagtc tccactatat gtattgtatt
cctcagtctc cactatataa 300acccacgtgt aagataatcc aaac
324237343DNAArtificial Sequencesynthetic 240nt
promoter with spacer 237tgaataatcc aaaagaaccc acgtgtaaga taatactcgg
catattgtat tggctgcatg 60aataatccaa aagaacccac acattattaa agcaagagag
gccaaaatac tacgtgtagc 120ccaaatgtac acttgtcatt gccaaaagaa cccacgtgta
gcccattccc acacattatt 180aaagcaagag aggccaagta agcccatgca aagttaacac
tcacgacccc attcccacgt 240gtagcccaaa ttagtaagat aatccaaaat accgtatatg
tacacttgtc attgccaagt 300aagataatac cgtatataaa cccacgtgta agataatcca
aac 343238344DNAArtificial Sequencesynthetic 240nt
promoter with spacer 238tttgcatgaa taatactcgg catattgtat tggctgcatt
tgcatttgca tttgcatgaa 60taatccaaaa gaacccacca aaattaaaat taaagcaaga
gaggccaagt aagataatac 120tcggcatatt gtattggctg catgaataat actcggcata
ttgtattggc tgcatgaata 180atccaaaata ctacgtgtag cccattcctc agtctccact
atatgtacac tcacgacccc 240attcctcagt ctccactata taaacccacg tgtaagccca
tgcaaagtta acactcacga 300ccccaaaata ccgtatataa acccacgtgt aagataatcc
aaac 344239362DNAArtificial Sequencesynthetic 240nt
promoter with spacer 239taccgtatat aaacccacac attattaaag caagagaggc
caagtaagat aatccaaatg 60tacacttgtc attgccaagt aaaattagta aaatactcgg
catattggct gcatttgcat 120gaataatacc gtatataaac ccaccaaaat taaaataccg
tatataaacc cacacattat 180taaaattaaa atactacgtg tagcccatgc aaagttaaca
cttgtcattg ccaaaagaac 240ccacgtgtaa gcccatgcaa agttaacact tgtcattgcc
aaaagaaccc acgtgtaagc 300ccattcccac acattattaa aataccgtat ataaacccac
gtgtagccca atctcaccaa 360ac
362240363DNAArtificial Sequencesynthetic 240nt
promoter with spacer 240aagcaagaga ggccaagtaa gataatccaa atgtattccc
acgtgtagcc catgcaaagt 60taacacttgt cattgccaag taagcccaat ctcaccatcc
ccattcctca gtctccacta 120tatgtacact tgtcattgcc aagtaaaatt aaaataccgt
atataaaccc accatcccca 180tgcaaagtta acactcacga ccccaaaatt aaagcaagag
aggccaagta aaatactacg 240tgtagcccaa tctcaccaaa tgtattccca ccatccccat
tcccacgtgt agcccatgca 300aagttaacac tcaccaaatt agtaaaatac cgtatataaa
cccacgtgta agataatcca 360aac
363241368DNAArtificial Sequencesynthetic 240nt
promoter with spacer 241cacattatta aaattaaagc aagagaggcc aagtaagccc
aaatgtacac ttgtcattgc 60caaattaaaa tactcggcat attgtattgt attcccacac
attattaaaa tactcggcat 120attgtattgg ctgcatttgc atttgcatga ataatactac
gtgtaagata atccaaaata 180ctacgtgtag cccatgcaaa gttaacactc accaaaagaa
cccacgtgta agcccatgca 240aagttaacac tcaccaaatg tattcccaca cattattaaa
attaaaatta gtaaaatacc 300gtatatgtat tcccacgtgt aagcccattc ctcagtctcc
actatataaa cccacgtgta 360gcccaaac
368242369DNAArtificial Sequencesynthetic 240nt
promoter with spacer 242agcccaatct caccaaaaga acccacgtgt agcccatgca
aagttaacac ttgtcattgc 60caaaagaacc cacgtgtaag cccattccca cacattatta
aagcaagaga ggccaagtaa 120gcccaaatgt acacttgtca ttgccaagta aaatactacg
tgtagcccat gcaaagttaa 180cactcaccat ccccaatctc acgaccccat tcccacgtgt
aagataatcc aaattaaagc 240aagagaggcc aaatgtattc ctcagtctcc actatataaa
cccaccatcc ccatgcaaag 300ttaacacttg tcattgccaa attagtaaga taataccgta
tataaaccca cgtgtaagat 360aatccaaac
369243379DNAArtificial Sequencesynthetic 240nt
promoter with spacer 243aataatactc ggcatattgg ctgcatgaat aatactcggc
atattgtatt gtattggctg 60catttgcatg aataatactc ggcatattgt attgtattgt
attcccacac attattaaag 120caagagaggc caagtaaaat actcggcata ttggctgcat
gaataatcca aattaaagca 180agagaggcca aaatactacg tgtagcccaa attaaaatac
tcggcatatt gtattcccac 240caaatgtatt cctcagtctc cactatatgt attgtattcc
tcagtctcca ctatataaac 300ccacgtgtag cccatgcaaa gttaacactt gtcattgcca
agtaaaatac cgtatataaa 360cccacgtgta agcccaaac
379244386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 244cattcccacc atccccaaaa gaacccacac attattaaag
caagagaggc caaaatacta 60cgtgtagccc attcctcagt ctccactata tgtattggct
gcatttgcat ttgcatttgc 120atttgcattt gcatttgcat ttgcatgaat aataccgtat
ataaacccac caaaagaacc 180cacacattat taaaatactc ggcatattgt attcccacgt
gtagcccaat ctcaccaaat 240gtattggctg catgaataat actacgtgta agcccaatct
cacgacccca atctcaccat 300ccccatgcaa agttaacact tgtcattgcc aaatgtattc
ccacgtgtaa gataataccg 360tatataaacc cacgtgtagc ccaaac
386245386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 245attaaagcaa gagaggccaa gtaaaatact acgtgtagcc
caatctcacc atccccattc 60ctcagtctcc actatatgta ttcccacaca ttattaaagc
aagagaggcc aagtaaaata 120ccgtatataa acccaccatc cccattccca cgtgtagccc
atgcaaagtt aacacttgtc 180attgccaaat gtacactcac catccccaaa ttagtaaaat
accgtatata aacccaccaa 240atgtacactc acgaccccat tcccaccatc cccatgcaaa
gttaacactc accatcccca 300ttcccaccaa aagaacccac gtgtagccca atctcacgac
cccattcctc agtctccact 360atataaaccc acgtgtaagc ccaaac
386246386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 246ctcagtctcc actatataaa cccacacatt attaaagcaa
gagaggccaa gtaaaatact 60acgtgtaagc ccaaatgtac actcaccaaa agaacccacc
atccccaaaa gaacccacgt 120gtagcccatg caaagttaac actcacgacc ccatgcaaag
ttaacactca ccatccccat 180tcctcagtct ccactatatg tacacttgtc attgccaagt
aagcccaatc tcacgacccc 240aatctcacga ccccaaatgt attcctcagt ctccactata
tgtattgtat tcccaccaaa 300ttagtaaaat actacgtgta gcccaatctc accatcccca
ttcctcagtc tccactatat 360aaacccacgt gtaagataat ccaaac
386247386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 247ttgtattcct cagtctccac tatataaacc cacacattat
taaagcaaga gaggccaaaa 60tactacgtgt agcccaaatg tattcccacg tgtagcccaa
aagaacccac gtgtaagccc 120attcccacgt gtaagcccat tcccacacat tattaaagca
agagaggcca agtaaaatac 180cgtatatgta ttcctcagtc tccactatat gtacacttgt
cattgccaag taaaatactc 240ggcatattgt attgtattcc tcagtctcca ctatataaac
ccaccatccc caaaattagt 300aagcccatgc aaagttaaca cttgtcattg ccaagtaaga
taatccaaat taaaataccg 360tatataaacc cacgtgtagc ccaaac
386248386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 248gataatactc ggcatattgg ctgcatgaat aatccaaaat
tagtaaaatt aaagcaagag 60aggccaagta aaatactacg tgtaagataa tactacgtgt
agcccattcc tcagtctcca 120ctatataaac ccacgtgtag cccatgcaaa gttaacactc
acgaccccaa atgtacactt 180gtcattgcca agtaaaatta gtaaaattag taagataatc
caaaatacta cgtgtagccc 240aatctcacca tccccattcc tcagtctcca ctatataaac
ccaccatccc cattcccaca 300cattattaaa attagtaaaa taccgtatat aaacccacac
attattaaaa taccgtatat 360aaacccacgt gtaagataat ccaaac
386249386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 249attaaagcaa gagaggccaa gtaaaatact acgtgtaaga
taatactacg tgtagcccaa 60tctcacgacc ccaaaatact cggcatattg gctgcatgaa
taatactacg tgtagcccaa 120atgtattgta ttcctcagtc tccactatat gtattcctca
gtctccacta tataaaccca 180cgtgtagccc atgcaaagtt aacactcacc atccccaatc
tcacgacccc attcctcagt 240ctccactata taaacccaca cattattaaa ataccgtata
taaacccacg tgtaagccca 300tgcaaagtta acactcacca tccccaaatg tacacttgtc
attgccaaat taaaataccg 360tatataaacc cacgtgtagc ccaaac
386250386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 250taaacccaca cattattaaa gcaagagagg ccaagtaaaa
ttaaaatact acgtgtaagc 60ccaatctcac gaccccaatc tcaccaaatg tacacttgtc
attgccaagt aaaatactcg 120gcatattggc tgcatttgca tgaataatac tacgtgtagc
ccattcctca gtctccacta 180tataaaccca cgtgtagccc atgcaaagtt aacacttgtc
attgccaagt aagataatac 240tacgtgtaag ataatccaaa agaacccacg tgtagcccat
gcaaagttaa cactcaccaa 300atgtattcct cagtctccac tatataaacc cacacattat
taaaataccg tatataaacc 360caccaaatta gtaagataat ccaaac
386251386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 251ccactatata aacccacaca ttattaaagc aagagaggcc
aaattagtaa aattaaagca 60agagaggcca aaatactacg tgtaagccca aatgtacact
caccaaaaga acccaccaaa 120agaacccacg tgtaagataa tactacgtgt aagcccaatc
tcaccatccc catgcaaagt 180taacactcac catccccatt cccacgtgta gcccaatctc
acgaccccat gcaaagttaa 240cactcacgac cccattcctc agtctccact atataaaccc
acgtgtaagc ccatgcaaag 300ttaacactca cgaccccaat ctcaccaaat gtattgtatt
cctcagtctc cactatataa 360acccaccatc cccattccca ccaaac
386252386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 252tagcccaatc tcaccaaaat accgtatatg tattcctcag
tctccactat atgtattggc 60tgcatgaata atccaaaatt aaagcaagag aggccaagta
aaatactacg tgtaagccca 120atctcacgac cccattccca ccaaaagaac ccacgtgtag
cccaatctca ccaaaattag 180taagataatc caaaatacta cgtgtaagcc catgcaaagt
taacacttgt cattgccaaa 240tgtacacttg tcattgccaa attaaagcaa gagaggccaa
gtaagcccat gcaaagttaa 300cacttgtcat tgccaaaata ctacgtgtaa gcccattcct
cagtctccac tatataaacc 360cacgtgtaag cccaatctca ccaaac
386253386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 253attattaaag caagagaggc caagtaaaat actacgtgta
gcccattccc acgtgtagcc 60catgcaaagt taacactcac catccccatg caaagttaac
acttgtcatt gccaaattaa 120aatactcggc atattggctg catttgcatt tgcatttgca
tttgcatttg catgaataat 180ccaaaagaac ccaccatccc cattcctcag tctccactat
atgtacactc acgaccccaa 240tctcaccatc cccatgcaaa gttaacactc accaaaagaa
cccacgtgta agcccaatct 300caccaaatta aagcaagaga ggccaaatta aaatactacg
tgtaagataa taccgtatat 360aaacccacgt gtaagataat ccaaac
386254386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 254accccattcc caccatcccc aatctcacga ccccatgcaa
agttaacact tgtcattgcc 60aagtaagccc attcctcagt ctccactata tgtacacttg
tcattgccaa atgtattccc 120accatcccca ttcctcagtc tccactatat aaacccacac
attattaaag caagagaggc 180caaattagta aaattagtaa gcccattccc accaaaatac
cgtatatgta ttgtattgta 240ttcccacgtg taagataata ctacgtgtag cccattccca
cgtgtaagcc catgcaaagt 300taacactcac catccccaat ctcacgaccc caaattagta
agataatacc gtatataaac 360ccacgtgtag cccaatctca ccaaac
386255386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 255gtaaaattaa agcaagagag gccaaaatac tacgtgtagc
ccaatctcac gaccccaaat 60taaaataccg tatatgtaca ctcacgaccc caatctcacg
accccatgca aagttaacac 120tcacgacccc attcccacgt gtaagcccat gcaaagttaa
cactcaccat ccccattccc 180accaaatgta ttcctcagtc tccactatat aaacccacac
attattaaaa tactacgtgt 240aagcccaatc tcacgacccc atgcaaagtt aacacttgtc
attgccaagt aagcccaaat 300taaaataccg tatatgtatt ggctgcatga ataatccaaa
attagtaaaa taccgtatat 360aaacccacgt gtaagataat ccaaac
386256386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 256attaaaatta aagcaagaga ggccaagtaa gataatacta
cgtgtagccc attcccacca 60tccccaatct caccaaaatt aaaattagta aaatactcgg
catattgtat tgtattggct 120gcatttgcat ttgcatgaat aatactcggc atattgtatt
cctcagtctc cactatataa 180acccacacat tattaaagca agagaggcca agtaagccca
tgcaaagtta acacttgtca 240ttgccaaaat accgtatata aacccacgtg tagcccaatc
tcacgacccc aaaataccgt 300atataaaccc acgtgtagcc catgcaaagt taacacttgt
cattgccaag taaaataccg 360tatataaacc cacgtgtagc ccaaac
386257386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 257ttggctgcat ttgcatgaat aatccaaatt aaagcaagag
aggccaaatt aaaatactac 60gtgtagccca tgcaaagtta acactcacca tccccaatct
cacgacccca ttcctcagtc 120tccactatat aaacccacgt gtagcccaat ctcaccatcc
ccattcctca gtctccacta 180tataaaccca ccatccccaa tctcaccatc cccaatctca
ccatccccat gcaaagttaa 240cactcaccat ccccaaaata ctcggcatat tgtattcctc
agtctccact atatgtacac 300ttgtcattgc caagtaagcc caaatgtatt ggctgcattt
gcatgaataa taccgtatat 360aaacccacgt gtaagataat ccaaac
386258386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 258agagaggcca agtaaaatac cgtatatgta ttcccacaca
ttattaaaat actacgtgta 60gcccaaaaga acccacacat tattaaagca agagaggcca
agtaagccca aaatactacg 120tgtagcccat gcaaagttaa cacttgtcat tgccaagtaa
gcccattccc acacattatt 180aaaattaaag caagagaggc caaatgtaca cttgtcattg
ccaaatgtat tgtattcctc 240agtctccact atataaaccc acgtgtagcc catgcaaagt
taacacttgt cattgccaag 300taaaatactc ggcatattgt attcctcagt ctccactata
tgtattcctc agtctccact 360atataaaccc acgtgtaagc ccaaac
386259386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 259tgcaaagtta acacttgtca ttgccaagta agataatcca
aattagtaag cccaaatgta 60cactcaccat ccccaatctc accaaatgta ttcccaccat
ccccatgcaa agttaacact 120cacgacccca atctcacgac cccaaaatta aagcaagaga
ggccaaaaga acccacacat 180tattaaagca agagaggcca agtaaaatta aaatactcgg
catattgtat tggctgcatg 240aataatacta cgtgtagccc aatctcacca aaagaaccca
cgtgtaagcc caaaagaacc 300cacgtgtaag cccatgcaaa gttaacactc accatcccca
ttcctcagtc tccactatat 360aaacccacgt gtaagataat ccaaac
386260386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 260attaaaatac tcggcatatt ggctgcatga ataataccgt
atataaaccc acacattatt 60aaagcaagag aggccaaaat actcggcata ttgtattgta
ttggctgcat gaataatcca 120aaataccgta tatgtacact tgtcattgcc aagtaaaatt
agtaagataa tactacgtgt 180agcccaatct caccaaaatt agtaagataa tactcggcat
attgtattcc cacgtgtaag 240cccaatctca ccatccccat tcccaccatc cccaaatgta
cactcaccaa aagaacccac 300gtgtaagccc atgcaaagtt aacacttgtc attgccaaat
taaaataccg tatataaacc 360cacgtgtaag cccaatctca ccaaac
386261386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 261attgccaagt aagataatcc aaatgtattc ccaccaaaat
actcggcata ttggctgcat 60gaataatacc gtatataaac ccacacatta ttaaagcaag
agaggccaag taaaatactc 120ggcatattgg ctgcatttgc atgaataatc caaattaaag
caagagaggc caaattagta 180aaatactacg tgtaagccca aaattaaaat actcggcata
ttgtattgta ttgtattggc 240tgcatttgca tgaataatcc aaatgtacac ttgtcattgc
caaaagaacc cacgtgtagc 300ccatgcaaag ttaacacttg tcattgccaa atgtattgta
ttcctcagtc tccactatat 360aaacccacca aaagaaccca ccaaac
386262386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 262acattattaa aattaaagca agagaggcca aatgtacact
tgtcattgcc aagtaagata 60atccaaatta gtaagataat ccaaaagaac ccacacatta
ttaaagcaag agaggccaaa 120ttaaaatact acgtgtaagc ccaaattagt aaaattaaaa
ttagtaagat aatccaaatt 180aaaatactac gtgtagccca tgcaaagtta acactcacga
ccccattcct cagtctccac 240tatataaacc caccaaaata ctacgtgtaa gcccattcct
cagtctccac tatataaacc 300cacgtgtagc ccatgcaaag ttaacacttg tcattgccaa
atgtattcct cagtctccac 360tatataaacc cacgtgtagc ccaaac
386263386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 263acattattaa agcaagagag gccaaattaa aattagtaaa
atactacgtg taagataata 60ctcggcatat tgtattcctc agtctccact atataaaccc
acgtgtaagc ccatgcaaag 120ttaacactca cgaccccatt cctcagtctc cactatataa
acccacgtgt agcccatgca 180aagttaacac tcacgacccc atgcaaagtt aacacttgtc
attgccaagt aagataatac 240cgtatatgta ttgtattccc acgtgtagcc caatctcacg
accccaaaag aacccacaca 300ttattaaagc aagagaggcc aaaatactac gtgtagccca
ttcctcagtc tccactatat 360aaacccacgt gtaagataat ccaaac
386264386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 264ggcatattgg ctgcatgaat aatccaaatt aaagcaagag
aggccaagta aaattagtaa 60aatactacgt gtaagataat ccaaaattag taagcccaaa
atactcggca tattggctgc 120atttgcatga ataatccaaa tgtattgtat tgtattcctc
agtctccact atatgtacac 180tcacgacccc aaattaaaat actacgtgta agataatcca
aattagtaag cccaaaagaa 240cccacgtgta gcccaaaaga acccaccatc cccaaattag
taagcccatg caaagttaac 300acttgtcatt gccaagtaaa ataccgtata tgtattgtat
tgtattcctc agtctccact 360atataaaccc acgtgtaagc ccaaac
386265386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 265gaccccaatc tcaccatccc catgcaaagt taacacttgt
cattgccaag taagcccaaa 60agaacccacc aaatgtattg tattcccaca cattattaaa
gcaagagagg ccaaatgtat 120tcccaccaaa agaacccacc atccccaatc tcacgacccc
aatctcacca aattagtaag 180ataatactac gtgtaagccc attcctcagt ctccactata
taaacccacc atccccaatc 240tcacgacccc attcctcagt ctccactata taaacccaca
cattattaaa ataccgtata 300taaacccacg tgtagcccat gcaaagttaa cactcaccat
ccccattcct cagtctccac 360tatataaacc cacgtgtagc ccaaac
386266386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 266cccatgcaaa gttaacactc acgaccccaa tctcaccatc
cccaatctca ccaaaagaac 60ccaccatccc catgcaaagt taacactcac gaccccattc
ccacacatta ttaaagcaag 120agaggccaaa ttagtaagat aataccgtat atgtacactc
accaaattaa agcaagagag 180gccaagtaag ataatactac gtgtagccca aaattaaagc
aagagaggcc aagtaagata 240atccaaatta gtaaaatact acgtgtagcc caatctcacc
atccccaaaa gaacccacgt 300gtaagcccat gcaaagttaa cacttgtcat tgccaaaatt
agtaagataa taccgtatat 360aaacccacgt gtaagataat ccaaac
386267386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 267ccacgtgtaa gataatactc ggcatattgg ctgcatttgc
atttgcattt gcatttgcat 60gaataatacc gtatatgtat tcccacgtgt aagataatcc
aaattagtaa aatactcggc 120atattggctg catgaataat actacgtgta agataatacc
gtatataaac ccacacatta 180ttaaagcaag agaggccaag taagataatc caaattagta
aaatactacg tgtaagccca 240ttcccacgtg tagcccatgc aaagttaaca ctcaccaaaa
tactcggcat attggctgca 300tgaataatcc aaattagtaa gcccaatctc accatcccca
ttcctcagtc tccactatat 360aaacccacgt gtaagataat ccaaac
386268386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 268tccactatat gtacactcac gaccccaaat gtacacttgt
cattgccaaa tgtattggct 60gcatgaataa tccaaatgta ttcccacaca ttattaaagc
aagagaggcc aagtaagata 120ataccgtata tgtattggct gcatgaataa tccaaaatta
gtaaaattag taagataata 180ctacgtgtag cccattcctc agtctccact atataaaccc
acgtgtagcc catgcaaagt 240taacactcac gaccccaatc tcacgacccc aaaagaaccc
accaaaagaa cccacacatt 300attaaagcaa gagaggccaa atgtacactc acgaccccaa
attagtaaaa taccgtatat 360aaacccacgt gtaagataat ccaaac
386269386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 269ttaaaattaa agcaagagag gccaaatgta cactcacgac
cccattcctc agtctccact 60atataaaccc accatcccca tgcaaagtta acactcacca
aattagtaag cccatgcaaa 120gttaacactt gtcattgcca agtaaaatta gtaaaattag
taaaatacta cgtgtagccc 180aaaattagta aaattagtaa gcccattccc accaaaatac
tacgtgtagc ccaaaagaac 240ccacacatta ttaaaatacc gtatataaac ccacgtgtag
cccatgcaaa gttaacactt 300gtcattgcca agtaagataa tccaaaatta aagcaagaga
ggccaagtaa gataataccg 360tatataaacc cacgtgtagc ccaaac
386270386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 270caaaagaacc cacacattat taaagcaaga gaggccaaaa
tactacgtgt agcccattcc 60cacacattat taaagcaaga gaggccaagt aagataatcc
aaattagtaa gcccaaaatt 120aaaatactcg gcatattgta ttgtattccc acgtgtagcc
catgcaaagt taacacttgt 180cattgccaaa atactcggca tattgtattc ccacacatta
ttaaagcaag agaggccaag 240taagcccatg caaagttaac acttgtcatt gccaagtaaa
attagtaagc ccaatctcac 300gaccccaatc tcacgacccc aaattaaaat actcggcata
ttgtattcct cagtctccac 360tatataaacc cacgtgtagc ccaaac
386271386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 271gtacactcac caaaagaacc cacacattat taaagcaaga
gaggccaagt aaaattaaaa 60ttagtaagcc catgcaaagt taacacttgt cattgccaaa
attagtaaaa ttaaaatacc 120gtatataaac ccacgtgtaa gataatacta cgtgtagccc
aatctcacga ccccaatctc 180acgaccccat tcccacacat tattaaagca agagaggcca
agtaagccca tgcaaagtta 240acacttgtca ttgccaagta agataatcca aaagaaccca
cgtgtagccc atgcaaagtt 300aacacttgtc attgccaagt aaaatactac gtgtagccca
ttcctcagtc tccactatat 360aaacccacgt gtaagataat ccaaac
386272386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 272tcagtctcca ctatataaac ccacacatta ttaaagcaag
agaggccaag taagataata 60ctacgtgtag cccatgcaaa gttaacactc accatcccca
tgcaaagtta acacttgtca 120ttgccaaaag aacccacgtg taagcccatg caaagttaac
actcacgacc ccaaattagt 180aagataatcc aaatgtacac tcaccatccc caaaattagt
aaaattagta aaatactacg 240tgtagcccat tcctcagtct ccactatata aacccaccaa
atgtattcct cagtctccac 300tatatgtatt cccaccaaaa taccgtatat aaacccacac
attattaaaa taccgtatat 360aaacccacca aaagaaccca ccaaac
386273386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 273tccactatat aaacccacac attattaaag caagagaggc
caagtaaaat actacgtgta 60gcccaatctc acgaccccaa tctcacgacc ccaatctcac
gaccccatgc aaagttaaca 120cttgtcattg ccaagtaaaa taccgtatat aaacccacgt
gtaagataat ccaaattaaa 180ataccgtata taaacccacc atccccaatc tcaccatccc
catgcaaagt taacactcac 240gaccccatgc aaagttaaca cttgtcattg ccaaatgtac
acttgtcatt gccaaaatac 300cgtatatgta ttgtattcct cagtctccac tatatgtatt
ggctgcatga ataataccgt 360atataaaccc acgtgtaagc ccaaac
386274386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 274attgccaaaa gaacccacca aatgtacact tgtcattgcc
aaaagaaccc acacattatt 60aaagcaagag aggccaaaag aacccacaca ttattaaaat
tagtaagata atccaaaata 120ctacgtgtag cccaatctca cgaccccatg caaagttaac
acttgtcatt gccaagtaaa 180ataccgtata taaacccacg tgtagcccat gcaaagttaa
cactcacgac cccaatctca 240cgaccccatt cctcagtctc cactatataa acccacgtgt
aagataatcc aaatgtacac 300ttgtcattgc caaaatacta cgtgtaagat aatccaaatt
agtaagataa taccgtatat 360aaacccacgt gtaagataat ccaaac
386275386DNAArtificial Sequencesynthetic 240nt
promoter with spacer 275cacattatta aagcaagaga ggccaaatta aaatactacg
tgtagcccat gcaaagttaa 60cactcacgac cccatgcaaa gttaacactc accatcccca
ttcccaccat ccccattccc 120accatcccca atctcacgac cccattcctc agtctccact
atataaaccc acgtgtagcc 180catgcaaagt taacacttgt cattgccaag taaaattagt
aaaataccgt atatgtattg 240gctgcatttg catgaataat accgtatatg tacactcacg
accccaatct cacgacccca 300tgcaaagtta acactcacca aaatactacg tgtaagccca
ttcctcagtc tccactatat 360aaacccacgt gtaagataat ccaaac
386276314DNAArtificial SequenceProPer 240nt with
spacer and enhancer 276aataatccaa attaaagcaa gagtaggaaa gtaagataat
ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg tagaagcgca
cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc agaaccccat tcctcagtct
ccactatata aacccaccat 240ctttcatctc accaaaccca ccacacaact cacaactcac
tctcacacct taaagaacca 300atcaccacca aaaa
314277314DNAArtificial SequenceProPer 240nt with
spacer and enhancer 277tataatccaa attaaagcaa gacctcgcaa gtaagataat
ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg tatgaggaca
cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc acgaccccat tcctcagtct
ccactatata aacccaccat 240cagcggtctc accaaaccca ccacacaact cacaactcac
tctcacacct taaagaacca 300atcaccacca aaaa
314278314DNAArtificial SequenceProPer 240nt with
spacer and enhancer 278tataatccaa attaaagcaa gacagtctaa gtaagataat
ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg taatgcgtca
cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc aaaaccccat tcctcagtct
ccactatata aacccaccat 240cttgagtctc accaaaccca ccacacaact cacaactcac
tctcacacct taaagaacca 300atcaccacca aaaa
314279314DNAArtificial SequenceProPer 240nt with
spacer and enhancer 279aataatccaa attaaagcaa gatgcgaaaa gtaagataat
ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg tattgtgcca
cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc actaccccat tcctcagtct
ccactatata aacccaccat 240catttatctc accaaaccca ccacacaact cacaactcac
tctcacacct taaagaacca 300atcaccacca aaaa
314280314DNAArtificial SequenceProPer 240nt with
spacer and enhancer 280tataatccaa attaaagcaa gacttttgca gtaagataat
ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg tactcactca
cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc aagaccccat tcctcagtct
ccactatata aacccaccat 240cttcggtctc accaaaccca ccacacaact cacaactcac
tctcacacct taaagaacca 300atcaccacca aaaa
314281314DNAArtificial SequenceProPer 240nt with
spacer and enhancer 281aataatccaa attaaagcaa gaatatgtaa gtaagataat
ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg taatctgcca
cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc atcaccccat tcctcagtct
ccactatata aacccaccat 240ctccactctc accaaaccca ccacacaact cacaactcac
tctcacacct taaagaacca 300atcaccacca aaaa
314282314DNAArtificial SequenceProPer 240nt with
spacer and enhancer 282tataatccaa attaaagcaa gatcgtggaa gtaagataat
ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg tacttatcca
cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc agtaccccat tcctcagtct
ccactatata aacccaccat 240cccacttctc accaaaccca ccacacaact cacaactcac
tctcacacct taaagaacca 300atcaccacca aaaa
314283314DNAArtificial SequenceProPer 240nt with
spacer and enhancer 283aataatccaa attaaagcaa gagtacggca gtaagataat
ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg tagcaattca
cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc aggaccccat tcctcagtct
ccactatata aacccaccat 240catctatctc accaaaccca ccacacaact cacaactcac
tctcacacct taaagaacca 300atcaccacca aaaa
314284314DNAArtificial SequenceProPer 240nt with
spacer and enhancer 284aataatccaa attaaagcaa gagcgacata gtaagataat
ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg taaagtctca
cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc actaccccat tcctcagtct
ccactatata aacccaccat 240caaacatctc accaaaccca ccacacaact cacaactcac
tctcacacct taaagaacca 300atcaccacca aaaa
314285314DNAArtificial SequenceProPer 240nt with
spacer and enhancer 285aataatccaa attaaagcaa gattagagaa gtaagataat
ccaaatgtac acttgtcatt 60gccaaaatta gtaaaatact cggcatattg taaggcccca
cattattaaa ataccgtata 120tgtattggct gcatttgcat gaataatact acgtgtaagc
ccaaaagaac ccacgtgtag 180cccatgcaaa gttaacactc atcaccccat tcctcagtct
ccactatata aacccaccat 240cgtaactctc accaaaccca ccacacaact cacaactcac
tctcacacct taaagaacca 300atcaccacca aaaa
3142861037DNAArtificial Sequencesynthetic 1064
promoter with spacer 286atgtgaaggc tgtaaaatga tcgattgatc ctaatagatt
atgaaaattt tataaaaatt 60aaagcaagag tatatgtatt tttaatgttt ttatataact
gaaaatactc ggcatgtttg 120agttgcaaat gtacactttt attattttgg ccaccatccc
caatctcacc atcgtttata 180gattattttg gccaagtatt ttggtgtcat attgggacaa
ttttcgttta tagatgatat 240gaatatccca cgtgtaaaat gttcatgagt attggattgg
ataaaatacc gaaattctct 300caccattcaa gtataactga ttcttttaac cgtatatcgt
ttataaaatg tttttatata 360actgaaatga tcctaattaa ttgtaaaaga tcaactctca
ccattccttc gattatgaac 420ggcatgtttg aaaatatgta ttattaaaat attttagaaa
tcgattgtat tggctgtaaa 480ggctgtaaaa attttggcca aggtaagata ttttttcctc
attgccttta cctattttat 540aaaaatgttc actttttgtt catgtttgaa aacaaatgat
cgattctgtc taaatgatat 600gaaaacaaat taaaggctgt aaaagaataa tccaaaagac
tatttttaac caaattctaa 660tactacgtgt aaaatacatt aacactatgt attatggatt
ggataaaatt tttggtgtaa 720gattatgaaa ttgttcactt ttggataata gaatcagtct
ttagattcta attagtaagc 780ccatgtttat atgttttaga agaacccacg tgtaaaggct
gtaaaattga tcctaataga 840aatcgattgt attggccaag gtaagcccaa tctcacgacc
ccatgcaaat tctgtctttt 900aacacttgtc aaagttaatg ttcactttta taaaataccg
taatcgattc aattctgtct 960ttacctattt atagaataat ccaaaatact cggcatgaac
ggcatgagta taaaaagaac 1020ccacgtgtag cccaaac
10372871129DNAArtificial Sequencesynthetic 1064
promoter with spacer 287ccaaggtaag atcaactctc cactatataa aagatcaact
ctccatttgc ttttttatga 60aaattctaat accgtaatcc taatagatta tgaaatgatc
ctaattaatt aaagcaagag 120aataaaattt tcgattatgg ataatagatt atgtattgga
ttggattgga taatcgattt 180tatgaataat tagtaaaaat gatcgattat gaaattaatt
gttcatgagt tgcatgagtt 240gcaaattttc catgcaaatt ctgtctaatt ttcgacgttg
ggatctgata gttcactttt 300gcaaagttaa ttgaaggctg taaaattttg gataaaattt
cgttcgaaga ttcaagtatt 360aatttcgacg ttaattaaag gctgtaagat catagattct
aaatgatatg tgatattgtt 420tgaagaaccc acgtgtagcc catgcaaatg tttgaaattt
cgaagaaata ccgtatatcc 480ccaatctcac gacccctaat accgaaacca gggacaacgt
taactgattc tctccatcgt 540tttggtgtca aagttaacac tatgtacact cacgacccct
aatagaaatt gtaaaatact 600acgtgtagcc catgcaaatt taatgtacac ttttttggtg
gtttcgttta ttttagatga 660tcctaatact acgtgtaaaa taccgtaatc gattggataa
tccaaatgta ttggccacca 720aatttatatc cccatgcaaa ttgtaaaaga aattaaccaa
cggcatatcc cacgtgtaaa 780gcaagagtct tttagaagaa tatgtgatat gtgatagttc
atcgttcgaa gataatagaa 840gatcatagtt tttgcttttg caaagactat ataatccaaa
ataccgaaat gtaggtcatg 900aaaataccga tcaactctcc atcgttcgaa gaaatggtgt
catgtacact tttaatgtat 960ttaatgtttg aaaataaaaa ttaacactat ataaaatatc
gttcgacgtt gggacaacgt 1020taatgtttat ttttccattc ccatgcaaag aaatttttat
atcgttcgaa atcgattatg 1080aatattttcg tttatacctc agtatataaa atactcggca
tatccaaac 11292881240DNAArtificial Sequencesynthetic 1064
promoter with spacer 288aaagcaagag tattggataa tcctaatttt gtgaaggctg
taagagagta tatgtacact 60ttttggtgtt tgaaattaaa gcaagagagt ctttagaaga
aatcgattgt attattatga 120ataatacatt atgtgaataa tccaaattca atttcgttcg
aagaaattaa aattttatca 180tatcgttcga ttggccaagt aagcccaatc tcacgacccc
atgtacacta tgttttttaa 240cactatgtac actcacccct aatactacgt gtaaaatatt
ttgcatgttt tcgaaaccca 300cacatttgag aggccaagta aaggctgtaa aaagattgta
aaaagactat tttttagggt 360gtctaaatgt acactatatg aataatccta cactcaccat
ttgaaggctg catttgcttt 420taactgatat tggataatta gtaaaatttt ttatcataga
tgatagttca tgtattgtaa 480aattctaatt aaagcaagag tcttgccttt acctatttat
acctcattgc caaggtaagc 540ccaaaagaac ccacacattt gaaatactcg gcatgtattc
ccacacatta ttaaccgtaa 600tcgattctaa tagattgaaa tgatagggtg tcaaagacta
tgtgaataaa cccacgtgta 660ggtcatgaaa tttttataga atcagtcttt agggatctga
tagggatctg atagggtgtc 720aaagactatg ttttaaccga tcaactctcc atttgctttt
agatgatagt tcatgagttg 780cattttcctt cgattttaac tgatattggc tgtaaaaaga
gtataaaaat actacgtgta 840aaaagatcaa ctctcacgac cccatttggt gtcaaagtta
attaaaatac attaatgttg 900agaggccaag gtaagatcat agttttttat aatccaaatt
aaccagggat ctgatatgaa 960atactacgtg taaaagacta tataaaagac tatatcccca
atctcacgac cccaatctca 1020cgacccctaa tagattgaaa tactcggcat gtatttgaaa
atgatcctac actcacccca 1080aaagaaccca cacattatga aatcgattct aaatgttttt
tgtgatattt gcaaagttaa 1140cactcacgac ccctaattaa ttgtaaaggc tgtaaagcaa
gagtctccat gcaaattttt 1200tgcttttata actgataggg acaattctct caccccaaac
12402891266DNAArtificial Sequencesynthetic 1064
promoter with spacer 289tgttcatcgt agtcttttaa ccgaaatttt gcttttggtg
taagataaac caacgttaat 60gtattgggac aacgttaatg tttgaaaaca aatttaactg
atagttttcg aagatcatat 120tgggatgtga tagggatgtg atagttcatg ttttttttgg
ataatcctac actatatgaa 180tatgaatatc ccacacattt gaaatgtttt tatgtttttg
ttcacttgtc atatcgttta 240tcatatcgta gtcttgccaa atttaattta tttttattaa
ttagtaagat tatgaacggc 300caaggtaaga ttattatgaa tatttatcat agttcacttt
tggtgtaaag gctgtaagag 360tcttttatat aactgaaaat gatagttttt cggtcataga
tgatagttca cttgtcatta 420aagcaagagt ataattagta aaaattttta ttaacacttt
taaccgtaat cctacacttg 480tcatagtttt ggctgtaaaa gactatgtac actcacccca
tgttttggct gtaaaattga 540agaacccaca cattatgaaa tactacgtgt aggtcatagt
tttggtggtt tcgaaaccca 600cacatttgaa aatttttgct ttttggtgta gcccattccc
accatcgtag tctttaccta 660ttttatagat gatcgatttt cctcattgcc aaattttttg
gtgtcaaagt taaccaaatt 720taattgtaaa atttcgacgt tgggacaatt ctctccattt
tatacctcat tgcctttacc 780tcattaaagc aagagagagt ctttagggat gtgaaggctg
catgagttgc atttgagaga 840gtctccatcc ccattttcgt tcgacgttaa cactcacgac
cccatgcaaa tgatcgattt 900tgtttatttt atgaacggcc aaggtaagag aggccaagta
tatccatttg ctttttgctt 960ttgtgaatat cgtagtctcc actattttgt tcatcgtttt
agaagaataa tccattccca 1020ccatccccat ttgaaaataa ttgtaagatt caattctaat
agatgatatt gggacaattc 1080tgtctaatta gtaagcccat tccttcgaag aacccacgtg
tagcccatgc aaattttcct 1140cattattaac cgtaatccta cactatgttg agaggccaag
gtaagagtat ttaatgttta 1200tttgcatttg aagaacccac gtgtagccca ttttataaaa
tacctcagta ttcctcattg 1260ccaaac
12662901269DNAArtificial Sequencesynthetic 1064
promoter with spacer 290ccatgttcac tttttttttc cttcgacgtt gggacaacgg
ccaagtaagc ccatgcaaag 60ttaattagta agatcaactc tccactatat aactgatagt
tttccttcga aaccaaaata 120ccgaaattct ctcacgaccc caaaagacta tgtatttgaa
atactcggca tgtttatttg 180aaataccgta tatgaaaatt gatcgattgt attgtttatc
atatttaatt tttcgttttc 240gtttatggat aatccaaatg tacacttttg cttttggtgt
agcccaaatt aaaatggtgt 300aggtcatgaa taaaaagaga ggccaaggta agagaatcag
tcttgccttt acctcattat 360taaaagaaat tttccatccc caatctcacc aaattaaagg
ctgcatgttt ttccatcgtt 420ttttcggtca tatttgaaat cgattctaat taaagcaaga
gtcttttgtg aagaataatc 480gattcaattc ttttggtgta ggtcattatg gataatccaa
aagaataatc caaatttcga 540cgttaatgta tttttccatt caagtattat tatgaaatcg
attgggacaa cgttgggaca 600attctttaga atatcgttcg attctgtctt tagaatcagt
cttgccttta gaaatcgatt 660gatcctacac tatatcccca tgcaaatggt gtagcccaaa
atatgtatta accgtaatcc 720aaaatactac gtgtaagccc aaatgtttga aaacaaatgg
tgtctaatag atgatagttt 780tggtggtttc ggtcatattt atataaaata ctcggcatgt
ttgaagaaat ttcgttcgaa 840ataccgaaat actacgtgta aaaagaaatt ttcgttcgac
gttaatttcg acgttaactg 900aaataatttt cggtcatgag tattggccac cattcaattt
ttggtgtctt tacctcagtc 960ttttggtggt ttcgacgttg ggatgtgaag gctgtaaaag
atcatatttt tatgaatatt 1020ttggtggttt cgacgttggg atgtgaagaa cccacgtgta
aaattttaac cgaaaccaaa 1080agaaattgtt ttaaccgtat ataatcgatt tttttagaag
aacccacaca tttgagtata 1140tcccacacat taaagcaaga gaatatcccc atgcaaagaa
cccaccattc aatttttaga 1200atcagtcttt tataccgatc atagattctt ttatagaatc
agtataaaat actacgtgta 1260agcccaaac
12692911360DNAArtificial Sequencesynthetic 1064
promoter with spacer 291gcaaattttt tcctcattaa aggctgtaaa agagtattgg
ctgtaaaaga acccacacat 60taaaggctgc atttttagat tctctcacca ttcaagtaaa
aatactacgt gtaaaagagt 120atataaaaga gaataaaaat tgatcgatta tgaatatcgt
agtctttaga ttgatcgatt 180ctctccatgc aaagaaattc aattcttttg tgaataatcc
aaattaaccg aaacccacca 240tccccaatct cacgacccct aatagatgat agttcatgtt
tgaaggctgc atgaaatggt 300gtaaaaatac attttatcat atccccaatc tcacgacccc
attttaggga tgtgaatatg 360tagcccatgc aaattaaaag aacccacgtg taaagcaaga
gaatcagtct tttggtggtt 420tcgaagataa taccgtatat ccaaaagatt gggacaacgg
ccaccatcgt ttatgaataa 480tccaaattta tggataatcc atgcaaagaa attgtaaaag
aacccacgtg taaaatactc 540ggcatatttt ggtgtcaaag agtcttgcct ttagaagaaa
tacctcatta tgtgaaggct 600gtaaaagact atttttaacc gtataaacca acggcatgta
ttttcggtca tattttggtg 660gtttcggtca ttaaccgatc aactctccac tatgtgaata
ttgttttgtg aagattgatc 720ctacactttt ggccaccatt cccatgtagg tcatagttca
tgaaaacaaa ttgatcgatt 780ctctcaccaa cgttgggaca acggcatgtt catcgttttt
atgaataaaa tttcgttcga 840agattcaagt aaaggctgta aaatactacg tgtaggtcat
agaaatcgat tttggataaa 900cccaccatcg ttcgacgttg ggacaacgtt gggacaacgg
ccaaattttt taaccgtata 960tccaaatgat cgattatgaa cggcatatcc catgtattaa
aagaatcagt attcctcatt 1020gccaaggtaa gcccaatctc acgaccccta atacattatg
aacggccaaa ataattgaaa 1080acaaatttcg aaacccacgt gtaaaaattt attaaccgaa
attcttttgc tttttaattt 1140cgacgttggg acaattcaat tcaagtaaaa tttcggtcat
atttgaaatt gaaatcgatt 1200gatcctacac tattttgcat ttgcttttta tgtatttttt
aactgattct gtctaataga 1260agaatattgg attgggacaa cgttaactga ttctctccat
gcaaagaaat acctcattgc 1320ctttacctca gtataactga aattaatttt atatccaaac
13602921432DNAArtificial Sequencesynthetic 1064
promoter with spacer 292tctccatttg agaggccaag gtaagagaga atatcgtagt
ctccattcaa ttctctccac 60tatttttcct tcgaagaacc cacacatttt tgcaaattta
ttaacactat gtatttttgt 120gaaggctgta aagcaagaga atatccatgc aaattaattg
atcctacact tgtcaaagaa 180atacatttga aggctgtaaa agaatatttt ttcggtcatg
aaatactacg tgtaaaatac 240attatggatt gtttatcata gttcatgttt ttggtgtaaa
atgatcctac actcaccatc 300gttttagatt gtaaaagatc atattggcca ccatcccaca
cattaacact cacgacccct 360aatagatgat cgattttata cctcattaat tgttcatcgt
tcgaaattaa ccgatcaact 420ctccactatt ttggctgcat gagttgcatt tttatagaat
cagtctttta ttaattttgt 480ttttaaccga tcaactctcc atcgtttatg gataatcgat
tatgaatatt gtaaaagaaa 540taatagatga tatgtagccc attcctcagt cttttgttca
tcgtttatag aaatgtattg 600ggacaatttt ggccaccatc gttcgaaatt tttggtgtca
aagagtattt tggtgtttta 660taaaagaaat tgatcctaca ctcaccccaa atttaatttt
tagaagaatc agtattggga 720tctgattctg tcttgccaag taaagcaaga gagtatataa
ctgaaattaa aaatatccat 780ccccaatctc acgaccccaa aataccgaaa cccacgtgta
gcccaaaaga ctatataact 840gatattggct gtaaaaatgg tgtcttgcct ttagaatcag
tcttgccttt agattgtatt 900atgtaggtca tagttcactt gtcattgcct ttacctattt
aaccgtatat gtttttatgg 960ataatagaaa tactcggcat gagttgcatg tttttggtgg
tttcggtcat tatgaaataa 1020tccatgcaaa gagtatatga atatgaaatt tttatcatag
ttcatgaaaa tttttcgaag 1080aataaaaaga acccaccatc gtttttatag aataatccta
attaaaattg atcctacact 1140cacccctaat agaatcagta tataactgaa attttgcatt
tgagagtata taactgaaat 1200actacgtgta agattgggac aattcaattc aagtattgga
ttgaaaacaa atttatcata 1260gaataatcga ttttggataa tcctacactt ttttaaccgt
atataaaaat accgatcaac 1320tctcaccatt ttccttcgac gttgggacaa ttctctccat
gcaaagttaa tgtattaaaa 1380tactacgtgt aggtcatgag tatataaacc agggacaacg
gcatatccaa ac 14322931509DNAArtificial Sequencesynthetic 1064
promoter with spacer 293ctatataaac caaaatggtg tttgagagag aatattttat
tatgaacggc atgagttgca 60tttgcaaatg atagttcatg agtattccca ccaacgttaa
ctgatagggt gtaggtcata 120tccaaaatac cgtaatccaa atttaacact attttcggtc
atagattatt tatgtgaata 180attaaagcaa gagagtcttt tatcatatcc ccaaattaaa
ggctgtaagc ccaaattttt 240aacactcacg accccatttt tgtttttcct tcgaaataat
cctacactat gtacactcac 300gaccccatgt ttatatcccc aaatggtggt ttcgacgttg
ggacaacggc caccatcgtt 360ttggtgtcaa agttaacact tttggtgttt ataccgtaat
cctaatagat tcaattctct 420caccaacggc atgagttgca tttgcttttt gcatttggtg
taaaagacta tttgaaatac 480tacgtgtagg tcattaattt aaccgtatat cgtttataga
tgatcctaca ctcaccccaa 540tctcacgacc cctaatttat accgtaatcc aaattgtaaa
agattcaatt caattttttg 600gtgtagccca ttttatgaaa tggtgtaaaa gaataatact
acgtgtaaaa gaaatttatc 660atagattatg aaatttaacc gtaatcctaa tagaaattct
aaatgtttat accgatcata 720gttttgtgaa taaaaagact attttataga ttcaatttta
taaaagagag aataaaagac 780tatgtacact tgtcaaagtt aactgattct ctcaccccat
gcaaattctt ttaactgaaa 840tttataatag atgatagggt gtcattgcct ttacctcagt
attccttcga agaaataccg 900atcatagttc actttttcct cagtctccac tatgtatttt
gtgaataaac ccacgtgtaa 960aatacattat gaaatatgta ttcccattcc tcattaaccg
taatccatgc aaagagtctt 1020ttatatgaaa tttatagatt gtattccttc gaagaaattt
cgtttataga tgatcgatta 1080tgaatatcgt tcgaagaaat tctaattgta ttccttcgat
tatttaacac tattttataa 1140tactcggcat atcccattca attttccatt cccattcctt
cgaagaaatt tatataaaat 1200accgatcata gttttagatg atcgattatg gataatagat
gatagttcat gttcatcgtt 1260tatatgaaaa tacattatgg ataatcgatt ctgtcttttt
tggctgcatg ttcatcgtag 1320tctccactat gttttataga tgatattttt tgtgaataat
cctacacttg tcatagttca 1380ctttttgttc atgagttgca aagactatgt tttataattt
ttattattat gaacggcatg 1440aaaatttcga ttctctcacg acccctaatt aaccgtatat
aaaccaggga caacggcata 1500tccccaaac
15092941509DNAArtificial Sequencesynthetic 1064
promoter with spacer 294aatctcaccc cattccttcg aagaacccac caaaatggtg
tcatagaaga atatcccatg 60caaattaaaa tttttcctca ttaaagcaag agaggccaag
taagagtctt tacctcattg 120ccaagtaaaa ttttaacact tgtcaaagtt aaccagggat
gtgatagttc atgtacacta 180tttgcatttg aaatcgattt ttggattggc tgtaaaggct
gtaaaaagac tatatcccac 240acatttgcat gttgagagtc tttacctatt tttcgattca
atttttaggg acaacgttgg 300gatgtgaata atactacgtg taaaatacat ttgaaaacaa
attaaaggct gtaaaagata 360atcgattctt tagaagaata aacccacgtg taagcccaat
ctcaccatcc cacgtgtaag 420cccatgcaaa tgatatgtac actcacgacc cctaattttt
ggtggtttcg aaataataca 480ttatgaaaat actacgtgta agcccaatct cacgacccca
aaataatcca aaagagagta 540ttggctgtaa aatattttgc ttttttgctt ttggtgtttg
aagaaatttt tttaacactt 600gtcaaagtta accgaaaccc acgtgtaagc ccatttgaag
atcaactctc acgaccccaa 660tctcacgacc cctaattaac acttttggtg taaaaagatt
atgaatatcg ttttagaata 720atcctaatag aaattgaaat ttcgacgttg ggatgtgaat
atccccattt gaaataccga 780tcaactctca ccatttgctt ttagaaatcg attcttttta
actgataggg tgtaaaattt 840atatgtattt taaccaggga caacgttggg atgtgaataa
acccaccatt cccacgtgta 900ggtcatagat tggataaacc aaaatactac gtgtaaaaga
gtatttaact gaaatacctc 960attgccttta cctattttta acactatata atccatcgtt
tatatccaaa agagagtctt 1020ttgtgatatt tttccatcgt agtctttaga agaaataccg
taatcctaca cttttggata 1080attagtaaga tcatagtttt gtgaataaac ccaccattcc
tcagtctcca tcgtttataa 1140ctgaaatttt atcatatttt agattggcca aggtaagaga
atattttagg gtgtttatta 1200attaaaatta accaaaagag agtataaaaa tactcggcat
atcgtttttt agggatgtga 1260ataaaccaac gttaactgaa aacaaattct gtctaaatgg
tggtttcgaa gaaatactcg 1320gcatgagttg caaattcaag tatatgtatt cctcattaat
taaagcaaga gagtctttag 1380attattttgg tgtcaaagat caactctcac gaccccattt
tccttcgacg ttaattgtat 1440tgtaaaatgg tgtcattaat taattgtaaa agagtatata
aaatgatagg gtgttttcca 1500tccccaaac
15092951509DNAArtificial Sequencesynthetic 1064
promoter with spacer 295cattcaattt tttatcatag atgatagttt ttcgaaatcg
attctaatag aaatactacg 60tgtaagagta ttaatgtttt tccattcaat tcaattttag
aagataaaag aatatcccca 120aatttaaccg taatcgattc aattctgtct tgccaagtat
atgaataatc caaattagta 180aagcaagaga gtctccattt gaaggctgta aaggctgtaa
aggctgtaaa atactacgtg 240taaaggctgt aaaaagacta tatgaataat cctaatagat
tatgaaattt cgttcgacgt 300tgggatctga ttctgtcttg cctttaccta tttaattgat
cgatttttcg acgttaatgt 360acactatatg ttcacttttg tgaagaaatt tttttagatg
atcgattatg aaatactacg 420tgtaaaagaa taattaaaag aaatcgattt ttattatgaa
aacaaatttt ggtgtcatag 480aagataattt atagattgta aaattaacac tatgttcatg
agtattggcc aagtaagccc 540attcccattt ggataaaaag actatatccc acgtgtaaaa
ataatcctac acttgtcaaa 600gagaataaaa gattatgtga ataattgaag gctgtaaaat
actacgtgta aagcaagaga 660gtattggctg catgagtata actgataggg tgtttataga
ttatgaaaat atcgttttcg 720tttttggtgt catatcccac gtgtaaaatg tttgaaaaca
aatttttcgg tcatagtttt 780tgctttttgg tgttttatat aatccaaatg tttatcatag
ttcacttgtc atagtttttg 840ttcatgttca tcgtagtctc cactatataa aatacctcag
tattggctgt aaaagactat 900tttgcatttt tggtgtcata tccccattcc tcattaacca
gggatctgat tctctcacga 960ccccatttgc atttggtggt ttcgttcgaa atgtaggtca
ttatgtattc ctcagtattg 1020taaaaatttc gattgggatc tgattcaatt ctaatagatt
gtaaaaatgg tgtctaaatg 1080ttcacttgtc aaagaaccca cgtgtaggtc atatcccatg
caaagattga tcctaatact 1140acgtgtaaaa ttttccatgt acacttttgg tgttttaggg
tgtcatgagt attatggata 1200atcgattgaa attttaggga caacggcatg aataatccaa
atttcgatta tgaacggcca 1260agtattggcc accaacggcc aagtatttta gattattaaa
agaaatacct cagtataaac 1320ccacgtgtaa aattgtatta tgaaaacaaa ttaaaggctg
catttgaagg ctgtaaagca 1380agagaataaa agaataatcg attgaaaaca aatttatcat
agttcacttg tcaaagttaa 1440ttagtaagag agaggccacc atttgcaaat tgatcctaca
ctatataatc caaattagta 1500agcccaaac
15092961509DNAArtificial Sequencesynthetic 1064
promoter with spacer 296gggacaattt atgaataatc ctaatagatt atggataatc
catgttcatg tatttttagg 60gtgtttgaaa ttttgctttt ggtgtttttg gtgtcaaagt
taattttcgg tcattgccaa 120ggtaagatca actctccact atataaaaga aatgtaggtc
atgaatatgt tgagttgcat 180gttgagagta ttaaccaacg gccaccaacg ttgggacaac
gttgggatct gattctctcc 240atttggtggt ttcgattatg aaatggtgtt tatagaagat
aatccatgca aagttaatgt 300ttgagtattc ctcagtatat gtagcccatg caaatgttca
tcgtagtctt ttgtgatagg 360gacaacggcc aaggtaagcc catttgcttt tggtgttttt
agaagatcat agttcacttg 420tcaaagagag agaataatcg attatgaaat actacgtgta
aaatgatagg gtgtcaaaga 480acccaccaaa ttaaagcaag agaggccacc attcaagtaa
aatgatcgat tttagggatc 540tgatagtttt gtttttggct gcatgaaaac aaattagtaa
gagagtctcc actatttaac 600cgaaaccagg gatctgattc aagtataaaa gactatgtag
gtcatagaag aatcagtctt 660ttttagatta ttttaaccga aacccacgtg taggtcattg
ccaaaatact cggcatatcg 720tttattttat aatcctacac tattttgtga ataattaaag
gctgtaagat tatgaaaata 780ctacgtgtaa gcccattccc acacattttc gaagaaccca
cgtgtaggtc atgaaattct 840ctcaccatcc caccattcaa gtattgggac aattctaatt
aactgaaatt aaaatatgta 900cactcacccc taatacatta tggattgtaa gcccatgttt
gaaggctgca tgagttgcaa 960attctgtcta atagattcta atagaaataa tcgattggga
tgtgaataat tttccatgta 1020ggtcatattt atcatagaaa ttgtattggc caaggtaaga
ttgtaaaata atcgattatt 1080aaaggctgta aaatacctat ttataataga ttctttagat
tgggatgtga aggctgtaaa 1140ggctgtaaaa tacctattta tatccatcgt tcgacgttaa
ccgtataaaa gatcaactct 1200caccattttc cttcgaagaa taatttttta tcatagttca
cttgtcaaag ttaatgtagg 1260tcatagattc aagtaaaaag actatgtagg tcatgagtat
tcccatgcaa attgatccta 1320atttaatgtt catgtattgt aagatcatag aagaaatttt
ggccaccatt caatttcggt 1380catgtattcc cacgtgtagg tcatgtttat aactgattct
gtctaaatgt tgagttgcaa 1440atgtaggtca ttgccaagta aaataccgta tatgttttcg
tttatataaa atactcggca 1500tatccaaac
15092971509DNAArtificial Sequencesynthetic 1064
promoter with spacer 297actattttaa cactattttg gtgtcatagt ttttggataa
tcgattatta attagtaagc 60ccaatctcac gacccctaat agaagattct gtctaatacc
gtataactga tatgtattcc 120catgtattcc ttcgaaaccc acgtgtaaaa gaacccacca
ttttagggtg taaaagacta 180tataaaattt tcggtcatgt attaaccagg gatctgatat
gtattggata aacccaccat 240ttgagagtat tttggctgta aaagagaata aaattagtaa
gagagaatat cgtttattaa 300aatgatagtt ttccttcgac gttaattaac caaaagacta
tgtacacttg tcattgcctt 360tacctcatta acactatgta ttttggataa tcctaataga
ataaaatggt gtaaaattaa 420ccagggatct gattctttta taccgtatat ccatcgttcg
aaatatcgta gtcttgccaa 480gtatataaac cagggacaac gttaattaaa gcaagagtat
tatgaacggc atgttgagta 540ttccttcgat tctaaatgta ggtcatagaa taatcctaca
ctattttata cctattttta 600taccgtaatc gattctgtct aatactacgt gtagcccaat
ctcaccattt gaaattttag 660atgatcctaa ttgaaattta tcatagttca tcgttcgaaa
ccaaaagaac ccacgtgtaa 720gagagtcttt ttccatcgtt tttaaccgta taaaataccg
aaatattttt tcggtcattg 780ccaaggtaag cccaatctca ccccatttgg ataatcctaa
tagatgatcg attttccatg 840tagcccaaaa gactatttga gaggccacca aaagaaatcg
attatgaaat taaaaagaaa 900tactacgtgt aaaggctgta aaagactatg tgaaggctgt
aaaattttgt gaaggctgta 960aaagaatcag tctccactat ttggtggttt cgaagattga
aatatcgttt atagattgaa 1020atgtattaac cgtatataaa ccagggatgt gatagttttt
taaccaggga tgtgaataat 1080ccaaatgatc ctaattgaaa atacctcagt ctccattcaa
ttcttttgct tttggtggtt 1140tcgaagaaat taaccgtata aaaagaaatt agtaaaggct
gcattttgtg aataaaccca 1200cgtgtagccc aatctcaccc catgcaaaga tcaactctcc
atccccattt gaaatcgatt 1260tttatgtatt gtattcctca ttatgaacgg catatttgag
ttgcatgaac ggcatgtttg 1320aaggctgtaa aaattaatgt tcatcgtagt cttgccttta
cctatttgct ttttagggac 1380aacggccaag gtaagattct tttaactgat agttcacttg
tcaaagttaa tgtttgaaaa 1440caaattaacc gaaattaaag gctgtaaaag aatcagtctc
cactatataa ttagtaagag 1500aggccaaac
15092981509DNAArtificial Sequencesynthetic 1064
promoter with spacer 298aaggctgcat ttgaaggctg taagatcata gttcatcgtt
tatacctcat tgcctttacc 60tattttccat cgttcgaaat tttcgtttat ggataataga
ttgtaaaaag aaatcgatta 120tgaataaaat aaaagattat taattaattg aaggctgcat
gtttttaggg tgtctaatac 180tcggcatatc gtagtctttt aacactattt tggtgtcttt
tataactgaa aacaaatttt 240atagattctc tcacgacccc taatagaata tcgttcgatt
caattcaagt aagcccatgc 300aaagttaatt tatgaataaa agactatttt ccatcgtttt
tatgaacggc caccatcgta 360gtctccacta tgtatttgag tattccttcg attctttacc
tcagtattcc cacacatttg 420aaatggtgtc atgagtattg taaagcaaga gaggccacca
tcgttttggt gtaagagtat 480tcctcagtat ataaacccac acattttatt aaccgaaacc
agggacaacg gcatgagtat 540taacactttt gtgatagggt gtaggtcatg ttgagagagt
ataatccaaa ttctgtctaa 600atggtgtttt tggtggtttc gaaaccaggg atctgattct
aaatgatatg tgaagaaatt 660aattttcgtt tataccgtat aactgattct aaatgttcac
ttttggataa ttaatttata 720tgtattgtat ttaattttcg aaattttcca tccccatgca
aagttaatgt ttgaaatacc 780gtatataaac ccacgtgtaa aagaatatga aaacaaattt
tcgaagaaat tttttaacca 840aattaaccgt aatccaaaag aaatttttat ggataataga
tgatcctaca ctttttcgat 900tggccaccaa attttagaag aaatcgatta tgaacggcca
aggtaagagt cttttggtgt 960aaaggctgta aaatactacg tgtaaaatac tacgtgtaag
cccatttgaa aacaaatgtt 1020catcgtagtc ttttgttcac ttgtcaaagt taacacttgt
caaagttaat gtttgagagt 1080attgtttgag agaatcagta ttaaaaatac tacgtgtagc
ccatgttttt cgattatgaa 1140tatgtacact ttttggctgt aagagtcttg ccaaggtaag
agtattggct gtaaaatgtt 1200ttggtgtttt cgaaatgttt gaaatcgatt atgaaataat
cctacactat ttaattgtaa 1260aatactacgt gtaaaggctg taaaatatgt tttttatttt
ataaaatttt cgaagaaccc 1320accattcaat tcaattctct ccactattta attgtaaagg
ctgtaaaaga ctatataaaa 1380gactatttat atgtattgga ttgtaaaaga acccacgtgt
aagcccatgc aaattttcgt 1440tttggattgg gacaatttcg aagaaatttt tcgtttataa
tagaagatat tggattggat 1500aatccaaac
15092991509DNAArtificial Sequencesynthetic 1064
promoter with spacer 299aactgattct gtcttttggc caccattcaa gtaagagtct
tttgttttgg tgtttatacc 60gatcaactct ccactatgtg aatatcgtag tctccatgca
aatttttggt gtaaaaagaa 120tcagtatatc gtagtcttta cctcagtctt gcctttacct
attttatacc gaaatttcga 180aacccacaca ttatgaacgg catgaaatta attaaagcaa
gagaggccaa attgaaggct 240gcatgttgag agtattggat aatagattca attctgtcta
atacattatg ttttggataa 300tcgattatga ataatccaaa ataccgaaac ccaccaaaag
aacccaccat cgtagtctcc 360attcctcatt aaaaatacct cagtatataa aatgtttatc
atagttcatc gttttggcca 420agtattaaaa taatcgatta tgaaaataat agatgatagg
gtgtctaata ctcggcatga 480aattttcctc agtataaaat taaaaagatt ctgtctttag
attcttttta tatgtattgg 540ccaccatttg caaagttaat gtacactttt gtgaataatc
catcgtagtc tttttccatt 600tttatatgtg aataaaccaa aagatatgaa cggcatgttt
attatgaata tgaaatattg 660gctgcatttg aaaataatcg attggataat cgattttcct
tcgacgttgg gacaacggcc 720aagtattgga ttggccaagg taagcccaat ctcacgaccc
ctaattgtat tcccatttga 780aaatgatcct acactcaccc cattcaattc aatttttata
ccgtataact gattcttttg 840gtgtaagagt ctccactatt taaccgtaat cctacactat
tttagaagaa atatgtacac 900ttttggataa tagaaatact acgtgtaaaa tactacgtgt
agcccaaaag actatataaa 960atactcggca tgaatatgaa taattaaagg ctgtaaaata
ctacgtgtaa aatactacgt 1020gtaaaagaaa tactacgtgt aaaattctaa tagaaattaa
aatgttgagt tgcaaagtta 1080accagggaca atttatatcg ttttttgtga tagggtgttt
ataaacccac gtgtagccca 1140aaattgtaaa aagataatcc aaaatactac gtgtaaaatt
gatcgatttt tggtgtcaaa 1200gttaattaac cgtataactg aaatcgatta ttaaccaaaa
tatttggata aacccaccat 1260ttggtgtctt tacctcatta ttttccatgt tcatcgtttt
agaagattat gaatatgtag 1320cccatgcaaa ttttcgttcg attattaaaa ttgatcgatt
gaaaacaaat ttcgaagaac 1380ccacacatta atgtattatg tagcccaaat ggtgtcatag
aataatagaa tatccaaaat 1440ttttataact gaaatcgatt gttcatgaac ggcatgagta
tataaaccca ccaaattgta 1500agcccaaac
15093001509DNAArtificial Sequencesynthetic 1064
promoter with spacer 300cttcgaagat caactctcac gacccctaat taaaggctgt
aaagcaagag aggccaccat 60cccaccattt gaaatacatt atgaaaacaa attttcctca
ttgccaaatt ctgtcttgcc 120tttacctatt ttggtggttt cggtcatgaa atcgattatg
aaatcgattg aaaattgtat 180tggattgatc ctacactcac gacccctaat tagtaagaga
gaggccaagg taagatcata 240gttcatcgta gtctccacta tttgagaggc caagtatttt
ggctgtaagc ccattcctca 300ttatgaaaac aaattttcga ttgatcgatt ttttgctttt
ggtgtttatg gataatttcg 360ttcgaaacca gggatgtgaa tatcgtttta taaaaatact
acgtgtagcc cattcaattc 420tttttatacc tcagtatata aaatacatta attaattttt
atggataatc catccccaat 480ctcaccccta atagaagaac ccacacatta tgtgaagaat
atcgtagtct tgccaaggta 540agcccaaaat ttaaccgtaa tcgatttttt aaccaaaata
ccgtatataa ttaaaataca 600ttaaccgtaa tcctacacta tttttaatgt tttccatgta
ggtcatgttg agaggccaaa 660ttagtaaaat aatccattcc tcattgcctt tagaagaaat
aaaatacctc attatttgag 720aggccaaggt aagcccatgt attggctgta aaaatgatcg
attgaaaata tttataccga 780aacccacgtg taagattcaa ttcaagtaag atcaactctc
acgaccccta atttttggtg 840tctaatagat tggctgtaaa atacctcagt ctccactatt
tttggtgttt tcgaaattta 900tacctcatta accagggaca acgttaaccg taatccaaat
gatatttaat tttaaccagg 960gatgtgatag ttcatgagtt gcaaatggtg tcatatccca
tttggtggtt tcgaaatgtt 1020tgaaataaaa agataatcca aaataaaaga gagtattttg
gtggtttcga agaaatgata 1080gggatgtgaa gaacccacgt gtaagcccaa tctcacgacc
cctaattagt aagcccaatc 1140tcaccccatt cccacgtgta aaaatggtgt tttggctgta
aaatggtgta aaagaataat 1200cgattggctg taagcccaat ctcaccccta atagaatatt
gttcatgaat aatccaaaat 1260ttatagaata attaaccagg gatgtgaagg ctgcatgagt
tgcatttgct ttttatataa 1320ctgaaatacc tcagtataaa ccaaaataat cctacactca
cgaccccatg caaagagaga 1380gtctccattc aatttcgacg ttaaccaggg atgtgatatt
ggccaaattt aatttatgta 1440ttgtattgtt ttaactgaaa tgtacactat atcgttttta
taactgaaat aaaataccgt 1500aatccaaac
15093011063DNAArtificial SequenceProPer full
promoter with spacer and enhancer 301atcgcagata tttggtgtct aaatgtttat
tgtcctgtat gttcatttgg gacaaattag 60ctgaacagcc agggacaacg ttgggatctg
atagggtgtc aaagagtatt ataaactggg 120acaatttcgg tcatgagttg caaattcaag
tatatagctc tgccgggggg attttcgaag 180aatatcccat ttgacgagtc acctggctca
ttaatgtttt tagattatga aattttatca 240tagtaagggg acagttattt ggtgtaaagg
ctgtaaaaag aaattgttca cttttgtttt 300cgtttatgtg aaggccctaa aagattgtgt
aagactattt tggtgttttg gataaaatga 360tagtttttat agattctttt gcttttagaa
gaaatacatt tgaaattttt tccatgttga 420gtataaacgc atttatcgta ttgaagatca
tagaaatatt ttaactgaaa aataatttat 480aactgattca attctctcca tttttatacc
tatttaaccg taatcgtggc gagatcccgt 540caggaccagc caagcatcct aattaaccaa
cggcatgtat tggataatta accgatcaac 600tctcacccct aatagaatca gagcgacact
tcgacgttaa ttgatcctac actacggggg 660tcatatccat cgttttaatt tttcgccacc
attcaattct gtcttgcctt tagggatgtg 720aatatgaacg gccaaggtcc gggtgcaaaa
ataatccaaa ttaaagcaag aaaccgggag 780taagataatc caaatgtaca cttgtcattg
ccaaaattag taaaatactc ggcatattgt 840actaggccac attattaaaa taccgtatat
gtattggctg catttgcatg aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc
ccatgcaaag ttaacactca ataccccatt 960cctcagtctc cactatataa acccaccatc
ggcactctca ccaaacccac cacacaactc 1020acaactcact ctcacacctt aaagaaccaa
tcaccaccaa aaa 10633021063DNAArtificial
SequenceProPer full promoter with spacer and enhancer 302acagcagata
tttggtgtct aaatgtttat tgttggctat gttcatgtga ttctggttac 60gcaatgcttc
agggacaacg ttgggatctg atagggtgtc aaagagtatt gtgctctggg 120acaatttcgg
tcatgagttg caaattcaag tatatacatt agtcgcacca gatttcgaag 180aatatcccat
ttgacgaccc acaatgctca ttaatgtttt tagattatga aattttatca 240tagttagaac
acaatggttt ggtgtaaagg ctgtaaaaag aaattgttca cttttgtttt 300cgtttatgtg
aaggctataa aagattgtat aagactattt tggtgttttg gataaaatga 360tagtttttat
agattctttt gcttttagaa gaaatacatt tgaaattttt tccatgttga 420gtataaagac
cattgtataa ttgaagatca tagaaatatt ttaactgaaa actcatttat 480aactgattca
attctctcca tttttatacc tatttaaccg taatcgtacc ctcggtacta 540cgctaggaag
cagccgtcct aattaaccaa cggcatgtat tggataatta accgatcaac 600tctcacccct
aatagaatca tctcgaaagt tcgacgttaa ttgatcctac actagtttgg 660tcatatccat
cgttttaatt ttttgccacc attcaattct gtcttgcctt tagggatgtg 720aatatgaacg
gccaagacgg agagtaaaaa ataatccaaa ttaaagcaag aaactggcag 780taagataatc
caaatgtaca cttgtcattg ccaaaattag taaaatactc ggcatattgt 840acaaggccac
attattaaaa taccgtatat gtattggctg catttgcatg aataatacta 900cgtgtaagcc
caaaagaacc cacgtgtagc ccatgcaaag ttaacactca tgaccccatt 960cctcagtctc
cactatataa acccaccatc acgactctca ccaaacccac cacacaactc 1020acaactcact
ctcacacctt aaagaaccaa tcaccaccaa aaa
10633031063DNAArtificial SequenceProPer full promoter with spacer and
enhancer 303tgtgcagata tttggtgtct aaatgtttat ttcaaaatat gttcatgacg
ccgctgaacg 60aagaaaggac agggacaacg ttgggatctg atagggtgtc aaagagtatt
agtcgatggg 120acaatttcgg tcatgagttg caaattcaag tatatggggt atctcagcaa
tctttcgaag 180aatatcccat ttgaagcatt ttactcctca ttaatgtttt tagattatga
aattttatca 240tagtataggg gtgttttttt ggtgtaaagg ctgtaaaaag aaattgttca
cttttgtttt 300cgtttatgtg aaggccctaa aagattgtac aagactattt tggtgttttg
gataaaatga 360tagtttttat agattctttt gcttttagaa gaaatacatt tgaaattttt
tccatgttga 420gtataaaagg gttcttctcc ttgaagatca tagaaatatt ttaactgaaa
acccatttat 480aactgattca attctctcca tttttatacc tatttaaccg taatcgctaa
gttcgtcaag 540gaaataactt ttaacctcct aattaaccaa cggcatgtat tggataatta
accgatcaac 600tctcacccct aatagaatca gtcgatgtct tcgacgttaa ttgatcctac
actacgtcgg 660tcatatccat cgttttaatt ttttgccacc attcaattct gtcttgcctt
tagggatgtg 720aatatgaacg gccaagctag ttgtcgaaaa ataatccaaa ttaaagcaag
atagtctcag 780taagataatc caaatgtaca cttgtcattg ccaaaattag taaaatactc
ggcatattgt 840aattttgcac attattaaaa taccgtatat gtattggctg catttgcatg
aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc ccatgcaaag ttaacactca
ctaccccatt 960cctcagtctc cactatataa acccaccatc agctatctca ccaaacccac
cacacaactc 1020acaactcact ctcacacctt aaagaaccaa tcaccaccaa aaa
10633041063DNAArtificial SequenceProPer full promoter with
spacer and enhancer 304cgtgcagata tttggtgtct aaatgtttat tctggcatat
gttcatttgt catgatttat 60ccgccccttc agggacaacg ttgggatctg atagggtgtc
aaagagtatt ttctgttggg 120acaatttcgg tcatgagttg caaattcaag tatatagttc
gccgtgctgc gttttcgaag 180aatatcccat ttgagactgg tcgttcctca ttaatgtttt
tagattatga aattttatca 240tagtcgcacc cattgtcttt ggtgtaaagg ctgtaaaaag
aaattgttca cttttgtttt 300cgtttatgtg aaggctgtaa aagattgttc aagactattt
tggtgttttg gataaaatga 360tagtttttat agattctttt gcttttagaa gaaatacatt
tgaaattttt tccatgttga 420gtataaactt gatcgttcgg ttgaagatca tagaaatatt
ttaactgaaa agaaatttat 480aactgattca attctctcca tttttatacc tatttaaccg
taatcgaagc cgaaatgagc 540caacgcctac gctatttcct aattaaccaa cggcatgtat
tggataatta accgatcaac 600tctcacccct aatagaatca atttcccgct tcgacgttaa
ttgatcctac actaggtggg 660tcatatccat cgttttaatt tttagccacc attcaattct
gtcttgcctt tagggatgtg 720aatatgaacg gccaagcaag ctttgaaaaa ataatccaaa
ttaaagcaag aaacaccaag 780taagataatc caaatgtaca cttgtcattg ccaaaattag
taaaatactc ggcatattgt 840agaacgccac attattaaaa taccgtatat gtattggctg
catttgcatg aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc ccatgcaaag
ttaacactca taaccccatt 960cctcagtctc cactatataa acccaccatc tcggatctca
ccaaacccac cacacaactc 1020acaactcact ctcacacctt aaagaaccaa tcaccaccaa
aaa 10633051063DNAArtificial SequenceProPer full
promoter with spacer and enhancer 305agagcagata tttggtgtct aaatgtttat
tggttcgtat gttcatagtc cgagtatgta 60cgagaaagcc agggacaacg ttgggatctg
atagggtgtc aaagagtatt gtacgttggg 120acaatttcgg tcatgagttg caaattcaag
tatatgctca aggaagctca agtttcgaag 180aatatcccat ttgagacgac cgacgcctca
ttaatgtttt tagattatga aattttatca 240tagttcaccc cgaagatttt ggtgtaaagg
ctgtaaaaag aaattgttca cttttgtttt 300cgtttatgtg aaggctataa aagattgtat
aagactattt tggtgttttg gataaaatga 360tagtttttat agattctttt gcttttagaa
gaaatacatt tgaaattttt tccatgttga 420gtataaaagg cacgggagtg ttgaagatca
tagaaatatt ttaactgaaa aataatttat 480aactgattca attctctcca tttttatacc
tatttaaccg taatcgcagt gatggacgcg 540ggcatcggta ggactatcct aattaaccaa
cggcatgtat tggataatta accgatcaac 600tctcacccct aatagaatca gcaaataagt
tcgacgttaa ttgatcctac actatcaggg 660tcatatccat cgttttaatt tttggccacc
attcaattct gtcttgcctt tagggatgtg 720aatatgaacg gccaaggact ccattaaaaa
ataatccaaa ttaaagcaag aactcttcag 780taagataatc caaatgtaca cttgtcattg
ccaaaattag taaaatactc ggcatattgt 840acagccccac attattaaaa taccgtatat
gtattggctg catttgcatg aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc
ccatgcaaag ttaacactca gcaccccatt 960cctcagtctc cactatataa acccaccatc
tcttttctca ccaaacccac cacacaactc 1020acaactcact ctcacacctt aaagaaccaa
tcaccaccaa aaa 10633061063DNAArtificial
SequenceProPer full promoter with spacer and enhancer 306cgagcagata
tttggtgtct aaatgtttat tgtagtatat gttcattttt aacttatgtt 60aggtgtcaac
agggacaacg ttgggatctg atagggtgtc aaagagtatt aataagtggg 120acaatttcgg
tcatgagttg caaattcaag tatattccta ctcgtagtgg gttttcgaag 180aatatcccat
ttgaacgtaa tcataactca ttaatgtttt tagattatga aattttatca 240tagtagacat
gctttgattt ggtgtaaagg ctgtaaaaag aaattgttca cttttgtttt 300cgtttatgtg
aaggcggtaa aagattgttc aagactattt tggtgttttg gataaaatga 360tagtttttat
agattctttt gcttttagaa gaaatacatt tgaaattttt tccatgttga 420gtataaaagg
cacgaatcaa ttgaagatca tagaaatatt ttaactgaaa agcgatttat 480aactgattca
attctctcca tttttatacc tatttaaccg taatcgggtg caccaactga 540attgctttaa
tgatactcct aattaaccaa cggcatgtat tggataatta accgatcaac 600tctcacccct
aatagaatca gtagcggttt tcgacgttaa ttgatcctac actattaggg 660tcatatccat
cgttttaatt tttggccacc attcaattct gtcttgcctt tagggatgtg 720aatatgaacg
gccaagcgca cgacgcaaaa ataatccaaa ttaaagcaag acctgcaaag 780taagataatc
caaatgtaca cttgtcattg ccaaaattag taaaatactc ggcatattgt 840aatcgctcac
attattaaaa taccgtatat gtattggctg catttgcatg aataatacta 900cgtgtaagcc
caaaagaacc cacgtgtagc ccatgcaaag ttaacactca caaccccatt 960cctcagtctc
cactatataa acccaccatc gaatttctca ccaaacccac cacacaactc 1020acaactcact
ctcacacctt aaagaaccaa tcaccaccaa aaa
10633071063DNAArtificial SequenceProPer full promoter with spacer and
enhancer 307gatgcagata tttggtgtct aaatgtttat tcgaggatat gttcatgggg
accgtcaagg 60tagatgacac agggacaacg ttgggatctg atagggtgtc aaagagtatt
ccctgatggg 120acaatttcgg tcatgagttg caaattcaag tatatcggaa caggtcatgg
gctttcgaag 180aatatcccat ttgatcgttt aaaggactca ttaatgtttt tagattatga
aattttatca 240tagtagcgcg tgtccggttt ggtgtaaagg ctgtaaaaag aaattgttca
cttttgtttt 300cgtttatgtg aaggccgtaa aagattgtaa aagactattt tggtgttttg
gataaaatga 360tagtttttat agattctttt gcttttagaa gaaatacatt tgaaattttt
tccatgttga 420gtataaacgt gaggttgtcc ttgaagatca tagaaatatt ttaactgaaa
atctatttat 480aactgattca attctctcca tttttatacc tatttaaccg taatcgcaca
tttaagggat 540ggcgacagca cccgcttcct aattaaccaa cggcatgtat tggataatta
accgatcaac 600tctcacccct aatagaatca gttttggtgt tcgacgttaa ttgatcctac
actatccggg 660tcatatccat cgttttaatt tttggccacc attcaattct gtcttgcctt
tagggatgtg 720aatatgaacg gccaagcaat gccttgaaaa ataatccaaa ttaaagcaag
atctctaaag 780taagataatc caaatgtaca cttgtcattg ccaaaattag taaaatactc
ggcatattgt 840agttctccac attattaaaa taccgtatat gtattggctg catttgcatg
aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc ccatgcaaag ttaacactca
agaccccatt 960cctcagtctc cactatataa acccaccatc atagctctca ccaaacccac
cacacaactc 1020acaactcact ctcacacctt aaagaaccaa tcaccaccaa aaa
10633081063DNAArtificial SequenceProPer full promoter with
spacer and enhancer 308catgcagata tttggtgtct aaatgtttat ttacccttat
gttcattcca cgctagggga 60gtgaatcacc agggacaacg ttgggatctg atagggtgtc
aaagagtatt ctcccgtggg 120acaatttcgg tcatgagttg caaattcaag tatatgacag
ggctgggaca cttttcgaag 180aatatcccat ttgattaatc aagagactca ttaatgtttt
tagattatga aattttatca 240tagtgattct ctgttacttt ggtgtaaagg ctgtaaaaag
aaattgttca cttttgtttt 300cgtttatgtg aaggccctaa aagattgtgc aagactattt
tggtgttttg gataaaatga 360tagtttttat agattctttt gcttttagaa gaaatacatt
tgaaattttt tccatgttga 420gtataaaggc gggctgggtc ttgaagatca tagaaatatt
ttaactgaaa agccatttat 480aactgattca attctctcca tttttatacc tatttaaccg
taatcgcagc ggaggaatgt 540cactgacaaa tgaacgtcct aattaaccaa cggcatgtat
tggataatta accgatcaac 600tctcacccct aatagaatca gttccgtcat tcgacgttaa
ttgatcctac actacgacgg 660tcatatccat cgttttaatt tttagccacc attcaattct
gtcttgcctt tagggatgtg 720aatatgaacg gccaagttac gtcaacaaaa ataatccaaa
ttaaagcaag aaaaccctag 780taagataatc caaatgtaca cttgtcattg ccaaaattag
taaaatactc ggcatattgt 840atcgagacac attattaaaa taccgtatat gtattggctg
catttgcatg aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc ccatgcaaag
ttaacactca ataccccatt 960cctcagtctc cactatataa acccaccatc agccttctca
ccaaacccac cacacaactc 1020acaactcact ctcacacctt aaagaaccaa tcaccaccaa
aaa 10633091063DNAArtificial SequenceProPer full
promoter with spacer and enhancer 309gtggcagata tttggtgtct aaatgtttat
tcctagatat gttcattgcg gaattgcttc 60tcaccgtgac agggacaacg ttgggatctg
atagggtgtc aaagagtatt tcgacgtggg 120acaatttcgg tcatgagttg caaattcaag
tatattcggt ccaccttgtt agtttcgaag 180aatatcccat ttgacatggg cgcgcgctca
ttaatgtttt tagattatga aattttatca 240tagttagcgc ctgcctattt ggtgtaaagg
ctgtaaaaag aaattgttca cttttgtttt 300cgtttatgtg aaggccgtaa aagattgtta
aagactattt tggtgttttg gataaaatga 360tagtttttat agattctttt gcttttagaa
gaaatacatt tgaaattttt tccatgttga 420gtataaagcc gaactgacac ttgaagatca
tagaaatatt ttaactgaaa atacatttat 480aactgattca attctctcca tttttatacc
tatttaaccg taatcgggat tagacgatat 540ttccgcgtca tgccggtcct aattaaccaa
cggcatgtat tggataatta accgatcaac 600tctcacccct aatagaatca gaatatttat
tcgacgttaa ttgatcctac actaacatgg 660tcatatccat cgttttaatt tttggccacc
attcaattct gtcttgcctt tagggatgtg 720aatatgaacg gccaagatcc catttcaaaa
ataatccaaa ttaaagcaag atttcagtag 780taagataatc caaatgtaca cttgtcattg
ccaaaattag taaaatactc ggcatattgt 840atgtgttcac attattaaaa taccgtatat
gtattggctg catttgcatg aataatacta 900cgtgtaagcc caaaagaacc cacgtgtagc
ccatgcaaag ttaacactca cgaccccatt 960cctcagtctc cactatataa acccaccatc
tgtagtctca ccaaacccac cacacaactc 1020acaactcact ctcacacctt aaagaaccaa
tcaccaccaa aaa 10633101063DNAArtificial
SequenceProPer full promoter with spacer and enhancer 310atggcagata
tttggtgtct aaatgtttat tgggtggtat gttcatcttc aatttagtgg 60tgtcaccggc
agggacaacg ttgggatctg atagggtgtc aaagagtatt aaattttggg 120acaatttcgg
tcatgagttg caaattcaag tatatcgacc cgcccccaat tatttcgaag 180aatatcccat
ttgagtggat agacgtctca ttaatgtttt tagattatga aattttatca 240tagtaccggt
aaccctgttt ggtgtaaagg ctgtaaaaag aaattgttca cttttgtttt 300cgtttatgtg
aaggcgttaa aagattgttg aagactattt tggtgttttg gataaaatga 360tagtttttat
agattctttt gcttttagaa gaaatacatt tgaaattttt tccatgttga 420gtataaagac
cgtggtatag ttgaagatca tagaaatatt ttaactgaaa agcaatttat 480aactgattca
attctctcca tttttatacc tatttaaccg taatcgaata atcccaaaaa 540gtatcgttag
tttggctcct aattaaccaa cggcatgtat tggataatta accgatcaac 600tctcacccct
aatagaatca ttcgtcgctt tcgacgttaa ttgatcctac actatcttgg 660tcatatccat
cgttttaatt tttcgccacc attcaattct gtcttgcctt tagggatgtg 720aatatgaacg
gccaagtgat ccgcataaaa ataatccaaa ttaaagcaag atatttcaag 780taagataatc
caaatgtaca cttgtcattg ccaaaattag taaaatactc ggcatattgt 840agcgactcac
attattaaaa taccgtatat gtattggctg catttgcatg aataatacta 900cgtgtaagcc
caaaagaacc cacgtgtagc ccatgcaaag ttaacactca tgaccccatt 960cctcagtctc
cactatataa acccaccatc ggtgctctca ccaaacccac cacacaactc 1020acaactcact
ctcacacctt aaagaaccaa tcaccaccaa aaa
1063311398DNAArtificial SequenceWO0116340-GUS data 311tccactatgt
aggtcatatc catcatttta atttttgggc accattcaat tccatcttgc 60ctttagggat
gtgaatatga acggccaagg taagagaata aaaataatcc aaattaaagc 120aagagaggcc
aagtaagata atccaaatgt acacttgtca tcgccgaaat tagtaaaata 180cgcggcatat
tgtattccca cacattatta aaataccgta tatgtattgg ctgcatttgc 240atgaataata
ctacgtgtaa gcccaaaaga acccacgtgt agcccatgca aagttaacac 300tcacgacccc
attcctcagt ctccactata taaacccacc atccccaatc ttaccaaacc 360caccacacga
ctcacaactc gactctcaca ccttaaag
398312417DNAArtificial SequenceWO0116340-SEQ-006UTR 312tccactatgt
aggtcatatc catcatttta atttttgggc accattcaat tccatcttgc 60ctttagggat
gtgaatatga acggccaagg taagagaata aaaataatcc aaattaaagc 120aagagaggcc
aagtaagata atccaaatgt acacttgtca tcgccgaaat tagtaaaata 180cgcggcatat
tgtattccca cacattatta aaataccgta tatgtattgg ctgcatttgc 240atgaataata
ctacgtgtaa gcccaaaaga acccacgtgt agcccatgca aagttaacac 300tcacgacccc
attcctcagt ctccactata taaacccacc atccccaatc ttaccaaacc 360caccacacga
ctcacaactc gactctcaca ccttaaagaa ccaatcacca ccaaaaa
4173131676DNAArtificial SequenceWO0116340-SEQ-006 313tccactatgt
aggtcatatc catcatttta atttttgggc accattcaat tccatcttgc 60ctttagggat
gtgaatatga acggccaagg taagagaata aaaataatcc aaattaaagc 120aagagaggcc
aagtaagata atccaaatgt acacttgtca tcgccgaaat tagtaaaata 180cgcggcatat
tgtattccca cacattatta aaataccgta tatgtattgg ctgcatttgc 240atgaataata
ctacgtgtaa gcccaaaaga acccacgtgt agcccatgca aagttaacac 300tcacgacccc
attcctcagt ctccactata taaacccacc atccccaatc ttaccaaacc 360caccacacga
ctcacaactc gactctcaca ccttaaagaa ccaatcacca ccaaaaaatg 420gcaaagctga
tgagcctagc agccgtagca acgcagttcc tcttcctgat cgtggtggac 480gcatccgtcc
gaaccacagt gattatcgac gaggagacca accaaggccg cggtggaggc 540aaggtggcag
ggacagcagc agtctgcgag cagcagatcc agcagcgaga cttcctgagg 600agctgccagc
agttcatgtg ggagaaagtc cagaggggcg gccacagcca ctattacaac 660cagggccgtg
gaggaggcga acagagccag tacttcgaac agctgtttgt gacgacctta 720agcaattgcg
caccgcggtg caccatgcca ggggacttga agcgtgccat cggccaaatg 780aggcaggaaa
tccagcagca gggacagcag cagggacagc agcaggaagt tcagaggtgg 840atccagcaag
ctaaacaaat cgctaaggac ctccccggac agtgccgcac ccagcctagc 900caatgccagt
tccagggcca gcagcaatct gcatggtttt gaaggggtga tcgattatga 960gatcgtacaa
agacactgct aggtgttaag gatggataat aataataata atgagatgaa 1020tgtgttttaa
gttagtgtaa cagctgtaat aaagagagag agagagagag agagagagag 1080agagagagag
agagagagag agaggctgat gaaatgttat gtatgtttct tggtttttaa 1140aataaatgaa
agcacatgct cgtgtggttc tatcgaatta ttcggcggtt cctgtgggaa 1200aaagtccaga
agggcggccg cagctactac tacaaccaag gccgtggagg agggcaacag 1260agccagcact
tcgatagctg ctgcgatgat cttaagcaat tgaggagcga gtgcacatgc 1320aggggactgg
agcgtgcaat cggccagatg aggcaggaca tccagcagca gggacagcag 1380caggaagttg
agaggtggtc ccatcaatct aaacaagtcg ctagggacct tccgggacag 1440tgcggcaccc
agcctagccg atgccagctc caggggcagc agcagtctgc atggttttga 1500agtggtgatc
gatgagatcg tataaagaca ctgctaggtg ttaaggatgg gataataaga 1560tgtgttttaa
gtcattaacc gtaataaaaa gagagagagg ctgatggaat gttatgtatg 1620tatgtttctt
ggtttttaaa attaaatgga aagcacatgc tcgtgtgggt tctatc
16763141121DNAArtificial SequenceWO2002102970-pConlinin-1 314caacggttcc
ggcggtatag agttgggtaa ttcgaaaccg cacagatcca attcgattag 60cagatatttg
gtgtctaaat gtttattttg tgatatgttc atgtttgaaa tggtggtttc 120gaaaccaggg
acaacgttgg gatctgatag ggtgtcaaag agtattatgg attgggacaa 180tttcggtcat
gagttgcaaa ttcaagtata tcgttcgatt atgaaaattt tcgaagaata 240tcccatttga
gagagtcttt acctcattaa tgtttttaga ttatgaaatt ttatcatagt 300tcatcgtagt
ctttttggtg taaaggctgt aaaaagaaat tgttcacttt tgttttcgtt 360tatgtgaagg
ctgtaaaaga ttgtaaaaga ctattttggt gttttggata aaatgatagt 420ttttatagat
tcttttgctt ttagaagaaa tacatttgaa attttttcca tgttgagtat 480aaaataccga
aatcgattga agatcataga aatattttaa ctgaaaacaa atttataact 540gattcaattc
tctccatttt tatacctatt taaccgtaat cgattctaat agatgatcga 600ttttttatat
aatcctaatt aaccaacggc atgtatggat aattaaccga tcaactctca 660cccctaatag
aatcagtatt ttccttcgac gttaattgat cctacactat gtaggtcata 720tccatcgttt
taatttttgg ccaccattca attctgtctt gcctttaggg atgtgaatat 780gaacggccaa
ggtaagagaa taaaaataat ccaaattaaa gcaagagagg ccaagtaaga 840taatccaaat
gtacacttgt cattgccaaa attagtaaaa tactcggcat attgtattcc 900cacacattat
taaaataccg tatatgtatt ggctgcattt gcatgaataa tactacgtgt 960aagcccaaaa
gaacccacgt gtagcccatg caaagttaac actcacgacc ccattcctca 1020gtctccacta
tataaaccca ccatccccaa tctcaccaaa cccaccacac aactcacaac 1080tcactctcac
accttaaaga accaatcacc accaaaaaat g
11213151017DNAArtificial SequenceWO2002102970-pConlinin-2 315aactgatata
tattactttg ttggttggtt aatagattaa cctatttttc ataaaattat 60aattaataaa
aaaattgagt ttttgaaatt ttgagctttc ttgtattatg ttggaacttc 120ttgttccatt
gcaataaaat cagttataaa aaaattacaa acgaagtgca ctcagtaatt 180aaccacctca
aacagactct cacttactca tagtaggatc aatattttcc ttcggcgata 240atcgttcctc
cactatgtag gtcattattt taatttttgg tgatttatta tgtgtctaat 300tttaaaaatt
aattattcga taaatattac ttttatgtat tgttagtttg ttttggaatt 360ttaaagtttg
agttggtctt aagagttatc ttgtttaacc gatattaatt gtaatactag 420aaaaataaag
cttataaaaa accttttatt tgtacataga taggggaatc gaagaagaaa 480aaaattcaaa
gtttaaatta tttattttat atttatgtta tttactttaa attttctaat 540ttctattaaa
tattaatcat atacgtcaaa gcgtaatata atgggcacct tacacaaaca 600ttcgatagaa
gggatgtgaa tatgaaggga ccaaagtgag atcttgccct cagctcctag 660tgcgcctctt
gctgttgctc cacgtgttaa tccaagtggc gagaaaagga gaataataac 720gcaaaaaaac
aggccaagta agataatcca agtgtacact tgtcatcgcc aaacttacta 780aaatacgcgg
caaattgtat acccacacat tattaccata ccatatattg gctgcatttg 840catgtataat
actacgtgta agctcagaaa attccacgtg tcgcccatgc aaaattaaca 900ctcacgaccc
attcctaaat ctccactata taaaccccca ctcccccatc ttaccaaacc 960caccacacaa
ctcacaactt agaaaaacca atcataacca aaatggcaaa gctgatg
1017316673DNAArtificial SequenceWO02102970-SEQ-001-cDNA-Conlinin-1
316gaaaaaccaa tcataaccaa aatggcaaag ctgatgagcc tagcagccgt agcaacggca
60ttcctcttcc tcattgtggt ggacgcatcc gtccgaacca cagtgatcat cgacgaggac
120accaaccaag gccgcggtgg ccaaggtggg caaggacagc agcagcaatg cgagaagcag
180atccaggagc aagactacct gaggagctgc cagcagttcc tgtgggagaa agtccagaag
240ggcggccgca gctactacta caaccaaggc cgtggaggag ggcaacagag ccagcacttc
300gatagctgct gcgatgatct taagcaattg aggagcgagt gcacatgcag gggactggag
360cgtgcaatcg gccagatgag gcaggacatc cagcagcagg gacagcagca ggaagttgag
420aggtgggtcc agcaagctaa acaagtcgct agggaccttc cgggacagtg cggcacccag
480cctagccgat gccagctcca ggggcagcag cagtctgcat ggttttgaag tggtgatcga
540tgagatcgta taaagacact tgctaggtgt taaggatggg ataataagat gtgttttaag
600tcattaaccc gtaattaaaa ggagagagag cttgatggaa tggtattgat gttccttggg
660ttttaaaaaa aaa
67331721DNAArtificial SequenceWO02102970-UTR-SEQ-001-cDNA-Conlinin-1
317gaaaaaccaa tcataaccaa a
21318676DNAArtificial SequenceWO02102970-SEQ-003-cDNA-Conlinin-2
318aagaaccaat caccaccaaa aaatggcaaa gctgatgagc ctggcagccg tagcaacggc
60attcctcttc ctgatcgtgg tggacgcatc cgtccgaacc acagtgatta tcgacgagga
120gaccaaccaa ggccgcggtg gaggccaagg tggccaggga cagcagcagt cttgcgagca
180gcagatccag cagcaagact tcctgaggag ctgccagcag ttcatgtggg agaaagtcca
240gaggggcggc cgcagccact attacaacca gggccgtgga ggaggcgaac agagccagta
300cttcgacagc tgttgtgacg accttaagca attgagcacc gggtgcacat gcaggggact
360tgagcgtgcc atcggccaaa tgaggcagga aatccagcag cagggacagc agcaggaagt
420tcagaggtgg atccagcaag ctaaacaaat cgctaaggac ctccccggac agtgccgacc
480cagcctagcc aatgccagtt ccagggccag cagcaatctg catggttttg aaggggtgat
540cgattatgag atcgtacaaa gacactgcta ggtgttaagg atggataata ataataataa
600tgagatggat gtgttttaag ttaatgtaac agcttaataa agagagagag agagagagag
660agagagagtc aaaaaa
67631922DNAArtificial SequenceWO02102970-UTR-SEQ-003-cDNA-Conlinin-2
319aagaaccaat caccaccaaa aa
223201039DNAArtificial SequenceWO2009130291-SEQ-026-pCnl1 320ttagcagata
tttggtgtct aaatgtttat tttgtgatat gttcatgttt gaaatggtgg 60tttcgaaacc
agggacaacg ttgggatctg atagggtgtc aaagagtatt atggattggg 120acaatttcgg
tcatgagttg caaattcaag tatatcgttc gattatgaaa attttcgaag 180aatatcccat
ttgagagagt ctttacctca ttaatgtttt tagattatga aattttatca 240tagttcatcg
tagtcttttt ggtgtaaagg ctgtaaaaag aaattgttca cttttgtttt 300cgtttatgtg
aaggctgtaa aagattgtaa aagactattt tggtgttttg gataaaatga 360tagtttttat
agattctttt gcttttagaa gaaatacatt tgaaattttt tccatgttga 420gtataaaata
ccgaaatcga ttgaagatca tagaaatatt ttaactgaaa acaaatttat 480aactgattca
attctctcca tttttatacc tatttaaccg taatcgattc taatagatga 540tcgatttttt
atataatcct aattaaccaa cggcatgtat tggataatta accgatcaac 600tctcacccct
aatagaatca gtattttcct tcgacgttaa ttgatcctac actatgtagg 660tcatatccat
cgttttaatt tttggccacc attcaattct gtcttgcctt tagggatgtg 720aatatgaacg
gccaaggtaa gagaataaaa ataatccaaa ttaaagcaag agaggccaag 780taagataatc
caaatgtaca cttgtcattg ccaaaattag taaaatactc ggcatattgt 840attcccacac
attattaaaa taccgtatat gtattggctg catttgcatg aataatacta 900cgtgtaagcc
caaaagaacc cacgtgtagc ccatgcaaag ttaacactca cgaccccatt 960cctcagtctc
cactatataa acccaccatc cccaatctca ccaaacccac cacacaactc 1020acaactcact
ctcacacct
1039321109DNAArtificial SequenceWO2009130291-SEQ-015-part 321cccaccatcc
ccaatctcac caaacccacc acacaactca caactcactc tcacacctta 60aagaaccaat
caccaccaaa aaaccatggg aaaaggatct gagggaaga
1093221080DNAArtificial SequenceWO2009130291-SEQ-016-reverse part
322ttagcagata tttggtgtct aaatgtttat tttgtgatat gttcatgttt gaaatggtgg
60tttcgaaacc agggacaacg ttgggatctg atagggtgtc aaagagtatt atggattggg
120acaatttcgg tcatgagttg caaattcaag tatatcgttc gattatgaaa attttcgaag
180aatatcccat ttgagagagt ctttacctca ttaatgtttt tagattatga aattttatca
240tagttcatcg tagtcttttt ggtgtaaagg ctgtaaaaag aaattgttca cttttgtttt
300cgtttatgtg aaggctgtaa aagattgtaa aagactattt tggtgttttg gataaaatga
360tagtttttat agattctttt gcttttagaa gaaatacatt tgaaattttt tccatgttga
420gtataaaata ccgaaatcga ttgaagatca tagaaatatt ttaactgaaa acaaatttat
480aactgattca attctctcca tttttatacc tatttaaccg taatcgattc taatagatga
540tcgatttttt atataatcct aattaaccaa cggcatgtat tggataatta accgatcaac
600tctcacccct aatagaatca gtattttcct tcgacgttaa ttgatcctac actatgtagg
660tcatatccat cgttttaatt tttggccacc attcaattct gtcttgcctt tagggatgtg
720aatatgaacg gccaaggtaa gagaataaaa ataatccaaa ttaaagcaag agaggccaag
780taagataatc caaatgtaca cttgtcattg ccaaaattag taaaatactc ggcatattgt
840attcccacac attattaaaa taccgtatat gtattggctg catttgcatg aataatacta
900cgtgtaagcc caaaagaacc cacgtgtagc ccatgcaaag ttaacactca cgaccccatt
960cctcagtctc cactatataa acccaccatc cccaatctca ccaaacccac cacacaactc
1020acaactcact ctcacacctt aaagaaccaa tcaccaccaa aaaaccatgg gaaaaggatc
1080323635DNAArtificial SequenceAJ414732-Truska-Linum mRNA Cnl1
323aagaaccaat caccaccaaa aaatggcaaa gctgatgagc ctggcagccg tagcaacggc
60attcctcttc ctgatcgtgg tggacgcatc cgtccgaacc acagtgatta tcgacgagga
120gaccaaccaa ggccgcggtg gaggccaagg tggccaggga cagcagcagt cttgcgagca
180gcagatccag cagcaagact tcctgaggag ctgccagcag ttcatgtggg agaaagtcca
240gaggggcggc cgcagccact attacaacca gggccgtgga ggaggcgaac agagccagta
300cttcgacagc tgttgtgacg accttaagca attgagcacc gggtgcacat gcaggggact
360tgagcgtgcc atcggccaaa tgaggcagga aatccagcag cagggacagc agcaggaagt
420tcagaggtgg atccagcaag ctaaacaaat cgctaaggac ctccccggac agtgccgcac
480ccagcctagc caatgccagt tccagggcca gcagcaatct gcatggtttt gaaggggtga
540tcgattatga gatcgtacaa agacactgct aggtgttaag gatggataat aataataata
600atgagatgga tgtgttttaa gttaatgtaa cagct
63532488DNAArtificial Sequencepromotor p1039+38 UTR 324ccccaatctc
accaaaccca ccacacaact cacaactcac tctcacacct tctagaggat 60ctgatatctg
cggccgcggc gcgccacc
8832538DNAArtificial Sequencepromotor p1039+38 differing part
325tctagaggat ctgatatctg cggccgcggc gcgccacc
3832652DNAArtificial Sequencepromotor p1039+2 UTR 326ccccaatctc
accaaaccca ccacacaact cacaactcac tctcacacct cc
523271064DNAArtificial SequenceSEQ ID NO 1 plus A 327ttagcagata
tttggtgtct aaatgtttat tttgtgatat gttcatgttt gaaatggtgg 60tttcgaaacc
agggacaacg ttgggatctg atagggtgtc aaagagtatt atggattggg 120acaatttcgg
tcatgagttg caaattcaag tatatcgttc gattatgaaa attttcgaag 180aatatcccat
ttgagagagt ctttacctca ttaatgtttt tagattatga aattttatca 240tagttcatcg
tagtcttttt ggtgtaaagg ctgtaaaaag aaattgttca cttttgtttt 300cgtttatgtg
aaggctgtaa aagattgtaa aagactattt tggtgttttg gataaaatga 360tagtttttat
agattctttt gcttttagaa gaaatacatt tgaaattttt tccatgttga 420gtataaaata
ccgaaatcga ttgaagatca tagaaatatt ttaactgaaa acaaatttat 480aactgattca
attctctcca tttttatacc tatttaaccg taatcgattc taatagatga 540tcgatttttt
atataatcct aattaaccaa cggcatgtat tggataatta accgatcaac 600tctcacccct
aatagaatca gtattttcct tcgacgttaa ttgatcctac actatgtagg 660tcatatccat
cgttttaatt tttggccacc attcaattct gtcttgcctt tagggatgtg 720aatatgaacg
gccaaggtaa gagaataaaa ataatccaaa ttaaagcaag agaggccaag 780taagataatc
caaatgtaca cttgtcattg ccaaaattag taaaatactc ggcatattgt 840attcccacac
attattaaaa taccgtatat gtattggctg catttgcatg aataatacta 900cgtgtaagcc
caaaagaacc cacgtgtagc ccatgcaaag ttaacactca cgaccccatt 960cctcagtctc
cactatataa acccaccatc cccaatctca ccaaacccac cacacaactc 1020acaactcact
ctcacacctt aaagaaccaa tcaccaccaa aaaa
10643281072DNAArtificial SequenceSEQ ID NO 1 Kozak ATG 328ttagcagata
tttggtgtct aaatgtttat tttgtgatat gttcatgttt gaaatggtgg 60tttcgaaacc
agggacaacg ttgggatctg atagggtgtc aaagagtatt atggattggg 120acaatttcgg
tcatgagttg caaattcaag tatatcgttc gattatgaaa attttcgaag 180aatatcccat
ttgagagagt ctttacctca ttaatgtttt tagattatga aattttatca 240tagttcatcg
tagtcttttt ggtgtaaagg ctgtaaaaag aaattgttca cttttgtttt 300cgtttatgtg
aaggctgtaa aagattgtaa aagactattt tggtgttttg gataaaatga 360tagtttttat
agattctttt gcttttagaa gaaatacatt tgaaattttt tccatgttga 420gtataaaata
ccgaaatcga ttgaagatca tagaaatatt ttaactgaaa acaaatttat 480aactgattca
attctctcca tttttatacc tatttaaccg taatcgattc taatagatga 540tcgatttttt
atataatcct aattaaccaa cggcatgtat tggataatta accgatcaac 600tctcacccct
aatagaatca gtattttcct tcgacgttaa ttgatcctac actatgtagg 660tcatatccat
cgttttaatt tttggccacc attcaattct gtcttgcctt tagggatgtg 720aatatgaacg
gccaaggtaa gagaataaaa ataatccaaa ttaaagcaag agaggccaag 780taagataatc
caaatgtaca cttgtcattg ccaaaattag taaaatactc ggcatattgt 840attcccacac
attattaaaa taccgtatat gtattggctg catttgcatg aataatacta 900cgtgtaagcc
caaaagaacc cacgtgtagc ccatgcaaag ttaacactca cgaccccatt 960cctcagtctc
cactatataa acccaccatc cccaatctca ccaaacccac cacacaactc 1020acaactcact
ctcacacctt aaagaaccaa tcaccaccaa aaaaccatgg ga 1072
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