METHOD FOR ISOLATION OF TRANSCRIPTION TERMINATION SEQUENCES

Abstract

The invention relates to efficient, high-throughput methods, systems, and DNA constructs for identification and isolation of transcription termination sequences. The invention relates further to specific terminator sequences identified by said methods isolated from rice.

Description

RELATED APPLICATIONS

[0001] The present application is a divisional application of U.S. patent application Ser. No. 13/894,840 filed May 15, 2013, which is a divisional application of U.S. patent application Ser. No. 11/659,126 filed Feb. 1, 2007, which is a national stage application (under 35 U.S.C. 371) of PCT/EP2005/008285 filed Jul. 30, 2005, which claims benefit of U.S. Provisional application No. 60/598,001 filed Aug. 2, 2004 and U.S. Provisional application No. 60/696,209 filed Jul. 1, 2005. The entire contents of each of these applications are hereby incorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Sequence_Listing_--074021_--0224_--01. The size of the text file is 330 KB, and the text file was created on Sep. 2, 2015.

FIELD OF THE INVENTION

[0003] The invention relates to efficient, high-throughput methods, systems, and DNA constructs for identification and isolation of transcription termination sequences.

BACKGROUND OF THE INVENTION

[0004] The aim of plant biotechnology is the generation of plants with advantageous novel properties, such as pest and disease resistance, resistance to environmental stress (e.g., water-logging, drought, heat, cold, light-intensity, day-length, chemicals, etc.), improved qualities (e.g., high yield of fruit, extended shelf-life, uniform fruit shape and color, higher sugar content, higher vitamins C and A content, lower acidity, etc.), or for the production of certain chemicals or pharmaceuticals (Dunwell 2000). Furthermore resistance against abiotic stress (drought, salt) and/or biotic stress (insects, fungal, nematode infections) can be increased. Crop yield enhancement and yield stability can be achieved by developing genetically engineered plants with desired phenotypes.

[0005] For all fields of biotechnology, beside promoter sequences, transcription terminator sequences are a basic prerequisite for the recombinant expression of specific genes. In animal systems, a machinery of transcription termination has been well defined (Zhao et al., 1999; Proudfoot, 1986; Kim et al., 2003; Yonaha and Proudfoot, 2000; Cramer et al., 2001; Kuerstem and Goodwin, 2003). Effective termination of RNA transcription is required to prevent unwanted transcription of trait-unrelated (downstream) sequences, which may interfere with trait performance (see below for more details). Especially arrangement of multiple gene expression cassettes in local proximity (e.g., within one T-DNA) is often causing suppression of gene expression of one or more genes in said construct in comparison to independent insertions (Padidam and Cao, 2001). This is causing problems especially in cases were strong gene expression from all cassettes is required

[0006] Previously efficiency of transcription termination had to be analyzed either by in vitro or in vivo transcription analysis of individual transcription termination sequences, which is a laborious and time-consuming procedure based on trial-and-error (Yonaha and Proudfoot, 1999, 2000; Yarnell and Roberts, 1999). To simplify this process, single nucleotide-recognizing probe such as beacon has been used for in vitro transcription (Liu et al., 2002).

[0007] In plants, understanding transcription termination and re-initiation is at the infant stage. There are no clearly defined polyadenylation signal sequences. Hasegawa et al. (2003) were not able to identify conserved polyadenylation signal sequences in both in vitro and in vivo systems in Nicotiana sylvestris and to determine the actual length of the primary (non-polyadenylated) transcript. There are vague ideas that weak terminator can generate read-through, which affects the expression of the genes located in neighboring expression cassettes (Padidam and Cao, 2001). Appropriate control of transcription termination will prevent read-through into sequences (e.g., other expression cassettes) localized downstream and will further allow efficient recycling of RNA polymerase II, which will improve gene expression.

[0008] Prediction of functional, efficient transcription termination sequences by bioinformatics is not feasible alternative since virtually no conserved sequences exist which would allow for such a prediction. Prediction of the efficiency in transcription termination of such sequences is even more beyond. Furthermore, experimental determination of the actual length and sequence of the primary transcript is difficult since these structures are highly instable being rapidly converted into polyadenylated transcripts (Hasegawa et al., 2003).

[0009] Production of genetically modified cells and organisms (such as plants) requires appropriate recombinant DNA in order to introduce genes of interest. The recombinant DNA contains more than one expression cassette, in general. The expression cassette is composed of promoter, gene of interest, and terminator. The expression of the gene of interest in the expression cassette can be negatively affected by inappropriate termination of transcription from the neighboring cassette. Transcriptional read-through and/or multiple use of the same transcription termination sequence may have one or more of the following disadvantages:

[0010] 1. Unwanted expression of downstream sequences may cause undesirable effects (e.g., changes in metabolic profile, gene silencing etc.).

[0011] 2. Unwanted expression of downstream sequences raises higher hurdles in de-regulation proceedings.

[0012] 3. Multiple use of identical transcription termination sequences may lead to failure of the whole transgenic expression approach by epigenic silencing. Because the present panel of evaluated transcription termination sequences is currently very limited, multiple use of the same transcription termination sequence in one transgenic organism is often unavoidable, which has proofed to result in unintended silencing of the entire transgenic expression constructs (Matzke 1994; Matzke 1989)

[0013] 4. Enablement of constructs comprising multiple gene expression cassettes without undesired interaction of transcription of different cassettes. Such interactions may--depending on the orientation of the cassettes--include unintended expression (e.g., in case of expression cassettes having the same direction of their reading frames) or unintended gene silencing (e.g., in case of inverted orientation of the cassettes).

[0014] In consequence, there is an unsolved demand (especially in the plant biotech area) for tight and alternative transcription termination sequences. There is no easy and reliable screening system to identify "tight" terminators that efficiently terminate transcription. It is therefore an objective of the present invention, to provide a method to easily identify such termination sequences and to provide tight and alternative transcription termination sequences for use on plants. This objective is achieved by this invention.

BRIEF DESCRIPTION OF THE INVENTION

[0015] Accordingly, a first embodiment of the invention related to a method for identification and isolation of transcription termination sequences for comprising the steps of:

[0016] i) providing a screening construct or screening vector comprising

[0017] a) a promoter sequence, and

[0018] b) one or more insertion sites--preferably a restriction or recombination site--for insertion of DNA sequences, and

[0019] c) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic, wherein insertion of an efficient transcription terminator into said insertion site changes expression of said additional sequences by said promoter sequence in comparison to no insertion, and

[0020] ii) providing one or more DNA sequences to be assessed for their transcription termination capability, and

[0021] iii) inserting one or more copies of said DNA sequences into said insertion site of said screening construct or screening vector, and

[0022] iv) introducing said screening construct or screening vector with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence, and

[0023] v) identifying and/or selecting screening construct or screening vector with a changed readily detectable characteristic in comparison to no insertion, and

[0024] vi) isolating the inserted DNA sequences from said identified and/or selected screening construct or screening vector for use as transcription termination sequences and--optionally--determining their sequence.

[0025] There are various options for localization of said insertion site in relation to said additional sequences. For example the insertion site may preferably be at a position selected from group of:

[0026] i) upstream of the additional sequences between said promoter and said additional sequences, and

[0027] ii) downstream of the additional sequences, and

[0028] iii) in between said additional sequences.

[0029] Depending on the localization of the insertion site to said additional sequences several especially preferred embodiments result. In one preferred embodiment method for identification and isolation of transcription termination sequences comprises the steps of:

[0030] i) providing a screening construct or screening vector comprising in 5' to 3' direction

[0031] a) a promoter sequence, and

[0032] b) one or more insertion sites--preferably a restriction or recombination site--for insertion of DNA sequences, and

[0033] c) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic,

[0034] wherein insertion of an efficient transcription terminator into said insertion site suppresses expression of said additional sequences by said promoter sequence in comparison to no insertion, and

[0035] ii) providing one or more DNA sequences to be assessed for their transcription termination capability, and

[0036] iii) inserting one or more copies of said DNA sequences into said insertion site of said screening construct or screening vector, and

[0037] iv) introducing said screening construct or screening vector with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence, and

[0038] v) identifying and/or selecting screening constructs or screening vectors with a changed readily detectable characteristic in comparison to no insertion, and

[0039] vi) isolating the inserted DNA sequences from said identified and/or selected screening constructs or screening vectors for use as transcription termination sequences and--optionally--determining their sequence.

[0040] One or more of the sequences to be assessed for their efficiency in transcription termination may be inserted into the screening vector or screening construct. In the case of insertion of two or more copies, in a preferred embodiment said DNA sequences to be assessed for their transcription termination efficiency are inserted into said insertion site in form of an inverted repeat. Thus, preferably the method for identification and isolation of transcription termination sequences comprises the steps of:

[0041] i) providing a screening construct or screening vector comprising in 5' to 3' direction

[0042] a) a promoter sequence, and

[0043] b) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic, and

[0044] c) one or more insertion sites--preferably a restriction or recombination site--for insertion of DNA sequences,

[0045] ii) providing one or more DNA sequences to be assessed for their transcription termination capability, and

[0046] iii) inserting at least two copies of a specific DNA sequence of said DNA sequences in form of an inverted repeat into said insertion site of said screening construct or screening vector, wherein insertion of an inverted repeat of an efficient transcription terminator into said insertion site allows expression of said additional sequences by said promoter sequence in comparison to no insertion, and

[0047] iv) introducing said screening constructs or screening vectors with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence, and

[0048] v) identifying and/or selecting screening constructs or screening vectors with said readily detectable characteristic in comparison to no insertion, and

[0049] vi) isolating the inserted DNA sequences from said identified and/or selected screening constructs or screening vectors for use as transcription termination sequences and--optionally--determining their sequence.

[0050] In another preferred embodiment of the invention, the method for identification and isolation of transcription termination sequences comprises the steps of:

[0051] i) providing a screening construct or screening vector comprising in 5' to 3' direction

[0052] a) a promoter sequence, and

[0053] b) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic, and embedded into said additional sequences one or more insertion sites--preferably a restriction or recombination site--for insertion of DNA sequences,

[0054] wherein insertion of an efficient transcription terminator into said insertion site suppresses full-length transcription of said additional sequences by said promoter sequence in comparison to no insertion, and

[0055] ii) providing one or more DNA sequences to be assessed for their transcription termination capability, and

[0056] iii) inserting one or more copies of said DNA sequences into said insertion site of said screening construct or screening vector, and

[0057] iv) introducing said screening constructs or screening vectors with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence, and

[0058] v) identifying and/or selecting screening constructs or screening vectors with a changed readily detectable characteristic in comparison to no insertion, and

[0059] vi) isolating the inserted DNA sequences from said identified and/or selected screening constructs or screening vectors for use as transcription termination sequences and--optionally--determining their sequence.

[0060] Preferably, the additional sequence is selected from the group consisting of positive selection marker, negative selection marker, counter selection marker, reporter genes, and toxic genes. In case of toxic genes, wherein said toxic gene may for example be a construct for gene silencing of an essential endogenous gene.

[0061] Preferably, the DNA sequence to be assessed for their transcription termination efficiency is provided by a method selected from the group consisting of:

i) provision of a selected sequence by amplification from a host genome, and ii) provision of a library of sequences by fragmentation of a host genome.

[0062] More preferably, the DNA sequence to be assessed for their transcription termination efficiency is derived from a plant cell.

[0063] There various methods for insertion of said sequences into said insertion site. Preferably, the DNA sequences to be assessed for their transcription termination efficiency are inserted into said insertion site by a method selected from the group consisting of:

i) recombinational cloning, and ii) insertion by sequence specific restriction and ligation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG. 1-A A1: Graphic of preferred Method A: Insertion site (IS) for transcription terminator (TT) to be assessed is localized between promoter (P1) and additional sequences (AS), which are able to cause a readily detectable characteristic. In case of an efficient transcription terminator (+), transcription from the promoter P1 is stopped at said transcription terminator (symbolized by arrow below construct). No expression of the additional sequences occurs and no change in characteristic is caused (symbolized by crossed circle). In case of no efficient transcription termination (-), transcription from the promoter P1 read-through said alleged transcription terminator leading to expression of the additional sequences (symbolized by arrow below construct), thereby causing the change in the characteristic (symbolized by lightening symbol).

[0065] A2: Graphic of preferred Method A based on terminator as additional sequences. Insertion site (IS) for transcription terminator (TT) to be assessed is localized between promoter (P1) and additional sequences which in this case are constituted by an inverted repeat of a known transcription terminator (T). The second copy of said terminator (symbolized by upside letter) downstream of the promoter is in its functional orientation. In case of an efficient transcription terminator (+), transcription from the promoter P1 is stopped at said transcription terminator (symbolized by arrow below construct) and normal expression of the marker protein (M1) occurs (symbolized by black lightening symbol). In case of no efficient transcription termination (-), transcription from the promoter P1 read-through said alleged transcription terminator leading to expression of the inverted repeat of the known transcription terminator, causing gene silencing (GS) of the Marker M1 expression (symbolized by crossed circle). Preferably, the construct comprises a further expression cassette leading to expression of Marker M2, which functions as a positive control for general presence of the screening construct, bringing about a preferably different second phenotype (symbolized by white lightening symbol).

[0066] FIG. 1-B A3: Graphic of preferred Method A based on terminator as additional sequences. Insertion site (IS) for transcription terminator (TT) to be assessed is localized between two expression cassette for different marker genes, which are oriented head to head to each other. The cassette for the marker M2 is terminated by a known transcription terminator. In case of an efficient termination of transcription by the inserted test terminator (+), transcription from the promoter P1 is stopped at said transcription terminator (symbolized by arrow below construct) and normal expression of the marker proteins (M1) and (M2) occurs (symbolized by black and white lightening symbols). In case of no efficient transcription termination (-), transcription from the promoter P1 read-through said alleged transcription terminator leading to expression of an RNA strand complementary to the one expressed from promoter P2, thereby causing hybridization of the transcript of the first marker gene (M1) with the constitutively expressed transcript from the second marker gene (M2). This causes a gene silencing (GS) of both Marker genes M1 and M2 (symbolized by crossed circle).

[0067] A4: Graphic of preferred Method A based on terminator as additional sequences. Insertion site (IS) for transcription terminator (TT) to be assessed is localized between promoter (P1) and additional sequences which in this case are constituted by non-transcribed DNA sequence preferably a fragment or a full length sequence of a known transcription terminator. The second expression cassette is oriented head to head to the first cassette and carries a second copy of said DNA sequence, terminator or terminator fragment. In case of an efficient termination of transcription by the inserted test terminator (+), transcription from the promoter P1 is stopped at said transcription terminator (symbolized by arrow below construct) and normal expression of the marker protein (M1) occurs (symbolized by black lightening symbol). In case of no efficient transcription termination (-), transcription from the promoter P1 read-through said alleged transcription terminator leading to expression of the known transcription terminator or transcription terminator fragment, causing hybridization of the transcript of the first marker gene (M1) with the constitutively expressed transcript from the second marker gene (M2) which carries the identical 3' UTR sequence, thereby causing a dose dependent repression of expression of both Marker genes M1 and M2 (symbolized by crossed circle). Preferably, the construct comprises a second marker gene (M2). As the effect of expression repression of both marker genes is dependent on the degree of hybridization between the two classes of transcripts it is possible to screen for intermediary phenotypes, allowing the selection of "weak" candidate terminator sequences or "tight" candidate terminator sequences e.g. by using different screening conditions.

[0068] FIG. 2: B1: Graphic of preferred Method B: Insertion site (IS) for transcription terminator (TT) to be assessed is downstream of the additional sequences (AS), which are able to cause a readily detectable characteristic. The transcription terminator (TT) to be assessed is inserted in form of an inverted repeat, wherein the first copy (symbolized by upside letter) downstream of the promoter is in its functional orientation. In case of an efficient transcription terminator (+), transcription from the promoter P1 is stopped at the first copy of the transcription terminator (symbolized by arrow below construct) and normal expression of the additional sequences (AS) occurs (symbolized by black lightening symbol). In case of no efficient transcription termination (-), transcription from the promoter P1 read-through both copies of said alleged transcription terminator leading to expression of the inverted repeat of said transcription terminator, causing gene silencing (GS) of the additional sequences (AS) expression (symbolized by crossed circle).

[0069] B2: Preferably, the construct comprises a further expression cassette, wherein AS2 is encoding for a different characteristic than AS1. Both expressions are silenced in case of an inefficient transcription terminator. In case of an efficient terminator, expression of both characteristics (symbolized by black and white lightening symbol, respectively) occurs.

[0070] FIG. 3 Graphic of preferred Method C: Insertion site (IS) for transcription terminator (TT) to be assessed is localized in an intron (IN) localized in the additional sequences (AS), which are able to cause a readily detectable characteristic. In case of an efficient transcription terminator (+), transcription from the promoter P1 is stopped at said transcription terminator (symbolized by arrow below construct). No full-length expression of the additional sequences occurs and no change in characteristic is caused (symbolized by crossed circle). In case of no efficient transcription termination (-), transcription from the promoter P1 read-through said alleged transcription terminator leading to full-length expression of the additional sequences (symbolized by arrow below construct), thereby causing the change in the characteristic (symbolized by lightening symbol).

[0071] FIG. 4 Schematic presentation of the screening constructs:

[0072] A: Lo523 negative control construct: Binary vector corresponding to the screening construct without insertion of an additional transcription terminator sequence. Upon use of this construct for transformation of plant cells the nptII gene will be transcribed. As there is no functional terminator present downstream of this gene transcription proceeds through the nos terminator IR leading to a transcript with hairpin structure, which causes silencing of nptII gene expression. These cells cannot grow on selective medium containing Kanamycin. By visualization of the constitutively expressed GFP marker gene these non-growing cells can be distinguished from non-transformed cells.

[0073] B: Screening construct B: Binary vector containing a first expression cassette with a constitutively expressed reporter gene for selection of transformed from untransformed cells/plants followed by a second expression cassette containing a nptII selection marker driven by a strong constitutive promoter. Downstream of the nptII gene an IR of the Agrobacterium nos terminator sequence is inserted, consisting of a first repeat in antisense direction followed by a short spacer sequence derived from the GUS reporter gene and the second repeat of the nos terminator which is inserted in its functional 5' to 3'direction. The fragments to be tested for transcription terminator activity are to be inserted between the nptII gene and said nos terminator IR.

[0074] D: Lo546 positive control construct C: Binary vector derived from construct C where a long fragment of the rbCs E9 terminator sequence is inserted between nptII gene and nos terminator IR. The E9 terminator is believed to act as a highly efficient terminator and will therefore terminate transcription of the nptII gene resulting in normal expression levels of the selection marker which enable the growth of the transformed cells in presence of Kanamycin.

[0075] FIG. 5 pENTR construct--position 1 visual marker (reporter gene): Gateway Entry vector A containing the GFP reporter gene under control of a constitutive promoter. The vector is used in combination with the Gateway Entry vector B Lo376 (SEQ ID NO: 76) and the Gateway Entry vector C, Lo522a (Lo522b respectively), for recombination based construction of positive control constructs and in combination with Lo503a (Lo503b respectively) for recombination based construction of binary negative control constructs and the screening construct.

[0076] FIG. 6 pENTR constructs for positive controls: The Gateway Entry vectors C contain the nptII selection marker gene under control of the constitutive STPT promoter. 3' to the selection marker a long fragment of the rbCs E9 terminator sequence is inserted in front of a nos terminator IR. The depicted constructs are used in combination with the Gateway Entry vectors A, Lo484, B Lo376 and C, Lo522 (Lo522b respectively), for recombination based construction of binary positive control constructs.

[0077] FIG. 7 pENTR constructs for negative controls: The Gateway Entry vectors C contain the nptII selection marker gene under control of the constitutive STPT promoter. 3' to the selection marker a nos terminator IR is inserted. This results in transcription of the nptII Gene and the inverted sequence fragment of the nos terminator followed by the nos terminator sequence in functional 5'-3'orientation, which will by default cause a hairpin structure at the 3'end of the transcript. The depicted constructs are used in combination with the Gateway Entry vectors A, Lo484, B Lo376 and C, Lo522 (Lo522b respectively), for recombination based construction of binary positive control constructs.

[0078] FIG. 8 pSUN1 constructs--negative controls: Binary construct derived from the recombination based insertion of the expression cassettes from the respective pENTR vectors into the Gateway Destination vector Lo442 pSUN1-R4R3 (SEQ ID NO: 77).

[0079] FIG. 9 pSUN1 constructs--positive controls: Binary construct derived from the recombination based insertion of the expression cassettes from the respective pENTR vectors into the Gateway Destination vector Lo442 pSUN1-R4R3 (SEQ ID NO: 77).

[0080] FIG. 10 Binary constructs Lo239-pSUN3-GWs-B1-BnAK700::GUS::nosT-B2 (negative control) and Lo657-pSUN3-GWs-B1-BnAK700::GUS::E9::nosT::B2 (positive control) representing FIGS. 4-E and F. Lo239 is derived from the Gateway based recombination of the pENTR Lo235 carrying the GUS marker cassette with the pSUN destination vector Lo125 pSUN3-GWs-NPTII carrying the nptII cassette. After modification of Lo235 by insertion of a long fragment of the rbCs E9 terminator downstream of the GUS marker gene the resulting Lo654 pENTR-BnAK700::GUS::E9::nosT is used for the Gateway based recombination with Lo125 pSUN3-GWs-NPTII to create the positive control construct Lo657-pSUN3-GWs-B1-BnAK700::GUS::E9::nosT:: B2.

[0081] FIG. 11 Screening results of negative and positive control constructs: The constructs described in FIG. 10 were used for floral dip transformation of Arabidopsis thaliana plants. The harvested seeds were tested for expression of the marker gene nptII by selection on Kanamycin. Whereas the seeds from plants which have been used for transformation with the positive control construct are viable on the selective medium, showing expression of the nptII gene (GUS gene expression is not shown but has been detected by X-Gluc reaction) the negative control construct yields only seeds which are not viable on Kanamycin and show no expression of the Gus marker gene.

[0082] FIG. 12 Diagram of the constructs for identification of transcription terminators of interest (TOI).

[0083] (a) The construct for in vitro transcription assays. Gene 1 and 2 prefer to be the sequences that are not homologous to the plant genome.

[0084] (b-d) The constructs for in vivo assays. The regions indicated as "dsRNA" are the sequences that generate double-stranded RNA (dsRNA). These dsRNAs down-regulate an essential gene for plant cells (b), negative selection marker gene (SMG) (c), or a reporter gene such as fluorescence protein (FP)

[0085] (d) In the construct (d), the DNA downstream of TOI can be another reporter gene or any sequences.

[0086] (e) Screening system in yeast: The system allows efficient screening of random sequences. Control vectors contain NOS terminator, truncated NOS, no terminator, or a DNA fragment with unidentified sequence.

[0087] FIG. 13 A: Control constructs (A-D) and a construct containing potential terminator candidate (E). GUS can be replaced with any reporter gene or a non-plant homologous DNA fragment including ATG and stop codon. In the control vectors, four variations are made: (A) 260 bp nopaline synthase terminator (NOS) including polyadenylation site, a cleavage sequence, and approximately 80 bp of nucleotides downstream of the cleavage sequence, (B) reverse orientation of NOS, (C) NOS DNA fragment either including polyadenylation signal and cleavage site or lacking the polyadenylation signal and downstream sequence, and (D) no terminator. Nopaline synthase terminator is replaced with various genomic fragments, which can be selected as potential terminator candidates. Lu^F represents a fragment of luciferase gene (approximately 200 to 300 bp) as a read through region. Lu^F can be replaced with a non-plant homologous DNA fragment (e.g. yeast intergenic sequences). Octopine synthase terminator is located in the end of the cassette to stabilize the transcripts including read through products. These constructs can be built in pUC based vector or a binary vector. TOI stands for terminator of interest. Although the NOS terminator has proven in the screening systems to be of only moderate efficiency, it can be used as kind of control terminator for the evaluation systems described herein.

[0088] B: An in vitro screening system. Two single strand fluorescence probes such as beacon probes that hybridize the read through region and polyadenylated RNA. The black bar represents polyadenylated RNA. The gray bar represents read through product. Probe 1 (black star) hybridizes the complementary sequence of the polyadenylated RNA. Probe 2 (gray star) hybridizes the complementary sequence of the read through region.

[0089] C: Control constructs (A, C) and constructs containing potential terminator candidates (B, D). RG represents any reporter gene. SMG represents any selectable marker gene. Approximately 260 bp Nopaline Synthase terminator (NOS) is used as a control terminator (A, C). Plant genomic fragment (<1 kb) is cloned between GUS or bar and dsRNA fragment to identify terminator of interest (TOI). Expression of dsRNA for essential gene (EG) causes lethal in plants due to down-regulation of essential gene in the transgenic plants (A, B). Expression of dsRNA for SMG causes lethal under specific selection pressure due to down-regulation of selectable marker gene in the transgenic plants (C, D). However, strong and tight terminator can limit the expression of dsRNA due to low levels or no read through resulting in producing transgenic plants. These constructs can be built in pUC based vector or a binary vector.

[0090] D: Control construct (A) is the vector into which control and test sequences were cloned. Positive control vector (B) comprises the NOS terminator sequence downstream of the GUS reporter gene. Negative control vector (C) comprises sequence obtained from an internal portion of a plant-expressible open-reading frame, and therefore should not possess transcriptional termination activity. Vector (D) represents vectors that comprise putative TOI candidates to be tested for terminator activity. These constructs were built into a pUC vector and used for transient analyses of TOI candidate sequences.

[0091] E: Control binary vectors (A, C) comprise no insertion of putative terminator sequences downstream of the primary reporter gene, GUS. Test vectors (B, D) comprise putative TOI candidates to be tested for transcriptional termination activity in stably transformed plants. Vectors (A, B) comprise a NOS terminator downstream of the secondary reporter gene, DsRed2; these constructs will be used to determine if efficiency of GUS termination by putative TOIs impacts expression of DsRed2. Vectors (C, D) contain no transcriptional termination sequence for the secondary reporter gene DsRed2, and will be used to test for bidirectional transcriptional termination activity by TOI sequences that are juxtaposed between the 3' ends of the DsRed2 and GUS genes.

[0092] FIG. 14 Maize leaf tissue following transient TOI assays. No GUS staining was observed in vectors that do not comprise a functional transcriptional terminator downstream of the GUS coding sequence (A & C). The presence of a functional terminator rescued GUS expression in the (+) control (B) vector as well as all four TOI candidate sequences (D-G).

[0093] FIG. 15 Control construct (A) and a construct containing potential terminator candidates (B). The constructs are composed of strong constitutive promoter (e.g. maize ubiquitin promoter), FP1 (gene encoding fluorescent protein1), known (A: e.g. NOS) or novel (B) terminator, IRES (e.g. EMCV), FP2 (gene encoding fluorescent protein2), and octopine synthase terminator.

[0094] FIG. 16 Terminator of interest (TOI) construct (A) and control constructs (B and C). A TOI is embedded within an intron of a lethal gene or a reporter gene (A). Control constructs will also be built without a TOI embedded in the intron (B) and with a known terminator, NOS, embedded in the intron (C). The lethal gene can be diphtheria toxin fragment A (DT-A) or any known lethal gene for plants in the art. The reporter gene can be green fluorescent protein or any known reporter gene functioning in plants in the art.

[0095] FIG. 17 The modified PIV2 intron. PIV intron contains (1) a consensus 5' recognition sequence (2) high AU content after the 5' splice site, (3) high AU content before the 3' splice site, (4) a consensus 3' recognition sequence, (5) a consensus branchpoint sequence CURAY, (6) a polyU tract between the branchpoint sequence and the 3' splice site.

[0096] FIG. 18 Terminator of interest (TOI) construct (1), and control constructs (2 and 3). Construct 4 expresses a dsRNA molecule which will target mRNA containing the RNAi target region of constructs 1 (leaky TOI) and construct 3 for degradation. ZmUbi (maize ubiquitin promoter), GFP (green fluorescence protein), OCS (Octopine synthase terminator), NOS (Nopaline synthase terminator), dsRNA (double-stranded RNA), siRNA (small interfering RNA), RNAi (RNA interference; silencing). For constructs 1, 2, and 3, the destination of the RNA produced is shown (translated or degraded) if construct 4 is also expressed in the same plant.

[0097] FIG. 19 Schematic drawing of the inserts in vector pTOI3 (SEQ ID NO: 73) and pTOI4 (SEQ ID NO: 73).

[0098] FIG. 20 A map of the pUC based expression vector that was used in transient analyses. Control and putative TOI sequences were cloned into the RsrII-SacI sites of this vector.

[0099] FIG. 21 Maps of binary vectors to be used for analysis of TOI activity in stably-transformed plants. A--vectors comprise Nos terminator downstream of DsRed2 reporter gene, and will be used to determine if efficiency of GUS termination by putative TOIs affects expression of DsRed2. TOI sequences were inserted at the AvrII site. B--vectors comprise no terminator for DsRed2 and will be used to assess bidirectional transcriptional termination activity by TOI candidates. TOI sequences will be inserted at SacI site.

GENERAL DEFINITIONS

[0100] 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. In the description that follows, a number of terms used in recombinant DNA technology are utilized extensively. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

[0101] 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.

[0102] 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 up or down (higher or lower).

[0103] As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0104] The term "nucleotide" refers to a base-sugar-phosphate combination. Nucleotides are monomeric units of a nucleic acid sequence (DNA and RNA). The term nucleotide includes ribonucleoside triphosphatase ATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof Such derivatives include, for example, [quadratureS]dATP, 7-deaza-dGTP and 7-deaza-dATP. The term nucleotide as used herein also refers to dideoxyribonucleosidetriphosphates (ddNTPs) and their derivatives. Illustrated examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According to the present invention, a "nucleotide" may be unlabeled or detectably labeled by well-known techniques. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.

[0105] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers or hybrids thereof in either single- or double-stranded, sense or antisense form.

[0106] 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. The term "nucleic acid" is used interchangeably herein with "gene", "cDNA, "mRNA", "oligonucleotide," and "polynucleotide".

[0107] The phrase "nucleic acid sequence" as used herein refers to a consecutive list of abbreviations, letters, characters or words, which represent nucleotides. In one embodiment, a nucleic acid can be a "probe" which is a relatively short nucleic acid, usually less than 100 nucleotides in length. Often a nucleic acid probe is from about 50 nucleotides in length to about 10 nucleotides in length. A "target region" of a nucleic acid is a portion of a nucleic acid that is identified to be of interest. A "coding region" of a nucleic acid is the portion of the nucleic acid which is transcribed and translated in a sequence-specific manner to produce into a particular polypeptide or protein when placed under the control of appropriate regulatory sequences. The coding region is said to encode such a polypeptide or protein.

[0108] The term "probe", as used herein, refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to a nucleotide sequence of interest. A probe may be single-stranded or double-stranded. It is contemplated that any probe used in the present invention will be labeled with any "reporter molecule," so that it is detectable in any detection system including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, calorimetric, gravimetric, magnetic, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

[0109] The term "oligonucleotide" refers to a synthetic or natural molecule comprising a covalently linked sequence of nucleotides which are joined by a phosphodiester bond between the 3' position of the deoxyribose or ribose of one nucleotide and the 5' position of the deoxyribose or ribose of the adjacent nucleotide.

[0110] The term "sense" is understood to mean a nucleic acid having a sequence which is homologous or identical to a target sequence, for example a sequence which binds to a protein transcription factor and which is involved in the expression of a given gene. According to a preferred embodiment, the nucleic acid comprises a gene of interest and elements allowing the expression of the said gene of interest. The sense RNA can be employed for gene silencing in a co-suppression or sense-suppression gene silencing approach. Expression of genes that when transcribed produce RNA transcripts that are identical or at least very similar to transcripts of endogenous genes can mediate gene silencing in an as yet not fully understood way of inhibition of gene expression referred to as co-suppression (disclosed by Napoli 1990; Jorgensen 1996; Goring 1991; Smith 1990; Van der Krol 1990). The expressed RNA can represent the endogenous target entirely or in part. Translation is nor required, transcription is sufficient. Application in plants is described (Napoli 1990; U.S. Pat. No. 5,034,323).

[0111] The term "antisense" is understood to mean a nucleic acid having a sequence complementary to a target sequence, for example a messenger RNA (mRNA) sequence the blocking of whose expression is sought to be initiated by hybridization with the target sequence. To maximize the antisense effects in a plant host, the use of homologous genes is preferred. With homologous is meant obtainable from the same plant species as the plant host. Heterologous, for the purpose of this specification shall mean obtainable from a different plant or non-plant species. Heterologous shall also comprise synthetic analogs of genes, modified in their mRNA encoding nucleic acid sequence to diverge at least 5% of the host gene. Gene silencing by antisense RNA is numerously described in the art (including various applications in plants; e.g. Sheehy 1988; U.S. Pat. No. 4,801,340; Mol 1990). A variation of the antisense approach is the use of quadrature-anomeric nucleic acid sequences. Such α-anomeric sequences are forming specific double-stranded hybrids with complementary RNA, wherein in contrast to "normal" antisense RNA (or β-nucleic acids) both strands are in parallel to each other (Gautier 1987).

[0112] The term "dsRNAi" or "double-stranded RNA interference" is intended to mean the method of gene silencing by expression of a RNA molecule corresponding to an endogenous gene together with its complementary RNA strand, thus providing two RNA sequences which may form by hybridization a double-stranded RNA structure. The two RNA strands may be on separate molecules or may be part of one molecule, thus forming a so-called self-complementary hairpin structure. Self-complementary hairpin forming RNA structure may be expressed for example from a DNA comprising an "inverted repeat" of a double-stranded DNA fragment. In this context the term "inverted repeat" is intended to mean the orientation of two fragments of double-stranded DNA (which are substantially identical or--preferably identical in sequence) in one double stranded DNA molecule in an inverted orientation (i.e. in a "head" to "head" or "tail" to tail" orientation so that the sense-strand of the first fragment is fused to the antisense strand of the second and vice versa). Preferably, the hairpin forming dsRNA may include a linker (e.g., an intron sequence for example the intron of the ST-LS1 gene from potato; Vancanneyt 1990) connecting the two complementary strands (e.g., as described in WO 99/53050). The method of dsRNAi is well described in the art for various organisms including animal and plant organism (e.g., Matzke 2000; Fire 1998; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364). These references and the methods disclosed therein are explicitly incorporated by reference. The phenotype of a dsRNAi expressing cell or organism is similar to that of a knock-out mutant, often resulting in complete gene silencing (Waterhouse 1998). The term "double-stranded RNA" or "dsRNA" as used herein is intended to mean one or more ribonucleic acid sequences, which because of complementary sequences are theoretically (i.e. according to the base-pairing rules of Watson and Crick) and/or practically (e.g., because of hybridization experiments in vitro and/or in vivo) capable to form double-stranded RNA structures. The person skilled in the art is aware of the fact that formation of a double-stranded RNA structure is an equilibrium between single-stranded and double-stranded forms. Preferably, the relation between double-stranded (i.e. hybridized) and single-stranded (i.e. non-hybridized or dissociated) forms is at least 1:10, preferably at least 1:1, more preferably at least 10:1. One strand of the dsRNA is essentially identical to the sequence of the endogenous gene. Essentially identical in this context means that a 100% identity is not required for efficient gene silencing, but that the dsRNA sequence may comprise insertions, deletions and point mutations in comparison to the target sequence. Preferably the homology between the dsRNA sequence and at least part of the target sequence is at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100%. Alternatively, an essential identity is one which allows for hybridization of the two sequences under high stringency conditions. The part of the target sequence which is having the homology with the dsRNA has a length of at least 23 bases, preferably at least 50 bases, more preferably at least 100 bases. Said part of the target gene may resemble various part of the gene, but is preferably part which encodes for the mRNA sequence transcribed from said gene.

[0113] The term "ribozyme" is intended to mean catalytic RNA-molecules, which are capable to induce sequence-specific cleavage of a target RNA (Tanner 1999). Preparation and use of ribozymes is disclosed in the art (Haseloff 1988; Haselhoff & Gerlach 1988; Steinecke 1992; de Feyter R 1996). Preferred are "hammerhead"-ribozymes (Haselhoff & Gerlach 1988). Disclosed are methods for gene silencing based on customized ribozymes (EP 0 291 533, EP 0 321 201, EP 0 360 257). Use in plants and plant cells is also disclosed (Steinecke 1992; de Feyter 1996). Suitable target sequences are ribozymes can be derived as described (Steinecke 1995) by secondary structure calculation of ribozyme and target sequences and the interaction thereof (Bayley 1992; Lloyd 1994). For example derivatives of the Tetrahymena L-19 IVS RNA can be employed and adapted to virtually any target sequence (U.S. Pat. No. 4,987,071; U.S. Pat. No. 5,116,742). Alternatively, ribozymes can be selected by screening of diversified ribozyme libraries (Bartel 1993).

[0114] The term "gene" refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the polypeptide in some manner. A gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (upstream) and following (downstream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons).

[0115] As used herein the term "coding region" when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5'-side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 3'-side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA). In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5'- and 3'-end of the sequences, which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5'-flanking region may contain regulatory sequences such as promoters and enhancers, which control or influence the transcription of the gene. The 3'-flanking region may contain sequences, which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.

[0116] The term "amplification" refers to any in vitro method for increasing a number of copies of a nucleotide sequence with the use of a polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a DNA and/or RNA molecule or primer thereby forming a new molecule complementary to a template. The formed nucleic acid molecule and its template can be used as templates to synthesize additional nucleic acid molecules. As used herein, one amplification reaction may consist of many rounds of replication. DNA amplification reactions include, for example, polymerase chain reaction (PCR). One PCR reaction may consist of 5 to 100 "cycles" of denaturation and synthesis of a DNA molecule.

[0117] The terms "polypeptide", "peptide", "oligopeptide", "polypeptide", "gene product", "expression product" and "protein" are used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues.

[0118] The term "isolated" as used herein means that a material has been removed from its original environment. For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides can be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and would be isolated in that such a vector or composition is not part of its original environment.

[0119] Preferably, the term "isolated" when used in relation to a nucleic acid, as in "an isolated nucleic acid sequence" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA which are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs which encode a multitude of proteins. For example, an isolated nucleic acid sequence encoding for a specific trait includes, by way of example, such nucleic acid sequences in cells which ordinarily contain said nucleic acid sequence, wherein said nucleic acid sequence is in a chromosomal or extrachromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid sequence may be present in single-stranded or double-stranded form. When an isolated nucleic acid sequence is to be utilized to express a protein, the nucleic acid sequence will contain at a minimum at least a portion of the sense or coding strand (i.e., the nucleic acid sequence may be single-stranded). Alternatively, it may contain both the sense and anti-sense strands (i.e., the nucleic acid sequence may be double-stranded).

[0120] As used herein, the term "purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated. An "isolated nucleic acid sequence" is therefore a purified nucleic acid sequence. "Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated.

[0121] As used herein, the terms "complementary" or "complementarity" are used in reference to nucleotide sequences related by the base-pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'. Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0122] A "complement" of a nucleic acid sequence as used herein refers to a nucleotide sequence whose nucleic acids show total complementarity to the nucleic acids of the nucleic acid sequence.

[0123] The term "wild-type", "natural" or of "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.

[0124] The term "transgenic" or "recombinant" when used in reference to a cell refers to a cell which contains a transgene, or whose genome has been altered by the introduction of a transgene. The term "transgenic" when used in reference to a tissue or to a plant refers to a tissue or plant, respectively, which comprises one or more cells that contain a transgene, or whose genome has been altered by the introduction of a transgene. Transgenic cells, tissues and plants may be produced by several methods including the introduction of a "transgene" comprising nucleic acid (usually DNA) into a target cell or integration of the transgene into a chromosome of a target cell by way of human intervention, such as by the methods described herein.

[0125] The term "transgene" as used herein refers to any nucleic acid sequence which is introduced into the genome of a cell by experimental manipulations. A transgene may be an "endogenous DNA sequence," or a "heterologous DNA sequence" (i.e., "foreign DNA"). The term "endogenous DNA sequence" refers to a nucleotide sequence which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence. The term "heterologous DNA sequence" refers to a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Heterologous DNA also includes an endogenous DNA sequence which contains some modification. Generally, although not necessarily, heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, selectable marker proteins (e.g., proteins which confer drug resistance), etc. Preferably, the term "transgenic" or "recombinant" with respect to a regulatory sequence (e.g., a promoter of the invention) means that said regulatory sequence is covalently joined and adjacent to a nucleic acid to which it is not adjacent in its natural environment.

[0126] The term "foreign gene" refers to any nucleic acid (e.g., gene sequence) which is introduced into the genome of a cell by experimental manipulations and may include gene sequences found in that cell so long as the introduced gene contains some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring gene.

[0127] "Recombinant polypeptides" or "recombinant proteins" refer to polypeptides or proteins produced by recombinant DNA techniques, i.e., produced from cells transformed by an exogenous recombinant DNA construct encoding the desired polypeptide or protein. Recombinant nucleic acids and polypeptide may also comprise molecules which as such does not exist in nature but are modified, changed, mutated or otherwise manipulated by man.

[0128] The terms "heterologous nucleic acid sequence" or "heterologous DNA" are used interchangeably to refer to a nucleotide sequence which is ligated to a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Generally, although not necessarily, such heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed.

[0129] The terms "organism", "host", "target organism" or "host organism" are referring to any prokaryotic or eukaryotic organism that can be a recipient of the screening construct or screening vector. A "host," as the term is used herein, includes prokaryotic or eukaryotic organisms that can be genetically engineered. For examples of such hosts, see Maniatis 1989. Included are entire organisms but also organs, parts, cells, cultures, and propagatable material derived therefrom. Preferred are microorganisms, non-human animal and plant organisms. Preferred microorganisms are bacteria, yeasts, algae or fungi.

[0130] Preferred bacteria are bacteria of the genus Escherichia, Corynebacterium, Bacillus, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Clostridium, Proionibacterium, Butyrivibrio, Eubacterium, Lactobacillus, Phaeodactylum, Colpidium, Mortierella, Entomophthora, Mucor, Crypthecodinium or cyanobacteria, for example of the genus Synechocystis. Especially preferred are microorganisms which are capable of infecting plants and thus of transferring the constructs according to the invention. Preferred microorganisms are those from the genus Agrobacterium and, in particular, the species Agrobacterium tumefaciens.

[0131] Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia. Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria or other fungi. Plant organisms are furthermore, for the purposes of the invention, other organisms which are capable of photosynthetic activity such as, for example, algae or cyanobacteria, and also mosses. Preferred algae are green algae such as, for example, algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella.

[0132] Preferred eukaryotic cells and organism comprise plant cells and organisms, animal cells, and non-human animal organism, including eukaryotic microorganism such as yeast, algae, or fungi.

[0133] "Non-human animal organisms" includes but is not limited to non-human vertebrates and invertebrates. Preferred are fish species, non-human mammals such as cow, horse, sheep, goat, mouse, rat or pig, birds such as chicken or goose. Preferred animal cells comprise for example CHO, COS, HEK293 cells. Invertebrate organisms include for example nematodes and insects. Insect cells include for example Drosophila S2 and Spodoptera Sf9 or Sf21 cells.

[0134] Preferred nematodes are those which are capable to invade plant, animal or human organism. Preferred namtodes include for example nematodes of the genus Ancylostoma, Ascaridia, Ascaris, Bunostomum, Caenorhabditis, Capillaria, Chabertia, Cooperia, Dictyocaulus, Haemonchus, Heterakis, Nematodirus, Oesophagostomum, Ostertagia, Oxyuris, Parascaris, Strongylus, Toxascaris, Trichuris, Trichostrongylus, Tfhchonema, Toxocara or Uncinaria. Especially preferred are plant parasitic nematodes such as Bursaphalenchus, Criconemella, Diiylenchus, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Melodoigyne, Nacobbus, Paratylenchus, Pratylenchus, Radopholus, Rotelynchus, Tylenchus or Xiphinema. Preferred insects comprise those of the genus Coleoptera, Diptera, Lepidoptera, and Homoptera.

[0135] Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria or other fungi described in Indian Chem Engr. Section B. Vol 37, No 1, 2 (1995) on page 15, table 6. Especially preferred is the filamentic Hemiascomycete Ashbya gossypii.

[0136] Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia, especially preferred are Saccharomyces cerevisiae and Pichia pastoris (ATCC Accession No. 201178).

[0137] The term "plant" or "plant organism" as used herein refers to a plurality of plant cells which are largely 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. Host or target organisms which are preferred as transgenic organisms are especially plants. 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, seeds, shoots and seedlings and parts, propagation material and cultures derived therefrom, for example cell cultures. The term "mature plants" is understood as meaning plants at any developmental stage beyond the seedling. The term "seedling" is understood as meaning a young, immature plant in an early developmental stage.

[0138] Annual, biennial, monocotyledonous and dicotyledonous plants are preferred host organisms for the generation of transgenic plants. The expression of genes is furthermore advantageous in all ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawns. Plants which may be mentioned by way of example but not by limitation are angiosperms, bryophytes such as, for example, Hepaticae (liverworts) and Musci (mosses); Pteridophytes such as ferns, horsetail and club mosses; gymnosperms such as conifers, cycads, ginkgo and Gnetatae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms) and Euglenophyceae.

[0139] Preferred are plants which are used for food or feed purpose such as the families of the Leguminosae such as pea, alfalfa and soya; Gramineae such as rice, maize, wheat, barley, sorghum, millet, rye, triticale, or oats; the family of the Umbelliferae, especially the genus Daucus, very especially the species carota (carrot) and Apium, very especially the species Graveolens dulce (celery) and many others; the family of the Solanaceae, especially the genus Lycopersicon, very especially the species esculentum (tomato) and the genus Solanum, very especially the species tuberosum (potato) and melongena (egg plant), and many others (such as tobacco); and the genus Capsicum, very especially the species annuum (peppers) and many others; the family of the Leguminosae, especially the genus Glycine, very especially the species max (soybean), alfalfa, pea, lucerne, beans or peanut and many others; and the family of the Cruciferae (Brassicacae), especially the genus Brassica, very especially the species napus (oil seed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli); and of the genus Arabidopsis, very especially the species thaliana and many others; the family of the Compositae, especially the genus Lactuca, very especially the species sativa (lettuce) and many others; the family of the Asteraceae such as sunflower, Tagetes, lettuce or Calendula and many other; the family of the Cucurbitaceae such as melon, pumpkin/squash or zucchini, and linseed. Further preferred are cotton, sugar cane, hemp, flax, chillies, and the various tree, nut and wine species. Very especially preferred are are Arabidopsis thaliana, Nicotiana tabacum, Tagetes erecta, Calendula officinalis, Glycine max, Zea mays, Oryza sativa, Triticum aestivum, Pisum sativum, Phaseolus vulgaris, Hordium vulgare, Brassica napus.

[0140] The term "cell" refers to a single cell. The term "cells" refers to a population of cells. The population may be a pure population comprising one cell type. Likewise, the population may comprise more than one cell type. In the present invention, there is no limit on the number of cell types that a cell population may comprise. The cells may be synchronize or not synchronized, preferably the cells are synchronized.

[0141] The term "organ" with respect to a plant (or "plant organ") means parts of a plant and may include (but shall not limited to) for example roots, fruits, shoots, stem, leaves, anthers, sepals, petals, pollen, seeds, etc.

[0142] The term "tissue" with respect to a plant (or "plant tissue") means arrangement of multiple plant cells including differentiated and undifferentiated tissues of plants. Plant tissues may constitute part of a plant organ (e.g., the epidermis of a plant leaf) but may also constitute tumor tissues and various types of cells in culture (e.g., single cells, protoplasts, embryos, calli, protocorm-like bodies, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.

[0143] 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.

[0144] The term "structural gene" as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide.

[0145] The term "nucleotide sequence of interest" refers to any nucleotide sequence, the manipulation of which may be deemed desirable for any reason (e.g., confer improved qualities), by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product, (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).

[0146] The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and--optionally--the subsequent translation of mRNA into one or more polypeptides.

[0147] The term "expression cassette" or "expression construct" as used herein is intended to mean the combination of any nucleic acid sequence to be expressed in operable linkage with a promoter sequence and--optionally--additional elements (like e.g., terminator and/or polyadenylation sequences) which facilitate expression of said nucleic acid sequence.

[0148] The term "promoter," "promoter element," or "promoter sequence" as used herein, refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. A promoter is typically, though not necessarily, located 5' (i.e., upstream) of a nucleotide sequence of interest (e.g., proximal to the transcriptional start site of a structural gene) whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. A repressible promoters rate of transcription decreases in response to a repressing agent. An inducible promoters rate of transcription increases in response to an inducing agent. A constitutive promoters rate of transcription is not specifically regulated, though it can vary under the influence of general metabolic conditions.

[0149] Promoters may be tissue specific or cell specific. The term "tissue specific" as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., petals) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., roots). Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into every tissue of the resulting transgenic plant, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant. The detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected. The term "cell type specific" as applied to a promoter refers to a promoter which is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term "cell type specific" when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., GUS activity staining or immunohistochemical staining. Briefly, tissue sections are embedded in paraffin, and paraffin sections are reacted with a primary antibody which is specific for the polypeptide product encoded by the nucleotide sequence of interest whose expression is controlled by the promoter. A labeled (e.g., peroxidase conjugated) secondary antibody which is specific for the primary antibody is allowed to bind to the sectioned tissue and specific binding detected (e.g., with avidin/biotin) by microscopy.

[0150] Promoters may be constitutive or regulatable. The term "constitutive" when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a transgene in substantially any cell and any tissue. In contrast, a "regulatable" promoter is one which is capable of directing a level of transcription of an operably linked nuclei acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, light, etc.) which is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus.

[0151] The term "transcription terminator" or "transcription terminator sequence" as used herein is intended to mean a sequence which leads to or initiates a stop of transcription of a nucleic acid sequence initiated from a promoter. Preferably, a transcription terminator sequences is furthermore comprising sequences which cause polyadenylation of the transcript. A transcription terminator may, for example, comprise one or more polyadenylation signal sequences, one or more polyadenylation attachment sequences, and downstream sequence of various lengths which causes termination of transcription. It has to be understood that also sequences downstream of sequences coding for the 3'-untranslated region of an expressed RNA transcript may be part of a transcription terminator although the sequence itself is not expressed as part of the RNA transcript. Furthermore, a transcription terminator may comprise additional sequences, which may influence its functionality, such a 3'-untranslated sequences (i.e. sequences of a gene following the stop-codon of the coding sequence). Transcription termination may involve various mechanisms including but not limited to induced dissociation of RNA polymerase II from their DNA template. As virtually all biological reactions transcription termination is never of 100% efficiency. The term "transcription termination efficiency" or "efficiency or transcription termination" as used herein is indicating the ratio between the frequencies of stops (or termination) of transcription in the region of said transcription terminator to the frequency of read-through transcription beyond said transcription terminator. The term "tight" or "efficient" in relation to transcription termination sequence as used herein is understood as a transcription termination sequence for which the efficiency of transcription termination is at least 10 (i.e. stop/read-through ratio of 10:1), preferably at least 100 (i.e. stop/read-through ratio of 100:1), more preferably 1000 (i.e. stop/read-through ratio of 1000:1). Transcription may end at one or more specific base pairs within said transcription terminator sequence. In consequence, there might be variability in the length of the transcript. However, preferably transcription termination has a low variability and end to at least 50%, preferably at least 80%, more preferably at least 90% at one specific base pair as judged by the resulting transcript length (excluding the poly-A tail).

[0152] The term "operable linkage" or "operably linked" is to be understood as meaning, for example, the sequential arrangement of a regulatory element (e.g. a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements (such as e.g., a terminator) in such a way that each of the regulatory elements can fulfill its intended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence. The expression may result depending on the arrangement of the nucleic acid sequences in relation to sense or antisense RNA. To this end, direct linkage in the chemical sense is not necessarily required. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions which are further away, or indeed from other DNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs. Operable linkage, and an expression construct, can be generated by means of customary recombination and cloning techniques as described (e.g., in Maniatis 1989; Silhavy 1984; Ausubel 1987; Gelvin 1990). However, further sequences which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences. The insertion of sequences may also lead to the expression of fusion proteins. Preferably, the expression construct, consisting of a linkage of promoter and nucleic acid sequence to be expressed, can exist in a vector-integrated form and be inserted into a plant genome, for example by transformation.

[0153] The term "transformation" as used herein refers to the introduction of genetic material (e.g., a transgene) into a cell. Transformation of a cell may be stable or transient. The term "transient transformation" or "transiently transformed" refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell's genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELISA) which detects the presence of a polypeptide encoded by one or more of the transgenes. Alternatively, transient transformation may be detected by detecting the activity of the protein (e.g., β-glucuronidase) encoded by the transgene (e.g., the uid A gene) as demonstrated herein [e.g., histochemical assay of GUS enzyme activity by staining with X-gluc which gives a blue precipitate in the presence of the GUS enzyme; and a chemiluminescent assay of GUS enzyme activity using the GUS-Light kit (Tropix)]. The term "transient transformant" refers to a cell which has transiently incorporated one or more transgenes.

[0154] In contrast, the term "stable transformation" or "stably transformed" refers to the introduction and integration of one or more transgenes into the genome of a cell, preferably resulting in chromosomal integration and stable heritability through meiosis. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences which are capable of binding to one or more of the transgenes. Alternatively, stable transformation of a cell may also be detected by the polymerase chain reaction of genomic DNA of the cell to amplify transgene sequences. The term "stable transformant" refers to a cell which has stably integrated one or more transgenes into the genomic DNA. Thus, a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene. Transformation also includes introduction of genetic material into plant cells in the form of plant viral vectors involving epichromosomal replication and gene expression which may exhibit variable properties with respect to meiotic stability. Stable transformation also includes introduction of genetic material into cells in the form of viral vectors involving epichromosomal replication and gene expression which may exhibit variable properties with respect to meiotic stability.

[0155] Cloning and transformation techniques for manipulation of ciliates and algae are well known in the art (WO 98/01572; Falciatore 1999; Dunahay 1995).

[0156] Principally speaking transformation techniques suitable for plant cells or organisms (as described below) can also be employed for animal or yeast organism and cells. Preferred are direct transformation techniques such as calcium phosphate or liposome mediated transformation, or electroporation.

[0157] The terms "infecting" and "infection" with a bacterium refer to co-incubation of a target biological sample, (e.g., cell, tissue, etc.) with the bacterium under conditions such that nucleic acid sequences contained within the bacterium are introduced into one or more cells of the target biological sample.

[0158] The term "Agrobacterium" refers to a soil-borne, Gram-negative, rod-shaped phytopathogenic bacterium which causes crown gall. The term "Agrobacterium" includes, but is not limited to, the strains Agrobacterium tumefaciens, (which typically causes crown gall in infected plants), and Agrobacterium rhizogenes (which causes hairy root disease in infected host plants). Infection of a plant cell with Agrobacterium generally results in the production of opines (e.g., nopaline, agropine, octopine etc.) by the infected cell. Thus, Agrobacterium strains which cause production of nopaline (e.g., strain LBA4301, C58, A208) are referred to as "nopaline-type" Agrobacteria; Agrobacterium strains which cause production of octopine (e.g., strain LBA4404, Ach5, B6) are referred to as "octopine-type" Agrobacteria; and Agrobacterium strains which cause production of agropine (e.g., strain EHA105, EHA101, A281) are referred to as "agropine-type" Agrobacteria.

[0159] The terms "bombarding, "bombardment," and "biolistic bombardment" refer to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/or entry of the particles into the target biological sample. Methods for biolistic bombardment are known in the art (e.g., U.S. Pat. No. 5,584,807, the contents of which are herein incorporated by reference), and are commercially available (e.g., the helium gas-driven microprojectile accelerator (PDS-1000/He) (BioRad).

[0160] The terms "homology" or "identity" when used in relation to nucleic acids refers to a degree of complementarity. Homology or identity between two nucleic acids is understood as meaning the identity of the nucleic acid sequence over in each case the entire length of the sequence, which is calculated by comparison with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) with the parameters being set as follows:

TABLE-US-00001 Gap Weight: 12 Length Weight: 4 Average Match: 2,912 Average Mismatch: -2,003

[0161] Alternatively, a partially complementary sequence is understood to be one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid and is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe (i.e., an oligonucleotide which is capable of hybridizing to another oligonucleotide of interest) will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

[0162] When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described infra. When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe which can hybridize to the single-stranded nucleic acid sequence under conditions of low stringency as described infra.

[0163] The terms "hybridization" and "hybridizing" as used herein includes "any process by which a strand of nucleic acid joins with a complementary strand through base pairing." (Coombs 1994). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids.

[0164] As used herein, the term "Tm" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson 1985). Other references include more sophisticated computations which take structural as well as sequence characteristics into account for the calculation of Tm.

[0165] Low stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE (43.8 g/L NaCl, 6.9 g/L NaH₂PO₄.H₂O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH), 1% SDS, 5×Denhardt's reagent [50×Denhardt's contains the following per 500 mL: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.2×SSPE, and 0.1% SDS at room temperature when a DNA probe of about 100 to about 1000 nucleotides in length is employed.

[0166] High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE, 1% SDS, 5×Denhardt's reagent and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1×SSPE, and 0.1% SDS at 68° C. when a probe of about 100 to about 1000 nucleotides in length is employed.

[0167] The term "equivalent" when made in reference to a hybridization condition as it relates to a hybridization condition of interest means that the hybridization condition and the hybridization condition of interest result in hybridization of nucleic acid sequences which have the same range of percent (%) homology. For example, if a hybridization condition of interest results in hybridization of a first nucleic acid sequence with other nucleic acid sequences that have from 80% to 90% homology to the first nucleic acid sequence, then another hybridization condition is said to be equivalent to the hybridization condition of interest if this other hybridization condition also results in hybridization of the first nucleic acid sequence with the other nucleic acid sequences that have from 80% to 90% homology to the first nucleic acid sequence.

[0168] When used in reference to nucleic acid hybridization the art knows well that numerous equivalent conditions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above-listed conditions. Those skilled in the art know that whereas higher stringencies may be preferred to reduce or eliminate non-specific binding between the nucleotide sequence of interest and other nucleic acid sequences, lower stringencies may be preferred to detect a larger number of nucleic acid sequences having different homologies to the nucleotide sequence of interest.

[0169] The term "recognition sequence" refers to a particular sequences which a protein, chemical compound, DNA, or RNA molecule (e.g., restriction endonuclease, a modification methylase, or a recombinase) recognizes and binds. With respect to a recombinase a recognition sequence will usually refer to a recombination site. For example, the recognition sequence for Cre recombinase is loxP which is a 34 base pair sequence comprised of two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see Sauer 1994; FIG. 1). Other examples of recognition sequences are the attB, attP, attL, and attR sequences which are recognized by the recombinase enzyme λ Integrase. attB is an approximately 25 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region. attP is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins integration host factor (IHF), FIS and excisionase (Xis) (see Landy 1993). Such sites may also be engineered according to the present invention to enhance production of products in the methods of the invention. When such engineered sites lack the P1 or H1 domains to make the recombination reactions irreversible (e.g., attR or attP), such sites may be designated attR' or attP' to show that the domains of these sites have been modified in some way.

[0170] The term "recombinase" is referring to an enzyme which catalyzes the exchange of DNA segments at specific recombination sites.

[0171] The term "recombinational cloning" is referring to a method, whereby segments of nucleic acid molecules or populations of such molecules are exchanged, inserted, replaced, substituted or modified, in vitro or in vivo, by action of a site-specific recombinase.

[0172] The term "Recombination proteins" refers to polypeptide including excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (Landy 1993).

[0173] Repression cassette: is a nucleic acid segment that contains a repressor of a Selectable marker present in the subcloning vector.

[0174] The term "site-specific recombinase" as used herein is referring to a type of recombinase which typically has at least the following four activities (or combinations thereof): (1) recognition of one or two specific nucleic acid sequences; (2) cleavage of said sequence or sequences; (3) topoisomerase activity involved in strand exchange; and (4) ligase activity to reseal the cleaved strands of nucleic acid (see Sauer 1994). Conservative site-specific recombination is distinguished from homologous recombination and transposition by a high degree of specificity for both partners. The strand exchange mechanism involves the cleavage and rejoining of specific DNA sequences in the absence of DNA synthesis (Landy 1989).

[0175] The term "vector" is referring to a nucleic acid molecule (preferably DNA) that provides a useful biological or biochemical property to an inserted nucleic acid sequence, preferably allows replication and/or transformation or transfection into host cells and organisms. Examples include plasmids, phages, autonomously replicating sequences (ARS), centromeres, and other sequences which are able to replicate or be replicated in vitro or in a host cell, or to convey a desired nucleic acid segment to a desired location within a host cell. A Vector can have one or more restriction endonuclease recognition sites at which the sequences can be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a nucleic acid fragment can be spliced in order to bring about its replication and cloning. Vectors can further provide primer sites, e.g., for PCR, transcriptional and/or translational initiation and/or regulation sites, recombinational signals, replicons, Selectable markers, etc. Clearly, methods of inserting a desired nucleic acid fragment which do not require the use of homologous recombination, transpositions or restriction enzymes (such as, but not limited to, UDG cloning of PCR fragments (U.S. Pat. No. 5,334,575, entirely incorporated herein by reference), TA Cloning® brand PCR cloning (Invitrogen Corp., Carlsbad, Calif.), and the like) can also be applied to clone a fragment into a cloning vector to be used according to the present invention. The cloning vector can further contain one or more selectable markers suitable for use in the identification of cells transformed with the cloning vector.

[0176] The term "primer" refers to a single stranded or double stranded oligonucleotide that is extended by covalent bonding of nucleotide monomers during amplification or polymerization of a nucleic acid molecule (e.g. a DNA molecule). In a preferred aspect, the primer comprises one or more recombination sites or portions of such recombination sites. Portions of recombination sties comprise at least 2 bases, at least 5 bases, at least 10 bases or at least 20 bases of the recombination sites of interest. When using portions of recombination sites, the missing portion of the recombination site may be provided by the newly synthesized nucleic acid molecule. Such recombination sites may be located within and/or at one or both termini of the primer. Preferably, additional sequences are added to the primer adjacent to the recombination site(s) to enhance or improve recombination and/or to stabilize the recombination site during recombination. Such stabilization sequences may be any sequences (preferably G/C rich sequences) of any length. Preferably, such sequences range in size from 1 to about 1,000 bases, 1 to about 500 bases, and 1 to about 100 bases, 1 to about 60 bases, 1 to about 25, 1 to about 10, 2 to about 10 and preferably about 4 bases. Preferably, such sequences are greater than 1 base in length and preferably greater than 2 bases in length.

[0177] The term "template" refers to double stranded or single stranded nucleic acid molecules which are to be amplified, synthesized or sequenced. In the case of double stranded molecules, denaturation of its strands to form a first and a second strand is preferably performed before these molecules will be amplified, synthesized or sequenced, or the double stranded molecule may be used directly as a template. For single stranded templates, a primer complementary to a portion of the template is hybridized under appropriate conditions and one or more polypeptides having polymerase activity (e.g. DNA polymerases and/or reverse transcriptases) may then synthesize a nucleic acid molecule complementary to all or a portion of said template. Alternatively, for double stranded templates, one or more promoters may be used in combination with one or more polymerases to make nucleic acid molecules complementary to all or a portion of the template. The newly synthesized molecules, according to the invention, may be equal or shorter in length than the original template. Additionally, a population of nucleic acid templates may be used during synthesis or amplification to produce a population of nucleic acid molecules typically representative of the original template population.

[0178] The term "adapter" refers to an oligonucleotide or nucleic acid fragment or segment (preferably DNA) which comprises one or more recognition sites (e.g., recombination sites or recognition sites for restriction endonucleases) which can be added to a circular or linear DNA molecule as well as other nucleic acid molecules described herein. Such adapters may be added at any location within a circular or linear molecule, although the adapters are preferably added at or near one or both termini of a linear molecule. Preferably, adapters are positioned to be located on both sides (flanking) a particularly nucleic acid molecule of interest. The synthesis of adapters (e.g., by oligonucleotide synthesis, annealing procedures, and or PCR) is a standard technique well known to the person skilled in the art. In accordance with the invention, adapters may be added to nucleic acid molecules of interest by standard recombinant techniques (e.g. restriction digest and ligation). For example, adapters may be added to a circular molecule by first digesting the molecule with an appropriate restriction enzyme, adding the adapter at the cleavage site and reforming the circular molecule which contains the adapter(s) at the site of cleavage. Alternatively, adapters may be ligated directly to one or more and preferably both termini of a linear molecule thereby resulting in linear molecule(s) having adapters at one or both termini. In one aspect of the invention, adapters may be added to a population of linear molecules, (e.g. a cDNA library or genomic DNA which has been cleaved or digested) to form a population of linear molecules containing adapters at one and preferably both termini of all or substantial portion of said population.

[0179] Other terms used in the fields of recombinant DNA technology and molecular and cell biology as used herein will be generally understood by one of ordinary skill in the applicable arts.

DETAILED DESCRIPTION OF THE INVENTION

[0180] A first subject matter of the invention relates to a method for identification and isolation of transcription termination sequences for comprising the steps of:

[0181] i) providing a screening construct or screening vector comprising

[0182] a) a promoter sequence, and

[0183] b) one or more insertion sites--preferably a restriction or recombination site--for insertion of DNA sequences, and

[0184] c) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic, wherein insertion of an efficient transcription terminator into said insertion site changes expression of said additional sequences by said promoter sequence in comparison to no insertion, and

[0185] ii) providing one or more DNA sequences to be assessed for their transcription termination capability, and

[0186] iii) inserting one or more copies of said DNA sequences into said insertion site of said screening construct or screening vector, and

[0187] iv) introducing said screening construct or screening vector with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence, and

[0188] v) identifying and/or selecting screening construct or screening vector with a changed readily detectable characteristic in comparison to no insertion, and

[0189] vi) isolating the inserted DNA sequences from said identified and/or selected screening construct or screening vector for use as transcription termination sequences and--optionally--determining their sequence.

[0190] By the method of the invention new transcription terminator sequences can be readily identified. The method can be based either on an in vitro or in vivo screening system. The screening method of the invention allows for selection of DNA sequences, screening constructs or screening vectors, and/or cells or organism (preferably plant cells and plant organisms) containing efficient transcription terminator sequences. Previously terminators had to be evaluated sequence-by-sequence. Testing of termination efficiency (tightness) was laborious. The methods of the present invention are time-efficient and very sensitive so that only very tight terminators will be identified. Tight terminators identified by this screening system will be used for the expression cassettes, which will reduce read through between cassettes and increase stability of the transgene expression. Discovery of various terminators of interest will provide opportunity to understand better termination of transcription in planta.

[0191] The term "readily detectable characteristic" as used herein is to be understood in the broad sense and may include any change of a characteristic, preferably a phenotypic characteristic. "Change" in this context may include increasing or decreasing said characteristic. In consequence, expression of said additional sequences under control of said promoter may cause increasing or decreasing a phenotypic characteristic. For example expression may cause a herbicide resistance (increased resistance) or may cause an toxic effect by expression of e.g., a toxic gene (decreased viability). Since depending on the localization of the insertion site in relation to said additional sequences (as described below in detail) an efficient transcription terminator may result in increased (preferably initiated) or decreased (preferably silenced) expression of said additional sequences both type of changes can be advantageously employed.

1. Localization of the Insertion Site

[0192] The insertion site may have various localizations with respect to the additional sequences which bring about the readily detectable characteristic:

1.1 Variation A:

[0193] For example the insertion site may be localized upstream (i.e. in 5' direction) of the additional sequences so that the insertion site is between the promoter sequences and said additional sequences (hereinafter "Variation A"). In case an efficient transcription terminator sequences in inserted into said insertion site transcription, transcription will stop before said additional sequences and no read-through transcription into this additional sequences will occur. In this case an efficient transcription terminator will result in decreased or preferably completely suppressed expression of the additional sequences. Depending whether presence or absence of transcription (i.e. expression) of said additional sequences brings about said readily detectable characteristic (which both is possible depending on the type of additional sequence employed) said readily detectable characteristic diminishes or is expressed. In any case an efficient transcription terminator will cause a changed readily detectable characteristic, which may be suppressed or increased in comparison to a scenario where no sequence is inserted into the insertion site. Thus in a preferred embodiment of the invention the method for identification and isolation of transcription termination sequences comprises the steps of:

[0194] i) providing a screening construct or screening vector comprising in 5' to 3' direction

[0195] a) a promoter sequence, and

[0196] b) one or more insertion sites--preferably a restriction or recombination site--for insertion of DNA sequences, and

[0197] c) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic,

[0198] wherein insertion of an efficient transcription terminator into said insertion site suppresses expression of said additional sequences by said promoter sequence in comparison to no insertion, and

[0199] ii) providing one or more DNA sequences to be assessed for their transcription termination capability, and

[0200] iii) inserting one or more copies of said DNA sequences into said insertion site of said screening construct or screening vector, and

[0201] iv) introducing said screening construct or screening vector with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence, and

[0202] v) identifying and/or selecting screening constructs or screening vectors with a changed readily detectable characteristic in comparison to no insertion, and

[0203] vi) isolating the inserted DNA sequences from said identified and/or selected screening constructs or screening vectors for use as transcription termination sequences and--optionally--determining their sequence.

[0204] As described below the additional sequences localized downstream of the insertion site may bring about said readily detectable characteristic only by expression (i.e. transcription) of an RNA (e.g., in cases where said additional sequences are forming an antisense RNA or dsRNA molecule) or by expression (i.e. transcription and translation of a protein). In the latter case it has to be ensured that appropriate translation can occur. This can be ensured by for example avoiding upstream ATG-codons, cloning the sequences in-frame with upstream coding sequences or--preferably--employing IRES sites which may allow efficient translation even in cases where the ATG codon is not close to the 5'-end of the transcript (Vagner 2001; for sequences see e.g., ifr31w3.toulouse.inserm.fr/IRESdatabase/).

1.2 Variation B:

[0205] In another preferred embodiment of the invention the insertion site may also be arranged downstream (i.e. in 3'-direction) of the additional sequences (hereinafter "Variation B"). For this variation the DNA sequences to be inserted into the insertion sites for evaluation for their transcription termination capability are preferably inserted in form of an inverted repeat. In case the inserted DNA sequence is an efficient transcription terminator only the first copy (i.e. first part) of the inverted repeat will be transcribed and normal expression of the additional sequences will occur. Depending whether presence or absence of transcription (i.e. expression) of said additional sequences brings about said readily detectable characteristic (which both is possible depending on the type of additional sequence employed) said readily detectable characteristic diminishes or is expressed. In any case an efficient transcription terminator will cause a changed readily detectable characteristic, which may be suppressed or increased in comparison to a scenario where no sequence is inserted into the insertion site. However, if only a weak transcription terminator or a sequence with no transcription termination capability at all is inserted the entire inverted repeat (i.e. both copies of the inserted sequence) will be transcribed causing transcription of a RNA comprising a double-stranded hairpin structure (formed by the RNA transcribed from inverted repeat of the inserted DNA sequences). This RNA by means of double-stranded RNA interference (dsRNAi) will cause gene silencing of its own expression (self-suppression or self-silencing) resulting in gene silencing of the expression cassette comprising said additional sequences. In this case an efficient transcription terminator will result in increased expression of the additional sequences and the detectable characteristic will change in just the other direction as in case of an efficient transcription terminator. Thus in a preferred embodiment of the invention the method for identification and isolation of transcription termination sequences comprises the steps of:

[0206] i) providing a screening construct or screening vector comprising in 5' to 3' direction

[0207] a) a promoter sequence, and

[0208] b) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic, and

[0209] c) one or more insertion sites--preferably a restriction or recombination site--for insertion of DNA sequences,

[0210] ii) providing one or more DNA sequences to be assessed for their transcription termination capability, and

[0211] iii) inserting at least two copies of a specific DNA sequence of said DNA sequences in form of an inverted repeat into said insertion site of said screening construct or screening vector, wherein insertion of an inverted repeat of an efficient transcription terminator into said insertion site allows expression of said additional sequences by said promoter sequence in comparison to no insertion, and

[0212] iv) introducing said screening constructs or screening vectors with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence, and

[0213] v) identifying and/or selecting screening constructs or screening vectors with said readily detectable characteristic in comparison to no insertion, and

[0214] vi) isolating the inserted DNA sequences from said identified and/or selected screening constructs or screening vectors for use as transcription termination sequences and--optionally--determining their sequence.

[0215] In a preferred embodiment of this variation two different promoters and two different additional sequences are employed. These two expression cassettes are arranged in a "tail-to-tail" orientation so that transcription initiated from said promoters in running against each other. Preferably the insertion site for the inverted repeat in between the two end (tails) of the two expression cassettes. Insertion of an inverted repeat of weak transcription terminator will result of gene silencing of both additional sequences, while insertion of an efficient transcription terminator results in expression of both additional sequences. In consequence a double-check of the transcription termination efficiency becomes feasible. Preferably one of the sequences is selected from the group of negative selection marker (thus an efficient transcription terminator will result in for example a herbicide or antibiotic resistance). The other additional sequence may be selected from the group of reporter genes (for example GFP or GUS; thus an efficient transcription terminator will result in an easily detectable color).

1.3 Variation C:

[0216] In a third preferred embodiment of the invention the insertion site may also be arranged within the additional sequences (for example and preferably embedded into an intron, which is located in said additional sequences) (hereinafter "Variation C"). The full-length transcript of said additional sequence is only made, if the sequence inserted into said insertion site does not control tight transcription termination and expression of said additional sequences will cause a change of said readily detectable characteristic. In case of an efficient transcription terminator inserted into said insertion site no full-length transcript will be produced. Thus in a preferred embodiment of the invention the method for identification and isolation of transcription termination sequences comprises the steps of:

[0217] i) providing a screening construct or screening vector comprising in 5' to 3' direction

[0218] a) a promoter sequence, and

[0219] b) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic, and embedded into said additional sequences one or more insertion sites--preferably a restriction or recombination site--for insertion of DNA sequences,

[0220] wherein insertion of an efficient transcription terminator into said insertion site suppresses full-length transcription of said additional sequences by said promoter sequence in comparison to no insertion, and

[0221] ii) providing one or more DNA sequences to be assessed for their transcription termination capability, and

[0222] iii) inserting one or more copies of said DNA sequences into said insertion site of said screening construct or screening vector, and

[0223] iv) introducing said screening constructs or screening vectors with said inserted DNA sequences into an in vitro or in vivo transcription system suitable to induce expression from said promoter sequence, and

[0224] v) identifying and/or selecting screening constructs or screening vectors with a changed readily detectable characteristic in comparison to no insertion, and

[0225] vi) isolating the inserted DNA sequences from said identified and/or selected screening constructs or screening vectors for use as transcription termination sequences and--optionally--determining their sequence.

[0226] Localisation within the additional sequences may be realized by various ways. In an improved embodiment of the invention, the insertion site for the transcription terminator sequences is localized within an intron comprised in said additional sequences. Efficient transcription termination will lead to incomplete transcription of the intron and the additional sequences, thereby preventing the phenotype caused by said additional sequences to occur. The additional sequence to be expressed may be--for example--a toxic gene (such as diphtheria toxin A) or a reporter gene (such as GFP). Additional examples are given below. In case of efficient transcription termination the phenotype corresponding to said sequences is not established. In case of toxic genes only stably transformed cell lines can be established which comprise an efficient transcription terminator sequence.

[0227] In another preferred embodiment the insertion site for the transcription termination sequences is localized between a first 5'-part of the additional sequences, which--for example--encodes for a reporter gene (such as GPF) and a 3'-part, which is preferably a non-protein encoding sequence with no homologous sequences in plants (such as for example part of luciferase gene as used in the examples below or sequences of bacteriophage λ). In addition to this screening construct another expression cassette is employed which is expressing an antisense or--preferably--a double-stranded RNA sequence corresponding to said 3'-part sequence. In case of an efficient transcription terminator, transcription of the additional sequence will stop at the terminator site and the 5'-end sequence will not be translated. In case of an inefficient (leaky) transcription terminator transcription will read-through into said 3'-part sequence thereby establishing a target for the antisense- or double-stranded RNA. This will cause degradation of the entire construct, including the region encoding for the marker sequence, thereby "silencing" the related phenotype (e.g., marker signal).

2. The Screening Construct or Screening Vector of the Invention

[0228] The screening constructs and screening vectors to be employed for the method of the invention may have various forms. In principle, any form is suitable which allows for expression or transcription of an RNA molecule. In consequence the screening construct or screening vector may be for example an RNA or a DNA molecule, it further may be single-stranded or double-stranded, and it may be linear or circular. Any combination of the before mentioned alternatives is included.

[0229] Screening constructs can be advantageously employed in scenarios were no replication is required, such as for example the in vitro screening system described below. However, preferably, a screening vector is employed. Said screening vector may be a RNA vector (such as for example a RNA virus vector) or--preferably--a DNA vector. More preferably the screening vector is a circular double-stranded DNA plasmid vector.

[0230] As essential feature the screening construct or screening vector of the invention comprises

[0231] a) a promoter sequence, and

[0232] b) additional sequences which causes upon expression under said promoter sequence a readily detectable characteristic.

2.1 Promoters for the Invention

[0233] The promoter is preferably chosen to be functional in the in vitro or in vivo system where evaluation of said transcription termination sequences is going to be carried out. Preferably, this system is similar or identical to the system where the transcription termination sequence should function in later expression constructs. For example, if a transcription terminator sequence is desired for plant organisms, a transcription system based on plant cells (either an in vitro system such as wheat germ extracts or a in vivo system such as a plant cell or a plant) is employed. In such a case the promoter sequences is preferably a sequence which is able to initiate transcription in plants, preferably an endogenous plant promoter or a promoter derived from a plant pathogen (such as a plant virus or Agrobacterium). Various promoters are known to the person skilled in the art for the various transcriptions systems or hosts for which the method of the invention can be employed.

[0234] As an illustration, promoters (and if necessary other transcriptional and translational regulatory signals) suitable for a mammalian host may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene that has a high level of expression. Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes. Illustrative eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer 1982), the TK promoter of Herpes virus (McKnight 1982), the SV40 early promoter (Benoist 1981), the Rous sarcoma virus promoter (Gorman 1982), the cytomegalovirus promoter (Foecking 1980), and the mouse mammary tumor virus promoter (see, generally, Etcheverry 1996). Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA polymerase promoter, can be used to control expression of the gene of interest in mammalian cells if the prokaryotic polymerase is expressed by an eukaryotic promoter (Zhou 1990; Kaufman 1991).

[0235] For expression in plants, plant-specific promoters are preferred. The term "plant-specific promoter" is understood as meaning, in principle, any promoter which is capable of governing the expression of genes, in particular foreign genes, in plants or plant parts, plant cells, plant tissues or plant cultures. In this context, expression can be, for example, constitutive, inducible or development-dependent. The following are preferred:

a) Constitutive Promoters

[0236] "Constitutive" promoters refers to those promoters which ensure expression in a large number of, preferably all, tissues over a substantial period of plant development, preferably at all times during plant development. A plant promoter or promoter originating from a plant virus is especially preferably used. The promoter of the CaMV (cauliflower mosaic virus) 35S transcript (Franck 1980; Odell 1985; Shewmaker 1985; Gardner 1986) or the 19S CaMV promoter (U.S. Pat. No. 5,352,605; WO 84/02913) is especially preferred. Another suitable constitutive promoter is the rice actin promoter (McElroy 1990), Rubisco small subunit (SSU) promoter (U.S. Pat. No. 4,962,028), the legumin B promoter (GenBank Acc. No. X03677), the promoter of the nopaline synthase from Agrobacterium, the TR dual promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S 1995), the ubiquitin 1 promoter (Christensen 1989, 1992; Bruce et al. 1989), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the promoters of the vacuolar ATPase subunits, the pEMU promoter (Last 1991); the MAS promoter (Velten 1984) and maize H3 histone promoter (Lepetit 1992; Atanassova 1992), the promoter of the Arabidopsis thaliana nitrilase-1 gene (GeneBank Acc. No.: U38846, nucleotides 3862 to 5325 or else 5342) or the promoter of a proline-rich protein from wheat (WO 91/13991), and further promoters of genes whose constitutive expression in plants.

b) Tissue-Specific or Tissue-Preferred Promoters

[0237] Furthermore preferred are promoters with specificities for seeds, such as, for example, the phaseolin promoter (U.S. Pat. No. 5,504,200; Bustos et al. 1989; Murai 1983; Sengupta-Gopalan 1985), the promoter of the 2S albumin gene (Joseffson 1987), the legumine promoter (Shirsat 1989), the USP (unknown seed protein) promoter (Baumlein 1991a), the napin gene promoter (U.S. Pat. No. 5,608,152; Stalberg 1996), the promoter of the sucrose binding proteins (WO 00/26388) or the legumin B4 promoter (LeB4; Baumlein (1991b), the Arabidopsis oleosin promoter (WO 98/45461), and the Brassica Bce4 promoter (WO 91/13980). Further preferred are a leaf-specific and light-induced promoter such as that from cab or Rubisco (Simpson 1985; Timko 1985); an anther-specific promoter such as that from LAT52 (Twell 1989b); a pollen-specific promoter such as that from Zml3 (Guerrero et al. (1993) Mol Gen Genet 224:161-168); and a microspore-preferred promoter such as that from apg (Twell et al. 1983).

c) Chemically Inducible Promoters

[0238] The expression cassettes may also contain a chemically inducible promoter (review article: Gatz 1997), by means of which the expression of the exogenous gene in the plant can be controlled at a particular point in time. Such promoters such as, for example, the PRP1 promoter (Ward 1993), a salicylic acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186), a tetracyclin-inducible promoter (Gatz 1991, 1992), an abscisic acid-inducible promoter (EP-A1 0 335 528) or an ethanol-cyclohexanone-inducible promoter (WO 93/21334) can likewise be used. Also suitable is the promoter of the glutathione-S transferase isoform II gene (GST-II-27), which can be activated by exogenously applied safeners such as, for example, N,N-diallyl-2,2-dichloroacetamide (WO 93/01294) and which is operable in a large number of tissues of both monocots and dicots. Further exemplary inducible promoters that can be utilized in the instant invention include that from the ACE1 system which responds to copper (Mett 1993); or the In2 promoter from maize which responds to benzenesulfonamide herbicide safeners (Hershey 1991; Gatz 1994). A promoter that responds to an inducing agent to which plants do not normally respond can be utilized. An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena 1991).

[0239] Particularly preferred are constitutive promoters. Furthermore, further promoters may be linked operably to the nucleic acid sequence to be expressed, which promoters make possible the expression in further plant tissues or in other organisms, such as, for example, E. coli bacteria. Suitable plant promoters are, in principle, all of the above-described promoters.

2.2 Additional Sequences for the Invention

[0240] The "additional sequence", which causes upon expression under said promoter sequence a readily detectable characteristic, can be selected from a broad variety of sequences. Selection may depend on various factors, for example, whether insertion of an efficient transcription terminator into said insertion site is expected to result in decreased expression (Variation A or C) or increased expression (Variation B) or the additional sequences in its functional form (i.e., which brings about the readily detectable characteristic).

[0241] For expected decreased expression (Variation A and C) it is preferred to employ a sequence which encodes for a selectable marker selected from the group consisting of a reporter gene, a counter selection marker, or a toxic gene. In an preferred embodiment of the in vivo screening systems of the invention, the expression of a toxic gene as the additional sequences will cause a inhibition of growth, propagation and/or or regeneration of said cells or organisms (e.g., plant cells or plants). In consequence, only cells or organisms will survive if an efficient ("tight") transcription termination sequence is inserted in front of said toxic phenotype causing sequence thereby preventing expression of this growth, propagation and/or or regeneration inhibiting sequences. The surviving cells can be isolated and the transcription terminator sequence can be identified and isolated, e.g., by amplification using PCR followed by sequencing.

[0242] For expected increased expression (Variation B) it is preferred to employ a sequence which encodes for a selectable marker selected from the group consisting of a reporter gene, a negative selection marker, or a positive selection marker.

[0243] The term "selection marker" refers to any nucleic acid or amino acid sequence which is useful to select and separate cells or organism comprising said selection marker from others not comprising it. Selection marker may comprise sequences which

[0244] i) allow for separation of cells or organism comprising said marker by conferring a resistance against an otherwise toxic compound (named herein within "negative selection marker"),

[0245] ii) allow for separation of cells or organism comprising said marker by conferring a growth advantage to said cells or organism (named herein within "positive selection marker").

[0246] Selection marker may further comprise sequences which allow for separation of cells or organism not comprising said marker by conferring a growth disadvantage to cells or organism comprising said marker (named herein within "counter selection marker" or "toxic gene").

[0247] Selection markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like. Examples of Selectable markers include but are not limited to:

[0248] (a) a DNA segment that encodes a product that provides resistance in a recipient cell or organism against otherwise toxic compounds ("Negative Selection Marker"); (e.g., antibiotics). Negative Selection Markers confer a resistance to a biocidal compound such as a metabolic inhibitor (e.g., 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin or glyphosate). Especially preferred Negative Selection Markers are those which confer resistance to herbicides. Examples which may be mentioned are:

[0249] Phosphinothricin acetyltransferases (PAT; also named Bialophos ° resistance; bar; de Block 1987; EP 0 333 033; U.S. Pat. No. 4,975,374)

[0250] 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) conferring resistance to Glyphosate® (N-(phosphonomethyl)glycine) (Shah 1986)

[0251] Glyphosate® degrading enzymes (Glyphosate® oxidoreductase; gox),

[0252] Dalapon® inactivating dehalogenases (deh)

[0253] sulfonylurea- and imidazolinone-inactivating acetolactate synthases (for example mutated ALS variants with, for example, the S4 and/or Hra mutation

[0254] Bromoxynil® degrading nitrilases (bxn)

[0255] Kanamycin- or. G418-resistance genes (NPTII; NPTI) coding e.g., for neomycin phosphotransferases (Fraley et al. 1983)

[0256] 2-Desoxyglucose-6-phosphate phosphatase (DOG.sup.R1-Gene product; WO 98/45456; EP 0 807 836) conferring resistance against 2-desoxyglucose (Randez-Gil et al. 1995).

[0257] hygromycin phosphotransferase (HPT), which mediates resistance to hygromycin (Vanden Elzen et al. 1985).

[0258] dihydrofolate reductase (Eichholtz et al. 1987)

[0259] Additional negative selectable marker genes of bacterial origin that confer resistance to antibiotics include the aadA gene, which confers resistance to the antibiotic spectinomycin, gentamycin acetyl transferase, streptomycin phosphotransferase (SPT), aminoglycoside-3-adenyl transferase and the bleomycin resistance determinant (Hayford 1988; Jones 1987; Svab 1990; Hille 1986).

[0260] Especially preferred are negative selection markers which confer resistance against the toxic effects imposed by D-amino acids like e.g., D-alanine and D-serine (WO 03/060133; Erikson 2004). Especially preferred as negative selection marker in this contest are the daol gene (EC: 1.4.3.3: GenBank Acc.-No.: U60066) from the yeast Rhodotorula gracilis (Rhodosporidium toruloides) and the E. coli gene dsdA (D-serine dehydratase (D-serine deaminase) [EC: 4.3.1.18; GenBank Acc.-No.: J01603). Selection Marker suitable in prokaryotic or non-plant eukaryotic systems can also be based on the Selection Markers described above for plants (beside that expression cassettes are based on other host-specific promoters). For mammal cells preferred are resistance against neomycin (G418), hygromycin, Bleomycin, Zeocin Gatignol 1987; Drocourt 1990), puromycin (see, for example, Kaufman 1990a, 1990b). Corresponding selectable marker genes are known in the art (see, for example, Srivastava 1991; Romanos 1995; Markie 1996; Pfeifer 1997; Tucker 1997; Hashida-Okado 1998). For prokaryotes preferred are resistances against Ampicillin, Kanamycin, Spectinomycin, or Tetracyclin. Selectable marker genes can be cloned or synthesized using published nucleotide sequences, or marker genes can be obtained commercially.

[0261] (b) a DNA segment that encodes a product that is toxic in a recipient cell or organism ("Counter Selection Marker"). A counter selection marker is especially suitable to select organisms with defined deletions originally comprising said marker (Koprek 1999). Examples for negative selection marker comprise thymidin kinases (TK), cytosine deaminases (Gleave 1999; Perera 1993; Stougaard 1993), cytochrome P450 proteins (Koprek 1999), haloalkan dehalogenases (Naested 1999), iaaH gene products (Sundaresan 1995), cytosine deaminase codA (Schlaman & Hooykaas 1997), or tms2 gene products (Fedoroff & Smith 1993). In general the term "counter selection marker" within the scope of this invention is to be understood in the broad sense including all proteins which either

[0262] i) cause a toxic effect per se on the cell or organism (e.g., a plant cell), or

[0263] ii) convert a non-toxic compound X into a toxic compound Y.

[0264] The term "non toxic compound X" as used herein in intended to mean compounds which in comparison to its conversion product Y--under otherwise identical conditions (i.e. conditions which are identical beside the difference in compound X and Y)--demonstrate a reduced, preferably an absent, biological activity, preferably toxicity. Preferably the toxicity of compound Y is at least two-times, preferably at least five-times, more preferably at least ten-times, most preferably at least one hundred-times the toxicity of the corresponding compound X. Conversion of X to Y can occur by various mechanism including but not limited to hydrolysis, deamination, saporation, dephosphorylation, phosphorylation, oxidation or an other way of activation, metabolisation, or conversion. Compound X can for example be an inactive precursor of a plant growth regulator or a herbicide. "Toxicity" or "toxic effect" as used herein means a measurable, negative effect on the physiology of a cell or an organism and may comprise symptoms including but not limited to decreased or impaired growth, decreased or impaired photosynthesis, decreased or impaired cell division, decreased or impaired regeneration or proliferation etc.

[0265] The counter selection marker may be an endogenous gene or a heterologous gene or transgene from another organism. The following counter selection marker are given by way of example:

[0266] 1. Cytosine deaminases (CodA or CDase), wherein compounds like e.g., 5-fluorocytosine (5-FC) is employed as non-toxic compound X. Cytosine deaminases catalyze deamination of cytosine to uracil (Kilstrup 1989; Anderson 1989). 5-FC is concerted to the toxic metabolite ("Y") 5-fluorouracil (5-FU) (Polak 1975). 5-FC is of low toxicity Toxizitat (Bennett 1990). In contrast, 5-FU exhibits a strong cytotoxic effect inhibiting RNA- and DNA-synthesis (Calabrisi 1990; Damon 1989).

[0267] Cells of plants and higher mammals do not exhibit a significant CDase-activity and are unable to deaminate 5-FC (Polak 1976; Koechlin 1966). In the context of the present invention, a CDase is introduced as a transgene into the target cell. Introduction can be done prior the screening (e.g., generating a stably transformed cell line or organism). Such cells or organism can then be used as master cell lines or master organism.

[0268] Corresponding CDase sequences, transgenic organisms (including plants) comprising said sequences, and negative selection schemes based on e.g., treatment of these cells or organisms with 5-FC (as non-toxic substance X) are known in the art (WO 93/01281; U.S. Pat. No. 5,358,866; Gleave 1999; Perera 1993; Stougaard 1993; EP-A1 595 837; Mullen 1992; Kobayashi 1995; Schlaman 1997; Xiaohui Wang 2001; Koprek 1999; Gallego 1999; Salomon 1998; Thykjaer 1997; Serino 1997; Risseeuw 1997; Blanc 1996; Corneille 2001). Cytosindeaminases and genes encoding the same can be isolated from various organisms, preferably microorganism, like for example Cryptococcus neoformans, Candida albicans, Torulopsis glabrata, Sporothrix schenckii, Aspergillus, Cladosporium, and Phialophora (Bennett 1990) and from bacteria like e.g., E. coli and Salmonella typhimurium (Andersen 1989). All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred are the sequences as described by GenBank Acc.-No: S56903, and the modified sequences described in EP-A1 595 873, which were modified to enable expression in eukaryotes.

[0269] 2. Cytochrome P-450 enzymes, especially the bacterial cytochrome P-450 SU1 gene product (CYP105A1) from Streptomyces griseolus (strain ATCC 11796), wherein substances like the sulfonylurea pro-herbicide R7402 (2-methylethyl-2-3-dihydro-N-[(4,6-dimethoxypyrimidine-2-yl)aminocarbonyl- ]-1,2-benzoisothiazol-7-sulfonamid-1,1-dioxide) as the non-toxic substance X are employed. Corresponding sequences are negative selection schemes employing e.g., R7402 are described in the art (O'Keefe 1994; Tissier 1999; Koprek 1999; O'Keefe 1991). All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred is the sequence described by GenBank Acc.-No: M32238.

[0270] 3. Indoleacetic acid hydrolases like e.g., the tms2 gene product from Agrobacterium tumefaciens, wherein substances like auxinamide compounds or naphthalacetamide (NAM) are employed as non-toxic compound X (NAM being converted to naphthyacetic acid, a phytotoxic compound). Corresponding sequences and the realisation of negative selection schemes (employing NAM as non-toxic compound X) are described in the art (Fedoroff 1993; Upadhyaya 2000; Depicker 1988; Karlin-Neumannn 1991; Sundaresan 1995; Cecchini 1998; Zubko 2000). All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred is the sequence described by GenBank Acc.-No: NC_--003308 (Protein_id="NP_--536128.1), AE009419, AB016260 (Protein_id="BAA87807.1) and NC002147.

[0271] 4. Haloalkane dehalogenases (dhlA gene product) e.g., from Xanthobacter autotropicus GJ10. This dehalogenase hydrolizes dihaloalkanes like 1,2-dichloroethane (DCE) to halogenated alcohols and inorganic halides (Naested 1999; Janssen 1994; Janssen 1989). All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred is the sequence described by GenBank Acc.-No: M26950.

[0272] 5. Thymidine kinases (TK), especially virale TKs from virus like Herpes Simplex virus, SV40, Cytomegalovirus, Varicella zoster virus, especially preferred is TK from Type 1 Herpes Simplex virus (TK HSV-1), wherein substances like e.g., acyclovir, ganciclovir or 1,2-deoxy-2-fluoro-β-D-arabinofuranosil-5-iodouracile (FIAU) are employed as non-toxic compound X. Corresponding compounds are realization of negative selection schemes (e.g., employing acyclovir, ganciclovir or FIAU) are known in the art (Czako 1994; Wigler 1977; McKnight 1980; McKnight 1980; Preston 1981; Wagner 1981; St. Clair 1987). All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred is the sequence described by GenBank Acc.-No: J02224, V00470, and V00467.

[0273] 6. Guanine phosphoribosyl transferases, hypoxanthine phosphoribosyl transferases or Xanthin guanin phosphoribosyl transferases, wherein compounds like 6-thioxanthin or allopurinol are employed as non-toxic substance X. Preferred is the guanine phosphoribosyl transferase (gpt) from e.g. E. Coli (Besnard 1987; Mzoz 1993; Ono 1997), hypoxanthin phosphoribosyltransferases (HPRT; Jolly 1983; Fenwick 1985), xanthin guanin phosphoribosyl transferases (e.g., from Toxoplasma gondii; Knoll 1998; Donald 1996). All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred is the sequence described by GenBank Acc.-No.: U10247 (Toxoplasma gondii HXGPRT), M13422 (E. coli gpt) and X00221 (E. coli gpt).

[0274] 7. Purine nucleoside phosphorylases (PNP; DeoD gene product) e.g., from E. coli, wherein compounds like for example 6-methylpurine deoxyribonucleoside are employed as non-toxic compound X. Suitable compounds and methods for carrying out counter-selection schemes (e.g., employing 6-methylpurine deoxyribonucleoside as non-toxic compound X) are well known to the person skilled in the art (Sorscher 1994). All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred is the sequence described by GenBank Acc.-No.: M60917.

[0275] 8. Phosphonate monoesterhydrolases, which are suitable to convert physiologically inactive ester derivatives of e.g., the herbicide Glyphosate (e.g., glycerylglyphosate) to the active form of the herbicide. Suitable compounds and methods for carrying out counter-selection schemes (e.g., employing glycerylglyphosate as non-toxic compound X) are well known to the person skilled in the art (U.S. Pat. No. 5,254,801; Dotson 1996; Dotson 1996). All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred is the sequence described by GenBank Acc.-No.: U44852.

[0276] 9. Aux-1 and--preferably--Aux-2 gene products e.g. aus derived from the Ti-plasmids of Agrobacterium strains (Beclin 1993; Gaudin 1995). The activity of both enzymes causes production of indole acetamide (IAA) in the plant cell. Aux-1 is encoding a indole acetamide synthase (IAMS) converting tryptophan to indole acetamide (VanOnckelen 1986). Aux-2 is encoding indole acetamide hydrolase (IAMH) converting indole acetamide (a compound without phytohormon activity) to the active auxin indole acetic acid (Inze 1984; Tomashow 1984; Schroder 1984). IAMH is furthermore capable to convert various indole amide-type substrates such as naphthyl acetamide, which is converted into the plant growth regulator naphthyl acetic acid (NAA). Use of IAMH as counter selection marker is for example disclosed in U.S. Pat. No. 5,180,873. Corresponding enzymes are also described for A. rhizogenes, A. vitis (Canaday 1992) and Pseudomonas savastanoi (Yamada 1985). The use as counter selection marker for selectively killing certain plant tissues (e.g., pollen; U.S. Pat. No. 5,426,041) or transgenic plants (U.S. Pat. No. 5,180,873) is described. Compounds and methods for counter selections (e.g. by employing naphthyl acetamide) are known to the person skilled in the art (see above). All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred is the sequence described by GenBank Acc.-No.: M61151, AF039169 and AB025110.

[0277] 10. Adenine phosphoribosyl transferases (APRT), wherein compounds such as 4-aminopyrazolo pyrimidine are employed as non-toxic compound X. Suitable compounds and methods for carrying out counter-selection schemes are well known to the person skilled in the art (Wigler 1979; Taylor 1985). All these references, the sequences and methods described therein are explicitly incorporated by reference.

[0278] 11. Methoxinin dehydrogenases, wherein compounds such as 2-amino-4-methoxybutanicacid (Methoxinin) are employed as non-toxic compound X, which is converted into the toxic compound methoxyvinylglycine (Margraff 1980).



[0279] 12. Rhizobitoxine synthases, wherein compound such as 2-amino-4-methoxybutanicacid (Methoxinin) are employed as non-toxic compound X, which is converted into the toxic compound 2-amino-4-[2-amino-3-hydroxypropyl]-trans-3-butanicacid (Rhizobitoxin) (Owens 1973).

[0280] 13. 5-Methylthioribose (MTR) kinases, wherein compounds such as 5-trifluoromethyl thioribose (MTR-analogue, "subversives substrate") are employed as non-toxic compound X, which is converted into the toxic compound Y carbothionyldifluoride. MTR-kinase is a key enzyme of the methionine salvage pathway. Corresponding enzyme activities are described in plants, bacteria, and protozoa but not in mammals. MTR kinases from various species can be identified according to defined sequence motives (Sekowska 2001; biomedcentral.com/1471-2180/1/15). Corresponding sequences are methods for counter selection (e.g., employing 5-trifluoromethyl thioribose) are known to the person skilled in the art and readily obtainable from sequence databases (e.g., Sekowska 2001; Cornell 1996). All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred is the sequence described by GenBank Acc.-No.: AF212863 or AC079674 and other MTK kinase enzymes as described in WO 03/078629 and DE10212892 hereby incorporated by reference.

[0281] 14. Alcoholdehydrogenases (Adh) especially plant Adh-1 gene products, where preferably compounds such as allylalcohol are employed as non-toxic compound X, which is converted into the toxic compound (Y) acrolein. Suitable, corresponding compounds and methods for carrying out counter-selection schemes (e.g., employing allyl alcohol) are well known to the person skilled in the art (Wisman 1991; Jacobs 1988; Schwartz 1981). Sequences can be readily derived from sequence databases. All these references, the sequences and methods described therein are explicitly incorporated by reference. Especially preferred is the sequence described by GenBank Acc.-No.: X77943, M12196, AF172282, X04049 or AF253472.

[0282] 15. Furthermore preferred as counter selection marker are "toxic genes" or "toxic sequences" which per se exhibit and toxic effect on a cell expressing said genes or sequences. Example may include but are not limited to sequences encoding toxic protein such as diphtheria toxin A, Ribonukleases (RNAse e.g., Barnase), ribosome-inhibiting proteins (RIP; such as ricine), magainins, DNAse, phytotoxins, proteins which are able to evoke a hypersensitive reaction, and proteases. Evoking a hypersensitive response (HR) is possible when a pathogen-derived elicitor protein and a corresponding plant-derived receptor protein are expressed simultaneously. Couples of such corresponding elicitor/receptor genes and their applicability to evoke a HR in a transgenic plant, are known in the art, e.g. for Cladosporium fulvum avr-genes and Lycopersicon esculentum Cf-genes (WO 91/15585) or for Psuedomonas syringae avr-genes and Arabidopsis thaliana RPM1-genes (Grant 1995).

[0283] Additional toxic sequences are those suppressing essential endogenous genes (such as housekeeping genes). The person skilled in the art is aware of various sequences and methods which can be employed to suppress ("silence") gene expression of endogenous genes. The terms "suppression" or "silencing" in relation to a gene, its gene product, or the activity of said gene product is to be understood in the broad sense comprising various mechanism of impairing or reducing the functionality on various levels of expression. Included are for example a quantitative reduction of transcription and translation up to an essentially complete absence of the transcription and/or translation product (i.e. lacking detectability by employing detection methods such as Northern or Western blot analysis, PCR, etc.)

[0284] Suitable method of gene silencing may include but shall not be limited to gene silencing by

[0285] (i) antisense suppression (see above for details),

[0286] (ii) sense suppression (co-suppression) (see above for details),

[0287] (iii) double-stranded RNA interference (see above for details),

[0288] (iv) expression of ribozymes against an endogenous RNA transcript (see above for details),

[0289] (v) expression of protein or DNA-binding factors: Expression of an endogenous gene can be "silenced" also by expression of certain DNA or protein binding factors which interfere with expression or activity of the gene of its gene product. For example artificial transcription factors of the zinc finger type can be adapted to any target sequence and can thus be employed for gene silencing (e.g., by being directed against the promoter region of the target gene). Methods for production of such factors are described (Dreier 2001; Dreier 2000; Beerli 2000a, 2000b; Segal 2000; Kang 2000; Beerli 1998; Kim 1997; Klug 1999; Tsai 1998; Mapp 2000; Sharrocks 1997; Zhang 2000). Furthermore factors can be employed which directly inhibit the gene product (by interacting with the resulting protein). Such protein binding factors may for example be aptameres (Famulok 1999), antibodies, antibody fragments, or single chain antibodies. Their generation is described (Owen 1992; Franken 1997; Whitelam 1996).

[0290] (vi) Gene silencing mediating viral expression systems: Gene silencing of endogenous genes can also be mediated employing specific viral expressions systems (Amplikon; Angell 1999). These systems and methods (termed "VIGS"; viral induced gene silencing) are mediating expression of sequences resembling the endogenous gene from a viral vector system. By classifying the expression as "viral" the entire expression (including expression of the homologous endogenous gene) is shot down by plant viral defense mechanism. Corresponding methods are described in the art (Ratcliff 2001; Fagard 2000; Anandalakshmi 1998; Ruiz 1998).

[0291] Essential endogenous genes suitable as targets for the method of the invention may for example be genes selected from those coding for enzymes that are essential for cell viability. These so called "housekeeping genes" may for example be selected from genes encoding for proteins such as ATP synthase, cytochrome c, pyruvate kinase, aminoacyl transferase, or phosphate, di-, tricarboxylkate and 2-oxo-glutarate translocators. A list of target enzymes is given in Table 1 by way of example but the invention is not limited to the enzymes mentioned in this table. More detailed listings can be assembled from series as Biochemistry of Plants (Eds. Stumpf & Conn, 1988-1991, Vols. 1-16 Academic Press) or Encyclopedia of Plant Physiology (New Series, 1976, Springer-Verlag, Berlin).

TABLE-US-00002

[0291] TABLE 1 EXAMPLES OF TARGET ENZYMES Enzvme ATP synthase (mitochondrion) adenine nucleotide translocator (mitochondrion) phosphate translocator (mitochondrion) tricarboxylate translocator (mitochondrion) dicarboxylate translocator (mitochondrion) 2-oxo-glutarate translocator (mitochondrion) cytochrome C (mitochondrion) pyruvate kinase glyceraldehyde-3P-dehydrogenase NADPH-cytochrome P450 reductase fatty acid synthase complex glycerol-3P-acyltransferase hydroxymethyl-glutaryl CoA reductase aminoacyl transferase transcription factors elongation factors phytoen desaturase nitrate reductase p-hydroxyphenylpyruvate dioxygenase (HPPD) transketolase (preferably enzymes described and claimed in EP-A1 723 017) ferredoxin oxidoreductase (preferably enzymes described and claimed in EP-A1 1 333 098) S-adenosylmethionin: Mg-protoporphyrin-IX-O-methyltransferase (preferably enzymes described and claimed in EP-A1 1 198 578) dihydrorotase (EC 3.5.2.3) (preferably enzymes described and claimed in EP-A1 1 210 437) phosphoribosyl pyrophosphate synthase (preferably enzymes described and claimed in EP-A1 1 294 925) aspartate carbamyl transferase (preferably enzymes described and claimed in EP-A1 1 259 623) dehydrochinate dehydratase/shikimate dehydrogenase (preferably enzymes described and claimed in EP-A1 1 315 808



[0292] As housekeeping genes are in general highly conserved, heterologous probes from other (plant) species can be used to isolate the corresponding gene from the species that is to be made resistant. Such gene isolations are well within reach of those skilled in the art and, in view of the present teaching require no undue experimentation.

[0293] (c) a DNA segment that encodes a product conferring to the recipient cell or organism an advantage by increased or improved regeneration, growth, propagation, multiplication ("Positive Selection Marker"). Genes like isopentenyltransferase from Agrobacterium tumefaciens (strain:P022; Genbank Acc.-No.: AB025109) may--as a key enzyme of the cytokinin biosynthesis--facilitate regeneration of transformed plants (e.g., by selection on cytokinin-free medium). Corresponding selection methods are described (Ebinuma et al. 2000a, 2000b). Additional Positive Selection Markers, which confer a growth advantage to a transformed plant in comparison with a non-transformed one, are described e.g., in EP-A 0 601 092. Growth stimulation selection markers may include (but shall not be limited to) β-glucuronidase (in combination with e.g., a cytokinin glucuronide), mannose-6-phosphate isomerase (in combination with mannose), UDP-galactose-4-epimerase (in combination with e.g., galactose), wherein mannose-6-phosphate isomerase in combination with mannose is especially preferred.

[0294] (d) a DNA segment that encodes a product that can be readily identified ("reporter genes" or "reporter proteins" or "reporter molecules"; e.g., phenotypic markers such as β-galactosidase, green fluorescent protein (GFP), and cell surface proteins). The term "reporter gene", "reporter protein", or "reporter molecule" is intended to mean any readily quantifiable protein (or the sequence encoding therefore), which via--for example--color or enzyme activity, makes possible an assessment of presence of said protein or expression of said reporter gene. Reporter genes encode readily quantifiable proteins and, via their color or enzyme activity, make possible an assessment of the transformation efficacy, the site of expression or the time of expression. Very especially preferred in this context are genes encoding reporter proteins (Schenborn 1999) such as the green fluorescent protein (GFP) (Sheen 1995; Haseloff 1997; Reichel 1996; Tian 1997; WO 97/41228; Chui 1996; Leffel 1997), the NAN reporter gene (Kavanagh 2002; WO 03/052104), chloramphenicol transferase, a luciferase (Ow 1986; Millar 1992), the aequorin gene (Prasher 1985), β-galactosidase, R locus gene (encoding a protein which regulates the production of anthocyanin pigments (red coloring) in plant tissue and thus makes possible the direct analysis of the promoter activity without addition of further auxiliary substances or chromogenic substrates (Dellaporta 1988; Ludwig 1990), with β-glucuronidase (GUS) being very especially preferred (Jefferson 1987b; 1987a). β-glucuronidase (GUS) expression is detected by a blue color on incubation of the tissue with 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid, bacterial luciferase (LUX) expression is detected by light emission; firefly luciferase (LUC) expression is detected by light emission after incubation with luciferin; and galactosidase expression is detected by a bright blue color after the tissue is stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. Reporter genes may also be used as scorable markers as alternatives to antibiotic resistance markers. Such markers are used to detect the presence or to measure the level of expression of the transferred gene. The use of scorable markers in plants to identify or tag genetically modified cells works well only when efficiency of modification of the cell is high.

[0295] (e) a DNA segment that encodes a product that inhibits a cell function in a recipient cell;

[0296] (f) a DNA segment that inhibits the activity of any of the DNA segments of (a)-(e) above;

[0297] (g) a DNA segment that binds a product that modifies a substrate (e.g. restriction endonucleases);

[0298] (h) a DNA segment that encodes a specific nucleotide recognition sequence which can be recognized by a protein, an RNA, a DNA or a chemical,

[0299] (i) a DNA segment that, when deleted or absent, directly or indirectly confers resistance or sensitivity to cell killing by particular compounds within a recipient cell;

[0300] (j) a DNA segment that encodes a product that suppresses the activity of a gene product in a recipient cell;

[0301] (k) a DNA segment that encodes a product that is otherwise lacking in a recipient cell (e.g, tRNA genes, auxotrophic markers), and;

[0302] (l) a DNA segment that can be used to isolate or identify a desired molecule (e.g., specific protein binding sites).

[0303] In a preferred embodiment of the invention, in cases where an efficient transcription terminator would lead to a decreased expression of the additional sequences (especially Method A, where the transcription terminator is inserted between said additional sequences and the promoter), said additional sequences may by an inverted repeat of a known transcription terminator sequence (such as for example the nos terminator) which is localized in a way that the second copy (the copy more downstream from the promoter sequence) is in its "normal" orientation (in which it is constituting a functional transcription terminator). It has to be noted, that such decreased expression of an inverted repeat transcription terminator leads to increased expression (or better not silenced expression) of sequences localized upstream of the insertion site. In case these sequences are encoding for example a marker, an increased resistance or signal can be observed.

[0304] Preferably, this transcription terminator (hereinafter "the second transcription terminator") is different from the sequence to be assessed for its transcription termination efficiency. In this case, it is preferred that further sequences are employed which are preferably localized between the promoter and the insertion site and are encoding e.g., for a selection marker or a reporter gene. In such a scenario, an efficient transcription terminator sequence would stop transcription and would not cause transcription of the inverted repeat of said second transcription terminator. In consequence normal expression of the sequences between promoter and insertion site would occur (leading to expression of the selection marker or the reporter gene). In cases, where the sequence inserted into the insertion site is not an effective transcription terminator, transcription will read-through into the inverted repeat of said second transcription terminator. Such a construct would cause its own gene silencing be dsRNAi. In consequence no expression of the sequences between promoter and insertion site would occur (silencing the expression of the selection marker or the reporter gene). Self-affecting gene silencing based on an inverted repeat sequence of an transcription terminator (e.g., NOS terminator) are described (Brummell 2003).

2.3 Other Elements of the Screening Construct or Screening Vector

[0305] The screening construct or screening vector may comprise further elements (e.g., genetic control sequences) in addition to a promoter and the additional sequences. The term "genetic control sequences" is to be understood in the broad sense and refers to all those sequences which have an effect on the materialization or the function of the screening construct or screening vector according to the invention. For example, genetic control sequences modify the transcription and translation in prokaryotic or eukaryotic organisms. Genetic control sequences furthermore also encompass the 5'-untranslated regions, introns or noncoding 3'-region of genes, such as, for example, the actin-1 intron, or the Adh1-S introns 1, 2 and 6 (general reference: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). It has been demonstrated that they may play a significant role in the regulation of gene expression. Thus, it has been demonstrated that 5'-untranslated sequences can enhance the transient expression of heterologous genes. Examples of translation enhancers which may be mentioned are the tobacco mosaic virus 5'-leader sequence (Gallie, 1987) and the like. Furthermore, they may promote tissue specificity (Rouster, 1998).

[0306] The screening construct or screening vector may advantageously comprise one or more enhancer sequences, linked operably to the promoter, which make possible an increased recombinant expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or additional transcription terminator sequences, may also be inserted at the 3'-end of the nucleic acid sequences to be expressed recombinantly.

[0307] In some embodiments of the invention (for example Variation A or C) or screening construct or screening vector can also include a known transcription termination sequence (preferably after the additional sequence), and optionally, a polyadenylation signal sequence. Polyadenylation signals which are suitable as control sequences are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular the OCS (octopine synthase) terminator and the NOS (nopaline synthase) terminator. An expression vector does not necessarily need to contain transcription termination and polyadenylation signal sequences, because these elements can be provided by the cloned gene or gene fragment.

[0308] The screening construct or screening vector of the invention may comprise further functional elements. The term functional element is to be understood in the broad sense and refers to all those elements which have an effect on the generation, amplification or function of the screening construct or screening vector according to the invention. Functional elements may include for example (but shall not be limited to) selectable marker genes (including negative, positive, and counter selection marker, see above for details), reporter genes, and

[0309] 1) Origins of replication, which ensure amplification of the expression cassettes or vectors according to the invention in, for example, E. coli. Examples which may be mentioned are ORI (origin of DNA replication), the pBR322 ori or the P15A ori (Maniatis 1989). Additional examples for replication systems functional in E. coli, are ColE1, pSC101, pACYC184, or the like. In addition to or in place of the E. coli replication system, a broad host range replication system may be employed, such as the replication systems of the P-1 Incompatibility plasmids; e.g., pRK290. These plasmids are particularly effective with armed and disarmed Ti-plasmids for transfer of T-DNA to the plant species host. An expression vector can also include a SV40 origin. This element can be used for episomal replication and rescue in cell lines expressing SV40 large T antigen.

[0310] 2) Elements which are necessary for Agrobacterium-mediated plant transformation, such as, for example, the right and/or--optionally--left border of the T-DNA or the vir region.

[0311] 3) Cloning Sites: The cloning site can preferably be a multicloning site. Any multicloning site can be used, and many are commercially available.

[0312] 4) S/MAR (scaffold/matrix attachment regions). Matrix attachment regions (MARs) are operationally defined as DNA elements that bind specifically to the nuclear matrix (nuclear scaffold proteins) in vitro and are proposed to mediate the attachment of chromatin to the nuclear scaffold in vivo. It is possible, that they also mediate binding of chromatin to the nuclear matrix in vivo and alter the topology of the genome in interphase nuclei. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable expression in transgenic organisms (especially plants) or cell lines, most likely by minimizing gene silencing (for reveiw: Allen 2000). Various S/MARS sequences and there effect on gene expression are described (Sidorenko 2003; Allen 1996; Villemure 2001; Mlynarova 2002). S/MAR elements may be preferably employed to reduce unintended gene silencing (Mlynarova 2003). An example for a S/MAR being the chicken lysozyme A element (Stief 1989).

[0313] 5) Sequences which further modify transcription, translation, and/or transport of an expressed protein. For example the expressed protein may be a chimeric protein comprising a secretory signal sequence. The secretory signal sequence is operably linked to a gene of interest such that the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide of interest into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the nucleotide sequence encoding the amino acid sequence of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleotide sequence of interest (U.S. Pat. No. 5,037,743, U.S. Pat. No. 5,143,830). Expression vectors can also comprise nucleotide sequences that encode a peptide tag to aid the purification of the polypeptide of interest. Peptide tags that are useful for isolating recombinant polypeptides include poly-Histidine tags (which have an affinity for nickel-chelating resin), c-myc tags, calmodulin binding protein (isolated with calmodulin affinity chromatography), substance P, the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which binds with anti-FLAG antibodies; see, for example, Luo 1996; Morganti 1996, and Zheng 1997). Nucleic acid molecules encoding such peptide tags are available, for example, from Sigma-Aldrich Corporation (St. Louis, Mo.).

[0314] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

2.4. Suitable Vectors for the Invention

[0315] As used herein, the terms "vector" and "vehicle" are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term "screening vector" as used herein refers to a recombinant DNA molecule comprising at least the above defined elements of said promoter and said additional sequences functional for evaluation of the transcription termination efficiency of an inserted sequence.

[0316] The methods of the invention are not limited to the vectors disclosed herein. Any vector which is capable of expressing a nucleic acid sequences, and preferably introducing a nucleic acid sequence of interest into a cell (e.g., a plant cell) is contemplated to be within the scope of this invention. Typically, vectors comprise the above defined essential elements of the invention in combination with elements which allow cloning of the vector into a bacterial or phage host. The vector preferably, though not necessarily, contains an origin of replication which is functional in a broad range of prokaryotic hosts. A selectable marker is generally, but not necessarily, included to allow selection of cells bearing the desired vector. Examples of vectors may be plasmids, cosmids, phages, viruses or Agrobacteria. More specific examples are given below for the individual transformation technologies.

[0317] Preferred are those vectors which make possible a stable integration of the expression construct into the host genome. In the case of injection or electroporation of DNA into cells (e.g., plant cells), the plasmid used need not meet any particular requirements. Simple plasmids such as those of the pUC series can be used. If intact plants are to be regenerated from the transformed cells, it is necessary for an additional selectable marker gene to be present on the plasmid. A variety of possible plasmid vectors are available for the introduction of foreign genes into plants, and these plasmid vectors contain, as a rule, a replication origin for multiplication in E. coli and a marker gene for the selection of transformed bacteria. Examples are pBR322, pUC series, M13mp series, pACYC184 and the like.

[0318] Preferred vectors for use in the invention include prokaryotic vectors, eukaryotic vectors or vectors which may shuttle between various prokaryotic and/or eukaryotic systems (e.g. shuttle vectors). Preferred eukaryotic vectors comprise vectors, which replicate in yeast cells, plant cells, fish cells, eukaryotic cells, mammalian cells, or insect cells. Preferred prokaryotic vectors comprise vectors which replicate in gram negative and/or gram positive bacteria, more preferably vectors which replicate in bacteria of the genus Escherichia, Salmonella, Bacillus, Streptomyces, Agrobacterium, Rhizobium, or Pseudomonas. Most preferred are vectors which replicates in both E. coli and Agrobacterium. Eukaryotic vectors for use in the invention include vectors which propagate and/or replicate and yeast cells, plant cells, mammalian cells (particularly human cells), fungal cells, insect cells, fish cells and the like. Particular vectors of interest include but are not limited to cloning vectors, sequencing vectors, expression vectors, fusion vectors, two-hybrid vectors, gene therapy vectors, and reverse two-hybrid vectors. Such vectors may be used in prokaryotic and/or eukaryotic systems depending on the particular vector.

[0319] In accordance with the invention, any vector may be used to construct a screening vector of the invention. In particular, vectors known in the art and those commercially available (and variants or derivatives thereof) may in accordance with the invention be engineered to include one or more recombination sites for use in the methods of the invention. Such vectors may be obtained from, for example, Invitrogen, Vector Laboratories Inc., InVitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter, OriGenes Technologies Inc., Stratagene, PerkinElmer, Pharmingen, Life Technologies, Inc., and Research Genetics. Such vectors may then for example be used for cloning or subcloning nucleic acid molecules of interest. General classes of vectors of particular interest include prokaryotic and/or eukaryotic cloning vectors, expression vectors, fusion vectors, two-hybrid or reverse two-hybrid vectors, shuttle vectors for use in different hosts, mutagenesis vectors, transcription vectors, vectors for receiving large inserts and the like.

[0320] Other vectors of interest include viral origin vectors (M13 vectors, bacterial phage λ vectors, adenovirus vectors, and retrovirus vectors), high, low and adjustable copy number vectors, vectors which have compatible replicons for use in combination in a single host (pACYC184 and pBR322) and eukaryotic episomal replication vectors (pCDM8).

[0321] Particular vectors of interest include prokaryotic expression vectors such as pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA, B, and C, pRSET A, B, and C (Invitrogen, Inc.), pGEMEX-1, and pGEMEX-2 (Promega, Inc.), the pET vectors (Novagen, Inc.), pTrc99A, pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia, Inc.), pKK233-2 and pKK388-1 (Clontech, Inc.), and pProEx-HT (Life Technologies, Inc.) and variants and derivatives thereof Vector donors can also be made from eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTetSplice (Life Technologies, Inc.), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), p3'SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBsueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Inc.) and variants or derivatives thereof.

[0322] Other vectors of particular interest include pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificial chromosomes), BAC's (bacterial artificial chromosomes), P1 (E. coli phage), pQE70, pQE60, pQE9 (quagan), pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (InVitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 and pSV-SPORT1 (Life Technologies, Inc.) and variants or derivatives thereof.

[0323] Additional vectors of interest include pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1(-)/Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZquadrature, pGAPZ, pGAPZquadrature, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SP1), pVgRXR, pcDNA2.1. pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1/Amp, pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/RSV, pREP4, pREP7, pREP8, pREP9, pREP 10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac from Invitrogen; λExCell, λgt11, pTrc99A, pKK223-3, pGEX-1λT, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia; pSCREEN-1 b(+), pT7Blue(R), pT7Blue-2, pCITE-4abc(+), pOCUS-2, pTAg, pET-32LIC, pET-30LIC, pBAC-2cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC, pT7Blue-2, ASCREEN-1, λBlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb, pET-19b, pET-20b(+), pET-2labcd(+), pET-22b(+), pET-23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3cp, pBACgus-2cp, pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg, and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic, pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, pβgal-Basic, pβgal-Control, pβgal-Promoter, pβgal-Enhancer, pCMVβ, pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1/2/3, pYEX-S1, pBacPAKHis, pBacPAK8/9, pAcUW31, BacPAK6, pTriplEx, λgt10, λgt11, pWE15, and ΔTriplEx from Clontech; Lambda ZAP II, pBK-CMV, pBK-RSV, pBluescript II KS+/-, pBluescript II SK+/-, pAD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct, pBS+/-, pBC KS+/-, pBC SK+/-, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCALk-c, pET-3abcd, pET-11'abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI/MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44, pOG45, pFRTβGAL, pNEβGAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, and pRS416 from Stratagene.

[0324] Two-hybrid and reverse two-hybrid vectors of particular interest include pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and variants or derivatives thereof.

[0325] Preferred vectors for expression in E. coli are pQE70, pQE60 und pQE-9 (QIAGEN, Inc.); pBluescript vectors, phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia Biotech, Inc.).

[0326] Preferred vectors for expression in eukaryotic, animals systems comprise pWLNE0, pSV2CAT, pOG44, pXT1 and pSG (Stratagene Inc.); pSVK3, pBPV, pMSG und pSVL (Pharmacia Biotech, Inc.). Examples for inducible vectors are pTet-tTak, pTet-Splice, pcDNA4/TO, pcDNA4/TO/LacZ, pcDNA6/TR, pcDNA4/TO/Myc-His/LacZ, pcDNA4/TO/Myc-His A, pcDNA4/TO/Myc-His B, pcDNA4/TO/Myc-His C, pVgRXR (Invitrogen, Inc.) or the pMAM-Serie (Clontech, Inc.; GenBank Accession No.: U02443). Preferred vectors for the expression in yeast comprise for example pYES2, pYD1, pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, PHIL-D2, PHIL-SI, pPIC3SK, pPIC9K, and PA0815 (Invitrogen, Inc.).

[0327] Preferred vector for plant transformation are described herein below and preferably comprise vectors for Agrobacterium-mediated transformation. Agrobacterium tumefaciens and A. rhizogenes are plant-pathogenic soil bacteria, which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant (Kado 1991). Vectors of the invention may be based on the Agrobacterium Ti- or Ri-plasmid and may thereby utilize a natural system of DNA transfer into the plant genome.

[0328] As part of this highly developed parasitism Agrobacterium transfers a defined part of its genomic information (the T-DNA; flanked by about 25 bp repeats, named left and right border) into the chromosomal DNA of the plant cell (Zupan 2000). By combined action of the so-called vir genes (part of the original Ti-plasmids) said DNA-transfer is mediated. For utilization of this natural system, Ti-plasmids were developed which lack the original tumor inducing genes ("disarmed vectors"). In a further improvement, the so called "binaTy vector systems", the T-DNA was physically separated from the other functional elements of the Ti-plasmid (e.g., the vir genes), by being incorporated into a shuttle vector, which allowed easier handling (EP-A1 0 120 516; U.S. Pat. No. 4,940,838). These binary vectors comprise (beside the disarmed T-DNA with its border sequences), prokaryotic sequences for replication both in Agrobacterium and E. coli. It is an advantage of Agrobacterium-mediated transformation that in general only the DNA flanked by the borders is transferred into the genome and that preferentially only one copy is inserted. Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are known in the art (Miki 1993; Gruber 1993; Moloney 1989). The use of T-DNA for the transformation of plant cells has been studied and described intensively (EP-A1 120 516; Hoekema 1985; Fraley 1985; and An 1985). Various binary vectors are known, some of which are commercially available such as, for example, pBIN19 (Clontech Laboratories, Inc. USA).

[0329] Hence, for Agrobacterium-mediated transformation the screening construct may be integrated into or the screening vector may consist of specific plasmids, such as shuttle or intermediate vectors, or binary vectors. If a Ti or Ri plasmid is to be used for the transformation, at least the right border, but in most cases the right and left border, of the Ti or Ri plasmid T-DNA is linked to the transgenic expression construct to be introduced in the form of a flanking region. Binary vectors are preferably used. Binary vectors are capable of replication both in E. coli and in Agrobacterium. They may comprise a selection marker gene and a linker or polylinker (for insertion of e.g. the expression construct to be transferred) flanked by the right and left T-DNA border sequence. They can be transferred directly into Agrobacterium (Holsters 1978). The selection marker gene permits the selection of transformed Agrobacteria and is, for example, the nptII gene, which confers resistance to kanamycin. The Agrobacterium which acts as host organism in this case should already contain a plasmid with the vir region. The latter is required for transferring the T-DNA to the plant cell. An Agrobacterium transformed in this way can be used for transforming plant cells. The use of T-DNA for transforming plant cells has been studied and described intensively (EP-A1 0 120 516; Hoekema 1985; An 1985; see also below). Common binary vectors are based on "broad host range"-plasmids like pRK252 (Bevan 1984) or pTJS75 (Watson 1985) derived from the P-type plasmid RK2. Most of these vectors are derivatives of pBIN19 (Bevan 1984). Various binary vectors are known, some of which are commercially available such as, for example, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA). Additional vectors were improved with regard to size and handling (e.g. pPZP; Hajdukiewicz 1994). Improved vector systems are described also in WO 02/00900.

[0330] In a preferred embodiment, Agrobacterium strains for use in the practice of the invention include octopine strains, e.g., LBA4404 or agropine strains, e.g., EHA101 or EHA105. Suitable strains of A. tumefaciens for DNA transfer are for example EHA101pEHA101 (Hood 1986), EHA105[pEHA105] (Li 1992), LBA4404[pAL4404] (Hoekema 1983), C58C1[pMP90] (Koncz 1986), and C58C1[pGV2260] (Deblaere 985). Other suitable strains are Agrobacterium tumefaciens C58, a nopaline strain. Other suitable strains are A. tumefaciens C58C1 (Van Laerebeke 1974), A136 (Watson et al. 1975) or LBA4011 (Klapwijk 1980). In a preferred embodiment, the Agrobacterium strain used to transform the plant tissue pre-cultured with the plant phenolic compound contains a L,L-succinamopine type Ti-plasmid, preferably disarmed, such as pEHA101. In another preferred embodiment, the Agrobacterium strain used to transform the plant tissue pre-cultured with the plant phenolic compound contains an octopine-type Ti-plasmid, preferably disarmed, such as pAL4404. Generally, when using octopine-type Ti-plasmids or helper plasmids, it is preferred that the virF gene be deleted or inactivated. In a preferred embodiment, the Agrobacterium strain used to transform the plant tissue pre-cultured with the plant phenolic compound such as acetosyringone. The method of the invention can also be used in combination with particular Agrobacterium strains, to further increase the transformation efficiency, such as Agrobacterium strains wherein the vir gene expression and/or induction thereof is altered due to the presence of mutant or chimeric virA or virG genes (e.g. Hansen 1994; Chen 1991; Scheeren-Groot 1994).

[0331] A binary vector or any other vector can be modified by common DNA recombination techniques, multiplied in E. coli, and introduced into Agrobacterium by e.g., electroporation or other transformation techniques (Mozo 1991). Agrobacterium is grown and used as described in the art. The vector comprising Agrobacterium strain may, for example, be grown for 3 days on YP medium (5 g/L yeast extract, 10 g/L peptone, 5 g/L Nail, 15 g/L agar, pH 6.8) supplemented with the appropriate antibiotic (e.g., 50 mg/L spectinomycin). Bacteria are collected with a loop from the solid medium and resuspended. For the purpose of this invention, Agrobacterium compatible vectors are provided by inserting site-specific recombination sites as described--for example--in the Examples.

[0332] After constructing a vector, the vector can be propagated in a host cell to synthesize nucleic acid molecules for the generation of a nucleic acid polymer. Vectors, often referred to as "shuttle vectors," are capable of replicating in at least two unrelated expression systems. To facilitate such replication, the vector should include at least two origins of replication, one effective in each replication system. Typically, shuttle vectors are capable of replicating in a eukaryotic system and a prokaryotic system. This enables detection of protein expression in eukaryotic hosts, the "expression cell type," and the amplification of the vector in the prokaryotic hosts, the "amplification cell type." As an illustration, one origin of replication can be derived from SV40, while another origin of replication can be derived from pBR322. Those of skill in the art know of numerous suitable origins of replication.

[0333] After constructing a vector, the vector is typically propagated in a host cell. Vector propagation is conveniently carried out in a prokaryotic host cell, such as E. coli or Bacillus subtilus. Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DB2, DB3.1, DH1, DH41, DH5, DH51, DH5IF, DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Suitable strains of Bacillus subtilus include BR151, YB886, M1119, M1120, and B170 (see, for example, Hardy 1985). Standard techniques for propagating vectors in prokaryotic hosts are well-known to those of skill in the art (see, for example, Ausubel 1995; Wu 1997).

3. The Sequences to be Assessed as Transcription Terminators

[0334] The sequences to be assessed using the method of the invention for their efficiency as transcription terminator sequences may be derived from various sources. In one embodiment of the invention sequences believed to function as transcription terminators may be assessed for their efficiency. Such sequences can be derived from for example regions downstream of the coding sequence of a gene (e.g., comprising the region encoding the 3'-untranslated region and additional downstream genomic sequences), preferably from a region which is surrounding the end of the mRNA transcript. Various of such sequences can be derived from comparison of genomic and cDNA libraries. The corresponding nucleic acid sequences to be inserted into the insertion site of the screening vector or screening construct can be obtained for example by isolation from the corresponding host organism (by the various cloning methods known to the person skilled in the art, e.g., by polymerase chain reaction employing appropriate primer oligonucleotides) or directly by DNA synthesis.

[0335] In a preferred embodiment the DNA molecules to be inserted for evaluation is a double-stranded, linear DNA molecule. The ends of said molecules may by blunt (i.e., without 5'- and/or 3' overhangs) or "sticky" (i.e., with 5'- and/or 3' overhangs). Preferably, the ends of the DNA molecule may have overhangs which allow insertion into cleavage sites of restriction endonuclease to facilitate insertion into an insertion site. The length and molecular weight of the DNA molecule may vary. In an preferred embodiment the molecule has a size of about 50 to about 5,000 base pairs, preferably from about 60 to about 2,000 base pairs, more preferably from about 70 to about 1,000 base pairs, most preferably from about 80 to about 500 base pairs.

[0336] Beside this educated approach (based on sequences for which some transcription termination efficiency can be presumed) in another preferred embodiment of the invention libraries of DNA sequences are screened to obtain efficient transcription terminator sequences. This embodiment does not require any previous sequence information and is based preferably solely on the phenotype of efficient transcription termination (which is difficult to correlate in practice with sequence information). The library of DNA sequences employed may be a synthetic library or a library of naturally occurring DNA molecules or a mixture of synthetic and naturally occurring DNA molecules. Preferably, the library of DNA molecules is a library of naturally occurring molecules, which may be derived from genomic DNA and/or cDNA of one or more organism. More preferably, the library is derived from the genomic DNA of an organism, preferably a plant organism.

[0337] In a preferred embodiment the DNA molecules of the DNA library are double-stranded, linear DNA molecules. The ends of said molecules may by blunt (i.e., without 5'- and/or 3' overhangs) or "sticky" (i.e., with 5'- and/or 3' overhangs). The length and molecular weight of the DNA molecules of the library may vary. In an preferred embodiment the molecules have a size of about 50 to about 5,000 base pairs, preferably from about 60 to about 2,000 base pairs, more preferably from about 70 to about 1,000 base pairs, most preferably from about 80 to about 500 base pairs.

[0338] The library of DNA molecules may be derived from the genomic and/or cDNA by various means known to the person skilled in the art. For example, the library may be derived by random shearing of DNA of exhaustive or partial digestion with endonucleases. Preferably, the library is derived by exhaustive digestion with a restriction endonuclease, which has preferably a 4 base recognition site (like, e.g., Sau3A). Following fragmentation (e.g., by restriction), DNA molecules of the preferred molecular weight (as determined above) may be isolated by for example molecular weight exclusion chromatography (size exclusion chromatography using for example Superose® columns, Amersham Bioscience, Inc.) or gel electrophoresis as known in the art (see for example Ellegren 1989).

[0339] In another preferred embodiment of the invention selected sequences can be assessed for their performance as transcription terminator sequences. Such sequences can be, for example, regions downstream of the coding sequence of a gene (e.g., comprising the region encoding the 3'-untranslated region and additional downstream genomic sequences), preferably from a region which is surrounding the end of the mRNA transcript. Such sequences can be derived by in silico search of genome databases, such as for example of Arabidopsis thaliana or rice.

[0340] In one preferred embodiment, a partial or--preferably--entire plant genome (such as the rice or Arabidopsis genome) is screened for potential plant derived terminator candidates. The following criteria are used to identify and determine suitable candidates for transcription terminator sequences which may be further analyzed in the method of the invention:

[0341] 1. Identification and/or isolation of intergenic regions between paired genes meeting predefined intergenic distance criteria. These genes may preferably have a head-to-tail orientation (i.e. transcription is running in the same direction), or--preferably--a tail-to-tail orientation (i.e. in opposite direction against each other). In the head-to-tail scenario the term "intergenic region" as used herein means the sequence in between (but excluding) the stop-codon of the "tail"-sequence and the start-codon of the "head" sequence, or--if known--the start of the promoter region of the "head" sequence. In the tail-to-tail orientation the term "intergenic region" as used herein means the sequence in between (but excluding) the two stop-codons of the coding sequence. Preferably, intergenic regions from paired genes in tail-to-tail orientation are identified which have a length from about 400 to 3,000 base pairs, preferably from about 700 to about 2,000 base pairs. Identification can be done by various means, including entire genome sequencing (e.g., in case of previously unknown sequences) or in silico screening of already known sequences (such as the Arabidopsis or rice genome). Existing database can be employed for this purpose such as the most updated data from The Institute of Genome Research (TIGR; PUB_tigr_rice_genome_v4.nt (v03212003), PUB_tigr_rice_cds_Oct022003.nt, pubOSest0603 (ncbi)).

[0342] 2. Identification and/or isolation of intergenic sequences which are flanked on both sides by genes having a high expression level. The term "high expression" or "high expression level" as used in this context means an expression level which is at least 5%, preferably at least 10%, more preferably at least 30%, most preferably at least 50% of the expression level of actin in the same mRNA source (i.e. cell or tissue). Expression may be judged by various means including but not limited to number of ESTs in a non-normalized EST library, Northern-blot analysis, RT-PCR etc. Low expression of one or both genes has been identified as an indicator for gene silencing by read-through transcription. Expression level can be profiled either by experiment (e.g., in vitro or in vivo for example by using expression profiling by chip or micro-array technology) or--preferably--in silico by simply counting the number of ESTs for each gene in non-normalized EST/cDNA-libraries which is indicative for expression level.

[0343] 3. Identification and/or isolation of intergenic sequences which are flanked on both sides by genes having an expression pattern which is preferably independent from the expression pattern of the other paired gene. The term "independent expression pattern" in this context means--for example--that the expression of the first gene is different in its tissue and/or developmental regulation from the expression of the second gene. Dependency and correlation of expression patterns of paired genes has been identified as an indicator for read-through transcription. Expression profiles can be analyzed either by experiment by comparing expression level of said paired genes in various organs or tissues (e.g., in vitro or in vivo for example by using expression profiling by chip technology) or--preferably--in silico by simply counting the number of ESTs for each gene in non-normalized EST/cDNA-libraries which is indicative for expression levels.

[0344] 4. Identification and/or isolation of intergenic sequences which are flanked on one or--preferably--both sides by genes having a low variability in length of the mRNA transcript derived from said paired genes. Such variability is for example indicated by existence of more than one transcript end in EST, cDNA libraries or databases. Variability in transcript length has been identified as an indicator for low stringency in transcription termination. Variability in transcript length can be analyzed either by experiment (e.g., by RT PCR) or--preferably--in silico by simply analyzing the 3'-ends of EST or cDNA clones in the database.

[0345] While the intergenic localization (step 1) is a prerequisite, in a preferred embodiment of the invention each of the criteria 2, 3, 4, and the length of the intergenic region (part of criteria 1) for a certain intergenic sequence is resulting in a criteria score. Addition of said scores (which may be multiplied by certain weight-indicators reflecting the different impact of the criteria) is resulting in a final score which is indicative for the potential of the sequence as a transcription terminator and isolator (see below). This score and the potential can be verified by evaluating the sequence of said intergenic region in one or more screening systems of the invention. The highest weight is given to criteria 2 (high expression profile), followed by criteria 2 (independent expression profile), and criteria 3 (low variability in transcript length). The preferred length for the intergenic regions are indicated below, but seem to have more impact on handling (i.e. in later cloning and transformation procedures) than on functionality of said region.

[0346] An intergenic region identified thereby is not only suitable in mono-gene expression cassettes, but is especially suitable in multi-gene expression cassettes not only providing transcription termination for two genes in one sequence, but also allowing efficient "isolation" of said two expression cassettes by minimizing their interference by read-through transcription (thus providing an "isolator"), which has proven to be a serious problem especially in multi-gene expression constructs. The term "isolator" when referring to a sequence (which is preferably localized in between two expression cassettes) as used herein is intended to mean the capability of said sequence to minimize or prevent the influence of one expression cassette on transcription from the other expression cassette, thus isolating the two expression cassettes from each other. Preferred embodiments and additional information for carrying out this method for providing intergenic sequences is given in Example 1.2 below.

[0347] Accordingly, another embodiment of the invention is directed to a method for identification and/or isolation intergenic regions--preferably with high transcription termination and/or isolator potential--said method including at least one, preferably at least two, more preferably at least three, most preferably all of the following selection criteria

[0348] 1. Identification and/or isolation or isolation of intergenic regions between paired genes meeting predefined intergenic distance criteria.

[0349] 2. Identification and/or isolation of intergenic sequences which are flanked on both sides by genes having a high expression level.

[0350] 3. Identification and/or isolation of intergenic sequences which are flanked on both sides by genes having an expression pattern which is preferably independent from the expression pattern of the other paired gene a high expression level.

[0351] 4. Identification and/or isolation of intergenic sequences which are flanked on one or--preferably--both sides by genes having a low variability in length of the mRNA transcript derived from said paired genes.

[0352] Preferably the paired genes flanking the intergenic region have tail-to-tail orientation Preferably, intergenic regions from paired genes in tail-to-tail orientation are identified which have a length from about 400 to 3,000 base pairs, preferably from about 700 to about 2,000 base pairs.

[0353] Thus, a preferred embodiment of the invention is related to a method for identification and/or isolation of intergenic regions with transcription termination potential said method including at least the steps of

[0354] a) identification and/or isolation or isolation of intergenic regions between paired genes having an intergenic distance of about 400 to 3,000 base pairs, and

[0355] b) identification and/or isolation of intergenic sequences which are flanked on both sides by genes having a high expression level.

[0356] More preferably said method further comprising the steps of

[0357] c) identification and/or isolation of intergenic sequences which are flanked on both sides by genes having an expression pattern which is preferably independent from the expression pattern of the other paired gene, and

[0358] d) identification and/or isolation of intergenic sequences which are flanked on one or--preferably--both sides by genes having a low variability in length of the mRNA transcript derived from said paired genes.

[0359] Most preferably, the intergenic region

[0360] a) is localized between genes which have a tail-to-tail localization (i.e. from which expression from said genes is directed in opposite direction against each other) and/or

[0361] b) has a length measured from the respective stop codons of the flanking genes from about 700 to about 2,000 base pairs.

[0362] Based on said method sequences from the rice genome were identified and found to be promising as transcription terminator sequences. Said sequences are described by SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, and 46.

[0363] Another embodiment of the invention is related to the use of said sequences to terminate transcription in a transgenic expression construct. More preferably is the use of said sequences as isolators in multi-gene expression constructs.

[0364] Another embodiment of the invention is related to a transgenic expression construct comprising in 5'-3'-direction

[0365] a) a promoter sequence functional in plants, and

[0366] b) a nucleic acid sequence of interest of to be expressed operably linked to said promoter a), and

[0367] c) at least one sequence selected from the group consisting of

[0368] i) the sequences described by SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, and 46, and

[0369] ii) the sequences having a homology of at least 60%, preferably 80%, more preferably 90%, most preferably 95% with a sequences described by described by SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46, capable to terminate transcription in a plant cell or organism, and

[0370] iii) the sequences hybridizing under low stringency conditions, preferably under high stringency conditions with a sequences described by described by SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 capable to terminate transcription in a plant cell or organism, and

[0371] iv) a fragment of at least 50 consecutive base pairs, preferably at least 100 consecutive base pairs, more preferably at least 250 consecutive base pairs, most preferably at least 500 consecutive base pairs of a sequence described under i), ii), and iii),

[0372] wherein said sequence c) is heterolog with respect to said promoter a) and/or said nucleic acid of interest b) and is mediating termination of expression of induced from said promoter a).

[0373] Another embodiment of the invention is related to a transgenic expression construct comprising at least two expression cassettes having a structure comprising in 5'-3'-direction

[0374] a1) a first promoter sequence functional in plants, and

[0375] b1) a first nucleic acid sequence of interest of to be expressed operably linked to said promoter a1), and

[0376] c) at least one sequence selected from the group consisting of

[0377] i) the sequences described by SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, and 46, and

[0378] ii) the sequences having a homology of at least 60%, preferably 80%, more preferably 90%, most preferably 95% with a sequences described by described by SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46, capable to terminate transcription in a plant cell or organism, and

[0379] iii) the sequences hybridizing under low stringency conditions, preferably under high stringency conditions with a sequences described by described by SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46, capable to terminate transcription in a plant cell or organism, and

[0380] iv) a fragment of at least 50 consecutive base pairs, preferably at least 100 consecutive base pairs, more preferably at least 250 consecutive base pairs, most preferably at least 500 consecutive base pairs of a sequence described under i), ii), and iii),

[0381] and,

[0382] b2) a second nucleic acid sequence of interest of to be expressed, and

[0383] a2) a second promoter sequence functional in plants operably linked to said nucleic acid sequence of interest b2), wherein said sequence c) is heterolog with respect to at least one element selected from promoter a1), promoter a2), nucleic acid of interest b1) and nucleic acid of interest b2), and is mediating termination of expression of induced from said promoters a1) and a2).

[0384] Since no protein expression is caused from the above described transcription terminator sequences, a higher degree of variation is acceptable without changing the functionality.

[0385] The method of the invention can also be employed to identify regions responsible for transcription termination within larger sequences. This would allow to delete unnecessary sequences and to provide small sequences for transcription termination, which is an important goal in construction gene expression vectors. Large sequences leading to large vectors are linked to inefficient transformation and instability of constructs. Preferably, such identification can be realized by inserting fragments of a larger sequence into a screening vector or screening construct. Such fragments can be derived, for example, by nuclease mediated shorting of 5'- and/or 3'-ends of the larger sequence (by restrictions enzymes or unspecific nucleases such as Bal31). Corresponding methods are well known to the person skilled in the art.

[0386] The larger sequence for which one may seek to identify the essential region for transcription termination may for example be the natural region downstream of the coding sequence of the gene, which is the source for the promoter employed in the transgenic expression construct (e.g., comprising the region encoding the 3'-untranslated region and additional downstream genomic sequences), preferably from a region which is surrounding the end of the mRNA transcript. It is advantageous to combine a promoter with its natural transcription terminator (and the heterogeneous sequence of interest in between) to obtain optimal expression results. While formerly either very long 3'-untranslated regions had to be employed to ensure efficient transcriptions termination, or laborious testing of shortened sequences had to be performed, the method of the present invention is allowing for fast and efficient restriction of a potential transcription terminator to its essential elements.

4. Insertion of the DNA Molecules into the Screening Vector

[0387] The DNA molecules to be assessed for their transcription termination efficiency may be inserted into the Screening Vector by various means. Preferably, the insertion is realized by one or more methods selected from the group consisting of:

[0388] a) Insertion into a restriction site: Various sequence specific endonuclease are known to the person skilled in art which can be employed for carrying out the method of the invention. Suitable endonuclease may be for example type II restriction endonucleases or artificial (e.g., chimeric) nucleases. Preferred are restriction endonucleases which are chosen in a way that only the insertion site is cleaved by said restriction endonuclease. Such restriction endonuclease may preferably include rare cutting endonucleases which have a recognition site of at least 8 base pairs (such as for example NotI) or even homing endonucleases, which have very long recognition sequences (Belfort 1997; Jasin 1996; Internet: http://rebase.neb.com/rebase/rebase.homing.html; Roberts 2001). Examples for preferred homing endonucleases include but are not limited to F-SceI, I-CeuI, I-ChuI, I-DmoI, I-CpaI, I-CpalI, I-CreI, I-CsmI, F-TevI, F-TevII, I-TevI, I-TevII, I-AniI, I-CvuI, I-LlaI, I-NanI, I-MsoI, I-NitI, I-NjaI, I-PakI, I-Port, I-PpoI, I-ScaI, I-Ssp6803I, PI-PkoI, PI-PkoII, PI-PspI, PI-TfuI, PI-TliI. Most preferred are I-CeuI, I-SceI, I-PpoI, PI-PspI, and PI-SceI.

[0389] b) Insertion into a recombination site: In a preferred embodiment of the invention, the insertion of DNA segments into the insertion site of the screening construct or screening vector is achieved by the use of recombination proteins, including recombinases and associated co-factors and proteins. Numerous recombination systems from various organisms can also be used, based on the teaching and guidance provided herein. See, e.g., Hoess 1986; Abremski 1986; Campbell, 1992; Qian 1992; Araki 1992). Many of these belong to the integrase family of recombinases (Argos 1986). Perhaps the best studied of these are the Integrase/att system from bacteriophage λ (Landy 1993), the Cre/loxP system from bacteriophage P1 (Hoess 1990), and the FLP/FRT system from the Saccharomyces cerevisiae 2μ circle plasmid (Broach 1982)). Detailed method for recombinase mediated cloning, appropriate recombination sites (to be employed as insertion sites), and corresponding recombinases are described e.g., in U.S. Pat. No. 5,888,732, hereby incorporated entirely by reference. A preferred system is the Gateway® cloning system (Invitrogen, Inc.). Corresponding ready-to-use mixture of lambda integrase with its corresponding co-factors can be obtained from Invitrogen Inc. (Gateway® LR Clonase® Plus enzyme).

[0390] Also procedures comprising combination of both method can be employed. However, it is not essential that the sequence to be assessed is inserted directly (i.e. in an one-step cloning procedure) into a quasi ready-to-go screening construct or screening vector. It may for example--first be linked to the additional sequence and then inserted into an appropriate construct or vector thus constituting the final screening construct or screening vector to be employed in the evaluation procedure. In principle, the ways and possibilities to assembly the various parts of a screening construct or screening vector are uncountable but well known and established to the person skilled in the art.

[0391] For the purpose of insertion into the insertion site the DNA sequence to be inserted may be linked to adapters providing the appropriate recognition sequences for restriction endonuclease or recombinase, respectively. However, in the case of restriction endonucleases adapters are not required in cases where a digestion of genomes is employed as a library of DNA sequences. Here the restriction enzyme employed for the digest should create DNA ends compatible with those at the cleaved insertion site.

5. The In Vitro Screening System

[0392] When performed as an in vitro screening system, the expression of the additional sequences (which may preferably be located downstream (i.e. in 3'-direction) of the insertion site) may be--for example--easily detected at the RNA levels using sensitive fluorescence probes that recognize single strand nucleotides. Such features are also to be understood as readily detectable characteristics. In case an efficient transcription terminator sequence is inserted in front of these sequences a reduced, preferably no significant or at all observable signal will be obtained.

[0393] Within the in vitro screening system, transcription of sequences located downstream of the transcription termination sequence inserted into the insertion site can be detected at the RNA levels using commercially available in vitro transcription systems (such as wheat germ nuclear extracts, HeLa nuclear extracts, rabbit reticulocyte extracts, or nuclear extracts from plant of interest) preferably in combination with single strand recognizing florescence probes (e.g. beacon probes). Various suitable in vitro transcription/translation systems are known in the art and commercially available (e.g., ActivePro®, PROTEINscript® II, Retic Lysate IVT® (treated) and Retic Lysate IVT®-96, Wheat Germ IVT®; all available from Ambion, Inc., Austin, USA). In this case no plant transformation is involved. In consequence, the screening construct or screening vector can be constructed on a simple base (e.g., pUC based). Preferably, individual screening constructs or screening vectors comprising different transcription termination sequences are placed in 96 well plates for in vitro transcription. The fluorescent probe hybridizes when read through occurs. The tighter transcription termination occurs, the less fluorescent products in the read through region are detected. The amounts of read through products can be normalized by the expression of sequences located upstream of the transcription termination sequences but still under control of the promoter.

6. The In Vivo Screening System

[0394] In a preferred embodiment, the method of the invention is realized in vivo, preferably in the target organism in which an efficient transcription terminator is sought for. The in vivo screening system allows for evaluation of multiple DNA sequences for their performance as transcription terminator sequences in parallel. Thus, a library of DNA sequences can be employed and inserted into the screening construct or screening vector yielding a library of screening constructs or screening vectors comprising various different DNA sequences. Said library of screening constructs or screening vectors is inserted into cells or organisms in a way that each individual cell or organism preferably comprises only one screening constructs or screening vectors of said library (comprising one specific DNA sequence to be assessed for the transcription termination capability). In consequence--as described below in more detail--this preferred embodiment does not necessarily require the sorting of the various screening constructs or screening vectors prior to evaluation for the transcription termination capability, which makes the method even more efficient. Thus, in a preferred embodiment the method of the invention therefore relates to a method for identification and isolation of transcription termination sequences for comprising the steps of:

[0395] i) providing a screening construct or screening vector comprising

[0396] a) a promoter sequence, and

[0397] b) one or more insertion sites--preferably a restriction or recombination site--for insertion of DNA sequences, and

[0398] c) at least one additional sequence which causes upon expression under said promoter sequence a readily detectable characteristic, wherein insertion of an efficient transcription terminator into said insertion site changes expression of said additional sequences by said promoter sequence in comparison to no insertion, and

[0399] ii) providing one or more DNA sequences to be assessed for their transcription termination capability, and

[0400] iii) inserting one or more copies of said DNA sequences into said insertion site of said screening construct or screening vector, and

[0401] iv) introducing said screening construct or screening vector with said inserted DNA sequences into cells or organisms suitable to induce expression from said promoter sequence, and

[0402] v) identifying and/or selecting cells or organisms with a changed readily detectable characteristic in comparison to no insertion, and

[0403] vi) isolating the inserted DNA sequences from said identified and/or selected screening construct or screening vector for use as transcription termination sequences and--optionally--determining their sequence.

[0404] All the above specified preferred variations (such as Variation A, B, or C) can also be advantageously combined with said in vivo system. In the in vivo screening systems of the invention, the expression of the sequence located downstream (at the 3'-end) of the insertion site will preferably cause an easily detectable phenotype. "Causing" includes both initiating or suppressing an easily detectable phenotype. For example, the sequence located downstream (at the 3'-end) of the insertion site may either code for a phenotype causing protein, or it may code for RNA (e.g., antisense or double stranded RNA) which causes suppression of expression of a phenotype causing protein. Multiple examples are given above.

[0405] In an preferred embodiment of the in vivo screening systems of the invention, the expression of the sequence located downstream (at the 3'-end) of the insertion site will cause a phenotype which is inhibiting growth, propagation and/or or regeneration of said cells or organisms (e.g., plant cells or plants), and which is therefore understood within the context of this invention to be "toxic" to said cells and/organisms (e.g., plant cells or plants). In consequence, only cells (or organisms) will survive if a tight transcription termination sequence is inserted in front of said toxic phenotype causing sequence thereby preventing expression of this growth, propagation and/or or regeneration inhibiting sequences. The surviving cells can be isolated and the transcription terminator sequence can be identified and isolated, e.g., by amplification using PCR followed by sequencing.

[0406] For conducting the screening in the in vivo system the screening construct or screening vector in transformed preferably into a cell, tissue or organism. The generation of a transformed organism or a transformed cell requires introducing the DNA in question into the host cell in question. A multiplicity of methods is available for this procedure, which is termed transformation (see also Keown 1990). For example, the DNA can be introduced directly by microinjection or by bombardment via DNA-coated microparticles. Also, the cell can be permeabilized chemically, for example using polyethylene glycol, so that the DNA can enter the cell by diffusion. The DNA can also be introduced by protoplast fusion with other DNA-containing units such as minicells, cells, lysosomes or liposomes. Another suitable method of introducing DNA is electroporation, where the cells are permeabilized reversibly by an electrical pulse.

[0407] The host cell or organism can be any prokaryotic or eukaryotic organism. Preferred are mammalian cells, non-human mammalian organism, plant cells and plant organisms as defined above.

[0408] The screening construct or screening vector of the invention is preferably introduced into a eukaryotic cell. It may be preferably inserted into the genome (e.g., plastids or chromosomal DNA) but may also be exist extra-chromosomal or epichromosomal. Preferred eukaryotic cells are mammalian cell, fungal cell, plant cell, insect cell, avian cell, and the like. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570, ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHOK1; ATCC CCL61; CHO DG44 (Chasin 1986), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).

[0409] A screening construct or screening vector can be introduced into host cells using a variety of standard techniques including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, and the like. Transfected cells can be selected and propagated to provide recombinant host cells that comprise the gene of interest stably integrated in the host cell genome.

[0410] The screening vector may be a baculovirus expression vector to be employed in a baculovirus system. The baculovirus system provides an efficient means to introduce cloned genes of interest into insect cells. Suitable expression vectors are based upon the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as Drosophila heat shock protein (hsp) 70 promoter, Autographa californice nuclear polyhedrosis virus immediate-early gene promoter (ie-1) and the delayed early 39K promoter, baculovirus p10 promoter, and the Drosophila metallothionein promoter. A second method of making recombinant baculovirus utilizes a transposon-based system (Luckow 1993). This system, which utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This system utilizes a transfer vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to move the DNA encoding the polypeptide of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "bacemid" (see, Hill-Perkins 1990; Bonning 1994; and Chazenbalk 1995). In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer 1985). Using a technique known in the art, a transfer vector containing a gene of interest is transformed into E. coli, and screened for bacmids, which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is then isolated using common techniques. The recombinant virus or bacinid is used to transfect host cells. Suitable insect host cells include cell lines derived from IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL 1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), as well as Drosophila Schneider-2 cells, and the HIGH FIVE® cell line (Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commercially available serum-free media can be used to grow and to maintain the cells. Suitable media are Sf900 II® (Life Technologies) or ESF 921® (Expression Systems) for the Sf9 cells; and ExcellO405® (JRH Biosciences, Lenexa, Kans.) or Express Five® (Life Technologies) for the T. ni cells. When recombinant virus is used, the cells are typically grown up from an inoculation density of approximately 2-5×10⁵ cells to a density of 1-2×10⁶ cells at which time a recombinant viral stock is added at a multiplicity of infection of 0.1 to 10, more typically near 3. Established techniques for the baculovirus systems are provided by Bailey 1991, Patel 1995, Ausubel 1995 (at pages 16-37 to 16-57), Richardson 1995, and by Lucknow, 1996.

[0411] Fungal cells, including yeast cells, can also be used as host cells for transformation with the screening construct or screening vector of the invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Suitable promoters for expression in yeast include promoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like. Many yeast cloning vectors have been designed and are readily available to be employed. These vectors include YIp-based vectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such as YEp13 and YCp vectors, such as YCp19. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, U.S. Pat. No. 4,599,311, U.S. Pat. No. 4,931,373, U.S. Pat. No. 4,870,008, U.S. Pat. No. 5,037,743, and U.S. Pat. No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). An illustrative vector system for use in Saccharomyces cerevisiae is the POTI vector system (U.S. Pat. No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Additional suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., U.S. Pat. No. 4,599,311, U.S. Pat. No. 4,615,974, and U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446, 5,063,154, 5,139,936, and 4,661,454.

[0412] Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson 1986, and U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al. (U.S. Pat. No. 4,935,349). Methods for transforming Acremonium chrysogenum are disclosed (U.S. Pat. No. 5,162,228). Methods for transforming Neurospora are disclosed (U.S. Pat. No. 4,486,533).

[0413] For example, the use of Pichia methanolica as host for the production of recombinant proteins is disclosed (U.S. Pat. No. 5,716,808, U.S. Pat. No. 5,736,383, Raymond 1998, WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565). DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P. methanolica, the promoter and terminator in the plasmid can be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formnate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. For large-scale, industrial processes where it is desirable to minimize the use of methanol host cells can be used in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells can be used that are deficient in vacuolar protease genes (PEP4 and PRB1). Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. P. methanolica cells can be transformed by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.

[0414] Standard methods for introducing nucleic acid molecules into bacterial, yeast, insect, mammalian, and plant cells are provided, for example, by Ausubel (1995). General methods for expressing and recovering foreign protein produced by a mammalian cell system are provided by, for example, in Etcheverry 1996. Established methods for isolating recombinant proteins from a baculovirus system are described (Richardson 1995).

[0415] Especially preferred in transfer of the screening construct or screening vector into plant cells, tissues and/or organism. Methods for introduction of a transgenic expression construct or vector into plant tissue may include but are not limited to, e.g., electroinjection (Nan 1995; Griesbach 1992); fusion with liposomes, lysosomes, cells, minicells or other fusible lipid-surfaced bodies (Fraley 1982); polyethylene glycol (Krens 1982); chemicals that increase free DNA uptake; transformation using virus, and the like. Furthermore, the biolistic method with the gene gun, electroporation, incubation of dry embryos in DNA-containing solution, and microinjection may be employed.

[0416] Protoplast based methods can be employed (e.g., for rice), where DNA is delivered to the protoplasts through liposomes, PEG, or electroporation (Shimamoto 1989; Datta 1990b). Transformation by electroporation involves the application of short, high-voltage electric fields to create "pores" in the cell membrane through which DNA is taken-up. These methods are--for example--used to produce stably transformed monocotyledonous plants (Paszkowski 1984; Shillito 1985; Fromm 1986) especially from rice (Shimamoto 1989; Datta 1990b; Hayakawa 1992).

[0417] Particle bombardment or "biolistics" is a widely used method for the transformation of plants, especially monocotyledonous plants. In the "biolistics" (microprojectile-mediated DNA delivery) method microprojectile particles are coated with DNA and accelerated by a mechanical device to a speed high enough to penetrate the plant cell wall and nucleus (WO 91/02071). The foreign DNA gets incorporated into the host DNA and results in a transformed cell. There are many variations on the "biolistics" method (Sanford 1990; Fromm 1990; Christou 1988; Sautter 1991). The method has been used to produce stably transformed monocotyledonous plants including rice, maize, wheat, barley, and oats (Christou 1991; Gordon-Kamm 1990; Vasil 1992; Wan 1994).

[0418] In addition to these "direct" transformation techniques, transformation can also be effected by bacterial infection by means of Agrobacterium tumefaciens or Agrobacterium rhizogenes. These strains contain a plasmid (Ti or Ri plasmid) which is transferred to the plant following Agrobacterium infection. Part of this plasmid, termed T-DNA (transferred DNA), is integrated into the genome of the plant cell (see above for description of vectors). To transfer the DNA to the plant cell, plant explants are cocultured with a transgenic Agrobacterium tumefaciens or Agrobacterium rhizogenes. Starting from infected plant material (for example leaf, root or stem sections, but also protoplasts or suspensions of plant cells), intact plants can be generated using a suitable medium which may contain, for example, antibiotics or biocides for selecting transformed cells. The plants obtained can then be screened for the presence of the DNA introduced, in this case the expression construct according to the invention. As soon as the DNA has integrated into the host genome, the genotype in question is, as a rule, stable and the insertion in question is also found in the subsequent generations. As a rule, the expression construct integrated contains a selection marker which imparts a resistance to a biocide (for example a herbicide) or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin and the like to the transformed plant. The selection marker permits the selection of transformed cells from untransformed cells (McCormick 1986). The plants obtained can be cultured and hybridized in the customary fashion. Two or more generations should be grown in order to ensure that the genomic integration is stable and hereditary. The abovementioned methods are described in detail in the relevant art (for example, in Jenes 1993, and in Potrykus 1991).

[0419] One of skill in the art knows that the efficiency of transformation by Agrobacterium may be enhanced by using a number of methods known in the art. For example, the inclusion of a natural wound response molecule such as acetosyringone (AS) to the Agrobacterium culture has been shown to enhance transformation efficiency with Agrobacterium tumefaciens (Shahla 1987). Alternatively, transformation efficiency may be enhanced by wounding the target tissue to be transformed. Wounding of plant tissue may be achieved, for example, by punching, maceration, bombardment with microprojectiles, etc. (see, e.g., Bidney 1992).

[0420] A number of other methods have been reported for the transformation of plants (especially monocotyledonous plants) including, for example, the "pollen tube method" (WO 93/18168; Luo 1988), macro-injection of DNA into floral tillers (Du 1989; de la Pena 1987), injection of Agrobacterium into developing caryopses (WO 00/63398), and tissue incubation of seeds in DNA solutions (Topfer 1989). Direct injection of exogenous DNA into the fertilized plant ovule at the onset of embryogenesis was disclosed in WO 94/00583. WO 97/48814 disclosed a process for producing stably transformed fertile wheat and a system of transforming wheat via Agrobacterium based on freshly isolated or pre-cultured immature embryos, embryogenic callus and suspension cells.

[0421] It may be desirable to target the nucleic acid sequence of interest to a particular locus on the plant genome. Site-directed integration of the nucleic acid sequence of interest into the plant cell genome may be achieved by, for example, homologous recombination using Agrobacterium-derived sequences. Generally, plant cells are incubated with a strain of Agrobacterium which contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described (U.S. Pat. No. 5,501,967, the entire contents of which are herein incorporated by reference). One of skill in the art knows that homologous recombination may be achieved using targeting vectors which contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene, or the coding regions of the gene. Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known.

[0422] Where homologous recombination is desired, the targeting vector used may be of the replacement- or insertion-type (U.S. Pat. No. 5,501,967). Replacement-type vectors generally contain two regions which are homologous with the targeted genomic sequence and which flank a heterologous nucleic acid sequence, e.g., a selectable marker gene sequence. Replacement-type vectors result in the insertion of the selectable marker gene which thereby disrupts the targeted gene. Insertion-type vectors contain a single region of homology with the targeted gene and result in the insertion of the entire targeting vector into the targeted gene.

[0423] Transformed cells, i.e. those which contain the introduced DNA integrated into the DNA of the host cell, can be selected from untransformed cells if a selectable marker is part of the introduced DNA. A selection marker gene may confer positive or negative selection.

[0424] A positive selection marker gene may be used in constructs for random integration and site-directed integration. Positive selection marker genes include antibiotic resistance genes, and herbicide resistance genes and the like. Transformed cells which express such a marker gene are capable of surviving in the presence of concentrations of the antibiotic or herbicide in question which kill an untransformed wild type. Examples are the bar gene, which imparts resistance to the herbicide phosphinotricin (bialaphos; Vasil 1992; Weeks 1993; Rathore 1993), the nptII gene, which imparts resistance to kanamycin, the hpt gene, which imparts resistance to hygromycin, or the EPSP gene, which imparts resistance to the herbicide glyphosate, geneticin (G-418) (aminoglycoside) (Nehra 1994), glyphosate (Della-Cioppa et al. 1987) and the ALS gene (chlorsulphuron resistance). Further preferred selectable and screenable marker genes are disclosed above.

[0425] A negative selection marker gene may also be included in the constructs. The use of one or more negative selection marker genes in combination with a positive selection marker gene is preferred in constructs used for homologous recombination. Negative selection marker genes are generally placed outside the regions involved in the homologous recombination event. The negative selection marker gene serves to provide a disadvantage (preferably lethality) to cells that have integrated these genes into their genome in an expressible manner. Cells in which the targeting vectors for homologous recombination are randomly integrated in the genome will be harmed or killed due to the presence of the negative selection marker gene. Where a positive selection marker gene is included in the construct, only those cells having the positive selection marker gene integrated in their genome will survive. The choice of the negative selection marker gene is not critical to the invention as long as it encodes a functional polypeptide in the transformed plant cell. The negative selection gene may for instance be chosen from the aux-2 gene from the Ti-plasmid of Agrobacterium, the tk-gene from SV40, cytochrome P450 from Streptomyces griseolus, the Adh gene from Maize or Arabidopsis, etc. Any gene encoding an enzyme capable of converting a substance which is otherwise harmless to plant cells into a substance which is harmful to plant cells may be used. Further preferred negative selection markers are disclosed above.

[0426] However, insertion of an expression cassette or a vector into the chromosomal DNA can also be demonstrated and analyzed by various other methods (not based on selection marker) known in the art like including, but not limited to, restriction mapping of the genomic DNA, PCR-analysis, DNA-DNA hybridization, DNA-RNA hybridization, DNA sequence analysis and the like. More specifically such methods may include e.g., PCR analysis, Southern blot analysis, fluorescence in situ hybridization (FISH), and in situ PCR.

[0427] As soon as a transformed plant cell has been generated, an intact plant can be obtained using methods known to the skilled worker. Accordingly, the present invention provides transgenic plants. The transgenic plants of the invention are not limited to plants in which each and every cell expresses the nucleic acid sequence of interest under the control of the promoter sequences provided herein. Included within the scope of this invention is any plant which contains at least one cell which expresses the nucleic acid sequence of interest (e.g., chimeric plants). It is preferred, though not necessary, that the transgenic plant comprises the nucleic acid sequence of interest in more than one cell, and more preferably in one or more tissue.

[0428] Once transgenic plant tissue which contains an expression vector has been obtained, transgenic plants may be regenerated from this transgenic plant tissue using methods known in the art. The term "regeneration" as used herein, means growing a whole plant from a plant cell, a group of plant cells, a plant part or a plant piece (e.g., from a protoplast, callus, protocorm-like body, or tissue part).

[0429] Species from the following examples of genera of plants may be regenerated from transformed protoplasts: Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciohorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Lolium, Zea, Triticum, Sorghum, and Datura.

[0430] For regeneration of transgenic plants from transgenic protoplasts, a suspension of transformed protoplasts or a Petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and plant hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. These three variables may be empirically controlled to result in reproducible regeneration.

[0431] Plants may also be regenerated from cultured cells or tissues. Dicotyledonous plants which have been shown capable of regeneration from transformed individual cells to obtain transgenic whole plants include, for example, apple (Malus pumila), blackberry (Rubus), Blackberry/raspberry hybrid (Rubus), red raspberry (Rubus), carrot (Daucus carota), cauliflower (Brassica oleracea), celery (Apium graveolens), cucumber (Cucumis sativus), eggplant (Solanum melongena), lettuce (Lactuca sativa), potato (Solanum tuberosum), rape (Brassica napus), wild soybean (Glycine canescens), strawberry (Fragaria ananassa), tomato (Lycopersicon esculentum), walnut (Juglans regia), melon (Cucumis melo), grape (Vitis vinifera), and mango (Mangifera indica). Monocotyledonous plants which have been shown capable of regeneration from transformed individual cells to obtain transgenic whole plants include, for example, rice (Oryza sativa), rye (Secale cereale), and maize (Zea mays).

[0432] In addition, regeneration of whole plants from cells (not necessarily transformed) has also been observed in: apricot (Prunus armeniaca), asparagus (Asparagus officinalis), banana (hybrid Musa), bean (Phaseolus vulgaris), cherry (hybrid Prunus), grape (Vitis vinifera), mango (Mangifera indica), melon (Cucumis melo), ochra (Abelmoschus esculentus), onion (hybrid Allium), orange (Citrus sinensis), papaya (Carrica papaya), peach (Prunus persica), plum (Prunus domestica), pear (Pyrus communis), pineapple (Ananas comosus), watermelon (Citrullus vulgaris), and wheat (Triticum aestivum).

[0433] The regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner. After the expression vector is stably incorporated into regenerated transgenic plants, it can be transferred to other plants by vegetative propagation or by sexual crossing. For example, in vegetatively propagated crops, the mature transgenic plants are propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. In seed propagated crops, the mature transgenic plants are self crossed to produce a homozygous inbred plant which is capable of passing the transgene to its progeny by Mendelian inheritance. The inbred plant produces seed containing the nucleic acid sequence of interest. These seeds can be grown to produce plants that would produce the selected phenotype. The inbred plants can also be used to develop new hybrids by crossing the inbred plant with another inbred plant to produce a hybrid.

[0434] Confirmation of the transgenic nature of the cells, tissues, and plants may be performed by PCR analysis, antibiotic or herbicide resistance, enzymatic analysis and/or Southern blots to verify transformation. Progeny of the regenerated plants may be obtained and analyzed to verify whether the transgenes are heritable. Heritability of the transgene is further confirmation of the stable transformation of the transgene in the plant. The resulting plants can be bred in the customary fashion. Two or more generations should be grown in order to ensure that the genomic integration is stable and hereditary. Corresponding methods are described, (Jenes 1993; Potrykus 1991).

7. Conduction the Screening, Isolation and Use of the Transcription Terminator Sequences

[0435] Once one or more DNA sequences or even a library of sequences to be assessed for their transcription termination efficiency was inserted into the screening construct or screening vector these vectors are submitted to the appropriate in vitro or in vivo screening system.

[0436] The readily detectable characteristic or the change thereof can be monitored by various means well known to the person skilled in the art depending on the additional sequence employed. The "output" of the screening system (i.e. the number of different transcription terminator sequences) and their efficiency can be controlled by setting certain cut-off limits. For example a certain intensity of color or fluorescence (in case the characteristic is a color), a certain resistance against a toxic compound (in case the characteristic is a resistance).

[0437] Screening constructs, screening vectors, or cells or organisms comprising those, derived from the screening process can be employed to isolate and analyze the transcription termination sequences comprised therein. Isolation can be done by various means including but not limited to PCR mediated amplification of the sequence inserted into the insertion site using primers specific for the known regions flanking said insertion site.

[0438] The isolated transcription terminator sequence can be used for various purposes in biotechnology, preferably in constructing gene expression constructs which require a tight transcription termination control i.e. a low read-through frequency. Such expression cassettes (consisting for example in 5'/3'-direction of a promoter, a gene of interest, and the isolated transcription termination sequence) can be produced by means of customary recombination and cloning techniques as are described (for example, in Maniatis 1989; Silhavy 1984; and in Ausubel 1987). The person skilled in the art is aware of numerous sequences which may be utilized as gene of interest in this context, e.g. to increase quality of food and feed, to produce chemicals, fine chemicals or pharmaceuticals (e.g., vitamins, oils, carbohydrates; Dunwell 2000), conferring resistance to herbicides, or conferring male sterility. Furthermore, growth, yield, and resistance against abiotic and biotic stress factors (like e.g., fungi, viruses, nematodes, or insects) may be enhanced. Advantageous properties may be conferred either by overexpressing proteins or by decreasing expression of endogenous proteins by e.g., expressing a corresponding antisense (Sheehy 1988; U.S. Pat. No. 4,801,340; Mol 1990) or double-stranded RNA (Matzke 2000; Fire 1998; Waterhouse 1998; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364).

[0439] It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are readily apparent and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

TABLE-US-00003 Sequences 1. SEQ ID NO: 1 Binary expression vector Lo546b-pSUN1-R4-Lo484::Lo376::Lo522b 2. SEQ ID NO: 2 Binary expression vector Lo546a-pSUN1-R4-Lo484::Lo376::Lo522a 3. SEQ ID NO: 3 Nucleic acid construct Lo522b-pENTR-C1-STPT-nptll-IRnos 4. SEQ ID NO: 4 Nucleic acid construct Lo522a-pENTR-C1-STPT-nptll-IRnos 5. SEQ ID NO: 5 Binary expression vector Lo523b-pSUN1-R4-Lo484::Lo376::Lo503b 6. SEQ ID NO: 6 Binary expression vector Lo523a-pSUN1-R4-Lo484::Lo376::Lo503a 7. SEQ ID NO: 7 Nucleic acid construct Lo503b-pENTR-C1-STPT-nptll-IRnos 8. SEQ ID NO: 8 Nucleic acid construct Lo503a-pENTR-C1-STPT-nptll-IRnos 9. SEQ ID NO: 9 Nucleic acid construct Lo484-pENTR-A1-inv-35s-GFP-E9 10. SEQ ID NO: 10-46: Nucleic acid sequence from rice (Oryza sativa sbsp. japonica) encoding sequences suitable as transcription terminators and expression cassette isolators. 11. SEQ ID NO: 47: Primer 1 (SacI, AvrII, SpeI, OCS 5') 5'-CG GAGCTC CCTAGG ACTAGT tcgaccggcatgccc-3' 12. SEQ ID NO: 48 Primer 2 (NotI, OCS 3') 5'-CC GCGGCCGC agcttggacaatcag-3' 13. SEQ ID NO: 49 Primer 3 (AvrII, XmaI, RsrII, Lu^F 5') 5'-CG CCTAGG CCCGGG CGGACCG cattaagaagggccc-3' 14. SEQ ID NO: 50 Primer 4 (SpeI Lu^F 3') 5'-CG ACTAGT agagagttctcagagc-3' 15. SEQ ID NO: 51 Primer 5 (RsrII, BspEI, target gene seq 5') 5' CG CGGACCG TCCGGA-N-3' [N represents a gene-specific sequence of preferably 10 to 20 bases] 16. SEQ ID NO: 52 Primer 6 (SpeI, AgeI, target gene seq 3') 5' CG ACTAGT ACCGGT-N-3' [N represents a gene-specific sequence of preferably 10 to 20 bases] 17. SEQ ID NO: 53 pTOI3 18. SEQ ID NO: 54 pTOI4 19. SEQ ID NO: 55 Oligonucleotideprimer Loy482-NosT-upper-SalI 5'-AAATTTGTCGACCGATCGGTCAAACATT-3' 20. SEQ ID NO: 56 Oligonucleotideprimer Loy483-NosT-Lower-HindIII 5'-AAATTTAAGCTTCCCGATCTAGTAACATAGATGACA-3' 21. SEQ ID NO: 57 Oligonucleotideprimer Loy494-Gus_upper_SalI_Spacer 5'-TTTTAGTCGACACGCTGGACTGGCATGAACT-3' 22. SEQ ID NO: 58 Oligonucleotideprimer Loy492-NosT-lower-BglII SpeI 5'-TTTTAAGATCTACTAGTCCGATCTAGTAACATAGATGACA-3' 23. SEQ ID NO: 59 Oligonucleotideprimer Loy493_Gus_upper_SalI_Spacer 5'-TTTAAGTCGACAAGTCGGCGGCTTTTCTGCT-3' 24. SEQ ID NO: 60 Oligonucleotideprimer Loy492-NosT-lower-BglII SpeI 5'-TTTTAAGATCTACTAGTCCGATCTAGTAACATAGATGACA-3' 25. SEQ ID NO: 61 Oligonucleotideprimer JMTOIprim1 5'-GGTTCCAAGGTACCAAAACAATGGGCGCTGATGATGTTGTTGAT-3' 26. SEQ ID NO: 62 Oligonucleotideprimer JMTOIprim2 5'-AAGGTAGAAGCAGAAACTTACCTGGATACGTCACTTTGACCA-3' 27. SEQ ID NO: 63 Oligonucleotideprimer JMTOIprim3 5'-TGGTCAAAGTGACGTATCCAGGTAAGTTTCTGCTTCTACCTT-3' 28. SEQ ID NO: 64 Oligonucleotideprimer JMTOIprim4 5'-GGTTCCAAGGATCCATTTATTTTGAAAAAAATATTTG-3' 29. SEQ ID NO: 65 Oligonucleotideprimer JMTOIprim5 5'-GGTTCCAAGGATCCAGTATATAGCAATTGCTTTTC-3' 30. SEQ ID NO: 66 Oligonucleotideprimer JMTOIprim6 5'-CGAGAACCTTCGTCAGTCCTGCACATCAACAAATTTTGGTCATAAAAAAAAAAATATTAGAA AAGTTATAAATTAAAATATAC-3' 31. SEQ ID NO: 67 Oligonucleotideprimer JMTOIprim7 5'-CTAATATTTTTTTTTTTATGACCAAAATTTGTTGATGTGCAGGAC-TGACGAAGGTTCTCGC AC-3' 32. SEQ ID NO: 68 Oligonucleotideprimer JMTOIprim8 5'-TTGGAACCACTAGTTTATCGCCTGACACGATTTCCTGC-3' 33. SEQ ID NO: 69 Oligonucleotideprimer JMTOIprim9 5'-GGTTCCAAGGATCCGATCGTTCAAACATTTGGCAA-3' 34. SEQ ID NO: 70 Oligonucleotideprimer JMTOIprim10 5'-GGTTCCAAGGATCCGATCTAGTAACATAGATGACA-3' 35. SEQ ID NO: 71 Screening construct pJMTOI1 36. SEQ ID NO: 72 Screening construct pJMTOI2 37. SEQ ID NO: 73 Screening construct pJMTOI3 38. SEQ ID NO: 74 Screening construct pJMTOI4 39. SEQ ID NO: 75 Screening construct pJMTOI5 40. SEQ ID NO: 76 Lo376-pENTR-B2 41. SEQ ID NO: 77 Lo442 pSUN1-R4R3-M20 (OCS10) (destination vector) 42. SEQ ID NO: 78 Binary vector Lo239-pSUN3-GWs-B1-BnAK700::GUS::nosT-B2 (10414 bp) 43. SEQ ID NO: 79 Binary vector Lo657-pSUN3-GWs-B1-BnAK700::GUS::E9::nosT::B2 (11153 bp) 44. SEQ ID NO: 80 GFP-Primer5: 5'-CGGCCTAGGGGCGCCCGGACCGagctgttcaccggca-3' 45. SEQ ID NO: 81 GFP-Primer 6: 5'-CGG ACT AGT gat gta gcc ctc agg-3' 46. SEQ ID NO: 82 Primer 7: 5'-CGA GCT CGT GCC TTT TGG ATC G-3' 47. SEQ ID NO: 83 Primer 8: 5'-CGG TCC GAA CGT GGT TGG-3' 48. SEQ ID NO: 84 Primer 9: 5'-CGA GCT CGG CCC TAT GAA TTG G-3' 49. SEQ ID NO: 85 Primer 10: 5'-CGG TCC GTC TCC TTC TGC ACA C-3' 50. SEQ ID NO: 86 Primer 11: 5'-CGA GCT CGA TGC ATT CCT TGG AT-3' 51. SEQ ID NO: 87 Primer 12: 5'-CCT AGG GTT TGG AGG TAT CAA G-3' 52. SEQ ID NO: 88 Primer 13: 5'-CGA GCT CCG TCC GAT GTG ATT CCG TC-3' 53. SEQ ID NO: 89 Primer 14: 5'-CCT AGG GGC AGT GTC GGC GGT T-3' 54. SEQ ID NO: 90 Primer 15: 5'-CGA GCT CCA GAG TGA CAG ACA GTG A-3' 55. SEQ ID NO: 91 Primer 16: 5'-CCT AGG TCT TCA ACT GTC CCC A-3' 56. SEQ ID NO: 92 Oryza sativa terminator BPST.3 (1,137 bp). This sequence is a functional equivalent of the sequence described by SEQ ID NO: 45. 57. SEQ ID NO: 93 Oryza sativa terminator BPST.4 (reverse complementary sequence of BPST.3) (1,137 bp). This sequence is a functional equivalent of the sequence described by SEQ ID NO: 45 58. SEQ ID NO: 94 Artificial sequence, vector pRJB058 (6,849 bp)) 59. SEQ ID NO: 95 insert from pRJB062: Nos terminator (Nos-T) sequence inserted into SacI-RsrII fragment of pRJB058 (257 bp) 60. SEQ ID NO: 96 insert from pRJB064: ORF sequence inserted into SacI digested and T4 DNA Polymerase filled in fragment of pRJB058 (1,089 bp) 61 SEQ ID NO: 97 insert from pRJB066: Oryza sativa BPST.1 sequence inserted into SacI digested and T4 DNA polymerase filled in fragment of pRJB058 (1,420 bp) 62. SEQ ID NO: 98 insert from pRJB065: Oryza sativa BPST.2 sequence inserted into SacI digested and T4 DNA polymerase filled in fragment of pRJB058 (1,414 bp) 63. SEQ ID NO: 99 insert from pRJB067: Oryza sativa BPST.3 sequence inserted into SacI digested and T4 DNA polymerase filled in fragment of pRJB058 (1,165 bp) 64. SEQ ID NO: 100 insert from pRJB068: Oryza sativa BPST.4 (reverse completentary sequence of BPST.3) sequence inserted into SacI digested and T4 DNA polymerase filled in fragment of pRJB058 (1,165 bp) 65. SEQ ID NO: 101 BPST.5-MCS: Oryza sativa BPST.5 sequence with EcoRI and AvrII sites (1,305 bp) 66. SEQ ID NO: 102 BPST.6-MCS: Oryza sativa BPST.6 sequence with EcoRI and AvrII sites (1,350 bp) 67. SEQ ID NO: 103 BPST.7-MCS: Oryza sativa BPST.7 sequence with EcoRI and SacI sites (1,532 bp) 68. SEQ ID NO: 104 BPST.8-MCS: Oryza sativa BPST.8 sequence (reverse complementary sequence of BPST.7) with EcoRI site (1,532 bp) 69. SEQ ID NO: 105 binary vector pRLI024 derived from pRJB058 (16,914 bp) 70. SEQ ID NO: 106 binary vector pRLI031 derived from pRLI024 (15,919 bp) 71. SEQ ID NO: 107 Oryza sativa terminator BPST.7 (1,499 bp). This sequence is a functional equivalent of the sequence described by SEQ ID NO: 11. 72. SEQ ID NO: 108 Oryza sativa terminator BPST.8 (reverse complementary sequence of BPST.3) (1,499 bp). This sequence is a functional equivalent of the sequence described by SEQ ID NO: 11.

EXAMPLES

Chemicals

[0440] Unless indicated otherwise, chemicals and reagents in the Examples were obtained from Sigma Chemical Company (St. Louis, Mo.), restriction endonucleases were from New England Biolabs (Beverly, Mass.) or Roche (Indianapolis, Ind.), oligonucleotides were synthesized by MWG Biotech Inc. (High Point, N.C.), and other modifying enzymes or kits regarding biochemicals and molecular biological assays were from Clontech (Palo Alto, Calif.), Pharmacia Biotech (Piscataway, N.J.), Promega Corporation (Madison, Wis.), or Stratagene (La Jolla, Calif.). Materials for cell culture media were obtained from Gibco/BRL (Gaithersburg, Md.) or DIFCO (Detroit, Mich.). The cloning steps carried out for the purposes of the present invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, growing bacteria, multiplying phages and sequence analysis of recombinant DNA, are carried out as described by Sambrook (1989). The sequencing of recombinant DNA molecules is carried out using ABI laser fluorescence DNA sequencer following the method of Sanger (Sanger 1977).

Example 1

Development of Genomic Libraries for Identification of Transcription Terminators

[0441] Genomic DNA from a target plant is prepared according to Qiagen plant DNA preparation kit (cat#12143). One quadratureg of the genomic DNA is digested with four base pair cutting enzyme (e.g. Sau3A) overnight at 37° C. or mechanical shearing in a Hamilton syringe or sonication followed by electroporation (0.8% Agarose gel) and gel purification using the QIAEX II Gel Extraction Kit (cat#20021). Fragmented genomic DNAs (500 to 1,000 bp) are cloned in to the screening constructs or screening vectors described herein. The resulting library of constructs and vectors is batch transformed into plant cells (see below).

Example 2

In Silico Identification of Sequences to Evaluation as Transcription Terminators

[0442] Beside other approaches described herein to provide sequences for evaluation of their suitability as transcription terminator sequences (e.g., genomic sequences as provided by Example 1), sequences can be provided by in silico search of genome databases, such as for example of Arabidopsis thaliana or rice. Accordingly, the whole rice genome sequences are screened for potential plant derived terminator candidates using the most updated data from the Institute of Genome Research (TIGR; PUB_tigr_rice_genome_v4.nt (v03212003), PUB_tigr_rice_cds_Oct022003.nt, pubOSest0603 (ncbi)). This screening system comprises three major components:

[0443] 1) identification of paired genes meeting predefined (for example 700 to 2,000 bp) intergenic distance criteria;

[0444] 2) determination of the expression levels and expression patterns of the identified paired genes;

[0445] 3) selection of intergenic sequences for terminator candidates by genome mapping.

[0446] The genome mapping requires the following activities: (1) manual verification of the gene model, reading frame of the coding sequences (CDS), and the intergenic structures underlying the genomic sequences and (2) selection of potential transcription terminators of interest candidates based on the EST sequence alignment and CDS.

2.1 Identification of Paired Genes of Interest

[0447] Given the recently released rice japonica genome sequences and the 56,056 annotated rice CDS, the coordinators of the beginning and ending of those 56,056 annotated rice CDS from genomic regions are retrieved and the intergenic distances are calculated. A frequency distribution table of intergenic distances at 200 bp/interval is generated such that appropriate intergenic distance can be defined. In order to capture maximal values of potential terminator candidates, the distance between genes in the range of 700 to 2,000 bp is used, leading to identify 16,058 pairs of rice genes (consisting of paired genes in head-to-tail, tail-to-head, and tail-to-tail orientation).

[0448] First, each pair of the identified rice CDS is blasted against rice EST databases to retrieve EST homolog sequences. The identified sequences that are homologous to ESTs are mapped onto the same rice genomic regions from which the rice CDS are derived using the splice alignment gene identification application, GeneSeger® (Version 1.9 (Oct. 22, 2002), Department of Zoology & Genetics, Iowa State University, Ames, Iowa 50011-3260). The underlying gene model, including the 5' end exon, the 3' end exon, CDS reading frame, and intergenic structure between two genes is carefully verified by graphically displaying the GeneSeger® genome mapping results using MyGV (Version 1.0 (from NewLink Genetics, 2901 South Loop Drive, Suite 3900, Ames, Iowa 50010) application. Potential gene terminator candidates are

[0449] 1) the paired CDS reading frames must be either head-to-tail, tail-to-tail, or tail-to-head orientated. The tail-to-tail orientation (i.e. from which expression from said genes is directed in opposite direction against each other) is the most desirable, as the intergenic sequences do not contain the promoter sequences and the intergenic sequence length can be minimized;

[0450] 2) the annotated CDS and its gene model must be verified and supported by the EST sequences according to sequence alignment.

[0451] Of these intergenic regions preferably regions are identified for further analysis which are localized between genes which have a tail-to-tail localization (i.e. from which expression from said genes is directed in opposite direction against each other).

2.2 Determination of Gene Expression Levels and Expression Patterns

[0452] Each pair of the identified rice CDS (i.e. corresponding to the genes flanking the intergenic region) is used to identify the corresponding EST sequences of high identity to rice EST database using blastn searching with expectation value set to 1.0e^-20. Those identified EST sequences, which presumably are considered as the same sequences as the rice CDS, are used to retrieve the gene expression profiling data derived from either the cDNA library clone distribution or microarray expression. Overall, a gene with a cluster/variant size of more than 100 clones derived from the cDNA libraries is considered as highly expressed, and so does the signal intensity beyond the top 25% quantile from the microarray expression studies. Highly expressed abundance for both of the paired genes is required as criteria for gene selection. Furthermore, the co-expression pattern of the paired genes can be assessed using the clone distribution across cDNA libraries or using the microarray expression data across different experiments. A linear correlation coefficient is calculated to determine the pattern of the gene expression. A pair of genes demonstrating unique expression pattern is desirable. Using those criteria, 5,279 pairs of rice CDs sequences are selected.

2.3 Determination of Transcript Length Variability

[0453] Preferably, the 3'-end of the EST sequence alignments corresponding to the genes flanking the intergenic region must demonstrate a low degree of variability with respect to transcript length. This is found to be predictive for a strong terminator signal.

[0454] Based on the above criteria, 37 rice potential intergenic genomic sequences (SEQ ID NO: 10 to 46) are selected for testing in the screening systems of the invention in order to identify terminators of interest. All of these sequences are localized in between genes which are orientated in the above mentioned preferred tail-to-tail orientation.

Example 3

In Vitro Screening System for Identifying Terminators of Interest

[0455] A high throughput screening method is developed to identify transcription terminators at the mRNA levels. The method includes in vitro transcription using single strand fluorescence probes such as beacon probes that hybridize polyadenylated RNA region and the read through region. The fluorescence amount of the read through products are compared with the amount of polyadenylated RNA. The stronger and tighter terminators will show the lesser amounts of read through products. Control vectors are constructed to establish the screening system (see FIG. 11 and agenda to this figure above).

[0456] A promoter for these constructs is preferably a strong constitutive promoter (e.g. maize ubiqutin promoter). In order to measure uncoupled transcription, SP6 or T7 phase promoter can be used for in vitro transcription. The coding sequence in the expression cassette can be any reporter gene or genomic DNA including start at the 5' end and stop codon at the 3' end, which do not have sequence homology to plant genome (e.g. intergenic sequences from yeast genome). Nopaline synthase terminator can be replaced with any other known terminator to use as a control or uncharacterized genomic DNA fragment to identify potential terminator candidates.

3.1 Vector Construction

[0457] Vector pBPSMM268 contains the GUS::potato intron gene followed by the NOS terminator region. To this vector, maize Ubiquitin promoter::intron is added by digestion of pMM268 with StuI and SmaI, followed by blunt ligation of the Ubiquitin promoter::intron fragment obtained from StuI digestion of pBPSCER043, which produces vector pTOI01.

[0458] In order to ensure efficient transcript processing of mRNAs that do not undergo transcriptional termination at putative transcription terminators, the OCS terminator region is cloned into pTOI01. pTOI02 is generated by digestion of pTOI01 with SacI and NotI, and ligation of the SacI/NotI fragment generated from the PCR amplification of the OCS terminator from vector p1 bxSuperGusQC using primers 1 and 2 (SEQ ID NO 47 and 48).

TABLE-US-00004 Primer 1 (SacI, AvrII, SpeI, OCS 5'; SEQ ID NO: 47): 5'-CG GAGCTC CCTAGG ACTAGT tcgaccggcatgccc-3' Primer 2 (NotI, OCS 3'; SEQ ID NO: 48): 5'- CC GCGGCCGC agcttggacaatcag-3'

[0459] A fragment of the firefly luciferase gene is cloned downstream of the transcription terminator sequences to be assessed insertion site in order to act as a unique sequence that is only transcribed in the presence of a poorly functioning terminator. pTOI03 is generated from the digestion of pTOI02 with AvrII and SpeI, and ligating in the AvrII/SpeI fragment generated from the PCR amplification of a 240 bp fragment of the firefly luciferase gene (Lu^F) from vector pGL3 (R2.2) basic vector (Promega cat# E6441) using primers 3 and 4 (SEQ ID NO: 49 and 50).

TABLE-US-00005 Primer 3 (AvrII, XmaI, RsrII, Lu^F 5'; SEQ ID NO: 49): 5'-CG CCTAGG CCCGGG CGGACCG cattaagaagggccc-3' Primer 4 (SpeI Lu^F 3'; SEQ ID NO: 50): 5'-CG ACTAGT agagagttctcagagc-3'

[0460] Vector pTOI03 is the base vector that is used to generate constructs testing putative transcription terminator sequences. Vector pTOI04 comprises pTOI03 with the addition of the NOS in forward orientation, and is generated by insertion of the NOS containing SacI fragment from pBPSCR043 into the unique SacI site of pTOI03. (Positive control--NOS). Vector pTOI05 comprises pTOI03 with the addition of the NOS in reverse orientation, and is generated by the insertion of the inverted NOS SacI fragment from pBPSCR043 into the unique SacI site of pTOI03. (Negative control--inverted NOS).

[0461] Vectors pTOI06-pTOI10 are generated by the PCR amplification of putative terminator sequences from rice genomic DNA (selected from the sequences described by SEQ ID NO: 10 to 46) such that a SacI site is generated on the 5' end of the sequence and a RsrII site is generated on the 3' end. (Note: if the sequence of individual genomic elements precludes the use of these two restriction enzymes, then the alternative enzymes AvrII or XmaI can be used for cloning purposes.) The source of transcription terminators can be from both the in silico screening system and the genomic libraries containing 500 to 1,000 bp fragments.

3.2 Preparation of BMS Suspension Cultures Cells

[0462] Black Mexican Sweetcorn (BMS) suspension cultured cells are propagated in Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose and 2 mg/L 2,4-dichlorophenoxyacetic acid. Every week 5 mL of a culture of stationary cells are transferred to 125 mL of fresh medium and cultured on a rotary shaker operated at 130 rpm at 27° C. in a 500 mL flask in the dark.

3.3 Preparation of the Nuclear Extract

[0463] The HeLa nuclear extract are purchased from Promega (HeLaScribe® Nuclear Extract; cat# E3092). Nuclear extracts are prepared from BMS cells as described (Moreno et al., 1997). BMS suspension cultured cells at logarithmic phase are harvested three days after the start of a fresh culture by spinning down at 2 krpm for 500 mL tubes for 10 min at 4° C. (1,200 rpm for 800 mL glass conical bottles at 170×g). The cell pellet is loosened and resuspended in cold HBSS (Hank's Balanced Salt Solution; Sigma cat# H9269). The cells are transferred into 50 mL Corning tube and spanned down at 1,200 rpm at 4° C. Packed cell volume (PCV) is measured by eye. The pellet is loosened and resuspended in 5×PCV hypotonic buffer followed by swelling the cells on ice for 10 min. The cells are spanned down at 1,200 rpm for 10 min. The supernatants are removed. One volume of PCV hypotonic buffer including 0.1% NP-40 is added to the pellet followed by resuspending the cells. The resuspended cells are transferred into chilled dounce homogenizer and measured the total volume before adding 1×PMSF (500×: 8.71 mg/mL). The cells are dounced for 10 to 15 strokes and checked the cells to yield 80 to 90% cell lysis. It is critical to avoid overdouncing the cells. Trypan blue is added to a small portion of the cells to check cell lysis under microscope. Blue cells indicate cell lysis. The cell lysis is quickly transferred into Corex 30 mL tube. 0.1 volume of sucrose restore buffer is added and gently mixed. The rotor and centrifuge have to be pre-cold. The nuclei are immediately spanned down at 10 krpm for 2 min in Beckman JA-20 rotor with brake. The supernatants containing cytoplasm are carefully removed and saved by adding glycerol to 20% (v/v) and stored at -70° C. The pellet is detached using a pipette and transferred into the nuclear resuspension buffer (3 mL/10⁹ cells) in an ultracentrifuge tube followed by adding N-quadrature-tosyl-L-lysine chloromethyl ketone (TLCK) protease inhibitor (250×: 10 mg/mL in 1 mM HCl), leupeptin (2,000×: 1 mg/mL in dH₂O), aprotinin (Sigma cat# A1153; 1,000×: 1 mg/mL in dH₂O), and pepstatin A (Sigma cat# P4265; 2,000×: 1 mg/mL in MeOH) to 1x. The tubes are balanced, rocked gently for 30 min, and spanned at 35 krpm in Ti454 (or 42 krpm in Ti70.a) for 90 min at 2° C. (150,000×g). The supernatants are transferred into another ultracentrifuge tube and measured the volume by eye. 0.33 g (NH₄)₂SO₄/mL is sprinkled into the extract for over 30 min with stirring or rocking until salt is dissolved after each addition on ice. The solution turns milky as the protein precipitates and is stirred or rocked for an additional 20 min at 4° C. followed by spinning down at 35 krpm for 30 min in Ti45 (or 32 krpm in Ti70.1). The pellet is resuspended in less than 1 mL of dialysis buffer (10⁹ cells/mL). The resuspended cells are dialyzed for one hour against more than 200 volume of dialysis buffer (2 L). The buffer should be changed during dialysis for an additional four hours. The dialyzed extract is spanned down at 35 krpm for one hour followed by storing small aliquots at -80° C.

Hypotonic Buffer

10 mM HEPES, pH 7.9 (KOH)

[0464] 0.75 mM spermidine 0.15 mM spermine

0.1 mM EDTA

0.1 mM EGTA, pH7.5 (KOH)

1 mM DTT

10 mM KCl

[0465] (add protease inhibitors and DTT before use)

10× Sucrose Restore Buffer

500 mM HEPES, pH 7.9 (KOH)

[0466] 7.5 mM spermidine 1.5 mM spermine

10 mM KCl

2 mM EDTA

10 mM DTT

[0467] 1×Sucrose Restore Buffer=1 volume 10×salts+9 volume 75% (w/v) sucrose

Nuclear Resuspension Buffer

20 mM HEPES, pH 7.9 (KOH)

[0468] 0.75 mM spermidine 0.15 mM sermine

0.2 mM EDTA

2 mM EGTA, pH 7.5 (KOH)

1 mM DTT

[0469] 25% glycerol 10% saturated ammonium sulfate (add protease inhibitors and DTT before use)

Dialysis Buffer

20 mM HEPES, pH7.9 (KOH)

[0470] 20% glycerol

100 mM KCl

0.2 mM EDTA

0.2 mM EGTA, pH 7.5 (KOH)

2 mM DTT

[0471] (add protease inhibitors and DTT before use)

3.4 An In Vitro Assay System

[0472] Primer sequences for molecular beacon probes are chosen (1) between GUS and NOS for detecting polyadenylated products and (2) within the truncated firefly luciferase gene for detecting read through products. The probes are designed by using Beacon Designer 3.0. Two different reporter dyes are chosen for this assay (e.g. Texas Red, Rhodamine Red, Tamra, Joe, Tox, Oregon green, etc.).

[0473] The constructs are linearized by restriction enzyme digestion with NotI enzyme at 37° C. overnight followed by electroporation (0.8% Agarose gel) and gel purification using the QIAEX II Gel Extraction Kit (cat#20021). One μg of the linearized single template is added into the reaction solution in a total volume of 25 μL (15 μL of a mixture of HeLa and BMS nuclear extracts at 1:1 ratio [v/v], 400 μM ATP, CTP, GTP, UTP, 400 nM final concentration of two beacon probes, 5 mM MgCl₂, mg/mL BSA). The reaction solution is incubated for 2 hour at room temperature. The reaction progress is monitored using a Cytofluor multiwell plate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. [Razik and Quatrano, 1997; Yammaguchi et al., 1998; Liu et al., 2002]. If a particular transcription terminator sequences to be assessed provides efficient transcriptional termination, the expression of sequences complimentary to probe 1 is much greater than the expression of probe 2-specific sequences. If a sequence does not terminate efficiently the ratio of probe 1: probe 2 expression is lower. A ratio of the yield obtained between polyadenylated RNA and the read through products is calculated to determine potential terminator candidates (see FIG. 11B and agenda to this figure above). In addition to using single strand fluorescence probes, the ratio of the yield can be detected using Reverse Transcriptase (RT)-PCR following the protocols in the art.

Example 4

In Vivo Screening System for Identifying Terminators of Interest

4.1 Vector Construction

4.1.1 DUC Expression Vectors

[0474] Vector pBPSMM268 contains the GUS::potato intron gene followed by the NOS terminator region. To this vector, maize Ubiquitin promoter::intron was added by digestion of pMM268 with StuI and SmaI, followed by blunt ligation of the Ubiquitin promoter::intron fragment obtained from StuI digestion of pBPSCER043, which produced vector pRJB051.

[0475] A fragment of the green fluoresecent protein (GFP-f) gene was cloned downstream of the transcription terminator sequences to be assessed insertion site in order to act as a unique sequence that is only transcribed in the presence of a poorly functioning terminator. pRJB058 was generated from the digestion of pRJB051 with AvrII and SpeI, and ligation of the AvrII/SpeI fragment generated from the PCR amplification of a 260 bp fragment of the GFP gene (GFP-F) from vector pALGFP1 using GFP-primers 5 and 6 (SEQ ID NOs: 80 and 81).

TABLE-US-00006 GFP-Primer 5 (AvrII, KasI, RsrII, AKR GFP19 5'; SEQ ID NO: 80): 5'-CGG CCT AGG GGC GCC CGG ACC Gag ctg ttc acc ggc a-3' GFP-Primer 6 (SpeI, S GFP 281 3'; SEQ ID NO: 81): 5'-CGG ACT AGT gat gta gcc ctc agg-3'

[0476] Vector pRJB058 is the base vector that was used to generate constructs testing putative transcription terminator sequences. Vector pRJB062 (SEQ ID NO: 95) comprises pRJB058 (SEQ ID NO: 94) with the addition of the NOS in forward orientation, and was generated by insertion of the NOS containing SacI fragment from pBPSCR043 into the unique SacI site of pTOI03. (Positive control--NOS).

[0477] Vector pRJB063 (reverse complementary sequence of SEQ ID NO: 95) comprises pRJB058 with the addition of the NOS in reverse orientation, and is generated by the insertion of the inverted NOS SacI fragment from pBPSCR043 into the unique SacI site of pTOI03. (Negative control--inverted NOS).

[0478] Vector pRJB064 (SEQ ID NO: 96) comprises pRJB048 with the addition of the 1.1 Kb ORF fragment from pRJB018. This vector will serve as a negative control for specific transcriptional termination by putative TOIs, as the sequence comprises an internal fragment from a known open reading frame, and should therefore possess minimal intrinsic transcriptional termination activity. Vector pRJB064 was generated by ligation of the 1.1 Kb HpaI/StuI fragment from pRJB018 into SacI digested and 3'-5-exonuclease-treated pRJB058.

[0479] Vectors pRJB065 (SEQ ID NO: 98) and pRJB066 (SEQ ID NO: 97) comprise pRJB058 with the addition of the rice genomic DNA BPST.2 (reverse complementary sequence of SEQ ID NO:33) and BPST.1 (SEQ ID NO:33), respectively. The 1.4 Kb PCR product produced from amplification of rice genomic DNA with primers 7 and 8 (SEQ ID Nos 82 and 83):

TABLE-US-00007 Primer 7: 5'-CGA GCT CGT GCC TTT TGG ATC G-3' Primer 8: 5'-CGG TCC GAA CGT GGT TGG-3'

[0480] The PCR product was TOPO cloned to produce pTOPO BPST.1 (SEQ ID NO:33) and BPST.2 (reverse complementary sequence of SEQ ID NO:33). The 1.4 Kb fragment resulting from EcoRI digestion and T4 DNA polymerase fill in reaction of pTOPO BPST.1 and BPST.2 was ligated into SacI digested and 3'-5-exonuclease-treated pRJB058. pRJB065 (SEQ ID NO: 98) represents the resulting vector comprising the BPST.2 putative terminator, and pRJB066 (SEQ ID NO: 97) represents the ligation product comprising the BPST.1 sequence.

[0481] Vectors pRJB067 (SEQ ID NO: 99) and pRJB068 (SEQ ID NO: 100) comprise pRJB058 with the addition of the rice genomic DNA BPST.3 (SEQ ID NO:92) and BPST.4 (reverse complementary sequence, SEQ ID NO:92), respectively. The 1.1 Kb PCR product produced from amplification of rice genomic DNA with primers 9 and 10 (SEQ ID NO: 84 and 85):

TABLE-US-00008 Primer 9: 5'-CGA GCT CGG CCC TAT GAA TTG G-3' Primer 10: 5'-CGG TCC GTC TCC TTC TGC ACA C-3'

[0482] The PCR product was TOPO cloned to produce pTOPO BPST.3 and BPST.4. The 1.1 Kb fragment resulting from EcoRI digestion and T4 DNA polymerase fill in reaction of pTOPO BPST.3 and BPST.4 was ligated into SacI digested and 3'-5-exonuclease-treated pRJB058. pRJB067 (SEQ ID NO: 99) represents the resulting vector comprising the BPST.3 putative terminator, and pRJB068 (SEQ ID NO: 100) represents the ligation product comprising the BPST.4 sequence.

[0483] BPST.5 (SEQ ID NO:18) produced a 1.2 Kb PCR product from amplification of rice genomic DNA with primers 11 and 12 (SEQ ID NO: 86 and 87):

TABLE-US-00009 Primer11: 5'-CGA GCT CGA TGC ATT CCT TGG AT-3' Primer12: 5'-CCT AGG GTT TGG AGG TAT CAA G-3'

[0484] BPST.6 (SEQ ID NO:10) produced a 1.3 Kb PCR product from amplification of rice genomic DNA with primers 13 and 14 (SEQ ID NO:88 and 89):

TABLE-US-00010 Primer 13: 5'-CGA GCT CCG TCC GAT GTG ATT CCG TC-3' Primer 14: 5'-CCT AGG GGC AGT GTC GGC GGT T-3'

[0485] BPST.7 (SEQ ID NO:107) and BPST.8 (reverse complementary sequence of SEQ ID NO:108) produced a1.5 Kb PCR product from amplification of rice genomic DNA with primers 15 and 16 (SEQ ID NO:90 and 91):

TABLE-US-00011 Primer 15: 5'-CGA GCT CCA GAG TGA CAG ACA GTG A-3' Primer 16: 5'-CCT AGG TCT TCA ACT GTC CCC A-3'

[0486] Additional TOI candidates will be isolated and cloned into pUC expression vectors as described above.

4.1.2 Binary Vectors

[0487] For evaluation of transcriptional termination by the putative TOI sequences in stably transformed maize plants, binary vectors were prepared for Agrobacterium-mediated maize transformation. The full-length T-DNA sequences for vectors pRLI024 and pRLI031 are provided in the attached sequence listing (SEQ ID NO: 105 and 106, respectively). The other vectors were derived therefrom by exchanging the terminator regions.

[0488] Vector pRLI024 (SEQ ID NO:105) was generated by ligation of the 4.9 Kb PvuII fragment from pRJB058 into pLM150 that had been digested with PmeI, generating pJB077. The 3.1 Kb DsRed2 expression cassette was liberated from vector pLM299 via FseI/PacI digestion, and ligated into FseI/PacI digested pRJB077 to generate pRLI024 (SEQ ID NO:105).

[0489] Vector pRLI025 was generated by ligation of the 4.9 Kb PvuII fragment from pRJB062 (SEQ ID NO:95) into pLM150 that had been digested with PmeI, generating pJB078. The 3.1 Kb DsRed2 expression cassette was liberated from vector pLM299 via FseI/PacI digestion, and ligated into FseI/PacI digested pRJB078 to generate pRLI025.

[0490] Vector pRLI026 was generated by ligation of the 4.9 Kb PvuII fragment from pRJB064 (SEQ ID NO:96) into pLM150 that had been digested with PmeI, generating pJB079. The 3.1 Kb DsRed expression cassette was liberated from vector pLM299 via FseI/PacI digestion, and ligated into FseI/PacI digested pRJB079 to generate pRLI026.

[0491] Vector pRLI027 was generated by ligation of the 4.9 Kb PvuII fragment from pRJB066 (SEQ ID NO: 97) into pLM150 that had been digested with PmeI, generating pJB080. The 3.1 Kb DsRed expression cassette was liberated from vector pLM299 via FseI/PacI digestion, and ligated into FseI/PacI digested pRJB080 to generate pRLI027.

[0492] Vector pRLI028 was generated by ligation of the 4.9 Kb PvuII fragment from pRJB065 (SEQ ID NO: 98) into pLM150 that had been digested with PmeI, generating pJB081. The 3.1 Kb DsRed expression cassette was liberated from vector pLM299 via FseI/PacI digestion, and ligated into FseI/PacI digested pRJB081 to generate pRLI028.

[0493] Vector pRLI029 was generated by ligation of the 4.9 Kb PvuII fragment from pRJB067 (SEQ ID NO:99) into pLM150 that had been digested with PmeI, generating pRLI022. The 3.1 Kb DsRed expression cassette was liberated from vector pLM299 via FseI/PacI digestion, and ligated into FseI/PacI digested pRLI022 to generate pRLI029.

[0494] Vector pRLI030 was generated by ligation of the 4.9 Kb PvuII fragment from pRJB068 (SEQ ID NO:100) into pLM150 that had been digested with PmeI, generating pRLI023. The 3.1 Kb DsRed expression cassette was liberated from vector pLM299 via FseI/PacI digestion, and ligated into FseI/PacI digested pRLI023 to generate pRLI030.

[0495] An alternative series of binary vectors was generated in order to evaluate putative TOIs with regard to their ability to direct bi-directional transcriptional termination. For these vectors, the TOI sequences were cloned between two reporter expression cassettes in tail-to-tail orientation.

[0496] Vector pLI024 (SEQ ID NO: 105) was digested with SacI to remove the 950 bp Nos-T and intervening sequences from between the DsRed ORF and the TOI MCS. The vector was recircularized to generate pLI031 (SEQ ID NO:106).

[0497] Vector pLI025 was digested with SacI to remove the 950 bp Nos-T and intervening sequences from between the DsRed ORF and the TOI MCS. The vector was recircularized to generate pLI032.

[0498] Vector pLI026 was digested with SacI to remove the 950 bp Nos-T and intervening sequences from between the DsRed ORF and the TOI MCS. The vector was recircularized to generate pLI033.

[0499] Vector pLI027 was digested with SacI to remove the 950 bp Nos-T and intervening sequences from between the DsRed ORF and the TOI MCS. The vector was recircularized to generate pLI034.

[0500] Vector pLI028 was digested with SacI to remove the 950 bp Nos-T and intervening sequences from between the DsRed ORF and the TOI MCS. The vector was recircularized to generate pLI035.

[0501] Vector pLI029 was digested with SacI to remove the 950 bp Nos-T and intervening sequences from between the DsRed ORF and the TOI MCS. The vector was recircularized to generate pLI036.

[0502] Vector pLI030 was digested with SacI to remove the 950 bp Nos-T and intervening sequences from between the DsRed ORF and the TOI MCS. The vector was recircularized to generate pLI037.

[0503] Additional TOI candidates will be isolated and cloned into binary vectors as described above.

4.2 Assays for Identifying Terminators of Interest

[0504] The test construct comprised a GUS reporter gene driven by the maize ubiquitin promoter, and enzyme sites to insert putative TOI and control sequences. The TOI functionality screen was based on the principle that in the absence of a functional terminator region the GUS mRNA will not be efficiently processed, and therefore will not be available to support high levels of translation of GUS protein. The results of these transient analyses are shown in Table 2 and Figure. 14. The experimental rationale was supported by the finding that the vector lacking an insertion at the TOI cloning site does not drive detectable GUS expression (pRJB058; SEQ ID NO: 94). The insertion of the nopaline synthase (Nos) terminator was able to rescue GUS expression (pRJB062; SEQ ID NO: 95). The insertion of sequence derived from an internal portion of an exogenous protein-coding gene (ORF sequence) did not result in GUS expression (pRJB064; SEQ ID NO: 96), signifying that the GUS expression seen with pRJB062 was due to intrinsic transcriptional termination activity by Nos, and not a non-specific effect due to insertion of any DNA sequence at that site. BPST.1 (SEQ ID NO:33) and BPST.2 (reverse complementary sequence of SEQ ID NO:33) showed GUS expression levels comparable to that seen with the Nos (+) control vector. BPST.3 (SEQ ID NO:92) and BPST.4 (reverse complementary sequence, SEQ ID NO:92) consistently resulted in significant GUS expression at levels that appeared to be slightly lower than observed with Nos.

TABLE-US-00012 TABLE 2 Transient GUS expression testing for terminator candidates Terminator candidates GUS expression* BPST (null)-(pRJB058; SEQ ID NO: 94) - 0% BPST (+) - Nos (pRJB062; SEQ ID NO: 95) ++++ 100% BPST (-) - ORF (pRJB064; SEQ ID NO: 96) - 0% BPST.1 (pRJB066; SEQ ID NO: 97) ++++ 100% BPST.2 (pRJB065; SEQ ID NO: 98) ++++ 100% BPST.3 (pRJB067; SEQ ID NO: 99) +++ 80% BPST.4 (pRJB068; SEQ ID NO: 100) +++ 80% *GUS histochemical assays: a range of GUS activities (- no expression to ++++ high expression).

[0505] No GUS staining was observed in vectors that do not comprise a functional transcriptional terminator downstream of the GUS coding sequence (pRJB058 and pRJB064; SEQ ID NO: 94 and 96, respectively). The presence of a functional terminator rescued GUS expression in the (+) control (pRJB062; SEQ ID NO: 95) vector as well as all four TOI candidate sequences (pRJB065-pRJB068: SEQ ID indicated in Table 2) (FIG. 14).

4.3 Analysis of Terminator Candidates in Stably Transformed Maize

[0506] The binary vectors pBPSLI027, pBPSLI028, pBPSLI029, and pBPSLI030 will be transformed into maize using Agrobacterium-mediated transformation (Example 11.4). The levels and patterns of GUS expression controlled by BPST.1 (SEQ ID NO:33), BPST.2 (reverse complementary sequence of SEQ ID NO:33), BPST.3 (SEQ ID NO: 92), or BPST.4 (reverse complementary sequence of SEQ ID NO:92) terminator will be compared with those controlled by NOS-t. BPST.1, BPST.2, BPST.3 and BPST.4 should show similar levels to that observed in transient assays (Table 2). This result will indicate that a transient assay can be used as a model system and is therefore one of the important screening systems to identify functional transcriptional terminators. However, the results obtained with the transient assays should be validated by the production of stable transformed transgenic plants.

Example 5

In Vivo Screening System Using Gene Silencing for Identifying Potential Transcription Terminators

[0507] A high throughput screening system is developed to identify and isolate tight transcriptional termination sequences. This method is time-efficient and does not involve RNA analysis. Since dsRNA molecules are efficient in even a small amount, only very tight terminators can be identified. (FIG. 13C)

[0508] As described above in more detail, the RNA may be preferably selected from

[0509] a) RNAs encoding for an antisense or preferably double-stranded RNAs, which downregulates expression of essential plant genes. In principle, any gene is suitable which has a lethal phenotype in a homozygous knockout (e.g. Phytoen desaturase, Nitrate reductase, HPPD, Acetohydroxyacid synthase, etc.)

[0510] b) RNAs encoding for toxic proteins, which expression causes lethal effect to the transgenic plants (negative selection markers like e.g., TK, codA, tyrA, Diphtheria toxin etc.). Furthermore, any endogenous gene suitable as herbicidal target can be employed in the above-mentioned approach (Table 1).

5.1 Vector Construction for RNAi

[0511] For experiments using RNAi down-regulation mechanism to evaluate transcription termination, vector pTOI03 is modified such that Lu^F is replaced with an appropriate dsRNA cassette. An appropriate RNAi cassette comprises a 200 to 300 bp sequence that is specific to the targeted gene, followed by a spacer sequence of approximately 150-200 bp, followed by an inverted repeat of the gene specific sequence, such when transcribed, a hairpin structure is formed with the gene-specific sequence forming the dsRNA stem. The RNAi target can be any gene that provides lethality or a screenable phenotype under down-regulation (e.g. AHAS, bar, target genes of herbicides (see Table 1), or other essential endogenous genes such as housekeeping genes). Gene-specific sequences are generated via PCR with appropriate restriction sites for forward and reverse orientation via amplification with primers 5 and 6 (SEQ ID NO: 51 and 52):

TABLE-US-00013 Primer 5 (RsrII, BspEI, target gene seq 5'; SEQ ID NO: 51): 5' CG CGGACCG TCCGGA-N-3' [N: gene-specific sequence of preferably 10 to 20 bases] Primer 6 (SpeI, AgeI, target gene seq 3'; SEQ ID NO: 52): 5' CG ACTAGT ACCGGT-N-3' [N: gene-specific sequence of preferably 10 to 20 bases]

[0512] The RNAi vector (pTOI11) is produced via a four-way ligation between (1) pTOI03 digested with RsrII and SpeI, (2) target gene PCR product digested with RsrII and AgeI, (3) the spacer sequence with AgeI and BspEI ends, and (4) target gene PCR product digested with BspEI and SpeI.

5.2 Assays for Identifying Terminators of Interest

[0513] These experiments are performed by bombardment of plant tissues or culture cells (Example 9.1), by PEG-mediated (or similar methodology) introduction of DNA to plant protoplasts (Example 9.2), or by Agrobacterium-mediated transformation (Example 9.3). The target tissues for these experiments can be plant tissues (e.g. leaf or root), cultured cells (e.g. maize BMS), or plant tissues (e.g. immature embryos) for Agrobacterium protocols.

[0514] The sequences used as potential transcription terminator sequences can either be derived from the in silico transcription terminator sequence screen or from a library of random genomic fragments. Only plants can survive in subsequent regeneration in the case of stable transformation, which have an efficient terminator inserted in front of the sequence encoding the toxic RNA (thereby blocking its expression). The surviving plants are isolated and the terminator sequence amplified using PCR and sequencing.

Example 6

In Vivo Screening System Using Bicistronic RNA Detection

[0515] A system is developed that utilizes the internal ribosome entry site (IRES) from encephalomiocarditis virus (EMCV), a picornovirus, or any functional IRES in plants. EMCV IRES has been shown in plants to efficiently direct translation of internally encoded proteins in parallel with canonical cap-mediated translation (Urwin et al., 2000). This method allows the screening of potential terminator sequences in plant tissue and will provide a screen to compare relative termination efficiency between multiple sequences. For these experiments, potentially bicistronic elements are generated containing two distinguishable fluorescent proteins (FP1 and FP2) separated by the transcription terminator to be assessed and IRES. If a particular transcription terminator sequences to be assessed provides efficient transcriptional termination, the expression of FP1 is much greater than the expression of FP2. If a sequence does not terminate efficiently the ratio of FP1:FP2 expression is lower.

6.1 Vector Construction

[0516] Test constructs comprises the following elements, described in order 5' to 3'. A strong constitutive promoter for the target tissue is used to drive expression of the RNA, such as Ubiquitin or ScBV for expression in maize leaf tissue. The most proximal open reading frame encodes for FP1 (e.g. DsRed1), followed by restriction sites for insertion of potential transcription terminator sequence elements. Immediately downstream of the insertion site for the transcription terminator sequences to be assessed is the encephalomyocarditis virus (EMCV) IRES element, followed by the open reading frame for FP2 (e.g. GFP), and followed by a known plant transcriptional terminator. This downstream terminator needs to be present in order to stabilize transcripts that are not efficiently terminated by the transcription terminator sequence, thereby allowing detection of mRNAs that encode FP2. If Agrobacterium mediated transformation experiments will be performed, then the vectors will have to include the LB and RB T-DNA elements flanking the expression cassette and selectable marker genes. (FIG. 15).

Example 7

System Based on Inverted Repeat of Nos Terminator

7.1 Generation of the Positive and Negative Binary Vector Control Constructs for the Screening of Terminator Activitiy

[0517] To test the transcription termination efficiency of a sequence a construct was generated with a strong constitutive promoter (STPT promoter) upstream of the nptII marker gene followed by a short MCS in which putative terminator sequences are cloned and an inverted repeat of the 3'-UTR region from the nos-gene of Agrobacterium tumefaciens, with the first repeat element being in the antisense orientation relative to the STPT promoter. This arrangement without a putative terminator sequence serves as a negative control: Both orientations of the nosT are incorporated into the resulting transcript, since the inverted nos element does not lead to transcript truncation and polyadenylation, which gives rise to a transcript with 3'hairpin structure leading to dsRNA-mediated, sequence-specific RNA-degradation and thus silencing of the nptII-gene. As a positive control the Ribulose-bisphosphat Carboxlase E9 terminator region is cloned in between the nptII-gene and the nos inverted repeat (nos-IR), which leads to the proper termination of nptII-transcripts and thus to the expression of nptII.

7.2 Cloning of pENTR-A1-inv-35s-GFP-E9

Insertion of Terminator E9:

[0518] Lo394-pENTR-A1-inv is opened with BamHI and KpnI. The E9 terminator is isolated from Lo424-pENTR-A1-inv-P2-E9 with BamHI and KpnI and ligated into the opened Lo394 in direct orientation. The resulting construct is named Lo483-pENTR-A1-inv-E9. The orientation of sites is attL4-E9-attR1.

7.3 Insertion of the CaMV 35s Promoter Together with the Gene for mGFP5er

[0519] The 35s promoter of CaMV and the Aequorea victoria gene for the green fluorescent protein mGFP5er-gene were isolated from Lo409-pENTR-35s-GFP5er-GUS with HindIII/SmaI and ligated into Lo483-pENTR-A1-Inv-E9 opened with HindIII/EcoRV in direct orientation. The orientation of sites is attL4-35s-mGFP5er-E9-attR1. The resulting construct is named Lo484-pENTR-A1-inv-35s-GFP-E9 (SEQ ID NO: 9).

7.4 Cloning of pENTR-C1-STPT-nptII-nosIR Insertion of the STPT-Promoter and the nptII-Gene:

[0520] Lo393-pENTR-C1 was opened with BamHI. The STPT promoter with the nptII-gene was cut out of Lo441-pENTR1A-STPT-nptII-CatpA with BamHI and ligated undirected in the opened Lo393. Clones with the correct orientation were identified via colony-PCR followed by control digests. The orientation of sites is attR2-STPT-nptII-attL3. The resulting construct is named Lo485-pENTR-C1-STPT-nptII.

7.5 Insertion of the Terminator nosT in Sense Orientation

[0521] The 257 bp 3'-UTR region of the nopaline synthase (nos) gene from Agrobacterium tumefaciens was amplified using PCR from Lo114-pSUN3-Gus-nos with the overhang primers Loy482-NosT-upper-SalI and Loy483-NosT-Lower-HindIII (SEQ ID NO: 55 and 56).

TABLE-US-00014 Loy482-NosT-upper-SalI (SEQ ID NO: 55): 5'-AAATTTGTCGACCGATCGGTCAAACATT-3' Loy483-NosT-Lower-HindIII (SEQ ID NO: 56): 5'-AAATTTAAGCTTCCCGATCTAGTAACATAGATGACA-3'

[0522] The resulting 282 bp PCR fragment was digested with SalI/HindIII and cloned in direct orientation into Lo485-pENTR-C1-STPT-nptII opened with SalI/HindIII. The orientation of sites is attR2-STPT-nptII-nosT-attL3. The resulting construct is named Lo486-pENTR-C1-STPT-nptII-nos.

7.6 Insertion of a Second Nos-Terminator Element in Antisense Orientation Between the nptII Gene and the Sense nosT Together with a Stuffer Sequence Derived from the 3'-Region of the Gus-Gene

[0523] The NosT was amplified together with a part of the 3'-region of the gus-gene out of Lo400-pENTR1A (B)-Ln-Prom2-TypDra-nosT. Therefore primer were designed which added a SalI site at the gus sequence and a SpeI-site together with a BglII-site at the end of nosT. Two kinds of constructs were prepared one including a 129 bp gus-spacer sequence between the inverted repeat of nosT and the other consisting of 155 bp spacer between the IR. The shorter version was amplified with the primers Loy494-Gus_upper_SalI_Spacer and Loy492-NosT-lower-BglII_SpeI (SEQ ID NO: 57 and 58).

TABLE-US-00015 Loy494-Gus_upper_SalI_Spacer (SEQ ID NO: 57): 5'-TTTTAGTCGACACGCTGGACTGGCATGAACT-3 ' Loy492-NosT-lower-BglII_SpeI (SEQ ID NO: 58): 5'-TTTTAAGATCTACTAGTCCGATCTAGTAACATAGATGACA-3 '

[0524] The longer version was amplified with Loy493_Gus_upper_SalI_Spacer together with Loy492-NosT-lower-BgIII_SpeI.

TABLE-US-00016 Loy493_Gus_upper_SalI_Spacer (SEQ ID NO: 59): 5'-TTTAAGTCGACAAGTCGGCGGCTTTTCTGCT-3' Loy492-NosT-lower-BglII_SpeI (SEQ ID NO: 60): 5'-TTTTAAGATCTACTAGTCCGATCTAGTAACATAGATGACA-3'.

[0525] The resulting PCR-fragments were digested with SalI/BglII and ligated into Lo486-pENTR-C1-STPT-nptII-nos opened with SalI/BglII. This resulted in the nptII open-reading frame being followed by a nos 3'-UTR in the antisense orientation relative to the STPT promoter, a 129 bp-spacer region respectively 155 bp-spacer region of gus-sequence in the antisense orientation, and a second nosT in the sense orientation. The orientation of sites is attR2-STPT-nptII-as nosT-spacer-s nosT-attL3. The resulting constructs were named Lo503a-pENTR-C1-STPT-nptII-IRnos (SEQ ID NO: 8) with the shorter spacer between the IR) and Lo503b-pENTR-C1-STPT-nptII-IRnos (SEQ ID NO: 7) with the longer spacer between the IR).

7.7 Generation of the Negative Control Construct

[0526] 7.7.1 Triple-LR Reaction to Create the Binary Expression Vector which Will Serve as the Negative Control

[0527] The triple-LR-reaction is carried out with the plasmids Lo484-pENTR-A1-inv-35s-GFP-E9, Lo376-pENTR-B2 (without insert; SEQ ID NO: 76) and Lo503a-pENTR-C1-STPT-nptII-IRnos, or with Lo503b-pENTR-C1-STPT-nptII-IRnos and Lo442-pSUN1-R4R3 (SEQ ID NO: 77), respectively according to the instructions of the manufacturer, using LR plus recombinase mix. The resulting binary plant transformation vectors were named Lo523a-pSUN1-R4-Lo484::Lo376::Lo503a (SEQ ID NO: 6) and Lo523b-pSUN1-R4-Lo484::Lo376::Lo503b (SEQ ID NO: 5), respectively.

7.8 Generation of the Positive Control Construct

7.8.1 Insertion of the E9 Terminator Upstream of IRnos

[0528] The E9 terminator was isolated from Lo444-pGST-δ-KpnI-LUC.sup.+ with BamHI/EcoRV. Lo503 is opened with SpeI, the 5'-protruding ends were completely filled in with Pfu turbo polymerase and cut again with BglII. The BglII/EcoRV fragment of the E9 terminator is ligated into the opened vectors Lo503a and Lo503b (which were digested first with SpeI and blunted, and subsequently with BamHI), respectively, in direct orientation. The orientation of sites is attR2-STPT-nptII-E9-IRnos-attL3. The resulting constructs were named Lo522a-pENTR-C1-STPT-nptII-IRnos (SEQ ID NO: 4) with the shorter spacer between the IR) and Lo522b-pENTR-C1-STPT-nptII-IRnos (SEQ ID NO: 3) with the longer spacer between the IR).

76.8.2 Triple-LR Reaction to Create the Binary Expression Vector which Will Serve as the Positive Control

[0529] The triple-LR-reaction is carried out with the plasmids Lo484-pENTR-A1-inv-35s-GFP-E9, Lo376-pENTR-B2 (without insert; SEQ ID NO: 76) and Lo522a-pENTR-C1-STPT-nptII-E9-IRnos or with Lo522b-pENTR-C1-STPT-nptII-E9-IRnos and Lo442-pSUN1-R4R3 ((SEQ ID NO: 77), respectively, according to the instructions of the manufacturer, using LR plus recombinase mix. The resulting binary plant transformation vectors were named Lo546a-pSUN1-R4-Lo484::Lo376::Lo522a (SEQ ID NO: 2) and Lo546b-pSUN1-R4-Lo484::Lo376::Lo522b (SEQ ID NO: 1), respectively.

Example 8

System Based on Inverted Repeat of Nos Terminator Using Two Expression Cassettes in Head to Head Orientation

8.1 Generation of the Positive and Negative Binary Vector Control Constructs for the Screening of Terminator Activity

[0530] To test the transcription termination efficiency of a putative terminator sequence a construct was generated with a strong seed specific promoter (BnAK promoter) upstream of the β-Glucuronidase (GUS) marker gene followed by a short MCS in which putative terminator sequences are cloned and the 3'-UTR region from the nos-gene of Agrobacterium tumefaciens. The second expression cassette in this construct contains the promoter of the nos-gene from Agrobacterium tumefaciens followed by the nptII marker gene and the 3' nos UTR. Both expression cassettes are oriented in a head to head manner. In transgenic plants this arrangement without a putative terminator sequence serves as a negative control: As the nos terminator is used for both the right hand and left hand expression cassette both orientations of the nosT are incorporated into the resulting transcripts, giving rise to GUS and nptII transcripts carrying complementary 3' sequences, leading to hybridization of the two mRNA species, thus resulting in sequence-specific RNA-degradation and total or partial silencing of the β-Glucuronidase and the nptII-gene. As a positive control the Ribulose-bisphosphate Carboxiase E9 terminator region is cloned in between the GUS-gene and the nosT, leading to correct truncation of the transcript and enabling high expression of GUS. Transcripts from the nptII marker gene, carrying the 3' Tnos UTR are not interacting with the GUS transcripts as there is no complementary sequence present and the nptII expression is not impaired (FIG. 1 A3).

8.2 Cloning of Lo239-pSUN3-GWs-B1-BnAK700::GUS::nosT-B2

[0531] Insertion of promoter: The seed specific promoter BnAK700 is isolated by HindIII/BamHI digestion of Lo229 and inserted into the vector Lo215 pENTR-MCS::GUS::nosT to create Lo235 pENTR-B-BnAK700::GUS::nosT.

[0532] Lo 239 (SEQ ID NO: 78) is created by LR reaction of Lo235 with the Gateway destination vector Lo125 pSUN3-GWs-NPTII (FIG. 10).

8.3 Cloning of Lo657-pSUN3-GWs-B1-BnAK700::GUS::E9::nosT::B2

Insertion of the E9 Terminator:

[0533] The Gateway Entry vector Lo235 is cut with Ecl136II to create blunt ends. The E9 insert is isolated by restriction of Lo489 with EcoRV and KpnI followed by fill in with Klenow fragment. The blunt ended insert is ligated to the linearized vector to create Lo654 pENTR-BnAK700::GUS::E9::nosT.

[0534] Lo657 (SEQ ID NO: 79) is created by LR reaction of Lo654 with the Gateway destination vector Lo125 pSUN3-GWs-NPTII (FIG. 10).

Example 9

Development of an In Vivo Screening System to Identify Terminators by Embedding Sequences of Interest within an Intron of a Lethal Gene or Reporter Gene

[0535] A terminator of interest (TOI) is embedded within an intron of a lethal gene including, but not limited to, diphtheria toxin fragment A (DT-A) or a reporter gene including, but not limited to, green fluorescence protein (GFP) (see FIG. 16A). If efficiency of transcription termination is low ("leaky" TOI), there is some transcription of the full length lethal or reporter gene. The intron with the embedded TOI is removed from the transcribed RNA allowing for translation of the full length lethal or reporter protein. In the example using the lethal gene, expression of full length DT-A kills the cells. If the TOI does not allow read through RNA products because of efficient transcription termination ("tight" TOI), only a partial protein is translated and the plant cells are viable. In the example using GFP, a leaky TOI yields full length GFP and cells that fluoresce green. A tight TOI produces only a partial GFP protein and cells don't fluoresce. Control constructs are constructed without a TOI embedded in the intron (FIG. 16B) and with a known terminator, NOS, embedded in the intron (FIG. 16C). See also agenda to FIG. 16 above.

[0536] Preferably, a strong constitutive promoter is used for these constructs such as the maize ubiquitin promoter (Zmubi). An octopine synthase terminator (OCS) is added to the end of the cassette to stabilize the read through products. The intron sequence to be used is a potato intron (PIV2) modified here to improve intron splicing efficiency. The modified PIV2 intron contains the following elements to promote efficient intron recognition and splicing in plants (FIG. 17):

[0537] (1) Transition at the 5' splice site from moderate AU content (exon) to high AU content (intron).

[0538] (2) Transition at the 3' splice site from high AU content (intron) to moderate AU content (exon).

[0539] (3) A consensus 5' splice recognition sequence CAG/GUAAGU. `/` identifies the splice site.

[0540] (4) A consensus 3' recognition sequence GCAG/G.

[0541] (5) A consensus branchpoint sequence CURAY upstream of the 3' splice site.

[0542] (6) A polyU tract just downstream of the branchpoint sequence and upstream of the 3' splice site.

[0543] A BamHI restriction site is added near the center of the intron for insertion of the transcription terminator of interest (TOI). A BamHI site is compatible with Sau3AI and is therefore ideal for insertions of genomic DNA fragments generated by a partial Sau3AI digest. The BamHI site can be substituted with other restriction sites to accommodate TOI libraries generated by other means. The cassettes in FIG. 15 can be placed in a binary vector for Agrobacterium-mediated plant transformation or in a pUC based vector for biolistic transformation. See also agenda to FIG. 16 above.

9.1 Vector Construction

[0544] Construct 2 (DT-A version) in FIG. 16 is constructed using the parental vector pTOI03 described above. pTOI03 is digested with KpnI and SpeI to remove the GUSint gene but leaving the ZmUbi promoter and OCS terminator. The 3' end of the first half of the DT-A gene is fused to the 5' end of the first half of the PIV2 intron by overlap PCR using the following primers:

TABLE-US-00017 JMTOIprim1 (SEQ ID NO: 61) 5'-GGTTCCAAGGTACCAAAACAATGGGCGCTGATGATGTTGTTGAT-3' JMTOIprim2 (SEQ ID NO: 62) 5'-AAGGTAGAAGCAGAAACTTACCTGGATACGTCACTTTGACCA-3' JMTOIprim3 (SEQ ID NO: 63) 5'-TGGTCAAAGTGACGTATCCAGGTAAGTTTCTGCTTCTACCTT-3' JMTOIprim4 (SEQ ID NO: 64) 5'-GGTTCCAAGGATCCATTTATTTTGAAAAAAATATTTG-3'

[0545] This overlap PCR places a KpnI site on the 5' end of the DT-A portion of the fused sequences followed by an ATG start codon preceded by the bases AAAACA to enhance translation. It also generates a consensus 5'splice site between the DT-A and intron sequences and a BamHI site 133 bp downstream of the 5' splice site. The 3' end of the second half of the PIV2 intron is fused to the 5' end of the second half of the DT-A gene by overlap PCR using the following primers:

TABLE-US-00018 JMTOIprim5 (SEQ ID NO: 65) 5'-GGTTCCAAGGATCCAGTATATAGCAATTGCTTTTC-3' JMTOIprim6 (SEQ ID NO: 66) 5'-CGAGAACCTTCGTCAGTCCTGCACATCAACAAATTTTGGTCATA AAAAAAAAAATATTAGAAAAGTTATAAATTAAAATATAC-3' JMTOIprim7 (SEQ ID NO: 67) 5'-CTAATATTTTTTTTTTTATGACCAAAATTTGTTGATGTGCAGGA CTGACGAAGGTTCTCGCAC-3' JMTOIprim8 (SEQ ID NO: 68) 5'-TTGGAACCACTAGTTTATCGCCTGACACGATTTCCTGC-3'

[0546] This overlap PCR places a BamHI site on the 5' end of the PCR product. A tract of 11 consecutive Us at positions+36 to +26 relative to the 3' splice site and 2 bases downstream of a natural CTAAT consensus branchpoint sequence in the PIV2 intron is added as well as a consensus 3' splice site between the PIV2 and DT-A sequences. This overlap PCR also generates a TAA stop codon at the end of the DT-A open reading frame followed by a SpeI restriction enzyme site. The first overlap PCR product is digested with KpnI and BamHI, the second PCR product is digested with BamHI and SpeI, and both PCR products are ligated to pTOI03 digested with KpnI and SpeI in a 3-way ligation to make pJMTOI1 (SEQ ID NO: 71). The construct comprises the following features:

TABLE-US-00019 Feature Location (base) ZmUbi promoter (1) . . . (1988) DT-A 5' end of coding sequence (2007) . . . (2268) Intron (2269) . . . (2488) DTA-A 3' end of coding sequence (2489) . . . (2811) OCS terminator (2818) . . . (3030)

[0547] Construct 3 (DT-A version) in FIG. 16C will be constructed by first placing BamHI sites on the ends of the NOS terminator using the following PCR primers:

TABLE-US-00020 JMTOIprim9 (SEQ ID NO: 69) 5'-GGTTCCAAGGATCCGATCGTTCAAACATTTGGCAA-3' JMTOIprim10 (SEQ ID NO: 70) 5'-GGTTCCAAGGATCCGATCTAGTAACATAGATGACA-3'

[0548] This PCR product is digested with BamHI and ligated into the unique BamHI site within the PIV2 intron of pJMTOI1. Plasmids generated from the ligation are screened to identify those with the correct NOS orientation, yielding pJMTOI2 (SEQ ID NO: 72). The construct comprises the following features:

TABLE-US-00021 Feature Location (base) ZmUbi promoter (1) . . . (1988) DT-A 5' end of coding sequence (2007) . . . (2268) Intron 5' end (2269) . . . (2380) Nos terminator (2387) . . . (2639) Intron 3' end (2646) . . . (2747) DTA-A 3' end of coding sequence (2748) . . . (3070) OCS terminator (3077) . . . (3289)

Example 10

An In Vivo Selection of Efficient Terminators

[0549] In this example, a terminator of interest (TOI) is placed between a reporter gene such as the green fluorescence protein (GFP) and a sequence with little or no homology to plant genes and that is a target of dsRNA mediated RNA silencing (FIG. 18). Each of these elements is under control of a single promoter, in this example, the maize ubiquitin promoter (ZmUbi). When expressed in plants, and if the TOI does not terminate transcription (leaky TOI), the entire transcript (including the region encoding GFP) is degraded by RNA silencing. If the TOI is functional as a terminator, the RNA will not be a target of RNA silencing and GFP will be produced leading to plants that fluoresce green (FIG. 18). TOIs may be obtained by fragmentation of genomic DNA or by a more selective procedure.

[0550] A BamHI restriction site will be placed at the junction between GFP and the spacer in construct 3 of FIG. 18 for insertion of the TOI (construct 1) or the NOS terminator (construct 2; pJMTOI3, SEQ ID NO: 73). A BamHI site is compatible with Sau3AI and is therefore ideal for insertions of genomic DNA fragments generated by a partial Sau3AI digest. The BamHI site can be substituted with other restriction sites to accommodate TOI libraries generated by other means. The cassettes in FIG. 18 can be placed in a binary vector for Agrobacterium-mediated plant transformation. Construct 2 (pJMTOI3) comprises the following features:

TABLE-US-00022 Feature Location (base) ZmUbi promoter (1) . . . (1988) GFP (2001) . . . (2696) NOS terminator (2703) . . . (2955) Spacer (2962) . . . (3161) RNAi target (3162) . . . (3461) OCS terminator (3468) . . . (3680)

[0551] To perform the terminator screen described in this example, a plant line must be established that can effectively silence RNAs that contain the RNAi target region. In this example, Arabidopsis will be used as the host plant although the screen can be used in any plant species that can be transformed. A plant that can effectively silence can be obtained with the following steps using established transformation and genetic screening techniques.

[0552] 1) Wild-type Arabidopsis is transformed with construct 3 in FIG. 18 (pJMTOI4, SEQ ID NO: 74). A T₁ plant is selected that is single copy for construct 3 and has strong green fluorescence. Construct 3 (pJMTOI4) comprises the following features:

TABLE-US-00023

[0552] Feature Location (base) ZmUbi promoter (1) . . . (1988) GFP (2001) . . . (2696) Spacer (2703) . . . (2902) RNAi target (2903) . . . (3202) OCS terminator (3209) . . . (3421)



[0553] 2) This fluorescent T₁ plant is transformed with construct 4 in FIG. 18 (pJMTOI5; SEQ ID NO: 75). A T₁ plant from this transformation is selected that is single copy and hemizygous for construct 3 and single copy for construct 4, and that no longer fluoresces green (silencing plant). Construct 4 (pJMTOI5) comprises the following features:

TABLE-US-00024

[0553] Feature Location (base) ZmUbi promoter (1) . . . (1988) RNAi target sense (2001) . . . (2300) Spacer (2309) . . . (2595) RNAi target anti-sense (2602) . . . (2901) NOS terminator (2908) . . . (3160)



[0554] 3) A T₂ plant with respect to construct 4 (pJMTOI5) is obtained that is homozygous for construct 4 and null for construct 3. To generate additional silencing plants, T₃ plants can be obtained from the plant isolated in step 3.

[0555] After an Arabidopsis silencing line containing construct 4 in FIG. 3 has been established, this line can be transformed with plasmid TOI libraries containing construct 1 in FIG. 3 and with the control constructs 2 and 3 (pJMTOI3 and (pJMTOI4, respectively) in FIG. 18. T₁ plants that fluoresce to a similar extent as plants transformed with construct 2 will be selected for further analysis. If a selected plant has a single integrant, quantitative RT-PCR targeting GFP and the RNAi target region can determine if the experimental TOI is acting as an efficient terminator. RT-PCR of plants transformed with construct 2 (pJMTOI3) and construct 3 (pJMTOI4) would serve as controls. If a selected plant has multiple integrants of construct 1, single integrants can be obtained from the T₂ generation. The TOIs from selected plants can be amplified and cloned by PCR.

Example 11

Assays for Identifying Terminators of Interest

[0556] These experiments are performed by bombardment of plant tissues or culture cells (Example 11.1), by PEG-mediated (or similar methodology) introduction of DNA to plant protoplasts (Example 11.2), or by Agrobacterium-mediated transformation (Example 11.3). The target tissue for these experiments can be plant tissues (e.g. leaf tissue has been described to best support IRES-mediated translation (Urwin et al., 2000), cultured plant cells (e.g. maize BMS), or plant embryos for Agrobacterium protocols.

[0557] The sequences used as potential transcription terminator sequences can either be derived from the in silico transcription terminator sequence screen or from a library of random genomic fragments. Ratio of expression of two different reporter genes is measured by quantification of expression of reporter genes or RT-PCR using the protocols in the art in order to determine potentially strong and tight terminator candidates.

11.1 Transient Assay Using Microprojectile Bombardment

[0558] The plasmid constructs are isolated using Qiagen plasmid kit (cat#12143). DNA is precipitated onto 0.6 μM gold particles (Bio-Rad cat#165-2262) according to the protocol described by Sanford et al. (1993) and accelerated onto target tissues (e.g. two week old maize leaves, BMS cultured cells, etc.) using a PDS-1000/He system device (Bio-Rad). All DNA precipitation and bombardment steps are performed under sterile conditions at room temperature.

[0559] Two mg of gold particles (2 mg/3 shots) are resuspended in 100% ethanol followed by centrifugation in a Beckman Microfuge 18 Centrifuge at 2000 rpm in an Eppendorf tube. The pellet is rinsed once in sterile distilled water, centrifuged, and resuspended in 25 μL of 1 μg/μL total DNA. The following reagents are added to the tube: 220 μL H₂O, 250 μL 2.5M CaCl₂, 50 μL 0.1M spermidine, free base. The DNA solution is briefly vortexed and placed on ice for 5 min followed by centrifugation at 500 rpm for 5 min in a Beckman Microfuge 18 Centrifuge. The supernatant is removed. The pellet is resuspended in 600 μL ethanol followed by centrifugation for 1 min at 14,000 rpm. The final pellet is resuspended in 36 μL of ethanol and used immediately or stored on ice for up to 4 hr prior to bombardment. For bombardment, two-week-old maize leaves are cut in approximately 1 cm in length and located on 2 inches diameter sterilized Whatman filter paper. In the case of BMS cultured cells, 5 mL of one-week-old suspension cells are slowly vacuum filtered onto the 2 inches diameter filter paper placed on a filter unit to remove excess liquid. The filter papers holding the plant materials are placed on osmotic induction media (N6 1-100-25, 0.2 M mannitol, 0.2 M sorbitol) at 27° C. in darkness for 2-3 hours prior to bombardment. A few minutes prior to shooting, filters are removed from the medium and placed onto sterile opened Petri dishes to allow the calli surface to partially dry. To keep the position of plant materials, a sterilized wire mesh screen is laid on top of the sample. Each plate is shot with 10 μL of gold-DNA solution once at 2,200 psi for the leaf materials and twice at 1100 psi for the BMS cultured cells. Following bombardment, the filters holding the samples are transferred onto MS basal media and incubated for 2 days in darkness at 27° C. prior to transient assays. Transient expression levels of the reporter gene are determined quantification of expression of reporter genes or RT-PCR using the protocols in the art in order to determine potentially strong and tight terminator candidates.

11.2 Transient Assay Using Protoplasts

[0560] Isolation of protoplasts is conducted by following the protocol developed by Sheen (1990). Maize seedlings are kept in the dark at 25° C. for 10 days and illuminated for 20 hours before protoplast preparation. The middle part of the leaves are cut to 0.5 mm strips (about 6 cm in length) and incubated in an enzyme solution containing 1% (w/v) cellulose RS, 0.1% (w/v) macerozyme R10 (both from Yakult Honsha, Nishinomiya, Japan), 0.6 M mannitol, 10 mM Mes (pH 5.7), 1 mM CaCl₂, 1 mM MgCl₂, 10 mM β-mercaptoethanol, and 0.1% BSA (w/v) for 3 hr at 23° C. followed by gentle shaking at 80 rpm for 10 min to release protoplasts. Protoplasts are collected by centrifugation at 100×g for 2 min, washed once in cold 0.6 M mannitol solution, centrifuged, and resuspended in cold 0.6 M mannitol (2×10⁶/mL).

[0561] A total of 50 μg plasmid DNA in a total volume of 100 μL sterile water is added into 0.5 mL of a suspension of maize protoplasts (1×10⁶ cells/mL) and mix gently. 0.5 mL PEG solution (40% PEG 4000, 100 mM CaNO₃, 0.5 mannitol) is added and pre-warmed at 70° C. with gentle shaking followed by addition of 4.5 mL MM solution (0.6 M mannitol, 15 mM MgCl₂, and 0.1% MES). This mixture is incubated for 15 minutes at room temperature. The protoplasts are washed twice by pelleting at 600 rpm for 5 min and resuspending in 1.0 mL of MMB solution [0.6 M mannitol, 4 mM Mes (pH 5.7), and brome mosaic virus (BMV) salts (optional)] and incubated in the dark at 25° C. for 48 hr. After the final wash step, collect the protoplasts in 3 mL MMB medium, and incubate in the dark at 25° C. for 48 hr. Transient expression levels of the reporter gene are determined quantification of expression of reporter genes or RT-PCR using the protocols in the art in order to determine potentially strong and tight terminator candidates.

11.3 Agrobacterium-Mediated Transformation in Dicotyledonous and Monocotyledonous Plants

[0562] 11.3.1 Transformation and Regeneration of Transgenic Arabidopsis thaliana (Columbia) Plants

[0563] To generate transgenic Arabidopsis plants, Agrobacterium tumefaciens (strain C58C1 pGV2260) is transformed with the various vector constructs described above. The Agrobacterial strains are subsequently used to generate transgenic plants. To this end, a single transformed Agrobacterium colony is incubated overnight at 28° C. in a 4 mL culture (medium: YEB medium with 50 μg/mL kanamycin and 25 μg/mL rifampicin). This culture is subsequently used to inoculate a 400 mL culture in the same medium, and this is incubated overnight (28° C., 220 rpm) and spun down (GSA rotor, 8,000 rpm, 20 min). The pellet is resuspended in infiltration medium (1/2 MS medium; 0.5 g/L MES, pH 5.8; 50 g/L sucrose). The suspension is introduced into a plant box (Duchefa), and 100 ml of SILWET L-77 (heptamethyltrisiloxan modified with polyalkylene oxide; Osi Specialties Inc., Cat. P030196) is added to a final concentration of 0.02%. In a desiccator, the plant box with 8 to 12 plants is exposed to a vacuum for 10 to 15 minutes, followed by spontaneous aeration. This is repeated twice or 3 times. Thereupon, all plants are planted into flowerpots with moist soil and grown under long-day conditions (daytime temperature 22 to 24° C., nighttime temperature 19° C.; relative atmospheric humidity 65%). The seeds are harvested after 6 weeks.

[0564] As an alternative, transgenic Arabidopsis plants can be obtained by root transformation. White root shoots of plants with a maximum age of 8 weeks are used. To this end, plants which are kept under sterile conditions in 1 MS medium (1% sucrose; 100 mg/L inositol; 1.0 mg/L thiamine; 0.5 mg/L pyridoxine; 0.5 mg/L nicotinic acid; 0.5 g MES, pH 5.7; 0.8% agar) are used. Roots are grown on callus-inducing medium for 3 days (1× Gamborg's B5 medium; 2% glucose; 0.5 g/L mercaptoethanol; 0.8% agar; 0.5 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid); 0.05 mg/L kinetin). Root sections 0.5 cm in length are transferred into 10 to 20 mL of liquid callus-inducing medium (composition as described above, but without agar supplementation), inoculated with 1 mL of the above-described overnight Agrobacterium culture (grown at 28° C., 200 rpm in LB) and shaken for 2 minutes. After excess medium has been allowed to run off, the root explants are transferred to callus-inducing medium with agar, subsequently to callus-inducing liquid medium without agar (with 500 mg/L betabactyl, SmithKline Beecham Pharma GmbH, Munich), incubated with shaking and finally transferred to shoot-inducing medium (5 mg/L 2-isopentenyladenine phosphate; 0.15 mg/L indole-3-acetic acid; 50 mg/L kanamycin; 500 mg/L betabactyl). After 5 weeks, and after 1 or 2 medium changes, the small green shoots are transferred to germination medium (1 MS medium; 1% sucrose; 100 mg/L inositol; 1.0 mg/L thiamine; 0.5 mg/L pyridoxine; 0.5 mg/L nicotinic acid; 0.5 g MES, pH 5.7; 0.8% agar) and regenerated into plants.

11.3.2 Transformation and Regeneration of Crop Plants

[0565] The Agrobacterium-mediated plant transformation using standard transformation and regeneration techniques may also be carried out for the purposes of transforming crop plants (Gelvin 1995; Glick 1993). For example, oilseed rape can be transformed by cotyledon or hypocotyl transformation (Moloney 1989; De Block 1989). The use of antibiotics for the selection of Agrobacteria and plants depends on the binary vector and the Agrobacterium strain used for the transformation. The selection of oilseed rape is generally carried out using kanamycin as selectable plant marker. The Agrobacterium-mediated gene transfer in linseed (Linum usitatissimum) can be carried out using for example a technique described by Mlynarova (1994). The transformation of soybean can be carried out using, for example, a technique described in EP-A1 0 424 047 or in EP-A1 0 397 687, U.S. Pat. No. 5,376,543, U.S. Pat. No. 5,169,770. The transformation of maize or other monocotyledonous plants can be carried out using, for example, a technique described in U.S. Pat. No. 5,591,616. The transformation of plants using particle bombardment, polyethylene glycol-mediated DNA uptake or via the silicon carbonate fiber technique is described, for example, by Freeling & Walbot (1993) "The maize handbook" ISBN 3-540-97826-7, Springer Verlag New York).

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Sequence CWU 1

1

108112287DNAArtificial sequenceBinary expression vector Lo546b-pSUN1-R4- Lo484::Lo376::Lo522b 1caactttgta tagaaaagtt ggccatgatt acgccaagct tgcatgcctg caggtcccca 60gattagcctt ttcaatttca gaaagaatgc taacccacag atggttagag aggcttacgc 120agcaggtctc atcaagacga tctacccgag caataatctc caggaaatca aataccttcc 180caagaaggtt aaagatgcag tcaaaagatt caggactaac tgcatcaaga acacagagaa 240agatatattt ctcaagatca gaagtactat tccagtatgg acgattcaag gcttgcttca 300caaaccaagg caagtaatag agattggagt ctctaaaaag gtagttccca ctgaatcaaa 360ggccatggag tcaaagattc aaatagagga cctaacagaa ctcgccgtaa agactggcga 420acagttcata cagagtctct tacgactcaa tgacaagaag aaaatcttcg tcaacatggt 480ggagcacgac acacttgtct actccaaaaa tatcaaagat acagtctcag aagaccaaag 540ggcaattgag acttttcaac aaagggtaat atccggaaac ctcctcggat tccattgccc 600agctatctgt cactttattg tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca 660tcattgcgat aaaggaaagg ccatcgttga agatgcctct gccgacagtg gtcccaaaga 720tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa 780gcaagtggat tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc 840ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagaa cacgggggac 900tctagaggat ccaaggagat ataacaatga agactaatct ttttctcttt ctcatctttt 960cacttctcct atcattatcc tcggccgaat tcagtaaagg agaagaactt ttcactggag 1020ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg 1080gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg 1140gaaaactacc tgttccatgg ccaacacttg tcactacttt ctcttatggt gttcaatgct 1200tttcaagata cccagatcat atgaagcggc acgacttctt caagagcgcc atgcctgagg 1260gatacgtgca ggagaggacc atcttcttca aggacgacgg gaactacaag acacgtgctg 1320aagtcaagtt tgagggagac accctcgtca acaggatcga gcttaaggga atcgatttca 1380aggaggacgg aaacatcctc ggccacaagt tggaatacaa ctacaactcc cacaacgtat 1440acatcatggc cgacaagcaa aagaacggca tcaaagccaa cttcaagacc cgccacaaca 1500tcgaagacgg cggcgtgcaa ctcgctgatc attatcaaca aaatactcca attggcgatg 1560gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc 1620ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac 1680atggcatgga tgaactatac aaacatgatg agctttaaga gaacggatcc ccatctgcgg 1740ccgcctcgag catatgctag aggatcctct agctagagct ttcgttcgta tcatcggttt 1800cgacaacgtt cgtcaagttc aatgcatcag tttcattgcg cacacaccag aatcctactg 1860agtttgagta ttatggcatt gggaaaactg tttttcttgt accatttgtt gtgcttgtaa 1920tttactgtgt tttttattcg gttttcgcta tcgaactgtg aaatggaaat ggatggagaa 1980gagttaatga atgatatggt ccttttgttc attctcaaat taatattatt tgttttttct 2040cttatttgtt gtgtgttgaa tttgaaatta taagagatat gcaaacattt tgttttgagt 2100aaaaatgtgt caaatcgtgg cctctaatga ccgaagttaa tatgaggagt aaaacacttg 2160tagttgtacc attatgctta ttcactaggc aacaaatata ttttcagacc tagaaaagct 2220gcaaatgtta ctgaatacaa gtatgtcctc ttgtgtttta gacatttatg aactttcctt 2280tatgtaattt tccagaatcc ttgtcagatt ctaatcattg ctttataatt atagttatac 2340tcatggattt gtagttgagt atgaaaatat tttttaatgc attttatgac ttgccaattg 2400attgacaaca tgcatcaatc gaccgggtac ccaagtttgt acaaaaaagc aggctggtac 2460ccggggatcc tctaggtcga ccagatctga tatctgcggc cgcctcgagc atatgggcat 2520gcaagcttgg cgtaatcatg gacccagctt tcttgtacaa agtggggtac ccggggatcc 2580tgatagctta tactcaaatt caacaagtta tatataaatg tatagatact acaatatcat 2640taacaaaagt caccttaaat aaatacacat atcttttatg ttctctattg ttttgcgtac 2700gctaacacaa tttctcatat gcaaaaggat gaatgagtaa caaattacct cataagaaca 2760atcatctttg cttacatact aatacaataa tcactcaatc aaccaataac atcaatcaca 2820taggtttaca tacaataatc actcaatcaa cttcataaga agaatcatgt ttacttaatt 2880catcaattat ccccaaaaac accactatta agtataaact acaacatatt tgtagtgatg 2940ggtcaacatt tttatcatat ttaaactcgg gttccctcaa atcgagaaat agtgaacatg 3000taatattaat tttaaatcgc aattacagaa attaattgaa tttggtcaaa tggacagaat 3060tttatagatt gggtggaact agaaaaaaaa aaaaaaagag tatagggtga attgagtaca 3120tgaaagtaca tggtaatcct agttaaacgc ataatacatg tgggttcatt tgtatttttt 3180tgtaacttac gagtaaactg gctacaacaa aaaaaaatta gaagattttt ttgttttgta 3240gaaaacccta attttagtta tagttgtata actttgataa aattataaaa ttgtattacg 3300aaaaaagtaa taagatattc aaaaaagcct agaataacgt atatgactat gagcatgaaa 3360ctgcaagtca aatgctgaca gacaaccata aacaaaagaa attaaataga gataccttta 3420aaataagtaa aatttcattt ataaaaaatc tactttcttg tgaatctgtc acgttcaata 3480atttgaagac cactcaacat acaaggtaaa taatgaaaaa taaaatctac caaaatttca 3540atcattatta tcttccaaaa aaacaaaatt atacagatga tgatggtgat atggaacttc 3600gattggctaa tattcactgt gtctctaaaa accatccact tatcaagata agatggaccc 3660tacactcatc caatctaaac cagtatctca agattcttat ctaattacat cattctctac 3720cgttagatga aattgaccat taaccctacc ataactccat acaccgcgag atactggatt 3780aaccaaatcg agatcatcgt agccgtccga tcaacaagta ccatctcttg aaatactcga 3840aatcctcata agtccgtccc tctttgctct cactatcaaa actctgaatt tcgatttcat 3900ctagagtcga gcccgggcga tatcggatct cgactctagt cgagggccca tgggagcttg 3960gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 4020ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 4080gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 4140ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 4200cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 4260tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 4320gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 4380cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 4440gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 4500cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 4560ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 4620agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 4680cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 4740cgagttcttc tgagcgggac ccaagctagc ttcgacggat cctctagcat atgctcgagg 4800cggccgcaga tatcagatcc tctagctaga gctttcgttc gtatcatcgg tttcgacaac 4860gttcgtcaag ttcaatgcat cagtttcatt gcgcacacac cagaatccta ctgagtttga 4920gtattatggc attgggaaaa ctgtttttct tgtaccattt gttgtgcttg taatttactg 4980tgttttttat tcggttttcg ctatcgaact gtgaaatgga aatggatgga gaagagttaa 5040tgaatgatat ggtccttttg ttcattctca aattaatatt atttgttttt tctcttattt 5100gttgtgtgtt gaatttgaaa ttataagaga tatgcaaaca ttttgttttg agtaaaaatg 5160tgtcaaatcg tggcctctaa tgaccgaagt taatatgagg agtaaaacac ttgtagttgt 5220accattatgc ttattcacta ggcaacaaat atattttcag acctagaaaa gctgcaaatg 5280ttactgaata caagtatgtc ctcttgtgtt ttagacattt atgaactttc ctttatgtaa 5340ttttccagaa tccttgtcag attctaatca ttgctttata attatagtta tactcatgga 5400tttgtagttg agtatgaaaa tattttttaa tgcattttat gacttgccaa ttgattgaca 5460acatgcatca atcgacctgc agcccaagct tgatctagtc cgatctagta acatagatga 5520caccgcgcgc gataatttat cctagtttgc gcgctatatt ttgttttcta tcgcgtatta 5580aatgtataat tgcgggactc taatcataaa aacccatctc ataaataacg tcatgcatta 5640catgttaatt attacatgct taacgtaatt caacagaaat tatatgataa tcatcgcaag 5700accggcaaca ggattcaatc ttaagaaact ttattgccaa atgtttgaac gatcggggaa 5760attcgagctc ggtagcaatt cccgaggctg tagccgacga tggtgcgcca ggagagttgt 5820tgattcattg tttgcctccc tgctgcggtt tttcaccgaa gttcatgcca gtccagcgtt 5880tttgcagcag aaaagccgcc gacttgtcga ccgatcggtc aaacatttgg caataaagtt 5940tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt 6000acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta 6060tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa 6120actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa gcttggcgta 6180atcatggcaa ctttattata caaagttggc attataaaaa agcattgctt atcaatttgt 6240tgcaacgaac aggtcactat cagtcaaaat aaaatcatta ttcaacttta ttatacatag 6300ttgataattc actggccgga tctgcttggt aataattgtc attagattgt ttttatgcat 6360agatgcactc gaaatcagcc aattttagac aagtatcaaa cggatgttaa ttcagtacat 6420taaagacgtc cgcaatgtgt tattaagttg tctaagcgtc aatttgttta caccacaata 6480tatcctgcca ccagccagcc aacagctccc cgaccggcag ctcggcacaa aatcaccacg 6540cgttaccacc acgccggccg gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt 6600cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc gaggccgcca aggcccgagg 6660cgtgaagttt ggcccccgcc ctaccctcac cccggcacag atcgcgcacg cccgcgagct 6720gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc 6780gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg 6840cggtgccttc cgtgaggacg cattgaccga ggccgacgcc ctggcggccg ccgagaatga 6900acgccaagag gaacaagcat gaaaccgcac caggacggcc aggacgaacc gtttttcatt 6960accgaagaga tcgaggcgga gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac 7020gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc 7080tggccggcca gcttggccgc tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta 7140tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa 7200caaatacgca aggggaacgc atgaaggtta tcgctgtact taaccagaaa ggcgggtcag 7260gcaagacgac catcgcaacc catctagccc gcgccctgca actcgccggg gccgatgttc 7320tgttagtcga ttccgatccc cagggcagtg cccgcgattg ggcggccgtg cgggaagatc 7380aaccgctaac cgttgtcggc atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg 7440gccggcgcga cttcgtagtg atcgacggag cgccccaggc ggcggacttg gctgtgtccg 7500cgatcaaggc agccgacttc gtgctgattc cggtgcagcc aagcccttac gacatatggg 7560ccaccgccga cctggtggag ctggttaagc agcgcattga ggtcacggat ggaaggctac 7620aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg catcggcggt gaggttgccg 7680aggcgctggc cgggtacgag ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct 7740acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg 7800cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa actcatttga gttaatgagg 7860taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag 7920cagcaaggct gcaacgttgg ccagcctggc agacacgcca gccatgaagc gggtcaactt 7980tcagttgccg gcggaggatc acaccaagct gaagatgtac gcggtacgcc aaggcaagac 8040cattaccgag ctgctatctg aatacatcgc gcagctacca gagtaaatga gcaaatgaat 8100aaatgagtag atgaatttta gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag 8160gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg 8220gctgggttgt ctgccggccc tgcaatggca ctggaacccc caagcccgag gaatcggcgt 8280gagcggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg atgacctggt 8340ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag aagcacgccc 8400cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc aaccgccggc 8460agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag attttttcgt 8520tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg tggccgtttt 8580ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc ttccagacgg 8640gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt acgacctggt 8700actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag ggaagggaga 8760caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct gccggcgagc 8820cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa acaccacgca 8880cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg tatccgaggg 8940tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc cggagtacat 9000cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga acccggacgt 9060gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt ttctctaccg 9120cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga cgatctacga 9180acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca agctgatcgg 9240gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg gcccgatcct 9300agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct aatgtacgga 9360gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaaggtc tctttcctgt 9420ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc cgtacattgg 9480gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata taaaagagaa 9540aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta aaacccgcct 9600ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag cgcctaccct 9660tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg ccgctggccg 9720ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag ccgcgccgtc 9780gccactcgac cgccggcgcc cacatcaagg caccctgcct cgcgcgtttc ggtgatgacg 9840gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg 9900ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag 9960ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga 10020gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag 10080aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 10140tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 10200aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 10260aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 10320tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 10380ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 10440cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 10500ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 10560ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 10620gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 10680agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 10740cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 10800aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 10860aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 10920ctcacgttaa gggattttgg tcatgcatga tatatctccc aatttgtgta gggcttatta 10980tgcacgctta aaaataataa aagcagactt gacctgatag tttggctgtg agcaattatg 11040tgcttagtgc atctaacgct tgagttaagc cgcgccgcga agcggcgtcg gcttgaacga 11100atttctagct agacattatt tgccgactac cttggtgatc tcgcctttca cgtagtggac 11160aaattcttcc aactgatctg cgcgcgaggc caagcgatct tcttcttgtc caagataagc 11220ctgtctagct tcaagtatga cgggctgata ctgggccggc aggcgctcca ttgcccagtc 11280ggcagcgaca tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa 11340cgtaagcact acatttcgct catcgccagc ccagtcgggc ggcgagttcc atagcgttaa 11400ggtttcattt agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga gttcctccgc 11460cgctggacct accaaggcaa cgctatgttc tcttgctttt gtcagcaaga tagccagatc 11520aatgtcgatc gtggctggct cgaagatacc tgcaagaatg tcattgcgct gccattctcc 11580aaattgcagt tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt gcacaacaat 11640ggtgacttct acagcgcgga gaatctcgct ctctccaggg gaagccgaag tttccaaaag 11700gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg taaccagcaa 11760atcaatatca ctgtgtggct tcaggccgcc atccactgcg gagccgtaca aatgtacggc 11820cagcaacgtc ggttcgagat ggcgctcgat gacgccaact acctctgata gttgagtcga 11880tacttcggcg atcaccgctt cccccatgat gtttaacttt gttttagggc gactgccctg 11940ctgcgtaaca tcgttgctgc tccataacat caaacatcga cccacggcgt aacgcgcttg 12000ctgcttggat gcccgaggca tagactgtac cccaaaaaaa cagtcataac aagccatgaa 12060aaccgccact gcgttccatg gacatacaaa tggacgaacg gataaacctt ttcacgccct 12120tttaaatatc cgattattct aataaacgct cttttctctt aggtttaccc gccaatatat 12180cctgtcaaac actgatagtt taaactgaag gcgggaaacg acaatcagat ctagtaggaa 12240acagctatga ccatgattac gccaagctat cgattacgcc aagctat 12287212261DNAArtificial sequenceBinary expression vector Lo546a-pSUN1-R4- Lo484::Lo376::Lo522a 2caactttgta tagaaaagtt ggccatgatt acgccaagct tgcatgcctg caggtcccca 60gattagcctt ttcaatttca gaaagaatgc taacccacag atggttagag aggcttacgc 120agcaggtctc atcaagacga tctacccgag caataatctc caggaaatca aataccttcc 180caagaaggtt aaagatgcag tcaaaagatt caggactaac tgcatcaaga acacagagaa 240agatatattt ctcaagatca gaagtactat tccagtatgg acgattcaag gcttgcttca 300caaaccaagg caagtaatag agattggagt ctctaaaaag gtagttccca ctgaatcaaa 360ggccatggag tcaaagattc aaatagagga cctaacagaa ctcgccgtaa agactggcga 420acagttcata cagagtctct tacgactcaa tgacaagaag aaaatcttcg tcaacatggt 480ggagcacgac acacttgtct actccaaaaa tatcaaagat acagtctcag aagaccaaag 540ggcaattgag acttttcaac aaagggtaat atccggaaac ctcctcggat tccattgccc 600agctatctgt cactttattg tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca 660tcattgcgat aaaggaaagg ccatcgttga agatgcctct gccgacagtg gtcccaaaga 720tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa 780gcaagtggat tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc 840ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagaa cacgggggac 900tctagaggat ccaaggagat ataacaatga agactaatct ttttctcttt ctcatctttt 960cacttctcct atcattatcc tcggccgaat tcagtaaagg agaagaactt ttcactggag 1020ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg 1080gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg 1140gaaaactacc tgttccatgg ccaacacttg tcactacttt ctcttatggt gttcaatgct 1200tttcaagata cccagatcat atgaagcggc acgacttctt caagagcgcc atgcctgagg 1260gatacgtgca ggagaggacc atcttcttca aggacgacgg gaactacaag acacgtgctg 1320aagtcaagtt tgagggagac accctcgtca acaggatcga gcttaaggga atcgatttca 1380aggaggacgg aaacatcctc ggccacaagt tggaatacaa ctacaactcc cacaacgtat 1440acatcatggc cgacaagcaa aagaacggca tcaaagccaa cttcaagacc cgccacaaca 1500tcgaagacgg cggcgtgcaa ctcgctgatc attatcaaca aaatactcca attggcgatg 1560gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc 1620ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac 1680atggcatgga tgaactatac aaacatgatg agctttaaga gaacggatcc ccatctgcgg 1740ccgcctcgag catatgctag aggatcctct agctagagct ttcgttcgta tcatcggttt 1800cgacaacgtt cgtcaagttc aatgcatcag tttcattgcg cacacaccag aatcctactg 1860agtttgagta ttatggcatt gggaaaactg tttttcttgt accatttgtt gtgcttgtaa 1920tttactgtgt tttttattcg gttttcgcta tcgaactgtg aaatggaaat ggatggagaa 1980gagttaatga atgatatggt ccttttgttc attctcaaat taatattatt tgttttttct 2040cttatttgtt gtgtgttgaa tttgaaatta taagagatat gcaaacattt tgttttgagt 2100aaaaatgtgt caaatcgtgg cctctaatga ccgaagttaa tatgaggagt aaaacacttg 2160tagttgtacc attatgctta ttcactaggc aacaaatata ttttcagacc tagaaaagct 2220gcaaatgtta ctgaatacaa gtatgtcctc ttgtgtttta gacatttatg aactttcctt 2280tatgtaattt tccagaatcc ttgtcagatt ctaatcattg ctttataatt atagttatac 2340tcatggattt gtagttgagt atgaaaatat tttttaatgc attttatgac ttgccaattg 2400attgacaaca tgcatcaatc gaccgggtac ccaagtttgt acaaaaaagc aggctggtac 2460ccggggatcc tctaggtcga ccagatctga tatctgcggc cgcctcgagc atatgggcat 2520gcaagcttgg cgtaatcatg gacccagctt tcttgtacaa agtggggtac ccggggatcc 2580tgatagctta tactcaaatt

caacaagtta tatataaatg tatagatact acaatatcat 2640taacaaaagt caccttaaat aaatacacat atcttttatg ttctctattg ttttgcgtac 2700gctaacacaa tttctcatat gcaaaaggat gaatgagtaa caaattacct cataagaaca 2760atcatctttg cttacatact aatacaataa tcactcaatc aaccaataac atcaatcaca 2820taggtttaca tacaataatc actcaatcaa cttcataaga agaatcatgt ttacttaatt 2880catcaattat ccccaaaaac accactatta agtataaact acaacatatt tgtagtgatg 2940ggtcaacatt tttatcatat ttaaactcgg gttccctcaa atcgagaaat agtgaacatg 3000taatattaat tttaaatcgc aattacagaa attaattgaa tttggtcaaa tggacagaat 3060tttatagatt gggtggaact agaaaaaaaa aaaaaaagag tatagggtga attgagtaca 3120tgaaagtaca tggtaatcct agttaaacgc ataatacatg tgggttcatt tgtatttttt 3180tgtaacttac gagtaaactg gctacaacaa aaaaaaatta gaagattttt ttgttttgta 3240gaaaacccta attttagtta tagttgtata actttgataa aattataaaa ttgtattacg 3300aaaaaagtaa taagatattc aaaaaagcct agaataacgt atatgactat gagcatgaaa 3360ctgcaagtca aatgctgaca gacaaccata aacaaaagaa attaaataga gataccttta 3420aaataagtaa aatttcattt ataaaaaatc tactttcttg tgaatctgtc acgttcaata 3480atttgaagac cactcaacat acaaggtaaa taatgaaaaa taaaatctac caaaatttca 3540atcattatta tcttccaaaa aaacaaaatt atacagatga tgatggtgat atggaacttc 3600gattggctaa tattcactgt gtctctaaaa accatccact tatcaagata agatggaccc 3660tacactcatc caatctaaac cagtatctca agattcttat ctaattacat cattctctac 3720cgttagatga aattgaccat taaccctacc ataactccat acaccgcgag atactggatt 3780aaccaaatcg agatcatcgt agccgtccga tcaacaagta ccatctcttg aaatactcga 3840aatcctcata agtccgtccc tctttgctct cactatcaaa actctgaatt tcgatttcat 3900ctagagtcga gcccgggcga tatcggatct cgactctagt cgagggccca tgggagcttg 3960gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 4020ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 4080gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 4140ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 4200cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 4260tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 4320gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 4380cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 4440gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 4500cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 4560ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 4620agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 4680cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 4740cgagttcttc tgagcgggac ccaagctagc ttcgacggat cctctagcat atgctcgagg 4800cggccgcaga tatcagatcc tctagctaga gctttcgttc gtatcatcgg tttcgacaac 4860gttcgtcaag ttcaatgcat cagtttcatt gcgcacacac cagaatccta ctgagtttga 4920gtattatggc attgggaaaa ctgtttttct tgtaccattt gttgtgcttg taatttactg 4980tgttttttat tcggttttcg ctatcgaact gtgaaatgga aatggatgga gaagagttaa 5040tgaatgatat ggtccttttg ttcattctca aattaatatt atttgttttt tctcttattt 5100gttgtgtgtt gaatttgaaa ttataagaga tatgcaaaca ttttgttttg agtaaaaatg 5160tgtcaaatcg tggcctctaa tgaccgaagt taatatgagg agtaaaacac ttgtagttgt 5220accattatgc ttattcacta ggcaacaaat atattttcag acctagaaaa gctgcaaatg 5280ttactgaata caagtatgtc ctcttgtgtt ttagacattt atgaactttc ctttatgtaa 5340ttttccagaa tccttgtcag attctaatca ttgctttata attatagtta tactcatgga 5400tttgtagttg agtatgaaaa tattttttaa tgcattttat gacttgccaa ttgattgaca 5460acatgcatca atcgacctgc agcccaagct tgatctagtc cgatctagta acatagatga 5520caccgcgcgc gataatttat cctagtttgc gcgctatatt ttgttttcta tcgcgtatta 5580aatgtataat tgcgggactc taatcataaa aacccatctc ataaataacg tcatgcatta 5640catgttaatt attacatgct taacgtaatt caacagaaat tatatgataa tcatcgcaag 5700accggcaaca ggattcaatc ttaagaaact ttattgccaa atgtttgaac gatcggggaa 5760attcgagctc ggtagcaatt cccgaggctg tagccgacga tggtgcgcca ggagagttgt 5820tgattcattg tttgcctccc tgctgcggtt tttcaccgaa gttcatgcca gtccagcgtg 5880tcgaccgatc ggtcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt 5940cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg 6000taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt 6060taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg 6120tcatctatgt tactagatcg ggaagcttgg cgtaatcatg gcaactttat tatacaaagt 6180tggcattata aaaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca 6240aaataaaatc attattcaac tttattatac atagttgata attcactggc cggatctgct 6300tggtaataat tgtcattaga ttgtttttat gcatagatgc actcgaaatc agccaatttt 6360agacaagtat caaacggatg ttaattcagt acattaaaga cgtccgcaat gtgttattaa 6420gttgtctaag cgtcaatttg tttacaccac aatatatcct gccaccagcc agccaacagc 6480tccccgaccg gcagctcggc acaaaatcac cacgcgttac caccacgccg gccggccgca 6540tggtgttgac cgtgttcgcc ggcattgccg agttcgagcg ttccctaatc atcgaccgca 6600cccggagcgg gcgcgaggcc gccaaggccc gaggcgtgaa gtttggcccc cgccctaccc 6660tcaccccggc acagatcgcg cacgcccgcg agctgatcga ccaggaaggc cgcaccgtga 6720aagaggcggc tgcactgctt ggcgtgcatc gctcgaccct gtaccgcgca cttgagcgca 6780gcgaggaagt gacgcccacc gaggccaggc ggcgcggtgc cttccgtgag gacgcattga 6840ccgaggccga cgccctggcg gccgccgaga atgaacgcca agaggaacaa gcatgaaacc 6900gcaccaggac ggccaggacg aaccgttttt cattaccgaa gagatcgagg cggagatgat 6960cgcggccggg tacgtgttcg agccgcccgc gcacgtctca accgtgcggc tgcatgaaat 7020cctggccggt ttgtctgatg ccaagctggc ggcctggccg gccagcttgg ccgctgaaga 7080aaccgagcgc cgccgtctaa aaaggtgatg tgtatttgag taaaacagct tgcgtcatgc 7140ggtcgctgcg tatatgatgc gatgagtaaa taaacaaata cgcaagggga acgcatgaag 7200gttatcgctg tacttaacca gaaaggcggg tcaggcaaga cgaccatcgc aacccatcta 7260gcccgcgccc tgcaactcgc cggggccgat gttctgttag tcgattccga tccccagggc 7320agtgcccgcg attgggcggc cgtgcgggaa gatcaaccgc taaccgttgt cggcatcgac 7380cgcccgacga ttgaccgcga cgtgaaggcc atcggccggc gcgacttcgt agtgatcgac 7440ggagcgcccc aggcggcgga cttggctgtg tccgcgatca aggcagccga cttcgtgctg 7500attccggtgc agccaagccc ttacgacata tgggccaccg ccgacctggt ggagctggtt 7560aagcagcgca ttgaggtcac ggatggaagg ctacaagcgg cctttgtcgt gtcgcgggcg 7620atcaaaggca cgcgcatcgg cggtgaggtt gccgaggcgc tggccgggta cgagctgccc 7680attcttgagt cccgtatcac gcagcgcgtg agctacccag gcactgccgc cgccggcaca 7740accgttcttg aatcagaacc cgagggcgac gctgcccgcg aggtccaggc gctggccgct 7800gaaattaaat caaaactcat ttgagttaat gaggtaaaga gaaaatgagc aaaagcacaa 7860acacgctaag tgccggccgt ccgagcgcac gcagcagcaa ggctgcaacg ttggccagcc 7920tggcagacac gccagccatg aagcgggtca actttcagtt gccggcggag gatcacacca 7980agctgaagat gtacgcggta cgccaaggca agaccattac cgagctgcta tctgaataca 8040tcgcgcagct accagagtaa atgagcaaat gaataaatga gtagatgaat tttagcggct 8100aaaggaggcg gcatggaaaa tcaagaacaa ccaggcaccg acgccgtgga atgccccatg 8160tgtggaggaa cgggcggttg gccaggcgta agcggctggg ttgtctgccg gccctgcaat 8220ggcactggaa cccccaagcc cgaggaatcg gcgtgagcgg tcgcaaacca tccggcccgg 8280tacaaatcgg cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg 8340cccagcggca acgcatcgag gcagaagcac gccccggtga atcgtggcaa gcggccgctg 8400atcgaatccg caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc 8460cgcccaaggg cgacgagcaa ccagattttt tcgttccgat gctctatgac gtgggcaccc 8520gcgatagtcg cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag 8580ctggcgaggt gatccgctac gagcttccag acgggcacgt agaggtttcc gcagggccgg 8640ccggcatggc cagtgtgtgg gattacgacc tggtactgat ggcggtttcc catctaaccg 8700aatccatgaa ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac 8760acgttgcgga cgtactcaag ttctgccggc gagccgatgg cggaaagcag aaagacgacc 8820tggtagaaac ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt acgaagaagg 8880ccaagaacgg ccgcctggtg acggtatccg agggtgaagc cttgattagc cgctacaaga 8940tcgtaaagag cgaaaccggg cggccggagt acatcgagat cgagctagct gattggatgt 9000accgcgagat cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt 9060tgatcgatcc cggcatcggc cgttttctct accgcctggc acgccgcgcc gcaggcaagg 9120cagaagccag atggttgttc aagacgatct acgaacgcag tggcagcgcc ggagagttca 9180agaagttctg tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt 9240tgaaggagga ggcggggcag gctggcccga tcctagtcat gcgctaccgc aacctgatcg 9300agggcgaagc atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag 9360caggggaaaa aggtcgaaaa ggtctctttc ctgtggatag cacgtacatt gggaacccaa 9420agccgtacat tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac attgggaacc 9480ggtcacacat gtaagtgact gatataaaag agaaaaaagg cgatttttcc gcctaaaact 9540ctttaaaact tattaaaact cttaaaaccc gcctggcctg tgcataactg tctggccagc 9600gcacagccga agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg 9660ccgcttcgcg tcggcctatc gcggccgctg gccgctcaaa aatggctggc ctacggccag 9720gcaatctacc agggcgcgga caagccgcgc cgtcgccact cgaccgccgg cgcccacatc 9780aaggcaccct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc 9840ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc 9900gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc 9960ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata 10020tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg 10080cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 10140actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 10200gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 10260ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 10320acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 10380ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 10440cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 10500tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 10560gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 10620ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 10680acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 10740gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 10800ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 10860tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc 10920atgatatatc tcccaatttg tgtagggctt attatgcacg cttaaaaata ataaaagcag 10980acttgacctg atagtttggc tgtgagcaat tatgtgctta gtgcatctaa cgcttgagtt 11040aagccgcgcc gcgaagcggc gtcggcttga acgaatttct agctagacat tatttgccga 11100ctaccttggt gatctcgcct ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg 11160aggccaagcg atcttcttct tgtccaagat aagcctgtct agcttcaagt atgacgggct 11220gatactgggc cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt 11280tgccggttac tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcgc 11340cagcccagtc gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat 11400cctgttcagg aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat 11460gttctcttgc ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga 11520tacctgcaag aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat 11580aacgccacgg aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct 11640cgctctctcc aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg 11700tttcatcaag ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc 11760cgccatccac tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct 11820cgatgacgcc aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccccca 11880tgatgtttaa ctttgtttta gggcgactgc cctgctgcgt aacatcgttg ctgctccata 11940acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact 12000gtaccccaaa aaaacagtca taacaagcca tgaaaaccgc cactgcgttc catggacata 12060caaatggacg aacggataaa ccttttcacg cccttttaaa tatccgatta ttctaataaa 12120cgctcttttc tcttaggttt acccgccaat atatcctgtc aaacactgat agtttaaact 12180gaaggcggga aacgacaatc agatctagta ggaaacagct atgaccatga ttacgccaag 12240ctatcgatta cgccaagcta t 1226136256DNAArtificial sequenceNucleic acid construct Lo522b-pENTR-C1-STPT- nptII-IRnos 3ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac 600taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg 660tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt 720ctcgttcaac tttcttgtac aaagtggggt acccggggat cctgatagct tatactcaaa 780ttcaacaagt tatatataaa tgtatagata ctacaatatc attaacaaaa gtcaccttaa 840ataaatacac atatctttta tgttctctat tgttttgcgt acgctaacac aatttctcat 900atgcaaaagg atgaatgagt aacaaattac ctcataagaa caatcatctt tgcttacata 960ctaatacaat aatcactcaa tcaaccaata acatcaatca cataggttta catacaataa 1020tcactcaatc aacttcataa gaagaatcat gtttacttaa ttcatcaatt atccccaaaa 1080acaccactat taagtataaa ctacaacata tttgtagtga tgggtcaaca tttttatcat 1140atttaaactc gggttccctc aaatcgagaa atagtgaaca tgtaatatta attttaaatc 1200gcaattacag aaattaattg aatttggtca aatggacaga attttataga ttgggtggaa 1260ctagaaaaaa aaaaaaaaag agtatagggt gaattgagta catgaaagta catggtaatc 1320ctagttaaac gcataataca tgtgggttca tttgtatttt tttgtaactt acgagtaaac 1380tggctacaac aaaaaaaaat tagaagattt ttttgttttg tagaaaaccc taattttagt 1440tatagttgta taactttgat aaaattataa aattgtatta cgaaaaaagt aataagatat 1500tcaaaaaagc ctagaataac gtatatgact atgagcatga aactgcaagt caaatgctga 1560cagacaacca taaacaaaag aaattaaata gagatacctt taaaataagt aaaatttcat 1620ttataaaaaa tctactttct tgtgaatctg tcacgttcaa taatttgaag accactcaac 1680atacaaggta aataatgaaa aataaaatct accaaaattt caatcattat tatcttccaa 1740aaaaacaaaa ttatacagat gatgatggtg atatggaact tcgattggct aatattcact 1800gtgtctctaa aaaccatcca cttatcaaga taagatggac cctacactca tccaatctaa 1860accagtatct caagattctt atctaattac atcattctct accgttagat gaaattgacc 1920attaacccta ccataactcc atacaccgcg agatactgga ttaaccaaat cgagatcatc 1980gtagccgtcc gatcaacaag taccatctct tgaaatactc gaaatcctca taagtccgtc 2040cctctttgct ctcactatca aaactctgaa tttcgatttc atctagagtc gagcccgggc 2100gatatcggat ctcgactcta gtcgagggcc catgggagct tggattgaac aagatggatt 2160gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca 2220gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 2280ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct 2340atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 2400gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 2460tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 2520tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg 2580gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc 2640agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac 2700ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat 2760cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 2820tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 2880cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg 2940acccaagcta gcttcgacgg atcctctagc atatgctcga ggcggccgca gatatcagat 3000cctctagcta gagctttcgt tcgtatcatc ggtttcgaca acgttcgtca agttcaatgc 3060atcagtttca ttgcgcacac accagaatcc tactgagttt gagtattatg gcattgggaa 3120aactgttttt cttgtaccat ttgttgtgct tgtaatttac tgtgtttttt attcggtttt 3180cgctatcgaa ctgtgaaatg gaaatggatg gagaagagtt aatgaatgat atggtccttt 3240tgttcattct caaattaata ttatttgttt tttctcttat ttgttgtgtg ttgaatttga 3300aattataaga gatatgcaaa cattttgttt tgagtaaaaa tgtgtcaaat cgtggcctct 3360aatgaccgaa gttaatatga ggagtaaaac acttgtagtt gtaccattat gcttattcac 3420taggcaacaa atatattttc agacctagaa aagctgcaaa tgttactgaa tacaagtatg 3480tcctcttgtg ttttagacat ttatgaactt tcctttatgt aattttccag aatccttgtc 3540agattctaat cattgcttta taattatagt tatactcatg gatttgtagt tgagtatgaa 3600aatatttttt aatgcatttt atgacttgcc aattgattga caacatgcat caatcgacct 3660gcagcccaag cttgatctag tccgatctag taacatagat gacaccgcgc gcgataattt 3720atcctagttt gcgcgctata ttttgttttc tatcgcgtat taaatgtata attgcgggac 3780tctaatcata aaaacccatc tcataaataa cgtcatgcat tacatgttaa ttattacatg 3840cttaacgtaa ttcaacagaa attatatgat aatcatcgca agaccggcaa caggattcaa 3900tcttaagaaa ctttattgcc aaatgtttga acgatcgggg aaattcgagc tcggtagcaa 3960ttcccgaggc tgtagccgac gatggtgcgc caggagagtt gttgattcat tgtttgcctc 4020cctgctgcgg tttttcaccg aagttcatgc cagtccagcg tttttgcagc agaaaagccg 4080ccgacttgtc gaccgatcgg tcaaacattt ggcaataaag tttcttaaga ttgaatcctg 4140ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa 4200ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat 4260tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc 4320gcgcggtgtc atctatgtta ctagatcggg aagcttggcg taatcatggc aactttatta 4380tacaaagttg gcattataaa aaagcattgc ttatcaattt gttgcaacga acaggtcact 4440atcagtcaaa ataaaatcat tatttggagc tccatggtag cgttaacgcg gccatcccct 4500atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa 4560atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg 4620cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc 4680cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg 4740tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt 4800ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa 4860actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg 4920atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat 4980atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt 5040cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc 5100aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct 5160ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag 5220tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag

5280gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat 5340ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta 5400ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat 5460cagaattggt taattggttg taacactggc agagcattac gctgacttga cgggacggcg 5520caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc 5580ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 5640tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 5700ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 5760tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 5820tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 5880actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 5940cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagcatt 6000gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 6060tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 6120ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 6180ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 6240cttttgctca catgtt 625646230DNAArtificial sequenceNucleic acid construct Lo522a-pENTR-C1-STPT- nptII-IRnos 4ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac 600taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg 660tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt 720ctcgttcaac tttcttgtac aaagtggggt acccggggat cctgatagct tatactcaaa 780ttcaacaagt tatatataaa tgtatagata ctacaatatc attaacaaaa gtcaccttaa 840ataaatacac atatctttta tgttctctat tgttttgcgt acgctaacac aatttctcat 900atgcaaaagg atgaatgagt aacaaattac ctcataagaa caatcatctt tgcttacata 960ctaatacaat aatcactcaa tcaaccaata acatcaatca cataggttta catacaataa 1020tcactcaatc aacttcataa gaagaatcat gtttacttaa ttcatcaatt atccccaaaa 1080acaccactat taagtataaa ctacaacata tttgtagtga tgggtcaaca tttttatcat 1140atttaaactc gggttccctc aaatcgagaa atagtgaaca tgtaatatta attttaaatc 1200gcaattacag aaattaattg aatttggtca aatggacaga attttataga ttgggtggaa 1260ctagaaaaaa aaaaaaaaag agtatagggt gaattgagta catgaaagta catggtaatc 1320ctagttaaac gcataataca tgtgggttca tttgtatttt tttgtaactt acgagtaaac 1380tggctacaac aaaaaaaaat tagaagattt ttttgttttg tagaaaaccc taattttagt 1440tatagttgta taactttgat aaaattataa aattgtatta cgaaaaaagt aataagatat 1500tcaaaaaagc ctagaataac gtatatgact atgagcatga aactgcaagt caaatgctga 1560cagacaacca taaacaaaag aaattaaata gagatacctt taaaataagt aaaatttcat 1620ttataaaaaa tctactttct tgtgaatctg tcacgttcaa taatttgaag accactcaac 1680atacaaggta aataatgaaa aataaaatct accaaaattt caatcattat tatcttccaa 1740aaaaacaaaa ttatacagat gatgatggtg atatggaact tcgattggct aatattcact 1800gtgtctctaa aaaccatcca cttatcaaga taagatggac cctacactca tccaatctaa 1860accagtatct caagattctt atctaattac atcattctct accgttagat gaaattgacc 1920attaacccta ccataactcc atacaccgcg agatactgga ttaaccaaat cgagatcatc 1980gtagccgtcc gatcaacaag taccatctct tgaaatactc gaaatcctca taagtccgtc 2040cctctttgct ctcactatca aaactctgaa tttcgatttc atctagagtc gagcccgggc 2100gatatcggat ctcgactcta gtcgagggcc catgggagct tggattgaac aagatggatt 2160gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca 2220gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 2280ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct 2340atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 2400gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 2460tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 2520tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg 2580gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc 2640agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac 2700ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat 2760cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 2820tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 2880cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg 2940acccaagcta gcttcgacgg atcctctagc atatgctcga ggcggccgca gatatcagat 3000cctctagcta gagctttcgt tcgtatcatc ggtttcgaca acgttcgtca agttcaatgc 3060atcagtttca ttgcgcacac accagaatcc tactgagttt gagtattatg gcattgggaa 3120aactgttttt cttgtaccat ttgttgtgct tgtaatttac tgtgtttttt attcggtttt 3180cgctatcgaa ctgtgaaatg gaaatggatg gagaagagtt aatgaatgat atggtccttt 3240tgttcattct caaattaata ttatttgttt tttctcttat ttgttgtgtg ttgaatttga 3300aattataaga gatatgcaaa cattttgttt tgagtaaaaa tgtgtcaaat cgtggcctct 3360aatgaccgaa gttaatatga ggagtaaaac acttgtagtt gtaccattat gcttattcac 3420taggcaacaa atatattttc agacctagaa aagctgcaaa tgttactgaa tacaagtatg 3480tcctcttgtg ttttagacat ttatgaactt tcctttatgt aattttccag aatccttgtc 3540agattctaat cattgcttta taattatagt tatactcatg gatttgtagt tgagtatgaa 3600aatatttttt aatgcatttt atgacttgcc aattgattga caacatgcat caatcgacct 3660gcagcccaag cttgatctag tccgatctag taacatagat gacaccgcgc gcgataattt 3720atcctagttt gcgcgctata ttttgttttc tatcgcgtat taaatgtata attgcgggac 3780tctaatcata aaaacccatc tcataaataa cgtcatgcat tacatgttaa ttattacatg 3840cttaacgtaa ttcaacagaa attatatgat aatcatcgca agaccggcaa caggattcaa 3900tcttaagaaa ctttattgcc aaatgtttga acgatcgggg aaattcgagc tcggtagcaa 3960ttcccgaggc tgtagccgac gatggtgcgc caggagagtt gttgattcat tgtttgcctc 4020cctgctgcgg tttttcaccg aagttcatgc cagtccagcg tgtcgaccga tcggtcaaac 4080atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata 4140taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt 4200atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac 4260aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat 4320cgggaagctt ggcgtaatca tggcaacttt attatacaaa gttggcatta taaaaaagca 4380ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg 4440gagctccatg gtagcgttaa cgcggccatc ccctatagtg agtcgtatta catggtcata 4500gctgtttcct ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat 4560aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt 4620gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct 4680gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat 4740cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt 4800gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt 4860ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc 4920cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt 4980gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt 5040aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt 5100gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa 5160atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt 5220gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga 5280atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct 5340tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg 5400cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac 5460tggcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa atcccttaac 5520gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 5580tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 5640gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 5700agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 5760aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 5820gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 5880gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 5940tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 6000agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 6060cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 6120gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 6180gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 6230511614DNAArtificial sequenceBinary expression vector Lo523b-pSUN1-R4- Lo484::Lo376::Lo503b 5caactttgta tagaaaagtt ggccatgatt acgccaagct tgcatgcctg caggtcccca 60gattagcctt ttcaatttca gaaagaatgc taacccacag atggttagag aggcttacgc 120agcaggtctc atcaagacga tctacccgag caataatctc caggaaatca aataccttcc 180caagaaggtt aaagatgcag tcaaaagatt caggactaac tgcatcaaga acacagagaa 240agatatattt ctcaagatca gaagtactat tccagtatgg acgattcaag gcttgcttca 300caaaccaagg caagtaatag agattggagt ctctaaaaag gtagttccca ctgaatcaaa 360ggccatggag tcaaagattc aaatagagga cctaacagaa ctcgccgtaa agactggcga 420acagttcata cagagtctct tacgactcaa tgacaagaag aaaatcttcg tcaacatggt 480ggagcacgac acacttgtct actccaaaaa tatcaaagat acagtctcag aagaccaaag 540ggcaattgag acttttcaac aaagggtaat atccggaaac ctcctcggat tccattgccc 600agctatctgt cactttattg tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca 660tcattgcgat aaaggaaagg ccatcgttga agatgcctct gccgacagtg gtcccaaaga 720tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa 780gcaagtggat tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc 840ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagaa cacgggggac 900tctagaggat ccaaggagat ataacaatga agactaatct ttttctcttt ctcatctttt 960cacttctcct atcattatcc tcggccgaat tcagtaaagg agaagaactt ttcactggag 1020ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg 1080gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg 1140gaaaactacc tgttccatgg ccaacacttg tcactacttt ctcttatggt gttcaatgct 1200tttcaagata cccagatcat atgaagcggc acgacttctt caagagcgcc atgcctgagg 1260gatacgtgca ggagaggacc atcttcttca aggacgacgg gaactacaag acacgtgctg 1320aagtcaagtt tgagggagac accctcgtca acaggatcga gcttaaggga atcgatttca 1380aggaggacgg aaacatcctc ggccacaagt tggaatacaa ctacaactcc cacaacgtat 1440acatcatggc cgacaagcaa aagaacggca tcaaagccaa cttcaagacc cgccacaaca 1500tcgaagacgg cggcgtgcaa ctcgctgatc attatcaaca aaatactcca attggcgatg 1560gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc 1620ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac 1680atggcatgga tgaactatac aaacatgatg agctttaaga gaacggatcc ccatctgcgg 1740ccgcctcgag catatgctag aggatcctct agctagagct ttcgttcgta tcatcggttt 1800cgacaacgtt cgtcaagttc aatgcatcag tttcattgcg cacacaccag aatcctactg 1860agtttgagta ttatggcatt gggaaaactg tttttcttgt accatttgtt gtgcttgtaa 1920tttactgtgt tttttattcg gttttcgcta tcgaactgtg aaatggaaat ggatggagaa 1980gagttaatga atgatatggt ccttttgttc attctcaaat taatattatt tgttttttct 2040cttatttgtt gtgtgttgaa tttgaaatta taagagatat gcaaacattt tgttttgagt 2100aaaaatgtgt caaatcgtgg cctctaatga ccgaagttaa tatgaggagt aaaacacttg 2160tagttgtacc attatgctta ttcactaggc aacaaatata ttttcagacc tagaaaagct 2220gcaaatgtta ctgaatacaa gtatgtcctc ttgtgtttta gacatttatg aactttcctt 2280tatgtaattt tccagaatcc ttgtcagatt ctaatcattg ctttataatt atagttatac 2340tcatggattt gtagttgagt atgaaaatat tttttaatgc attttatgac ttgccaattg 2400attgacaaca tgcatcaatc gaccgggtac ccaagtttgt acaaaaaagc aggctggtac 2460ccggggatcc tctaggtcga ccagatctga tatctgcggc cgcctcgagc atatgggcat 2520gcaagcttgg cgtaatcatg gacccagctt tcttgtacaa agtggggtac ccggggatcc 2580tgatagctta tactcaaatt caacaagtta tatataaatg tatagatact acaatatcat 2640taacaaaagt caccttaaat aaatacacat atcttttatg ttctctattg ttttgcgtac 2700gctaacacaa tttctcatat gcaaaaggat gaatgagtaa caaattacct cataagaaca 2760atcatctttg cttacatact aatacaataa tcactcaatc aaccaataac atcaatcaca 2820taggtttaca tacaataatc actcaatcaa cttcataaga agaatcatgt ttacttaatt 2880catcaattat ccccaaaaac accactatta agtataaact acaacatatt tgtagtgatg 2940ggtcaacatt tttatcatat ttaaactcgg gttccctcaa atcgagaaat agtgaacatg 3000taatattaat tttaaatcgc aattacagaa attaattgaa tttggtcaaa tggacagaat 3060tttatagatt gggtggaact agaaaaaaaa aaaaaaagag tatagggtga attgagtaca 3120tgaaagtaca tggtaatcct agttaaacgc ataatacatg tgggttcatt tgtatttttt 3180tgtaacttac gagtaaactg gctacaacaa aaaaaaatta gaagattttt ttgttttgta 3240gaaaacccta attttagtta tagttgtata actttgataa aattataaaa ttgtattacg 3300aaaaaagtaa taagatattc aaaaaagcct agaataacgt atatgactat gagcatgaaa 3360ctgcaagtca aatgctgaca gacaaccata aacaaaagaa attaaataga gataccttta 3420aaataagtaa aatttcattt ataaaaaatc tactttcttg tgaatctgtc acgttcaata 3480atttgaagac cactcaacat acaaggtaaa taatgaaaaa taaaatctac caaaatttca 3540atcattatta tcttccaaaa aaacaaaatt atacagatga tgatggtgat atggaacttc 3600gattggctaa tattcactgt gtctctaaaa accatccact tatcaagata agatggaccc 3660tacactcatc caatctaaac cagtatctca agattcttat ctaattacat cattctctac 3720cgttagatga aattgaccat taaccctacc ataactccat acaccgcgag atactggatt 3780aaccaaatcg agatcatcgt agccgtccga tcaacaagta ccatctcttg aaatactcga 3840aatcctcata agtccgtccc tctttgctct cactatcaaa actctgaatt tcgatttcat 3900ctagagtcga gcccgggcga tatcggatct cgactctagt cgagggccca tgggagcttg 3960gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 4020ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 4080gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 4140ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 4200cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 4260tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 4320gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 4380cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 4440gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 4500cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 4560ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 4620agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 4680cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 4740cgagttcttc tgagcgggac ccaagctagc ttcgacggat cctctagcat atgctcgagg 4800cggccgcaga tatcagatct actagtccga tctagtaaca tagatgacac cgcgcgcgat 4860aatttatcct agtttgcgcg ctatattttg ttttctatcg cgtattaaat gtataattgc 4920gggactctaa tcataaaaac ccatctcata aataacgtca tgcattacat gttaattatt 4980acatgcttaa cgtaattcaa cagaaattat atgataatca tcgcaagacc ggcaacagga 5040ttcaatctta agaaacttta ttgccaaatg tttgaacgat cggggaaatt cgagctcggt 5100agcaattccc gaggctgtag ccgacgatgg tgcgccagga gagttgttga ttcattgttt 5160gcctccctgc tgcggttttt caccgaagtt catgccagtc cagcgttttt gcagcagaaa 5220agccgccgac ttgtcgaccg atcggtcaaa catttggcaa taaagtttct taagattgaa 5280tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt 5340aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc 5400gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt 5460atcgcgcgcg gtgtcatcta tgttactaga tcgggaagct tggcgtaatc atggcaactt 5520tattatacaa agttggcatt ataaaaaagc attgcttatc aatttgttgc aacgaacagg 5580tcactatcag tcaaaataaa atcattattc aactttatta tacatagttg ataattcact 5640ggccggatct gcttggtaat aattgtcatt agattgtttt tatgcataga tgcactcgaa 5700atcagccaat tttagacaag tatcaaacgg atgttaattc agtacattaa agacgtccgc 5760aatgtgttat taagttgtct aagcgtcaat ttgtttacac cacaatatat cctgccacca 5820gccagccaac agctccccga ccggcagctc ggcacaaaat caccacgcgt taccaccacg 5880ccggccggcc gcatggtgtt gaccgtgttc gccggcattg ccgagttcga gcgttcccta 5940atcatcgacc gcacccggag cgggcgcgag gccgccaagg cccgaggcgt gaagtttggc 6000ccccgcccta ccctcacccc ggcacagatc gcgcacgccc gcgagctgat cgaccaggaa 6060ggccgcaccg tgaaagaggc ggctgcactg cttggcgtgc atcgctcgac cctgtaccgc 6120gcacttgagc gcagcgagga agtgacgccc accgaggcca ggcggcgcgg tgccttccgt 6180gaggacgcat tgaccgaggc cgacgccctg gcggccgccg agaatgaacg ccaagaggaa 6240caagcatgaa accgcaccag gacggccagg acgaaccgtt tttcattacc gaagagatcg 6300aggcggagat gatcgcggcc gggtacgtgt tcgagccgcc cgcgcacgtc tcaaccgtgc 6360ggctgcatga aatcctggcc ggtttgtctg atgccaagct ggcggcctgg ccggccagct 6420tggccgctga agaaaccgag cgccgccgtc taaaaaggtg atgtgtattt gagtaaaaca 6480gcttgcgtca tgcggtcgct gcgtatatga tgcgatgagt aaataaacaa atacgcaagg 6540ggaacgcatg aaggttatcg ctgtacttaa ccagaaaggc gggtcaggca agacgaccat 6600cgcaacccat ctagcccgcg ccctgcaact cgccggggcc gatgttctgt tagtcgattc 6660cgatccccag ggcagtgccc gcgattgggc ggccgtgcgg gaagatcaac cgctaaccgt 6720tgtcggcatc gaccgcccga cgattgaccg cgacgtgaag gccatcggcc ggcgcgactt 6780cgtagtgatc gacggagcgc cccaggcggc ggacttggct gtgtccgcga tcaaggcagc 6840cgacttcgtg ctgattccgg tgcagccaag cccttacgac atatgggcca ccgccgacct 6900ggtggagctg gttaagcagc gcattgaggt cacggatgga aggctacaag cggcctttgt 6960cgtgtcgcgg gcgatcaaag gcacgcgcat cggcggtgag gttgccgagg cgctggccgg 7020gtacgagctg cccattcttg agtcccgtat cacgcagcgc gtgagctacc caggcactgc 7080cgccgccggc acaaccgttc ttgaatcaga acccgagggc gacgctgccc gcgaggtcca 7140ggcgctggcc gctgaaatta aatcaaaact catttgagtt aatgaggtaa agagaaaatg 7200agcaaaagca caaacacgct aagtgccggc cgtccgagcg cacgcagcag caaggctgca 7260acgttggcca gcctggcaga cacgccagcc atgaagcggg tcaactttca gttgccggcg 7320gaggatcaca ccaagctgaa gatgtacgcg gtacgccaag gcaagaccat taccgagctg 7380ctatctgaat acatcgcgca gctaccagag taaatgagca aatgaataaa tgagtagatg 7440aattttagcg gctaaaggag gcggcatgga aaatcaagaa caaccaggca ccgacgccgt 7500ggaatgcccc atgtgtggag gaacgggcgg ttggccaggc gtaagcggct gggttgtctg 7560ccggccctgc aatggcactg gaacccccaa gcccgaggaa tcggcgtgag cggtcgcaaa 7620ccatccggcc

cggtacaaat cggcgcggcg ctgggtgatg acctggtgga gaagttgaag 7680gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg tgaatcgtgg 7740caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc cggtgcgccg 7800tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc gatgctctat 7860gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg tctgtcgaag 7920cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca cgtagaggtt 7980tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact gatggcggtt 8040tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa gcccggccgc 8100gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga tggcggaaag 8160cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt tgccatgcag 8220cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga agccttgatt 8280agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga gatcgagcta 8340gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct gacggttcac 8400cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct ggcacgccgc 8460gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg cagtggcagc 8520gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc aaatgacctg 8580ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt catgcgctac 8640cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca gatgctaggg 8700caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga tagcacgtac 8760attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa cccaaagccg 8820tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa aggcgatttt 8880tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc ctgtgcataa 8940ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg gtcgctgcgc 9000tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc aaaaatggct 9060ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc actcgaccgc 9120cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg aaaacctctg 9180acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 9240agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc 9300acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg 9360agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 9420aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 9480gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 9540ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 9600ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 9660cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 9720ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 9780tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 9840gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 9900tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 9960gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 10020tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 10080ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 10140agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 10200gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 10260attttggtca tgcatgatat atctcccaat ttgtgtaggg cttattatgc acgcttaaaa 10320ataataaaag cagacttgac ctgatagttt ggctgtgagc aattatgtgc ttagtgcatc 10380taacgcttga gttaagccgc gccgcgaagc ggcgtcggct tgaacgaatt tctagctaga 10440cattatttgc cgactacctt ggtgatctcg cctttcacgt agtggacaaa ttcttccaac 10500tgatctgcgc gcgaggccaa gcgatcttct tcttgtccaa gataagcctg tctagcttca 10560agtatgacgg gctgatactg ggccggcagg cgctccattg cccagtcggc agcgacatcc 10620ttcggcgcga ttttgccggt tactgcgctg taccaaatgc gggacaacgt aagcactaca 10680tttcgctcat cgccagccca gtcgggcggc gagttccata gcgttaaggt ttcatttagc 10740gcctcaaata gatcctgttc aggaaccgga tcaaagagtt cctccgccgc tggacctacc 10800aaggcaacgc tatgttctct tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg 10860gctggctcga agatacctgc aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg 10920cgcttagctg gataacgcca cggaatgatg tcgtcgtgca caacaatggt gacttctaca 10980gcgcggagaa tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa 11040gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc aatatcactg 11100tgtggcttca ggccgccatc cactgcggag ccgtacaaat gtacggccag caacgtcggt 11160tcgagatggc gctcgatgac gccaactacc tctgatagtt gagtcgatac ttcggcgatc 11220accgcttccc ccatgatgtt taactttgtt ttagggcgac tgccctgctg cgtaacatcg 11280ttgctgctcc ataacatcaa acatcgaccc acggcgtaac gcgcttgctg cttggatgcc 11340cgaggcatag actgtacccc aaaaaaacag tcataacaag ccatgaaaac cgccactgcg 11400ttccatggac atacaaatgg acgaacggat aaaccttttc acgccctttt aaatatccga 11460ttattctaat aaacgctctt ttctcttagg tttacccgcc aatatatcct gtcaaacact 11520gatagtttaa actgaaggcg ggaaacgaca atcagatcta gtaggaaaca gctatgacca 11580tgattacgcc aagctatcga ttacgccaag ctat 11614611588DNAArtificial sequenceBinary expression vector Lo523a-pSUN1-R4- Lo484::Lo376::Lo503a 6caactttgta tagaaaagtt ggccatgatt acgccaagct tgcatgcctg caggtcccca 60gattagcctt ttcaatttca gaaagaatgc taacccacag atggttagag aggcttacgc 120agcaggtctc atcaagacga tctacccgag caataatctc caggaaatca aataccttcc 180caagaaggtt aaagatgcag tcaaaagatt caggactaac tgcatcaaga acacagagaa 240agatatattt ctcaagatca gaagtactat tccagtatgg acgattcaag gcttgcttca 300caaaccaagg caagtaatag agattggagt ctctaaaaag gtagttccca ctgaatcaaa 360ggccatggag tcaaagattc aaatagagga cctaacagaa ctcgccgtaa agactggcga 420acagttcata cagagtctct tacgactcaa tgacaagaag aaaatcttcg tcaacatggt 480ggagcacgac acacttgtct actccaaaaa tatcaaagat acagtctcag aagaccaaag 540ggcaattgag acttttcaac aaagggtaat atccggaaac ctcctcggat tccattgccc 600agctatctgt cactttattg tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca 660tcattgcgat aaaggaaagg ccatcgttga agatgcctct gccgacagtg gtcccaaaga 720tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa 780gcaagtggat tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc 840ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagaa cacgggggac 900tctagaggat ccaaggagat ataacaatga agactaatct ttttctcttt ctcatctttt 960cacttctcct atcattatcc tcggccgaat tcagtaaagg agaagaactt ttcactggag 1020ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg 1080gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg 1140gaaaactacc tgttccatgg ccaacacttg tcactacttt ctcttatggt gttcaatgct 1200tttcaagata cccagatcat atgaagcggc acgacttctt caagagcgcc atgcctgagg 1260gatacgtgca ggagaggacc atcttcttca aggacgacgg gaactacaag acacgtgctg 1320aagtcaagtt tgagggagac accctcgtca acaggatcga gcttaaggga atcgatttca 1380aggaggacgg aaacatcctc ggccacaagt tggaatacaa ctacaactcc cacaacgtat 1440acatcatggc cgacaagcaa aagaacggca tcaaagccaa cttcaagacc cgccacaaca 1500tcgaagacgg cggcgtgcaa ctcgctgatc attatcaaca aaatactcca attggcgatg 1560gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc 1620ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac 1680atggcatgga tgaactatac aaacatgatg agctttaaga gaacggatcc ccatctgcgg 1740ccgcctcgag catatgctag aggatcctct agctagagct ttcgttcgta tcatcggttt 1800cgacaacgtt cgtcaagttc aatgcatcag tttcattgcg cacacaccag aatcctactg 1860agtttgagta ttatggcatt gggaaaactg tttttcttgt accatttgtt gtgcttgtaa 1920tttactgtgt tttttattcg gttttcgcta tcgaactgtg aaatggaaat ggatggagaa 1980gagttaatga atgatatggt ccttttgttc attctcaaat taatattatt tgttttttct 2040cttatttgtt gtgtgttgaa tttgaaatta taagagatat gcaaacattt tgttttgagt 2100aaaaatgtgt caaatcgtgg cctctaatga ccgaagttaa tatgaggagt aaaacacttg 2160tagttgtacc attatgctta ttcactaggc aacaaatata ttttcagacc tagaaaagct 2220gcaaatgtta ctgaatacaa gtatgtcctc ttgtgtttta gacatttatg aactttcctt 2280tatgtaattt tccagaatcc ttgtcagatt ctaatcattg ctttataatt atagttatac 2340tcatggattt gtagttgagt atgaaaatat tttttaatgc attttatgac ttgccaattg 2400attgacaaca tgcatcaatc gaccgggtac ccaagtttgt acaaaaaagc aggctggtac 2460ccggggatcc tctaggtcga ccagatctga tatctgcggc cgcctcgagc atatgggcat 2520gcaagcttgg cgtaatcatg gacccagctt tcttgtacaa agtggggtac ccggggatcc 2580tgatagctta tactcaaatt caacaagtta tatataaatg tatagatact acaatatcat 2640taacaaaagt caccttaaat aaatacacat atcttttatg ttctctattg ttttgcgtac 2700gctaacacaa tttctcatat gcaaaaggat gaatgagtaa caaattacct cataagaaca 2760atcatctttg cttacatact aatacaataa tcactcaatc aaccaataac atcaatcaca 2820taggtttaca tacaataatc actcaatcaa cttcataaga agaatcatgt ttacttaatt 2880catcaattat ccccaaaaac accactatta agtataaact acaacatatt tgtagtgatg 2940ggtcaacatt tttatcatat ttaaactcgg gttccctcaa atcgagaaat agtgaacatg 3000taatattaat tttaaatcgc aattacagaa attaattgaa tttggtcaaa tggacagaat 3060tttatagatt gggtggaact agaaaaaaaa aaaaaaagag tatagggtga attgagtaca 3120tgaaagtaca tggtaatcct agttaaacgc ataatacatg tgggttcatt tgtatttttt 3180tgtaacttac gagtaaactg gctacaacaa aaaaaaatta gaagattttt ttgttttgta 3240gaaaacccta attttagtta tagttgtata actttgataa aattataaaa ttgtattacg 3300aaaaaagtaa taagatattc aaaaaagcct agaataacgt atatgactat gagcatgaaa 3360ctgcaagtca aatgctgaca gacaaccata aacaaaagaa attaaataga gataccttta 3420aaataagtaa aatttcattt ataaaaaatc tactttcttg tgaatctgtc acgttcaata 3480atttgaagac cactcaacat acaaggtaaa taatgaaaaa taaaatctac caaaatttca 3540atcattatta tcttccaaaa aaacaaaatt atacagatga tgatggtgat atggaacttc 3600gattggctaa tattcactgt gtctctaaaa accatccact tatcaagata agatggaccc 3660tacactcatc caatctaaac cagtatctca agattcttat ctaattacat cattctctac 3720cgttagatga aattgaccat taaccctacc ataactccat acaccgcgag atactggatt 3780aaccaaatcg agatcatcgt agccgtccga tcaacaagta ccatctcttg aaatactcga 3840aatcctcata agtccgtccc tctttgctct cactatcaaa actctgaatt tcgatttcat 3900ctagagtcga gcccgggcga tatcggatct cgactctagt cgagggccca tgggagcttg 3960gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 4020ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 4080gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 4140ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 4200cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 4260tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 4320gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 4380cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 4440gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 4500cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 4560ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 4620agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 4680cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 4740cgagttcttc tgagcgggac ccaagctagc ttcgacggat cctctagcat atgctcgagg 4800cggccgcaga tatcagatct actagtccga tctagtaaca tagatgacac cgcgcgcgat 4860aatttatcct agtttgcgcg ctatattttg ttttctatcg cgtattaaat gtataattgc 4920gggactctaa tcataaaaac ccatctcata aataacgtca tgcattacat gttaattatt 4980acatgcttaa cgtaattcaa cagaaattat atgataatca tcgcaagacc ggcaacagga 5040ttcaatctta agaaacttta ttgccaaatg tttgaacgat cggggaaatt cgagctcggt 5100agcaattccc gaggctgtag ccgacgatgg tgcgccagga gagttgttga ttcattgttt 5160gcctccctgc tgcggttttt caccgaagtt catgccagtc cagcgtgtcg accgatcggt 5220caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta 5280tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt 5340tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag 5400aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac 5460tagatcggga agcttggcgt aatcatggca actttattat acaaagttgg cattataaaa 5520aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt 5580attcaacttt attatacata gttgataatt cactggccgg atctgcttgg taataattgt 5640cattagattg tttttatgca tagatgcact cgaaatcagc caattttaga caagtatcaa 5700acggatgtta attcagtaca ttaaagacgt ccgcaatgtg ttattaagtt gtctaagcgt 5760caatttgttt acaccacaat atatcctgcc accagccagc caacagctcc ccgaccggca 5820gctcggcaca aaatcaccac gcgttaccac cacgccggcc ggccgcatgg tgttgaccgt 5880gttcgccggc attgccgagt tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg 5940cgaggccgcc aaggcccgag gcgtgaagtt tggcccccgc cctaccctca ccccggcaca 6000gatcgcgcac gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag aggcggctgc 6060actgcttggc gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac 6120gcccaccgag gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc 6180cctggcggcc gccgagaatg aacgccaaga ggaacaagca tgaaaccgca ccaggacggc 6240caggacgaac cgtttttcat taccgaagag atcgaggcgg agatgatcgc ggccgggtac 6300gtgttcgagc cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct ggccggtttg 6360tctgatgcca agctggcggc ctggccggcc agcttggccg ctgaagaaac cgagcgccgc 6420cgtctaaaaa ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat 6480atgatgcgat gagtaaataa acaaatacgc aaggggaacg catgaaggtt atcgctgtac 6540ttaaccagaa aggcgggtca ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc 6600aactcgccgg ggccgatgtt ctgttagtcg attccgatcc ccagggcagt gcccgcgatt 6660gggcggccgt gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg 6720accgcgacgt gaaggccatc ggccggcgcg acttcgtagt gatcgacgga gcgccccagg 6780cggcggactt ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc 6840caagccctta cgacatatgg gccaccgccg acctggtgga gctggttaag cagcgcattg 6900aggtcacgga tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc 6960gcatcggcgg tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt cttgagtccc 7020gtatcacgca gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc gttcttgaat 7080cagaacccga gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa 7140aactcatttg agttaatgag gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc 7200cggccgtccg agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc 7260agccatgaag cgggtcaact ttcagttgcc ggcggaggat cacaccaagc tgaagatgta 7320cgcggtacgc caaggcaaga ccattaccga gctgctatct gaatacatcg cgcagctacc 7380agagtaaatg agcaaatgaa taaatgagta gatgaatttt agcggctaaa ggaggcggca 7440tggaaaatca agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg 7500gcggttggcc aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc actggaaccc 7560ccaagcccga ggaatcggcg tgagcggtcg caaaccatcc ggcccggtac aaatcggcgc 7620ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg caggccgccc agcggcaacg 7680catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg gccgctgatc gaatccgcaa 7740agaatcccgg caaccgccgg cagccggtgc gccgtcgatt aggaagccgc ccaagggcga 7800cgagcaacca gattttttcg ttccgatgct ctatgacgtg ggcacccgcg atagtcgcag 7860catcatggac gtggccgttt tccgtctgtc gaagcgtgac cgacgagctg gcgaggtgat 7920ccgctacgag cttccagacg ggcacgtaga ggtttccgca gggccggccg gcatggccag 7980tgtgtgggat tacgacctgg tactgatggc ggtttcccat ctaaccgaat ccatgaaccg 8040ataccgggaa gggaagggag acaagcccgg ccgcgtgttc cgtccacacg ttgcggacgt 8100actcaagttc tgccggcgag ccgatggcgg aaagcagaaa gacgacctgg tagaaacctg 8160cattcggtta aacaccacgc acgttgccat gcagcgtacg aagaaggcca agaacggccg 8220cctggtgacg gtatccgagg gtgaagcctt gattagccgc tacaagatcg taaagagcga 8280aaccgggcgg ccggagtaca tcgagatcga gctagctgat tggatgtacc gcgagatcac 8340agaaggcaag aacccggacg tgctgacggt tcaccccgat tactttttga tcgatcccgg 8400catcggccgt tttctctacc gcctggcacg ccgcgccgca ggcaaggcag aagccagatg 8460gttgttcaag acgatctacg aacgcagtgg cagcgccgga gagttcaaga agttctgttt 8520caccgtgcgc aagctgatcg ggtcaaatga cctgccggag tacgatttga aggaggaggc 8580ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac ctgatcgagg gcgaagcatc 8640cgccggttcc taatgtacgg agcagatgct agggcaaatt gccctagcag gggaaaaagg 8700tcgaaaaggt ctctttcctg tggatagcac gtacattggg aacccaaagc cgtacattgg 8760gaaccggaac ccgtacattg ggaacccaaa gccgtacatt gggaaccggt cacacatgta 8820agtgactgat ataaaagaga aaaaaggcga tttttccgcc taaaactctt taaaacttat 8880taaaactctt aaaacccgcc tggcctgtgc ataactgtct ggccagcgca cagccgaaga 8940gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta cgccccgccg cttcgcgtcg 9000gcctatcgcg gccgctggcc gctcaaaaat ggctggccta cggccaggca atctaccagg 9060gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc ccacatcaag gcaccctgcc 9120tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 9180cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 9240ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg 9300gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat 9360accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac 9420tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 9480aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 9540gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 9600ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 9660ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 9720gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 9780ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 9840cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 9900cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 9960gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 10020aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 10080tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 10140gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 10200tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgcatg atatatctcc 10260caatttgtgt agggcttatt atgcacgctt aaaaataata aaagcagact tgacctgata 10320gtttggctgt gagcaattat gtgcttagtg catctaacgc ttgagttaag ccgcgccgcg 10380aagcggcgtc ggcttgaacg aatttctagc tagacattat ttgccgacta ccttggtgat 10440ctcgcctttc acgtagtgga caaattcttc caactgatct gcgcgcgagg ccaagcgatc 10500ttcttcttgt ccaagataag cctgtctagc ttcaagtatg acgggctgat actgggccgg 10560caggcgctcc attgcccagt cggcagcgac atccttcggc gcgattttgc cggttactgc 10620gctgtaccaa atgcgggaca acgtaagcac tacatttcgc tcatcgccag cccagtcggg 10680cggcgagttc catagcgtta aggtttcatt tagcgcctca aatagatcct gttcaggaac 10740cggatcaaag agttcctccg ccgctggacc taccaaggca acgctatgtt ctcttgcttt 10800tgtcagcaag atagccagat caatgtcgat cgtggctggc tcgaagatac ctgcaagaat 10860gtcattgcgc tgccattctc caaattgcag ttcgcgctta gctggataac gccacggaat 10920gatgtcgtcg tgcacaacaa

tggtgacttc tacagcgcgg agaatctcgc tctctccagg 10980ggaagccgaa gtttccaaaa ggtcgttgat caaagctcgc cgcgttgttt catcaagcct 11040tacggtcacc gtaaccagca aatcaatatc actgtgtggc ttcaggccgc catccactgc 11100ggagccgtac aaatgtacgg ccagcaacgt cggttcgaga tggcgctcga tgacgccaac 11160tacctctgat agttgagtcg atacttcggc gatcaccgct tcccccatga tgtttaactt 11220tgttttaggg cgactgccct gctgcgtaac atcgttgctg ctccataaca tcaaacatcg 11280acccacggcg taacgcgctt gctgcttgga tgcccgaggc atagactgta ccccaaaaaa 11340acagtcataa caagccatga aaaccgccac tgcgttccat ggacatacaa atggacgaac 11400ggataaacct tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct 11460taggtttacc cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac 11520gacaatcaga tctagtagga aacagctatg accatgatta cgccaagcta tcgattacgc 11580caagctat 1158875583DNAArtificial sequenceNucleic acid construct Lo503b-pENTR-C1- STPT-nptII-IRnos 7ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac 600taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg 660tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt 720ctcgttcaac tttcttgtac aaagtggggt acccggggat cctgatagct tatactcaaa 780ttcaacaagt tatatataaa tgtatagata ctacaatatc attaacaaaa gtcaccttaa 840ataaatacac atatctttta tgttctctat tgttttgcgt acgctaacac aatttctcat 900atgcaaaagg atgaatgagt aacaaattac ctcataagaa caatcatctt tgcttacata 960ctaatacaat aatcactcaa tcaaccaata acatcaatca cataggttta catacaataa 1020tcactcaatc aacttcataa gaagaatcat gtttacttaa ttcatcaatt atccccaaaa 1080acaccactat taagtataaa ctacaacata tttgtagtga tgggtcaaca tttttatcat 1140atttaaactc gggttccctc aaatcgagaa atagtgaaca tgtaatatta attttaaatc 1200gcaattacag aaattaattg aatttggtca aatggacaga attttataga ttgggtggaa 1260ctagaaaaaa aaaaaaaaag agtatagggt gaattgagta catgaaagta catggtaatc 1320ctagttaaac gcataataca tgtgggttca tttgtatttt tttgtaactt acgagtaaac 1380tggctacaac aaaaaaaaat tagaagattt ttttgttttg tagaaaaccc taattttagt 1440tatagttgta taactttgat aaaattataa aattgtatta cgaaaaaagt aataagatat 1500tcaaaaaagc ctagaataac gtatatgact atgagcatga aactgcaagt caaatgctga 1560cagacaacca taaacaaaag aaattaaata gagatacctt taaaataagt aaaatttcat 1620ttataaaaaa tctactttct tgtgaatctg tcacgttcaa taatttgaag accactcaac 1680atacaaggta aataatgaaa aataaaatct accaaaattt caatcattat tatcttccaa 1740aaaaacaaaa ttatacagat gatgatggtg atatggaact tcgattggct aatattcact 1800gtgtctctaa aaaccatcca cttatcaaga taagatggac cctacactca tccaatctaa 1860accagtatct caagattctt atctaattac atcattctct accgttagat gaaattgacc 1920attaacccta ccataactcc atacaccgcg agatactgga ttaaccaaat cgagatcatc 1980gtagccgtcc gatcaacaag taccatctct tgaaatactc gaaatcctca taagtccgtc 2040cctctttgct ctcactatca aaactctgaa tttcgatttc atctagagtc gagcccgggc 2100gatatcggat ctcgactcta gtcgagggcc catgggagct tggattgaac aagatggatt 2160gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca 2220gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 2280ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct 2340atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 2400gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 2460tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 2520tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg 2580gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc 2640agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac 2700ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat 2760cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 2820tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 2880cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg 2940acccaagcta gcttcgacgg atcctctagc atatgctcga ggcggccgca gatatcagat 3000ctactagtcc gatctagtaa catagatgac accgcgcgcg ataatttatc ctagtttgcg 3060cgctatattt tgttttctat cgcgtattaa atgtataatt gcgggactct aatcataaaa 3120acccatctca taaataacgt catgcattac atgttaatta ttacatgctt aacgtaattc 3180aacagaaatt atatgataat catcgcaaga ccggcaacag gattcaatct taagaaactt 3240tattgccaaa tgtttgaacg atcggggaaa ttcgagctcg gtagcaattc ccgaggctgt 3300agccgacgat ggtgcgccag gagagttgtt gattcattgt ttgcctccct gctgcggttt 3360ttcaccgaag ttcatgccag tccagcgttt ttgcagcaga aaagccgccg acttgtcgac 3420cgatcggtca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 3480gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 3540catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 3600cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 3660tatgttacta gatcgggaag cttggcgtaa tcatggcaac tttattatac aaagttggca 3720ttataaaaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata 3780aaatcattat ttggagctcc atggtagcgt taacgcggcc atcccctata gtgagtcgta 3840ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta 3900cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag 3960taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc 4020caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg 4080tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg 4140caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga 4200atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact 4260caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg 4320tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg 4380taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa 4440taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca 4500agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg 4560tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt 4620tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg 4680tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga 4740tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa 4800ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc 4860aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat 4920caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 4980accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 5040ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt 5100aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 5160accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 5220gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 5280ggagcgaacg acctacaccg aactgagata cctacagcgt gagcattgag aaagcgccac 5340gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 5400gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 5460ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 5520aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 5580gtt 558385557DNAArtificial sequenceNucleic acid construct Lo503a-pENTR-C1-STPT- nptII-IRnos 8ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac 600taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg 660tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt 720ctcgttcaac tttcttgtac aaagtggggt acccggggat cctgatagct tatactcaaa 780ttcaacaagt tatatataaa tgtatagata ctacaatatc attaacaaaa gtcaccttaa 840ataaatacac atatctttta tgttctctat tgttttgcgt acgctaacac aatttctcat 900atgcaaaagg atgaatgagt aacaaattac ctcataagaa caatcatctt tgcttacata 960ctaatacaat aatcactcaa tcaaccaata acatcaatca cataggttta catacaataa 1020tcactcaatc aacttcataa gaagaatcat gtttacttaa ttcatcaatt atccccaaaa 1080acaccactat taagtataaa ctacaacata tttgtagtga tgggtcaaca tttttatcat 1140atttaaactc gggttccctc aaatcgagaa atagtgaaca tgtaatatta attttaaatc 1200gcaattacag aaattaattg aatttggtca aatggacaga attttataga ttgggtggaa 1260ctagaaaaaa aaaaaaaaag agtatagggt gaattgagta catgaaagta catggtaatc 1320ctagttaaac gcataataca tgtgggttca tttgtatttt tttgtaactt acgagtaaac 1380tggctacaac aaaaaaaaat tagaagattt ttttgttttg tagaaaaccc taattttagt 1440tatagttgta taactttgat aaaattataa aattgtatta cgaaaaaagt aataagatat 1500tcaaaaaagc ctagaataac gtatatgact atgagcatga aactgcaagt caaatgctga 1560cagacaacca taaacaaaag aaattaaata gagatacctt taaaataagt aaaatttcat 1620ttataaaaaa tctactttct tgtgaatctg tcacgttcaa taatttgaag accactcaac 1680atacaaggta aataatgaaa aataaaatct accaaaattt caatcattat tatcttccaa 1740aaaaacaaaa ttatacagat gatgatggtg atatggaact tcgattggct aatattcact 1800gtgtctctaa aaaccatcca cttatcaaga taagatggac cctacactca tccaatctaa 1860accagtatct caagattctt atctaattac atcattctct accgttagat gaaattgacc 1920attaacccta ccataactcc atacaccgcg agatactgga ttaaccaaat cgagatcatc 1980gtagccgtcc gatcaacaag taccatctct tgaaatactc gaaatcctca taagtccgtc 2040cctctttgct ctcactatca aaactctgaa tttcgatttc atctagagtc gagcccgggc 2100gatatcggat ctcgactcta gtcgagggcc catgggagct tggattgaac aagatggatt 2160gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca 2220gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 2280ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct 2340atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 2400gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 2460tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 2520tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg 2580gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc 2640agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac 2700ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat 2760cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 2820tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 2880cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg 2940acccaagcta gcttcgacgg atcctctagc atatgctcga ggcggccgca gatatcagat 3000ctactagtcc gatctagtaa catagatgac accgcgcgcg ataatttatc ctagtttgcg 3060cgctatattt tgttttctat cgcgtattaa atgtataatt gcgggactct aatcataaaa 3120acccatctca taaataacgt catgcattac atgttaatta ttacatgctt aacgtaattc 3180aacagaaatt atatgataat catcgcaaga ccggcaacag gattcaatct taagaaactt 3240tattgccaaa tgtttgaacg atcggggaaa ttcgagctcg gtagcaattc ccgaggctgt 3300agccgacgat ggtgcgccag gagagttgtt gattcattgt ttgcctccct gctgcggttt 3360ttcaccgaag ttcatgccag tccagcgtgt cgaccgatcg gtcaaacatt tggcaataaa 3420gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga 3480attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt 3540ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg 3600caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg gaagcttggc 3660gtaatcatgg caactttatt atacaaagtt ggcattataa aaaagcattg cttatcaatt 3720tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttggag ctccatggta 3780gcgttaacgc ggccatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc 3840agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat 3900catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata 3960ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt 4020ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga 4080agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta 4140cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc 4200attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag 4260cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag 4320tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg 4380tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt 4440ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt 4500tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt 4560ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat 4620accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac 4680ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga 4740tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta 4800cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt 4860cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 4920ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 4980tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 5040taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 5100caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 5160agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 5220gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 5280gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 5340ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 5400acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 5460tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 5520ggttcctggc cttttgctgg ccttttgctc acatgtt 555795047DNAArtificial sequenceNucleic acid construct Lo484-pENTR-A1-inv- 35s-GFP-E9 9ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggcccga gttaacgcta ccatggagct ccaaataatg 600attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgataagca atgctttttt 660ataatgccaa ctttgtatag aaaagttgcc atgattacgc caagcttgca tgcctgcagg 720tccccagatt agccttttca atttcagaaa gaatgctaac ccacagatgg ttagagaggc 780ttacgcagca ggtctcatca agacgatcta cccgagcaat aatctccagg aaatcaaata 840ccttcccaag aaggttaaag atgcagtcaa aagattcagg actaactgca tcaagaacac 900agagaaagat atatttctca agatcagaag tactattcca gtatggacga ttcaaggctt 960gcttcacaaa ccaaggcaag taatagagat tggagtctct aaaaaggtag ttcccactga 1020atcaaaggcc atggagtcaa agattcaaat agaggaccta acagaactcg ccgtaaagac 1080tggcgaacag ttcatacaga gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa 1140catggtggag cacgacacac ttgtctactc caaaaatatc aaagatacag tctcagaaga 1200ccaaagggca attgagactt ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca 1260ttgcccagct atctgtcact ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa 1320atgccatcat tgcgataaag gaaaggccat cgttgaagat gcctctgccg acagtggtcc 1380caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc 1440ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca 1500ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg agagaacacg 1560ggggactcta gaggatccaa ggagatataa caatgaagac taatcttttt ctctttctca 1620tcttttcact tctcctatca ttatcctcgg ccgaattcag taaaggagaa gaacttttca 1680ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg 1740tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca 1800ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttctct tatggtgttc 1860aatgcttttc aagataccca gatcatatga agcggcacga cttcttcaag agcgccatgc 1920ctgagggata cgtgcaggag aggaccatct tcttcaagga cgacgggaac tacaagacac 1980gtgctgaagt caagtttgag ggagacaccc tcgtcaacag gatcgagctt aagggaatcg 2040atttcaagga ggacggaaac atcctcggcc acaagttgga atacaactac aactcccaca 2100acgtatacat catggccgac aagcaaaaga acggcatcaa agccaacttc aagacccgcc 2160acaacatcga agacggcggc gtgcaactcg ctgatcatta tcaacaaaat actccaattg 2220gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct gccctttcga 2280aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga 2340ttacacatgg catggatgaa ctatacaaac atgatgagct ttaagagaac ggatccccat 2400ctgcggccgc ctcgagcata tgctagagga tcctctagct agagctttcg ttcgtatcat 2460cggtttcgac aacgttcgtc aagttcaatg catcagtttc attgcgcaca caccagaatc 2520ctactgagtt tgagtattat ggcattggga aaactgtttt tcttgtacca tttgttgtgc 2580ttgtaattta ctgtgttttt tattcggttt tcgctatcga actgtgaaat ggaaatggat 2640ggagaagagt taatgaatga tatggtcctt ttgttcattc tcaaattaat attatttgtt 2700ttttctctta tttgttgtgt gttgaatttg aaattataag agatatgcaa acattttgtt 2760ttgagtaaaa atgtgtcaaa tcgtggcctc taatgaccga agttaatatg aggagtaaaa 2820cacttgtagt tgtaccatta tgcttattca ctaggcaaca aatatatttt cagacctaga 2880aaagctgcaa atgttactga

atacaagtat gtcctcttgt gttttagaca tttatgaact 2940ttcctttatg taattttcca gaatccttgt cagattctaa tcattgcttt ataattatag 3000ttatactcat ggatttgtag ttgagtatga aaatattttt taatgcattt tatgacttgc 3060caattgattg acaacatgca tcaatcgacc gggtacccaa gtttgtacaa aaaagttgaa 3120cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag 3180actacataat actgtaaaac acaacatatg cagtcactat gaaccaacta cttagatggt 3240attagtgacc tgtagaattc gagctctaga gctgcagggc ggccatcccc tatagtgagt 3300cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat 3360gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa 3420acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa 3480attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat 3540caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac 3600atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga 3660cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt 3720tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt 3780caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg 3840tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa 3900tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg 3960aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc 4020atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg 4080atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc 4140tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc 4200ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg 4260ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat 4320gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa 4380agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 4440aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 4500cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 4560agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 4620tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 4680gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 4740gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcat tgagaaagcg 4800ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 4860gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 4920ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 4980ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 5040acatgtt 5047101313DNAOryza sativa 10cgtccgatgt gattccgtcc tcttgattgt tttccttccc cctaattttt gttgcatacg 60cggagagaag ctttagcttt aagaggttaa ctggcggcac ctcaatttct ttgggcttca 120gcatgggtta agaccaccta ataatcctgt cgcattcttg ctcttggaaa agtttcttcc 180aacattgcag tccagtcaga gaatggcctg taataattag tgacaaaatt atagcagtag 240atagaatctg ttcattcaag ttcttcaaat tactgggcca agcttcaatg tcattttttg 300cttaactcgt gctcgtgctg ttatagttta caactatctt ctttacctgc ccataaccaa 360tgtgaccatc gaatcaaacg tatgtggcgg tgaatcccgt tgcttgtgcg ttgctagatt 420tttgaggtcg ccttctgttc caaattccaa tcatttgaat tcgaagggat tacctgtcga 480ctgcgcatac gatcttttat tgagaatatt tgctaggtgt gggagttcct gcgtgtgttc 540gtttagtgga cgtgcgtatg tgagcatgtg ctatatgttg tgcttcgtga aaactcagct 600tcttttcatc aagatcggca cctctgctta tcacctaatc ccatcattcc aatagcaaca 660taacatagat gtgacacatc ctatacatga tattgggatg gagagagtat tcctaagatg 720catctctacc ctttctcttc gaatgcacat ttattgggag tcatttttaa tgtgcatttg 780tgcaaactgg taccatggaa ccctagtgct accctaacag atatgctcca tcgtcactac 840aaactcttgt ttacatccac aaactactgc ttggaagggc acgctgacat ttgatcagaa 900gaaagaatga catgggtgcc aaagttatgg atcagtgtgg tgaaattcga gaataaccga 960caagatactt ctcaatcttg tcacaataag agggttattt ttgtttagta cagtacaact 1020aacgataccg cacaaaaaac ggtgaacgcg atatcgcaca aaaaaaagag caacacgttt 1080gaattatatt caggtttgct actgcaatcc aacaccatct cataactaca ttatcctaat 1140cgagcctttt taagtaatga ggccagtaca attaaaaaag gtgccgtaga caatagcaaa 1200cccatgaaga actgaaaaca aatgaatgca aaactaaaat tgtcttagtt ctgtcgacaa 1260atgccaactg ctataatcgt acatgcgtct ccgaagaaac cgccgacact gcc 1313111498DNAOryza sativa 11cagagtgaca gacagtgata cttgctagcc ttttttatga gcaccctttt tggcctttta 60gttcctcatt agctaagaga tgtgatgagt gaggttttat tttttggctt tgtttggagt 120tttgctttgg aggaagatag agaagccttt agaatgagtt gtgatggagg atcacatagt 180ttgtctcagg tttacaatgt gcggtaaaaa aaaggaagga aataaagtga tgttagaata 240tacagttttt taatccattt ctacagcttc aaattctcga ttgagcggca cgttctttga 300aatgttcata gcaaagcaaa tcaaacgaaa tggatgagat ttgaaactaa gaaactgcac 360ttgacaaagc acgctgcgct ctctgaaatt catgttcagc agaacgcctt tcagaaattc 420atgttctgct gctgcaggaa ggcagctaag aactccatgc aataaaaata taaaatgcag 480tgcagttatt agttagtgta gtagtaatct aagaaacaac aggtttaaca gaatattacc 540ttcaaaaaca tcccctcaaa tcaagactcg gaaatcaaca ccattctgaa actagatgca 600atactgcgct cctaatggaa tcttcagtgg ttactcctac acgccactaa aaaaaacggg 660ctttggaaaa acgtcaccct gaaacaaaag ctcagtaaaa ataccactct aaaatcttgc 720agcatgcaat atcacaaaag cagatcaatc cgtaattgta tttatgctgc atataccgtg 780atattttctg tgatcctctg cactatcttc agctcatcag ctgcatattt tgaagccgga 840ctgatggctc atcgctttgt ttcggcttat tgtgatagcc atccatgcgt tgacgaaaga 900tttagatata tcacaatttt aaccaagttc agagcaatgt tagctttagc tgatgatata 960cacggcgcac tgattgccgc cgttattttc agagccttgt aaaacctgaa acaaattgtg 1020tctccaaatt cttactagta ctgcagacat agcggatttg ttttcaggat gaattatcac 1080aaaggtaagt caattcatag ccatattacg caagttcaaa actgcaggcc acaccatacg 1140tttctgaaag tagaaacaag agatcttgca gcagatcacc actaaagaag cagtaaccgc 1200aaaaaattat aacataatcc agaattaaca cttcgcagca ttactgaatt cactttaata 1260gcacttctca tcatgaacta gtacaacaac ataactgtcc agtggaaatg tgaaatgcat 1320acaccaagta atggtccata acatgaacta tatggataca acaacgttct tatttcgctc 1380atatatacat gaaataagtc ttgcacgtct tggttactta ataggtgcga taatcgccgt 1440aggcttttag aagaagaaaa aaaagtgagc ctgcaaagtt cctggggaca gttgaaga 1498121246DNAOryza sativa 12cccttgacag ctccagtttt actcttgtgc agtgtactga aattctggta ctgtagtaca 60gtcttccact ggtttggttt gtcgctgagg cagcagaagg cttatggtta cacgtacacg 120gtgacggtag gtcggtagct ggcttttggg tagaggaatc tgtatgtctg agtgaaataa 180tactggattc gccatttggt gcgtttcctt ttttgtttcc ttttggctgg cctgctttct 240tgcttcgtaa cttgaccaaa aagttctggc cgtcgcaaag agctggaact gtgaatacat 300tttctactgc ataaaacgtt acacgcatca tgcatcacct acacggttac acccgataga 360attcggttac agttcaatta caaggcactc ccgagtccca gaagctagca cgtcgagcag 420acaaacccta cagataaaat ttctcctcgc catgctttta agctttacgc taaccacatg 480cttataataa taacgagcat tgccgcgtca tttccagatt gaagtggcag tgaagggcaa 540caaaatgatc atcaccttgc tgaaattagt gggcagcagc aaccatagcc gtgcgagcac 600ccgtgaaagt tgatatcagc gcccccagct gtaacttgtc attgcaagca aagcttagcc 660ggtacctgca cacagacgcc atgaacaacg tgccatctta agggaaaagg ccataaatgg 720ttatactacc tacctttcca agaagactaa aggctaaaaa tatttggaat atttcataat 780attaatcagt tattatactt ccataagtat tccagatagc caaaggtcaa ctttaggtaa 840ttgcatcatg aaagcaaccc aaagactcat tttcccccca aatgcaaaca ttccacaagg 900cacaaataag caattccaac atgtgaggtt tgcttaacaa ctattgcttt gcagtatgca 960accatttaac tgtaatggct aaattgtgaa acacatacaa taaagtatgt tagctggttt 1020aaattattta ttattatttt tcttacgtaa gatactccat actagttatt tgatgtgatg 1080acataaatga ctatcttgaa gcatataata tgaataaatc agcaaaatgt tccgtcaaag 1140ttttgaaaaa aaagggtact gagtagaaaa tcagccaaaa atcacaaatg atttatgatc 1200aaataaaata ttatatgcaa gttctaggtg aactaagcaa ggcaca 124613771DNAOryza sativa 13cgtggcaatg gctgtcatgc tttggcacat actaatagca aggtagaatg gtacagctat 60ttcattattt ttgcccttgt atatttgtat cactacatga gtaaacgacg tttagttatc 120gatagttttg ttatgagtga tgaatgatct gcatcgtact gccaatgcct tgcattctca 180aatggttgca cacttgcact catacaaagt tagtacactc catcatatta taaattactt 240ttttttcaag ttaaatttcc tgaaagtttg ataaaattta tagaaaaaat ataacgacgc 300ttataacact aaattaattt cattacatct aacattaaac atattttgat ttttttttgt 360tttgtgttaa atatattact atgtttttct ataaacttga ttaaacatat aaaagtttaa 420cttcaaaaaa aagttaaaat gacttgtaat ataaaacgta ggcagtacaa tgcgaatgta 480gggtactcca tccagctgag gtaaaccaac tccaatatat atacaaacac aaacaacgta 540cccaattttt actgttaaaa tacaggcaca atgcctggta tcacacgtta ttaagtagac 600agactcgata accatgacac ggacagggac ttcttgccac tggtttacgc acggttaata 660ttacagacca cacatagaga gacggcttag ctatttgcaa ataagcttga caagatagat 720gatgctccaa aaggatgcga tctcagcagt tgagtactta cgctggttca t 77114755DNAOryza sativa 14ttctgagcac ctgacaaaat attcagtcct ggattggacg tatatattgt tggtaaactc 60ctgagttgct gatgagggaa aagtattgaa atgaaatctt gaggttttac ataggcgatt 120gtgttcatca cataaacgcc ttccttttcc ttgcccggaa tgcaagaacg agagatacct 180tgaaaatgtt ttatcctgtc atgaatgtac ctcaatcgca agatgtacag tgtggttgga 240ccggctggtt ggttaattat attggtcctg acatgttcct atcctaatta atctcctgtt 300tgtggggaaa acgaagaata agtatgtcat tttcatctag ctgggttgcc tgcatctgca 360tgctagctag ctactgtaaa tgatgcgtac gtatgctcat gaatgaattc gcaagcgatc 420gccgcaccat acatcctttg ttcttgccat taatcaagca agataagcta cctagtagtg 480ctataacttg cgttgattaa ttgcatcggt cgattacaat acaagctcgc tatacatact 540cgtccgctga ttgatcagtt tctgcacttc tgcagtgata aacacaaaca ctacctcgat 600cgatcgatca gttgtgcagt gggtttgggc cctccggcac cacccgcttg gacgcagaca 660caccatgatc ctcctcctcc tcctgcatca tgcgcctgct gctgtagtaa tggccgggcg 720gcgccgccat ctcctccgtc aaaaggccgc agccc 755151373DNAOryza sativa 15tgctctgatg tatgccttca aattcatcct tctgtgcaga aaatagctgt agagaaatgg 60aaactgggct gtttttatcc gtgcccccta atatgagaaa ctatgcctcc ctcagatttt 120tttggctaga ctaacagaaa tatgagaata gcccatatca ttgacattag atgcagagca 180tgggaaatgc caatgtggat tggcttgtgt gtaacaactg acaaacatgt gctgtgcaat 240tgtgaatact ggagttgtac aagtggtata ctgtaatact gttatagcat agcatcagaa 300tacaaagaca gaaatgaaca catagcatcg atgtctcttt tctttatttt tttctttgga 360cagcatgcct tgaattattt gccataatgt ctccgtcccg ctggcttcat cacaccttct 420ttgctgggtt tagtttcagt tcatcgtttg gattagctgt ttatgaatag agatgctgtt 480tctgtctatg ttcactctca gggatgaaca tagggcctgc actgtctatg ttcaaactca 540gagaggaaca ttgggcctgc actgcagcgg tgaaatggat ccgtgtgttc gcgccaccag 600aatgagcttg gggcgcaatg gaacatgtga tgtcagttaa agttgcaggt aaggcagtca 660ccggtcaaat cacggcactc gccaaccagg ccggagcata tcggctctgc cgtttgtttg 720ctcctatctg tacgtagcac gagctgtttt ccattggcaa cgcctgatcg atcaaggcgt 780tctactcccc tcagcacatg acgccaacgg gttgcatctg ctgacgacaa atcgctacgc 840agactccaga ttttacagct gtgttctgca tcaaccatgg ggaattggcg acccaaagtg 900ccaaaccatc acacgcaagt tgccatgatc gatgcgtgaa ccggaaattg atcctctaac 960ttgtccccat aatttatgag cctacgtatg agtaacccat agcatagtat cagagaacag 1020taagtcagat aagccttttc gatctgcgac catcaaaatc gaacaaacgg cagcacattt 1080tagccgaaac gaatcagaga tctgccccag atttgcaacc tacacacttg acaatctcaa 1140cttggctgcc tgcaatctgc cattcccctt ccactgcaac atggagatag ccccacccac 1200caacgcagcc accctacgga aaagcacacc gtaacgccgc ccaccaaccc ccctccgaat 1260ctcctccgat cccgacgcct atagatgaat aaatctcctc cctcttctca ctagacaaag 1320cccaaaccaa ggccaaaacc ctcctcccct cctctcctct ccattccgcc gcc 1373161208DNAOryza sativa 16tgtaccatat ctccagtgag gcttgagggc gaaaattcgg aagccctctg ttttacaacc 60tgcttgtatg ccatgttcca gttctgagga taccgggacc tatcaatttc gtgtcctctg 120ctgtgctaga ctcgatcaca ttggtgaacc atgaatatca tgtccctttc aaagaaatcg 180ttgtgcaagt actgatatac cgtactctat tttgttgatt gctttttgag cgtgtgttta 240tctgttcagt cgtcttgctc tctttttttt ttttttgggg gggggggggg tgggggtgga 300tttttcataa aattggtatg cggcactaac gatcatgata gcgtcgaaag aacacgattc 360ggtttattag ccccgaaaat tatctccatt tttcttttta acattttggc ggctgtcatg 420caaaattgcg caggaaatta gactagttag tttaatatca aaattggcca catttgagta 480ttactttttg ttccccgact gcatggtaaa aagaaagata taaatataca gaaatacttc 540tgaaaatcta ctgacatgat tttcattttt tttccatacg ttaacacacg aagtttaaga 600acctctctta actgctagcg ttccaaagca agaaaaggac aaaccgcatt ttaaaacaaa 660ctccatgtta ttttcaagga ccaattaatt tgcatatgtg agtgcattcg atgatcatgt 720ttacgctacc tccaattttg ttgaaatgtg tgatagtagc ttaaggaggt agcaggaccc 780aaagtgggtg tgcgtatcca caagtgcgtg taattgtgtt caccttgtac taggggaaaa 840aaaaagaaga gcaagacggt atggggtaaa ctgagaagaa aaaacggaga aaacctctat 900ttcttaaaat ccctttctca actcatttta ctcacgtcca tgctcaccac aaagaaatga 960attcgcagtc aaacatcact aaaataggta atgctccgca agttcatcca ctacacctac 1020ggaaaaaaaa aatgtggtgc ccaaatgccc aataatacaa actgttcccc tcccgtttct 1080ttggctgtcc tgttccatgc tctttgccga gccagaacaa aacacaattg agcaagccaa 1140ccatggaaag ctcagcacat taaggcaggt caagatggaa cctgcacctc atgtacctac 1200gctgtaag 1208171494DNAOryza sativa 17aggtttctag tctactactg ctagtacatt gcccgtggta ctcttgtttt gcatctaggc 60tagggccagt gtgaaccagc agactaccac tgtgagtcgc ctctgtaata aaatttgttc 120ggcctatggc catcctcagt acgttgtcaa tgtctcggtg ggctgttatg ctcaactgca 180atgctgcatc cactgaaacc tctttctaga tgtgttggta tgaaatgcgg tattgcgttc 240ccgatttccc ctgctactgt cttgaacctg ccgtggtaca atccttgttt gctgcacttc 300atttttagtt cgttctaatt ccgcctggaa tgacttttga gtgcttcacg ggctgactgc 360ctgaccgtgt tgctgtacat gcctacccca tcagttcaaa aaaaaacaat cctacctacg 420aaattcgttt tttattggga caggacatcg gctgagttta atttcaaaat ttttcaacaa 480actttcaact tttccatcac atcaaaactt tccctataca cgtaaacttt caacttttct 540gtcatatcgt ttcaatttca accaaacttt taattttgat gtgaactaaa cccacccatc 600attggatgga cttggcaaaa catggcttga ccattgagat cgttttgggg attttgttca 660gtcagctctt ccatggtgtc tatcagaaga gttggggccg tacaacgcaa attatttccc 720agatggtgtt gatatcatca atgatcatgg caatttggca aaccatttct tatttcccag 780atggggttgg tataataatt aatcatggca accccatttg tttggtgcta acttgatgaa 840acatgaatgc cctcaacctc aacagatggg ctccagtccg gccggcgccg gccagcactc 900ggtcaacaca atatgggcac gagtggggct caaggaagtg tggggcgcag cacaaggagc 960caagagacaa ggagtcatgt tggtcctttc agtttcaggc aaaaaaggaa ccgaaaggag 1020ccaaggatcg gaccctttcg aagctttttt gaccatttcg tccgacttgc aaggcaggcc 1080cagaaacaaa aataacccaa ctgtctagtg gtaatatcat ttgggatctg ttaggatttt 1140cagttcgaag aaactgtcct gattcagagt tcagacaaaa tacgccttca gtagttggtc 1200ctgccgcccc tcgtagtttc actatcgaaa gggatcgctg ctgtcgtaat acgtttggtt 1260ttggcaatac agaaatcaat ctgctcgcaa agtataatat cataatccaa agttccaaac 1320ccttttattc gaacatagta ccatatatca tacaaagcag ccacaacttg cagagaatgt 1380gggtttcgaa catcaacaaa caaacaaatg caatgttcag tctacccggc tctcaatctt 1440gatcatatgt acaaagcagg tatagacggg atctcatttc catggtgatt caat 1494181274DNAOryza sativa 18gatgcattcc ttggattgtt cccaatgtat tccagaaatc atagttttga tgccaaagtt 60ggtcttcggt atttgttact tggagatggc aaatcgactt gagcaatgtt aaagttttgg 120gcatttaaat tatagacctt tgcttggcac ggttagcttg tttcaaatcc gttgtttgtt 180gtggaatgtg tttcacatat gtggtaggtg aagaatctca ttatggttcg ctgtttcatt 240ctcttgcgtt tatcacccgc tgtctgctaa cttagggtgt gtttagtcct cgtcaaaatt 300ggaagtttgg ttgaaattgg aacaatgtga cgaaaaagtt gaaatttttt ttgtgggtag 360gaaagttttg atgtgataga aaagttggaa gtttaaaaaa aaagtttaga actaaactcg 420gccctagtca atttaccctt actattaggc attatccccg ctgtctgcta acctagtcaa 480tttacccttg tattattact aggcattatc cttgcctccg ttagtggttt gttcttgaga 540gcggccaggt aggaaaattc cacttgaaga ggagtgcgtt acgccggcct cttgccatta 600tcatcgtttg tattgatatg cagaaataga aagaaaaacc taggattttg atgaaaataa 660atcggatata gatatcataa aacacattgg aacttgagat ggagagaact ctagatattt 720tccatgagat tgttacctaa ttcctacttt cctacaaaat cttatgaaac tgccattcga 780aaggaatatc gtaggattct cgagtcccaa tccgttccag aaaggtaaat cggaacaatg 840gtacgaaaaa tctgtcccca ttggatcata agacttggac ggtctatccc atggtactaa 900gggttttgga aagaattttg cagaatttga atggatgtat cctatacaca agttttctgt 960gtacacatcg tacacatcaa ctaacaattt ttaccaaaaa tctaagaaca aattcacatg 1020ttctatcatg agttatcaca cattcacacg ttcatatctt ggttaaatct gaacagcagg 1080ctgtaaataa accacaaaag tctccaaatt atgggcctca acagtaatca atatcgtttc 1140ctcagacggc aattgccagt ttgccacaac agccatgttg gctaacattt gatacattcg 1200gagtactgct tgaatttgca ggctctccct aaccttatta tccctatagt ctttgactct 1260tgatacctcc aaac 127419806DNAOryza sativa 19aaaacgcaga gatgtgaatt tttaacacaa gaactatata ttagtcttgc aatcatctcc 60ttagcgacct agagtctctt ttcgtaattg atctgtattt aatagtagca gtctctaaaa 120tggacaccac tttgagccat cataggagtg tcttctccat tgaattgtga ggaaactggg 180caaagcttct tcatggcagc tgtaacagag agtttaagca aaagggagca ggagtttgca 240tttgccgaac tcgttgtcac tgcccctgag agcaatattc tgtcatcttt gtgaagatct 300acatcttctt gtgaccttac aatttgttaa atcaatgcaa gaatatggtt atctgcttac 360atgatcatca atagttttga gatttttgaa ttccctagaa aacaaattgt cagtgatctc 420catgattagt tgtataacat gtagatcagg cagtaacaat atggattgag caagctggat 480acttcagttt atttttacag tcacgaacaa ttatgcaagt tccaaagtac aaggcaaatg 540gaacatgcca ccaaaccgct atgagtaaaa gtatgaaaca cagaatggct ggatagcttt 600gcaatggttg atgcaaatat ggcgattcac ataacagaac agtgtgcaat gcacaaagtt 660ttttaacaaa atggagctac taaaccataa ctactacttt actttagtgg cagtgatcca 720ctctccagag tttacaccaa aaaaatctta agacctggca gcgtcacaaa caagtcaagc 780atagcgttct aaaactctca ggtatt 806201435DNAOryza sativa 20ctgaactaca gtctcagcga gttttattag caaggagaag ctgtaattag tggccactgt 60tgttcaatgt aaaaagaaag aagcgacacg gtatgatgta caagaaagca cagatgtgtg 120atagaatggt attagggaat gatgtgtgct gcatttggtg aaaagaatac acaactgtag 180gcctcataca aatgtggttg ttaatttgtg acagaactac tctgaattta atagagatca 240acctatacaa ataccatcaa agaaagtgtc ggcacgtgac caaaagttcc aaaaacacgg 300gataactcct gattaaacaa ggagaaaaaa aaattatttt ctgatttatc tatatagcct 360tcagttcaga gtttgatact tatactggaa aagtggaaat ggacccagaa cacgaatcaa 420ccggaattta agttcagttc cagcacaatt ccaggcatcc gcccgtttgt ctgatgtcta 480cgtactgcaa aaatgtcgct catgtctgct agacgactac caaattaggc caagaaaaaa 540aaaaccaagc attaagcctt gaacattcac aggacaaagt ctgcaaatgg tccaagcaaa 600ctgatgcaac catgcaaatt tcagtcacag ttacactgcg gaaagaaggc atacatgtca 660aggcaacatg gcatgccatc atcacgccag ttcactgata

cagcatccag tcataatgga 720gttgcacaga ccatattact acaagggcaa agtcaacttg cagcacattg ctgttcgttt 780tgagtagcct cagtcacagc ttagccgcct ccaacctggc gtttccagga gtcctccaca 840ataagctcgt caacgcccca gtcctcatag ctatatttga acacacagta aactattata 900atgaggttac aaaatcagga aacacataaa tgtcttttat tcaaatctca ccttccgtca 960ggtaagtacc gcctaattgt gaatgggatc ttgcgtgctc tcagctcttt catggcaatc 1020taatttgcac aaagacaata gtttagcata gcaagtaatt tgaaagatag aataaaatca 1080actgacaatt tctgacagaa agagagaaga aatctaagta gtatttataa tgagttgttg 1140ataagtgcag tcaacattca tctcatcttg gttttgtggg agtactctac caaaatattt 1200cagttaccat gcctaaaaca taccaaaata attttttatc cgggaaaagc aatctaagac 1260ccttgtggaa ttctaaaaac atgtgaaaat ttttcatgac atggaggaag agggacacgt 1320gtaaacatac aaaattttaa gattgagaaa catatgggag tcaacaaaaa ttgtattcat 1380attcccggaa gaaatgaatt cctccaatct ctccctttct ctggccatca tgata 143521889DNAOryza sativa 21tcgtcctagg tggtccattg ccctcaacta gacaatctat gcgcatcagg tatgtcaaat 60ggtttcaaca attttacctg ttctatttcg ttttaaaatt tgaaacaggg aagggcctgc 120tgaatttgat catgaagtga atttgatatc cagatttctt atcagcgcca actccaaagc 180acatgcagca attataagta ctgactgact gcaagaatag actgtacttc tatgaagttg 240gatctcttca cctatctgtt caccctaaac cttttcatgg tttataagaa tgcgggttgt 300ttcttgcaaa attttgggta gaatggtgca aaaaagaatt tgttcgataa tgaagatagc 360tgtggttttg ttagcttgtg ttatattgat gtaatttaag atcatgcgag tgtttatttt 420cccggcgaca ttcttttgtt tattcattat atgcagtatc catttatctt ggctctcacc 480tgcctgatga aaaacatgta tgtttatcca taccatatgt aacaccgtcg aatgttaatc 540acttctgcgt aacaatgcaa atgtgccaaa actagtatag aagtattccg gtcaaaaatg 600aaagaacatt aataaacaaa agaaaaaaaa catctggcca gtaatgcctc gtccgaaact 660agtaaactac gcgaaacaat caactgaaaa ggacaggtga atcagccaac caagactatt 720gccagtaaac gagaaagcca gtgcagtaac aaccatccat aaattgaagg taaacgggcg 780cattttccag caccatgcca accaggctga ttacaaatcc caacacatca gatgttttca 840gggaaatcaa tagaatatga tcgttgctcg cgacagccgg gaatcagta 889221721DNAOryza sativa 22cattgccatg cctttcatga tcttgcaata tggacttgtc tattatggat ttgtgtgtca 60ctataaattt ggtagtgtga gactgcttga tggtttgtgg gtttgttcat ggaaccatgg 120agatatggct ctatcctgta tttcgttgtt gcgctcagaa tgtatgggcc tgtgatgcac 180tgttccaagt ttaattgaga tgtgtcaatg ttcaagttac agaataaatt gctgatatgt 240tctcagttct catggacacc ttgggaggat tgtatggttc tgactactca tggaggttga 300gtaatgcagc gtttccgttc gtttgctctt tatttgataa agtataaaat gcttcgggag 360gattgtgtgg tttatgttag ctcatcgagg ttaagcaata cagcatttca gttcgtttgc 420tctttatttt gataaatatc aaatgttttg gatgatttgt gtggttctat gttattcatc 480aaggttgagt gatgtagcgt ttcggttcgt ttgcttcttt gttggataaa atatgaaagt 540gcggtttggg caaatgcact tgtgggacaa aattaaaaga taaagatagt tcccctcttt 600ttgttggata caacgtgact ttggctttgg ccaaatgcac ttgtgggact aaattaaaag 660ataacctctt gggcaaattc acttgtggga aaatatgatg tgtgattttg ggaaaatgcg 720cttgtgggac aaaataaaaa cataatcaaa atatgaagtg ctaattttct ttggcaaaat 780gcagaaaata ataataatct aaatatgaag tgtaaatttt ctttgggcaa atgcataaaa 840attgggaaaa gacaaaaaaa taaaacataa tcaaaatatg aactggaaaa ggtttaaaaa 900aaatacaaaa cacataatca aaatatgcaa atatgaacta taaattttct ttgggcaaac 960gcaatgtttt taggaagatg tgagcttttt ttccttaacc aaatcagcct aaaggcaaag 1020accatcagag aactgtcata atctgattat cctgatctca ctcgtcaact tttcttccag 1080gaagataata tgagaataca gaacaatgag gacaactcga cgcaacagtc agtggggagg 1140agcaagcttg gaggagtcaa atagctcaaa cagtcatgca caatacttga tatggcctgg 1200tctaccaaag cagtttttct ggatgtctct cttgggaaga tacatcattt tgttccccca 1260cgaaaggcta caacaccctg attaacacaa gcaaaagttt cacccaaata catcctacag 1320aatcattcct caactcccag agagcaaaag gtataaaaga accgtttcgg tatagtcaag 1380caagctatct aagcagaatc aatggaacaa tgcgaaattc cgagcatcag aaccagcagt 1440gcgtatggat ggctgtcatc agaacctgcg gagcccacga tcaaacatcc ttgcagatgg 1500gcactcatat gacatatgac ccctcccacc gcaggtgcca cagatcatgg ttgtcatgca 1560gttgcggctg atgtgccctg gcttgccaca caagcggcag gtaatgtcac ggaaagggcc 1620gccctgtatc tctgaagaaa tggtcgtttt ttgacagttc cgagcaagat gcccagagac 1680attgcacagg ttgcaaacag gctcgttcgt gcactcccga g 1721231599DNAOryza sativa 23acccatcaat taatctcttt tcttttctta atctattttt cttctctttg caatcactcc 60tcctatttgt gcgatagcgt gcatgggcca tgtgacaatc tcttgtgatg aatggatgat 120cacgcgtgct acctaaccta ccagatcgcc atatcaggct atcgctgatc gcgtgatctg 180tagtatcaca tccgtttgtg tgggggtaaa atcagccatc ttcctgtaat aaaatagaat 240gattttccat tagactcact gatcattggc tgtgaaattt gccggccgcc tgttcatcag 300ttatctcgat caaggcttca acttttactg tggtatgccg gctcctagct ggaaaatgtt 360gaggacaaaa actcttcttc aacccaaaga taacacaacc atcctgaatt tggttgtagg 420ggacgcaaat tcaaaaccat aattttctgg tcacaataat tgatgtgttc aagataacct 480gctctgctct gaattttctg tcatcttaat taattacagc tgccttgaat cgttcagaca 540atacacgata tcttagttga ctcggaaata aaatgaacga atgaatctaa agacgagcct 600tgcgtggatt gcaaattggc aaaggcagca cagatgctcc cgtcactcgc ttagaataac 660aagccacaaa cacaacacaa gaacaaagag aatttgcttt gtgcaagatt cttctaatgc 720gattacatca agaatctgtt actgtcagtg tcactgtcaa ccatttgcac aacgggcggg 780ccttgacgtt ccagcatttt tgacgacgaa aaagtggaga aaagagagga gacaaaataa 840cagtcttgcg tggcaccgcc tggagtccac gtcactttga acggctctgc tacaacgata 900cacccggcaa gacagcaact tgcattgtgt ttatcattgg tggctcaaaa tcacagcaaa 960acaagccatt gcaatatata atatgatgtc gacagacggc tgttcgcaaa gtaccatttt 1020tcccacgata agctgaatct atcatactcg actgccagta tttggtcagt ttccatacac 1080ggttagctga atcttgcctt cacaaattaa cagttttttt ccaccctgca agattgcatt 1140ataataaacc tgtaacattt aagctatcga cagagacgtc agtaaaagga acaaccagcc 1200agtttgcaat agcttatctg cttataatag gaagatgaac tctaactggc agtggcacag 1260atcactgtga gaaataagaa ataacatgac tggttccatt tagcacaggc acatcaaagt 1320ataccttttc ttcccagcta taccactaac tggaatgaat accatcgtat gtttcaatct 1380tgtcaagaaa cgaataaaaa tatcttcatg cggacagggt tccaacaata gtaaaaaagg 1440aaaaaagaat acagacgcat aattgccaat cctctacttt catctacata gctgtgcaac 1500aaatgcttct ctgagctgca ccacatttta aactcaatac ggtattctgt gcaggtttgc 1560caaaacaaaa aatgctaatg atctcgcccc atcaaccac 159924715DNAOryza sativa 24agtatcatgt tggcatgttc ggttgttctt gtttgttcgt gggtggaaac ttacattggt 60tggaacataa caatacagaa tagggaatac tagtcagttt ggcatctgtt tagtttgatg 120atatgacttg gtgatgatat atactcgaga tttgtttctc tcttctgcgt tttgaagaat 180ttaggagctg taaatctgag ggcattatat atcgagatta cttctgcttc tgttgatcct 240tttgcatgtt acttttcact gttaccaact agcagctgct cttatgcaca ggccctagcg 300aacactaacc accattgcac tgacaaatca caaacaaaaa agaactccca tgcaaatttt 360ggaatctata actcatcacc tctcattcgt aatagaatcg atcctgctag aaaagaggga 420caaaaagagg gggaaaaaca aacatcaggt actccaaaaa gggccaagct taccaggact 480acatctgaaa ttctgaatag acgtatatgc tacaattagt aaaccaatat caccactgct 540ctcccttgcc acaaaacaag ggaagcttca atttatgacc cgatttttgc tgataatctc 600tcacataaac ccaactcaac acataccctc ttcccgaatg ttgataattc caacccacga 660aaatatcaaa ctttttcacc gacgcatagc aaccgaggca tagcaactaa ctcgc 715251191DNAOryza sativa 25ggcagctggt cacgcttagc ggcggccggt ccggtgctag ccttctcctg agcttgtgtg 60ttggcgatgt tggtgtgtgg gtggtggatt ttttttcttt ttcctggtta cgaccctcca 120gggttataat attgtaattt tttcctgctc tatcaatata attttgcacc gtctcgtgcg 180agtcgttcaa aaaaaaataa aggtagtttt taaaaaaaaa aatctaattt acagatgtat 240tggatgagtt tgaccatgtg acatatgcat ggataaatat gttaaaatca ttacttaaat 300cttttgtttt tttatctact tgttctatct tccatacaaa taagtatcaa tataatatta 360gtataaaatt attatataaa ttgagaactt atgattgata tattaatata ttatatcagt 420tatttaaaac taaccgttta agtttctcaa ttcataaaac cacaatccaa aattacttta 480gacggtaata taaagggtct tataaagttt aggaggctag atatccgata atttaaaatc 540cagagcataa gatagactca catacaatgt ttatgtggta tacgcctata tggacttttt 600ccaagctgta acgggtggca aatttgccac ccaagccgca attctctaaa atatattccc 660tataaaaaaa tatttttcat ttcatagtag ccaccaatat ctggaatgct ggatgatact 720caaatgaact ctgctgttgt ctattgtatc agagcaaatt catcagctct aacaagaaag 780aaacaataag ctattaagca taccggccgt cctcttgctg tttttaacgc atttgtagac 840atatgtggtt tgagtaccaa ttcaatacaa agcaagagga gcagtacaaa ctaaatggta 900ctcacacaat gaagcaaggt gaacttccaa atatctgcaa ccattattta cagaagataa 960tagctattta gacagctatg agcctcacac atgtaccttt gaatcgccaa acaaatactt 1020ctagcgcatt tatatatgaa catataatgg agccaacaca tcatttacaa ccaacatccc 1080agtgagttat ttgtttggat caccactaac ttcgcaggca acctatgatt tgctctataa 1140tcagttccgc ctcggtgaca atccaaacct tcacgagttg cctgcaacca a 119126791DNAOryza sativa 26actttggtct accttttgta tgatagatat taagttaaga agtatgagac atcgatattg 60ttcttgcgga tgtgtggctg gctggaagtg tgtacatttg tgattattcg tcccttgttg 120taatcaccaa ggttctagca accgaaaata tttgattctt cgcatcgggt gagacataac 180ttgtctgaag gtgacatgca tatctatcta ccttactcaa gagggaaaac aaaattctgc 240atcttgaact gaactgaact gaactgaact tgccttctct tgttctgcat ttgattgatc 300tttgtgcaga agaaatgccc ccaaatttgt ccatattttc ttcccattac ctcagcattg 360cagatttggg ccttggcacg gttgttcaga acaagtgcgt cctgtttgca tactgaacct 420ctgaaatttt cagagttcag aatcaggaac tagctcaatc aactgaaaat gctaaagatt 480agaacaagcg aaccatgcca gcattaacat aaccgcaacg cggccagaca taaattaagg 540tagatctgta actacaatat tgcttaatta tgagcttaca taagctagca gcaactgcac 600tacagtacta caatactaca gactaatctt ggatgtcggc aaattcagtt ggggcatcat 660ctgacgactg atcatgcagc aatgacctac ttgactacat ctctggcttc ctgaactata 720gactatgagc aaggtgctca cctaacactg gaatatacta ctaagtagca ttctttacac 780aggatgctgt t 791271478DNAOryza sativa 27cgaaggccca ggttttcgct tttatgctgc agctagttcg ttctcatttg aggtgggaag 60gaaaactgcc tcaaaaaagg aaattttccc agcttacaag caccagcagc tgccttttct 120gtgggtgtga ttttgcgttg gtaattgtta gcagttcagc agtttaattt ctcttttacc 180ttattttgtt tctcatgtca ttaacaaccg ggtgtagcat ttaggaagtt ggatgatcat 240gagctgtacc aaaatatttg tattgctgga agaatatatt gatggtagaa ttgtagctta 300cacttcccaa aaatgtattt actggttgta caacagtcag cagtaaaatt aaatggaatt 360taagtcaggg tcattcgttg tcctgctggt gaactatact cgagtacgtc tagggctcat 420ctggtgaatg atcagaccaa tacgaaagaa ttccagctag taatttatca catcaaatta 480atcataactc acttttcagg gcttatttac ttcgttgcct aattttggta aggtattgta 540tagaccgatg gtttttactt tgagacctta ccaaatttta atagaagttt tgagagtaga 600aagttatgga attaccaaag tgaacaaact ggctggctgg atgcaaaacc ataggcgcag 660ccaaagacgc gagctgtttt tacactctag tacattacag taatcagcca gatctgtcaa 720accatttgtc tgcaccaaca attagagctc tgcatgctat tctggcacaa aacccatcag 780tatcagtaca tcacacgcat ccacacatac aaagcatccc agaagggatt gcaacccaaa 840acggaacagc acaaactacg gccagttcga agtgggtcaa gatatacact catgtcgcac 900aggctgtaac actaacacga gaagcaaact tgaagaatgc tgaggaaacg catctagcac 960tagagtggga acgagtcgag cagctgccat cgaactgatg accatcaggc aattagagtt 1020gcattcttgt aggacctgca tgtacaagtc atagctcatg acaagaggtt taaaagacaa 1080tattataacc ccttcgtcct aaattccagg tttcaaaatt ctactcacaa gcatacatca 1140tttaataagg gacattactg tcttttattc ttaaacttaa tctatgctaa acaacataaa 1200aggacaacta tattgggacg ggggagtagc taacagagtg aaaatcaaag gagtaacgaa 1260tttagtagtg tcactgtcga tacacctaat ttaacaccat aatatctcca actctccaca 1320acacaaggac tgctgataaa ataaatgcac ttctttagga gaataatgtg ctcatcaagg 1380ccatgtacaa aatatttctc caaaaagttc aacaaaggtt ccaggtaccc gtacagcaag 1440ctatatcagg aatggagtcc aatgcgttac cttcatat 1478281678DNAOryza sativa 28gaaagcagca atcgccctgg actgtgattc tgtgacagcg aagttttggg tggaagatgc 60agattgatgg ttctctgcaa ctgttggtgg agctaggttc agcagctatg ttcgcgagtg 120tattagcgct cttttggcgg tgctgggtag tagaatgtag aatcgtttct tgttgattcc 180ctgaactgat ccagatcaaa catcttctaa aggttctgcg atgcaatgct agctgcagac 240gggagatggt acagggggtc gttttgctac tttgccgccg atgttgtagg gtgtgtagac 300agactgtaga tgaataatgt cagtaacacc tgtggggggt tcagtgctag tgggctgcaa 360aacttgagca ctgcttcgat caaattgtgt ctttgattat gaaattatgg aatccagcac 420aggcaggtaa taatttcgta atagagcata aacaatttgt gaccttaggt gcgaagaaca 480taaagctgac caaggtgaag taatattgat gcattgtgcg ctgcatttaa tttggttgtt 540gaattcagag tacgtattta aataggtgtc cggaggggaa aatgtacaca aatgtttact 600ccgtttgctg tgcatctcgc agtgatacaa aaaaatacaa atcaaaccac gctaaaaaat 660ttagtcagtt gctcgtcttc agaattcagt cctcaaatat ttttaatgtt tcctccatgg 720ctgctcgtcc gttcattttg cactcgtttt gctctctgtg aactggagag ccccagaggt 780tctctttttg catgtaatct gagtttctga tcaaccattc agcctggcta agaaaaccat 840atggtggtgg cgttcttcta acttgtgtct gcgcggctgt tggcttcatc caattcatcg 900atcgccagtt actacttact tactaacagc gtccgggaac aggagttgag ccaaagaggc 960aggtttatct tccattaagc ttaagagaac tcggagatat ctcgaagttc ttggcacggc 1020acgtcctgtt ttcactttca aacttgagca ttcagtttag acgaggtact tgattttttt 1080tttgtgtgca cgccccagca ggtgttttaa gaaaaaaatt aggcaactta aaattgttgt 1140cccttctcct gtgttttgcg ctaataattc catcctgcac atggcatctc tgaggaatca 1200aagatcaact agcaacagca acaattagcc gcgatagatc agacatagac actggcactt 1260tcggatgaac aacagctagc tagattaact gaaagcaagc acggttacaa aggacacacc 1320cagcagaggc atcagttgga aacaagaagc tagcagctgc tggccatgaa aagccaaagc 1380atcacctagc tagcattcag cccctgcctg accgatcctg caataaacta aaacacatac 1440cctagctact gaccatgatc gatcagcacc taaaccaaac taccaaagct agctaaagct 1500aagcacgcat caccatatga aacataacat agcaaatcat acgggcgcct aaatgcgtgc 1560atccgtcaag acctacatta ttattgataa gagagctagc tagctaagac tgactcgatg 1620actacaactg agacgactac gacacactgg cattcacgga tgcaaaagtg tgtgtagt 167829781DNAOryza sativa 29gcgtattcca attttttgat ttacgaattc ttcccgtgag ctcaagcaaa gagtggctct 60gcagaacagt taagatctga tgcatagcca agttatatgg gttatgtatg tcaacaaatc 120attgctcccc cccccccccc cagagatgta ttagtctgta tttcggcatt actcatttgg 180aaatttgttt caaggcttgt gctttgatct atgtacttct atttgttatt ggaaaaggaa 240ttaatatggc gattggtgtt acttcgctat ttgattgggt tattattgta gcctgaatat 300tacattacaa accttcaatt ctgtagcaat agctttttat tagagaaatt ttacgggctc 360ttaacgagat accgagaggt accacatttt tctgtaatgt accaagaaca aaaaaagtga 420tactctcttt ataatccttg taaaaacaaa aggtaccaaa atgataactt ggaatactta 480tcaagagctg taaaaatgac ccttggagca acggtgtaat tatactttaa gacaaaacga 540tatagctata acccgaaaca aaagatcaaa tgtaagccta aaattagcta ttgcaaggag 600ctgaaaaggc acttatgttc caatcaaatt atgtaagaat catattcctt tattctttca 660tattggtgaa gccgtaccgt gtcagtatag gctgattttt ttacattaaa gcaaaaaaaa 720atgtctatgt tcacaaggta gagggtgatt catgtgtcct tttactcttc agtaacaccg 780g 781301260DNAOryza sativamisc_feature(183)..(183)may stand for any base (a,t,c,g) 30ttattttcca gttctggtgg tcacttccag ttgtgttttt actgtacaat tagggctctg 60ttgggaggca acatatccta tgcacacagg ccctcacgta tacacacgtg cacatcaact 120aaaaaatgtc accaaaaaat ctagaaaaaa tcatacatat actttcaatt gtattacacc 180tanggttaaa atcttaatgt caaattcatt atattttagc cgtaacaaaa aaaaaacaaa 240aaatctgaca gttttaaggt tacaattttg tcagcatttt atcttttttg ttattctcta 300tgtacaataa atttgaagat gcgactttgc atgtagatgt aatactattg aaagtacatg 360tatgaatttt actagaattt tttgtgataa tttttagttg gtatacacgg tgtgtatacg 420tgaggncctg tgtgcatagg atacgctccc ctgttgggac gactatttta agggattttt 480tcaataggaa ttagtccaat tcataccaaa atccttaaaa aaaaaatcct ctattctgaa 540ggccttaagg acctattcgt tttggagaaa tttcagagga tttggatttt taagaattaa 600ttcctatcat tgccatttgg tttataggaa tgtagacata ggaaaatcaa ggaattcttc 660ctttcctaca agttgtagag agaaaacata agaaattttt cgtcttctta aatctcttgg 720aaaattcctg tggattgaag tgtgcgtata tttctatttc tccactttaa attccttaaa 780aacaaatagg gtgcctaact atttcatctg gtcttgtatg acactgacat ggtttttcag 840atgcacattt agctaataca gtacaattca tattgttgaa gttttgcgca tatttttatg 900taggagtaaa tgagttttaa gtatcaattt aaatttaaaa tgatgctggt acatcttttc 960aaatttgatc aaaagctcca acgagtatta ctcctatttg cacataattg gcagcgttgt 1020gaattccaaa caagcaaata aagaaagaaa aagacagctt aaaacgaccg cataactaga 1080gtcacagcta agcgataatt cacaacatca cgatgcaaat cataaatcag tttcgttttt 1140ataaaaaccc tcgtaggttc cagcagactg ctcataaaaa cggagtattc aacatcgcag 1200agaccatttc tctgacccat agcaaaatca cagcgacaaa atccgctgtt tacatttaat 1260311529DNAOryza sativa 31ttcaagttgc atcgccactt atgaacactc catgaacact taattctgga ggcatgattg 60gtccgtggga ttaagacatc ttgatagact gcagtggagc ccagcccata atctgaagaa 120ctcattaccg actcttggct gaagtgcttc ggagtgattg atgccattct gtcatgtgat 180caagttcatg tccatactac atacgtcgtc tggtccttta gagcaacatg acttcaggac 240gttgctatca acggctattg atgataaact agtaataaaa tttgtcgatg aaattacttg 300ccgtgataaa tgattccgtt gtcgccaggt tacagttatt caacgtaaga acttcatatt 360cattctagaa accaacacta ttcatcacca ttttcctcat ccccgaaaaa ctttgaccag 420tacctctgtt tttgccaccc ccctccctcc ccggccacaa ggtgtagaat caagtgccat 480cgctgaccaa tttgagctgc tcccaattaa aatacaaact aaaataaaaa agtatagaaa 540aaatgaaatt acctatggat aatattatgt caatggcatt aaaatttgtg aattttggca 600tttggataat agtacctccg ccttaaaata tatggatttt tcggattagg cacgagtaat 660aagaaagttg gtggaattaa atagaaaaat attatgattg gttgagaaga ggaaaagttc 720gtgaaattaa acagaaaaat gttatgattg attagaagaa aagaatgtaa gtagttcgaa 780tggtgtaagg ttgtgattgg ttgagaagag aatgtaggta gagaaattgt tatatgttag 840gataaaattt gaaggataca agttgttacg ttaaggagag agtattatgt ctatgtacta 900tcaaaattca attatgattt tttttcaact taagctgttt tcgtcaattg gacgggatct 960tgctacgacc acgatcggtt tggtaaacga cctgccccaa tccttcccac gcacctcctc 1020tcccttcccg cgatcagtcg tcgcctacca accaactaca taatgctcta ccgacacctc 1080ataaagctct ctcctccccc accctacgct accgtctctc gtgcacgcac ttcacgacga 1140cttcgccagc aagggaattg tactccagag caacagctca accgacgacg agagctcaca 1200acattgcctt tgcaaaaaaa acaactcgcg tatttcccat tctttataca attcctgtta 1260tagtgtggta aaaaatttac acgtattgat ctctaggctg cctggattga tcggcgtcag 1320ccctctccag ggagctctcg atcctgcccg ttgtgacaaa aataacatac acaaggcaca 1380ataggatcga ctattctaac aaaaaaaaaa cgaaaaaaga aagttaagaa aaaaaaaacg 1440aacggcatgt tcttctccca tcattccatt ttgcacttga accgtctgga tgcgtctgat 1500gcttggatat catcaatggc atcgagaca 1529321277DNAOryza sativa 32cagaaggaga tcaagaatgt ctttggcgag acattgcttc gacattgctt cagtgtcaaa 60atacaccatc atcattgaga tcagtgttct taaggcgaca aggcgaggca aggcgaccca 120cccctgctca attgcctagg

cgacgcttta agcgtttaag gcgagggtaa ggcgacgcct 180tccacacaag cacaagaggc atactagaaa tatatgaggc aaaaaagaga ggaagaacat 240gaaggggcga ggaagatcca ttggagaagg gagcagcagg agggagacaa ccatcttctc 300tttccatctt tcccatgttt ccaatctctg ctcgccctat tctctactct ctttccatct 360ttcacccttc cctctcccta aattggtgca ctgctatatc tattctcttt ccatctttcg 420cccttccctc tccctaaatt gtcgcacatg cgatttccgt cctatcttca cgcgatttcc 480ttcccagcat gcataaatcc ctaagcggtg gggacgctct gctccctgat ggcgctcaga 540cgcctaggga cgctttttaa accatgattg agattcctcc acgacgatca cctccatcca 600gtttctcgct ccacattcta tccggcgatg agaatttgtt ttttatcaaa taactatcac 660aaagctgcta gcccttcccc aaatccaccc ctacctctct cactgttgct actgttgatt 720tttgcctaga catcagcatc actggataga acatgcaatg aggaactaga tggagtgagc 780ttggtaaagg aattaattgg tgattgccat actttagcag cagcaaagca atgtctgaca 840ctaagaacca cttgattttc ttgaggacag gagtctctgt tcctctagtt atttgtagga 900attggactag gagcttcaag aacatggtgt tatgcggttg aatttgccat aggcagatca 960atggagctcc catagtggca gcaatgaggg tgaataatgg acttcaagca taagttcaaa 1020gaccagaagt ggactatttc tgaatttagt ccgaaaaatg aactttggga cgaaaagtat 1080ggacttcatc catatcaaaa ccatgaccaa acataacttc tttcgaaatc catactgcac 1140aaacatacat gtgccttttt ttaaaagata ttgggtaaag tgtatttgtg ccatgaatgg 1200ctgctatcag gaatgcaatc tccacatgtt gtagcattgt taaaagctaa aactgcaaga 1260ttttgcacca agtaata 1277331381DNAOryza sativa 33tgccttttgg atcgaaagat gcagttgttt tgctgctgaa ggcatcctca tgtctgttac 60tttgatatgg tcttgaaata aatcttcgtt tacaagagaa caacactctg ttttatcttg 120ttttgtgtgc cagaagcctg ctgaacaatt ttatgtcaat ttctttgcta ctaagaaagt 180tatgcaccaa aatgaaggga aattctgtga agtttgtaat tttcaagtga ttggcattat 240tctgtttagt tcgtgcagtg ccatgttcct tatataatca cagaacatgt aaaaggccga 300aatattttcc ttgtcatttt ccatgcgaga acggcctttc gatgagactg gaaagtcctg 360caataactat tgtccaaaat aacacacggc tctctaccga ttcacatcaa aaccaaccca 420aacgctctct ctgttgcttg tgttctggac ttctctgagc gcagttcagg agtctcgagt 480gtgcctcaac agtacatgtt aaacataaaa ctgccattga agattaccac caaccatatt 540ggaagtttat catggcatat acttgccttt caaagaccct aattttcaaa gtgaacatgg 600gactttcaga ctacagataa cctcaatcga taacatcacc gaaaattcga tattactaca 660aagattttgg ttgaaaactt ctctgaaccc aatttaaata caagtcgccc tcggtgatgg 720ctactcgcca ttccacgctc acagttgcag ttcacacttg caaaatgaaa aaaaaaatct 780tctgaacagc aatcatcaac cggttcgacc tcaggatcat cacagaaatg aaaaggacga 840ggaatctcaa tttaagaaag ttcctatcca aatatccaac aaaaatctga ctgtctggct 900attattaatt caacaggaga tgctcgttaa agacaatata gttctgtaat ttgtaacaac 960acaagagcct gaggatataa cgagttacaa acatgctgcc gttgcagcat ggtcatcaat 1020atacaacatt acaacaacat atggggatcg cagaacactc ccattccacg attttacaac 1080atatgcttca caacacgtta taaatacatg gttgtaccga gctgtagaga gacagccagc 1140acgtcccaac tacaaatgca cactagccaa cagcaaacat aaaaactact ctctggtaag 1200tcactatata tacgcactat agcttcacat gactaaaatt ataacatgac ataatttctt 1260tcgtataaat tagccccagg aagcttctgt ccgaatcgcc atcttgggag caataaatat 1320actggcattc ttcacaatgg atgcattatt tacatatata tacttcttct ccaaccacgt 1380t 1381341172DNAOryza sativa 34gtaactccag caatttactc ccaaccaata caattgcaca attgtgccct aaatttaata 60ccagataagc taataagtga tctcttaaaa tgtctgtgat ctagggacct ggccatttga 120tggagaattt ggttagctat ataccttgtg aagagattga tacaaagatg ggctatgagt 180tcaagggaac tagttatggt gatcccagac catactcagc agatacttaa gtgtgcttcc 240aatgatttta gcttcatttt tgtatagtaa aagaaatggt ttgtaccgag ccacagtgat 300tgagcaatag agaagtagct aggtaggcat ttctgctctc atgtggaacg acatacaata 360caatacacac agaattttta gaaaagaaaa atacaggtgg cggtgtatca tagtcataga 420taaccatatt aagaaaaata tggatggcgg tgtatcaaaa accataaaat aaggtgtttc 480tgtctatttg gagttgtttt ggcatgcttt cctgaaaaca ccaggctctt gttttcagaa 540atccatgata agagagttga tcacttgctg gttttcttgt gggctatgat atcggccgac 600ggccaagtat ctaataaaag attcttgatc agggccatgt ggagtgatca taaaaccttc 660ttcttcagtg gcacttattg ggcaggacat tattaccaga ttaggttttg ttggaaacat 720ttccattact tgctaactga aggagcactc cctgcagaga gctggccttg agcacaaaat 780tccatctatt gctagactac cccaaatata aaaatgaggc aggaatcaga tcaaccccat 840ggtcaattca aaatatgacc agtcgatata gcaccttaag aacttaaaat attaacaaaa 900agggtagggg agttgcaatc agcgttgttt ggatggccca ggaattgaag ggctgagatc 960atagcagatc tcatactaga gcacccaagt aaacatctcc caaaagaaat gtacatgttt 1020atcagtgatt ttatttatct aattagcata ccaatacaaa ggaaaagaag tgcacagact 1080aaagaaaacc tcacttttct caaaatagaa aaaataaacc acaaaatctt cctggggcta 1140agtgagatct tacaacagtg tatgggagtt tt 117235790DNAOryza sativa 35aggcaagaac gaacatgtga aggcaacgtg ctacactaag atcttcacat gttggttttt 60tgtataaatc ttattcgcga ttgtcgcgcc tatcaagctt aagctatggc gatgtcgccc 120aggtgacctc ttatgtaaat atgtgcgaat aagacagcat tctggagttg ttcttcctgt 180ggacctctac ttgtagtata tggactaatg tgttgtatga gtacgtgaag cagaattcgg 240tattatcaag gcttgtaatg ttagatgctc ttatattaca ctatgaagtg tcaactttca 300agtggccacg tttacacata tatttgctat gctgacttgc tgagacatgc ttggttttag 360ggcggtttgc agcttaacat gtgttccttt caaagagcag tagatctgca gtttgcatgg 420tattcctgtc caaactgtct agtatactct atactgatac tctggtataa agctccctgc 480agtttgcaac tgatgtattc tggaaactgc taagacacct cagctacctt tgtttgtttt 540aaagactatc tcaactacct ggaactctgc attttgttta gtacggcaat taaggactga 600gttttcagca aacagacttc gatgctattt ttgattgaaa taaagcaggc aaagttcggt 660ctctcacata aacgaaacac attcatggat gatgtcgaaa gtgcaacgga atgatacaca 720tgacatgtaa catttagcct tagaatttta ttgattcgac gcagcaatcc aacttttgac 780ggacttggcc 790361283DNAOryza sativa 36atcgacgacg acgacggcca tggtgatcac tgctatagtt gtagcagaag gaggaggagg 60ctgctctgct ttcttgctgt gtgctgcgat cgagttgtcc atcagtgttg ctgttcttag 120agaagaagac tgacatctct ctctctctct atatatatag aaaaccctta atctatagag 180cattttgttt ctgcttatat tgctacatgc tctatctttt tggttgaact atctacatat 240atatctatat ttctatctac tcactccttt ctccttatgt aacatacctc tcaaaactct 300gaactggctt gtgctgatca tctgatgtat aatatataat atataaatat atatcgcccc 360ttggacacca tcgcatatca atctcctgga tatacttggt agactggctt aaatcatttc 420tgctcttctg ggtcattaca gcttgattat aattggataa ttatcaaaga gctgagtagc 480tgacacagtt tttgcattga gctgtcccag tccagagtct acttcgaagt ttagaacacc 540agtatcactg catctgcata tgcctgatgc gaggaaagcc aataaatatg gatctgatga 600gcatgccatg cagaaaggat aagatacaga cggcacgcat gcatgcccac atacacgtgc 660tgtatcaatc aagagaacat catccaaata tccaaaccaa aatgcaaagg atcaacatca 720tcatcaatga ccagtgccac actgcacaag ttaaagaggc acagcggatg agatctggtt 780cagttcaggt acccttgttg agagttggca gagttcttgc tgtatccaaa gtgaatcttc 840cccaagggga aagcaacaat ttacacggtg gtcaataaat aagagtgcat gtacaagtca 900aaaatatagt agtagtatac ataaggtcca caacatcagc taattaagtt actccttttt 960tggcttatta tttcatatgc actggtgtta aaattcctgc ggcggaacaa gacttttgct 1020ttttcggctg gaaagattga gctttccatg gagcatcagc agcacaacac aattgaacag 1080ctagctattc tttttaagtc gatcttgaaa catacattac tatgatgctg ggattaaaga 1140gctggagaag agcttctgtg tctctccatt tttttttaca tcattgaggg aatgatcttg 1200gccgaaatga ccggctttta ctttaccaca ggggaagaaa accttcttca gtcaggtgtg 1260ttgcctccac catgcattca ggg 1283371072DNAOryza sativa 37cagctcggta gcaaagcaca tattgaatca tgctgccgat acaaaaatcc tgctgtttag 60aagatacaag ctaggaatag aaaagcaagc gaagactcaa catcaacatt gataacatgt 120gggaaaaatg attgttgagg aaaccataga taaattggcc gaactcaact gcccattttt 180cccaaaactg gtttagcagg aaagtttaag cagcatgtac aatgaaattg tatgtgcagc 240tccatttata ctagtcctgc atgcaatgag ttctagacaa ccgaagttga taaaagtttc 300atgacactgc catacaaaca acaaacaaat ctgtttcagg tctaatgaga ggaaaatgat 360tgttggagaa aacagaataa gcttaaccca actgcccact ttcctctaat tgatatagca 420ggaaacactt tcggcaaaaa atgcatagca taaggcatta ggccgataca cactatcctg 480cagggtaaca tgacaacgga aaaaacaaat gtaaaatcca tgaaaaagta aaacatttct 540ttggtactgc tatctagaga gaaaactaaa agctacagaa gcacaataag gaataaataa 600agcaagaatg caatttacag atcaccaaaa cagacataat gtcataatat aaaaatatgt 660agagataaat gagaggaaaa tgattgttga aaaaagcaca aaagcttaac ccaactgccc 720actttcccca gatcaatata gcaggaaaca cttttggcac aagacgcaca aaaaagcgtt 780aagccgatac acactatcct gcaggatgaa gcatatacag tgggcagaat gattacctac 840tatactaatc tcatgtaaaa aaatgagaaa tatatcagga actagaaagt gagacacacc 900ataaatgtta ctgagcacca ggaagaaatt aatagtgacg aatcttgtat tagttgctga 960tgtttgcaac ttaaaaacta gaactcttta ctagtaataa tgttaataat gaacattaca 1020taactgaaaa aaacatgaag cgcatgcatc agatcaccta aacttattct at 1072381549DNAOryza sativa 38ttggtttaga gcttgagatg ttttaaaatc gttcattcct gagttataat agcccaagaa 60cctcttgcac tatgttcgat caatccaccc cagctacttt cagatttctc gagtaattgg 120tgcaattttg catgaacatg ctgccttgtt tggaattctt ttttgagtac cggagaatta 180ccatgttagt gtgttggttt atgttttgaa ggtggccttg ttattctgtt gctgtgttga 240tgtttggacg caattgctgg attggtactt ggtagcatgc tagaggtttt ttactctgct 300tcatttgagt tgctgggttg cagttgcagg catcggtgta aactcaactc gaagtcgttt 360gctgctgcct atagtccagg cggtgtaaat tcaaccttat tttttttgta aaatgaaatt 420ttaatttcag gagagcaaca gacagaccaa actcttaact gttatgacct gacaagttta 480atttctacca aaatttgtac cgtcgttgtc gaggaatttc ttcttgccgt tcgaattcga 540tttcttcggt attgggttgg tccggtctca atccgttccg attgtgtcag tcttgggcca 600taaagcccat gtgttacaag cctaacagcc caattcgact ggcccacgac ttttgcaggc 660gggcaccaat tccgggtgca ctccgacgcg gggcgcgtga tcaaatttct tttcacactt 720tatatcctat caaaatttaa taattttcaa attttgttat attttggtat tatataaatt 780cagtagtgct aaactagttt ggttcacgca gaaaaaaaac atttgaattc gaaaggattt 840gtttattatg ggaaataatc atataaggga acatatgtta tacatatata gaagcttttg 900cgtgtacacg ccaactaaga aaatatggct ggttctaaaa cttctacacg ttttaaaatc 960caataggaat gcaaagcata tgacatttca gctctacgtt tatctctatc tcgtattcac 1020aatcatatgt ttcaaaggta accttatcaa atcctttttg gtggaaattt ccgaaattac 1080aaaatacgaa caagcccttc agtactagcg ttggcgtact gcattttgcc catctcgctt 1140ggccaagaaa gggggacaag ggggaaacgc atgtcgcagc ctggcacgtt ccaaacttcc 1200aatcgcagag aaaaaaaaaa gagaagcaga tagagaggca tcgatcgtat ctacgagtac 1260aaaagtaacc aagcaaacca tctagcttta ttttctcctg tccccaaggc caaacagcac 1320agagctgagg cccccttaaa aattcatcag aacacatcca tcaaaaaact tattacaaca 1380cggcgagtat atatgctacc atatcctcga caactgccta tagtgccaag aacactcaac 1440aaccaccata caaacatttg acagaacaca tgcagatatc accgccggcg agttgaaact 1500attctacccc ctcctcccag tttcttttcc cttcccccgt ttcagcagt 154939766DNAOryza sativa 39gtaggaattc actaaaactg tatacttcgg actgggaagg gaaaacaaaa caaagtttat 60tcagtatatt gttttgttct tctttgccat ccacttcagc tgtaagttca atttaacacc 120ctttttgttg agcaagatat gctgtttttt cttggtgtca tttacatgtg gaatcagatg 180ccagaaactt actttgtcaa gattggaatg ttcacaatta aattactaag gtgttctcat 240ttttaacttg ctactgtagt cacattggcc acattagcac actatttcgt gttggctaaa 300ttcctgtttt tgtacagttg tcgtacatat gtaaagtatg gagtgaagga tcgtaatact 360ctagtgtagt tttcttaaaa tagttactga tattctgtgt tcacaaccac ctgctttata 420ttcctagtag gtatcaaata ctagtctgca aatgaacaag tgattaagtg acaataagta 480acatattcca aagaaagaac ttctacccat gctccatttt tcattcacag gcaccctctt 540tgttaaacac tgtatcctcg tgtatgtcat gttacaactt tttataaatt acataaattg 600tacaagtctc aactctcaat tcagtgagac agaaggattt ctcctccttt tttgaatgca 660ggatctgatt gatcaaacaa atttaccggt caattccttc aatcttgtct tcaacctcag 720agtagagctc cctgagcttt tccaagtttt cctctgatgt ttccca 766401691DNAOryza sativa 40gttatctgtt caaggagccc tgaaagtctg aaactgaaaa acaacatgaa taacagtgaa 60tgattagaac ccagaaacct gctaaattgt ttatcagtga acatcttcat aaaccgtagt 120ggaacccttc tgaaatcaat ctgaactgaa gctggcatct gcctgttaat acattataca 180taataaaatt accggatcat cattttgtgc agatcaacac actgtaactg tagtatgcag 240aaatcccgat ctgcagttgt tcctgtgatc ctgtctagca ttttgtaaag aagaaagcac 300tggagcatga aaaaaccaat ctgaagctgt tcctgtcaag cattttagtt gtttgtttgc 360gttgatacgt tctttgtttc tctgtggtat tgtcaaccag tcaaattgca atataatatc 420attgagcgtt atatttgacc aagttacttc tgaattcatg cagaccacta tcacttttga 480ccaattactt ctgaattcat gttcctttct ctattatcac tttgaccaat tacttcattt 540atgttcttca ccctacaatc acttttgagt tttgaccaac tacttctgaa ttcatgttct 600tctccctagc ttgttttaga gacttgactg ctgaatcaac atggccttcc ataatttgct 660acctaactga acctaaacca agacattctc ttgcacgata cattttgctt tcatgcttat 720agtttagaaa ccatgtctta tcttgcattg catactaagt tctcaaataa tttaatgaca 780gtttgacatg gctgagctgt atgcctgttt atgcaaagct ggcatgttca tcatacgaac 840tcttcagttc aaagttgaga gagccaacca gaaattgaaa gccaacgtca agaataaatc 900agctacctat tgttagaatg tctatggata tctctgcaac cttaacagcc agctgcagaa 960atcaatctga acttaatgct gtttccgtct agcatttatc tgtgacacct cacacgtaga 1020tattgctcct tgaatggtac ttatgttgct ttcatttcta aagttactaa atcatgacca 1080attcgatggc gatttcggtc ccagttcgtc acatttccac actgatgtct ttcagaggct 1140ttaaacttcc accttctttt cagagctccc aattaaatca tctttcttga tcagtcactt 1200ccatttttgc tactgatgat gccagtaata aaccaccatc atcggcatcg cacatgatga 1260caattgacaa accaaccacc aactttcgag atccaacaaa ttcacagaca acaaaagagc 1320aggtttctac cagattctga agagagcaaa ggaataatca gaagttgaaa gattctaaac 1380attagactgc ttgccatctt aatatatcca acattaacaa gggcggtagt agaatcatta 1440gtacacatga ttctacatca ctctctattt ttgctttttt ttttctaaca cattttagat 1500gatgcacctg aaactgaaca gaagctgctt tactactaca agggatgcag caccagaaag 1560ctgcaatttt tccccctttt tttttcagtt tttatacaca gtgaagaaaa gcagagagac 1620taaactctga aaactccaag gttatacact agacagcagc agcagcagca tcaccactca 1680cccatggcgc t 169141836DNAOryza sativa 41cctgctggta ttggagacgt taagaacatg acgttaggta atcctatctg tatttgaggc 60aaactaggga cctcctgctg ctggcttaat gtattgcact gcatgacatt acatgacctc 120catgtgttga acaagggctg aggctgttta actatagcac tagcagtacc gttttgcagc 180tcgagagagc tagtagtgta tgtatgaatt cctcaggtgc tattcgtagt agtaagcgcg 240aattgattat tatcagttca tgatggtgct actattcata ttcatgtttc tggttggcat 300gggcggtgct agtgcaaact gcagagtgct gtaaatattc cccaaatctg caatgaattg 360aacgagggtg catttgccct tatgactgtt gcggtgttgc aggtttttga atgagaagga 420aagattacta tgtagaaaac gctgtgttgt atttgtcggt cgcaggcatc tggtggtaaa 480ttctgttact gaagaaagtc cttgtcgcta tatatttgtg tgtttactta tgctcttgcc 540tggaataaaa ctcgaggatc ttttcctttg caaaactggg aaaatccaga agtatatgct 600atacgatcag ccgcattgca tttggttgat tgtattatag tgtacaaact cggcaacata 660gttgaaattt ctgtggcatc cttattaaat aaatacttat tatacatacg tataaataac 720tgctgcaagt aatctgcaag cagttgtttc tctcagctac tagtagtttg tgtgtgtgtg 780tttccgtcga aaacccctgt gaaataacac aagccagggt taccgcaaca atgatt 836421021DNAOryza sativa 42cggacttggg gagatctttt catgatgaaa agaaagttag gctcagtgtg taatatgtag 60ggaattgttg gcatatccaa atgtatttcc tcttgaatac attattttgc tcgcatgcat 120cctctccttt tccaattgta tgtttggttt atcgaatttt tgttgttccg acaaaaaaat 180taggcccttg cagagtaatg gttcaggatt tcaagctgag cacagaatag aaagtttaac 240aaaaaaaaga aagtgtagtc tgtagattgt aataccaata acatctgaaa accacaagct 300atgtaattgt acctttatgc gtgtgatgtt tttaactaca atcaaacaat agtcagtgaa 360accaagtcta agaggattga tcattgatgc cagttgatgt tgccttttca gattattcct 420ggaatctaaa tgaaccatgg gatgcaacaa caagagtaac ccttttaaaa ccttttttta 480tatatacaaa aagaaaatga tattagctca ggaagaaagg aggaacacaa acatcatctg 540aagccatcaa gccaatacta ttaaatgaat gggtaatatc taacattcta tgaccaaaag 600aattgtgtaa tagtgaaacc catactgttt gctgaatgta tttggaactg gaagcatcac 660atctggaaaa aaggaaaaac agagaaccaa accagaaaac aatgtcactg agatgactgc 720caagtataca ttccaatcca actgaacaaa tatgttacac aactaattaa acaaaaaaac 780atgctggtac ggtggtaaat aatatatgct tcatatgaac accaataaaa tttcttcaca 840tatgctgtta tcgtacaaaa tttgtttagt tgcaaagtga aattgcattg gaaggtcttt 900ccactttgtt gcaaatatct agccttaaag tttccctcag tctacaggtg ttcacaaagt 960tacttcagcc tcagaggtac ccactttgcc atcaaccagt ctggaaatat ctgctagatg 1020c 102143798DNAOryza sativa 43atgaccttag agcttttctt tcacctgtca tgacttgaat ggaaggcaaa taatctgttt 60cacctctttc cagatgtaat ttctacagat gtcttctggg tcattttatg gatgtacctt 120ctgaatgatt ttctgttgga ttccataatt ttgattatgt gaatatcaat aaaaacatgt 180ttcaggtttt tagattccct taaaaggtaa agggttgtca tttactgaat ccatggctat 240ctgaacttgt caagaatcca agatacacca gaagattgga attagttgat tctggcaaac 300cagaaaaaaa aggttaaatg gagatgcggg gtatcgatcc ccgtacctct cgcatgctaa 360gcgagcgctc taccatctga gctacatccc catgttgggg tcatttctta ttagttcatt 420ctactattgg acaacacaaa gaccaaatgt aggcagaaag gcagtggatc gaatgaaagt 480cacacgggcg caaatgccat ttacgaacaa atgagagcga actgaacttg tcatgttcaa 540aacattgtgc cccaaggatt gaacggtaca gtatttcagg aaagaataca ttgccttcta 600agcggtttga gtgataaaaa aattgttaaa aacataaaac acactgaatt aaatgtcaac 660ggttccatgg tttcaactat cccagaaaag cagggttggc atttcgagat cttccgtctc 720ttaaaactga atggatgccc atgcttcaaa atgaaaccct tgaggcagca actgaaagca 780tgaaaagaaa tcaccaat 79844865DNAOryza sativa 44ctgtcctata tatagccaaa atctggaatg cctccctggt cattttgttc gttacggctt 60gtgtagtgtt ttgattttgt ttcgatttct gtagtagtgt cgtgctgctg tgccttttgt 120ttttgtggct gtgacttgtg agcatccatg gaacttgtta cgctgaccaa taacattgtg 180actcgggtag tctttatgtg gtttgcgctc aacttgaaag agtcagaacc aattgctgta 240tatgtttttt ttttgggtga aatatgcgct gtagaagcaa atgatctgca tttctctggt 300gttttgatgc caaatcgact actaatcgag actttatcgt aggcaaggat aacaagttat 360gccttttgat actatcgagg catgttaacg tccattccag aaatattact tggctcagat 420atgcaataaa caggagaaaa tcagcagcat actcttcccg aattacccga attacgttca 480cacaccaaaa ttagggggta gaaaaaaagc ttgcaacgga gcaatgccga gcttgtgtgc 540cagttcaaaa gggcgcttca gtaccggatt ggaagtacag actacagttt ggggtataaa 600aacacacaga tgcaactgca aggcatataa gactaacttc aagtttgtag gaattaaaac 660aagtatccca tctggaattc atcctcaccg atcaacgagt atcgcagtta aacagcagaa 720ccgacaaata caagtaagct gacaaactat gaaaactgaa gtatctgaaa tacactaaca 780ttcttaaggc ataaagcacg agatatgatt attcataatg ccggtcccaa cgaagagtca 840tctgattgag cacttggaaa atcac 865451162DNAOryza sativa

45gccctatgaa ttggaatgta tttgaccata caaaaaccat acaaagtgta gggttctaat 60tagcaaatgt aagtttgtaa ttcttctgtc taaatgtagt gtgttgaggt ggtcagttgc 120cgtgactggc tctttcagat ggtaattaca aagaaatgta acattaatgt tgtaacaagg 180gtgccatttt gtttctgatt ccagatgaaa aggaaaaact attccatatt ctgcaggatt 240aaacgtttct gctgttcaca acaggtaaag tcactccact tcagggaaag acagcaaaat 300catttctttt acacagactt tagataaccc ttttttttgg ggttcttggt atatgccaac 360ttttgtagcc tgcaccagaa acaaaaatga agacttttgc taaagatgta aaagtggcat 420gatgtcctgg atgaccaaat aattcatgac aaatggatta aaagagccca atatctgaaa 480gagactggcc agcagccact aatgtcacca accacatatg taacacttgg tgcataattc 540aagagggagc atctcctcca gaatcaggat tgaaaggtac aacctcatag taaatcctcg 600gaatatagca tgtgcagcat aagaatatat cagtgttgtg ctgggtaaga aaccacatga 660accaattagg aataaataat catgctgaaa ttatagcaat gcttgcaatt tgcaaacgat 720aaagctagac gcgggttgct ggaataacaa tccatctcca acaaaatagt acagaatata 780actgaatggc cagctcagac cctaacagaa ttgaaaagct ggattcatca gcactccatt 840gagcaatcta gatcaggaaa gagcatagat gcataatgaa ctgagatccc ttcaaaatga 900ctaactaata tttttttttc ttataaaaga gtttacaaca gtacaaccac gaagatcagc 960actaccatta ctgattttgt taacatagag tgatttatca tgtgtgccag acaaacaaca 1020gatacattca tacatagcat aacttacagc acatgataca gactacggag aacggttaat 1080cttaaaataa aaacaaaaaa acaaggaggc aaagcttatt ttgcctggga ttcatctaaa 1140tgcagttgtg tgcagaagga ga 1162461297DNAOryza sativa 46tgttttgtca agctcccgct tgggaaaagc ttggtttttt tgttacgtgt tttgacctgg 60aacaacattt gacacatgtc ttttgatctc attgtctgtt tttcaagccc aataagaatt 120tgggtcgagc attgttttag gatcgaccat atacagtacc tctctttgca ttacaatgaa 180gagcagttaa tttgggtcac tttttacatc tttactgaag tagaaacgcg tcctctgtct 240gtgacagttt ttttgttctc cagattgttt gccgcttttt acttgcctga tacctgtgat 300agtagtacta gatgtagatg gttgtactgg gcgaaattca gcgaggctag gagttttgga 360ggattttggt tgaactagtt tctgcgagaa ttcaataaaa tttctgaatt atgatggagc 420cacggagaac aagcaatctg aaagaagata cgcaggtact gcgagtgtag tgcagccaga 480agcaagttat ccgtagcttg tgaaagcaca gttggcttct tctacccaaa atataagaac 540ctattaacca aaatataaga acctattaac gaatgagata ttttctagta ctaagtatat 600tttctagtac taagtccata ttcgtagtac taggaaatgc cccatccgat ttaagttcaa 660attcgtagta ttaggaaatg tcctatccgg ttctaggtta ttgtacttaa gaacagatgg 720agtaggaaac taggttattg tacttaagaa cggatggagt aggaaacaac ggtgtactca 780cacagagctg attgtctgaa caatcttcca gaggtcatca aactgctgat cagatcagaa 840aaaaagattg aaattttgga tctcaatacc atttttacct gtcttgaacg gattgcaaca 900atatgcattc agaactttag acatgcatac atactgcatt ggaaatgcgt ggaacaaaaa 960cccatacctg ccttatggga actataagaa atatgttgta cattagcaac aaagtgaatt 1020tccaatacaa acataagatc atcaatatta tccatgataa ataaaaatac agggctggta 1080taagcaacat acaatctccg cttgttcaga acctaaaatt tgtttacagt tgtaccacat 1140ggtatgctct ctacaactaa tgctactaag gaaatttcac agcatcgcga cagaacaaac 1200aacagatgac atatcagtaa ctaaaatcat gtgttctgat atgtccagct gatgaggcat 1260gcaaaacttc aggagatcag aaccctctac ttcaaag 12974735DNAArtificial sequenceOligonucleotide primer Primer 1(SacI, AvrII, SpeI, OCS 5') 47cggagctccc taggactagt tcgaccggca tgccc 354825DNAArtificial sequencePrimer 2 (NotI, OCS 3') 48ccgcggccgc agcttggaca atcag 254936DNAArtificial sequencePrimer 3 (AvrII, XmaI, RsrII, LuF 5') 49cgcctaggcc cgggcggacc gcattaagaa gggccc 365024DNAArtificial sequencePrimer 4 (SpeI LuF 3') 50cgactagtag agagttctca gagc 245116DNAArtificial sequencePrimer 5 (RsrII, BspEI, target gene seq 5') 51cgcggaccgt ccggan 165215DNAArtificial sequencePrimer 6 (SpeI, AgeI, target gene seq 3') 52cgactagtac cggtn 15534811DNAArtificial sequencepTOI3 53caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgataagcaa 60tgctttctta taatgccaac tttgtacaag aaagctgggt cggcgcgcca agcttgcatg 120cctgcaggca tgcaagcttc cgcggctgca gtgcagcgtg acccggtcgt gcccctctct 180agagataatg agcattgcat gtctaagtta taaaaaatta ccacatattt tttttgtcac 240acttgtttga agtgcagttt atctatcttt atacatatat ttaaacttta ctctacgaat 300aatataatct atagtactac aataatatca gtgttttaga gaatcatata aatgaacagt 360tagacatggt ctaaaggaca attgagtatt ttgacaacag gactctacag ttttatcttt 420ttagtgtgca tgtgttctcc tttttttttg caaatagctt cacctatata atacttcatc 480cattttatta gtacatccat ttagggttta gggttaatgg tttttataga ctaatttttt 540tagtacatct attttattct attttagcct ctaaattaag aaaactaaaa ctctatttta 600gtttttttat ttaatagttt agatataaaa tagaataaaa taaagtgact aaaaattaaa 660caaataccct ttaagaaatt aaaaaaacta aggaaacatt tttcttgttt cgagtagata 720atgccagcct gttaaacgcc gtcgacgagt ctaacggaca ccaaccagcg aaccagcagc 780gtcgcgtcgg gccaagcgaa gcagacggca cggcatctct gtcgctgcct ctggacccct 840ctcgagagtt ccgctccacc gttggacttg ctccgctgtc ggcatccaga aattgcgtgg 900cggagcggca gacgtgagcc ggcacggcag gcggcctcct cctcctctca cggcaccggc 960agctacgggg gattcctttc ccaccgctcc ttcgctttcc cttcctcgcc cgccgtaata 1020aatagacacc ccctccacac cctctttccc caacctcgtg ttgttcggag cgcacacaca 1080cacaaccaga tctcccccaa atccacccgt cggcacctcc gcttcaaggt acgccgctcg 1140tcctcccccc ccccccccct ctctaccttc tctagatcgg cgttccggtc catggttagg 1200gcccggtagt tctacttctg ttcatgtttg tgttagatcc gtgtttgtgt tagatccgtg 1260ctgctagcgt tcgtacacgg atgcgacctg tacgtcagac acgttctgat tgctaacttg 1320ccagtgtttc tctttgggga atcctgggat ggctctagcc gttccgcaga cgggatcgat 1380ttcatgattt tttttgtttc gttgcatagg gtttggtttg cccttttcct ttatttcaat 1440atatgccgtg cacttgtttg tcgggtcatc ttttcatgct tttttttgtc ttggttgtga 1500tgatgtggtc tggttgggcg gtcgttctag atcggagtag aattctgttt caaactacct 1560ggtggattta ttaattttgg atctgtatgt gtgtgccata catattcata gttacgaatt 1620gaagatgatg gatggaaata tcgatctagg ataggtatac atgttgatgc gggttttact 1680gatgcatata cagagatgct ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg 1740gtcgttcatt cgttctagat cggagtagaa tactgtttca aactacctgg tgtatttatt 1800aattttggaa ctgtatgtgt gtgtcataca tcttcatagt tacgagttta agatggatgg 1860aaatatcgat ctaggatagg tatacatgtt gatgtgggtt ttactgatgc atatacatga 1920tggcatatgc agcatctatt catatgctct aaccttgagt acctatctat tataataaac 1980aagtatgttt tataattatt tcgatcttga tatacttgga tgatggcata tgcagcagct 2040atatgtggat ttttttagcc ctgccttcat acgctattta tttgcttggt actgtttctt 2100ttgtcgatgc tcaccctgtt gtttggtgtt acttctgcag ggtacccccg ggtggtcagt 2160cccttatgtt acgtcctgta gaaaccccaa cccgtgaaat caaaaaactc gacggcctgt 2220gggcattcag tctggatcgc gaaaactgtg gaattgatca gcgttggtgg gaaagcgcgt 2280tacaagaaag ccgggcaatt gctgtgccag gcagttttaa cgatcagttc gccgatgcag 2340atattcgtaa ttatgcgggc aacgtctggt atcagcgcga agtctttata ccgaaaggtt 2400gggcaggcca gcgtatcgtg ctgcgtttcg atgcggtcac tcattacggc aaagtgtggg 2460tcaataatca ggaagtgatg gagcatcagg gcggctatac gccatttgaa gccgatgtca 2520cgccgtatgt tattgccggg aaaagtgtac gtaagtttct gcttctacct ttgatatata 2580tataataatt atcattaatt agtagtaata taatatttca aatatttttt tcaaaataaa 2640agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat tttaatttat 2700aacttttcta atatatgacc aaaatttgtt gatgtgcagg tatcaccgtt tgtgtgaaca 2760acgaactgaa ctggcagact atcccgccgg gaatggtgat taccgacgaa aacggcaaga 2820aaaagcagtc ttacttccat gatttcttta actatgccgg aatccatcgc agcgtaatgc 2880tctacaccac gccgaacacc tgggtggacg atatcaccgt ggtgacgcat gtcgcgcaag 2940actgtaacca cgcgtctgtt gactggcagg tggtggccaa tggtgatgtc agcgttgaac 3000tgcgtgatgc ggatcaacag gtggttgcaa ctggacaagg cactagcggg actttgcaag 3060tggtgaatcc gcacctctgg caaccgggtg aaggttatct ctatgaactg tgcgtcacag 3120ccaaaagcca gacagagtgt gatatctacc cgcttcgcgt cggcatccgg tcagtggcag 3180tgaagggcca acagttcctg attaaccaca aaccgttcta ctttactggc tttggtcgtc 3240atgaagatgc ggacttacgt ggcaaaggat tcgataacgt gctgatggtg cacgaccacg 3300cattaatgga ctggattggg gccaactcct accgtacctc gcattaccct tacgctgaag 3360agatgctcga ctgggcagat gaacatggca tcgtggtgat tgatgaaact gctgctgtcg 3420gctttaacct ctctttaggc attggtttcg aagcgggcaa caagccgaaa gaactgtaca 3480gcgaagaggc agtcaacggg gaaactcagc aagcgcactt acaggcgatt aaagagctga 3540tagcgcgtga caaaaaccac ccaagcgtgg tgatgtggag tattgccaac gaaccggata 3600cccgtccgca agtgcacggg aatatttcgc cactggcgga agcaacgcgt aaactcgacc 3660cgacgcgtcc gatcacctgc gtcaatgtaa tgttctgcga cgctcacacc gataccatca 3720gcgatctctt tgatgtgctg tgcctgaacc gttattacgg atggtatgtc caaagcggcg 3780atttggaaac ggcagagaag gtactggaaa aagaacttct ggcctggcag gagaaactgc 3840atcagccgat tatcatcacc gaatacggcg tggatacgtt agccgggctg cactcaatgt 3900acaccgacat gtggagtgaa gagtatcagt gtgcatggct ggatatgtat caccgcgtct 3960ttgatcgcgt cagcgccgtc gtcggtgaac aggtatggaa tttcgccgat tttgcgacct 4020cgcaaggcat attgcgcgtt ggcggtaaca agaaagggat cttcactcgc gaccgcaaac 4080cgaagtcggc ggcttttctg ctgcaaaaac gctggactgg catgaacttc ggtgaaaaac 4140cgcagcaggg aggcaaacaa tgaatcaaca actctcctgg cgcaccatcg tcggctacag 4200cctcgggaat tgctaccgag ctccctaggc ccgggcggac cgcattaaga agggccctgc 4260tcccttctac cctctggagg atggcaccgc tggcgagcag ctgcacaagg ccatgaagag 4320gtatgccctg gtgcctggca ccattgcctt caccgatgcc cacattgagg tggacatcac 4380ctatgccgag tacttcgaga tgtctgtgcg cctggccgag gccatgaaga ggtacggcct 4440gaacaccaac caccgcatcg tggtgtgctc tgagaactct ctactagttc gaccggcatg 4500ccctgcttta atgagatatg cgagacgcct atgatcgcat gatatttgct ttcaattctg 4560ttgtgcacgt tgtaaaaaac ctgagcatgt gtagctcaga tccttaccgc cggtttcggt 4620tcattctaat gaatatatca cccgttacta tcgtattttt atgaataata ttctccgttc 4680aatttactga ttgtccaagc tgcggccgcg gagcctgctt ttttgtacaa agttggcatt 4740ataaaaaagc attgctcatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa 4800atcattattt g 4811545053DNAArtificial sequencepTOI4 54caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgataagcaa 60tgctttctta taatgccaac tttgtacaag aaagctgggt cggcgcgcca agcttgcatg 120cctgcaggca tgcaagcttc cgcggctgca gtgcagcgtg acccggtcgt gcccctctct 180agagataatg agcattgcat gtctaagtta taaaaaatta ccacatattt tttttgtcac 240acttgtttga agtgcagttt atctatcttt atacatatat ttaaacttta ctctacgaat 300aatataatct atagtactac aataatatca gtgttttaga gaatcatata aatgaacagt 360tagacatggt ctaaaggaca attgagtatt ttgacaacag gactctacag ttttatcttt 420ttagtgtgca tgtgttctcc tttttttttg caaatagctt cacctatata atacttcatc 480cattttatta gtacatccat ttagggttta gggttaatgg tttttataga ctaatttttt 540tagtacatct attttattct attttagcct ctaaattaag aaaactaaaa ctctatttta 600gtttttttat ttaatagttt agatataaaa tagaataaaa taaagtgact aaaaattaaa 660caaataccct ttaagaaatt aaaaaaacta aggaaacatt tttcttgttt cgagtagata 720atgccagcct gttaaacgcc gtcgacgagt ctaacggaca ccaaccagcg aaccagcagc 780gtcgcgtcgg gccaagcgaa gcagacggca cggcatctct gtcgctgcct ctggacccct 840ctcgagagtt ccgctccacc gttggacttg ctccgctgtc ggcatccaga aattgcgtgg 900cggagcggca gacgtgagcc ggcacggcag gcggcctcct cctcctctca cggcaccggc 960agctacgggg gattcctttc ccaccgctcc ttcgctttcc cttcctcgcc cgccgtaata 1020aatagacacc ccctccacac cctctttccc caacctcgtg ttgttcggag cgcacacaca 1080cacaaccaga tctcccccaa atccacccgt cggcacctcc gcttcaaggt acgccgctcg 1140tcctcccccc ccccccccct ctctaccttc tctagatcgg cgttccggtc catggttagg 1200gcccggtagt tctacttctg ttcatgtttg tgttagatcc gtgtttgtgt tagatccgtg 1260ctgctagcgt tcgtacacgg atgcgacctg tacgtcagac acgttctgat tgctaacttg 1320ccagtgtttc tctttgggga atcctgggat ggctctagcc gttccgcaga cgggatcgat 1380ttcatgattt tttttgtttc gttgcatagg gtttggtttg cccttttcct ttatttcaat 1440atatgccgtg cacttgtttg tcgggtcatc ttttcatgct tttttttgtc ttggttgtga 1500tgatgtggtc tggttgggcg gtcgttctag atcggagtag aattctgttt caaactacct 1560ggtggattta ttaattttgg atctgtatgt gtgtgccata catattcata gttacgaatt 1620gaagatgatg gatggaaata tcgatctagg ataggtatac atgttgatgc gggttttact 1680gatgcatata cagagatgct ttttgttcgc ttggttgtga tgatgtggtg tggttgggcg 1740gtcgttcatt cgttctagat cggagtagaa tactgtttca aactacctgg tgtatttatt 1800aattttggaa ctgtatgtgt gtgtcataca tcttcatagt tacgagttta agatggatgg 1860aaatatcgat ctaggatagg tatacatgtt gatgtgggtt ttactgatgc atatacatga 1920tggcatatgc agcatctatt catatgctct aaccttgagt acctatctat tataataaac 1980aagtatgttt tataattatt tcgatcttga tatacttgga tgatggcata tgcagcagct 2040atatgtggat ttttttagcc ctgccttcat acgctattta tttgcttggt actgtttctt 2100ttgtcgatgc tcaccctgtt gtttggtgtt acttctgcag ggtacccccg ggtggtcagt 2160cccttatgtt acgtcctgta gaaaccccaa cccgtgaaat caaaaaactc gacggcctgt 2220gggcattcag tctggatcgc gaaaactgtg gaattgatca gcgttggtgg gaaagcgcgt 2280tacaagaaag ccgggcaatt gctgtgccag gcagttttaa cgatcagttc gccgatgcag 2340atattcgtaa ttatgcgggc aacgtctggt atcagcgcga agtctttata ccgaaaggtt 2400gggcaggcca gcgtatcgtg ctgcgtttcg atgcggtcac tcattacggc aaagtgtggg 2460tcaataatca ggaagtgatg gagcatcagg gcggctatac gccatttgaa gccgatgtca 2520cgccgtatgt tattgccggg aaaagtgtac gtaagtttct gcttctacct ttgatatata 2580tataataatt atcattaatt agtagtaata taatatttca aatatttttt tcaaaataaa 2640agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat tttaatttat 2700aacttttcta atatatgacc aaaatttgtt gatgtgcagg tatcaccgtt tgtgtgaaca 2760acgaactgaa ctggcagact atcccgccgg gaatggtgat taccgacgaa aacggcaaga 2820aaaagcagtc ttacttccat gatttcttta actatgccgg aatccatcgc agcgtaatgc 2880tctacaccac gccgaacacc tgggtggacg atatcaccgt ggtgacgcat gtcgcgcaag 2940actgtaacca cgcgtctgtt gactggcagg tggtggccaa tggtgatgtc agcgttgaac 3000tgcgtgatgc ggatcaacag gtggttgcaa ctggacaagg cactagcggg actttgcaag 3060tggtgaatcc gcacctctgg caaccgggtg aaggttatct ctatgaactg tgcgtcacag 3120ccaaaagcca gacagagtgt gatatctacc cgcttcgcgt cggcatccgg tcagtggcag 3180tgaagggcca acagttcctg attaaccaca aaccgttcta ctttactggc tttggtcgtc 3240atgaagatgc ggacttacgt ggcaaaggat tcgataacgt gctgatggtg cacgaccacg 3300cattaatgga ctggattggg gccaactcct accgtacctc gcattaccct tacgctgaag 3360agatgctcga ctgggcagat gaacatggca tcgtggtgat tgatgaaact gctgctgtcg 3420gctttaacct ctctttaggc attggtttcg aagcgggcaa caagccgaaa gaactgtaca 3480gcgaagaggc agtcaacggg gaaactcagc aagcgcactt acaggcgatt aaagagctga 3540tagcgcgtga caaaaaccac ccaagcgtgg tgatgtggag tattgccaac gaaccggata 3600cccgtccgca agtgcacggg aatatttcgc cactggcgga agcaacgcgt aaactcgacc 3660cgacgcgtcc gatcacctgc gtcaatgtaa tgttctgcga cgctcacacc gataccatca 3720gcgatctctt tgatgtgctg tgcctgaacc gttattacgg atggtatgtc caaagcggcg 3780atttggaaac ggcagagaag gtactggaaa aagaacttct ggcctggcag gagaaactgc 3840atcagccgat tatcatcacc gaatacggcg tggatacgtt agccgggctg cactcaatgt 3900acaccgacat gtggagtgaa gagtatcagt gtgcatggct ggatatgtat caccgcgtct 3960ttgatcgcgt cagcgccgtc gtcggtgaac aggtatggaa tttcgccgat tttgcgacct 4020cgcaaggcat attgcgcgtt ggcggtaaca agaaagggat cttcactcgc gaccgcaaac 4080cgaagtcggc ggcttttctg ctgcaaaaac gctggactgg catgaacttc ggtgaaaaac 4140cgcagcaggg aggcaaacaa tgaatcaaca actctcctgg cgcaccatcg tcggctacag 4200cctcgggaat tgctaccgag ctccgatcgt tcaaacattt ggcaataaag tttcttaaga 4260ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag 4320catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga 4380gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat 4440aaattatcgc gcgcggtgtc atctatgtta ctagatccgg accgcattaa gaagggccct 4500gctcccttct accctctgga ggatggcacc gctggcgagc agctgcacaa ggccatgaag 4560aggtatgccc tggtgcctgg caccattgcc ttcaccgatg cccacattga ggtggacatc 4620acctatgccg agtacttcga gatgtctgtg cgcctggccg aggccatgaa gaggtacggc 4680ctgaacacca accaccgcat cgtggtgtgc tctgagaact ctctactagt tcgaccggca 4740tgccctgctt taatgagata tgcgagacgc ctatgatcgc atgatatttg ctttcaattc 4800tgttgtgcac gttgtaaaaa acctgagcat gtgtagctca gatccttacc gccggtttcg 4860gttcattcta atgaatatat cacccgttac tatcgtattt ttatgaataa tattctccgt 4920tcaatttact gattgtccaa gctgcggccg cggagcctgc ttttttgtac aaagttggca 4980ttataaaaaa gcattgctca tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata 5040aaatcattat ttg 50535528DNAArtificial sequenceOligonucleotideprimer Loy482-NosT-upper-SalI 55aaatttgtcg accgatcggt caaacatt 285636DNAArtificial sequenceOligonucleotideprimer Loy483-NosT-Lower-HindIII 56aaatttaagc ttcccgatct agtaacatag atgaca 365731DNAArtificial sequenceOligonucleotideprimer Loy494- Gus_upper_SalI_ Spacer 57ttttagtcga cacgctggac tggcatgaac t 315840DNAArtificial sequenceOligonucleotideprimer Loy492-NosT-lower- BglII SpeI 58ttttaagatc tactagtccg atctagtaac atagatgaca 405931DNAArtificial sequenceOligonucleotideprimer Loy493_Gus_upper_SalI_ Spacer 59tttaagtcga caagtcggcg gcttttctgc t 316040DNAArtificial sequenceOligonucleotideprimer Loy492-NosT-lower- BglII SpeI 60ttttaagatc tactagtccg atctagtaac atagatgaca 406144DNAArtificial sequenceOligonucleotideprimer JMTOIprim1 61ggttccaagg taccaaaaca atgggcgctg atgatgttgt tgat 446242DNAArtificial sequenceOligonucleotideprimer JMTOIprim2 62aaggtagaag cagaaactta cctggatacg tcactttgac ca 426342DNAArtificial sequenceOligonucleotideprimer JMTOIprim3 63tggtcaaagt gacgtatcca ggtaagtttc tgcttctacc tt 426437DNAArtificial sequenceOligonucleotideprimer JMTOIprim4 64ggttccaagg atccatttat tttgaaaaaa atatttg 376535DNAArtificial sequenceOligonucleotideprimer JMTOIprim5 65ggttccaagg atccagtata tagcaattgc ttttc 356683DNAArtificial sequenceOligonucleotideprimer JMTOIprim6 66cgagaacctt cgtcagtcct gcacatcaac aaattttggt cataaaaaaa aaaatattag 60aaaagttata aattaaaata tac 836763DNAArtificial sequenceOligonucleotideprimer JMTOIprim7 67ctaatatttt tttttttatg accaaaattt gttgatgtgc aggactgacg aaggttctcg 60cac 636838DNAArtificial

sequenceOligonucleotideprimer JMTOIprim8 68ttggaaccac tagtttatcg cctgacacga tttcctgc 386935DNAArtificial sequenceOligonucleotideprimer JMTOIprim9 69ggttccaagg atccgatcgt tcaaacattt ggcaa 357035DNAArtificial sequenceOligonucleotideprimer JMTOIprim10 70ggttccaagg atccgatcta gtaacataga tgaca 35713030DNAArtificial sequencepJMTOI1 71tgcagtgcag cgtgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa 60gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 120ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 180atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 240tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 300tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 360tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 420gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata gtttagatat 480aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 540actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 600gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 660ggcacggcat ctctgtcgct gcctctggac ccctctcgag agttccgctc caccgttgga 720cttgctccgc tgtcggcatc cagaaattgc gtggcggagc ggcagacgtg agccggcacg 780gcaggcggcc tcctcctcct ctcacggcac cggcagctac gggggattcc tttcccaccg 840ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ccctctctac 1020cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact tctgttcatg 1080tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac acggatgcga 1140cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg gggaatcctg 1200ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg tttcgttgca 1260tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg tttgtcgggt 1320catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt 1380ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt ttggatctgt 1440atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga aatatcgatc 1500taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga tgctttttgt 1560tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct agatcggagt 1620agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca 1680tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga taggtataca 1740tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc tattcatatg 1800ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat tatttcgatc 1860ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt agccctgcct 1920tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg 1980tgttacttct gcagggtacc aaaacaatgg gcgctgatga tgttgttgat tcttctaaat 2040cttttgtgat ggaaaacttt tcttcgtacc acgggactaa acctggttat gtagattcca 2100ttcaaaaagg tatacaaaag ccaaaatctg gtacacaagg aaattatgac gatgattgga 2160aagggtttta tagtaccgac aataaatacg acgctgcggg atactctgta gataatgaaa 2220acccgctctc tggaaaagct ggaggcgtgg tcaaagtgac gtatccaggt aagtttctgc 2280ttctaccttg taagtttctg cttctacctt tgatatatat ataataatta tcattaatta 2340gtagtaatat aatatttcaa atattttttt caaaataaat ggatccagta tatagcaatt 2400gcttttctgt agtttataag tgtgtatatt ttaatttata acttttctaa tatttttttt 2460tttatgacca aaatttgttg atgtgcagga ctgacgaagg ttctcgcact aaaagtggat 2520aatgccgaaa ctattaagaa agagttaggt ttaagtctca ctgaaccgtt gatggagcaa 2580gtcggaacgg aagagtttat caaaaggttc ggtgatggtg cttcgcgtgt agtgctcagc 2640cttcccttcg ctgaggggag ttctagcgtt gaatatatta ataactggga acaggcgaaa 2700gcgttaagcg tagaacttga gattaatttt gaaacccgtg gaaaacgtgg ccaagatgcg 2760atgtatgagt atatggctca agcctgtgca ggaaatcgtg tcaggcgata aactagttcg 2820accggcatgc cctgctttaa tgagatatgc gagacgccta tgatcgcatg atatttgctt 2880tcaattctgt tgtgcacgtt gtaaaaaacc tgagcatgtg tagctcagat ccttaccgcc 2940ggtttcggtt cattctaatg aatatatcac ccgttactat cgtattttta tgaataatat 3000tctccgttca atttactgat tgtccaagct 3030723289DNAArtificial sequencepJMTOI2 72tgcagtgcag cgtgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa 60gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 120ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 180atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 240tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 300tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 360tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 420gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata gtttagatat 480aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 540actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 600gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 660ggcacggcat ctctgtcgct gcctctggac ccctctcgag agttccgctc caccgttgga 720cttgctccgc tgtcggcatc cagaaattgc gtggcggagc ggcagacgtg agccggcacg 780gcaggcggcc tcctcctcct ctcacggcac cggcagctac gggggattcc tttcccaccg 840ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ccctctctac 1020cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact tctgttcatg 1080tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac acggatgcga 1140cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg gggaatcctg 1200ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg tttcgttgca 1260tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg tttgtcgggt 1320catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt 1380ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt ttggatctgt 1440atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga aatatcgatc 1500taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga tgctttttgt 1560tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct agatcggagt 1620agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca 1680tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga taggtataca 1740tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc tattcatatg 1800ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat tatttcgatc 1860ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt agccctgcct 1920tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg 1980tgttacttct gcagggtacc aaaacaatgg gcgctgatga tgttgttgat tcttctaaat 2040cttttgtgat ggaaaacttt tcttcgtacc acgggactaa acctggttat gtagattcca 2100ttcaaaaagg tatacaaaag ccaaaatctg gtacacaagg aaattatgac gatgattgga 2160aagggtttta tagtaccgac aataaatacg acgctgcggg atactctgta gataatgaaa 2220acccgctctc tggaaaagct ggaggcgtgg tcaaagtgac gtatccaggt aagtttctgc 2280ttctaccttg taagtttctg cttctacctt tgatatatat ataataatta tcattaatta 2340gtagtaatat aatatttcaa atattttttt caaaataaat ggatccgatc gttcaaacat 2400ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata 2460atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat 2520gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa 2580aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg 2640gatccagtat atagcaattg cttttctgta gtttataagt gtgtatattt taatttataa 2700cttttctaat attttttttt ttatgaccaa aatttgttga tgtgcaggac tgacgaaggt 2760tctcgcacta aaagtggata atgccgaaac tattaagaaa gagttaggtt taagtctcac 2820tgaaccgttg atggagcaag tcggaacgga agagtttatc aaaaggttcg gtgatggtgc 2880ttcgcgtgta gtgctcagcc ttcccttcgc tgaggggagt tctagcgttg aatatattaa 2940taactgggaa caggcgaaag cgttaagcgt agaacttgag attaattttg aaacccgtgg 3000aaaacgtggc caagatgcga tgtatgagta tatggctcaa gcctgtgcag gaaatcgtgt 3060caggcgataa actagttcga ccggcatgcc ctgctttaat gagatatgcg agacgcctat 3120gatcgcatga tatttgcttt caattctgtt gtgcacgttg taaaaaacct gagcatgtgt 3180agctcagatc cttaccgccg gtttcggttc attctaatga atatatcacc cgttactatc 3240gtatttttat gaataatatt ctccgttcaa tttactgatt gtccaagct 3289733680DNAArtificial sequencepJMTOI3 73tgcagtgcag cgtgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa 60gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 120ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 180atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 240tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 300tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 360tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 420gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata gtttagatat 480aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 540actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 600gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 660ggcacggcat ctctgtcgct gcctctggac ccctctcgag agttccgctc caccgttgga 720cttgctccgc tgtcggcatc cagaaattgc gtggcggagc ggcagacgtg agccggcacg 780gcaggcggcc tcctcctcct ctcacggcac cggcagctac gggggattcc tttcccaccg 840ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ccctctctac 1020cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact tctgttcatg 1080tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac acggatgcga 1140cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg gggaatcctg 1200ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg tttcgttgca 1260tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg tttgtcgggt 1320catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt 1380ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt ttggatctgt 1440atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga aatatcgatc 1500taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga tgctttttgt 1560tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct agatcggagt 1620agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca 1680tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga taggtataca 1740tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc tattcatatg 1800ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat tatttcgatc 1860ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt agccctgcct 1920tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg 1980tgttacttct gcagggtacc atggctcagt caaagcacgg tctaacaaaa gaaatgacaa 2040tgaaataccg tatggaaggg tgcgtcgatg gacataaatt tgtgatcacg ggagagggca 2100ttggatatcc gttcaaaggg aaacaggcta ttaatctgtg tgtggtcgaa ggtggaccat 2160tgccatttgc cgaagacata ttgtcagctg cctttatgta cggaaacagg gttttcactg 2220aatatcctca agacatagct gactatttca agaactcgtg tcctgctggt tatacatggg 2280acaggtcttt tctctttgag gatggagcag tttgcatatg taatgcagat ataacagtga 2340gtgttgaaga aaactgcatg tatcatgagt ccaaatttta tggagtgaat tttcctgctg 2400atggacctgt gatgaaaaag atgacagata actgggagcc atcctgcgag aagatcatac 2460cagtacctaa gcaggggata ttgaaagggg atgtctccat gtacctcctt ctgaaggatg 2520gtgggcgttt acggtgccaa ttcgacacag tttacaaagc aaagtctgtg ccaagaaaga 2580tgccggactg gcacttcatc cagcataagc tcacccgtga agaccgcagc gatgctaaga 2640atcagaaatg gcatctgaca gaacatgcta ttgcatccgg atctgcattg ccctgaggat 2700ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 2760cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 2820gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 2880acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 2940ctatgttact agatcggatc catcaccgtt tgtgtgaaca acgaactgaa ctggcagact 3000atcccgccgg gaatggtgat taccgacgaa aacggcaaga aaaagcagtc ttacttccat 3060gatttcttta actatgccgg aatccatcgc agcgtaatgc tctacaccac gccgaacacc 3120tgggtggacg atatcaccgt ggtgacgcat gtcgcgcaag actgtaacca cgcgtctgtt 3180gactggcagg tggtggccaa tggtgatgtc agcgttgaac tgcgtgatgc ggatcaacag 3240gtggttgcaa ctggacaagg cactagcggg actttgcaag tggtgaatcc gcacctctgg 3300caaccgggtg aaggttatct ctatgaactg tgcgtcacag ccaaaagcca gacagagtgt 3360gatatctacc cgcttcgcgt cggcatccgg tcagtggcag tgaagggcga acagttcctg 3420attaaccaca aaccgttcta ctttactggc tttggtcgtc aactagttcg accggcatgc 3480cctgctttaa tgagatatgc gagacgccta tgatcgcatg atatttgctt tcaattctgt 3540tgtgcacgtt gtaaaaaacc tgagcatgtg tagctcagat ccttaccgcc ggtttcggtt 3600cattctaatg aatatatcac ccgttactat cgtattttta tgaataatat tctccgttca 3660atttactgat tgtccaagct 3680743421DNAArtificial sequencepJMTOI4 74tgcagtgcag cgtgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa 60gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 120ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 180atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 240tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 300tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 360tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 420gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata gtttagatat 480aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 540actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 600gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 660ggcacggcat ctctgtcgct gcctctggac ccctctcgag agttccgctc caccgttgga 720cttgctccgc tgtcggcatc cagaaattgc gtggcggagc ggcagacgtg agccggcacg 780gcaggcggcc tcctcctcct ctcacggcac cggcagctac gggggattcc tttcccaccg 840ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ccctctctac 1020cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact tctgttcatg 1080tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac acggatgcga 1140cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg gggaatcctg 1200ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg tttcgttgca 1260tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg tttgtcgggt 1320catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt 1380ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt ttggatctgt 1440atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga aatatcgatc 1500taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga tgctttttgt 1560tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct agatcggagt 1620agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca 1680tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga taggtataca 1740tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc tattcatatg 1800ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat tatttcgatc 1860ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt agccctgcct 1920tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg 1980tgttacttct gcagggtacc atggctcagt caaagcacgg tctaacaaaa gaaatgacaa 2040tgaaataccg tatggaaggg tgcgtcgatg gacataaatt tgtgatcacg ggagagggca 2100ttggatatcc gttcaaaggg aaacaggcta ttaatctgtg tgtggtcgaa ggtggaccat 2160tgccatttgc cgaagacata ttgtcagctg cctttatgta cggaaacagg gttttcactg 2220aatatcctca agacatagct gactatttca agaactcgtg tcctgctggt tatacatggg 2280acaggtcttt tctctttgag gatggagcag tttgcatatg taatgcagat ataacagtga 2340gtgttgaaga aaactgcatg tatcatgagt ccaaatttta tggagtgaat tttcctgctg 2400atggacctgt gatgaaaaag atgacagata actgggagcc atcctgcgag aagatcatac 2460cagtacctaa gcaggggata ttgaaagggg atgtctccat gtacctcctt ctgaaggatg 2520gtgggcgttt acggtgccaa ttcgacacag tttacaaagc aaagtctgtg ccaagaaaga 2580tgccggactg gcacttcatc cagcataagc tcacccgtga agaccgcagc gatgctaaga 2640atcagaaatg gcatctgaca gaacatgcta ttgcatccgg atctgcattg ccctgaggat 2700ccatcaccgt ttgtgtgaac aacgaactga actggcagac tatcccgccg ggaatggtga 2760ttaccgacga aaacggcaag aaaaagcagt cttacttcca tgatttcttt aactatgccg 2820gaatccatcg cagcgtaatg ctctacacca cgccgaacac ctgggtggac gatatcaccg 2880tggtgacgca tgtcgcgcaa gactgtaacc acgcgtctgt tgactggcag gtggtggcca 2940atggtgatgt cagcgttgaa ctgcgtgatg cggatcaaca ggtggttgca actggacaag 3000gcactagcgg gactttgcaa gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc 3060tctatgaact gtgcgtcaca gccaaaagcc agacagagtg tgatatctac ccgcttcgcg 3120tcggcatccg gtcagtggca gtgaagggcg aacagttcct gattaaccac aaaccgttct 3180actttactgg ctttggtcgt caactagttc gaccggcatg ccctgcttta atgagatatg 3240cgagacgcct atgatcgcat gatatttgct ttcaattctg ttgtgcacgt tgtaaaaaac 3300ctgagcatgt gtagctcaga tccttaccgc cggtttcggt tcattctaat gaatatatca 3360cccgttacta tcgtattttt atgaataata ttctccgttc aatttactga ttgtccaagc 3420t 3421753160DNAArtificial sequencepJMTOI5 75tgcagtgcag cgtgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa 60gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 120ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 180atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 240tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 300tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 360tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 420gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata gtttagatat 480aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 540actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 600gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 660ggcacggcat ctctgtcgct gcctctggac ccctctcgag agttccgctc caccgttgga 720cttgctccgc tgtcggcatc cagaaattgc gtggcggagc ggcagacgtg agccggcacg 780gcaggcggcc tcctcctcct ctcacggcac cggcagctac gggggattcc tttcccaccg 840ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960ccgtcggcac

ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ccctctctac 1020cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact tctgttcatg 1080tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac acggatgcga 1140cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg gggaatcctg 1200ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg tttcgttgca 1260tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg tttgtcgggt 1320catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt 1380ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt ttggatctgt 1440atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga aatatcgatc 1500taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga tgctttttgt 1560tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct agatcggagt 1620agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca 1680tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga taggtataca 1740tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc tattcatatg 1800ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat tatttcgatc 1860ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt agccctgcct 1920tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg 1980tgttacttct gcagggtacc ctgtaaccac gcgtctgttg actggcaggt ggtggccaat 2040ggtgatgtca gcgttgaact gcgtgatgcg gatcaacagg tggttgcaac tggacaaggc 2100actagcggga ctttgcaagt ggtgaatccg cacctctggc aaccgggtga aggttatctc 2160tatgaactgt gcgtcacagc caaaagccag acagagtgtg atatctaccc gcttcgcgtc 2220ggcatccggt cagtggcagt gaagggcgaa cagttcctga ttaaccacaa accgttctac 2280tttactggct ttggtcgtca ggcgcgccta ctccccgagt ctgaaccgct cgatcgaacc 2340tcactcaaca ttgacgagag cgagtcgtct aactcttgca gcctctttgc gtcgtgaaag 2400ctctctggaa gaagaagatt ctcatcagga tccccgagaa actcggataa gagactcaca 2460gagtagccat gctcggtcac aacgctacaa tggtccaagc tcgtaagctc gaagccgaag 2520tccagatctg tgctggcgaa gtcgatgtcg gacttaagaa cttcctccaa atggcggtcg 2580cagtcttcaa attcgctcga gtgacgacca aagccagtaa agtagaacgg tttgtggtta 2640atcaggaact gttcgccctt cactgccact gaccggatgc cgacgcgaag cgggtagata 2700tcacactctg tctggctttt ggctgtgacg cacagttcat agagataacc ttcacccggt 2760tgccagaggt gcggattcac cacttgcaaa gtcccgctag tgccttgtcc agttgcaacc 2820acctgttgat ccgcatcacg cagttcaacg ctgacatcac cattggccac cacctgccag 2880tcaacagacg cgtggttaca gactagtgat cgttcaaaca tttggcaata aagtttctta 2940agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt 3000aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt 3060agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag 3120gataaattat cgcgcgcggt gtcatctatg ttactagatc 3160762665DNAArtificial sequenceLo376-pENTR-B2 76ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 660agcaggctgg tacccgggga tcctctaggt cgaccagatc tgatatctgc ggccgcctcg 720agcatatggg catgcaagct tggcgtaatc atggacccag ctttcttgta caaagttggc 780attataagaa agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat 840aaaatcatta tttgccatcc agctgatccg gtgcaggttg gcggcggcgt gcgtagcgaa 900gaatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg 960tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa 1020aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa 1080cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc 1140gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc 1200cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg 1260tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta 1320ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat 1380tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc 1440ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg 1500ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc 1560gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac 1620cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga 1680aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg 1740ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa 1800aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt 1860ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac 1920gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag 1980cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 2040tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 2100agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 2160ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 2220acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 2280ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 2340gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 2400gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 2460gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 2520tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 2580caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 2640tttgctggcc ttttgctcac atgtt 2665777686DNAArtificial sequenceLo442 pSUN1-R4R3-M20 (OCS10) (destination vector) 77ttccatggac atacaaatgg acgaacggat aaaccttttc acgccctttt aaatatccga 60ttattctaat aaacgctctt ttctcttagg tttacccgcc aatatatcct gtcaaacact 120gatagtttaa actgaaggcg ggaaacgaca atcagatcta gtaggaaaca gctatgacca 180tgattacgcc aagctatcga ttacgccaag ctatcaactt tgtatagaaa agttgaacga 240gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact 300acataatact gtaaaacaca acatatccag tcactatggt cgacctgcag actggctgtg 360tataagggag cctgacattt atattcccca gaacatcagg ttaatggcgt ttttgatgtc 420attttcgcgg tggctgagat cagccacttc ttccccgata acggagaccg gcacactggc 480catatcggtg gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc 540acgggggact ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc 600gggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt 660ataggtgtaa accttaaact gcatttcacc agcccctgtt ctcgtcggca aaagagccgt 720tcatttcaat aaaccgggcg acctcagcca tcccttcctg attttccgct ttccagcgtt 780cggcacgcag acgacgggct tcattctgca tggttgtgct taccgaaccg gagatattga 840catcatatat gccttgagca actgatagct gtcgctgtca actgtcactg taatacgctg 900cttcatagca tacctctttt tgacatactt cgggtataca tatcagtata tattcttata 960ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg cttttagtaa gccggatcct 1020ctagattacg ccccgcctgc cactcatcgc agtactgttg taattcatta agcattctgc 1080cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc atcagcacct 1140tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat 1200tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca 1260tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt 1320gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa 1380acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca 1440gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg cgggcaagaa 1500tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg 1560taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa 1620aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg atttttttct 1680ccattttagc ttccttagct cctgaaaatc tcgacggatc ctaactcaaa atccacacat 1740tatacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgcggcc gccatagtga 1800ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa 1860tatattgata tttatatcat tttacgtttc tcgttcaact ttattataca tagttgataa 1920ttcactggcc ggatctgctt ggtaataatt gtcattagat tgtttttatg catagatgca 1980ctcgaaatca gccaatttta gacaagtatc aaacggatgt taattcagta cattaaagac 2040gtccgcaatg tgttattaag ttgtctaagc gtcaatttgt ttacaccaca atatatcctg 2100ccaccagcca gccaacagct ccccgaccgg cagctcggca caaaatcacc acgcgttacc 2160accacgccgg ccggccgcat ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt 2220tccctaatca tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag 2280tttggccccc gccctaccct caccccggca cagatcgcgc acgcccgcga gctgatcgac 2340caggaaggcc gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg 2400taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc 2460ttccgtgagg acgcattgac cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa 2520gaggaacaag catgaaaccg caccaggacg gccaggacga accgtttttc attaccgaag 2580agatcgaggc ggagatgatc gcggccgggt acgtgttcga gccgcccgcg cacgtctcaa 2640ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc caagctggcg gcctggccgg 2700ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt 2760aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac 2820gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt caggcaagac 2880gaccatcgca acccatctag cccgcgccct gcaactcgcc ggggccgatg ttctgttagt 2940cgattccgat ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct 3000aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg 3060cgacttcgta gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa 3120ggcagccgac ttcgtgctga ttccggtgca gccaagccct tacgacatat gggccaccgc 3180cgacctggtg gagctggtta agcagcgcat tgaggtcacg gatggaaggc tacaagcggc 3240ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg ccgaggcgct 3300ggccgggtac gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga gctacccagg 3360cactgccgcc gccggcacaa ccgttcttga atcagaaccc gagggcgacg ctgcccgcga 3420ggtccaggcg ctggccgctg aaattaaatc aaaactcatt tgagttaatg aggtaaagag 3480aaaatgagca aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg cagcagcaag 3540gctgcaacgt tggccagcct ggcagacacg ccagccatga agcgggtcaa ctttcagttg 3600ccggcggagg atcacaccaa gctgaagatg tacgcggtac gccaaggcaa gaccattacc 3660gagctgctat ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg aataaatgag 3720tagatgaatt ttagcggcta aaggaggcgg catggaaaat caagaacaac caggcaccga 3780cgccgtggaa tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa gcggctgggt 3840tgtctgccgg ccctgcaatg gcactggaac ccccaagccc gaggaatcgg cgtgagcggt 3900cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg gtgatgacct ggtggagaag 3960ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg ccccggtgaa 4020tcgtggcaag cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc ggcagccggt 4080gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac cagatttttt cgttccgatg 4140ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt tttccgtctg 4200tcgaagcgtg accgacgagc tggcgaggtg atccgctacg agcttccaga cgggcacgta 4260gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct ggtactgatg 4320gcggtttccc atctaaccga atccatgaac cgataccggg aagggaaggg agacaagccc 4380ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt tctgccggcg agccgatggc 4440ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac gcacgttgcc 4500atgcagcgta cgaagaaggc caagaacggc cgcctggtga cggtatccga gggtgaagcc 4560ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta catcgagatc 4620gagctagctg attggatgta ccgcgagatc acagaaggca agaacccgga cgtgctgacg 4680gttcaccccg attacttttt gatcgatccc ggcatcggcc gttttctcta ccgcctggca 4740cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta cgaacgcagt 4800ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat cgggtcaaat 4860gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat cctagtcatg 4920cgctaccgca acctgatcga gggcgaagca tccgccggtt cctaatgtac ggagcagatg 4980ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag gtctctttcc tgtggatagc 5040acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat tgggaaccca 5100aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga gaaaaaaggc 5160gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg cctggcctgt 5220gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac ccttcggtcg 5280ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg ccgctcaaaa 5340atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc gtcgccactc 5400gaccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg acggtgaaaa 5460cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag 5520cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac 5580ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt 5640gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 5700cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 5760cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 5820aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 5880gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 5940tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 6000agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 6060ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 6120taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 6180gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 6240gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 6300ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 6360ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 6420gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 6480caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 6540taagggattt tggtcatgca tgatatatct cccaatttgt gtagggctta ttatgcacgc 6600ttaaaaataa taaaagcaga cttgacctga tagtttggct gtgagcaatt atgtgcttag 6660tgcatctaac gcttgagtta agccgcgccg cgaagcggcg tcggcttgaa cgaatttcta 6720gctagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct 6780tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta 6840gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg 6900acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc 6960actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca 7020tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga 7080cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg 7140atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc 7200agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact 7260tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg 7320atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata 7380tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac 7440gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg 7500gcgatcaccg cttcccccat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta 7560acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg 7620gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc 7680actgcg 76867810414DNAArtificial sequencebinary vector Lo239-pSUN3-GWs-B1- BnAK700::GUS::nosT-B2 78atcacaagtt tgtacaaaaa agcaggcttt aaaggaacca attcagtcga cacgaagctt 60tcgaggctaa cgttggtgga gatgttaagg cacggcatct tctcttcttc ttcttttttg 120tttgtttgtt tgtttcgctc tctatcactc actcgatcga tccccagtct tcttttcggg 180aaagctgtgt cagaccaatt atattccatc tttaatttgg ccctttttat tattcgctat 240ggcccaacat ttcaggccca atttatacga ttataaatat aaccacttga ttgtgctgcc 300gctcagctga atagtactcc ctccgttttt taatataaat cgttttacag ttatgcacgt 360aaattaagaa aaccattaac tttttatatt ttctaaacaa aaacatcatt aattatttac 420ttactcacaa ttcaaccaat agaaaaatag aagatatatt accattggtc atacaacatt 480aattattaat aaattttaca tagaaaaccg aaaacgacat ataatttgga acaaaaaaat 540ttctctaaaa ctacttatat taaaaaacgg agagagtata tttaactagt tcgtcttact 600gacaaattga agaggcagac caagacacgc ggctcctcga tggctgtctt atgccaagtg 660gcggtgcctg cttctgcatt aaataggtag aagaactcta attacagaag ctcgaggtag 720ataggatccc cgggtaggtc agtcccttat gttacgtcct gtagaaaccc caacccgtga 780aatcaaaaaa ctcgacggcc tgtgggcatt cagtctggat cgcgaaaact gtggaattga 840tcagcgttgg tgggaaagcg cgttacaaga aagccgggca attgctgtgc caggcagttt 900taacgatcag ttcgccgatg cagatattcg taattatgcg ggcaacgtct ggtatcagcg 960cgaagtcttt ataccgaaag gttgggcagg ccagcgtatc gtgctgcgtt tcgatgcggt 1020cactcattac ggcaaagtgt gggtcaataa tcaggaagtg atggagcatc agggcggcta 1080tacgccattt gaagccgatg tcacgccgta tgttattgcc gggaaaagtg tacgtatcac 1140cgtttgtgtg aacaacgaac tgaactggca gactatcccg ccgggaatgg tgattaccga 1200cgaaaacggc aagaaaaagc agtcttactt ccatgatttc tttaactatg ccggaatcca 1260tcgcagcgta atgctctaca ccacgccgaa cacctgggtg gacgatatca ccgtggtgac 1320gcatgtcgcg caagactgta accacgcgtc tgttgactgg caggtggtgg ccaatggtga 1380tgtcagcgtt gaactgcgtg atgcggatca acaggtggtt gcaactggac aaggcactag 1440cgggactttg caagtggtga atccgcacct ctggcaaccg ggtgaaggtt atctctatga 1500actgtgcgtc acagccaaaa gccagacaga gtgtgatatc tacccgcttc gcgtcggcat 1560ccggtcagtg gcagtgaagg gcgaacagtt cctgattaac cacaaaccgt tctactttac 1620tggctttggt cgtcatgaag atgcggactt gcgtggcaaa ggattcgata acgtgctgat 1680ggtgcacgac cacgcattaa tggactggat tggggccaac tcctaccgta cctcgcatta 1740cccttacgct gaagagatgc tcgactgggc agatgaacat ggcatcgtgg tgattgatga 1800aactgctgct gtcggcttta acctctcttt aggcattggt ttcgaagcgg gcaacaagcc 1860gaaagaactg tacagcgaag aggcagtcaa cggggaaact cagcaagcgc acttacaggc 1920gattaaagag ctgatagcgc gtgacaaaaa ccacccaagc gtggtgatgt ggagtattgc 1980caacgaaccg gatacccgtc cgcaaggtgc acgggaatat ttcgcgccac tggcggaagc 2040aacgcgtaaa ctcgacccga cgcgtccgat cacctgcgtc aatgtaatgt tctgcgacgc 2100tcacaccgat accatcagcg atctctttga tgtgctgtgc ctgaaccgtt attacggatg 2160gtatgtccaa agcggcgatt tggaaacggc agagaaggta

ctggaaaaag aacttctggc 2220ctggcaggag aaactgcatc agccgattat catcaccgaa tacggcgtgg atacgttagc 2280cgggctgcac tcaatgtaca ccgacatgtg gagtgaagag tatcagtgtg catggctgga 2340tatgtatcac cgcgtctttg atcgcgtcag cgccgtcgtc ggtgaacagg tatggaattt 2400cgccgatttt gcgacctcgc aaggcatatt gcgcgttggc ggtaacaaga aagggatctt 2460cactcgcgac cgcaaaccga agtcggcggc ttttctgctg caaaaacgct ggactggcat 2520gaacttcggt gaaaaaccgc agcagggagg caaacaatga atcaacaact ctcctggcgc 2580accatcgtcg gctacagcct cgggaattgc taccgagctc gaatttcccc gatcgttcaa 2640acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca 2700tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat 2760ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa 2820acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag 2880atcgggaatt ctagacccag ctttcttgta caaagtggtg ataattcact ggccgtcgtt 2940ttacaacgac tcagagcttg acaggaggcc cgatctagta acatagatga caccgcgcgc 3000gataatttat cctagtttgc gcgctatatt ttgttttcta tcgcgtatta aatgtataat 3060tgcgggactc taatcataaa aacccatctc ataaataacg tcatgcatta catgttaatt 3120attacatgct taacgtaatt caacagaaat tatatgataa tcatcgcaag accggcaaca 3180ggattcaatc ttaagaaact ttattgccaa atgtttgaac gatcggggat catccgggtc 3240tgtggcggga actccacgaa aatatccgaa cgcagcaaga tctagagctt gggtcccgct 3300cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata 3360ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg 3420gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat 3480ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg 3540acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg 3600agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta 3660cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc 3720gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga 3780gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca 3840gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc 3900gctgcctcgt cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc 3960gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt 4020gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca 4080tcttgttcaa tcatgcgaaa cgatccagat ccggtgcaga ttatttggat tgagagtgaa 4140tatgagactc taattggata ccgaggggaa tttatggaac gtcagtggag catttttgac 4200aagaaatatt tgctagctga tagtgacctt aggcgacttt tgaacgcgca ataatggttt 4260ctgacgtatg tgcttagctc attaaactcc agaaacccgc ggctgagtgg ctccttcaac 4320gttgcggttc tgtcagttcc aaacgtaaaa cggcttgtcc cgcgtcatcg gcgggggtca 4380taacgtgact cccttaattc tccgctcatg atcagattgt cgtttcccgc cttcagttta 4440aactatcagt gtttgacagg atcctgcttg gtaataattg tcattagatt gtttttatgc 4500atagatgcac tcgaaatcag ccaattttag acaagtatca aacggatgtt aattcagtac 4560attaaagacg tccgcaatgt gttattaagt tgtctaagcg tcaatttgtt tacaccacaa 4620tatatcctgc caccagccag ccaacagctc cccgaccggc agctcggcac aaaatcacca 4680cgcgttacca ccacgccggc cggccgcatg gtgttgaccg tgttcgccgg cattgccgag 4740ttcgagcgtt ccctaatcat cgaccgcacc cggagcgggc gcgaggccgc caaggcccga 4800ggcgtgaagt ttggcccccg ccctaccctc accccggcac agatcgcgca cgcccgcgag 4860ctgatcgacc aggaaggccg caccgtgaaa gaggcggctg cactgcttgg cgtgcatcgc 4920tcgaccctgt accgcgcact tgagcgcagc gaggaagtga cgcccaccga ggccaggcgg 4980cgcggtgcct tccgtgagga cgcattgacc gaggccgacg ccctggcggc cgccgagaat 5040gaacgccaag aggaacaagc atgaaaccgc accaggacgg ccaggacgaa ccgtttttca 5100ttaccgaaga gatcgaggcg gagatgatcg cggccgggta cgtgttcgag ccgcccgcgc 5160acgtctcaac cgtgcggctg catgaaatcc tggccggttt gtctgatgcc aagctggcgg 5220cctggccggc cagcttggcc gctgaagaaa ccgagcgccg ccgtctaaaa aggtgatgtg 5280tatttgagta aaacagcttg cgtcatgcgg tcgctgcgta tatgatgcga tgagtaaata 5340aacaaatacg caaggggaac gcatgaaggt tatcgctgta cttaaccaga aaggcgggtc 5400aggcaagacg accatcgcaa cccatctagc ccgcgccctg caactcgccg gggccgatgt 5460tctgttagtc gattccgatc cccagggcag tgcccgcgat tgggcggccg tgcgggaaga 5520tcaaccgcta accgttgtcg gcatcgaccg cccgacgatt gaccgcgacg tgaaggccat 5580cggccggcgc gacttcgtag tgatcgacgg agcgccccag gcggcggact tggctgtgtc 5640cgcgatcaag gcagccgact tcgtgctgat tccggtgcag ccaagccctt acgacatatg 5700ggccaccgcc gacctggtgg agctggttaa gcagcgcatt gaggtcacgg atggaaggct 5760acaagcggcc tttgtcgtgt cgcgggcgat caaaggcacg cgcatcggcg gtgaggttgc 5820cgaggcgctg gccgggtacg agctgcccat tcttgagtcc cgtatcacgc agcgcgtgag 5880ctacccaggc actgccgccg ccggcacaac cgttcttgaa tcagaacccg agggcgacgc 5940tgcccgcgag gtccaggcgc tggccgctga aattaaatca aaactcattt gagttaatga 6000ggtaaagaga aaatgagcaa aagcacaaac acgctaagtg ccggccgtcc gagcgcacgc 6060agcagcaagg ctgcaacgtt ggccagcctg gcagacacgc cagccatgaa gcgggtcaac 6120tttcagttgc cggcggagga tcacaccaag ctgaagatgt acgcggtacg ccaaggcaag 6180accattaccg agctgctatc tgaatacatc gcgcagctac cagagtaaat gagcaaatga 6240ataaatgagt agatgaattt tagcggctaa aggaggcggc atggaaaatc aagaacaacc 6300aggcaccgac gccgtggaat gccccatgtg tggaggaacg ggcggttggc caggcgtaag 6360cggctgggtt gtctgccggc cctgcaatgg cactggaacc cccaagcccg aggaatcggc 6420gtgagcggtc gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg 6480gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc 6540cccggtgaat cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg 6600gcagccggtg cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc 6660gttccgatgc tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt 6720ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac 6780gggcacgtag aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg 6840gtactgatgg cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga 6900gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga 6960gccgatggcg gaaagcagaa agacgacctg gtagaaacct gcattcggtt aaacaccacg 7020cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag 7080ggtgaagcct tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac 7140atcgagatcg agctagctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac 7200gtgctgacgg ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac 7260cgcctggcac gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac 7320gaacgcagtg gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc 7380gggtcaaatg acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc 7440ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg 7500gagcagatgc tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg tctctttcct 7560gtggatagca cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt 7620gggaacccaa agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaaagag 7680aaaaaaggcg atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc 7740ctggcctgtg cataactgtc tggccagcgc acagccgaag agctgcaaaa agcgcctacc 7800cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc 7860cgctcaaaaa tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg 7920tcgccactcg accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga 7980cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 8040tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc 8100agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca 8160gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 8220agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 8280gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 8340tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 8400aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 8460aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 8520ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 8580tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 8640agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 8700gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 8760tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 8820acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 8880tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 8940caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 9000aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 9060aactcacgtt aagggatttt ggtcatgcat gatatatctc ccaatttgtg tagggcttat 9120tatgcacgct taaaaataat aaaagcagac ttgacctgat agtttggctg tgagcaatta 9180tgtgcttagt gcatctaacg cttgagttaa gccgcgccgc gaagcggcgt cggcttgaac 9240gaatttctag ctagacatta tttgccgact accttggtga tctcgccttt cacgtagtgg 9300acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg tccaagataa 9360gcctgtctag cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag 9420tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac 9480aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt 9540aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc 9600gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga 9660tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct 9720ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca 9780atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa 9840aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc 9900aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta caaatgtacg 9960gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc 10020gatacttcgg cgatcaccgc ttcccccatg atgtttaact ttgttttagg gcgactgccc 10080tgctgcgtaa catcgttgct gctccataac atcaaacatc gacccacggc gtaacgcgct 10140tgctgcttgg atgcccgagg catagactgt accccaaaaa aacagtcata acaagccatg 10200aaaaccgcca ctgcgggggt tccatggaca tacaaatgga cgaacggata aaccttttca 10260cgccctttta aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca 10320atatatcctg tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tcagatctag 10380taggaaacag ctatgaccat gattacgcca agct 104147911152DNAArtificial sequencebinary vector Lo657- pSUN3-GWs-B1- BnAK700::GUS::E9::nosT::B2 79atcacaagtt tgtacaaaaa agcaggcttt aaaggaacca attcagtcga cacgaagctt 60tcgaggctaa cgttggtgga gatgttaagg cacggcatct tctcttcttc ttcttttttg 120tttgtttgtt tgtttcgctc tctatcactc actcgatcga tccccagtct tcttttcggg 180aaagctgtgt cagaccaatt atattccatc tttaatttgg ccctttttat tattcgctat 240ggcccaacat ttcaggccca atttatacga ttataaatat aaccacttga ttgtgctgcc 300gctcagctga atagtactcc ctccgttttt taatataaat cgttttacag ttatgcacgt 360aaattaagaa aaccattaac tttttatatt ttctaaacaa aaacatcatt aattatttac 420ttactcacaa ttcaaccaat agaaaaatag aagatatatt accattggtc atacaacatt 480aattattaat aaattttaca tagaaaaccg aaaacgacat ataatttgga acaaaaaaat 540ttctctaaaa ctacttatat taaaaaacgg agagagtata tttaactagt tcgtcttact 600gacaaattga agaggcagac caagacacgc ggctcctcga tggctgtctt atgccaagtg 660gcggtgcctg cttctgcatt aaataggtag aagaactcta attacagaag ctcgaggtag 720ataggatccc cgggtaggtc agtcccttat gttacgtcct gtagaaaccc caacccgtga 780aatcaaaaaa ctcgacggcc tgtgggcatt cagtctggat cgcgaaaact gtggaattga 840tcagcgttgg tgggaaagcg cgttacaaga aagccgggca attgctgtgc caggcagttt 900taacgatcag ttcgccgatg cagatattcg taattatgcg ggcaacgtct ggtatcagcg 960cgaagtcttt ataccgaaag gttgggcagg ccagcgtatc gtgctgcgtt tcgatgcggt 1020cactcattac ggcaaagtgt gggtcaataa tcaggaagtg atggagcatc agggcggcta 1080tacgccattt gaagccgatg tcacgccgta tgttattgcc gggaaaagtg tacgtatcac 1140cgtttgtgtg aacaacgaac tgaactggca gactatcccg ccgggaatgg tgattaccga 1200cgaaaacggc aagaaaaagc agtcttactt ccatgatttc tttaactatg ccggaatcca 1260tcgcagcgta atgctctaca ccacgccgaa cacctgggtg gacgatatca ccgtggtgac 1320gcatgtcgcg caagactgta accacgcgtc tgttgactgg caggtggtgg ccaatggtga 1380tgtcagcgtt gaactgcgtg atgcggatca acaggtggtt gcaactggac aaggcactag 1440cgggactttg caagtggtga atccgcacct ctggcaaccg ggtgaaggtt atctctatga 1500actgtgcgtc acagccaaaa gccagacaga gtgtgatatc tacccgcttc gcgtcggcat 1560ccggtcagtg gcagtgaagg gcgaacagtt cctgattaac cacaaaccgt tctactttac 1620tggctttggt cgtcatgaag atgcggactt gcgtggcaaa ggattcgata acgtgctgat 1680ggtgcacgac cacgcattaa tggactggat tggggccaac tcctaccgta cctcgcatta 1740cccttacgct gaagagatgc tcgactgggc agatgaacat ggcatcgtgg tgattgatga 1800aactgctgct gtcggcttta acctctcttt aggcattggt ttcgaagcgg gcaacaagcc 1860gaaagaactg tacagcgaag aggcagtcaa cggggaaact cagcaagcgc acttacaggc 1920gattaaagag ctgatagcgc gtgacaaaaa ccacccaagc gtggtgatgt ggagtattgc 1980caacgaaccg gatacccgtc cgcaaggtgc acgggaatat ttcgcgccac tggcggaagc 2040aacgcgtaaa ctcgacccga cgcgtccgat cacctgcgtc aatgtaatgt tctgcgacgc 2100tcacaccgat accatcagcg atctctttga tgtgctgtgc ctgaaccgtt attacggatg 2160gtatgtccaa agcggcgatt tggaaacggc agagaaggta ctggaaaaag aacttctggc 2220ctggcaggag aaactgcatc agccgattat catcaccgaa tacggcgtgg atacgttagc 2280cgggctgcac tcaatgtaca ccgacatgtg gagtgaagag tatcagtgtg catggctgga 2340tatgtatcac cgcgtctttg atcgcgtcag cgccgtcgtc ggtgaacagg tatggaattt 2400cgccgatttt gcgacctcgc aaggcatatt gcgcgttggc ggtaacaaga aagggatctt 2460cactcgcgac cgcaaaccga agtcggcggc ttttctgctg caaaaacgct ggactggcat 2520gaacttcggt gaaaaaccgc agcagggagg caaacaatga atcaacaact ctcctggcgc 2580accatcgtcg gctacagcct cgggaattgc taccgagatc tgcggccgcg gcgcgccaat 2640tgactagtag gcctatcgat taattaaggc cgcctcgagc atatgctaga ggatcctcta 2700gctagagctt tcgttcgtat catcggtttc gacaacgttc gtcaagttca atgcatcagt 2760ttcattgcgc acacaccaga atcctactga gtttgagtat tatggcattg ggaaaactgt 2820ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg ttttcgctat 2880cgaactgtga aatggaaatg gatggagaag agttaatgaa tgatatggtc cttttgttca 2940ttctcaaatt aatattattt gttttttctc ttatttgttg tgtgttgaat ttgaaattat 3000aagagatatg caaacatttt gttttgagta aaaatgtgtc aaatcgtggc ctctaatgac 3060cgaagttaat atgaggagta aaacacttgt agttgtacca ttatgcttat tcactaggca 3120acaaatatat tttcagacct agaaaagctg caaatgttac tgaatacaag tatgtcctct 3180tgtgttttag acatttatga actttccttt atgtaatttt ccagaatcct tgtcagattc 3240taatcattgc tttataatta tagttatact catggatttg tagttgagta tgaaaatatt 3300ttttaatgca ttttatgact tgccaattga ttgacaacat gcatcaatcg accggctcga 3360atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 3420gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 3480tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 3540tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 3600tgtcatctat gttactagat cgggaattct agacccagct ttcttgtaca aagtggtgat 3660aattcactgg ccgtcgtttt acaacgactc agagcttgac aggaggcccg atctagtaac 3720atagatgaca ccgcgcgcga taatttatcc tagtttgcgc gctatatttt gttttctatc 3780gcgtattaaa tgtataattg cgggactcta atcataaaaa cccatctcat aaataacgtc 3840atgcattaca tgttaattat tacatgctta acgtaattca acagaaatta tatgataatc 3900atcgcaagac cggcaacagg attcaatctt aagaaacttt attgccaaat gtttgaacga 3960tcggggatca tccgggtctg tggcgggaac tccacgaaaa tatccgaacg cagcaagatc 4020tagagcttgg gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc 4080gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc 4140tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc 4200cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag 4260gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg 4320aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga 4380ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg 4440caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc 4500tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc 4560cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg 4620gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg 4680gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag 4740cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga 4800gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atccagatcc ggtgcagatt 4860atttggattg agagtgaata tgagactcta attggatacc gaggggaatt tatggaacgt 4920cagtggagca tttttgacaa gaaatatttg ctagctgata gtgaccttag gcgacttttg 4980aacgcgcaat aatggtttct gacgtatgtg cttagctcat taaactccag aaacccgcgg 5040ctgagtggct ccttcaacgt tgcggttctg tcagttccaa acgtaaaacg gcttgtcccg 5100cgtcatcggc gggggtcata acgtgactcc cttaattctc cgctcatgat cagattgtcg 5160tttcccgcct tcagtttaaa ctatcagtgt ttgacaggat cctgcttggt aataattgtc 5220attagattgt ttttatgcat agatgcactc gaaatcagcc aattttagac aagtatcaaa 5280cggatgttaa ttcagtacat taaagacgtc cgcaatgtgt tattaagttg tctaagcgtc 5340aatttgttta caccacaata tatcctgcca ccagccagcc aacagctccc cgaccggcag 5400ctcggcacaa aatcaccacg cgttaccacc acgccggccg gccgcatggt gttgaccgtg 5460ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc 5520gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac cccggcacag 5580atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca 5640ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg 5700cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc 5760ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc 5820aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg 5880tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt 5940ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc 6000gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata 6060tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact 6120taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc gcgccctgca 6180actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcgattg 6240ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga 6300ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag cgccccaggc 6360ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc 6420aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga 6480ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg 6540catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc ttgagtcccg 6600tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc 6660agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa 6720actcatttga

gttaatgagg taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc 6780ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca 6840gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac 6900gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca 6960gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat 7020ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg 7080cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc 7140caagcccgag gaatcggcgt gagcggtcgc aaaccatccg gcccggtaca aatcggcgcg 7200gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc 7260atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa 7320gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac 7380gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc 7440atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc 7500cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt 7560gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga 7620taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta 7680ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc 7740attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc 7800ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa 7860accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca 7920gaaggcaaga acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc 7980atcggccgtt ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg 8040ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc 8100accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg 8160gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc 8220gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt 8280cgaaaaggtc tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg 8340aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa 8400gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt 8460aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag 8520ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg 8580cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg 8640cgcggacaag ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg caccctgcct 8700cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 8760agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 8820tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg 8880cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata 8940ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact 9000gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 9060atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 9120caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 9180cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 9240taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 9300ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 9360tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 9420gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 9480ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 9540aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 9600aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 9660agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 9720cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 9780gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgcatga tatatctccc 9840aatttgtgta gggcttatta tgcacgctta aaaataataa aagcagactt gacctgatag 9900tttggctgtg agcaattatg tgcttagtgc atctaacgct tgagttaagc cgcgccgcga 9960agcggcgtcg gcttgaacga atttctagct agacattatt tgccgactac cttggtgatc 10020tcgcctttca cgtagtggac aaattcttcc aactgatctg cgcgcgaggc caagcgatct 10080tcttcttgtc caagataagc ctgtctagct tcaagtatga cgggctgata ctgggccggc 10140aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc ggttactgcg 10200ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct catcgccagc ccagtcgggc 10260ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa atagatcctg ttcaggaacc 10320ggatcaaaga gttcctccgc cgctggacct accaaggcaa cgctatgttc tcttgctttt 10380gtcagcaaga tagccagatc aatgtcgatc gtggctggct cgaagatacc tgcaagaatg 10440tcattgcgct gccattctcc aaattgcagt tcgcgcttag ctggataacg ccacggaatg 10500atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga gaatctcgct ctctccaggg 10560gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt 10620acggtcaccg taaccagcaa atcaatatca ctgtgtggct tcaggccgcc atccactgcg 10680gagccgtaca aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat gacgccaact 10740acctctgata gttgagtcga tacttcggcg atcaccgctt cccccatgat gtttaacttt 10800gttttagggc gactgccctg ctgcgtaaca tcgttgctgc tccataacat caaacatcga 10860cccacggcgt aacgcgcttg ctgcttggat gcccgaggca tagactgtac cccaaaaaaa 10920cagtcataac aagccatgaa aaccgccact gcgggggttc catggacata caaatggacg 10980aacggataaa ccttttcacg cccttttaaa tatccgatta ttctaataaa cgctcttttc 11040tcttaggttt acccgccaat atatcctgtc aaacactgat agtttaaact gaaggcggga 11100aacgacaatc agatctagta ggaaacagct atgaccatga ttacgccaag ct 111528037DNAArtificial sequenceOligonucleotide primer 5 80cggcctaggg gcgcccggac cgagctgttc accggca 378124DNAArtificial sequenceOligonucleotide primer 6 81cggactagtg atgtagccct cagg 248222DNAArtificial sequenceOligonucleotide primer 7 82cgagctcgtg ccttttggat cg 228318DNAArtificial sequenceOligonucleotide primer 8 83cggtccgaac gtggttgg 188422DNAArtificial sequenceOligonucleotide primer 9 84cgagctcggc cctatgaatt gg 228522DNAArtificial sequenceOligonucleotide primer 10 85cggtccgtct ccttctgcac ac 228623DNAArtificial sequenceOligonucleotide primer 11 86cgagctcgat gcattccttg gat 238722DNAArtificial sequenceOligonucleotide primer 12 87cctagggttt ggaggtatca ag 228826DNAArtificial sequenceOligonucleotide primer 13 88cgagctccgt ccgatgtgat tccgtc 268922DNAArtificial sequenceOligonucleotide primter 14 89cctaggggca gtgtcggcgg tt 229025DNAArtificial sequenceOligonucleotide primer 15 90cgagctccag agtgacagac agtga 259122DNAArtificial sequenceOligonucleotide primer 16 91cctaggtctt caactgtccc ca 22921137DNAOryza sativaterminator(1)..(1137)BPST.3 92ggaatgtatt tgaccataca aaaaccgtac aaagtgtagg gttctaatta gcaaatgtaa 60gtttgtaatt cttctgtcta aatgtagtgt gttgaggtgg tcagttgccg tgactggctc 120tttcagatgg taattacaaa gaaatgtaac attaatgttg taacaagggt gccattttgt 180ttctgattcc agatgaaaag gaaaaactat tccatattct gcaggattaa acgtttctgc 240tgttcacaac aggtaaagtc actccacttc agggaaagac agcaaaatca tttcttttac 300acagacttta gataaccctt ttttttgggg ttcttggtat atgccaactt ttgtagcctg 360caccagaaac aaaaatgaag acttttgcta aagatgtaaa agtggcatga tgtcctggat 420gaccaaataa ttcatgacaa atggattaaa agagcccaat atctgaaaga gactggccag 480cagccactaa tgtcaccaac cacatatgta acacttggtg cataattcaa gagggagcat 540ctcctccaga atcaggattg aaaggtacaa cctcatagta aatcctcgga atatagcatg 600tgcagcataa gaatatatca gtgttgtgct gggtaagaaa ccacatgaac caattaggaa 660taaataatca tgctgaaatt atagcaatgc ttgcaatttg caaacgataa agctagacgc 720gggttgctgg aataacaatc catctccaac aaaatagtac agaatataac tgaatggcca 780gctcagaccc taacagaatt gaaaagctgg attcatcagc actccattga gcaatctaga 840tcaggaaaga gcatagatgc ataatgaact gagatccctt caaaatgact aactaatatt 900tttttttctt ataaaagagt ttacaacagt acaaccacga agatcagcac taccattact 960gattttgtta acatagagtg atttatcatg tgtgccagac aaacaacaga tacattcata 1020catagcataa cttacagcac atgatacaga ctacggagaa cggttaatct taaaataaaa 1080acaaaaaaac aaggaggcaa agcttatttt gcctgggatt catctaaatg cagttgt 1137931137DNAOryza sativaterminator(1)..(1137)BPST.4 93acaactgcat ttagatgaat cccaggcaaa ataagctttg cctccttgtt tttttgtttt 60tattttaaga ttaaccgttc tccgtagtct gtatcatgtg ctgtaagtta tgctatgtat 120gaatgtatct gttgtttgtc tggcacacat gataaatcac tctatgttaa caaaatcagt 180aatggtagtg ctgatcttcg tggttgtact gttgtaaact cttttataag aaaaaaaaat 240attagttagt cattttgaag ggatctcagt tcattatgca tctatgctct ttcctgatct 300agattgctca atggagtgct gatgaatcca gcttttcaat tctgttaggg tctgagctgg 360ccattcagtt atattctgta ctattttgtt ggagatggat tgttattcca gcaacccgcg 420tctagcttta tcgtttgcaa attgcaagca ttgctataat ttcagcatga ttatttattc 480ctaattggtt catgtggttt cttacccagc acaacactga tatattctta tgctgcacat 540gctatattcc gaggatttac tatgaggttg tacctttcaa tcctgattct ggaggagatg 600ctccctcttg aattatgcac caagtgttac atatgtggtt ggtgacatta gtggctgctg 660gccagtctct ttcagatatt gggctctttt aatccatttg tcatgaatta tttggtcatc 720caggacatca tgccactttt acatctttag caaaagtctt catttttgtt tctggtgcag 780gctacaaaag ttggcatata ccaagaaccc caaaaaaaag ggttatctaa agtctgtgta 840aaagaaatga ttttgctgtc tttccctgaa gtggagtgac tttacctgtt gtgaacagca 900gaaacgttta atcctgcaga atatggaata gtttttcctt ttcatctgga atcagaaaca 960aaatggcacc cttgttacaa cattaatgtt acatttcttt gtaattacca tctgaaagag 1020ccagtcacgg caactgacca cctcaacaca ctacatttag acagaagaat tacaaactta 1080catttgctaa ttagaaccct acactttgta cggtttttgt atggtcaaat acattcc 1137946849DNAArtificial sequencevector pRJB058 94caggaaacag ctatgaccat gtaatacgac tcactatagg ggatatcagc tggatggcaa 60ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc 120tttcttataa tgccaacttt gtacaagaaa gctgggtcgg cgcgccaagc ttgcatgcct 180gcaggcatgc aagcttccgc ggctgcagtg cagcgtgacc cggtcgtgcc cctctctaga 240gataatgagc attgcatgtc taagttataa aaaattacca catatttttt ttgtcacact 300tgtttgaagt gcagtttatc tatctttata catatattta aactttactc tacgaataat 360ataatctata gtactacaat aatatcagtg ttttagagaa tcatataaat gaacagttag 420acatggtcta aaggacaatt gagtattttg acaacaggac tctacagttt tatcttttta 480gtgtgcatgt gttctccttt ttttttgcaa atagcttcac ctatataata cttcatccat 540tttattagta catccattta gggtttaggg ttaatggttt ttatagacta atttttttag 600tacatctatt ttattctatt ttagcctcta aattaagaaa actaaaactc tattttagtt 660tttttattta atagtttaga tataaaatag aataaaataa agtgactaaa aattaaacaa 720atacccttta agaaattaaa aaaactaagg aaacattttt cttgtttcga gtagataatg 780ccagcctgtt aaacgccgtc gacgagtcta acggacacca accagcgaac cagcagcgtc 840gcgtcgggcc aagcgaagca gacggcacgg catctctgtc gctgcctctg gacccctctc 900gagagttccg ctccaccgtt ggacttgctc cgctgtcggc atccagaaat tgcgtggcgg 960agcggcagac gtgagccggc acggcaggcg gcctcctcct cctctcacgg caccggcagc 1020tacgggggat tcctttccca ccgctccttc gctttccctt cctcgcccgc cgtaataaat 1080agacaccccc tccacaccct ctttccccaa cctcgtgttg ttcggagcgc acacacacac 1140aaccagatct cccccaaatc cacccgtcgg cacctccgct tcaaggtacg ccgctcgtcc 1200tccccccccc cccccctctc taccttctct agatcggcgt tccggtccat ggttagggcc 1260cggtagttct acttctgttc atgtttgtgt tagatccgtg tttgtgttag atccgtgctg 1320ctagcgttcg tacacggatg cgacctgtac gtcagacacg ttctgattgc taacttgcca 1380gtgtttctct ttggggaatc ctgggatggc tctagccgtt ccgcagacgg gatcgatttc 1440atgatttttt ttgtttcgtt gcatagggtt tggtttgccc ttttccttta tttcaatata 1500tgccgtgcac ttgtttgtcg ggtcatcttt tcatgctttt ttttgtcttg gttgtgatga 1560tgtggtctgg ttgggcggtc gttctagatc ggagtagaat tctgtttcaa actacctggt 1620ggatttatta attttggatc tgtatgtgtg tgccatacat attcatagtt acgaattgaa 1680gatgatggat ggaaatatcg atctaggata ggtatacatg ttgatgcggg ttttactgat 1740gcatatacag agatgctttt tgttcgcttg gttgtgatga tgtggtgtgg ttgggcggtc 1800gttcattcgt tctagatcgg agtagaatac tgtttcaaac tacctggtgt atttattaat 1860tttggaactg tatgtgtgtg tcatacatct tcatagttac gagtttaaga tggatggaaa 1920tatcgatcta ggataggtat acatgttgat gtgggtttta ctgatgcata tacatgatgg 1980catatgcagc atctattcat atgctctaac cttgagtacc tatctattat aataaacaag 2040tatgttttat aattatttcg atcttgatat acttggatga tggcatatgc agcagctata 2100tgtggatttt tttagccctg ccttcatacg ctatttattt gcttggtact gtttcttttg 2160tcgatgctca ccctgttgtt tggtgttact tctgcagggt acccccgggt aggtcagtcc 2220cttatgttac gtcctgtaga aaccccaacc cgtgaaatca aaaaactcga cggcctgtgg 2280gcattcagtc tggatcgcga aaactgtgga attggtcagc gttggtggga aagcgcgtta 2340caagaaagcc gggcaattgc tgtgccaggc agttttaacg atcagttcgc cgatgcagat 2400attcgtaatt atgcgggcaa cgtctggtat cagcgcgaag tctttatacc gaaaggttgg 2460gcaggccagc gtatcgtgct gcgtttcgat gcggtcactc attacggcaa agtgtgggtc 2520aataatcagg aagtgatgga gcatcagggc ggctatacgc catttgaagc cgatgtcacg 2580ccgtatgtta ttgccgggaa aagtgtacgt aagtttctgc ttctaccttt gatatatata 2640taataattat cattaattag tagtaatata atatttcaaa tatttttttc aaaataaaag 2700aatgtagtat atagcaattg cttttctgta gtttataagt gtgtatattt taatttataa 2760cttttctaat atatgaccaa aatttgttga tgtgcaggta tcaccgtttg tgtgaacaac 2820gaactgaact ggcagactat cccgccggga atggtgatta ccgacgaaaa cggcaagaaa 2880aagcagtctt acttccatga tttctttaac tatgccggaa tccatcgcag cgtaatgctc 2940tacaccacgc cgaacacctg ggtggacgat atcaccgtgg tgacgcatgt cgcgcaagac 3000tgtaaccacg cgtctgttga ctggcaggtg gtggccaatg gtgatgtcag cgttgaactg 3060cgtgatgcgg atcaacaggt ggttgcaact ggacaaggca ctagcgggac tttgcaagtg 3120gtgaatccgc acctctggca accgggtgaa ggttatctct atgaactgtg cgtcacagcc 3180aaaagccaga cagagtgtga tatctacccg cttcgcgtcg gcatccggtc agtggcagtg 3240aagggcgaac agttcctgat taaccacaaa ccgttctact ttactggctt tggtcgtcat 3300gaagatgcgg acttgcgtgg caaaggattc gataacgtgc tgatggtgca cgaccacgca 3360ttaatggact ggattggggc caactcctac cgtacctcgc attaccctta cgctgaagag 3420atgctcgact gggcagatga acatggcatc gtggtgattg atgaaactgc tgctgtcggc 3480tttaacctct ctttaggcat tggtttcgaa gcgggcaaca agccgaaaga actgtacagc 3540gaagaggcag tcaacgggga aactcagcaa gcgcacttac aggcgattaa agagctgata 3600gcgcgtgaca aaaaccaccc aagcgtggtg atgtggagta ttgccaacga accggatacc 3660cgtccgcaag gtgcacggga atatttcgcg ccactggcgg aagcaacgcg taaactcgac 3720ccgacgcgtc cgatcacctg cgtcaatgta atgttctgcg acgctcacac cgataccatc 3780agcgatctct ttgatgtgct gtgcctgaac cgttattacg gatggtatgt ccaaagcggc 3840gatttggaaa cggcagagaa ggtactggaa aaagaacttc tggcctggca ggagaaactg 3900catcagccga ttatcatcac cgaatacggc gtggatacgt tagccgggct gcactcaatg 3960tacaccgaca tgtggagtga agagtatcag tgtgcatggc tggatatgta tcaccgcgtc 4020tttgatcgcg tcagcgccgt cgtcggtgaa caggtatgga atttcgccga ttttgcgacc 4080tcgcaaggca tattgcgcgt tggcggtaac aagaaaggga tcttcactcg cgaccgcaaa 4140ccgaagtcgg cggcttttct gctgcaaaaa cgctggactg gcatgaactt cggtgaaaaa 4200ccgcagcagg gaggcaaaca atgaatcaac aactctcctg gcgcaccatc gtcggctaca 4260gcctcgggaa ttgctaccga gctccctagg gggcgcccgg accgagctgt tcaccggcat 4320cgtgcccatc ctgatcgagc tgaatggcga tgtgaatggc cacaagttca gcgtgagcgg 4380cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg 4440caagctgcct gtgccctggc ccaccctggt gaccaccctg agctacggcg tgcagtgctt 4500ctcacgctac cccgatcaca tgaagcagca cgacttcttc aagagcgcca tgcctgaggg 4560ctacatcact agttaagact ggccgtcgtt ttacaacgtc gtgactggga aaacatccat 4620gctagcgtta acgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc 4680ggaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac 4740aaatccgccg ggagcggatt tgaacgttgt gaagcaacgg cccggagggt ggcgggcagg 4800acgcccgcca taaactgcca ggcatcaaac taagcagaag gccatcctga cggatggcct 4860ttttgcgttt ctacaaactc ttcctggcta gcggtacgcg tattaattgc gttgcgctca 4920ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 4980gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 5040cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 5100tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 5160aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 5220catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 5280caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 5340ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 5400aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 5460gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 5520cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 5580ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 5640tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 5700tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 5760cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 5820tggaacgacg cgtaactcac gttaagggat tttggtcatg agcttgcgcc gtcccgtcaa 5880gtcagcgtaa tgctctgcca gtgttacaac caattaacca attctgatta gaaaaactca 5940tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga 6000aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga 6060tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc 6120tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag 6180aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg 6240tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga 6300cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc 6360aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc 6420tggaatgctg tttttccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg 6480ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc 6540tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca 6600tcgggcttcc catacaagcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc 6660catttatacc catataaatc agcatccatg ttggaattta atcgcggcct cgacgtttcc 6720cgttgaatat ggctcataac

accccttgta ttactgttta tgtaagcaga cagttttatt 6780gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacgggc 6840cagagctgc 684995257DNAArtificial sequenceNos-T sequence inserted into SacI-RsrII fragment of pRJB058 95cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 60gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 120catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 180cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 240tatgttacta gatccgg 257961089DNAArtificial sequenceORF sequence inserted into SacI digested and T4 DNA Polymerase filled in fragment of pRJB058 96cgaacaacaa aaaaaccatc ccgaacaacc tggttgaaaa ctacctgacc ccgatgtctc 60tggcatactg gttcatggat gatggtggta aatgggatta caacaaaaac tctaccaaca 120aatcgatcgt actgaacacc cagtctttca ctttcgaaga agtagaatac ctggttaagg 180gtctgcgtaa caaattccaa ctgaactgtt acgtaaaaat caacaaaaac aaaccgatca 240tctacatcga ttctatgtct tacctgatct tctacaacct gatcaaaccg tacctgatcc 300cgcagatgat gtacaaactg ccgaacacta tctcctccga aactttcctg aaaggtacca 360aaggtgggat acgaaaagac cgaagaggag ggagaatgtt gaaacacaag cgccagagag 420atgatgggga gggcaggggt gaagtggggt ctgctggaga catgagagct gccaaccttt 480ggccaagccc gctcatgatc aaacgctcta agaagaacag cctggccttg tccctgacgg 540ccgaccagat ggtcagtgcc ttgttggatg ctgagccccc catacctcta ttccgagtat 600gatcctacca gacccttcag tgaagcttcg atgatgggct tactgaccaa cctggcagac 660agggagctgg ttcacatgat caactgggcg aagagggtgc caggctttgt ggatttgacc 720ctccatgatc aggtccacct tctagaatgt gcctggctag agatcctgat gattggtctc 780gtctggcgct ccatggaaca cccggggaag ctcctgtttg ctcctaactt gctcctggac 840aggaatcaag gtaaatgtgt ggaaggcatg gtggagatct tcgacatgct gctggctaca 900tcatctcggt tccgcatgat gaatctgcag ggagaggagt ttgtgtgcct caaatctatt 960attttgctta attctggagt gtacacattt ctgtccagca ccctgaagtc tctggaagag 1020aaggaccata tccaccgagt cctggacaag atcacagaca ctttgatcca cctgatggcc 1080aaggcaggc 1089971414DNAArtificial sequenceOryza sativa BPST.1 sequence inserted into SacI digested and T4 DNA Polymerase filled in fragment of pRJB058 97aattcgccct tcggaccgtg ccttttggat cgaaagatgc agttgttttg ctgctgaagg 60catcctcatg tctgttactt tgatatggtc ttgaaataaa tcttcgttta caagagaaca 120acactctgtt ttatcttgtt ttgtgtgcca gaagcctgct gaacaatttt atgtcaattt 180ctttgctact aagaaagtta tgcaccaaaa tgaagggaaa ttctgtgaag tttgtaattt 240tcaagtgatt ggcattattc tgtttagttc gtgcagtgcc atgttcctta tataatcaca 300gaacatgtaa aaggccgaaa tattttcctt gtcattttcc atgcgagaac ggcctttcga 360tgagactgga aagtcctgca ataactattg tccaaaataa cacacggctc tctaccgatt 420cacatcaaaa ccaacccaaa cgctctctct gttgcttgtg ttctggactt ctctgagcgc 480agttcaggag tctcgagtgt gcctcaacag tacatgttaa acataaaact gccattgaag 540attaccacca accatattgg aagtttatca tggcatatac ttgcctttca aagaccctaa 600ttttcaaagt gaacatggga ctttcagact acagataacc tcaatcgata acatcaccga 660aaattcgata ttactacaaa gattttggtt gaaaacttct ctgaacccaa tttaaataca 720agtcgccctc ggtgatggct actcgccatt ccacgctcac agttgcagtt cacacttgca 780aaatgaaaaa aaaaatcttc tgaacagcaa tcatcaaccg gttcgacctc aggatcatca 840cagaaatgaa aaggacgagg aatctcaatt taagaaagtt cctatccaaa tatccaacaa 900aaatctgact gtctggctat tattaattca acaggagatg ctcgttaaag acaatatagt 960tctgtaattt gtaacaacac aagagcctga ggatataacg agttacaaac atgctgccgt 1020tgcagcatgg tcatcaatat acaacattac aacaacatat ggggatcgca gaacactccc 1080attccacgat tttacaacat atgcttcaca acacgttata aatacatggt tgtaccgagc 1140tgtagagaga cagccagcac gtcccaacta caaatgcaca ctagccaaca gcaaacataa 1200aaactactct ctggtaagtc actatatata cgcactatag cttcacatga ctaaaattat 1260aacatgacat aatttctttc gtataaatta gccccaggaa gcttctgtcc gaatcgccat 1320cttgggagca ataaatatac tggcattctt cacaatggat gcattattta catatatata 1380cttcttctcc aaccacgttc gagctcgaag ggcg 1414981414DNAArtificial sequenceOryza sativa BPST.2 sequence inserted into SacI digested and T4 DNA Polymerase filled in fragment of pRJB058 98cgcccttcga gctcgaacgt ggttggagaa gaagtatata tatgtaaata atgcatccat 60tgtgaagaat gccagtatat ttattgctcc caagatggcg attcggacag aagcttcctg 120gggctaattt atacgaaaga aattatgtca tgttataatt ttagtcatgt gaagctatag 180tgcgtatata tagtgactta ccagagagta gtttttatgt ttgctgttgg ctagtgtgca 240tttgtagttg ggacgtgctg gctgtctctc tacagctcgg tacaaccatg tatttataac 300gtgttgtgaa gcatatgttg taaaatcgtg gaatgggagt gttctgcgat ccccatatgt 360tgttgtaatg ttgtatattg atgaccatgc tgcaacggca gcatgtttgt aactcgttat 420atcctcaggc tcttgtgttg ttacaaatta cagaactata ttgtctttaa cgagcatctc 480ctgttgaatt aataatagcc agacagtcag atttttgttg gatatttgga taggaacttt 540cttaaattga gattcctcgt ccttttcatt tctgtgatga tcctgaggtc gaaccggttg 600atgattgctg ttcagaagat tttttttttc attttgcaag tgtgaactgc aactgtgagc 660gtggaatggc gagtagccat caccgagggc gacttgtatt taaattgggt tcagagaagt 720tttcaaccaa aatctttgta gtaatatcga attttcggtg atgttatcga ttgaggttat 780ctgtagtctg aaagtcccat gttcactttg aaaattaggg tctttgaaag gcaagtatat 840gccatgataa acttccaata tggttggtgg taatcttcaa tggcagtttt atgtttaaca 900tgtactgttg aggcacactc gagactcctg aactgcgctc agagaagtcc agaacacaag 960caacagagag agcgtttggg ttggttttga tgtgaatcgg tagagagccg tgtgttattt 1020tggacaatag ttattgcagg actttccagt ctcatcgaaa ggccgttctc gcatggaaaa 1080tgacaaggaa aatatttcgg ccttttacat gttctgtgat tatataagga acatggcact 1140gcacgaacta aacagaataa tgccaatcac ttgaaaatta caaacttcac agaatttccc 1200ttcattttgg tgcataactt tcttagtagc aaagaaattg acataaaatt gttcagcagg 1260cttctggcac acaaaacaag ataaaacaga gtgttgttct cttgtaaacg aagatttatt 1320tcaagaccat atcaaagtaa cagacatgag gatgccttca gcagcaaaac aactgcatct 1380ttcgatccaa aaggcacggt ccgaagggcg aatt 1414991165DNAArtificial sequenceOryza sativa BPST.3 sequence inserted into SacI digested and T4 DNA Polymerase filled in fragment of pRJB058 99ggccctatga attggaatgt atttgaccat acaaaaaccg tacaaagtgt agggttctaa 60ttagcaaatg taagtttgta attcttctgt ctaaatgtag tgtgttgagg tggtcagttg 120ccgtgactgg ctctttcaga tggtaattac aaagaaatgt aacattaatg ttgtaacaag 180ggtgccattt tgtttctgat tccagatgaa aaggaaaaac tattccatat tctgcaggat 240taaacgtttc tgctgttcac aacaggtaaa gtcactccac ttcagggaaa gacagcaaaa 300tcatttcttt tacacagact ttagataacc cttttttttg gggttcttgg tatatgccaa 360cttttgtagc ctgcaccaga aacaaaaatg aagacttttg ctaaagatgt aaaagtggca 420tgatgtcctg gatgaccaaa taattcatga caaatggatt aaaagagccc aatatctgaa 480agagactggc cagcagccac taatgtcacc aaccacatat gtaacacttg gtgcataatt 540caagagggag catctcctcc agaatcagga ttgaaaggta caacctcata gtaaatcctc 600ggaatatagc atgtgcagca taagaatata tcagtgttgt gctgggtaag aaaccacatg 660aaccaattag gaataaataa tcatgctgaa attatagcaa tgcttgcaat ttgcaaacga 720taaagctaga cgcgggttgc tggaataaca atccatctcc aacaaaatag tacagaatat 780aactgaatgg ccagctcaga ccctaacaga attgaaaagc tggattcatc agcactccat 840tgagcaatct agatcaggaa agagcataga tgcataatga actgagatcc cttcaaaatg 900actaactaat attttttttt cttataaaag agtttacaac agtacaacca cgaagatcag 960cactaccatt actgattttg ttaacataga gtgatttatc atgtgtgcca gacaaacaac 1020agatacattc atacatagca taacttacag cacatgatac agactacgga gaacggttaa 1080tcttaaaata aaaacaaaaa aacaaggagg caaagcttat tttgcctggg attcatctaa 1140atgcagttgt gtgcagaagg agacg 11651001165DNAArtificial sequenceOryza sativa BPST.4 sequence inserted into SacI digested and T4 DNA Polymerase filled in fragment of pRJB058 100cgtctccttc tgcacacaac tgcatttaga tgaatcccag gcaaaataag ctttgcctcc 60ttgttttttt gtttttattt taagattaac cgttctccgt agtctgtatc atgtgctgta 120agttatgcta tgtatgaatg tatctgttgt ttgtctggca cacatgataa atcactctat 180gttaacaaaa tcagtaatgg tagtgctgat cttcgtggtt gtactgttgt aaactctttt 240ataagaaaaa aaaatattag ttagtcattt tgaagggatc tcagttcatt atgcatctat 300gctctttcct gatctagatt gctcaatgga gtgctgatga atccagcttt tcaattctgt 360tagggtctga gctggccatt cagttatatt ctgtactatt ttgttggaga tggattgtta 420ttccagcaac ccgcgtctag ctttatcgtt tgcaaattgc aagcattgct ataatttcag 480catgattatt tattcctaat tggttcatgt ggtttcttac ccagcacaac actgatatat 540tcttatgctg cacatgctat attccgagga tttactatga ggttgtacct ttcaatcctg 600attctggagg agatgctccc tcttgaatta tgcaccaagt gttacatatg tggttggtga 660cattagtggc tgctggccag tctctttcag atattgggct cttttaatcc atttgtcatg 720aattatttgg tcatccagga catcatgcca cttttacatc tttagcaaaa gtcttcattt 780ttgtttctgg tgcaggctac aaaagttggc atataccaag aaccccaaaa aaaagggtta 840tctaaagtct gtgtaaaaga aatgattttg ctgtctttcc ctgaagtgga gtgactttac 900ctgttgtgaa cagcagaaac gtttaatcct gcagaatatg gaatagtttt tccttttcat 960ctggaatcag aaacaaaatg gcacccttgt tacaacatta atgttacatt tctttgtaat 1020taccatctga aagagccagt cacggcaact gaccacctca acacactaca tttagacaga 1080agaattacaa acttacattt gctaattaga accctacact ttgtacggtt tttgtatggt 1140caaatacatt ccaattcata gggcc 11651011305DNAArtificial sequenceterminator sequence from Orzza sativa with 5' and 3'-multiple cloning site linkers 101aattcgccct tcgagctcga tgcattcctt ggattgttcc caatgtattc cagaaatcat 60agttttgatg ccaaagttgg tcttcggtat ttgttacttg gagatggcaa atcgacttga 120gcaatgttaa agttttgggc atttaaatta tagacctttg cttggcacgg ttagcttgtt 180tcaaatccgt tgtttgttgt ggaatgtgtt tcacatatgt ggtaggtgaa gaatctcatt 240atggttcgct gtttcattct cttgcgttta tcacccgctg tctgctaact tagggtgtgt 300ttagtcctcg tcaaaattgg aagtttggtt gaaattggaa caatgtgacg aaaaagttga 360aatttttttt gtgggtagga aagttttgat gtgatagaaa agttggaagt ttaaaaaaaa 420agtttagaac taaactcggc cctagtcaat ttacccttac tattaggcat tatccccgct 480gtctgctaac ctagtcaatt tacccttgta ttattactag gcattatcct tgcctccgtt 540agtggtttgt tcttgagagc ggccaggtag gaaaattcca cttgaagagg agtgcgttac 600gccggcctct tgccattatc atcgtttgta ttgatatgca gaaatagaaa gaaaaaccta 660ggattttgat gaaaataaat cggatataga tatcataaaa cacattggaa cttgagatgg 720agagaactct agatattttc catgagattg ttacctaatt cctactttcc tacaaaatct 780tatgaaactg ccattcgaaa ggaatatcgt aggattctcg agtcccaatc cgttccagaa 840aggtaaatcg gaacaatggt acgaaaaatc tgtccccatt ggatcataag acttggacgg 900tctatcccat ggtactaagg gttttggaaa gaattttgca gaatttgaat ggatgtatcc 960tatacacaag ttttctgtgt acacatcgta cacatcaact aacaattttt accaaaaatc 1020taggaacaaa ttcacatgtt ctatcatgag ttatcacaca ttcacacgtt catatcttgg 1080ttaaatctga acagcaggct gtaaataaac cacaaaagtc tccaaattat gggcctcaac 1140agtaatcaat atcgtttcct cagacggcaa ttgccagttt gccacaacag ccatgttggc 1200taacatttga tacattcgga gtactgcttg aatttgcagg ctctccctaa ccttattatc 1260cctatagtct ttgactcttg atacctccaa accctaggaa gggcg 13051021350DNAArtificial sequenceterminator sequence from Orzza sativa with 5' and 3'-multiple cloning site linkers 102gaattcgccc ttcgagctcc gtccgatgtg attccgtcct cttgattgtt ttccttcccc 60ctaatttttg ttgcatacgc ggagagaagc tttagcttta agaggttaac tggcggcacc 120tcaatttctt tgggcttcag catgggttaa gaccacctaa taatcctgtc gcattcttgc 180tcttggaaaa gtttcttcca acattgcagt ccagtcagag aatggcctgt aataattagt 240gacaaaatta tagcagtaga tagaatctgt tcattcaagt tcttcaaatt actgggccaa 300gcttcaatgt cattttttgc ttaactcgtg ctcgtgctgt tatagtttac aactatcttc 360tttacctgcc cataaccaat gtgaccatcg aatcaaacgt atgtggcggt gaatcccgtt 420gcttgtgcgt tgctagattt ttgaggtcgc cttctgttcc aaattccaat catttgaatt 480cgaagggatt acctgtcgac tgcgcatacg atcttttatt gagaatattt gctaggtgtg 540ggagttcctg cgtgtgttcg tttagtggac gtgcgtatgt gagcatgtgc tatatgttgt 600gcttcgtgaa aactcagctt cttttcatca agatcggcac ctctgcttat cacctaatcc 660catcattcca atagcaacat aacatagatg tgacacatcc tatacatgat attgggatgg 720agagagtatt cctaagatgc atctctaccc tttctcttcg aatgcacatt tattgggagt 780catttttaat gtgcatttgt gcaaactggt accatggaac cctagtgcta ccctaacaga 840tatgctccat cgtcactaca aactcttgtt tacatccaca aactactgct tggaagggca 900cgctgacatt tgatcagaag aaagaatgac atgggtgcca aagttatgga tcagtgtggt 960gaaattcgag aataaccgac aagatacttc tcaatcttgt cacaataaga gggttatttt 1020tgtttagtac agtacaacta acgataccgc acaaaaaacg gtgaacgcga tatcgcacaa 1080aaaaaagagc aacacgtttg aattatattc aggtttgcta ctgcaatcca acaccatctc 1140ataactacat tatcctaatc gagccttttt aagtaatgag gccagtacaa ttaaaaaagg 1200tgccgtagac aatagcaaac ccatgaagaa ctgaaaacaa atgaatgcaa aactaaaatt 1260gtcttagttc tgtcgacaaa tgccaactgc tataatcgta catgcgtctc cgaagaaacc 1320gccgacactg cccctaggaa gggcgaattc 13501031532DNAArtificial sequenceterminator sequence from Orzza sativa with 5' and 3'-multiple cloning site linkers 103gaattcgccc ttcgagctcc agagtgatag acagtgctac ttgctagcct tttttatgag 60cacccttttt ggccttttag ttcctcatta gctaagagat gtgatgagtg aggttttatt 120ttttggcttt gtttggagtt ttgctttgga ggaagataga gaagccttta gaatgagttg 180tgatggagga tcacatagtt tgtctcaggt ttacaatgtg cggtaaaaaa aaggaaggaa 240ataaagtgat gttagaatat acagtttttt aatccatttc tacagcttca aattctcgat 300tgagcggcac gttctttgaa atgttcatag caaagcaaat caaacgaaat ggatgagatt 360tgaaactaag aaactgcact tgacaaagca cgctgcgctc tctgaaattc atgttcagca 420gaacgccttt cagaaattca tgttctgctg ctgcaggaag gcagctaaga actccatgca 480ataaaaatat aaaatgcagt gcagttatta gttagtgtag tagtaatcta agaaacaaca 540ggtttaacag aatattacct tcaaaaacat cccctcaaat caagactcgg aaatcaacac 600cattctgaaa ctagatgcaa tactgcgctc ctaatggaat cttcagtggt tactcctaca 660cgccactaaa aaaaacgggc tttggaaaaa cgtcaccctg aaaacaagag ctcagtaaaa 720ataccactct aaaatcttgc agcatgcatt atcacaaaag cagatcaatc cgtaattgta 780tttatgctgc atataccgtg atattttctg tgatcctctg cactatcttc agctcatcag 840ctgcatattt tgaagccgga ctgatggctc atcgctttgt ttcggcttat tgtgatagcc 900atccatgcgt tgacgaaaga tttagatata tcacaatttt aaccaagttc agagcaatgt 960tagctttagc tgatgatata cacggcgcac tgattgccgc cgttattttc agagccttgt 1020aaaacctgaa acaaattgtg tctccaaatt cttactagta ctgcagacat agcggatttg 1080ttttcaggat gaattatcac aaaggtaagt caattcatag ccatattacg caagttcaaa 1140actgcaggcc acaccatacg tttctgaaag tagaaacaag agatcttgca gcagatcacc 1200actaaagaag cagtaaccgc aaaaaattat aacataatcc agaattaaca cttcgcagca 1260ttactgaatt cactttaata gcacttctca tcatgaacta gtacaacaac ataactgtcc 1320agtggaaatg tgaaatgcat acaccaagta atggtccata acatgaacta tatggataca 1380acaacgttct tatttcgctc atatatacat gaaataagtc ttgcacgtct tggttactta 1440ataggtgcga taatcgccgt aggcttttag aagaagaaaa aaaagtgagc ctgcaaagtt 1500cctggggaca gttgaagacc aagggcgaat tc 15321041532DNAArtificial sequenceterminator sequence from Orzza sativa with 5' and 3'-multiple cloning site linkers 104cgaattcgcc cttggtcttc aactgtcccc aggaactttg caggctcact tttttttctt 60cttctaaaag cctacggcga ttatcgcacc tattaagtaa ccaagacgtg caagacttat 120ttcatgtata tatgagcgaa ataagaacgt tgttgtatcc atatagttca tgttatggac 180cattacttgg tgtatgcatt tcacatttcc actggacagt tatgttgttg tactagttca 240tgatgagaag tgctattaaa gtgaattcag taatgctgcg aagtgttaat tctggattat 300gttataattt tttgcggtta ctgcttcttt agtggtgatc tgctgcaaga tctcttgttt 360ctactttcag aaacgtatgg tgtggcctgc agttttgaac ttgcgtaata tggctatgaa 420ttgacttacc tttgtgataa ttcatcctga aaacaaatcc gctatgtctg cagtactagt 480aagaatttgg agacacaatt tgtttcaggt tttacaaggc tctgaaaata acggcggcaa 540tcagtgcgcc gtgtatatca tcagctaaag ctaacattgc tctgaacttg gttaaaattg 600tgatatatct aaatctttcg tcaacgcatg gatggctatc acaataagcc gaaacaaagc 660gatgagccat cagtccggct tcaaaatatg cagctgatga gctgaagata gtgcagagga 720tcacagaaaa tatcacggta tatgcagcat aaatacaatt acggattgat ctgcttttgt 780gataatgcat gctgcaagat tttagagtgg tatttttact gagctcttgt tttcagggtg 840acgtttttcc aaagcccgtt ttttttagtg gcgtgtagga gtaaccactg aagattccat 900taggagcgca gtattgcatc tagtttcaga atggtgttga tttccgagtc ttgatttgag 960gggatgtttt tgaaggtaat attctgttaa acctgttgtt tcttagatta ctactacact 1020aactaataac tgcactgcat tttatatttt tattgcatgg agttcttagc tgccttcctg 1080cagcagcaga acatgaattt ctgaaaggcg ttctgctgaa catgaatttc agagagcgca 1140gcgtgctttg tcaagtgcag tttcttagtt tcaaatctca tccatttcgt ttgatttgct 1200ttgctatgaa catttcaaag aacgtgccgc tcaatcgaga atttgaagct gtagaaatgg 1260attaaaaaac tgtatattct aacatcactt tatttccttc ctttttttta ccgcacattg 1320taaacctgag acaaactatg tgatcctcca tcacaactca ttctaaaggc ttctctatct 1380tcctccaaag caaaactcca aacaaagcca aaaaataaaa cctcactcat cacatctctt 1440agctaatgag gaactaaaag gccaaaaagg gtgctcataa aaaaggctag caagtagcac 1500tgtctatcac tctggagctc gaagggcgaa tt 153210516914DNAArtificial sequencebinary vector derived from pRJB058 105gtgattttgt gccgagctgc cggtcgggga gctgttggct ggctggtggc aggatatatt 60gtggtgtaaa caaattgacg cttagacaac ttaataacac attgcggacg tttttaatgt 120actgaattgg atccgcccgg gcggtacccg gggatcctct agaactagtg gatcccccgg 180gctgcaggtc aacggatcac ctatcaacat cccagctaaa aacagtaaaa agggggaaaa 240cgtgggtgag ttgagtctgt cttgtggaaa aaacgtttta gtttctcctg gaattaacaa 300taaaaacagt tgaacaagat tgactgttcc tccgggaggg tttggaacat cgttacagat 360gtgagcgaaa ggtgaggaaa cagagcggag ggcttggagg tgacctcggt agtcgacgcc 420ggagttgagc ttgatgacga caccgtactg gcgtaccagg cctagtagtg aacaccgggc 480ctgaagctgt cgccgccgct gctcatcttg tgggctgtgc ccggtgtccc tgttgcggat 540tgcgggtggc agcctggcag gtgggtgcga cccgtttgga ctccctgatc tgggcccttt 600gtgtcagtac cgtctgtact ccgatgacat gcacaccgtc gtccacagtc aagtccacaa 660tctcccctct ttttttaacg gaatagttca aaatctcctt gacgcacgct atcgtgtacc 720agcgctcact ggacaccacg tttgtaatcc acgccgacac gtcggtccca cgtcgacagg 780ccccaccgtc cggtctgtag cgtgtacgta ttcgggcgac ggacgtgtcg tcgtcgtctt 840gcgagtccca ttcccatcac catctgagcc acacatcctc tgaacaaaag cagggaggcc 900tctacgcaca tccccctttc tcccactccg tgtccgtggc acccacccca aaccctcgcg 960ccgcctccga gacagccgcc gcaaccatgg ccaccgccgc cgccgcgtct accgcgctca 1020ctggcgccac taccgctgcg cccaaggcga ggcgccgggc gcacctcctg gccacccgcc 1080gcgccctcgc cgcgcccatc aggtgctcag cggcgtcacc cgccatgccg atggctcccc

1140cggccacccc gctccggccg tggggcccca ccgatccccg caagggcgcc gacatcctcg 1200tcgagtccct cgagcgctgc ggcgtccgcg acgtcttcgc ctaccccggc ggcgcgtcca 1260tggagatcca ccaggcactc acccgctccc ccgtcatcgc caaccacctc ttccgccacg 1320agcaagggga ggcctttgcg gcctccggct acgcgcgctc ctcgggccgc gtcggcgtct 1380gcatcgccac ctccggcccc ggcgccacca accttgtctc cgcgctcgcc gacgcgctgc 1440tcgattccgt ccccatggtc gccatcacgg gacaggtgcc gcgacgcatg attggcaccg 1500acgccttcca ggagacgccc atcgtcgagg tcacccgctc catcaccaag cacaactacc 1560tggtcctcga cgtcgacgac atcccccgcg tcgtgcagga ggctttcttc ctcgcctcct 1620ctggtcgacc ggggccggtg cttgtcgaca tccccaagga catccagcag cagatggcgg 1680tgcctgtctg ggacaagccc atgagtctgc ctgggtacat tgcgcgcctt cccaagcccc 1740ctgcgactga gttgcttgag caggtgctgc gtcttgttgg tgaatcccgg cgccctgttc 1800tttatgttgg cggtggctgc gcagcatctg gtgaggagtt gcgacgcttt gtggagctga 1860ctggaatccc ggtcacaact actcttatgg gcctcggcaa cttccccagc gacgacccac 1920tgtctctgcg catgctaggt atgcatggca cggtgtatgc aaattatgca gtggataagg 1980ccgatctgtt gcttgcactt ggtgtgcggt ttgatgatcg tgtgacaggg aagattgagg 2040cttttgcaag cagggctaag attgtgcacg ttgatattga tccggctgag attggcaaga 2100acaagcagcc acatgtgtcc atctgtgcag atgttaagct tgctttgcag ggcatgaatg 2160ctcttcttga aggaagcaca tcaaagaaga gctttgactt tggctcatgg aacgatgagt 2220tggatcagca gaagagggaa ttcccccttg ggtataaaac atctaatgag gagatccagc 2280cacaatatgc tattcaggtt cttgatgagc tgacgaaagg cgaggccatc atcggcacag 2340gtgttgggca gcaccagatg tgggcggcac agtactacac ttacaagcgg ccaaggcagt 2400ggttgtcttc agctggtctt ggggctatgg gatttggttt gccggctgct gctggtgctt 2460ctgtggccaa cccaggtgtt actgttgttg acatcgatgg agatggtagc tttctcatga 2520acgttcagga gctagctatg atccgaattg agaacctccc ggtgaaggtc tttgtgctaa 2580acaaccagca cctggggatg gtggtgcagt gggaggacag gttctataag gccaacagag 2640cgcacacata cttgggaaac ccagagaatg aaagtgagat atatccagat ttcgtgacga 2700tcgccaaagg gttcaacatt ccagcggtcc gtgtgacaaa gaagaacgaa gtccgcgcag 2760cgataaagaa gatgctcgag actccagggc cgtacctctt ggatataatc gtcccacacc 2820aggagcatgt gttgcctatg atccctaatg gtggggcttt caaggatatg atcctggatg 2880gtgatggcag gactgtgtac tgatctaaaa tccagcaagc aactgatcta aaatccagca 2940agcaccgcct ccctgctagt acaagggtga tatgttttta tctgtgtgat gttctcctgt 3000attctatctt tttttgtagg ccgtcagcta tctgttatgg taatcctatg tagcttccga 3060ccttgtaatt gtgtagtctg ttgttttcct tctggcatgt gtcataagag atcatttaag 3120tgccttttgc tacatataaa taagataata agcactgcta tgcagtggtt ctgaattggc 3180ttctgttgcc aaatttaagt gtccaactgg tccttgcttt tgttttcgct atttttttcc 3240ttttttagtt attattatat tggtaatttc aactcaacat atgatgtatg gaataatgct 3300agggctgcaa tttcaaacta ttttacaaac cagaatggca ttttcgtggt ttgaggggag 3360tgaaaaaaaa tgaggcattt gactgaatta gttacctgat ccattttcgt ggtttggatc 3420attggaatta aattccattc taataatagt aattttggca tatatcaatt aagttaattc 3480ggttttatgc aaaatatatt tgtatactat tattatcaag atgtcggaga tatttatatg 3540ctacattttt actatacagg agtgagatga agagtgtcat gtaagttaca cagtagaaac 3600aaattctatt aatgcataaa atcatttcca tcatccaccc tatgaatttg agatagacct 3660atatctaaac tttgaaaagt ggttgaatat caaattccaa attaaataag ttattttatt 3720gagtgaattc taatttctct aaaacgaagg gatctaaacg ccctctaaag ctaatttgga 3780aactcaaact ttcttagcat tggaggggat tgagaaaaaa tattaattca ttttcatctc 3840aatcattcaa tctccaaaga gatttgagtt ccttattagt ctgttccatg catcaaatcg 3900gctcaatgtg tcattatttg ccatgacgat tgacgagttg ttctggggcc tagcgctttc 3960cacgccgatg tgctggggcc tggtcctgga gaagacagct tgatatttaa agctatcaat 4020tgtttcaatt gattcccact tcatttttct aaatgtagaa aacggtgacg tataagaaaa 4080agaatgaatt aggactttta ttccgtacac taatctagag cggccgcaag cttgtacaac 4140gcgtaccggt taattaaggt accaagcttc cgcggctgca gtgcagcgtg acccggtcgt 4200gcccctctct agagataatg agcattgcat gtctaagtta taaaaaatta ccacatattt 4260tttttgtcac acttgtttga agtgcagttt atctatcttt atacatatat ttaaacttta 4320ctctacgaat aatataatct atagtactac aataatatca gtgttttaga gaatcatata 4380aatgaacagt tagacatggt ctaaaggaca attgagtatt ttgacaacag gactctacag 4440ttttatcttt ttagtgtgca tgtgttctcc tttttttttg caaatagctt cacctatata 4500atacttcatc cattttatta gtacatccat ttagggttta gggttaatgg tttttataga 4560ctaatttttt tagtacatct attttattct attttagcct ctaaattaag aaaactaaaa 4620ctctatttta gtttttttat ttaatagttt agatataaaa tagaataaaa taaagtgact 4680aaaaattaaa caaataccct ttaagaaatt aaaaaaacta aggaaacatt tttcttgttt 4740cgagtagata atgccagcct gttaaacgcc gtcgacgagt ctaacggaca ccaaccagcg 4800aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca cggcatctct gtcgctgcct 4860ctggacccct ctcgagagtt ccgctccacc gttggacttg ctccgctgtc ggcatccaga 4920aattgcgtgg cggagcggca gacgtgagcc ggcacggcag gcggcctcct cctcctctca 4980cggcaccggc agctacgggg gattcctttc ccaccgctcc ttcgctttcc cttcctcgcc 5040cgccgtaata aatagacacc ccctccacac cctctttccc caacctcgtg ttgttcggag 5100cgcacacaca cacaaccaga tctcccccaa atccacccgt cggcacctcc gcttcaaggt 5160acgccgctcg tcctcccccc ccccccccct ctctaccttc tctagatcgg cgttccggtc 5220catggttagg gcccggtagt tctacttctg ttcatgtttg tgttagatcc gtgtttgtgt 5280tagatccgtg ctgctagcgt tcgtacacgg atgcgacctg tacgtcagac acgttctgat 5340tgctaacttg ccagtgtttc tctttgggga atcctgggat ggctctagcc gttccgcaga 5400cgggatcgat ttcatgattt tttttgtttc gttgcatagg gtttggtttg cccttttcct 5460ttatttcaat atatgccgtg cacttgtttg tcgggtcatc ttttcatgct tttttttgtc 5520ttggttgtga tgatgtggtc tggttgggcg gtcgttctag atcggagtag aattctgttt 5580caaactacct ggtggattta ttaattttgg atctgtatgt gtgtgccata catattcata 5640gttacgaatt gaagatgatg gatggaaata tcgatctagg ataggtatac atgttgatgc 5700gggttttact gatgcatata cagagatgct ttttgttcgc ttggttgtga tgatgtggtg 5760tggttgggcg gtcgttcatt cgttctagat cggagtagaa tactgtttca aactacctgg 5820tgtatttatt aattttggaa ctgtatgtgt gtgtcataca tcttcatagt tacgagttta 5880agatggatgg aaatatcgat ctaggatagg tatacatgtt gatgtgggtt ttactgatgc 5940atatacatga tggcatatgc agcatctatt catatgctct aaccttgagt acctatctat 6000tataataaac aagtatgttt tataattatt tcgatcttga tatacttgga tgatggcata 6060tgcagcagct atatgtggat ttttttagcc ctgccttcat acgctattta tttgcttggt 6120actgtttctt ttgtcgatgc tcaccctgtt gtttggtgtt acttctgcag ggtacggatc 6180cggcgcgcca ctagtcccgg tcgccaccat ggcctcctcc gagaacgtca tcaccgagtt 6240catgcgcttc aaggtgcgca tggagggcac cgtgaacggc cacgagttcg agatcgaggg 6300cgagggcgag ggccgcccct acgagggcca caacaccgtg aagctgaagg tgaccaaggg 6360cggccccctg cccttcgcct gggacatcct gtccccccag ttccagtacg gctccaaggt 6420gtacgtgaag caccccgccg acatccccga ctacaagaag ctgtccttcc ccgagggctt 6480caagtgggag cgcgtgatga acttcgagga cggcggcgtg gcgaccgtga cccaggactc 6540ctccctgcag gacggctgct tcatctacaa ggtgaagttc atcggcgtga acttcccctc 6600cgacggcccc gtgatgcaga agaagaccat gggctgggag gcctccaccg agcgcctgta 6660cccccgcgac ggcgtgctga agggcgagac ccacaaggcc ctgaagctga aggacggcgg 6720ccactacctg gtggagttca agtccatcta catggccaag aagcccgtgc agctgcccgg 6780ctactactac gtggacgcca agctggacat cacctcccac aacgaggact acaccatcgt 6840ggagcagtac gagcgcaccg agggccgcca ccacctgttc ctgtagcggc cgccctgcag 6900ggagctcgaa tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc 6960ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa 7020taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc 7080aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat 7140cgcgcgcggt gtcatctatg ttactagatc gggaattcgg gcccggccgg ccagatcttg 7200attgtcgttt cccgccttca gtttctggca cgacaggttt cccgactgga aagcgggcag 7260tgagcgcaac gcaattaata cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa 7320aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt 7380cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt 7440gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc 7500gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcgt taacgctagc 7560atggatgttt tcccagtcac gacgttgtaa aacgacggcc agtcttaact agtgatgtag 7620ccctcaggca tggcgctctt gaagaagtcg tgctgcttca tgtgatcggg gtagcgtgag 7680aagcactgca cgccgtagct cagggtggtc accagggtgg gccagggcac aggcagcttg 7740ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg 7800ccgctcacgc tgaacttgtg gccattcaca tcgccattca gctcgatcag gatgggcacg 7860atgccggtga acagctcggt ccgggcgccc cctagggagc tcggtagcaa ttcccgaggc 7920tgtagccgac gatggtgcgc caggagagtt gttgattcat tgtttgcctc cctgctgcgg 7980tttttcaccg aagttcatgc cagtccagcg tttttgcagc agaaaagccg ccgacttcgg 8040tttgcggtcg cgagtgaaga tccctttctt gttaccgcca acgcgcaata tgccttgcga 8100ggtcgcaaaa tcggcgaaat tccatacctg ttcaccgacg acggcgctga cgcgatcaaa 8160gacgcggtga tacatatcca gccatgcaca ctgatactct tcactccaca tgtcggtgta 8220cattgagtgc agcccggcta acgtatccac gccgtattcg gtgatgataa tcggctgatg 8280cagtttctcc tgccaggcca gaagttcttt ttccagtacc ttctctgccg tttccaaatc 8340gccgctttgg acataccatc cgtaataacg gttcaggcac agcacatcaa agagatcgct 8400gatggtatcg gtgtgagcgt cgcagaacat tacattgacg caggtgatcg gacgcgtcgg 8460gtcgagttta cgcgttgctt ccgccagtgg cgcgaaatat tcccgtgcac cttgcggacg 8520ggtatccggt tcgttggcaa tactccacat caccacgctt gggtggtttt tgtcacgcgc 8580tatcagctct ttaatcgcct gtaagtgcgc ttgctgagtt tccccgttga ctgcctcttc 8640gctgtacagt tctttcggct tgttgcccgc ttcgaaacca atgcctaaag agaggttaaa 8700gccgacagca gcagtttcat caatcaccac gatgccatgt tcatctgccc agtcgagcat 8760ctcttcagcg taagggtaat gcgaggtacg gtaggagttg gccccaatcc agtccattaa 8820tgcgtggtcg tgcaccatca gcacgttatc gaatcctttg ccacgcaagt ccgcatcttc 8880atgacgacca aagccagtaa agtagaacgg tttgtggtta atcaggaact gttcgccctt 8940cactgccact gaccggatgc cgacgcgaag cgggtagata tcacactctg tctggctttt 9000ggctgtgacg cacagttcat agagataacc ttcacccggt tgccagaggt gcggattcac 9060cacttgcaaa gtcccgctag tgccttgtcc agttgcaacc acctgttgat ccgcatcacg 9120cagttcaacg ctgacatcac cattggccac cacctgccag tcaacagacg cgtggttaca 9180gtcttgcgcg acatgcgtca ccacggtgat atcgtccacc caggtgttcg gcgtggtgta 9240gagcattacg ctgcgatgga ttccggcata gttaaagaaa tcatggaagt aagactgctt 9300tttcttgccg ttttcgtcgg taatcaccat tcccggcggg atagtctgcc agttcagttc 9360gttgttcaca caaacggtga tacctgcaca tcaacaaatt ttggtcatat attagaaaag 9420ttataaatta aaatatacac acttataaac tacagaaaag caattgctat atactacatt 9480cttttatttt gaaaaaaata tttgaaatat tatattacta ctaattaatg ataattatta 9540tatatatatc aaaggtagaa gcagaaactt acgtacactt ttcccggcaa taacatacgg 9600cgtgacatcg gcttcaaatg gcgtatagcc gccctgatgc tccatcactt cctgattatt 9660gacccacact ttgccgtaat gagtgaccgc atcgaaacgc agcacgatac gctggcctgc 9720ccaacctttc ggtataaaga cttcgcgctg ataccagacg ttgcccgcat aattacgaat 9780atctgcatcg gcgaactgat cgttaaaact gcctggcaca gcaattgccc ggctttcttg 9840taacgcgctt tcccaccaac gctgaccaat tccacagttt tcgcgatcca gactgaatgc 9900ccacaggccg tcgagttttt tgatttcacg ggttggggtt tctacaggac gtaacataag 9960ggactgacct acccgggggt accctgcaga agtaacacca aacaacaggg tgagcatcga 10020caaaagaaac agtaccaagc aaataaatag cgtatgaagg cagggctaaa aaaatccaca 10080tatagctgct gcatatgcca tcatccaagt atatcaagat cgaaataatt ataaaacata 10140cttgtttatt ataatagata ggtactcaag gttagagcat atgaatagat gctgcatatg 10200ccatcatgta tatgcatcag taaaacccac atcaacatgt atacctatcc tagatcgata 10260tttccatcca tcttaaactc gtaactatga agatgtatga cacacacata cagttccaaa 10320attaataaat acaccaggta gtttgaaaca gtattctact ccgatctaga acgaatgaac 10380gaccgcccaa ccacaccaca tcatcacaac caagcgaaca aaaagcatct ctgtatatgc 10440atcagtaaaa cccgcatcaa catgtatacc tatcctagat cgatatttcc atccatcatc 10500ttcaattcgt aactatgaat atgtatggca cacacataca gatccaaaat taataaatcc 10560accaggtagt ttgaaacaga attctactcc gatctagaac gaccgcccaa ccagaccaca 10620tcatcacaac caagacaaaa aaaagcatga aaagatgacc cgacaaacaa gtgcacggca 10680tatattgaaa taaaggaaaa gggcaaacca aaccctatgc aacgaaacaa aaaaaatcat 10740gaaatcgatc ccgtctgcgg aacggctaga gccatcccag gattccccaa agagaaacac 10800tggcaagtta gcaatcagaa cgtgtctgac gtacaggtcg catccgtgta cgaacgctag 10860cagcacggat ctaacacaaa cacggatcta acacaaacat gaacagaagt agaactaccg 10920ggccctaacc atggaccgga acgccgatct agagaaggta gagagggggg ggggggggga 10980ggacgagcgg cgtaccttga agcggaggtg ccgacgggtg gatttggggg agatctggtt 11040gtgtgtgtgt gcgctccgaa caacacgagg ttggggaaag agggtgtgga gggggtgtct 11100atttattacg gcgggcgagg aagggaaagc gaaggagcgg tgggaaagga atcccccgta 11160gctgccggtg ccgtgagagg aggaggaggc cgcctgccgt gccggctcac gtctgccgct 11220ccgccacgca atttctggat gccgacagcg gagcaagtcc aacggtggag cggaactctc 11280gagaggggtc cagaggcagc gacagagatg ccgtgccgtc tgcttcgctt ggcccgacgc 11340gacgctgctg gttcgctggt tggtgtccgt tagactcgtc gacggcgttt aacaggctgg 11400cattatctac tcgaaacaag aaaaatgttt ccttagtttt tttaatttct taaagggtat 11460ttgtttaatt tttagtcact ttattttatt ctattttata tctaaactat taaataaaaa 11520aactaaaata gagttttagt tttcttaatt tagaggctaa aatagaataa aatagatgta 11580ctaaaaaaat tagtctataa aaaccattaa ccctaaaccc taaatggatg tactaataaa 11640atggatgaag tattatatag gtgaagctat ttgcaaaaaa aaaggagaac acatgcacac 11700taaaaagata aaactgtaga gtcctgttgt caaaatactc aattgtcctt tagaccatgt 11760ctaactgttc atttatatga ttctctaaaa cactgatatt attgtagtac tatagattat 11820attattcgta gagtaaagtt taaatatatg tataaagata gataaactgc acttcaaaca 11880agtgtgacaa aaaaaatatg tggtaatttt ttataactta gacatgcaat gctcattatc 11940tctagagagg ggcacgaccg ggtcacgctg cactgcagcc gcggaagctt gcatgcctgc 12000aggcatgcaa gcttggcgcg ccgacccagc tttcttgtac aaagttggca ttataagaaa 12060gcattgctta tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat 12120ttgccatcca gaaactatca gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa 12180agagcgttta ttagaataat cggatattta aaagggcgtg aaaaggttta tccgttcgtc 12240catttgtatg tgcatgccaa ccacagggtt cccctcggga gtgcttggca ttccgtgcga 12300taatgacttc tgttcaacca cccaaacgtc ggaaagcctg acgacggagc agcattccaa 12360aaagatccct tggctcgtct gggtcggcta gaaggtcgag tgggctgctg tggcttgatc 12420cctcaacgcg gtcgcggacg tagcgcagcg ccgaaaaatc ctcgatcgca aatccgacgc 12480tgtcgaaaag cgtgatctgc ttgtcgctct ttcggccgac gtcctggcca gtcatcacgc 12540gccaaagttc cgtcacagga tgatctggcg cgagttgctg gatctcgcct tcaatccggg 12600tctgtggcgg gaactccacg aaaatatccg aacgcagcaa gatcgtcgac caattcttga 12660agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt 12720tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 12780ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 12840taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 12900tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 12960gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 13020atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 13080ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata 13140cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 13200ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 13260aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 13320ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 13380gacgagcgtg acaccacgat gccggggggg gggggggggg acatgaggtt gccccgtatt 13440cagtgtcgct gatttgtatt gtctgaagtt gtttttacgt taagttgatg cagatcaatt 13500aatacgatac ctgcgtcata attgattatt tgacgtggtt tgatggcctc cacgcacgtt 13560gtgatatgta gatgataatc attatcactt tacgggtcct ttccggtgat ccgacaggtt 13620acggggcggc gacctcgcgg gttttcgcta tttatgaaaa ttttccggtt taaggcgttt 13680ccgttcttct tcgtcataac ttaatgtttt tatttaaaat accctctgaa aagaaaggaa 13740acgacaggtg ctgaaagcga gctttttggc ctctgtcgtt tcctttctct gtttttgtcc 13800gtggaatgaa caatggaacc cccccccccc ccccctgcag caatggcaac aacgttgcgc 13860aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 13920gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 13980gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 14040gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 14100gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 14160gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 14220atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 14280ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 14340ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 14400ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 14460ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 14520ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 14580tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 14640tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 14700tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 14760tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 14820gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 14880tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 14940ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 15000gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 15060gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt 15120acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat 15180gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc 15240cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 15300cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 15360caccgaaacg cgcgaggcag cagatccccc gatcaagtag atacactaca tatatctaca 15420atagacatcg agccggaagg tgatgtttac tttcctgaaa tccccagcaa ttttaggcca 15480gtttttaccc aagacttcgc ctctaacata aattatagtt accaaatctg gcaaaagggt 15540tgaccggggg gggggggaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca 15600agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag 15660gggtgttatg agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa 15720catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc 15780gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa 15840aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt 15900tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac 15960cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga 16020aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa 16080ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa 16140cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt

16200ctggaaagaa atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga 16260tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg 16320acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga 16380gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat 16440gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg 16500gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcggctttg ttgaataaat 16560cgaacttttg ctgagttgaa ggatcagatc acgcatcttc ccgacaacgc agaccgttcc 16620gtggcaaagc aaaagttcaa aatcaccaac tggtccacct acaacaaagc tctcatcaac 16680cgtggctccc tcactttctg gctggatgat ggggcgattc agggatcaca ggcagcaacg 16740ctctgtcatc gttacaatca acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg 16800gcagcttagt tgccgttctt ccgaatagca tcggtaacat gagcaaagtc tgccgcctta 16860caacggctct cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg agtg 1691410615919DNAArtificial sequencebinary vector derived from pRLI024 106gtgattttgt gccgagctgc cggtcgggga gctgttggct ggctggtggc aggatatatt 60gtggtgtaaa caaattgacg cttagacaac ttaataacac attgcggacg tttttaatgt 120actgaattgg atccgcccgg gcggtacccg gggatcctct agaactagtg gatcccccgg 180gctgcaggtc aacggatcac ctatcaacat cccagctaaa aacagtaaaa agggggaaaa 240cgtgggtgag ttgagtctgt cttgtggaaa aaacgtttta gtttctcctg gaattaacaa 300taaaaacagt tgaacaagat tgactgttcc tccgggaggg tttggaacat cgttacagat 360gtgagcgaaa ggtgaggaaa cagagcggag ggcttggagg tgacctcggt agtcgacgcc 420ggagttgagc ttgatgacga caccgtactg gcgtaccagg cctagtagtg aacaccgggc 480ctgaagctgt cgccgccgct gctcatcttg tgggctgtgc ccggtgtccc tgttgcggat 540tgcgggtggc agcctggcag gtgggtgcga cccgtttgga ctccctgatc tgggcccttt 600gtgtcagtac cgtctgtact ccgatgacat gcacaccgtc gtccacagtc aagtccacaa 660tctcccctct ttttttaacg gaatagttca aaatctcctt gacgcacgct atcgtgtacc 720agcgctcact ggacaccacg tttgtaatcc acgccgacac gtcggtccca cgtcgacagg 780ccccaccgtc cggtctgtag cgtgtacgta ttcgggcgac ggacgtgtcg tcgtcgtctt 840gcgagtccca ttcccatcac catctgagcc acacatcctc tgaacaaaag cagggaggcc 900tctacgcaca tccccctttc tcccactccg tgtccgtggc acccacccca aaccctcgcg 960ccgcctccga gacagccgcc gcaaccatgg ccaccgccgc cgccgcgtct accgcgctca 1020ctggcgccac taccgctgcg cccaaggcga ggcgccgggc gcacctcctg gccacccgcc 1080gcgccctcgc cgcgcccatc aggtgctcag cggcgtcacc cgccatgccg atggctcccc 1140cggccacccc gctccggccg tggggcccca ccgatccccg caagggcgcc gacatcctcg 1200tcgagtccct cgagcgctgc ggcgtccgcg acgtcttcgc ctaccccggc ggcgcgtcca 1260tggagatcca ccaggcactc acccgctccc ccgtcatcgc caaccacctc ttccgccacg 1320agcaagggga ggcctttgcg gcctccggct acgcgcgctc ctcgggccgc gtcggcgtct 1380gcatcgccac ctccggcccc ggcgccacca accttgtctc cgcgctcgcc gacgcgctgc 1440tcgattccgt ccccatggtc gccatcacgg gacaggtgcc gcgacgcatg attggcaccg 1500acgccttcca ggagacgccc atcgtcgagg tcacccgctc catcaccaag cacaactacc 1560tggtcctcga cgtcgacgac atcccccgcg tcgtgcagga ggctttcttc ctcgcctcct 1620ctggtcgacc ggggccggtg cttgtcgaca tccccaagga catccagcag cagatggcgg 1680tgcctgtctg ggacaagccc atgagtctgc ctgggtacat tgcgcgcctt cccaagcccc 1740ctgcgactga gttgcttgag caggtgctgc gtcttgttgg tgaatcccgg cgccctgttc 1800tttatgttgg cggtggctgc gcagcatctg gtgaggagtt gcgacgcttt gtggagctga 1860ctggaatccc ggtcacaact actcttatgg gcctcggcaa cttccccagc gacgacccac 1920tgtctctgcg catgctaggt atgcatggca cggtgtatgc aaattatgca gtggataagg 1980ccgatctgtt gcttgcactt ggtgtgcggt ttgatgatcg tgtgacaggg aagattgagg 2040cttttgcaag cagggctaag attgtgcacg ttgatattga tccggctgag attggcaaga 2100acaagcagcc acatgtgtcc atctgtgcag atgttaagct tgctttgcag ggcatgaatg 2160ctcttcttga aggaagcaca tcaaagaaga gctttgactt tggctcatgg aacgatgagt 2220tggatcagca gaagagggaa ttcccccttg ggtataaaac atctaatgag gagatccagc 2280cacaatatgc tattcaggtt cttgatgagc tgacgaaagg cgaggccatc atcggcacag 2340gtgttgggca gcaccagatg tgggcggcac agtactacac ttacaagcgg ccaaggcagt 2400ggttgtcttc agctggtctt ggggctatgg gatttggttt gccggctgct gctggtgctt 2460ctgtggccaa cccaggtgtt actgttgttg acatcgatgg agatggtagc tttctcatga 2520acgttcagga gctagctatg atccgaattg agaacctccc ggtgaaggtc tttgtgctaa 2580acaaccagca cctggggatg gtggtgcagt gggaggacag gttctataag gccaacagag 2640cgcacacata cttgggaaac ccagagaatg aaagtgagat atatccagat ttcgtgacga 2700tcgccaaagg gttcaacatt ccagcggtcc gtgtgacaaa gaagaacgaa gtccgcgcag 2760cgataaagaa gatgctcgag actccagggc cgtacctctt ggatataatc gtcccacacc 2820aggagcatgt gttgcctatg atccctaatg gtggggcttt caaggatatg atcctggatg 2880gtgatggcag gactgtgtac tgatctaaaa tccagcaagc aactgatcta aaatccagca 2940agcaccgcct ccctgctagt acaagggtga tatgttttta tctgtgtgat gttctcctgt 3000attctatctt tttttgtagg ccgtcagcta tctgttatgg taatcctatg tagcttccga 3060ccttgtaatt gtgtagtctg ttgttttcct tctggcatgt gtcataagag atcatttaag 3120tgccttttgc tacatataaa taagataata agcactgcta tgcagtggtt ctgaattggc 3180ttctgttgcc aaatttaagt gtccaactgg tccttgcttt tgttttcgct atttttttcc 3240ttttttagtt attattatat tggtaatttc aactcaacat atgatgtatg gaataatgct 3300agggctgcaa tttcaaacta ttttacaaac cagaatggca ttttcgtggt ttgaggggag 3360tgaaaaaaaa tgaggcattt gactgaatta gttacctgat ccattttcgt ggtttggatc 3420attggaatta aattccattc taataatagt aattttggca tatatcaatt aagttaattc 3480ggttttatgc aaaatatatt tgtatactat tattatcaag atgtcggaga tatttatatg 3540ctacattttt actatacagg agtgagatga agagtgtcat gtaagttaca cagtagaaac 3600aaattctatt aatgcataaa atcatttcca tcatccaccc tatgaatttg agatagacct 3660atatctaaac tttgaaaagt ggttgaatat caaattccaa attaaataag ttattttatt 3720gagtgaattc taatttctct aaaacgaagg gatctaaacg ccctctaaag ctaatttgga 3780aactcaaact ttcttagcat tggaggggat tgagaaaaaa tattaattca ttttcatctc 3840aatcattcaa tctccaaaga gatttgagtt ccttattagt ctgttccatg catcaaatcg 3900gctcaatgtg tcattatttg ccatgacgat tgacgagttg ttctggggcc tagcgctttc 3960cacgccgatg tgctggggcc tggtcctgga gaagacagct tgatatttaa agctatcaat 4020tgtttcaatt gattcccact tcatttttct aaatgtagaa aacggtgacg tataagaaaa 4080agaatgaatt aggactttta ttccgtacac taatctagag cggccgcaag cttgtacaac 4140gcgtaccggt taattaaggt accaagcttc cgcggctgca gtgcagcgtg acccggtcgt 4200gcccctctct agagataatg agcattgcat gtctaagtta taaaaaatta ccacatattt 4260tttttgtcac acttgtttga agtgcagttt atctatcttt atacatatat ttaaacttta 4320ctctacgaat aatataatct atagtactac aataatatca gtgttttaga gaatcatata 4380aatgaacagt tagacatggt ctaaaggaca attgagtatt ttgacaacag gactctacag 4440ttttatcttt ttagtgtgca tgtgttctcc tttttttttg caaatagctt cacctatata 4500atacttcatc cattttatta gtacatccat ttagggttta gggttaatgg tttttataga 4560ctaatttttt tagtacatct attttattct attttagcct ctaaattaag aaaactaaaa 4620ctctatttta gtttttttat ttaatagttt agatataaaa tagaataaaa taaagtgact 4680aaaaattaaa caaataccct ttaagaaatt aaaaaaacta aggaaacatt tttcttgttt 4740cgagtagata atgccagcct gttaaacgcc gtcgacgagt ctaacggaca ccaaccagcg 4800aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca cggcatctct gtcgctgcct 4860ctggacccct ctcgagagtt ccgctccacc gttggacttg ctccgctgtc ggcatccaga 4920aattgcgtgg cggagcggca gacgtgagcc ggcacggcag gcggcctcct cctcctctca 4980cggcaccggc agctacgggg gattcctttc ccaccgctcc ttcgctttcc cttcctcgcc 5040cgccgtaata aatagacacc ccctccacac cctctttccc caacctcgtg ttgttcggag 5100cgcacacaca cacaaccaga tctcccccaa atccacccgt cggcacctcc gcttcaaggt 5160acgccgctcg tcctcccccc ccccccccct ctctaccttc tctagatcgg cgttccggtc 5220catggttagg gcccggtagt tctacttctg ttcatgtttg tgttagatcc gtgtttgtgt 5280tagatccgtg ctgctagcgt tcgtacacgg atgcgacctg tacgtcagac acgttctgat 5340tgctaacttg ccagtgtttc tctttgggga atcctgggat ggctctagcc gttccgcaga 5400cgggatcgat ttcatgattt tttttgtttc gttgcatagg gtttggtttg cccttttcct 5460ttatttcaat atatgccgtg cacttgtttg tcgggtcatc ttttcatgct tttttttgtc 5520ttggttgtga tgatgtggtc tggttgggcg gtcgttctag atcggagtag aattctgttt 5580caaactacct ggtggattta ttaattttgg atctgtatgt gtgtgccata catattcata 5640gttacgaatt gaagatgatg gatggaaata tcgatctagg ataggtatac atgttgatgc 5700gggttttact gatgcatata cagagatgct ttttgttcgc ttggttgtga tgatgtggtg 5760tggttgggcg gtcgttcatt cgttctagat cggagtagaa tactgtttca aactacctgg 5820tgtatttatt aattttggaa ctgtatgtgt gtgtcataca tcttcatagt tacgagttta 5880agatggatgg aaatatcgat ctaggatagg tatacatgtt gatgtgggtt ttactgatgc 5940atatacatga tggcatatgc agcatctatt catatgctct aaccttgagt acctatctat 6000tataataaac aagtatgttt tataattatt tcgatcttga tatacttgga tgatggcata 6060tgcagcagct atatgtggat ttttttagcc ctgccttcat acgctattta tttgcttggt 6120actgtttctt ttgtcgatgc tcaccctgtt gtttggtgtt acttctgcag ggtacggatc 6180cggcgcgcca ctagtcccgg tcgccaccat ggcctcctcc gagaacgtca tcaccgagtt 6240catgcgcttc aaggtgcgca tggagggcac cgtgaacggc cacgagttcg agatcgaggg 6300cgagggcgag ggccgcccct acgagggcca caacaccgtg aagctgaagg tgaccaaggg 6360cggccccctg cccttcgcct gggacatcct gtccccccag ttccagtacg gctccaaggt 6420gtacgtgaag caccccgccg acatccccga ctacaagaag ctgtccttcc ccgagggctt 6480caagtgggag cgcgtgatga acttcgagga cggcggcgtg gcgaccgtga cccaggactc 6540ctccctgcag gacggctgct tcatctacaa ggtgaagttc atcggcgtga acttcccctc 6600cgacggcccc gtgatgcaga agaagaccat gggctgggag gcctccaccg agcgcctgta 6660cccccgcgac ggcgtgctga agggcgagac ccacaaggcc ctgaagctga aggacggcgg 6720ccactacctg gtggagttca agtccatcta catggccaag aagcccgtgc agctgcccgg 6780ctactactac gtggacgcca agctggacat cacctcccac aacgaggact acaccatcgt 6840ggagcagtac gagcgcaccg agggccgcca ccacctgttc ctgtagcggc cgccctgcag 6900ggagctcggt agcaattccc gaggctgtag ccgacgatgg tgcgccagga gagttgttga 6960ttcattgttt gcctccctgc tgcggttttt caccgaagtt catgccagtc cagcgttttt 7020gcagcagaaa agccgccgac ttcggtttgc ggtcgcgagt gaagatccct ttcttgttac 7080cgccaacgcg caatatgcct tgcgaggtcg caaaatcggc gaaattccat acctgttcac 7140cgacgacggc gctgacgcga tcaaagacgc ggtgatacat atccagccat gcacactgat 7200actcttcact ccacatgtcg gtgtacattg agtgcagccc ggctaacgta tccacgccgt 7260attcggtgat gataatcggc tgatgcagtt tctcctgcca ggccagaagt tctttttcca 7320gtaccttctc tgccgtttcc aaatcgccgc tttggacata ccatccgtaa taacggttca 7380ggcacagcac atcaaagaga tcgctgatgg tatcggtgtg agcgtcgcag aacattacat 7440tgacgcaggt gatcggacgc gtcgggtcga gtttacgcgt tgcttccgcc agtggcgcga 7500aatattcccg tgcaccttgc ggacgggtat ccggttcgtt ggcaatactc cacatcacca 7560cgcttgggtg gtttttgtca cgcgctatca gctctttaat cgcctgtaag tgcgcttgct 7620gagtttcccc gttgactgcc tcttcgctgt acagttcttt cggcttgttg cccgcttcga 7680aaccaatgcc taaagagagg ttaaagccga cagcagcagt ttcatcaatc accacgatgc 7740catgttcatc tgcccagtcg agcatctctt cagcgtaagg gtaatgcgag gtacggtagg 7800agttggcccc aatccagtcc attaatgcgt ggtcgtgcac catcagcacg ttatcgaatc 7860ctttgccacg caagtccgca tcttcatgac gaccaaagcc agtaaagtag aacggtttgt 7920ggttaatcag gaactgttcg cccttcactg ccactgaccg gatgccgacg cgaagcgggt 7980agatatcaca ctctgtctgg cttttggctg tgacgcacag ttcatagaga taaccttcac 8040ccggttgcca gaggtgcgga ttcaccactt gcaaagtccc gctagtgcct tgtccagttg 8100caaccacctg ttgatccgca tcacgcagtt caacgctgac atcaccattg gccaccacct 8160gccagtcaac agacgcgtgg ttacagtctt gcgcgacatg cgtcaccacg gtgatatcgt 8220ccacccaggt gttcggcgtg gtgtagagca ttacgctgcg atggattccg gcatagttaa 8280agaaatcatg gaagtaagac tgctttttct tgccgttttc gtcggtaatc accattcccg 8340gcgggatagt ctgccagttc agttcgttgt tcacacaaac ggtgatacct gcacatcaac 8400aaattttggt catatattag aaaagttata aattaaaata tacacactta taaactacag 8460aaaagcaatt gctatatact acattctttt attttgaaaa aaatatttga aatattatat 8520tactactaat taatgataat tattatatat atatcaaagg tagaagcaga aacttacgta 8580cacttttccc ggcaataaca tacggcgtga catcggcttc aaatggcgta tagccgccct 8640gatgctccat cacttcctga ttattgaccc acactttgcc gtaatgagtg accgcatcga 8700aacgcagcac gatacgctgg cctgcccaac ctttcggtat aaagacttcg cgctgatacc 8760agacgttgcc cgcataatta cgaatatctg catcggcgaa ctgatcgtta aaactgcctg 8820gcacagcaat tgcccggctt tcttgtaacg cgctttccca ccaacgctga ccaattccac 8880agttttcgcg atccagactg aatgcccaca ggccgtcgag ttttttgatt tcacgggttg 8940gggtttctac aggacgtaac ataagggact gacctacccg ggggtaccct gcagaagtaa 9000caccaaacaa cagggtgagc atcgacaaaa gaaacagtac caagcaaata aatagcgtat 9060gaaggcaggg ctaaaaaaat ccacatatag ctgctgcata tgccatcatc caagtatatc 9120aagatcgaaa taattataaa acatacttgt ttattataat agataggtac tcaaggttag 9180agcatatgaa tagatgctgc atatgccatc atgtatatgc atcagtaaaa cccacatcaa 9240catgtatacc tatcctagat cgatatttcc atccatctta aactcgtaac tatgaagatg 9300tatgacacac acatacagtt ccaaaattaa taaatacacc aggtagtttg aaacagtatt 9360ctactccgat ctagaacgaa tgaacgaccg cccaaccaca ccacatcatc acaaccaagc 9420gaacaaaaag catctctgta tatgcatcag taaaacccgc atcaacatgt atacctatcc 9480tagatcgata tttccatcca tcatcttcaa ttcgtaacta tgaatatgta tggcacacac 9540atacagatcc aaaattaata aatccaccag gtagtttgaa acagaattct actccgatct 9600agaacgaccg cccaaccaga ccacatcatc acaaccaaga caaaaaaaag catgaaaaga 9660tgacccgaca aacaagtgca cggcatatat tgaaataaag gaaaagggca aaccaaaccc 9720tatgcaacga aacaaaaaaa atcatgaaat cgatcccgtc tgcggaacgg ctagagccat 9780cccaggattc cccaaagaga aacactggca agttagcaat cagaacgtgt ctgacgtaca 9840ggtcgcatcc gtgtacgaac gctagcagca cggatctaac acaaacacgg atctaacaca 9900aacatgaaca gaagtagaac taccgggccc taaccatgga ccggaacgcc gatctagaga 9960aggtagagag gggggggggg ggggaggacg agcggcgtac cttgaagcgg aggtgccgac 10020gggtggattt gggggagatc tggttgtgtg tgtgtgcgct ccgaacaaca cgaggttggg 10080gaaagagggt gtggaggggg tgtctattta ttacggcggg cgaggaaggg aaagcgaagg 10140agcggtggga aaggaatccc ccgtagctgc cggtgccgtg agaggaggag gaggccgcct 10200gccgtgccgg ctcacgtctg ccgctccgcc acgcaatttc tggatgccga cagcggagca 10260agtccaacgg tggagcggaa ctctcgagag gggtccagag gcagcgacag agatgccgtg 10320ccgtctgctt cgcttggccc gacgcgacgc tgctggttcg ctggttggtg tccgttagac 10380tcgtcgacgg cgtttaacag gctggcatta tctactcgaa acaagaaaaa tgtttcctta 10440gtttttttaa tttcttaaag ggtatttgtt taatttttag tcactttatt ttattctatt 10500ttatatctaa actattaaat aaaaaaacta aaatagagtt ttagttttct taatttagag 10560gctaaaatag aataaaatag atgtactaaa aaaattagtc tataaaaacc attaacccta 10620aaccctaaat ggatgtacta ataaaatgga tgaagtatta tataggtgaa gctatttgca 10680aaaaaaaagg agaacacatg cacactaaaa agataaaact gtagagtcct gttgtcaaaa 10740tactcaattg tcctttagac catgtctaac tgttcattta tatgattctc taaaacactg 10800atattattgt agtactatag attatattat tcgtagagta aagtttaaat atatgtataa 10860agatagataa actgcacttc aaacaagtgt gacaaaaaaa atatgtggta attttttata 10920acttagacat gcaatgctca ttatctctag agaggggcac gaccgggtca cgctgcactg 10980cagccgcgga agcttgcatg cctgcaggca tgcaagcttg gcgcgccgac ccagctttct 11040tgtacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac gaacaggtca 11100ctatcagtca aaataaaatc attatttgcc atccagaaac tatcagtgtt tgacaggata 11160tattggcggg taaacctaag agaaaagagc gtttattaga ataatcggat atttaaaagg 11220gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct 11280cgggagtgct tggcattccg tgcgataatg acttctgttc aaccacccaa acgtcggaaa 11340gcctgacgac ggagcagcat tccaaaaaga tcccttggct cgtctgggtc ggctagaagg 11400tcgagtgggc tgctgtggct tgatccctca acgcggtcgc ggacgtagcg cagcgccgaa 11460aaatcctcga tcgcaaatcc gacgctgtcg aaaagcgtga tctgcttgtc gctctttcgg 11520ccgacgtcct ggccagtcat cacgcgccaa agttccgtca caggatgatc tggcgcgagt 11580tgctggatct cgccttcaat ccgggtctgt ggcgggaact ccacgaaaat atccgaacgc 11640agcaagatcg tcgaccaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata 11700ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 11760gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 11820acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 11880tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 11940agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 12000cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 12060aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg 12120gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 12180agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 12240aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 12300gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 12360ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgccgg gggggggggg 12420gggggacatg aggttgcccc gtattcagtg tcgctgattt gtattgtctg aagttgtttt 12480tacgttaagt tgatgcagat caattaatac gatacctgcg tcataattga ttatttgacg 12540tggtttgatg gcctccacgc acgttgtgat atgtagatga taatcattat cactttacgg 12600gtcctttccg gtgatccgac aggttacggg gcggcgacct cgcgggtttt cgctatttat 12660gaaaattttc cggtttaagg cgtttccgtt cttcttcgtc ataacttaat gtttttattt 12720aaaataccct ctgaaaagaa aggaaacgac aggtgctgaa agcgagcttt ttggcctctg 12780tcgtttcctt tctctgtttt tgtccgtgga atgaacaatg gaaccccccc cccccccccc 12840tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 12900ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 12960ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 13020cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 13080gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 13140actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 13200aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 13260caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 13320aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 13380accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 13440aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 13500ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 13560agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 13620accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 13680gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 13740tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 13800cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 13860cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 13920cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 13980ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 14040taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 14100gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg 14160cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg 14220ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga

14280cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 14340atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagcagat cccccgatca 14400agtagataca ctacatatat ctacaataga catcgagccg gaaggtgatg tttactttcc 14460tgaaatcccc agcaatttta ggccagtttt tacccaagac ttcgcctcta acataaatta 14520tagttaccaa atctggcaaa agggttgacc gggggggggg ggaaagccac gttgtgtctc 14580aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt 14640ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt 14700gctcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc 14760gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc 14820cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg 14880tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta 14940ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat 15000tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc 15060ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg 15120ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc 15180gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac 15240cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga 15300aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg 15360ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa 15420aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt 15480ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac 15540gggacggcgg ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc agatcacgca 15600tcttcccgac aacgcagacc gttccgtggc aaagcaaaag ttcaaaatca ccaactggtc 15660cacctacaac aaagctctca tcaaccgtgg ctccctcact ttctggctgg atgatggggc 15720gattcaggga tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg 15780cgagatcatc cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt 15840aacatgagca aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat 15900gggctgcctg tatcgagtg 159191071499DNAOryza sativaterminator(1)..(1499) 107cagagtgata gacagtgcta cttgctagcc ttttttatga gcaccctttt tggcctttta 60gttcctcatt agctaagaga tgtgatgagt gaggttttat tttttggctt tgtttggagt 120tttgctttgg aggaagatag agaagccttt agaatgagtt gtgatggagg atcacatagt 180ttgtctcagg tttacaatgt gcggtaaaaa aaaggaagga aataaagtga tgttagaata 240tacagttttt taatccattt ctacagcttc aaattctcga ttgagcggca cgttctttga 300aatgttcata gcaaagcaaa tcaaacgaaa tggatgagat ttgaaactaa gaaactgcac 360ttgacaaagc acgctgcgct ctctgaaatt catgttcagc agaacgcctt tcagaaattc 420atgttctgct gctgcaggaa ggcagctaag aactccatgc aataaaaata taaaatgcag 480tgcagttatt agttagtgta gtagtaatct aagaaacaac aggtttaaca gaatattacc 540ttcaaaaaca tcccctcaaa tcaagactcg gaaatcaaca ccattctgaa actagatgca 600atactgcgct cctaatggaa tcttcagtgg ttactcctac acgccactaa aaaaaacggg 660ctttggaaaa acgtcaccct gaaaacaaga gctcagtaaa aataccactc taaaatcttg 720cagcatgcat tatcacaaaa gcagatcaat ccgtaattgt atttatgctg catataccgt 780gatattttct gtgatcctct gcactatctt cagctcatca gctgcatatt ttgaagccgg 840actgatggct catcgctttg tttcggctta ttgtgatagc catccatgcg ttgacgaaag 900atttagatat atcacaattt taaccaagtt cagagcaatg ttagctttag ctgatgatat 960acacggcgca ctgattgccg ccgttatttt cagagccttg taaaacctga aacaaattgt 1020gtctccaaat tcttactagt actgcagaca tagcggattt gttttcagga tgaattatca 1080caaaggtaag tcaattcata gccatattac gcaagttcaa aactgcaggc cacaccatac 1140gtttctgaaa gtagaaacaa gagatcttgc agcagatcac cactaaagaa gcagtaaccg 1200caaaaaatta taacataatc cagaattaac acttcgcagc attactgaat tcactttaat 1260agcacttctc atcatgaact agtacaacaa cataactgtc cagtggaaat gtgaaatgca 1320tacaccaagt aatggtccat aacatgaact atatggatac aacaacgttc ttatttcgct 1380catatataca tgaaataagt cttgcacgtc ttggttactt aataggtgcg ataatcgccg 1440taggctttta gaagaagaaa aaaaagtgag cctgcaaagt tcctggggac agttgaaga 14991081499DNAOryza sativaterminator(1)..(1499) 108gagacaaact atgtgatcct ccatcacaac tcattctaaa ggcttctcta tcttcctcca 60aagcaaaact ccaaacaaag ccaaaaaata aaacctcact catcacatct cttagctaat 120gaggaactaa aaggccaaaa agggtgctca taaaaaaggc tagcaagtag cactgtctat 180cactctgtct tcaactgtcc ccaggaactt tgcaggctca cttttttttc ttcttctaaa 240agcctacggc gattatcgca cctattaagt aaccaagacg tgcaagactt atttcatgta 300tatatgagcg aaataagaac gttgttgtat ccatatagtt catgttatgg accattactt 360ggtgtatgca tttcacattt ccactggaca gttatgttgt tgtactagtt catgatgaga 420agtgctatta aagtgaattc agtaatgctg cgaagtgtta attctggatt atgttataat 480tttttgcggt tactgcttct ttagtggtga tctgctgcaa gatctcttgt ttctactttc 540agaaacgtat ggtgtggcct gcagttttga acttgcgtaa tatggctatg aattgactta 600cctttgtgat aattcatcct gaaaacaaat ccgctatgtc tgcagtacta gtaagaattt 660ggagacacaa tttgtttcag gttttacaag gctctgaaaa taacggcggc aatcagtgcg 720ccgtgtatat catcagctaa agctaacatt gctctgaact tggttaaaat tgtgatatat 780ctaaatcttt cgtcaacgca tggatggcta tcacaataag ccgaaacaaa gcgatgagcc 840atcagtccgg cttcaaaata tgcagctgat gagctgaaga tagtgcagag gatcacagaa 900aatatcacgg tatatgcagc ataaatacaa ttacggattg atctgctttt gtgataatgc 960atgctgcaag attttagagt ggtattttta ctgagctctt gttttcaggg tgacgttttt 1020ccaaagcccg ttttttttag tggcgtgtag gagtaaccac tgaagattcc attaggagcg 1080cagtattgca tctagtttca gaatggtgtt gatttccgag tcttgatttg aggggatgtt 1140tttgaaggta atattctgtt aaacctgttg tttcttagat tactactaca ctaactaata 1200actgcactgc attttatatt tttattgcat ggagttctta gctgccttcc tgcagcagca 1260gaacatgaat ttctgaaagg cgttctgctg aacatgaatt tcagagagcg cagcgtgctt 1320tgtcaagtgc agtttcttag tttcaaatct catccatttc gtttgatttg ctttgctatg 1380aacatttcaa agaacgtgcc gctcaatcga gaatttgaag ctgtagaaat ggattaaaaa 1440actgtatatt ctaacatcac tttatttcct tccttttttt taccgcacat tgtaaacct 1499

Claims

1. An expression construct comprising in 5' to 3' direction: a) a promoter sequence functional in plants; b) a nucleic acid sequence of interest to be expressed operably linked to the promoter sequence of a); and c) at least one sequence operably linked to the promoter sequence of a) and the nucleic acid sequence of b), wherein said at least one sequence is selected from the group consisting of: i) the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46; ii) sequences having a homology of at least 60%, 80%, 90% or 95% with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; iii) sequences hybridizing under high stringency conditions equivalent to hybridization at 68.degree. C. in a solution consisting of 5.times.SSPE, 1% SDS, 5.times.Denhardt's reagent and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1.times.SSPE and 0.1% SDS at 68.degree. C. with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; and iv) a fragment of at least 50, 100, 250 or 500 consecutive base pairs of any of the sequences of i), ii) and iii), wherein said at least one sequence of c) is heterologous to said promoter sequence of a) and/or said nucleic acid sequence of interest to be expressed of b), and is mediating termination of expression of the nucleic acid sequence of b) induced from said promoter sequence of a).

2. A vector comprising the expression construct of claim 1.

3. A transgenic host cell or non-human organism comprising: a) the expression construct of claim 1; or b) a vector comprising said expression construct.

4. A transgenic plant comprising: a) the expression construct of claim 1; or b) a vector comprising said expression construct.

5. A transgenic expression construct comprising at least two expression cassettes and having a structure comprising in 5'-3'-direction: a1) a first promoter sequence functional in plants; b1) a first nucleic acid sequence of interest to be expressed operably linked to said promoter sequence of a1); c) at least one sequence operably linked to the promoter sequence of a1) and the nucleic acid sequence of b1), wherein said at least one sequence is selected from the group consisting of: i) the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46; ii) sequences having a homology of at least 60%, 80%, 90% or 95% with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; iii) sequences hybridizing under high stringency conditions equivalent to hybridization at 68.degree. C. in a solution consisting of 5.times.SSPE, 1% SDS, 5.times.Denhardt's reagent and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1.times.SSPE and 0.1% SDS at 68.degree. C. with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; and iv) a fragment of at least 50, 100, 250 or 500 consecutive base pairs of any of the sequences of i), ii) and iii), b2) a second nucleic acid sequence of interest to be expressed; and a2) a second promoter sequence functional in plants operably linked to said nucleic acid sequence of interest to be expressed of b2), wherein said at least one sequence of c) is heterologous to at least one element selected from the promoter sequence of a1), the promoter sequence of a2), the nucleic acid sequence of interest to be expressed of b1), and the nucleic acid sequence of interest to be expressed of b2), and is mediating termination of expression induced from said promoter sequences of a1) and a2).

6. A vector comprising the transgenic expression construct of claim 5.

7. A transgenic host cell or non-human organism comprising: a) the transgenic expression construct of claim 5; or b) a vector comprising said transgenic expression construct.

8. A transgenic plant comprising: a) the transgenic expression construct of claim 5; or b) a vector comprising said transgenic expression construct.

9. A method for terminating transcription of a nucleic acid molecule in an expression construct, comprising operably linking a terminator sequence to a heterologous nucleic acid molecule, wherein said terminator sequence is selected from the group consisting of: i) the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46; ii) sequences having a homology of at least 60%, 80%, 90% or 95% with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; iii) sequences hybridizing under high stringency conditions equivalent to hybridization at 68.degree. C. in a solution consisting of 5.times.SSPE, 1% SDS, 5.times.Denhardt's reagent and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1.times.SSPE and 0.1% SDS at 68.degree. C. with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; and iv) a fragment of at least 50, 100, 250 or 500 consecutive base pairs of any of the sequences of i), ii) and iii).

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