KR20090084014A - Transformation vector for eliminating selection marker gene - Google Patents

Abstract

A vector containing a cassette for removing gene for selecting vector of plant transformation is provided to reduce price and time for evaluating harmfulness and safety and enable easy cloning. A cassette for removing gene for selecting vector of plant transformation comprises a Cre recombinase which is operatively linked to pBip-L promoter, argE(ornithine deacetylase) gene, and selection marker gene. The cassette comprises a sequence number 1(SEQ ID NO:1) or 2. A method for producing a transformed plant in which selection marker gene is removed comprises: a step of inserting a target gene into the vector for removing selection marker gene to produce recombinant plant expression vector; a step of transforming the recombinant plant expression vector in a plant; and a step of treating tunicamycin and N-acetyl phosphinothricin to select a transformant.

Description

Transformation vector for eliminating selection marker gene

The present invention relates to a transformation vector for removing a selection marker gene, and more particularly, to remove a selection marker gene of a plant transformation vector including a Cre (Cre recombinase) gene, an argE (ornithine deacetylase) gene, and a selection marker gene. Cassette for use, a vector for removing a selection marker gene comprising the cassette, a method for producing a transformed plant from which the selection marker gene is removed using the vector, and a transformed plant from which the selection marker gene prepared by the method is removed will be.

Soil microorganisms ( Agrobacterium) are used for the 'plant transformation method', a technique of introducing foreign genes into plants. tumefaciens etc.) and direct delivery methods (microparticle bombarding method, chemical method). These technologies all rely on Selection Marker Genes, such as antibiotic resistance genes and herbicide resistance genes. In other words, the genes to be introduced into the plant and the selection marker gene are carried in a single DNA fragment and transferred to the plant, so that only the plant cells to which the DNA fragment is delivered have resistance to this antibiotic or herbicide. Transplanted plants can be obtained from differentiated cells.

However, social attention has been focused on the potential harms of antibiotic or herbicide resistance, which are indispensable in the production of transgenic plants, causing significant obstacles to the commercialization of developed transgenic plants. In addition, the European Union has banned the use of antibiotic resistance genes used clinically in transgenic crops since 2004.

In addition, when introducing a number of foreign genes to improve the plant, once used the selection marker gene is not reusable, once used must be removed. That is, for this reason, it is necessary to develop a technique for removing the selection marker gene.

Therefore, in order to develop a transformed crop in which only a desired gene has been introduced, the production of a transgenic vector for removing a cover label and the development of a transformed crop using the same are very important for the early breeding of the transformed crop.

Negative selection and co-transformation is a method of inserting a selection marker gene and a foreign gene of interest to be introduced into different T-DNAs and simultaneously introducing them into plants. The selection marker and the gene of interest may be inserted at different positions on the genome of the plant and subsequently separated into progeny having the selection marker gene and the target gene to obtain a marker-free transgenic plant. Although it is easy to produce vectors, it is difficult to apply to long-term crops or nutrient breeding crops.

The present invention has been made in accordance with the above-described requirements, and as a selection marker gene removal technique using site-specific recombination, a method using Cre recombinase and a loxP site specifically recognized by this protein, The selection marker gene is located between the two loxP sites and the target gene is located outside of the loxP site, and then the selection marker can be removed by chemically inducing the expression of Cre recombinase. In addition, there is a need for a negative selection marker that can effectively remove cells with remaining selection marker genes by chemical induction. This method can be applied to nutritive crops in which simultaneous transformation methods are difficult to apply.

The inventors of the present invention discovered a Bip- L promoter derived from Arabidopsis which is strongly induced by a specific chemical (tunicamycin, Tm), and derived from microorganisms. Using the argE gene (ornithine deacetylase), it has secured a screening technology that selectively kills only transformed cells. In the present invention, the two techniques were combined to produce a vector for removing a selection marker (SM) gene to be used for making a transformant plant having no selection marker gene. The vector modified a lot of restriction enzyme sites through gene recombination to facilitate cloning of the target gene. Therefore, plant transformants can be prepared by easily cloning a gene of interest in multiple cloning sites.

In order to solve the above problems, the present invention provides a cassette for removing a selection marker gene of a plant transformation vector comprising a Cre (Cre recombinase) gene, an argith (ornithine deacetylase) gene, and a selection marker gene.

The present invention also provides a transformation vector for removing a selection marker gene comprising the cassette.

The present invention also provides a method for producing a transgenic plant from which a selection marker gene is removed using the vector.

The present invention also provides a transgenic plant from which the selection marker gene prepared by the above method is removed.

According to the transformation vector for removing the selected marker gene of the present invention, it is possible to reduce a lot of time and cost required for risk and safety evaluation for commercialization of the transgenic plant. In addition, since the cloning of the gene of interest in the vector is very easy, the production of a transgenic plant is easy.

In order to achieve the object of the present invention, the present invention is a selection marker of a plant transformation vector comprising a Cre (Cre recombinase) gene, argE (ornithine deacetylase) gene, and selection marker gene operably linked to the pBip-L promoter Provided is a cassette for gene removal.

The cassette of the present invention includes a Cre (Cre recombinase) gene, an ornithine deacetylase (argE) gene, and a selection marker gene. Specifically, Cre (Cre recombinase) gene is operably linked to the pBip-L promoter. BiP is a luminal binding protein, a type of HSP70 chaperone family located in the ER, and is called a glucose-regulated protein 78 in mammals (Gething, Semin. Cell. Dev). Biol., 10, pp 465-472, 1999). BiP plays an important role in the translocation, folding and assembly of proteins through the ER. The pBip-L promoter is a Arabidopsis-derived promoter whose expression is strongly induced by tunicamycin (Tm). In the present invention, 'operably linked' means that the Cre gene is normally expressed by the pBip-L promoter. Cre recombinase expressed by the Cre gene specifically recognizes loxP sites, and can remove selectable marker genes between two loxP sites.

The argith (ornithine deacetylase) gene of the present invention corresponds to a Negative Selectable Marker Gene, which encodes an enzyme that selectively kills only transformed cells. Treatment with pinotriscin can selectively kill only cells with argE. The gene may be derived from microorganisms. The reverse selection gene means a gene used when selectively selecting only plant cells without the selection marker gene. These backlabeled genes encode enzymes that metabolize chemicals that inhibit or kill the growth of plant cells. Plants that do not have back-labeled genes are not affected by the precursors of a given toxic substance, but in the case of plants that have them, growth is inhibited or killed by the action of enzymes that produce toxic substances from the precursors. Plant selection by such a reverse selection gene is called negative selection. Many such back-labeled genes have been reported to date. Examples of these genes include codA, argE, iaaH, and P450 su1, and the enzymes encoded by them convert non-toxic substrates into toxic products that hinder the growth of plants with back-labeled genes. The codA gene is derived from Escherichia coli, and cytosine deaminase produced by the expression of this gene disrupts DNA replication by damaging non-toxic 5-fluorocytosine (5-FC). This mechanism confers sensitivity to 5-fluorocytosine through a mechanism that converts it into toxic 5-fluorouracil (5-FU).

The selection marker gene of the present invention may be, but is not limited to, NPTII (neomycin phosphotransferase II) gene, HPT (hygromycin phosphotransferase) gene, bar gene, and the like. The selection marker gene refers to a gene that is transformed with a gene of interest in a plant so that only a transgenic plant can be selectively selected. A number of selection marker genes are currently reported, and representative examples of such genes include antibiotic resistance genes, herbicide resistance genes, virus resistance genes, pest resistance genes, male infertility genes, and fruit maturation prevention genes. That is, when antibiotics or herbicides are put into the medium during the plant transformation step using soil microorganisms, only plant cells to which the selection marker genes immediately adjacent to the target genes are delivered are selectively grown. As such, introduction of selection marker genes into plant cells plays a role in selecting a transgenic plant having a target gene.

Kanamycin phosphotransferase produced by the expression of the nptii gene confers resistance to the antibiotic kanamycin through the mechanism of phosphorylating and inactivating kanamycin. Hygromycin phosphotransferase produced by the expression of the hpt gene confers resistance to the antibiotic hygromycin through a mechanism that phosphorylates and inactivates hygromycin. Phosphothrisin acetyltransferase produced by the expression of the bar gene confers resistance to the herbicide 'Basta' (Basta, phosphinothricin) through the mechanism of acetylating and inactivating phosphinothricin. .

In a preferred embodiment of the present invention nptii and hpt genes were used.

The cassette according to an embodiment of the present invention may be a cassette for removing a selection marker gene of the plant transformation vector described in FIG. 11 or 12. The cassette described in FIG. 11 has the nptii gene as the selection marker gene, and the cassette described in FIG. 12 has the hpt (used interchangeably with hphi) gene as the selection marker gene.

The cassette described in FIG. 11 has loxP sites at both ends, and therein, a Cre (re recombinase) gene, an argith (ornithine deacetylase) gene, and a neomycin phosphotransferase II (NPTII) selection marker gene operably linked to a pBip-L promoter therein. Is present. The Cre gene has a Bip-L promoter as a promoter, a nos terminator as a terminator, the argE gene has a CaMV 35S promoter as a promoter, a nos terminator as a terminator, the nptii gene has a nos promoter as a promoter, a nos terminator as a terminator In the cassette, there is one HindIII restriction enzyme site.

The cassette described in FIG. 12 has loxP sites at both ends, and the Cre (Cre recombinase) gene, argE (ornithine deacetylase) gene, and hygromycin phosphotransferase (HPT) selection marker gene operably linked to the pBip-L promoter therein. exist. The Cre gene has a Bip-L promoter as a promoter, a nos terminator as a terminator, the argE gene has a CaMV 35S promoter as a promoter, a nos terminator as a terminator, and the hpt (or hphi) gene as a nos promoter as a promoter, nos as a terminator It has a terminator and there is a HindIII restriction enzyme site inside the cassette.

In the cassette according to the embodiment of the present invention, the cassette described in Figure 11 or 12 may be composed of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. In addition, variants of such sequences are included within the scope of the present invention. A variant is a nucleotide sequence that changes in nucleotide sequence but has similar functional properties as the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Specifically, the cassette has at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 It may include a base sequence.

The “% sequence homology” for a polynucleotide is determined by comparing the two optimally arranged sequences with the comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

To achieve another object of the present invention, the present invention provides a plant transformation vector comprising a Cre (Cre recombinase) gene, an argE (ornithine deacetylase) gene, and a selection marker gene operably linked to a pBip-L promoter. Provided is a transformation vector for removing selection marker gene comprising a cassette for removal of selection marker gene. Preferably, the transformation vector may include a cassette for removing a selection marker gene of the plant transformation vector described in FIG. 11 or 12. The Cre gene, argE gene, and NPTII or HPT selection marker gene are as described above.

The transformation vector according to an embodiment of the present invention may be a pSYCAN vector or a pSYCAH vector described in FIG. 19. The pSYCAN vector or pSYCAH vector includes a cassette for removing a selection marker gene of a plant transformation vector including a Cre gene, an argE gene, and an NPTII or HPT selection marker gene, and includes a restriction enzyme site existing in the vector. Many were removed to facilitate insertion of the target gene into multiple cloning sites. The pSYCAN vector or pSYCAH vector may be more preferably composed of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, respectively.

The term “vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838.

In the plant expression vector according to an embodiment of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the plant expression vector according to an embodiment of the present invention, the terminator may use a conventional terminator, and examples thereof include nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agro Terminators of the octopine gene of bacterium tumefaciens, but are not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

In order to achieve another object of the present invention, the present invention comprises the steps of preparing a recombinant plant expression vector by inserting the target gene in the transformation vector for the selection marker gene of the present invention;

Transforming the plant with the recombinant plant expression vector; And

Provided is a method for producing a transformed plant from which a selection marker gene has been removed, comprising selecting a transformant from which the selection marker gene has been removed.

The method for producing a transgenic plant from which the selection marker gene of the present invention has been removed includes preparing a recombinant plant expression vector by inserting a target gene into the transformation vector for removing the selection marker gene of the present invention. The transformation vector for removing the selection marker gene of the present invention may preferably be a pSYCAN vector or a pSYCAH vector, as described above.

There is no limitation to the gene of interest that can be incorporated into the vector of the present invention. In the exemplary embodiment of the present invention, β-glucuronidase (GUS), which is a reporter gene, is used instead of the target gene. GUS has an enzymatic activity that acts on the substrate X-Gluc to form a blue multiplex, which is very useful for identifying transformed plants.

Method for producing a transformed plant of the present invention comprises the step of transforming the plant with the recombinant plant expression vector. Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including both dicotyledonous plants as well as monocotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. Preferred methods according to the invention include Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

The method for producing a transgenic plant of the present invention includes selecting a transformant from which the selection marker gene has been removed, which can be performed by treating the transformed plant with tunicamycin and N-acetyl phosphinothricin. . That is, by treating the tunicamycin, the pBip-L promoter is strongly expressed, and Cre recombinase is expressed by the pBip-L promoter in which the expression is induced, which specifically recognizes the loxP site, thereby causing two loxPs. Remove the selection marker gene between the sites. In addition, N-acetyl phosphinothricin is treated to selectively kill only cells with argE.

In addition, the method of the present invention may comprise the step of regenerating the transgenic plant from said selected transgenic plant cells. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.

In order to achieve another object of the present invention, the present invention provides a transgenic plant from which the selection marker gene prepared by the method for producing a transgenic plant of the present invention has been removed. There are no restrictions on the plant of interest requiring transformation. Thus, any plant is effective as a plant of the present invention, which may be a dicotyledonous or monocotyledonous plant. The plant according to an embodiment of the present invention is Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, It may be a dicotyledonous plant, such as a daikon radish, watermelon, melon, cucumber, zucchini, gourd, strawberry, soybean, green bean, kidney bean, or pea, but preferably tobacco.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example

Example 1: Preparation of selection marker gene removal cassette from which restriction enzyme recognition site was removed

1) pLoxC3 Introduction of Bip- L: Cre Cassettes

1 shows the introduction of the Bip- L: Cre cassette into pLoxC3 . PLoxC3 with altered structure of multiple cloning site (MCS) of pLoxC2 in our laboratory The vector was constructed by primer annealing method (FIG. 1A). This vector is based on MCS Cre - recombinase ( Cre ) It has a recognition site LoxP site on both sides, was produced to enable the introduction of the cassette cloned Cre gene (Fig. 1B) to the Bip-L promoter (Fig. 1C). PBip- L: Cre cassette was cut into whole ORF with PstI and SalI (FIG. 1B) and introduced into pLoxC3 (FIG. 1C). And PBip -L: The clone containing a Cre cassette (# 2, 6, 7, and: a result of cutting the Cre PBip -L: Cre cassette pLoxC3 + Bip -L with PstI and SalI to verify that the correctly introduced in pLoxC3 8) was confirmed (FIG. 1D).

2) pLoxC3 + Cre P35S : argE Introduction of the cassette

2 is pLoxC3 + Cre P35S : argE Figure shows the introduction of the cassette. The selectable marker gene in the reverse pLoxC3 + Cre produced on the argE (P35S: argE) were introduced between EcoRI and Pst1 in pLoxC3 (Fig. 2C). And after cutting with PstI and EcoRI to confirm that the argE ( P35S : argE ) cassette (FIG. 2B) was introduced into pLoxC3 + Cre , clones containing the P35S : argE cassette (# 1, 2, 3) as shown in FIG. 2D , 4, 5, 7, 8).

3) pLoxC3 + Cre + argE Modification of MCS

3 is pLoxC3 + Cre + argE The figure shows the MCS variant of. Several restriction enzyme sites in the MCS of pLoxC3 were modified to introduce a positive selectable marker gene into the vector constructed above. To this end, pLoxC3 + Cre + argE was treated with SphI and EcoRI, and primer pairs were synthesized to remove these two restriction enzyme sites. In the meantime, EcoRI, KpnI, and SphI restriction enzyme sites were introduced as shown in FIG. 3. The vector created in this way is called ' pLoxC3 + Cre + argE_ Modified' Named.

4) Introduction of Nptii and hphi positive selection marker genes to pLoxC3 + Cre + argE _ Modified

Figures 4 and 5, respectively pLoxC3 + Cre + argE _ a positive selection marker gene and the Modified Nptii hphi This is a picture of introducing a gene. Subsequently, positive selection marker genes Nptii ( P35s : Nptii ) and hphi ( P35 : hphi ) for pLoxC3 + Cre + argE _ Modified were introduced between KpnI and EcoRI, respectively. As shown in Figures 4 and 5, pLoxC3 + Cre + argE_ Modified vector in Nptii (P35s: Nptii) and hphi (P35: hphi) After cutting the KpnI and EcoRI to verify that the introduced, Nptii (P35s: Nptii) Recombination clones containing (# 2, 3, 4, 8, 9) were confirmed by gel electrophoresis (Fig. 4D), and also the recombinant clones containing the hphi ( P35 : hphi ) (# 1, 2, 3, 6, 7, 8, 9) was also confirmed (Fig. 5D).

5) Removal of restriction enzyme recognition site in cassette

Figure 6 shows the major restriction enzyme recognition site of the selection marker gene removal cassette. Since the prepared selection label removal cassette has a total length of 6 kb and has various restriction enzyme recognition sites, there are many difficulties in actually using it for transformation vectors. In order to efficiently remove the restriction enzyme recognition site in the cassette, each part of the cassette must be separated separately, the restriction enzyme recognition site must be removed from each part, and then recombined again. To this end, parts of the cLoxC3 - CAN cassette and the cLoxC3 - CAH cassette were cloned (FIG. 7). 7 shows each part of the cassette.

(1) removal of non-ORF recognition sites

Among the restriction enzyme recognition sites of the respective parts of the cassette, non-sequence specific restriction enzyme recognition sites used for cloning were identified in E. coli. Klenow fragment and T4 DNA Removed using polymerase (Table 1).

Table 1. Removed non-ORF restriction enzyme recognition sites

▶ BiP -L :: CREi ( Pst I- Sal I) T4 DNA polymerase removal Sac I ▶ p35s :: argE ( Pst I- Kpn I) Klenow fragment removal Xba I Bam HI T4 DNA polymerase removal Sac I Sph I ▶ p35s :: nptII (Kpn I- Eco RI) Klenow fragment removal Xba I T4 DNA polymerase Remove Sac I ▶ p35s :: hphi (Kpn I- Eco RI) T4 DNA polymerase Remove Pst I

The cassette made through this is as follows (Fig. 8). 8 shows each part of the cassette after removal of the non-ORF recognition site.

(2) Removal of restriction enzyme recognition site in ORF

Restriction recognition sites in the ORF were removed using a site-specific single point mutation technique using PCR to prevent mutations in each codon (Table 2 and Figure 9). 9 shows each part of the cassette from which all restriction enzyme recognition sites were removed.

Table 2. Removal of restriction enzyme recognition sites in ORF using site-specific single point mutation technique

pBiP -L :: CREi

Bam HI CGG C GG ATC C GA → CGG C GA ATC C GA  Arg Arg Ile Arg Arg Arg Ile Arg

p35s :: argE

Hind III GGC AAG CTT TAC → GGC AAG CTA TAC  Gly Lys Leu Tyr    Gly Lys Leu Tyr Sal I GAT GTC GAC GTC → GAT GTC GAT GTC  Asp Val Asp Val    Asp Val Asp Val Sal I ACG GTC GAC GAG → ACG GTC GAT GAG  Thr Val Asp Glu    Thr Val Asp Glu

p35S :: nptII

Sph I GCG C GC ATG C CC → GCG C GT ATG C CC  Ala Arg Met Pro    Ala Arg Met Pro

(3) Removal of Recombinant and Restriction Enzyme Recognition Sites of Selective Marker Gene Removal Cassette

Each part from which the recognition site was removed was cloned back into the pLoxC3 vector. That is, the cassette was removed from the various restriction enzyme recognition sites shown in Figure 6 (Fig. 10). 10 shows a recombinant selection marker gene removal cassette.

And Klenow fragment and T4 DNA Using polymerase , the restriction enzyme recognition site used for recombination was removed again. That is, the selection marker gene removal cassette was minimized to minimize restriction enzyme recognition sites (FIGS. 11 and 12). 11 shows a selection marker gene removal cassette (cLoxC3-CAN) from which a restriction enzyme recognition site including an nptii gene is removed. 12 shows a selection marker gene removal cassette (cLoxC3-CAH) from which a restriction enzyme recognition site including an hpt (also called hphi) gene is removed. FIG. 13 shows the results of electrophoresis after treatment of the cassettes of FIGS. 6 and 11 and 12 with restriction enzymes. (A) Panel is pLoxC3 - CAN , (B) Panel is pLoxC3 - CAH And asterisks indicate the pLoxC3 vector backbone (2.9 kb). As can be seen in Figure 13, the final cassette can be seen that a lot of restriction enzyme recognition site was removed.

Example  2: pSYCAN  Vector and pSYCAH  Manufacture of vector

pJM121 including RK2 oriV, nptIII-L and TrfA of pBI121 (see, Xiang et al., 1999, Plant Molecular Biology , 40 , pp. 711-717). Specifically, PCR amplified fragments (indicated by red bars in the left map of FIG. 14) from pBI121 were digested with XbaI and XhoI, and the NotI restriction site was removed by Klenow filling. 14 is a map of the pJM121 vector with NotI restriction enzyme sites removed by Klenow filling.

pJM122 was prepared by introducing a T-DNA border sequence into pJM121. Specifically, T-DNA fragments PCR amplified from pBI121 (indicated by red bars in the left map of FIG. 15) were cleaved with SpeI, then introduced into pJM121 cleaved with XbaI, and the SpeI and XbaI sites removed at ligation. It became. 15 is a map of the pJM122 vector.

pNS vectors were prepared by introducing multiple cloning sites into pJM122. Specifically, pNS vectors were prepared by PCR amplification from pJM122 (indicated by red bars in the left map of FIG. 16). 16 is a map of the pNS vector.

P35S :: GUS :: Tnos fragments (indicated by red bars in the left map of FIG. 17) from the pBI121 vector were then introduced into the pNS vector by EcoRI / HindIII double cleavage and the NotI recognition site between BamHI and XbaI. PNS-GUS vector was prepared by inserting. 17 is a map of the pNS-GUS vector.

The cLoxC3-CAN cassette (FIG. 11) and the cLoxC3-CAH cassette (FIG. 12) were inserted into the NotI recognition site to prepare pNS-GUS CAN vectors and pNS-GUS CAH vectors, respectively. 18 is a map of the pNS-GUS CAN vector.

GUS :: Tnos fragments were removed by SacI / BamHI double digestion followed by T4 DNA polymerase erasing. The 35S promoter fragment was replaced by multiple cloning sites of pBluescriptII SK + by XbaI / KpnI double cleavage to complete pSYCAN and pSYCAH. 19 is a map of the pSYCAN vector and the pSYCAH vector.

Example  3: Construction of Test Transformation Vectors and Transgenic Plants

Of the two methods developed in the present invention, the most important advantage of the cassette removal system to remove the starter gene is that the starter gene can be removed in callus without generation. to be. Therefore, transgenic plants were selected in the callus state, and test vectors were prepared using tobacco, a model plant capable of confirming the removal of the selection marker gene, and a test transgenic plant was produced.

First, the model plant tobacco ( Nicotiana tabacum ), to test application of the selection marker removal system, transgenic transgenic Agrobacterium tumefaciens (LBA4401) bacteria, pNS-GUS CAN (see FIG. 18), which is a plant transformation vector for the cassette removal test prepared in the present invention, for tobacco transformation. And then Nicotiana through leaf disc transformation tabacum It was introduced in the Samsun line. After the transduction, the primary selection was induced by inducing callus growth for 3 weeks in MS medium containing kanamycin.

In the first selection, the stems induced by the callus were transferred to the root induction medium and cultured for one month to induce the roots and confirmed the transformation using PCR technique (FIG. 20). 20 shows genomic PCR results for the GUS coding sequence. A total of 24 plants were induced, but 6 transformants (Line number 2, 4, 5, 6, 15, 17) were identified by PCR.

Six identified individuals were transferred to root induction medium for passage culture, followed by two weeks of acclimation, and then treated with tunicamycin for cassette removal. The tunicamycin treatment was soaked for 4 hours in a 0.05% (v / v) DMSO aqueous solution containing 5 ug / ml of tunicamycin, and the excess solution was removed and purified again for 1 week. After the completion of the purification process, the whole plant is immersed in an aqueous solution containing 1 µg / ml N-acetyl phosphinothricin for 2 hours, and then the excess solution is removed for 1 week. We went through the process of necrosis and observed the progression of negative selection.

After transforming the transformants obtained through cassette removal and reverse selection for 3 weeks, the leaves were obtained and PCR techniques confirmed that the target gene was finally introduced and the selection marker gene was removed (FIG. 21). Figure 21 shows genomic PCR results for the selection marker cassette. Among the six transformants, 15 transformants were completely removed from the selection marker cassette, and the other five transformants were not completely removed from the selection marker cassette.

All of the obtained transformants showed no reaction to tunicamycin and N-acetyl phosphinothricin, because most of the selection marker cassettes were removed, and thus the cells were not killed much by N-acetyl phosphinothricin. do. The transformants also grew normally and induced stems and roots. Thus, tunicamycin and N-acetyl phosphinothricin did not cause any problems in plant differentiation and growth (FIG. 22). 22 shows the transformed plants. A to C are transformed tobacco (N. tabacum) lines 2, 4 and 15, respectively.

1 shows the introduction of the Bip- L: Cre cassette into pLoxC3 .

2 is pLoxC3 + Cre P35S : argE Figure shows the introduction of the cassette.

3 is pLoxC3 + Cre + argE The figure shows the MCS variant of.

Figures 4 and 5, respectively pLoxC3 + Cre + argE _ a positive selection marker gene and the Modified Nptii hphi This is a picture of introducing a gene.

Figure 6 shows the major restriction enzyme recognition site of the selection marker gene removal cassette.

7 shows each part of the cassette.

8 shows each part of the cassette after removal of the non-ORF recognition site.

9 shows each part of the cassette from which all restriction enzyme recognition sites were removed.

10 shows a recombinant selection marker gene removal cassette.

11 shows a selection marker gene removal cassette (cLoxC3-CAN) from which a restriction enzyme recognition site including an nptii gene is removed.

12 shows a selection marker gene removal cassette (cLoxC3-CAH) from which a restriction enzyme recognition site including an hpt (also called hphi) gene is removed.

FIG. 13 shows the results of electrophoresis after treatment of the cassettes of FIGS. 6 and 11 and 12 with restriction enzymes.

14 is a map of the pJM121 vector with NotI restriction enzyme sites removed by Klenow filling.

15 is a map of the pJM122 vector.

16 is a map of the pNS vector.

17 is a map of the pNS-GUS vector.

18 is a map of the pNS-GUS CAN vector.

19 is a map of the pSYCAN vector and the pSYCAH vector.

20 shows genomic PCR results for the GUS coding sequence.

Figure 21 shows genomic PCR results for the selection marker cassette.

22 shows the transformed plants.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Transformation vector for eliminating selection marker gene <130> PN07125 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 6244 <212> DNA <213> Artificial Sequence <220> <223> cLoxC3-CAN cassette <400> 1 gcggccgctg acggtatcgc gataagctat aacttcgtat aatgtatgct atacgaagtt 60 atgaattgtc gatcgacgat ctagtaacat agatgacacc gcgcgcgata atttatccta 120 gtttgcgcgc tatattttgt tttctatcgc gtattaaatg tataattgcg ggactctaat 180 cataaaaacc catctcataa ataacgtcat gcattacatg ttaattatta catgcttaac 240 gtaattcaac agaaattata tgataatcat cgcaagaccg gcaacaggat tcaatcttaa 300 gaaactttat tgccaaatgt ttgaacgatc ggggaaattc ggtcgagcta atcgccatct 360 tccagcaggc gcaccattgc ccctgtttca ctatccaggt tacggatata gttcatgaca 420 atatttacat tggtccagcc accagcttgc atgatctccg gtattgaaac tccagcgcgg 480 gccatatctc gcgcggctcc gacacgggca ctgtgtccag accaggccag gtatctctga 540 ccagagtcat ccttagcgcc gtaaatcaat cgatgagttg cttcaaaaat cccttccagg 600 gcgcgagttg atagctggct ggtggcagat ggcgcggcaa caccattttt tctgacccgg 660 caaaacaggt agttattcgg atcatcagct acaccagaga cggaaatcca tcgctcgacc 720 agtttagtta cccccaggct aagtgccttc tctacacctg cggtgctaac cagcgttttc 780 gttctgccaa tatggattaa cattctccca ccgtcagtac gtgagatatc tttaaccctg 840 atcctggcaa tttcggctat acgtaacagg gtgttataag caatccccag aaatgccaga 900 ttacgtatat cctggcagcg atcgctattt tccatgagtg aacgaacctg taactatcat 960 catcatcata gacacacgaa ataaagtaat cagattatca gttaaagcta tgtaatattt 1020 acaccataac caatcaatta aaaaatagat cagtttaaag aaagatcaaa gctcaaaaaa 1080 ataaaaagag aaaagggtcc taaccaagaa aatgaaggag aaaaactaga aatttacctg 1140 gtcgaaatca gtgcgttcga acgctagagc ctgttttgca cgttcaccgg catcaacgtt 1200 ttcttttcgg attcgccgca taaccagtga aacagcattg ctgtcacttg gtcgtggcag 1260 cccggaccga cgatgaagca tgtttagctg gcccaaatgt tgctggatag tttttactgc 1320 cagaccgcgc gcctgaagat atagaagata atcgcgaaca tcttcaggtt ctgcgggaaa 1380 ccatttccgg ttattcaact tgcaccatgc cgcccacgac cggcaaacgg acagaagcat 1440 tttccaggta tgctcagaaa acgcctggcg atccctgaac atgtccatca ggttcttgcg 1500 aacctcatca ctcgtcgcat cgaccggtaa tgcaggcaaa ttttggtgta cggtcagtaa 1560 attggacatg cgtgggtctt caacctttct cttcttcttt ggagccatct cagatccgga 1620 aacttttgcg tacgatctct ctttttgatt tgaacagaga gcttcctttt gttttctggt 1680 ttttaagctt tgaagaagaa gttaggatgc ttaaataggt ccagtgcccg aaggtgatgg 1740 tgaagtagtc gctttttatg gaagacgaag aaatttaact atgtcatcgg agatgacgtg 1800 gaccaattat cttacgacga tctataatgt caaccaatca gatttggtct tgtgttggag 1860 taatcgtggc cgttggttac ggtccccaga ttagcctttt caatttcaga aagaatgcta 1920 acccacagat ggttagagag gcttacgcag caggtctcat caagacgatc tacccgagca 1980 ataatctcca ggaaatcaaa taccttccca agaaggttaa agatgcagtc aaaagattca 2040 ggactaactg catcaagaac acagagaaag atatatttct caagatcaga agtactattc 2100 cagtatggac gattcaaggc ttgcttcaca aaccaaggca agtaatagag attggagtct 2160 ctaaaaaggt agttcccact gaatcaaagg ccatggagtc aaagattcaa atagaggacc 2220 taacagaact cgccgtaaag actggcgaac agttcataca gagtctctta cgactcaatg 2280 acaagaagaa aatcttcgtc aacatggtgg agcacgacac acttgtctac tccaaaaata 2340 tcaaagatac agtctcagaa gaccaaaggg caattgagac ttttcaacaa agggtaatat 2400 ccggaaacct cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg 2460 aaaaggaagg tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag 2520 atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa 2580 aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg 2640 taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt 2700 catttcattt ggagagaaca cgggggactc tagctagagg atcgatccat gaaaaacaaa 2760 ttaccgccat ttatcgagat ttaccgcgct ctgattgcca caccttcaat aagcgccacg 2820 gaagaggcac tcgatcaaag caatgcagat ttaatcactc tgctggcgga ctggtttaaa 2880 gatttgggct tcaatgtgga agtgcagcct gttccaggaa ctcgcaacaa attcaatatg 2940 ctggcaagta tcggacaggg ggctggcggc ttgttgctgg cggggcatac cgatacggtg 3000 ccatttgatg acggtcgctg gacgcgcgat ccgtttacac tgacggagca tgacggcaag 3060 ctatacggct taggcaccgc cgacatgaaa ggcttttttg cgtttatcct tgatgcgcta 3120 cgcgatgtcg atgtcacgaa actgaaaaaa ccgctctaca ttctggcgac tgctgatgaa 3180 gaaaccagta tggccggagc gcgttatttt gccgaaacta ccgccctgcg cccggattgc 3240 gccatcattg gcgaaccgac gtcactacaa ccggtacgcg cacataaagg tcatatctct 3300 aacgccatcc gtattcaggg ccagtcgggg cactccagcg atccagcacg cggagttaac 3360 gctatcgaac taatgcacga cgccatcggg catattttgc aattgcgcga taacctgaaa 3420 gaacgttatc actacgaagc gtttaccgtg ccatacccta cgctcaacct cgggcatatt 3480 cacggtggcg acgcttctaa ccgtatttgc gcttgctgtg agttgcatat ggatattcgt 3540 ccgctgcctg gcatgacact caatgaactt aatggtttgc tcaacgatgc attggctccg 3600 gtgagcgaac gctggccggg tcgtctgacg gtcgatgagc tgcatccgcc gatccctggc 3660 tatgaatgcc caccgaatca tcaactggtt gaagtggttg agaaattgct cggagcaaaa 3720 accgaagtgg tgaactactg taccgaagcg ccgtttattc aaacgttatg cccgacgctg 3780 gtgttggggc ctggctcaat taatcaggct catcaacctg atgaatatct ggaaacacgg 3840 tttatcaagc ccacccgcga actgataacc caggtaattc accatttttg ctggcattaa 3900 gcgaatttcc ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt 3960 gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt 4020 aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta 4080 tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc 4140 gcggtgtcat ctatgttact agatcgggat tagcgctgcg tccccagatt agccttttca 4200 atttcagaaa gaatgctaac ccacagatgg ttagagaggc ttacgcagca ggtctcatca 4260 agacgatcta cccgagcaat aatctccagg aaatcaaata ccttcccaag aaggttaaag 4320 atgcagtcaa aagattcagg actaactgca tcaagaacac agagaaagat atatttctca 4380 agatcagaag tactattcca gtatggacga ttcaaggctt gcttcacaaa ccaaggcaag 4440 taatagagat tggagtctct aaaaaggtag ttcccactga atcaaaggcc atggagtcaa 4500 agattcaaat agaggaccta acagaactcg ccgtaaagac tggcgaacag ttcatacaga 4560 gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacacac 4620 ttgtctactc caaaaatatc aaagatacag tctcagaaga ccaaagggca attgagactt 4680 ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct atctgtcact 4740 ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 4800 gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 4860 cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 4920 gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt 4980 cctctatata aggaagttca tttcatttgg agagaacgcg gggggcttct tgaggatcta 5040 gagctttcgc agatctgtcg atcgaccatg gggattgaac aagatggatt gcacgcaggt 5100 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc 5160 tgctctgatg ccgccgtgtt tcggctgtca gcgcaggggc gcccggttct ttttgtcaag 5220 accgacctgt ccggtgccct gaatgaactc caggacgagg cagcgcggct atcgtggctg 5280 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac 5340 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc 5400 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc 5460 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc 5520 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg 5580 ttcgccaggc tcaaggcgcg tatgcccgac ggcgaggatc tcgtcgtgac acatggcgat 5640 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc 5700 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa 5760 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat 5820 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt 5880 tcggatcgat cctctagcta gagtcgatcg acaagctccg aatttccccg atcgttcaaa 5940 catttggcaa taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat 6000 ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt 6060 tatgagatgg gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa 6120 caaaatatag cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga 6180 tcgggaattg ggatcataac ttcgtataat gtatgctata cgaagttatg ctagagcggc 6240 cgcc 6244 <210> 2 <211> 6400 <212> DNA <213> Artificial Sequence <220> <223> cLoxC3-CAH cassette <400> 2 gcggccgctg acggtatcgc gataagctat aacttcgtat aatgtatgct atacgaagtt 60 atgaattgca cgatctagta acatagatga caccgcgcgc gataatttat cctagtttgc 120 gcgctatatt ttgttttcta tcgcgtatta aatgtataat tgcgggactc taatcataaa 180 aacccatctc ataaataacg tcatgcatta catgttaatt attacatgct taacgtaatt 240 caacagaaat tatatgataa tcatcgcaag accggcaaca ggattcaatc ttaagaaact 300 ttattgccaa atgtttgaac gatcggggaa attcgcgcta atcgccatct tccagcaggc 360 gcaccattgc ccctgtttca ctatccaggt tacggatata gttcatgaca atatttacat 420 tggtccagcc accagcttgc atgatctccg gtattgaaac tccagcgcgg gccatatctc 480 gcgcggctcc gacacgggca ctgtgtccag accaggccag gtatctctga ccagagtcat 540 ccttagcgcc gtaaatcaat cgatgagttg cttcaaaaat cccttccagg gcgcgagttg 600 atagctggct ggtggcagat ggcgcggcaa caccattttt tctgacccgg caaaacaggt 660 agttattcgg atcatcagct acaccagaga cggaaatcca tcgctcgacc agtttagtta 720 cccccaggct aagtgccttc tctacacctg cggtgctaac cagcgttttc gttctgccaa 780 tatggattaa cattctccca ccgtcagtac gtgagatatc tttaaccctg atcctggcaa 840 tttcggctat acgtaacagg gtgttataag caatccccag aaatgccaga ttacgtatat 900 cctggcagcg atcgctattt tccatgagtg aacgaacctg taactatcat catcatcata 960 gacacacgaa ataaagtaat cagattatca gttaaagcta tgtaatattt acaccataac 1020 caatcaatta aaaaatagat cagtttaaag aaagatcaaa gctcaaaaaa ataaaaagag 1080 aaaagggtcc taaccaagaa aatgaaggag aaaaactaga aatttacctg gtcgaaatca 1140 gtgcgttcga acgctagagc ctgttttgca cgttcaccgg catcaacgtt ttcttttcgg 1200 attcgccgca taaccagtga aacagcattg ctgtcacttg gtcgtggcag cccggaccga 1260 cgatgaagca tgtttagctg gcccaaatgt tgctggatag tttttactgc cagaccgcgc 1320 gcctgaagat atagaagata atcgcgaaca tcttcaggtt ctgcgggaaa ccatttccgg 1380 ttattcaact tgcaccatgc cgcccacgac cggcaaacgg acagaagcat tttccaggta 1440 tgctcagaaa acgcctggcg atccctgaac atgtccatca ggttcttgcg aacctcatca 1500 ctcgttgcat cgaccggtaa tgcaggcaaa ttttggtgta cggtcagtaa attggacatg 1560 cgtgggtctt caacctttct cttcttcttt ggagccatcg agatcctgag aactcttctt 1620 cgatctcgtt tgttagttta tttggaagag tatgaagttc gtcgtctatt tatatgtgct 1680 tcagtatgat tcgtatttag cctcggtaga gtgtcctctc caatcacttc ttgacacgta 1740 agcttaatgg gttattttgt gttggcgcgc tccttacttt ttacacacgt aaagtaccca 1800 atcacaagca gacacgtgac attaaatcgt catcgcaaaa tctttcaaag gttttacgac 1860 ttcttcgtat gtatgtatag ttatccaaaa caattatcat tataatcaaa attaaattat 1920 taacaatgtt tttatcacat gcatgaatta ttaaaagatt tttttcgtgt accgattaat 1980 aaaataaaaa gatttgcgtc ataatttaca taaggataat ttcttttgtt attaatgaat 2040 tagtgtaatg ccaaaactac tgcatatgcg gtgaggtgta atatttggaa gaacatgact 2100 aatgcttaac cactccattt atttatcacg cagacatata tataagaaat atgccatttt 2160 ctaattactc tcagtgttgt atttgtattg gaatcaatgc acaataagaa ttggatattg 2220 gataacaaca aaacaggcaa accaacccta atggcggccg tcactctgca aagctattgc 2280 aggcgtcccc agattagcct tttcaatttc agaaagaatg ctaacccaca ggttagagag 2340 gcttacgcag caggtctcat caagacgatc tacccgagca ataatctcca ggaaatcaaa 2400 taccttccca agaaggttaa agatgcagtc aaaagattca ggactaactg catcaagaac 2460 acagagaaag atatatttct caagatcaga agtactattc cagtatggac gattcaaggc 2520 ttgcttcaca aaccaaggca agtaatagag attggagtct ctaaaaaggt agttcccact 2580 gaatcaaagg ccatggagtc aaagattcaa atagaggacc taacagaact cgccgtaaag 2640 actggcgaac agttcataca gagtctctta cgactcaatg acaagaagaa aatcttcgtc 2700 aacatggtgg agcacgacac acttgtctac tccaaaaata tcaaagatac agtctcagaa 2760 gaccaaaggg caattgagac ttttcaacaa agggtaatat ccggaaacct cctcggattc 2820 cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg tggctcctac 2880 aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc cgacagtggt 2940 cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt tccaaccacg 3000 tcttcaaagc aagtggattg atgtgatatc tccactgacg taagggatga cgcacaatcc 3060 cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt ggagagaaca 3120 cgggggactc tagctagagg atcgatccat gaaaaacaaa ttaccgccat ttatcgagat 3180 ttaccgcgct ctgattgcca caccttcaat aagcgccacg gaagaggcac tcgatcaaag 3240 caatgcagat ttaatcactc tgctggcgga ctggtttaaa gatttgggct tcaatgtgga 3300 agtgcagcct gttccaggaa ctcgcaacaa attcaatatg ctggcaagta tcggacaggg 3360 ggctggcggc ttgttgctgg cggggcatac cgatacggtg ccatttgatg acggtcgctg 3420 gacgcgcgat ccgtttacac tgacggagca tgacggcaag ctatacggct taggcaccgc 3480 cgacatgaaa ggcttttttg cgtttatcct tgatgcgcta cgcgatgtcg atgtcacgaa 3540 actgaaaaaa ccgctctaca ttctggcgac tgctgatgaa gaaaccagta tggccggagc 3600 gcgttatttt gccgaaacta ccgccctgcg cccggattgc gccatcattg gcgaaccgac 3660 gtcactacaa ccggtacgcg cacataaagg tcatatctct aacgccatcc gtattcaggg 3720 ccagtcgggg cactccagcg atccagcacg cggagttaac gctatcgaac taatgcacga 3780 cgccatcggg catattttgc aattgcgcga taacctgaaa gaacgttatc actacgaagc 3840 gtttaccgtg ccatacccta cgctcaacct cgggcatatt cacggtggcg acgcttctaa 3900 ccgtatttgc gcttgctgtg agttgcatat ggatattcgt ccgctgcctg gcatgacact 3960 caatgaactt aatggtttgc tcaacgatgc attggctccg gtgagcgaac gctggccggg 4020 tcgtctgacg gtcgatgagc tgcatccgcc gatccctggc tatgaatgcc caccgaatca 4080 tcaactggtt gaagtggttg agaaattgct cggagcaaaa accgaagtgg tgaactactg 4140 taccgaagcg ccgtttattc aaacgttatg cccgacgctg gtgttggggc ctggctcaat 4200 taatcaggct catcaacctg atgaatatct ggaaacacgg tttatcaagc ccacccgcga 4260 actgataacc caggtaattc accatttttg ctggcattaa gcgaatttcc ccgatcgttc 4320 aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat 4380 catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt 4440 atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga 4500 aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact 4560 agatcgggaa ttagcgcttg cgtccccaga ttagcctttt caatttcaga aagaatgcta 4620 acccacagat ggttagagag gcttacgcag caggtctcat caagacgatc tacccgagca 4680 ataatctcca ggaaatcaaa taccttccca agaaggttaa agatgcagtc aaaagattca 4740 ggactaactg catcaagaac acagagaaag atatatttct caagatcaga agtactattc 4800 cagtatggac gattcaaggc ttgcttcaca aaccaaggca agtaatagag attggagtct 4860 ctaaaaaggt agttcccact gaatcaaagg ccatggagtc aaagattcaa atagaggacc 4920 taacagaact cgccgtaaag actggcgaac agttcataca gagtctctta cgactcaatg 4980 acaagaagaa aatcttcgtc aacatggtgg agcacgacac acttgtctac tccaaaaata 5040 tcaaagatac agtctcagaa gaccaaaggg caattgagac ttttcaacaa agggtaatat 5100 ccggaaacct cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg 5160 aaaaggaagg tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag 5220 atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa 5280 aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg 5340 taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt 5400 catttcattt ggagagaaca cgggggaccg ccggtcgcgg aggccatgga tgcgatcgct 5460 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa 5520 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa 5580 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt 5640 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat 5700 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg 5760 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag 5820 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc acgactccgg 5880 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc 5940 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact 6000 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa 6060 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa gaaatagagt 6120 agatgccgac cggatctgtc gatcgacaag ctcgacggat cttcgagttt ctccataata 6180 atgtgtgagt agttcccaga taagggaatt agggttccta tagggtttcg ctcatgtgtt 6240 gagcatataa gaaaccctta gtatgtattt gtatttgtaa aatacttcta tcaataaaat 6300 ttctaattcc taaaaccaaa atccagtact aaagaattaa ttccccggga tcataacttc 6360 gtataatgta tgctatacga agttatgcta gagcggccgc 6400 <210> 3 <211> 9810 <212> DNA <213> Artificial Sequence <220> <223> pSYCAN vector <400> 3 tctagttctg gggaaccctg tggttggcat gcacatacaa atggacgaac ggataaacct 60 tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacc 120 cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctgc 180 gcggccgctg acggtatcgc gataagctat aacttcgtat aatgtatgct atacgaagtt 240 atgaattgtc gatcgacgat ctagtaacat agatgacacc gcgcgcgata atttatccta 300 gtttgcgcgc tatattttgt tttctatcgc gtattaaatg tataattgcg ggactctaat 360 cataaaaacc catctcataa ataacgtcat gcattacatg ttaattatta catgcttaac 420 gtaattcaac agaaattata tgataatcat cgcaagaccg gcaacaggat tcaatcttaa 480 gaaactttat tgccaaatgt ttgaacgatc ggggaaattc ggtcgagcta atcgccatct 540 tccagcaggc gcaccattgc ccctgtttca ctatccaggt tacggatata gttcatgaca 600 atatttacat tggtccagcc accagcttgc atgatctccg gtattgaaac tccagcgcgg 660 gccatatctc gcgcggctcc gacacgggca ctgtgtccag accaggccag gtatctctga 720 ccagagtcat ccttagcgcc gtaaatcaat cgatgagttg cttcaaaaat cccttccagg 780 gcgcgagttg atagctggct ggtggcagat ggcgcggcaa caccattttt tctgacccgg 840 caaaacaggt agttattcgg atcatcagct acaccagaga cggaaatcca tcgctcgacc 900 agtttagtta cccccaggct aagtgccttc tctacacctg cggtgctaac cagcgttttc 960 gttctgccaa tatggattaa cattctccca ccgtcagtac gtgagatatc tttaaccctg 1020 atcctggcaa tttcggctat acgtaacagg gtgttataag caatccccag aaatgccaga 1080 ttacgtatat cctggcagcg atcgctattt tccatgagtg aacgaacctg taactatcat 1140 catcatcata gacacacgaa ataaagtaat cagattatca gttaaagcta tgtaatattt 1200 acaccataac caatcaatta aaaaatagat cagtttaaag aaagatcaaa gctcaaaaaa 1260 ataaaaagag aaaagggtcc taaccaagaa aatgaaggag aaaaactaga aatttacctg 1320 gtcgaaatca gtgcgttcga acgctagagc ctgttttgca cgttcaccgg catcaacgtt 1380 ttcttttcgg attcgccgca taaccagtga aacagcattg ctgtcacttg gtcgtggcag 1440 cccggaccga cgatgaagca tgtttagctg gcccaaatgt tgctggatag tttttactgc 1500 cagaccgcgc gcctgaagat atagaagata atcgcgaaca tcttcaggtt ctgcgggaaa 1560 ccatttccgg ttattcaact tgcaccatgc cgcccacgac cggcaaacgg acagaagcat 1620 tttccaggta tgctcagaaa acgcctggcg atccctgaac atgtccatca ggttcttgcg 1680 aacctcatca ctcgtcgcat cgaccggtaa tgcaggcaaa ttttggtgta cggtcagtaa 1740 attggacatg cgtgggtctt caacctttct cttcttcttt ggagccatct cagatccgga 1800 aacttttgcg tacgatctct ctttttgatt tgaacagaga gcttcctttt gttttctggt 1860 ttttaagctt tgaagaagaa gttaggatgc ttaaataggt ccagtgcccg aaggtgatgg 1920 tgaagtagtc gctttttatg gaagacgaag aaatttaact atgtcatcgg agatgacgtg 1980 gaccaattat cttacgacga tctataatgt caaccaatca gatttggtct tgtgttggag 2040 taatcgtggc cgttggttac ggtccccaga ttagcctttt caatttcaga aagaatgcta 2100 acccacagat ggttagagag gcttacgcag caggtctcat caagacgatc tacccgagca 2160 ataatctcca ggaaatcaaa taccttccca agaaggttaa agatgcagtc aaaagattca 2220 ggactaactg catcaagaac acagagaaag atatatttct caagatcaga agtactattc 2280 cagtatggac gattcaaggc ttgcttcaca aaccaaggca agtaatagag attggagtct 2340 ctaaaaaggt agttcccact gaatcaaagg ccatggagtc aaagattcaa atagaggacc 2400 taacagaact cgccgtaaag actggcgaac agttcataca gagtctctta cgactcaatg 2460 acaagaagaa aatcttcgtc aacatggtgg agcacgacac acttgtctac tccaaaaata 2520 tcaaagatac agtctcagaa gaccaaaggg caattgagac ttttcaacaa agggtaatat 2580 ccggaaacct cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg 2640 aaaaggaagg tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag 2700 atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa 2760 aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg 2820 taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt 2880 catttcattt ggagagaaca cgggggactc tagctagagg atcgatccat gaaaaacaaa 2940 ttaccgccat ttatcgagat ttaccgcgct ctgattgcca caccttcaat aagcgccacg 3000 gaagaggcac tcgatcaaag caatgcagat ttaatcactc tgctggcgga ctggtttaaa 3060 gatttgggct tcaatgtgga agtgcagcct gttccaggaa ctcgcaacaa attcaatatg 3120 ctggcaagta tcggacaggg ggctggcggc ttgttgctgg cggggcatac cgatacggtg 3180 ccatttgatg acggtcgctg gacgcgcgat ccgtttacac tgacggagca tgacggcaag 3240 ctatacggct taggcaccgc cgacatgaaa ggcttttttg cgtttatcct tgatgcgcta 3300 cgcgatgtcg atgtcacgaa actgaaaaaa ccgctctaca ttctggcgac tgctgatgaa 3360 gaaaccagta tggccggagc gcgttatttt gccgaaacta ccgccctgcg cccggattgc 3420 gccatcattg gcgaaccgac gtcactacaa ccggtacgcg cacataaagg tcatatctct 3480 aacgccatcc gtattcaggg ccagtcgggg cactccagcg atccagcacg cggagttaac 3540 gctatcgaac taatgcacga cgccatcggg catattttgc aattgcgcga taacctgaaa 3600 gaacgttatc actacgaagc gtttaccgtg ccatacccta cgctcaacct cgggcatatt 3660 cacggtggcg acgcttctaa ccgtatttgc gcttgctgtg agttgcatat ggatattcgt 3720 ccgctgcctg gcatgacact caatgaactt aatggtttgc tcaacgatgc attggctccg 3780 gtgagcgaac gctggccggg tcgtctgacg gtcgatgagc tgcatccgcc gatccctggc 3840 tatgaatgcc caccgaatca tcaactggtt gaagtggttg agaaattgct cggagcaaaa 3900 accgaagtgg tgaactactg taccgaagcg ccgtttattc aaacgttatg cccgacgctg 3960 gtgttggggc ctggctcaat taatcaggct catcaacctg atgaatatct ggaaacacgg 4020 tttatcaagc ccacccgcga actgataacc caggtaattc accatttttg ctggcattaa 4080 gcgaatttcc ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt 4140 gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt 4200 aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta 4260 tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc 4320 gcggtgtcat ctatgttact agatcgggat tagcgctgcg tccccagatt agccttttca 4380 atttcagaaa gaatgctaac ccacagatgg ttagagaggc ttacgcagca ggtctcatca 4440 agacgatcta cccgagcaat aatctccagg aaatcaaata ccttcccaag aaggttaaag 4500 atgcagtcaa aagattcagg actaactgca tcaagaacac agagaaagat atatttctca 4560 agatcagaag tactattcca gtatggacga ttcaaggctt gcttcacaaa ccaaggcaag 4620 taatagagat tggagtctct aaaaaggtag ttcccactga atcaaaggcc atggagtcaa 4680 agattcaaat agaggaccta acagaactcg ccgtaaagac tggcgaacag ttcatacaga 4740 gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacacac 4800 ttgtctactc caaaaatatc aaagatacag tctcagaaga ccaaagggca attgagactt 4860 ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct atctgtcact 4920 ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 4980 gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 5040 cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 5100 gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt 5160 cctctatata aggaagttca tttcatttgg agagaacgcg gggggcttct tgaggatcta 5220 gagctttcgc agatctgtcg atcgaccatg gggattgaac aagatggatt gcacgcaggt 5280 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc 5340 tgctctgatg ccgccgtgtt tcggctgtca gcgcaggggc gcccggttct ttttgtcaag 5400 accgacctgt ccggtgccct gaatgaactc caggacgagg cagcgcggct atcgtggctg 5460 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac 5520 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc 5580 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc 5640 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc 5700 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg 5760 ttcgccaggc tcaaggcgcg tatgcccgac ggcgaggatc tcgtcgtgac acatggcgat 5820 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc 5880 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa 5940 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat 6000 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt 6060 tcggatcgat cctctagcta gagtcgatcg acaagctccg aatttccccg atcgttcaaa 6120 catttggcaa taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat 6180 ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt 6240 tatgagatgg gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa 6300 caaaatatag cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga 6360 tcgggaattg ggatcataac ttcgtataat gtatgctata cgaagttatg ctagagcggc 6420 cgctctagaa ctagtggatc ccccgggctg caggaattcg atatcaagct tatcgatacc 6480 gtcgacctcg agggggggcc cggtaccaaa accaccccag tacattaaaa acgtccgcaa 6540 tgtgttatta agttgtctaa gcgtcaattt gtttacacca caatatatcc tgccaccagc 6600 cagccaacag ctccccgacc ggcagctcgg cacaaaatca ccactcgata caggcagccc 6660 atcagtccac tagagtaaag cgctggctgc tgaaccccca gccggaactg accccacaag 6720 gccctagcgt ttgcaatgca ccaggtcatc attgacccag gcgtgttcca ccaggccgct 6780 gcctcgcaac tcttcgcagg cttcgccgac ctgctcgcgc cacttcttca cgcgggtgga 6840 atccgatccg cacatgaggc ggaaggtttc cagcttgagc gggtacggct cccggtgcga 6900 gctgaaatag tcgaacatcc gtcgggccgt cggcgacagc ttgcggtact tctcccatat 6960 gaatttcgtg tagtggtcgc cagcaaacag cacgacgatt tcctcgtcga tcaggacctg 7020 gcaacgggac gttttcttgc cacggtccag gacgcggaag cggtgcagca gcgacaccga 7080 ttccaggtgc ccaacgcggt cggacgtgaa gcccatcgcc gtcgcctgta ggcgcgacag 7140 gcattcctcg gccttcgtgt aataccggcc attgatcgac cagcccaggt cctggcaaag 7200 ctcgtagaac gtgaaggtga tcggctcgcc gataggggtg cgcttcgcgt actccaacac 7260 ctgctgccac accagttcgt catcgtcggc ccgcagctcg acgccggtgt aggtgatctt 7320 cacgtccttg ttgacgtgga aaatgacctt gttttgcagc gcctcgcgcg ggattttctt 7380 gttgcgcgtg gtgaacaggg cagagcgggc cgtgtcgttt ggcatcgctc gcatcgtgtc 7440 cggccacggc gcaatatcga acaaggaaag ctgcatttcc ttgatctgct gcttcgtgtg 7500 tttcagcaac gcggcctgct tggcctcgct gacctgtttt gccaggtcct cgccggcggt 7560 ttttcgcttc ttggtcgtca tagttcctcg cgtgtcgatg gtcatcgact tcgccaaacc 7620 tgccgcctcc tgttcgagac gacgcgaacg ctccacggcg gccgatggcg cgggcagggc 7680 agggggagcc agttgcacgc tgtcgcgctc gatcttggcc gtagcttgct ggaccatcga 7740 gccgacggac tggaaggttt cgcggggcgc acgcatgacg gtgcggcttg cgatggtttc 7800 ggcatcctcg gcggaaaacc ccgcgtcgat cagttcttgc ctgtatgcct tccggtcaaa 7860 cgtccgattc attcaccctc cttgcgggat tgccccgact cacgccgggg caatgtgccc 7920 ttattcctga tttgacccgc ctggtgcctt ggtgtccaga taatccacct tatcggcaat 7980 gaagtcggtc ccgtagaccg tctggccgtc cttctcgtac ttggtattcc gaatcttgcc 8040 ctgcacgaat accagcgacc ccttgcccaa atacttgccg tgggcctcgg cctgagagcc 8100 aaaacacttg atgcggaaga agtcggtgcg ctcctgcttg tcgccggcat cgttgcgcca 8160 catctaggta ctaaaacaat tcatccagta aaatataata ttttattttc tcccaatcag 8220 gcttgatccc cagtaagtca aaaaatagct cgacatactg ttcttccccg atatcctccc 8280 tgatcgaccg gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa 8340 gatcaataaa gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc 8400 cgtgggaaaa gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc 8460 gcggatcttt aaatggagtg tcttcttccc agttttcgca atccacatcg gccagatcgt 8520 tattcagtaa gtaatccaat tcggctaagc ggctgtctaa gctattcgta tagggacaat 8580 ccgatatgtc gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt 8640 cagggctttg ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga 8700 gcagattgct ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc 8760 cttccagcca tagcatcatg tccttttccc gttccacatc ataggtggtc cctttatacc 8820 ggctgtccgt catttttaaa tataggtttt cattttctcc caccagctta tataccttag 8880 caggagacat tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt 8940 ccggtgatat tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa 9000 gataccccaa gaagctaatt ataacaagac gaactccaat tcactgttcc ttgcattcta 9060 aaaccttaaa taccagaaaa cagctttttc aaagttgttt tcaaagttgg cgtataacat 9120 agtatcgacg gagccgattt tgaaaccaca attatgggtg atgctgccaa ctcgagcggg 9180 ccgggagggt tcgagaaggg ggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg 9240 cgcagccctg gttaaaaaca aggtttataa atattggttt aaaagcaggt taaaagacag 9300 gttagcggtg gccgaaaaac gggcggaaac ccttgcaaat gctggatttt ctgcctgtgg 9360 acagcccctc aaatgtcaat aggtgcgccc ctcatctgtc agcactctgc ccctcaagtg 9420 tcaaggatcg cgcccctcat ctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg 9480 cacttatccc caggcttgtc cacatcatct gtgggaaact cgcgtaaaat caggcgtttt 9540 cgccgatttg cgaggctggc cagctccacg tcgccggccg aaatcgagcc tgcccctcat 9600 ctgtcaacgc cgcgccgggt gagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt 9660 cagtgagggc caagttttcc gcgaggtatc cacaacgccg gcggccggcc gcggtgtctc 9720 gcacacggct tcgacggcgt ttctggcgcg tttgcagggc catagacggc cgccagccca 9780 gcggcgaggg caaccagccc ggtgagcgtc 9810 <210> 4 <211> 9967 <212> DNA <213> Artificial Sequence <220> <223> pSYCAH vector <400> 4 gctcaccggg ctggttgccc tcgccgctgg gctggcggcc gtctatggcc ctgcaaacgc 60 gccagaaacg ccgtcgaagc cgtgtgcgag acaccgcggc cggccgccgg cgttgtggat 120 acctcgcgga aaacttggcc ctcactgaca gatgaggggc ggacgttgac acttgagggg 180 ccgactcacc cggcgcggcg ttgacagatg aggggcaggc tcgatttcgg ccggcgacgt 240 ggagctggcc agcctcgcaa atcggcgaaa acgcctgatt ttacgcgagt ttcccacaga 300 tgatgtggac aagcctgggg ataagtgccc tgcggtattg acacttgagg ggcgcgacta 360 ctgacagatg aggggcgcga tccttgacac ttgaggggca gagtgctgac agatgagggg 420 cgcacctatt gacatttgag gggctgtcca caggcagaaa atccagcatt tgcaagggtt 480 tccgcccgtt tttcggccac cgctaacctg tcttttaacc tgcttttaaa ccaatattta 540 taaaccttgt ttttaaccag ggctgcgccc tgtgcgcgtg accgcgcacg ccgaaggggg 600 gtgccccccc ttctcgaacc ctcccggccc gctcgagttg gcagcatcac ccataattgt 660 ggtttcaaaa tcggctccgt cgatactatg ttatacgcca actttgaaaa caactttgaa 720 aaagctgttt tctggtattt aaggttttag aatgcaagga acagtgaatt ggagttcgtc 780 ttgttataat tagcttcttg gggtatcttt aaatactgta gaaaagagga aggaaataat 840 aaatggctaa aatgagaata tcaccggaat tgaaaaaact gatcgaaaaa taccgctgcg 900 taaaagatac ggaaggaatg tctcctgcta aggtatataa gctggtggga gaaaatgaaa 960 acctatattt aaaaatgacg gacagccggt ataaagggac cacctatgat gtggaacggg 1020 aaaaggacat gatgctatgg ctggaaggaa agctgcctgt tccaaaggtc ctgcactttg 1080 aacggcatga tggctggagc aatctgctca tgagtgaggc cgatggcgtc ctttgctcgg 1140 aagagtatga agatgaacaa agccctgaaa agattatcga gctgtatgcg gagtgcatca 1200 ggctctttca ctccatcgac atatcggatt gtccctatac gaatagctta gacagccgct 1260 tagccgaatt ggattactta ctgaataacg atctggccga tgtggattgc gaaaactggg 1320 aagaagacac tccatttaaa gatccgcgcg agctgtatga ttttttaaag acggaaaagc 1380 ccgaagagga acttgtcttt tcccacggcg acctgggaga cagcaacatc tttgtgaaag 1440 atggcaaagt aagtggcttt attgatcttg ggagaagcgg cagggcggac aagtggtatg 1500 acattgcctt ctgcgtccgg tcgatcaggg aggatatcgg ggaagaacag tatgtcgagc 1560 tattttttga cttactgggg atcaagcctg attgggagaa aataaaatat tatattttac 1620 tggatgaatt gttttagtac ctagatgtgg cgcaacgatg ccggcgacaa gcaggagcgc 1680 accgacttct tccgcatcaa gtgttttggc tctcaggccg aggcccacgg caagtatttg 1740 ggcaaggggt cgctggtatt cgtgcagggc aagattcgga ataccaagta cgagaaggac 1800 ggccagacgg tctacgggac cgacttcatt gccgataagg tggattatct ggacaccaag 1860 gcaccaggcg ggtcaaatca ggaataaggg cacattgccc cggcgtgagt cggggcaatc 1920 ccgcaaggag ggtgaatgaa tcggacgttt gaccggaagg catacaggca agaactgatc 1980 gacgcggggt tttccgccga ggatgccgaa accatcgcaa gccgcaccgt catgcgtgcg 2040 ccccgcgaaa ccttccagtc cgtcggctcg atggtccagc aagctacggc caagatcgag 2100 cgcgacagcg tgcaactggc tccccctgcc ctgcccgcgc catcggccgc cgtggagcgt 2160 tcgcgtcgtc tcgaacagga ggcggcaggt ttggcgaagt cgatgaccat cgacacgcga 2220 ggaactatga cgaccaagaa gcgaaaaacc gccggcgagg acctggcaaa acaggtcagc 2280 gaggccaagc aggccgcgtt gctgaaacac acgaagcagc agatcaagga aatgcagctt 2340 tccttgttcg atattgcgcc gtggccggac acgatgcgag cgatgccaaa cgacacggcc 2400 cgctctgccc tgttcaccac gcgcaacaag aaaatcccgc gcgaggcgct gcaaaacaag 2460 gtcattttcc acgtcaacaa ggacgtgaag atcacctaca ccggcgtcga gctgcgggcc 2520 gacgatgacg aactggtgtg gcagcaggtg ttggagtacg cgaagcgcac ccctatcggc 2580 gagccgatca ccttcacgtt ctacgagctt tgccaggacc tgggctggtc gatcaatggc 2640 cggtattaca cgaaggccga ggaatgcctg tcgcgcctac aggcgacggc gatgggcttc 2700 acgtccgacc gcgttgggca cctggaatcg gtgtcgctgc tgcaccgctt ccgcgtcctg 2760 gaccgtggca agaaaacgtc ccgttgccag gtcctgatcg acgaggaaat cgtcgtgctg 2820 tttgctggcg accactacac gaaattcata tgggagaagt accgcaagct gtcgccgacg 2880 gcccgacgga tgttcgacta tttcagctcg caccgggagc cgtacccgct caagctggaa 2940 accttccgcc tcatgtgcgg atcggattcc acccgcgtga agaagtggcg cgagcaggtc 3000 ggcgaagcct gcgaagagtt gcgaggcagc ggcctggtgg aacacgcctg ggtcaatgat 3060 gacctggtgc attgcaaacg ctagggcctt gtggggtcag ttccggctgg gggttcagca 3120 gccagcgctt tactctagtg gactgatggg ctgcctgtat cgagtggtga ttttgtgccg 3180 agctgccggt cggggagctg ttggctggct ggtggcagga tatattgtgg tgtaaacaaa 3240 ttgacgctta gacaacttaa taacacattg cggacgtttt taatgtactg gggtggtttt 3300 ggtaccgggc cccccctcga ggtcgacggt atcgataagc ttgatatcga attcctgcag 3360 cccgggggat ccactagttc tagagcggcc gctctagcat aacttcgtat agcatacatt 3420 atacgaagtt atgatcccgg ggaattaatt ctttagtact ggattttggt tttaggaatt 3480 agaaatttta ttgatagaag tattttacaa atacaaatac atactaaggg tttcttatat 3540 gctcaacaca tgagcgaaac cctataggaa ccctaattcc cttatctggg aactactcac 3600 acattattat ggagaaactc gaagatccgt cgagcttgtc gatcgacaga tccggtcggc 3660 atctactcta tttctttgcc ctcggacgag tgctggggcg tcggtttcca ctatcggcga 3720 gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc 3780 cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc caagctgcat 3840 catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg cggagcatat 3900 acgcccggag tcgtggcgat cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct 3960 gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc tcgtattggg 4020 aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca 4080 ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg 4140 cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg tcgtccatca 4200 cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca cgccatgtag 4260 tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg ctaagatcgg 4320 ccgcagcgat cgcatccatg gcctccgcga ccggcggtcc cccgtgttct ctccaaatga 4380 aatgaacttc cttatataga ggaagggtct tgcgaaggat agtgggattg tgcgtcatcc 4440 cttacgtcag tggagatatc acatcaatcc acttgctttg aagacgtggt tggaacgtct 4500 tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact gtcggcagag 4560 gcatcttcaa cgatggcctt tcctttatcg caatgatggc atttgtagga gccaccttcc 4620 ttttccacta tcttcacaat aaagtgacag atagctgggc aatggaatcc gaggaggttt 4680 ccggatatta ccctttgttg aaaagtctca attgcccttt ggtcttctga gactgtatct 4740 ttgatatttt tggagtagac aagtgtgtcg tgctccacca tgttgacgaa gattttcttc 4800 ttgtcattga gtcgtaagag actctgtatg aactgttcgc cagtctttac ggcgagttct 4860 gttaggtcct ctatttgaat ctttgactcc atggcctttg attcagtggg aactaccttt 4920 ttagagactc caatctctat tacttgcctt ggtttgtgaa gcaagccttg aatcgtccat 4980 actggaatag tacttctgat cttgagaaat atatctttct ctgtgttctt gatgcagtta 5040 gtcctgaatc ttttgactgc atctttaacc ttcttgggaa ggtatttgat ttcctggaga 5100 ttattgctcg ggtagatcgt cttgatgaga cctgctgcgt aagcctctct aaccatctgt 5160 gggttagcat tctttctgaa attgaaaagg ctaatctggg gacgcaagcg ctaattcccg 5220 atctagtaac atagatgaca ccgcgcgcga taatttatcc tagtttgcgc gctatatttt 5280 gttttctatc gcgtattaaa tgtataattg cgggactcta atcataaaaa cccatctcat 5340 aaataacgtc atgcattaca tgttaattat tacatgctta acgtaattca acagaaatta 5400 tatgataatc atcgcaagac cggcaacagg attcaatctt aagaaacttt attgccaaat 5460 gtttgaacga tcggggaaat tcgcttaatg ccagcaaaaa tggtgaatta cctgggttat 5520 cagttcgcgg gtgggcttga taaaccgtgt ttccagatat tcatcaggtt gatgagcctg 5580 attaattgag ccaggcccca acaccagcgt cgggcataac gtttgaataa acggcgcttc 5640 ggtacagtag ttcaccactt cggtttttgc tccgagcaat ttctcaacca cttcaaccag 5700 ttgatgattc ggtgggcatt catagccagg gatcggcgga tgcagctcat cgaccgtcag 5760 acgacccggc cagcgttcgc tcaccggagc caatgcatcg ttgagcaaac cattaagttc 5820 attgagtgtc atgccaggca gcggacgaat atccatatgc aactcacagc aagcgcaaat 5880 acggttagaa gcgtcgccac cgtgaatatg cccgaggttg agcgtagggt atggcacggt 5940 aaacgcttcg tagtgataac gttctttcag gttatcgcgc aattgcaaaa tatgcccgat 6000 ggcgtcgtgc attagttcga tagcgttaac tccgcgtgct ggatcgctgg agtgccccga 6060 ctggccctga atacggatgg cgttagagat atgaccttta tgtgcgcgta ccggttgtag 6120 tgacgtcggt tcgccaatga tggcgcaatc cgggcgcagg gcggtagttt cggcaaaata 6180 acgcgctccg gccatactgg tttcttcatc agcagtcgcc agaatgtaga gcggtttttt 6240 cagtttcgtg acatcgacat cgcgtagcgc atcaaggata aacgcaaaaa agcctttcat 6300 gtcggcggtg cctaagccgt atagcttgcc gtcatgctcc gtcagtgtaa acggatcgcg 6360 cgtccagcga ccgtcatcaa atggcaccgt atcggtatgc cccgccagca acaagccgcc 6420 agccccctgt ccgatacttg ccagcatatt gaatttgttg cgagttcctg gaacaggctg 6480 cacttccaca ttgaagccca aatctttaaa ccagtccgcc agcagagtga ttaaatctgc 6540 attgctttga tcgagtgcct cttccgtggc gcttattgaa ggtgtggcaa tcagagcgcg 6600 gtaaatctcg ataaatggcg gtaatttgtt tttcatggat cgatcctcta gctagagtcc 6660 cccgtgttct ctccaaatga aatgaacttc cttatataga ggaagggtct tgcgaaggat 6720 agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg 6780 aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt 6840 tgggaccact gtcggcagag gcatcttcaa cgatggcctt tcctttatcg caatgatggc 6900 atttgtagga gccaccttcc ttttccacta tcttcacaat aaagtgacag atagctgggc 6960 aatggaatcc gaggaggttt ccggatatta ccctttgttg aaaagtctca attgcccttt 7020 ggtcttctga gactgtatct ttgatatttt tggagtagac aagtgtgtcg tgctccacca 7080 tgttgacgaa gattttcttc ttgtcattga gtcgtaagag actctgtatg aactgttcgc 7140 cagtctttac ggcgagttct gttaggtcct ctatttgaat ctttgactcc atggcctttg 7200 attcagtggg aactaccttt ttagagactc caatctctat tacttgcctt ggtttgtgaa 7260 gcaagccttg aatcgtccat actggaatag tacttctgat cttgagaaat atatctttct 7320 ctgtgttctt gatgcagtta gtcctgaatc ttttgactgc atctttaacc ttcttgggaa 7380 ggtatttgat ttcctggaga ttattgctcg ggtagatcgt cttgatgaga cctgctgcgt 7440 aagcctctct aacctgtggg ttagcattct ttctgaaatt gaaaaggcta atctggggac 7500 gcctgcaata gctttgcaga gtgacggccg ccattagggt tggtttgcct gttttgttgt 7560 tatccaatat ccaattctta ttgtgcattg attccaatac aaatacaaca ctgagagtaa 7620 ttagaaaatg gcatatttct tatatatatg tctgcgtgat aaataaatgg agtggttaag 7680 cattagtcat gttcttccaa atattacacc tcaccgcata tgcagtagtt ttggcattac 7740 actaattcat taataacaaa agaaattatc cttatgtaaa ttatgacgca aatcttttta 7800 ttttattaat cggtacacga aaaaaatctt ttaataattc atgcatgtga taaaaacatt 7860 gttaataatt taattttgat tataatgata attgttttgg ataactatac atacatacga 7920 agaagtcgta aaacctttga aagattttgc gatgacgatt taatgtcacg tgtctgcttg 7980 tgattgggta ctttacgtgt gtaaaaagta aggagcgcgc caacacaaaa taacccatta 8040 agcttacgtg tcaagaagtg attggagagg acactctacc gaggctaaat acgaatcata 8100 ctgaagcaca tataaataga cgacgaactt catactcttc caaataaact aacaaacgag 8160 atcgaagaag agttctcagg atctcgatgg ctccaaagaa gaagagaaag gttgaagacc 8220 cacgcatgtc caatttactg accgtacacc aaaatttgcc tgcattaccg gtcgatgcaa 8280 cgagtgatga ggttcgcaag aacctgatgg acatgttcag ggatcgccag gcgttttctg 8340 agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg ggcggcatgg tgcaagttga 8400 ataaccggaa atggtttccc gcagaacctg aagatgttcg cgattatctt ctatatcttc 8460 aggcgcgcgg tctggcagta aaaactatcc agcaacattt gggccagcta aacatgcttc 8520 atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc tgtttcactg gttatgcggc 8580 gaatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta gcgttcgaac 8640 gcactgattt cgaccaggta aatttctagt ttttctcctt cattttcttg gttaggaccc 8700 ttttctcttt ttattttttt gagctttgat ctttctttaa actgatctat tttttaattg 8760 attggttatg gtgtaaatat tacatagctt taactgataa tctgattact ttatttcgtg 8820 tgtctatgat gatgatgata gttacaggtt cgttcactca tggaaaatag cgatcgctgc 8880 caggatatac gtaatctggc atttctgggg attgcttata acaccctgtt acgtatagcc 8940 gaaattgcca ggatcagggt taaagatatc tcacgtactg acggtgggag aatgttaatc 9000 catattggca gaacgaaaac gctggttagc accgcaggtg tagagaaggc acttagcctg 9060 ggggtaacta aactggtcga gcgatggatt tccgtctctg gtgtagctga tgatccgaat 9120 aactacctgt tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc caccagccag 9180 ctatcaactc gcgccctgga agggattttt gaagcaactc atcgattgat ttacggcgct 9240 aaggatgact ctggtcagag atacctggcc tggtctggac acagtgcccg tgtcggagcc 9300 gcgcgagata tggcccgcgc tggagtttca ataccggaga tcatgcaagc tggtggctgg 9360 accaatgtaa atattgtcat gaactatatc cgtaacctgg atagtgaaac aggggcaatg 9420 gtgcgcctgc tggaagatgg cgattagcgc gaatttcccc gatcgttcaa acatttggca 9480 ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct 9540 gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg 9600 ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata 9660 gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgtgcaat 9720 tcataacttc gtatagcata cattatacga agttatagct tatcgcgata ccgtcagcgg 9780 ccgcgcagat tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt 9840 ggcgggtaaa cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt 9900 gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca accacagggt tccccagaac 9960 tagagac 9967  

Claims (11)

A cassette for removing the selection marker gene of a plant transformation vector comprising a Cre (Cre recombinase) gene, an argith (ornithine deacetylase) gene, and a selection marker gene operably linked to a pBip-L promoter. The cassette for removing the selection marker gene of the vector for plant transformation according to claim 1 or 12. The cassette for removing a selection marker gene of a plant transformation vector according to claim 2, wherein the cassette of FIG. 11 or 12 is composed of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. For removal of selection marker genes including cassette for removal of selection marker gene of plant transformation vector comprising Cre (Cre recombinase) gene, argE (ornithine deacetylase) gene, and selection marker gene operably linked to pBip-L promoter Transformation vector. The vector according to claim 4, comprising a cassette for removing the selection marker gene of the plant transformation vector described in FIG. 11 or 12. The vector according to claim 5, which is a pSYCAN vector or a pSYCAH vector described in FIG. The vector according to claim 6, wherein the pSYCAN vector or pSYCAH vector consists of a nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, respectively. Preparing a recombinant plant expression vector by inserting a target gene into the transformation vector for removing the selection marker gene of claim 4; Transforming the plant with the recombinant plant expression vector; And A method for producing a transformed plant from which a selection marker gene has been removed, comprising selecting a transformant from which the selection marker gene has been removed. The method of claim 8, wherein the selection vector is a pSYCAN vector or a pSYCAH vector. The method of claim 8, wherein selecting the transformant from which the selection marker gene has been removed is characterized in that the transformed plant is treated with tunicamycin and N-acetyl phosphinothricin. A transgenic plant from which a selection marker gene prepared by the method of any one of claims 8 to 10 has been removed.

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