阅读说明：本技术 启动子区域分析方法和用于实施该方法的细胞 (Promoter region analysis method and cell for carrying out the method ) 是由 P.沙皮罗 V.查里 J.林-琼斯 J.拉默丁 于 2020-03-26 设计创作，主要内容包括：提供了评估启动子区域活性的方法。所述方法包括培养包含核酸的细胞,所述核酸包含编码可操作地偶联至启动子区域的酶供体(ED)的区域,在其中当所述启动子区域有活性时表达所述ED的条件下培养。所述方法还包括如果表达将所述ED与酶受体(EA)接触以形成具有酶活性的ED-EA复合物。所述方法还包括检测所述酶活性的水平以评估所述启动子区域的活性。所述启动子区域的活性可以是指示性的,因此可以用于评估目标细胞信号转导通路和/或目标内源性或外源性(例如,引入的)转录因子的活性。还提供了可用于例如实施本公开内容的方法的细胞、组合物和试剂盒。(Methods of assessing promoter region activity are provided. The method comprises culturing a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) operably coupled to a promoter region, under conditions wherein the ED is expressed when the promoter region is active. The method further comprises contacting the ED with an enzyme receptor (EA) if expressed to form an ED-EA complex having enzymatic activity. The method further comprises detecting the level of the enzyme activity to assess the activity of the promoter region. The activity of the promoter region may be indicative and thus may be used to assess the activity of the target cell signaling pathway and/or the target endogenous or exogenous (e.g., introduced) transcription factor. Also provided are cells, compositions, and kits useful, for example, in practicing the methods of the disclosure.)

1. A method of assessing promoter region activity comprising:

culturing a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) operably coupled to a promoter region under conditions wherein the ED is expressed when the promoter region is active;

contacting the ED with an enzyme receptor (EA) to form an ED-EA complex having enzymatic activity, if expressed;

detecting the level of the enzyme activity to assess the activity of the promoter region; and

assessing the level of activation of a transcription factor based on the detected level of the enzyme activity.

2. The method of claim, wherein the promoter region comprises a Transcription Factor Response Element (TFRE) for a transcription factor of interest, and wherein the activity of the promoter region is indicative of the activity of the transcription factor.

3. The method of claim 2, wherein the promoter region comprises at least one TFRE.

4. The method of claim 1, further comprising introducing an expression vector encoding the transcription factor into the cell and culturing the cell under conditions in which the transcription factor is expressed.

5. The method of claim 1, further comprising contacting the cell with an agent, and assessing the level of activity of the promoter region in response to contacting the cell with the agent based on the detected level of the enzyme activity.

6. The method of claim 5, wherein the activity of the promoter region is indicative of an activity of a target cell signaling pathway.

7. The method of claim 5, wherein contacting the cell with the agent comprises culturing the cell in the presence of the agent.

8. The method of claim 5, wherein assessing the level of activity of the promoter region in response to contacting the cell with the agent comprises comparing the level of enzyme activity detected in the absence of the agent to the level of enzyme activity detected in the presence of the agent.

9. The method of claim 5, wherein the agent is a small molecule.

10. The method of claim 5, wherein the reagent is a detection reagent.

11. The method of claim 5, wherein the cell is contacted with more than one detection reagent.

12. The method of claim 1, wherein the nucleic acid further encodes a carrier protein fused to the ED, such that an ED-carrier protein fusion is expressed when the promoter region is active.

13. The method of claim 14, wherein the carrier protein exhibits an enzymatic activity that is different from the enzymatic activity of the ED-EA complex.

14. The method of claim 1, wherein detecting the level of the enzymatic activity comprises providing a substrate for the ED-EA complex, wherein a detectable signal is generated upon hydrolysis of the substrate by the ED-EA complex.

15. The method of claim 1, wherein the ED comprises the amino acid sequence set forth in SEQ ID No.30 or a variant thereof that complexes with the EA to form an ED-EA complex.

16. A method of assessing promoter region activity comprising:

culturing a cell comprising a nucleic acid comprising a region encoding a carrier protein fused to an Enzyme Donor (ED) to form an ED-carrier protein fusion, wherein the ED-carrier protein fusion is operably coupled to a promoter region, under conditions wherein the ED-carrier protein fusion is expressed when the promoter region is active;

contacting the ED with an enzyme receptor (EA) to form an ED-EA complex having enzymatic activity, if expressed;

detecting the level of the enzyme activity to assess the activity of the promoter region; and

assessing the level of activation of a transcription factor based on the detected level of the enzyme activity.

17. The method of claim 16, wherein the promoter region comprises a Transcription Factor Response Element (TFRE) for a transcription factor of interest, and wherein the activity of the promoter region is indicative of the activity of the transcription factor.

18. The method of claim 17 wherein the carrier protein comprises a domain selected to affect the stability of the ED-carrier protein fusion.

19. The method of claim 18, wherein the domain is selected to increase the stability of an ED-carrier protein fusion as compared to the ED-carrier protein fusion lacking the domain.

20. The method of claim 18, wherein the domain is selected to destabilize the ED-carrier protein compared to an ED-carrier protein lacking the domain.

21. The method of claim 16, further comprising introducing an expression vector encoding the transcription factor into the cell and culturing the cell under conditions in which the transcription factor is expressed.

22. The method of claim 16, comprising contacting the cell with an agent, and assessing the level of activity of the promoter region in response to contacting the cell with the agent based on the detected level of the enzyme activity.

23. The method of claim 16, wherein the activity of the promoter region is indicative of an activity of a target cell signaling pathway.

24. The method of claim 22, wherein assessing the level of activity of the promoter region in response to contacting the cell with the agent comprises comparing the level of enzyme activity detected in the absence of the agent to the level of enzyme activity detected in the presence of the agent.

25. The method of claim 22, wherein the agent is a small molecule.

26. The method of claim 22, wherein the reagent is a detection reagent.

27. A kit, comprising:

a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) operably coupled to a promoter region; and

instructions for using the cell to perform the method of any one of claims 1 to 26.

28. The kit of claim 27, further comprising instructions for: assessing the level of activation of the transcription factor, or a pathway that activates the transcription factor, based on the detected level of the enzyme activity; contacting the cells with an agent during the culturing; and assessing activation of the promoter region in response to contacting the cell with the agent based on the detected level of the enzyme activity.

Technical Field

The present invention relates generally to methods of assessing promoter region activity, and more specifically, the promoter region is coupled to a detection agent such that expression of the promoter region directs expression of the detection agent that measures promoter region activity. The disclosed methods assess the activity of a cell signaling pathway and the effect of a test compound on a cell signaling pathway.

Reference to sequence listing

This application contains a sequence listing that has been filed as an ASCII text file and is incorporated by reference herein in its entirety. This text file was created at 22.03.2020, named "PBH _010_1Seq _ list. txt" and has a size of 49,795 bytes.

Background

Interest in exploring various aspects of the cell signaling pathway and the effects of different compounds or molecules on the regulation of the cell signaling pathway has been a key driver in understanding the disease and in finding therapeutic approaches. As more and more proteins in the cell signaling pathway and their functions are identified, interest in finding molecules that modulate the activity of these proteins is growing dramatically. The expression or inhibition of pathway proteins is helpful in understanding the effect of test compounds or test conditions on signal transduction pathways and in finding new pharmaceutical drugs.

Disclosure of Invention

The following brief summary is not intended to include all features and aspects of the present invention, nor is it intended that the present invention necessarily include all features and aspects discussed in this summary.

Many embodiments of the present invention provide a method for assessing promoter region activity. In many other embodiments, a method of assessing promoter region activity is disclosed, comprising culturing a cell comprising a nucleic acid comprising a region encoding a first β -galactosidase fragment operably coupled to a promoter region, under conditions wherein said first β -galactosidase fragment is expressed when said promoter region is active. In other embodiments, the method further comprises contacting the first β -galactosidase fragment with a second β -galactosidase fragment if expressed to form an active enzyme complex, and detecting the level of enzyme activity to assess the activity of the promoter region. In other embodiments, the activity of the promoter region may be indicative and thus may be used to assess the activity of a cell signaling pathway and/or an endogenous or exogenous (e.g., introduced) transcription factor.

In many embodiments, the invention provides a method of assessing promoter region activity comprising culturing a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) operably coupled to a promoter region, under conditions wherein the ED is expressed when the promoter region is active. In many other embodiments, the methods further comprise contacting the ED with an enzyme receptor (EA) to form an ED-EA complex having enzymatic activity if expressed, and detecting the level of enzymatic activity to assess the activity of the promoter region. In a further embodiment, the ED fragment comprises the amino acid sequence set forth in SEQ ID NO 30 or a variant thereof. In other embodiments, the activity of the promoter region may be indicative and thus may be used to assess the activity of a cell signaling pathway and/or an endogenous or exogenous (e.g., introduced) transcription factor.

In other embodiments, a method of assessing promoter region activity is disclosed, wherein the method comprises culturing a cell comprising a nucleic acid comprising a region encoding a first β -galactosidase fragment fused to a carrier protein, wherein said first β -galactosidase fragment is operably coupled to a promoter region, under conditions wherein a first β -galactosidase fragment-carrier protein fusion is expressed when said promoter region is active. In certain embodiments, the method further comprises contacting the first β -galactosidase fragment with a second β -galactosidase fragment, if expressed, to form an active enzyme complex, and detecting the level of said enzyme activity to assess the activity of said promoter region. In many embodiments, the activity of the promoter region can be indicative and, thus, can be used to assess the activity of a cell signaling pathway and/or an endogenous or exogenous (e.g., introduced) transcription factor. In many other embodiments, the carrier protein comprises a domain selected to affect the stability of the ED-carrier protein fusion, wherein the domain selected increases the stability of the ED-carrier protein fusion as compared to an ED-carrier protein fusion lacking the domain, or the domain selected destabilizes the ED-carrier protein as compared to an ED-carrier protein lacking the domain. In addition, the carrier protein domain targets the ED-carrier protein fusion for proteasomal degradation. The domains comprise a proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) degradation signal or a CL1 degradation signal.

In other embodiments, a method of assessing promoter region activity is disclosed, wherein the method comprises culturing a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) fragment fused to a carrier protein, wherein the ED fragment is operably coupled to a promoter region, under conditions wherein the ED is expressed when the promoter region is active. In certain embodiments, the methods further comprise contacting ED with an enzyme receptor (EA) fragment, if expressed, to form an ED-EA complex having enzymatic activity, and detecting the level of enzymatic activity to assess the activity of the promoter region. In many other embodiments, the carrier protein comprises a domain selected to affect the stability of the ED-carrier protein fusion, wherein the domain selected increases the stability of the ED-carrier protein fusion as compared to an ED-carrier protein fusion lacking the domain, or the domain selected destabilizes the ED-carrier protein as compared to an ED-carrier protein lacking the domain.

In many embodiments, the carrier protein may have a detectable enzymatic activity that is different from the enzymatic activity of the β -galactosidase enzyme, wherein when the carrier protein is expressed as described in the disclosed methods, the enzymatic activity of the carrier protein can be detected using known enzymatic activity detection methods. In many other embodiments, the carrier protein is expressed under conditions in which the promoter region is active, such that the detection method can be applied to detect the enzymatic activity of the carrier protein to assess the activity of the promoter region. In other embodiments, the expression of the carrier protein is co-expressed with the ED fragment when the promoter region is active, wherein the ED fragment forms a complex with the EA fragment to form an active enzyme complex with enzymatic activity, and the level of enzymatic activity of both carrier proteins and the β -galactosidase complex is detected to assess the activity of the promoter region. In other embodiments, the activity of the promoter region may be indicative and thus may be used to assess the activity of a cell signaling pathway and/or an endogenous or exogenous (e.g., introduced) transcription factor.

In various embodiments, the carrier protein may be a natural protein, a mutant protein, a synthetic protein, wherein the carrier protease activity is detected by well-known detection methods for such protein carriers. In other embodiments, the carrier protein is mutated such that the mutation renders the carrier protein inactive for enzymatic activity. In a still further embodiment, the mutant carrier protein may function solely as a carrier protein fused to a β -galactosidase fragment that is operably linked to the target promoter region but which does not exhibit any detectable enzymatic activity when the target promoter region is active.

In one embodiment, the invention provides a method of assessing the activity of a promoter region, wherein the method comprises culturing a cell comprising a nucleic acid comprising a region encoding a carrier protein fused to ED, wherein the ED-carrier protein fusion is operably coupled to the promoter region, under conditions wherein the ED fragment is expressed with expression of the carrier protein when the promoter region is active. In another embodiment, the method further comprises contacting the ED fragment with the EA fragment to form an active enzyme complex having enzymatic activity, and detecting the level of enzymatic activity to assess the activity of the promoter region. In yet another embodiment, the method detects the non- β -galactosidase enzyme activity of the carrier protein and the β -galactosidase enzyme activity of the ED-EA enzyme complex, such that the result is a two-point detection method that gives the enzyme activity of the carrier protein and the enzyme activity of the ED-EA fragment complex.

In many embodiments, the invention provides a method of assessing the activity of a promoter region, wherein the method comprises culturing a cell comprising a nucleic acid comprising a region encoding a mutant carrier protein fused to an ED fragment, wherein the ED-mutant carrier protein fusion is operably coupled to the promoter region, wherein the mutant carrier protein lacks detectable enzymatic activity. The cells are cultured under conditions in which the ED fragment is expressed when the promoter region is active. In certain embodiments, the methods further comprise contacting ED with EA if expressed to form an active enzyme complex having enzymatic activity, and detecting the level of enzymatic activity to assess the activity of the promoter region.

In many embodiments, the invention provides a method of assessing promoter region activity, wherein the method comprises culturing a cell comprising a nucleic acid comprising a region encoding a carrier protein without any intrinsic enzymatic activity fused to an ED fragment, wherein the ED-carrier protein fusion is operably coupled to the promoter region, wherein the carrier protein lacks detectable enzymatic activity. The cells are cultured under conditions in which the ED fragment is expressed when the promoter region is active. In certain embodiments, the methods further comprise contacting ED with EA if expressed to form an active enzyme complex having enzymatic activity, and detecting the level of enzymatic activity to assess the activity of the promoter region.

In many embodiments, the present invention provides a method for assessing promoter region activity. In other embodiments, a method of assessing promoter region activity is disclosed comprising culturing a cell comprising a nucleic acid comprising a region encoding a first β -galactosidase fragment operably coupled to a promoter region, introducing a detection reagent into the culture, wherein the first β -galactosidase fragment is expressed when the promoter region is active. In other embodiments, the method further comprises contacting the first β -galactosidase fragment with the second β -galactosidase fragment if expressed to form an active enzyme complex having enzymatic activity, and detecting the level of enzymatic activity to assess the activity of the promoter region. In another embodiment, the activity of the promoter region may be indicative and thus may be used to assess the activity of a cell signaling pathway and/or an endogenous or exogenous (e.g., introduced) transcription factor.

In many embodiments, the method further comprises contacting the cell with one or more detection reagents and detecting the effect of the detection reagents by assessing the activity of the promoter region in response to the detection reagents. In many other embodiments, the method further comprises contacting the cell with a first agent, wherein the cell is first contacted with a first agent that affects the activity of the promoter region of interest, and wherein the activity is assessed by detecting the enzymatic activity of the ED-EA complex; contacting the cell with a second agent, wherein the second agent affects the activity of the first agent, said activity being assessed by detecting the enzymatic activity of the ED-EA complex and comparing it to the activity of the promoter region when the cell is contacted with the first agent only. In various embodiments, the first agent can be an agonist and the second agent can be an antagonist.

In various embodiments, the cell may be contacted with more than one agent. One or more of the disclosed agents can be introduced into a cell sequentially, such as introducing a first agent and introducing a second agent, or in combination, such as introducing more than one agent into a cell culture at the same time, to determine the effect of different detection agents on the activity of a promoter of interest. The agent can be a detection agent, a small molecule, an agonist, an antagonist, a biologic, an approved drug, a research drug, a peptide, a protein, an antibody, a cell expressing a heterologous protein, a cell expressing an endogenous protein, a product secreted by a cell, a toxin, a natural product, a promoter, an inhibitor, or an inverse agonist.

In a certain embodiment, the promoter region comprises at least one Transcription Factor Response Element (TFRE) for a transcription factor of interest, wherein the activity of the promoter region is indicative of the activity of the transcription factor. In certain further embodiments, the level of activation of the transcription factor is assessed based on the detected level of enzymatic activity of the first β -galactosidase fragment expressed when the promoter region is active.

In certain embodiments, the promoter region comprises a first TFRE and a second TFRE. In certain other embodiments, the promoter region comprises a first TFRE and a second TFRE, wherein the first TFRE and the second TFRE are TFREs for the same transcription factor. In still further embodiments, the promoter region comprises a first TFRE and a second TFRE, wherein the first TFRE and the second TFRE are TFREs for different transcription factors.

In various embodiments, the promoter region comprises at least one TFRE. Furthermore, according to many embodiments, the promoter region comprises two or more TFREs. The TFREs contained in the promoter region may be the same TFRE such that the TFREs are for the same transcription factor, or may be different TFREs such that different TFREs in the promoter region are for different transcription factors.

In a certain embodiment, the promoter region comprises an endogenous promoter region for a gene of interest. In certain further embodiments, the level of activation of one or more pathways that activate the promoter region is assessed based on the detected level of enzymatic activity of the first β -galactosidase fragment expressed when the promoter region is active.

In other embodiments, the disclosed methods comprise introducing an expression vector encoding a transcription factor into a cell and culturing the cell under conditions in which the transcription factor is expressed, wherein the promoter region is coupled to the first β -galactosidase fragment. In certain further embodiments, the level of activation of the transcription factor encoded by the expression vector is assessed based on the detected level of enzymatic activity of said first β -galactosidase fragment expressed when said promoter region is active.

In other embodiments, the activity of the promoter region is indicative of the activity of a target cell signaling pathway, wherein the level of activity of the cell signaling pathway is assessed based on the level of enzyme activity detected. In many other embodiments, the activity of the transcription factor of interest is indicative of the activity of a cell signaling pathway of interest, wherein the level of activity of the cell signaling pathway is assessed based on the level of enzyme activity detected.

In many embodiments, the invention provides a method of assessing promoter region activity comprising contacting a cell with an agent (e.g., a detection agent), and assessing the level of activity of the promoter region in response to contacting the cell with the agent based on the level of enzyme activity of the carrier protein detected. In further embodiments, the invention provides a method of assessing the level of promoter region activity comprising basing a cell on an agent (e.g., a detection agent); and assessing the level of activity of the promoter region in response to contacting the cell with the agent based on the detected level of enzyme activity of the ED-EA complex and comparing the level of enzyme activity detected in the presence of the detection agent to the level of enzyme activity of the ED-EA complex in the absence of the detection agent.

In other embodiments, the invention provides a method of assessing promoter region activity comprising culturing a cell comprising a nucleic acid comprising a region encoding a first β -galactosidase fragment operably coupled to a promoter region, under conditions wherein said first β -galactosidase fragment is expressed when said promoter region is active; contacting the cell with a reagent (e.g., a detection reagent); contacting the first β -galactosidase fragment with a second β -galactosidase fragment if expressed to form an active enzyme complex having an enzymatic activity, and detecting the level of said enzymatic activity to assess the activity of said promoter region in response to contacting said cell with said agent, and comparing the level of enzymatic activity detected in the presence of said detection agent to the level of enzymatic activity in the absence of detection agent or a control condition.

In a still further embodiment, the invention provides a method of assessing promoter region activity comprising culturing a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) fragment operably linked to a promoter region, under conditions wherein the ED fragment is expressed when the promoter region is active; contacting the cell with a reagent (e.g., a detection reagent); contacting said ED with an EA fragment if expressed to form an active enzyme complex having enzymatic activity, and detecting the level of enzymatic activity of the ED-EA complex to assess the activity of said promoter region in response to contacting said cell with said agent, and comparing the level of enzymatic activity detected in the presence of said agent to the level of enzymatic activity in the absence of said agent.

Thus, in many embodiments, the methods disclose nucleic acids encoding a carrier protein fused to the ED, such that the ED-carrier protein fusion is expressed when the promoter protein is active. In one embodiment, the carrier protein exhibits an enzymatic activity that is different from the enzymatic activity of the ED-EA complex. In another embodiment, the carrier protein is a mutein that exhibits no detectable enzymatic activity when compared to the wild-type carrier protein. In yet another embodiment, a carrier protein lacking any intrinsic enzymatic activity is fused to the ED fragment, such that the ED-carrier protein fusion is expressed when the promoter fragment is active.

In further embodiments, the agent is a small molecule, protein, peptide, antibody, cell surface protein, detection agent, cell, product from a cell (e.g., a product secreted by a cell), agonist, inverse agonist, partial agonist, or antagonist. In many more embodiments, the cell surface protein present on the cell does not comprise a nucleic acid comprising a region encoding the ED fragment of β -galactosidase. In other embodiments, the cell surface protein present on the cell comprises a nucleic acid comprising a region encoding the ED fragment of β -galactosidase.

In many embodiments, the method further comprises detecting a level of enzymatic activity, which comprises providing a substrate for the ED-EA complex, wherein a detectable signal is generated upon hydrolysis of the substrate by the ED-EA complex. In many other embodiments, the detectable signal is a chemiluminescent signal or a biochemical luminescent signal. In other embodiments, the ED and EA are β -galactosidase fragments, wherein said ED fragments comprise the sequence set forth in SEQ ID No.30 or a variant thereof, which complex with EA to form an ED-EA complex having glycosidase hydrolase activity.

In other embodiments, the cell is a mammalian cell, rodent cell, human cell, immune cell, T cell, Jurkat cell, cancer cell, carcinoma cell, HepG2 cell, sarcoma cell, or other well-known cell types.

In various embodiments, the nucleic acid is a plasmid, a cellular chromosome, a nuclear chromosome, a mitochondrial chromosome. In other embodiments, the nucleic acid further encodes a carrier protein fused to the ED, such that the ED-carrier protein fusion is expressed when the promoter region is active.

In further embodiments, the carrier protein having detectable enzymatic activity can be a luciferase, a modified luciferase, a beta-lactamase, an alkaline phosphatase, a peroxidase, a fluorescent protein, or other carrier protein having detectable activity.

In many embodiments, the invention provides assays for studying agonists, antagonists, activators, and inhibitors for various pathways and promoter regions.

In still further embodiments, the invention also discloses cells, compositions, and kits useful, for example, in practicing the methods of the disclosure.

Other features will be apparent from the accompanying drawings and from the detailed description that follows.

Drawings

Example embodiments are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which like reference numerals refer to similar elements and in which:

FIG. 1: plasmid map of activated T cell Nuclear Factor (NFAT) Enzyme Fragment Complementation (EFC) reporter construct.

FIG. 2: the NFAT EFC reporter construct is expressed in U2OS NFAT EFC reporter cells in response to a cell stimulating mixture of phorbol 12-myristate 13-acetate (PMA) and ionomycin.

FIG. 3: plasmid map of NF-kB EFC reporter construct.

FIG. 4: single cell clones of U2OS NF-kB EFC reporter cells responded to the cytokine TNF α.

FIG. 5: inhibitors were screened using an assay based on U2OS NFkB EFC reporter cells.

FIG. 6: two assays were tested using the NFkB EFC reporter Gene assayTNF α activity and potency of the batches.

FIG. 7: the NFkB EFC reporter detects a strong response of endogenous CD40 receptors in cells to CD40 ligand (CD 40L).

FIG. 8: in a co-culture assay, Jurkat NFAT EFC reporter cells responded to OKT3 ligand expressed and presented on the surface of CHO-K1 cells.

FIG. 9: plasmid map of the IL 2-promoter-EFC reporter construct.

FIG. 10: the Jurkat IL 2-promoter EFC reporter cell line detects stimulation of a variety of different response elements activated by different signaling pathways.

FIG. 11: response of an IL 2-promoter EFC reporter construct (comprising a complex native promoter with multiple different response elements) to intracellular mimetics of two different signaling pathways.

FIG. 12: ROR γ T transcription factor activity was reduced by the inverse agonist GSK805 in U2OS cells expressing ROR γ T transcription factor and ROR γ T EFC reporter plasmids.

FIG. 13: the EFC-based NF-. kappa.B transcriptional reporter assay showed better sensitivity to the CD40L/CD40 receptor than the luciferase system.

FIG. 14: EFC-based NF κ B transcriptional reporter assays showed better sensitivity to TNF α than luciferase systems.

FIG. 15: an assay result of an NF κ B pathway reporter cell line according to one embodiment of the disclosure. RLU is relative light unit.

FIG. 16: an assay result of an NFAT pathway reporter cell line according to one embodiment of the present disclosure. RLU is relative light unit.

FIG. 17: an assay result for a STAT3 pathway reporter cell line according to one embodiment of the present disclosure. RLU is relative light unit.

FIG. 18: an assay result of an NFAT pathway reporter cell line according to one embodiment of the present disclosure. RLU is relative light unit.

FIG. 19: the assay results of the PD1 pathway reporter assay according to one embodiment of the present disclosure, which indicate that further modifications to the pathway reporter assay can be made to generate assays for other targets. RLU is relative light unit.

FIG. 20: an assay result of an NF- κ B pathway reporter assay using a carrier protein coupled to a promoter according to one embodiment of the disclosure.

FIG. 21: assay results of the U2OS RANK NF- κ B pathway reporter gene assay according to one embodiment of the disclosure.

FIG. 22: assay results of a HEK NF- κ B pathway reporter gene assay according to one embodiment of the disclosure.

FIG. 23: assay results of a HEK CD27-NF- κ B pathway reporter assay according to one embodiment of the disclosure.

Fig. 24a and 24 b: according to one embodiment of the present disclosure, the results of the assay were directed to U2OS NF-. kappa.B reporter cell line and U2OS RANK-NF-. kappa.B reporter cell line, respectively.

Other features of the present embodiments will be apparent from the accompanying drawings and from the detailed description that follows.

Detailed Description

Methods of assessing promoter region activity are provided. The method comprises culturing a cell comprising a nucleic acid comprising a region encoding a first β -galactosidase fragment operably coupled to a promoter region of interest, which is cultured under conditions wherein said first β -galactosidase fragment is expressed when said promoter region is active, wherein said promoter region can become active in response to said cell culture conditions. The method further comprises contacting the first β -galactosidase fragment with a second β -galactosidase fragment if expressed to form an active enzyme complex; detecting the level of enzyme activity provides an assessment of the activity of the promoter region. The activity of the promoter region may be indicative, and thus may be used to assess the activity of a target cell signaling pathway and/or an endogenous or exogenous (e.g., introduced) transcription factor.

Further, the method comprises culturing a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) operably coupled to a target promoter region, under conditions wherein the ED is expressed when the promoter region is active. The method further comprises contacting the ED with an enzyme receptor (EA) if expressed to form an ED-EA complex having enzymatic activity. The method further comprises detecting the level of the enzyme activity to assess the activity of the promoter region. The activity of the promoter region may be indicative and thus may be used to assess the activity of a cell signaling pathway and/or an endogenous or exogenous (e.g., introduced) transcription factor. Also provided are methods comprising contacting the cell with an agent (e.g., a detection agent), and assessing the level of activity of the promoter region in response to contacting the cell with the agent based on the level of detected enzyme activity. Also provided are cells, compositions, and kits useful, for example, in practicing the methods of the disclosure.

The invention also provides a method of assessing promoter region activity in a cell, wherein the cell comprises a nucleic acid comprising a carrier protein fused to an Enzyme Donor (ED) fragment, wherein the carrier protein-ED fusion is operably coupled to a target promoter region. The method further comprises culturing the cell under conditions wherein the ED is expressed when the promoter region is active; if expressed, the ED is contacted with an enzyme receptor (EA) to form an ED-EA complex having enzymatic activity, and the level of the enzymatic activity is measured to assess the activity of a promoter region of interest, wherein the activity of the promoter region is indicative of the activity of a transcription factor of interest and/or a cellular signaling pathway of interest.

Before the methods, cells, compositions, and kits of the present disclosure are described in greater detail, it is to be understood that the methods, cells, compositions, and kits are not limited to the specific embodiments described, as such may, of course, vary. 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 be limiting, since the scope of the methods, cells, compositions, and kits will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that range is encompassed within the method, cell, composition and kit. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the methods, cells, compositions, and kits, subject to any specific exclusion within the stated range. When the range includes one or both of the limits, ranges excluding one or both of the limits are also included in the methods, cells, compositions, and kits.

Certain ranges are presented herein, and numerical values are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number preceding it, as well as numbers that are near or approximate to the number preceding the term. In determining whether a number is near or approximate to a specifically recited number, the near or approximate non-recited number may be a number that provides substantial equivalents of the specifically recited number in the context in which it is presented.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which methods, cells, compositions, and kits belong. Representative illustrative methods, cells, compositions, and kits are now described, although any methods, cells, compositions, and kits similar or equivalent to those described herein can also be used in the practice or detection of the methods, cells, compositions, and kits.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference to disclose and describe the materials and/or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods, cells, compositions and kits are not entitled to antedate such publication because the publication is not entitled to antedate such publication by virtue of its inclusion in a publication which may differ from the actual publication date and requires independent identification.

It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for "mere," "only," etc. terminology in connection with the recitation of claim elements or the use of a "negative" limitation.

It is to be understood that certain features of the methods, cells, compositions and kits described in the context of separate embodiments may also be provided in combination in a single embodiment for clarity. Conversely, various features of the methods, cells, compositions, and kits that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments are specifically embraced by the present disclosure and disclosed herein to the extent that such combinations are embraced by operable processes and/or compositions, as if each and every combination were individually and explicitly disclosed. Moreover, all sub-combinations listed in the embodiments describing these variables are also specifically encompassed by the present methods, cells, compositions and kits and are disclosed herein as if each such sub-combination were individually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method may proceed in the order of the recited events or in any other order that is logically possible.

Method

As summarized above, the present disclosure provides methods of assessing promoter region activity. The method comprises culturing a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) operably coupled to a target promoter region, under conditions wherein the ED is expressed when the promoter region is active. The method further comprises contacting the ED with an enzyme receptor (EA) if expressed to form an ED-EA complex having enzymatic activity. The method further comprises detecting the level of enzyme activity to assess the activity of the promoter region of interest. The activity of the promoter region may be indicative of the activity of a transcription factor of interest and/or a cell signaling pathway of interest. Thus, the method may further comprise assessing the activity of the transcription factor of interest and/or the cellular signaling pathway of interest based on the detected level of enzyme activity.

Methods for assessing promoter region activity can be used in a variety of situations. For example, the methods can be used to determine the level of activity of a promoter region when the cell is in a target condition. Conditions of interest include, but are not limited to, pH, temperature, genetic conditions of the cell (e.g., one or more mutations (e.g., point mutations, deletions, insertions, and/or the like) in one or more chromosomes of the cell), conditions in which the cell is contacted with a reagent (e.g., a detection reagent), and the like. In some embodiments, the method of assessing promoter region activity comprises contacting the cell with an agent (e.g., a detection agent) during culturing, and assessing the activity of the promoter region (and optionally, transcriptional activity of a gene of interest and/or activity of a cell signaling pathway of interest) in response to contacting the cell with the agent based on the level of enzyme activity detected. Such methods can be used, for example, to determine whether the agent affects the level of activity of the promoter region (and optionally, the transcriptional activity of the gene of interest and/or the activity of the cell signaling pathway of interest). The method of assessing promoter region activity may also be used to assess the effect of an agonist, antagonist, detection agent, transcription factor, cell signaling pathway activator, or cell signaling pathway inhibitor.

Also provided are methods of assessing whether a detection agent affects the level of activity of a target cell signaling pathway. Such methods comprise culturing a cell in the presence of a detection agent, wherein the cell comprises a nucleic acid comprising a region encoding the ED operably coupled to a promoter region, under conditions in which the ED is expressed when the promoter region is active, wherein the activity of the promoter region is indicative of the level of activity of a cell signaling pathway of interest. The method further comprises contacting the ED with EA, if expressed, to form an ED-EA complex having enzymatic activity, and detecting the level of the enzymatic activity to assess whether the detection agent affects the level of activity of a target cell signaling pathway.

The methods are based in part on the unexpected discovery that Enzyme Fragment Complementation (EFC) based reporter assays/systems of the present disclosure exhibit increased sensitivity compared to existing reporter systems that rely on expression of: 1) full-length (single polypeptide) enzymes, such as full-length luciferase, beta-galactosidase, Chloramphenicol Acetyl Transferase (CAT); and 2) a fluorescent protein. In addition, the methods further demonstrate that the EFC-based reporter assays of the present disclosure observe increased sensitivity to ligand stimulation compared to corresponding luciferase-based assays that rely on expression of full-length (single polypeptide) luciferase, e.g., as demonstrated in the experimental section below. Thus, the assays of the present disclosure constitute an improvement over existing reporter assays, e.g., in terms of the ability of an agent (e.g., a detection agent) to perform more efficiently in the assay, which, in some embodiments, results in an assay that is more physiologically relevant, that is, more accurately reflects that the effect of the agent (e.g., a detection agent) on cells in a natural environment is more similar to the in vivo environment.

In some embodiments, the sensitivity of the method is in terms of half maximal Effective Concentration (EC)₅₀) The values represent, in the context of the present disclosure, the concentration of an agent (e.g., a detection agent) that induces a response (as indicated by the level of enzyme activity) in the cell between the baseline and the maximum after exposure of the cell to the agent for a particular exposure time. According to some embodiments, the methods of the present disclosure (e.g., a method of assessing the effect of a detection agent on a target promoter region or cell signaling pathway) exhibit an EC of 100 μ g/mL or less, 10 μ g/mL or less, 1 μ g/mL or less, 100ng/mL or less, 10ng/mL or less, 1ng/mL or less, 100pg/mL or less, or 10pg/mL or less₅₀The value is obtained. According to some embodiments, the methods of the present disclosure (e.g., one method of assessing the effect of a detection agent on a target promoter region or cell signaling pathway) exhibit an EC of 10 μ Μ or less, 1 μ Μ or less, 100nM or less, 10nM or less, 1nM or less, 100pM or less, 10pM or less, or 1pM or less₅₀The value is obtained.

According to some embodiments, the methods of the present disclosure exhibit greater efficacy compared to existing reporter systems that rely on expression of: 1) full-length (single polypeptide) enzymes, such as full-length luciferase, beta-galactosidase, Chloramphenicol Acetyl Transferase (CAT); and/or 2) a fluorescent protein. As mentioned above, EC₅₀A smaller value of (a) indicates a greater efficacy. In certain embodiments, the methods of the present disclosure are apparentPotency is shown to be 2:1 or greater, 5:1 or greater, 10:1 or greater, 15:1 or greater, 20:1 or greater, 25:1 or greater, 30:1 or greater, 35:1 or greater, 40:1 or greater, 45:1 or greater, or 50:1 or greater.

As used herein, a "promoter region" is a nucleic acid (e.g., DNA) region that comprises at least one element (e.g., a nucleotide sequence, such as a Transcription Factor Response Element (TFRE)) known to regulate transcription. For example, the promoter region may comprise at least one element known to be bound by the DNA-binding domain of a transcription factor. In certain embodiments, the at least one element is known to regulate the expression of one or more genes, depending on whether an activated transcription factor binds to the element. In this manner, the combination of the promoter region with the ED-EA reporter gene system can interrogate the level of activity of the promoter region, which in turn helps identify conditions that affect the expression of one or more genes known to be regulated by at least one element in the promoter region. It will be appreciated that the level of activity of the promoter region may be indicative of the level of activity of the transcription factor of interest and/or the cellular signaling pathway of interest (e.g., transcriptional up-regulation and/or down-regulation of one or more genes may be a downstream consequence of the signaling pathway of interest, e.g., the signaling pathway may modulate the activity of the transcription factor by post-translationally modifying it by phosphorylation, acetylation, ubiquitination and/or other covalent modifications), such that the promoter region and the ED-EA reporter system are able to interrogate the level of activity of the transcription factor of interest and/or the cellular signaling pathway of interest, which in turn facilitates identification of conditions that affect the level of activity of the transcription factor of interest and/or the cellular signaling pathway of interest. According to some embodiments, for example, the conditions comprise contacting the cell with a reagent (e.g., a detection reagent).

Another way in which a signal transduction pathway can regulate transcription factor activity is to regulate the concentration of active transcription factors at promoter sites associated with ED expression by altering their synthesis, degradation, and/or their subcellular localization, all of which may affect the ability of the transcription factors to regulate the promoter region.

Transcription factors of interest include, but are not limited to: endogenous transcription factors (i.e., transcription factors expressed by a cell from a native/non-introduced nucleic acid of the cell (e.g., a wild-type chromosome of the cell)); heterologous, transfected native transcription factors (i.e., wild-type forms of transcription factors that are not expressed by the cell); heterologous, transfected recombinant chimeric transcription factors (i.e., transcription factors comprising two or more heterologous domains, e.g., an activation domain of a transcription factor of interest fused to a heterologous DNA binding domain (e.g., GAL4 DNA binding domain) for binding to a universal TFRE (e.g., GAL4/UAS) of a nucleic acid); a heterologously transfected constitutively active transcription factor; or any combination of two or more such transcription factors. According to any such embodiment, the transcription factor may be activated or inactivated by endogenous or engineered cell signaling pathways, and may reflect its activity.

In certain embodiments, one or more proteins of a cellular pathway or signaling pathway may be genetically altered (e.g., overexpressed, knocked-down, or knocked-out) in order to better study the pathway, answer specific mechanistic questions, or serve as a positive or negative experimental control. In certain embodiments, a genetically altered cellular pathway or signaling pathway may be constitutively active or inactive as desired for the intended experimental purpose.

As described above, the methods of the present disclosure may include assessing the activity of a transcription factor of interest and/or a cell signaling pathway of interest, wherein the level of activity of the promoter region (and the corresponding level of expression of ED) provides a reading of the level of activity of the transcription factor of interest and/or the cell signaling pathway of interest. In some embodiments, the method comprises assessing the level of activity of a transcription factor of interest and/or a cellular signaling pathway of interest in response to the cell and an agent (e.g., a control agent, a detection agent, etc.) based on the level of enzyme activity detected. "detection agent" refers to an agent (small molecule, peptide, polypeptide, nucleic acid, etc.) for which it is unknown whether contact of a cell with the agent will alter the level of activity of a transcription factor of interest and/or a signal transduction pathway of a cell of interest prior to contact of the cell with the agent. The detection agent may further refer to, but is not limited to, a small molecule, agonist, antagonist, biologic, approved drug, research drug, peptide, protein, antibody, cell expressing a heterologous protein, cell expressing an endogenous protein, product secreted by a cell, toxin, natural product, promoter, inhibitor, or inverse agonist.

The detection reagent used according to the methods of the present disclosure may be cell impermeable (e.g., to query whether the detection reagent alters the level of activity of the target transcription factor and/or the target cell signaling pathway by interacting with (e.g., binding to) a molecule on the surface of the cell) or cell permeable (e.g., to query whether the detection reagent alters the level of activity of the cell signaling pathway by interacting with (e.g., binding to) a molecule on the surface of the cell or a molecule within the cell or a compartment thereof, e.g., a molecule within the cytoplasm, a molecule on the surface of an organelle, a molecule within an organelle, etc.).

As used herein, a "cell signaling pathway" comprises a molecule of a cell or a series of molecules of a cell (e.g., one or more cell surface molecules and/or one or more intracellular molecules) that is responsive to an external signal such that the external signal results in up-regulation of expression of one or more genes and/or down-regulation of expression of one or more genes. The up-regulation and/or down-regulation of the expression of one or more genes corresponds to an increase and/or decrease in the level of activity of the promoter of the one or more genes. Thus, the level of activity of a cellular signaling pathway can be assessed based on the level of activity of a promoter region (or a sub-region thereof) that is a downstream target (positive or negative) for signaling through the cellular signaling pathway.

It will be appreciated that a particular signal transduction pathway may be named/characterized according to the molecules present in the signal transduction pathway. For example, signal transduction pathways may be named according to receptors (e.g., cell surface receptors, cytoplasmic receptors, etc.) that initiate signal transduction upon binding to an external signal. As another example, a particular signal transduction pathway may be named/characterized based on a "downstream" molecule of an external signal receptor and an "upstream" molecule of a transcription factor in the signal pathway. As another example, a particular signaling pathway may be named/characterized according to transcription factors in the signaling pathway (e.g., nfkb, NFAT, STAT3, etc.).

In many embodiments, cell signaling pathways whose level of activity can be assessed (e.g., to determine whether a test compound affects the level of activity of a signaling pathway) include, but are not limited to, the Akt signaling pathway, the AMP-activated protein kinase (AMPK) signaling pathway, the apoptotic signaling pathway, the Epidermal Growth Factor Receptor (EGFR) signaling pathway, the estrogen signaling pathway, the Fibroblast Growth Factor Receptor (FGFR) signaling pathway, the growth factor receptor signaling pathway, the insulin signaling pathway, the JAK-STAT signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, the rapamycin Mechanical target (mTOR) signaling pathway, the NF- κ B signaling pathway, the Notch signaling pathway, the activated T cell Nuclear Factor (NFAT) signaling pathway, the p53 signaling pathway, the like, Transforming growth factor beta (TGF- β) signaling pathway, Toll-like receptor (TLR) signaling pathway, Vascular Endothelial Growth Factor (VEGF) signaling pathway, and Wnt signaling pathway. According to some embodiments, the cell signaling pathway is the nfkb signaling pathway. In certain embodiments, the cell signaling pathway is a STAT (e.g., STAT3 and/or STAT5) signaling pathway. According to some embodiments, the cell signaling pathway is an NFAT signaling pathway.

Reagents (e.g., detection reagents) with which the cells can be contacted include, but are not limited to, small molecules, polypeptides (including peptides), nucleic acids, and the like. In some embodiments, the agent is an agonist, an inverse agonist (i.e., an agent that binds to the same molecule (e.g., receptor) as an agonist but has an opposite effect as an agonist), a partial agonist (i.e., an agent that binds to the same molecule (e.g., receptor) as an agonist but has the same effect as an agonist but to a lesser extent) or an antagonist (i.e., an agent that binds to the same molecule (e.g., receptor) as an agonist and prevents the binding of the agonist to the molecule, e.g., does not affect the activity of the molecule). "Small molecule" refers to a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 750amu or less, 500amu or less, 400amu or less, 300amu or less, or 200amu or less. In certain aspects, the small molecule is not composed of repeating molecular units as present in a polymer.

The terms "polypeptide", "peptide" or "protein" are used interchangeably herein to refer to a linear series of amino acid residues interconnected by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The amino acids may include 20 "standard" genetically encodable amino acids, amino acid analogs, or combinations thereof. In some embodiments, when the detection reagent is a protein, the protein is a soluble protein, e.g., is not associated with (bound to or is part of) a cell. In other embodiments, the detection reagent may be an insoluble protein. Examples of insoluble proteins of interest include, but are not limited to, cell surface proteins. Thus, in some embodiments, the method may comprise exposing the cell to a second cell, and after contacting the cell with a cell surface protein, assessing whether the protein on the surface of the second cell affects the level of activity of a target cell signaling pathway in the cell. In some embodiments, the cell can be co-cultured with the second cell such that the cell is contacted with a cell surface protein of the second cell. According to some embodiments, the cell surface protein (e.g., cell surface ligand) of the second cell may be isolated and purified from the second cell and contacted with the cells (responder cells) in soluble, soluble and cross-linked form, or when coated onto a solid support (e.g., bead or tissue culture plate surface).

In some embodiments, when the agent is a nucleic acid, the agent is an oligonucleotide. As used herein, an "oligonucleotide" is a single-stranded polymer of nucleotides of 2 to 500 nucleotides (e.g., 2 to 200 nucleotides). Oligonucleotides may be synthetic, or may be enzymatically prepared, and in some embodiments are 5 to 50 nucleotides in length (e.g., 9 to 50 nucleotides in length). An oligonucleotide may comprise a ribonucleotide monomer (i.e., may be an oligoribonucleotide or "RNA oligoribonucleotide") or a deoxyribonucleotide monomer (i.e., may be an oligodeoxyribonucleotide or "DNA oligonucleotide"). For example, the oligonucleotide may be 5 to 9, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, or 150 to 200, up to 500 or more nucleotides in length. In some embodiments, when the agent is a nucleic acid, the agent is short interfering rna (sirna), microrna (mirna), morpholine, and/or the like. Methods of designing and delivering siRNA, miRNA, morpholine, etc. for targeting specific mrnas are well known and described, for example, in monsori et al, (2014) Adv Pharm bull.4(4): 313-; xin et al, (2017) Mol Cancer 16: 134; chakraborty et al, (2017) Mol Ther Nucleic Acids 8: 132-143; and Ahmadzada et al, (2018) Biophys Rev.10(1): 69-86. sirnas, mirnas, morpholines, etc. can be designed based on the known needs of the mRNA to be targeted and using available Tools, e.g., siRNA Wizard from invitogen, siDESIGN Center from Dharmacon, BLOCK-iTTMRNAi Designer from Invitrogen, miR-Synth available at MicroRNA.

In some embodiments, the cell is contacted with an agent (e.g., a detection agent), wherein the agent is part of a library of agents, e.g., a library of small molecules, a library of polypeptides, a library of sirnas, and the like. Such methods can further include performing the method in high throughput, wherein the cells are provided into wells of a tissue culture plate (e.g., 4, 6, 8, 12, 24, 48, 96, 384, 1536 well tissue culture plates, etc.), the cells are contacted with one or a set of detection reagents from a library of detection reagents (e.g., a small molecule library, a polypeptide library, a siRNA library, etc.), and the method includes identifying an agent that affects a level of activity of a signal transduction pathway of interest based on the level of enzyme activity detected.

According to some embodiments, the promoter region comprises a Transcription Factor Response Element (TFRE) of a transcription factor of interest, and the activity of the promoter region is indicative of the activity of the transcription factor. In some embodiments, the method comprises assessing the level of activation of the transcription factor based on the detected level of enzyme activity. In certain embodiments, a naturally-occurring transcription factor of interest may be expressed or overexpressed in a cell (e.g., if absent or present at a level below the determined ideal level). In certain embodiments, the transcription factor is a universal transcription factor, which is a mutant transcription factor that is constitutively active or inactive. In certain embodiments, the transcription factor may be knocked-down or knocked-out. In certain embodiments, the transcription factor is a chimeric transcription factor comprising an activation domain of a transcription factor of interest fused to a heterologous nucleic acid binding domain that binds to a TFRE. The TFRE may be one to which the DNA binding domain of the target wild-type transcription factor binds (e.g., wild-type STAT3 binds the TFRE in a method comprising assessing the level of STAT3 activity). In some embodiments, the TFRE is a "universal" TFRE, meaning that the TFRE is a TFRE in a reporter assay system that can be used to assess the level of activity of various transcription factors that are naturally associated with different TFREs. For example, in some embodiments, the cell expresses a chimeric transcription factor comprising a transcription factor activation domain of interest fused to a heterologous nucleic acid binding domain that binds to a universal TFRE. In this manner, the same nucleic acid can be used in an EFC reporter assay to assess the level of transcription factor activity that does not bind to the same TFRE in nature. A non-limiting example of a universal TFRE that can be used is GAL 4/upstream activation sequence (GAL4/UAS), in which the activation domain of a target transcription factor (e.g., NF κ B, STAT3, NFAT, ELK1, etc.) is fused to a GAL4 DNA binding domain, which enables assessment of activation of the target transcription factor without the need for a native TFRE for the target transcription factor. In some embodiments, the method comprises introducing into the cell an expression vector encoding the transcription factor, and culturing the cell under conditions in which the transcription factor is expressed.

In certain embodiments, the promoter region comprises a single Transcription Factor Response Element (TFRE). The single TFRE may be a TFRE that binds to and responds to activation of a single transcription factor (e.g., class a, which is an example of an NF-kB responsive element), as exemplified in some examples of the experimental section below. Such embodiments may be used, for example, to isolate a particular target TFRE, to determine conditions that affect the activity of that TFRE in isolation (i.e., without interference from other TFREs), and/or to determine conditions that affect activation or inactivation of transcription factors that bind to and respond to the TFRE. In certain other embodiments, the promoter region comprises more than one Transcription Factor Response Element (TFRE).

According to some embodiments, the promoter region comprises at least one TFRE. Furthermore, according to many embodiments, the promoter region comprises two or more TFREs. The TFREs contained in the promoter region may be the same TFRE such that the TFREs are for the same transcription factor, or may be different TFREs such that different TFREs in the promoter region are for different transcription factors. The TFREs may be introduced into the cell in combination or sequentially. In certain embodiments, the promoter region comprises a first TFRE and a second TFRE, wherein the first and second TFREs are different, e.g., the TFREs bind to and respond to activation and/or inactivation of different transcription factors. It may be desirable to use a promoter region comprising two or more TFREs, for example, a promoter region that mimics a naturally occurring wild-type promoter region having multiple TRFEs that bind to and respond to activation and/or inactivation of different transcription factors (e.g., class B, which is an example of an IL-2 gene promoter having at least 6 different transcription factor-specific response elements). As exemplified in some examples in the experimental section below. In some embodiments, the promoter region mimics a set of all TFREs present in a naturally occurring wild-type promoter region having a plurality of TFREs that bind to and respond to activation and/or inactivation of different transcription factors. In some embodiments, the promoter region comprises a TFRE that is an enhancer. An "enhancer" refers to a cis-acting DNA sequence that can bind to one or more proteins to enhance gene transcription, and which can be located at most 1Mb from the region encoding ED. The ability to use different specific promoter regions of class a or class B allows the present method to be applied to a wide range of different biological problems, as well as to screen for conditions that produce a desired result, e.g., activation or inactivation of a promoter of interest, activation or inactivation of expression of a gene of interest, activation or inactivation of a signal transduction pathway of a cell of interest, etc.

As summarized above, the method comprises culturing the cells under conditions wherein the ED is expressed when the promoter is active. By "active" is meant that the promoter region is in a detectably elevated state that allows the ED expression level to be above background. Such a state may be an "unbound" state, wherein a detectable increase in ED expression level above background occurs when no transcription factor is bound to the promoter region. Such a state may also be one in which detectable levels of ED expression above background occur when one or more transcription factors bind to the promoter region (e.g., when one or more of the transcription factors are themselves activated), and in which binding of the one or more transcription factors is desired for detectably elevated ED expression levels; and/or up-regulation (induction) or down-regulation of the level of ED expression compared to the level of ED expression when said promoter region is in an unbound state.

Culturing the cell such that the conditions under which the ED is expressed can be altered when the promoter is active. Such conditions may include in a suitable vessel (e.g., a cell culture plate or well thereof), in a suitable medium (e.g., a cell culture medium such as DMEM, RPMI, MEM, IMDM, DMEM/F-12, etc.), at a suitable temperature (e.g., 32 ℃ -42 ℃, such as 37 ℃) and pH (e.g., pH 7.0-7.7, such as pH 7.4), in the presence of a suitable CO₂The cells are cultured in a percentage (e.g., 3% to 10%, such as 5%) of the environment. Non-limiting examples of cell culture conditions that may be used are described in the experimental section below.

The cells used in the method may be any suitable cells. In certain embodiments, the cell type is selected based on the biological process of interest. For example, if one wishes to practice the method to investigate conditions that affect T cell activation, the cells may be activatable T cells, e.g., Jurkat cells. According to some embodiments, the cell is a type of cell that one of skill in the art uses to interrogate a target cell signaling pathway. In certain embodiments, the cell is a type of cell that one skilled in the art uses to interrogate cells containing certain specific cells and components of a molecule of interest, such as certain receptors in a pathway of interest or downstream signaling molecules, e.g., protein kinases, adapters, transcription factors, and the like.

According to some embodiments, the cell is a primary cell. "Primary cells" refers to cells obtained directly from a living tissue (e.g., tissue biopsy material) and established for growth in vitro. In some embodiments, the cell is from a cell line. Non-limiting examples of such cell lines include Jurkat, U2OS, HepG2, HeLa, MCF-7, PC-12, PBMC, HUVEC, HEK-293, COS-7, BHK-21, HEp-2, HT-1080, MDCK, and the like. According to some embodiments, the cell is an epithelial cell, a mesothelial cell, or an endothelial cell. In some embodiments, the cell is an immune cell. Non-limiting examples of immune cells that can be used include T cells, B cells, Natural Killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In certain embodiments, the immune cell is a T cell. Examples of T cells include naive T cells (T cells)_N) Cytotoxic T cell (T)_CTL) Memory T cell (T)_MEM) T memory stem cell (T)_SCM) Central memory T cell (T)_CM) Effector memory T cells (T)_EM) Tissue resident memory T cells (T)_RM) Effector T cell (T)_EFF) Regulatory T cells (T)_REG) Helper T cell (T)_H、T_H1、T_H2、T_H17) CD4+ T cells, CD8+ T cells, virus-specific T cells, and α β T cells (T cells)_αβ) And gamma delta T cells (T)_γδ)。

According to some embodiments, the cell is a cancer cell. "cancer cell" refers to a cell exhibiting a tumor cell phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cell proliferation, loss of density-dependent growth inhibition, anchorage-independent growth potential, any suitable indicator capable of promoting tumor growth and/or development and/or cell transformation in a non-human animal model of immune hypofunction. "cancer cell" is used interchangeably herein with "tumor cell", "malignant cell" or "cancerous cell" and includes solid tumors, semi-solid tumors, primary tumors, metastatic tumors, cancer cell lines, and the like. In certain aspects, the cancer cell is a carcinoma cell. Target cancer tumor cells include, but are not limited to, HepG2 cells. In certain aspects, the cancer cell is a sarcoma cell. Non-limiting examples of sarcoma cells include osteosarcoma cells, such as U2OS cells.

The nucleic acid used in the present method may be any nucleic acid suitable for operably coupling the promoter region to the region encoding the ED. In some embodiments, the nucleic acid is stably integrated into the chromosomal DNA of the cell, e.g., non-specifically or site-specifically. In some embodiments, the nucleic acid is episomal (or "episomal"). "episome" or "episomal" refers to a nucleic acid (e.g., DNA) molecule that independently replicates chromosomal DNA of a cell. A non-limiting example of an episome that can be used in the present method is a plasmid. When the nucleic acid is an episome (e.g., a plasmid), the episome may comprise one or more elements in addition to the promoter region and the region encoding the ED. For example, the plasmid may comprise an origin of replication, one or more regions encoding proteins that confer antibiotic resistance (e.g., ampicillin resistance (AmpR), hygromycin resistance, etc.) to the cell, one or more poly (a) signals, a pause signal, an SV40 late poly (a) signal, an SV40 enhancer, an SV40 early promoter, and the like, as well as any desired combinations of such elements. Plasmids introduced with nucleic acids for episomal or chromosomally integrated expression can be adjacent to and genetically linked to antibiotic selection markers that can be used to select only cells that stably express the nucleic acid. The plasmid into which the nucleic acid is introduced may be delivered by a viral vector or may be transfected by electroporation or any other suitable method using chemical reagents.

The nucleotide sequences of the plasmids (including the plasmids used in the experimental section below) and their elements/subsequences used to perform the methods of the present disclosure are provided in table 1 below.

TABLE 1 nucleotide sequences

In certain embodiments, the nucleic acid is a chromosome of the cell. For example, a chromosome of the cell can be modified (e.g., using genome editing techniques, such as homologous recombination, CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), etc.) such that the region encoding the ED is inserted into the chromosome. In some embodiments, the region encoding the ED is inserted into the genome of the cell such that the region encoding the ED is operably coupled to a native promoter region of the chromosome. The native promoter region may be a promoter region used to assess the transcriptional activity of one or more genes of interest and/or one or more cellular signaling pathways of interest. As just one example, if it is desired to assess the activity of the nfkb signaling pathway in a cell, a region encoding ED may be inserted site-specifically downstream of a promoter region comprising an nfkb binding site. In certain embodiments, the region encoding the ED is inserted into the chromosome along with a promoter region (i.e., an exogenous promoter region), wherein the region encoding the ED is operably coupled to the exogenous promoter region. In any embodiment wherein the nucleic acid is a chromosome of the cell, the chromosome can be a nuclear chromosome or a mitochondrial chromosome.

In certain embodiments, the nucleic acid further encodes a carrier protein fused to the ED, such that the ED-carrier protein fusion is expressed when the promoter region is active. The carrier protein selected for the ED may confer different desired physical or biological properties to the ED (e.g., stability, localization, bioinert, detection by another method than EFC, etc.). According to some embodiments, the carrier protein comprises a domain selected to affect the stability of the ED-carrier protein fusion. In certain embodiments, the domain is selected to increase the stability of the ED-carrier protein fusion as compared to an ED-carrier protein fusion lacking the domain. In other embodiments, the domain is selected to stabilize the ED-carrier protein fusion as compared to an ED-carrier protein fusion lacking the domain. For example, the domain can be a domain that targets the ED-carrier protein fusion for proteasomal degradation (e.g., ubiquitin-dependent proteasomal degradation). One example of a domain of the ED-carrier protein fusion that can be used to target degradation against the proteasome is the proline (P), glutamate (E), serine (S) and threonine (T) (PEST) degradation signals. Another example of such a domain is the CL1 degradation signal. The amino acid sequences of exemplary PEST and CL1 degradation signals are provided in table 2 below.

TABLE 2 amino acid sequence of degradation signals

In certain embodiments, the carrier protein comprises two or more domains selected to affect in combination the stability of the ED-carrier protein fusion. For example, the carrier protein may comprise a PEST degradation signal and a CL1 degradation signal to enhance targeting of the ED-carrier protein fusion for proteasomal degradation relative to targeting achieved using a single such signal.

In many embodiments, the promoter is further coupled to a carrier protein such that the presence of the carrier protein enhances the binding to an enzyme that is dependent on (1) full-length (single polypeptide), such as full-length luciferase, β -galactosidase, Chloramphenicol Acetyltransferase (CAT); and 2) the signal obtained in a more sensitive and efficient assay than existing reporter systems for the expression of fluorescent proteins. The carrier protein may be operably coupled to a promoter region. The carrier protein may have a detectable activity, such that expression of the carrier protein can be detected by well-known detection methods. In many other embodiments, the detectable activity of the carrier protein is different from the enzymatic activity of the beta-galactosidase.

In other embodiments, the carrier protein may be fused to an ED enzyme fragment of β -galactosidase operably coupled to a promoter region. In many embodiments, the carrier protein may be co-expressed with the ED enzyme fragment to enhance the output signal or data point when the promoter region is active. The carrier protein used in the methods of the present disclosure may have a detectable enzymatic activity that is different from that of β -galactosidase, wherein when the promoter region is active, the enzymatic activity of the carrier protein can be detected using detection methods well known for the enzymatic activity. The carrier protein having detectable enzymatic activity may be a luciferase, a modified luciferase, a fluorescent protein, a natural protein or a synthetic protein.

Furthermore, the carrier protein may also be a mutated carrier protein, wherein the mutation in the carrier protein results in the carrier protein's enzymatic activity being inhibited, such that the carrier protein does not express any detectable activity when the promoter region is active. A carrier protein having such mutations can be fused to an ED enzyme fragment operably coupled to a promoter region, wherein the ED fragment, if expressed, combines with an EA enzyme fragment to form an ED-EA enzyme complex having enzymatic activity, and the enzymatic activity is measured to assess the activity of the target promoter region, thereby assessing the activity of the transcription factor. Thus, the present invention discloses the use of a carrier protein having a different enzymatic activity than beta-galactosidase, and also discloses mutated carrier proteins which inactivate the enzymatic activity of said carrier protein.

In many embodiments, the carrier protein does not have any intrinsic enzymatic activity. The carrier protein further comprises a domain selected to affect the stability of the β -galactosidase fragment, e.g., an Enzyme Donor (ED) fragment-carrier protein fusion, wherein the selected domain increases the stability of the ED-carrier protein fusion as compared to an ED-carrier protein fusion lacking the domain. In addition, the carrier protein may further comprise a domain selected to destabilize the ED-carrier protein fusion compared to an ED-carrier protein fusion lacking said domain.

As summarized above, the methods of the present disclosure further comprise detecting a level of enzyme activity to assess the activity of the promoter region. According to some embodiments, detecting the level of enzymatic activity comprises providing a substrate for the ED-EA complex, wherein a detectable signal is generated upon hydrolysis of the substrate by the ED-EA complex. In certain embodiments, the detectable signal is a chemiluminescent signal.

Aspects of the methods include the use of reduced affinity enzyme complementation reporter systems, such as β -galactosidase fragment complementation (EFC) reporter systems. An "affinity-reduced" enzyme-complementation reporter system refers to a system consisting of two or more fragments (i.e., reporter subunits) of an enzyme that themselves lack any detectable activity observed in its parent enzyme (which can be detected directly or indirectly), but which when brought into sufficient proximity, e.g., by random interaction or binding member-mediated interaction, produce a detectable amount of the activity of the parent enzyme. One aspect of the reduced affinity enzyme complementation reporter system of the invention is that at least one reporter subunit used in the system is a variant of the corresponding domain in its wild-type parent enzyme such that its interaction with other subunits in the system is reversible under assay conditions, lacking the interaction mediated by the binding moiety of interest. In this system a small fragment of beta-galactosidase and a larger fragment of beta-galactosidase are used, wherein the two fragments have a lower affinity for each other. The small fragment enzyme donor ("ED") of β -galactosidase can have a naturally occurring sequence or a mutated sequence. According to some embodiments, the ED is a donor fragment of β -galactosidase. Various β -galactosidase fragments ED can be used. In certain embodiments, when the ED is a β -galactosidase donor fragment, the ED comprises an amino acid sequence shown in table 3 below, or a variant thereof (e.g., a variant thereof having 10 or fewer, 8 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conservative amino acid substitution, relative to the amino acid sequence shown in table 3), which complexes with the EA to form an enzyme having enzymatic activity. The amino acid sequence of an exemplary β -galactosidase donor fragment ED is provided in table 3 below.

Chemiluminescence assays can be used to detect the activity of β -galactosidase or the ED-EA complex that forms an active β -galactosidase complex. For example, cells containing β -gal fusions are lysed in a buffer mixture containing a Galacton Plus substrate from the galactoght Plus assay kit (Tropix, Bedford Mass.). Bronstein et al, J.Biolumin.Chemimun., 4:99-111 (1989). After addition of the light emission accelerator solution, the luminescence is measured in a photometer or scintillation counter. In many embodiments, the method of detecting enzymatic activity directed against β -galactosidase further comprises lysing the cells and detecting using any β -galactosidase substrate capable of producing a detectable product (e.g., a direct chromogenic, fluorescent, or chemiluminescent substrate or a substrate for a coupled assay with a bioluminescent read).

TABLE 3 amino acid sequence of exemplary beta-galactosidase donor fragment ED

As used herein, a "conservative substitution" is a substitution in which one amino acid is substituted with another amino acid having similar properties, such that one skilled in the art of peptide/protein chemistry would be able to expect the secondary structure and the hydrophilic properties of the peptide/protein or domain thereof to be substantially unchanged. Modifications may be made in the structure of the polynucleotides and polypeptides involved in particular embodiments and still obtain a functional molecule encoding a variant or derivative polypeptide having the desired characteristics, e.g., the ability to complex with an EA to form an enzyme having glycosidase activity. When it is desired to alter the amino acid sequence of the ED, EA or domains thereof to form an equivalent or even an improved variant ED or EA, one skilled in the art, for example, may alter one or more codons of the encoding DNA sequence.

"EA" refers to an enzyme receptor fragment used in an enzyme fragment complementation assay. In certain embodiments, the ED is a β -galactosidase donor fragment and the EA is a β -galactosidase receptor fragment. For example, the ED may be an ED comprising the amino acid sequence shown in table 3 (or a variant thereof that complexes with EA to form an enzyme having glycosidase activity), and EA is a commercially available EA that complexes with ED to form an enzyme having glycosidase activity. According to some embodiments, the composition is commercially available from Eurofins discovery X, Corporation/ProLink^TMSuch EAs are provided in a test kit.

The methods of the present disclosure include contacting ED with EA if expressed to form an ED-EA complex having enzymatic activity. In some embodiments, the cell is intact when the EA is contacted with the EA. For example, the ED may be contacted with the EA when the cell is living, when the cell is fixed, and the like. The cell is intact when the EA is contacted with the EA, which is typically a cell permeable enzyme fragment, so that the EA can cross the cell membrane to contact the ED expressed in the cell. When using a β -galactosidase based EFC system, β -galactosidase enzyme activity can be measured using a range of methods, including the use of live cell flow cytometry and histochemical staining with the chromogenic substrate 5-bromo-4-chloro-3-indolyl β -D-galactopyranoside (X-Gal). See, e.g., Nolan et al, Proc.Natl.Acad.Sci., USA,85: 2603-; and Lojda, Z., Enzyme Histochemistry: Alaberration Manual, Springer, Berlin (1979). Important substrates of β -gal that can be used in living cells are also encompassed in the methods and materials of the present disclosure. For example, the fluorogenic substrate resorufin β -galactosidase bisaminopropyl polyethylene glycol 1900(RGPEG) has been described. Minden (1996) BioTechniques 20(1): 122-. Such compounds can be delivered into cells by microinjection, electroporation, or various bulk loading techniques. Once inside the cell, the substrate cannot escape through the plasma membrane or gap junctions. Another important substrate that can be used in the methods and materials of the present disclosure is fluorescein di- β -D-galactopyranoside (FDG), which is particularly suitable for analysis by Fluorescence Activated Cell Sorting (FACS) and flow cytometry. Nolan et al, Proc.Natl.Acad.Sci, USA,85: 2603-.

In some embodiments, the method further comprises lysing the cells and contacting the ED with the EA, which comprises combining a cell lysate with the EA. Any suitable lysis reagent (e.g., lysis buffer) can be used to lyse the cells. Non-limiting examples of lysis buffers include NP-40 lysis buffer, RIPA (radioimmunoprecipitation assay) lysis buffer, SDS (sodium dodecyl sulfate) lysis buffer, ACK (ammonium-potassium chloride) lysis buffer, and the like. The lysis buffer may comprise a buffer salt (e.g., Tris-HCl) and/or an ionic salt (e.g., NaCl) to adjust the pH and osmolarity of the lysate. Detergents (such as Triton X-100 or SDS) may be added to disrupt cell membrane structure. The lysis buffer may contain additional useful components, such as protease inhibitors and the like. When the method involves lysing the cells and using a β -galactosidase based EFC system, the activity can be detected using a chemiluminescent assay to reconstitute the β -galactosidase. For example, cells containing reconstituted β -galactosidase (by EFC) can be lysed (with or without contacting with a cross-linking agent) in a buffer mixture containing a Galacton Plus substrate from the galactoright Plus assay kit (Tropix, Bedford Mass.). Bronstein et al, J.Biolumin.Chemimun., 4:99-111 (1989). After addition of the light emission accelerator solution, the luminescence is measured in a photometer or scintillation counter. In some embodiments, when the method comprises lysing the cells and using a β -galactosidase based EFC system, one commercially available from Eurofins discover X Corporation may be used/ProLinkTMorDetection kit for detecting the presence of a substance by chemical reactionAnd (3) photo-detecting enzyme activity.

The present disclosure also provides cells. The cells can be used to practice the methods of the present disclosure. The cells of the present disclosure may comprise any nucleic acid of the present disclosure comprising a region encoding an Enzyme Donor (ED) operably coupled to a promoter region, including any of the nucleic acids described in the methods section above and experimental section below, which are incorporated herein for brevity and not reiterated herein. In some embodiments, the cell has any of the characteristics of the cells described in the methods section above and experimental section below (e.g., can be of any cell type, etc.), which are incorporated herein for brevity and not reiterated herein.

Composition comprising a metal oxide and a metal oxide

As summarized above, the present disclosure also provides compositions. In certain embodiments, the compositions are useful, for example, in practicing the methods of the present disclosure. According to some embodiments, the compositions of the present disclosure include any nucleic acid and/or any cell of the present disclosure, including any nucleic acid and/or cell described in the methods section above and experimental sections below, which are incorporated herein for brevity and not reiterated herein.

The compositions of the present disclosure may comprise any nucleic acid and/or any cell of the present disclosure in a liquid culture medium. The liquid medium can be an aqueous liquid medium such as water, buffer solution, cell culture medium (e.g., DMEM, RPMI, MEM, IMDM, DMEM/F-12), and the like. One or more additives, such as antibiotics, salts (e.g., NaCl, MgCl)₂、KCl、MgSO₄) Buffer (Tris buffer, N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), 2- (N-morpholine) ethanesulfonic acid (MES), 2- (N-morpholine) ethanesulfonic acid sodium salt (MES), 3- (N-morpholine) propanesulfonic acid (MOPS), N-Tris [ hydroxymethyl ] methane]Methyl-3-aminopropanesulfonic acid (TAPS), and the like), solubilizing agents, detergents (e.g., nonionic detergents such as tween-20, and the like), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, and the like can be present in such compositions.

In certain embodiments, a composition is provided comprising any cell of the present disclosure in a buffered liquid culture medium. According to some embodiments, the liquid medium is a cell culture medium, e.g., DMEM, RPMI, MEM, IMDM, DMEM/F-12, and the like. In certain embodiments, a composition is provided comprising any of the nucleic acids of the present disclosure in lyophilized form or in a buffered liquid culture medium.

The compositions of the present disclosure may be present in any suitable container, such as a tube, vial, ampoule, one or more wells of a plate, e.g., 4, 6, 8, 12, 24, 48, 96, 384, 1536-well tissue culture plates, and the like.

Reagent kit

Aspects of the disclosure also include kits. In certain embodiments, the kit can be used, for example, to perform the methods of the present disclosure. According to some embodiments, the kits of the present disclosure comprise any of the nucleic acids, cells, and/or compositions of the present disclosure, including any of the nucleic acids, cells, and/or compositions described in the methods and compositions section above and the experimental section below, which are incorporated herein for brevity and not reiterated herein.

In certain embodiments, a kit is provided that comprises a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) operably coupled to a promoter region, and instructions for using the cell to perform any of the methods of the present disclosure. For example, the kit can comprise instructions for assessing the activity of the promoter region of the nucleic acid. The kit may contain instructions (and any useful reagents) for use in any of the culturing, contacting, detecting, etc. steps described in the methods section above and the experimental section below.

In many embodiments, a kit is provided that comprises a cell comprising a nucleic acid comprising a region encoding a carrier protein fused to ED operably coupled to a promoter region, and instructions for using the cell to perform any of the methods of the present disclosure.

The kits of the present disclosure can further comprise instructions for contacting the cell with an agent (e.g., a control agent, a detection agent, and/or the like) during culturing, and assessing the level of activity of the promoter region in response to contacting the cell with the agent (e.g., a small molecule, a protein (e.g., a cell surface protein), a nucleic acid, etc.) based on the level of enzyme activity detected. Such kits may further comprise instructions for contacting the cell with a control agonist that activates a target cell signaling pathway. Such kits may also comprise a control agonist. The level of activity of the promoter region can be used as a basis for assessing the effect of the agent on a transcription factor of interest and/or a cellular signaling pathway of interest. The instructions may include instructions for performing such an assessment.

According to some embodiments, the ED encoded by the nucleic acid present in the cells of the kit is the β -galactosidase donor fragment ED. For example, the ED may comprise the amino acid sequence of an exemplary β -galactosidase donor fragment ED shown in table 3, or a variant thereof capable of complexing with EA to form an enzyme having glycosidase activity. In certain embodiments, the kits of the present disclosure comprise an EA. For example, when the nucleic acid of the cell encodes β -galactosidase donor fragment ED, the EA comprised in the kit may be a β -galactosidase receptor fragment EA selected such that the ED-EA pair produces a functional enzyme with glycosidase activity by EFC.

According to some embodiments, the kit of the present disclosure further comprises instructions for lysing the cells prior to contacting the ED with the EA. In certain embodiments, the kits of the present disclosure comprise a lysis reagent. Non-limiting examples of lysis buffers that may be included in the kits of the present disclosure include NP-40 lysis buffer, RIPA (radioimmunoprecipitation assay) lysis buffer, SDS (sodium dodecyl sulfate) lysis buffer, ACK (ammonium-potassium chloride) lysis buffer, and the like. In other embodiments, the kits of the disclosure further comprise instructions for contacting ED with EA when the cell is intact. Such kits may comprise instructions for contacting ED with EA in fixed, intact cells (and detection by flow cytometry, etc.).

The components of the kit may be present in separate containers, or multiple components may be present in a single container. Suitable containers include individual tubes (e.g., vials), ampoules, wells of one or more plates, and the like.

The instructions provided with the kit may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, and the like. As such, the instructions may be present in the kit as a package insert, in a container label for the kit or components thereof (i.e., associated with a package or sub-package), and the like. In other embodiments, the instructions are present as an electronically stored data file on a suitable computer readable storage medium, such as a portable flash drive, DVD, CD-ROM, floppy disk, and the like. In other embodiments, no actual instructions are present in the kit, but are provided as obtained from a remote source (e.g., via the internet). An example of such an embodiment is a kit containing a website from which the instructions can be viewed and/or downloaded. As with the instructions, the manner in which the instructions are obtained is recorded on a suitable substrate.

The following examples are provided for illustration and not limitation.

Experiment of

Example 1 NFAT EFC reporter Gene construct

The NFAT EFC reporter construct comprises a promoter having 4 tandem repeats (4x) of NFAT transcription factor binding response elements, each element having the following sequence:

GGAGGAAAAACTGTTTCATACAGAAGGCGT (SEQ ID NO: 5). PEST is a protein destabilization sequence (a peptide sequence rich in proline (P), glutamate (E), serine (E), and threonine (T)) that enhances proteasome-mediated turnover of the prolelael (epl) protein by a reporter gene. ePL protein is a-45 amino acid fragment inactive by beta-galactosidase. It has previously been demonstrated that reducing the lifetime of the carrier protein can shorten the response time required for detection and possibly increase sensitivity to ligand concentration (Fan and Wood, Assay Drug Dev technol.2007 Feb; 5(1): 127-36).

Figure 1 shows a plasmid map of an NFAT EFC reporter construct for EFC-based assays of NFAT transcription factor activation and activation of signal transduction pathways affecting NFAT. Different promoter elements (e.g., the 4x responsive NFAT element in this example), different carrier proteins, and different destabilizing motifs (e.g., PEST in this example) can be used to target and tailor these constructs for different applications.

Figure 2 shows the Dose Response Curve (DRC) of U2OS NFAT EFC reporter cells for a "cell-stimulatory" mixture of phorbol 12-myristate 13-acetate (PMA) and ionomycin (data expressed as 1x fold, where the 1x mixture is 81nM PMA/1.34 μ M ionomycin cell stimulation). Stimulating the cells for 20h, then passingFLASH detection reagent (+ enzyme receptor (EA)) was used for detection, and cells were lysed for 1 h. 5K and 10K refer to the number of cells seeded per well in 384 well plates. S/B is the signal on the background (bottom) of the assay response curve (top).

It is known that a "cell-stimulating" mixture of phorbol 12-myristate 13-acetate (PMA) and ionomycin activates the "NFAT signal transduction pathway" and NFAT transcription factors. Stimulation of U2OS cells stably transfected with the NFAT EFC reporter construct (see figure 1) using different dilutions of the PMA/ionomycin mixture (where 1X ═ 81nM PMA/1.34 μ M ionomycin) for 20h revealed dose-dependent stimulation of the NFAT EFC reporter as evidenced by EFC after addition of + EA. The potency of the mixture was shown to be 0.047X (or 3.8 nPMA/63 nM ionomycin) using 5,000 cells. These data indicate that U2OS NFAT EFC reporter cells are suitable for developing assays for activating or inhibiting the "NFAT signaling pathway" and NFAT transcription factors. Inhibition can be detected as a decrease in the Relative Light Units (RLU) of the test compound in the presence of a stimulating dose of a cell stimulating cocktail or other activating ligand.

Example 2 NFkB EFC reportGene construct

The NF-kB EFC reporter plasmid contained a promoter with 5 tandem repeats of NF- κ B transcription factor binding response elements, with 3 GGGAATTTCC (SEQ ID NO:6) sequences interspersed with 2 alternating GGGGACTTTCC (SEQ ID NO:6) sequences.

FIG. 3 shows a plasmid map of the NF-. kappa.B EFC reporter plasmid used in the EFC-based assay for NF-. kappa.B transcription factor activation and NF-. kappa.B signaling pathway activation. Different carrier proteins and different destabilizing motifs (e.g., PEST in this example) can be used to tailor these constructs for different applications.

FIG. 4: single cell clones of U2OS NF-. kappa.B EFC reporter cells responded to the cytokine TNF. alpha. 2500 cells/well from 5 individual single cell clones of U2OS cells stably expressing the NF- κ B EFC reporter construct were seeded in 384-well plates. Cells were stimulated for 6h with the indicated concentrations of TNF α, followed byFLASH detection reagent (+5 × EA) detects cell lysates.

TNF α is known to stimulate the "NF-. kappa.B signaling pathway" and NF-. kappa.B transcription factors. Stimulation of U2OS cells stably transfected with the NF- κ B EFC reporter construct using different dilutions of TNF α 6h revealed dose-dependent stimulation of the NF- κ B EFC reporter as evidenced by EFC after addition of + EA (fig. 4). The potency of TNF α was shown to be 0.03-0.12nM for different clones. These data indicate that U2OS NF-kB EFC reporter cells are capable of sensitive response to the cytokine TNF α, indicating the possibility of developing assays for activating or inhibiting the NF-. kappa.B signaling pathway and NF-. kappa.B transcription factors. Inhibition assays can be developed by looking for RLU reduction of test compounds in the presence of stimulating doses of TNF α or other activating ligands (see figure 5).

FIG. 5: inhibitors were screened using an assay based on U2OS nfkb EFC reporter cells. U2OS NFkB EFC reporter cells were treated with 2nM TNF α (agonist), with or without various concentrations (and 3 batches) of the anti-TNF α inhibitor adalimumab. In this example, the 3 batches of cefamax tested were all from lot #1047318, simulating a particular activity reduction or activity loss by a forced degradation protocol, where the samples were unstressed (e.g., control), stressed at 70 ℃ for 15min, and stressed at 70 ℃ for 30 min.

U2OS NF-. kappa.B EFC reporter cells can also be used to measure inhibition and to study NF-. kappa.B signaling inhibitors. U2OS NF-. kappa.B EFC reporter assay was used to demonstrate adalimumab ((R))) Capable of inhibiting TNF α -stimulated NF- κ B signaling (FIG. 5). This dose-dependence of the inhibitory response allows the efficacy of different batches of adalimumab to be measured. In FIG. 5Show thatThe samples inhibited the change in potency after various degrees of forced degradation by heating. It is worth noting that forced degradation is a method for developing assays for biologicals (protein therapeutics) to simulate batches with different potencies, e.g., for QC/batch factory assays, or loss of potency for various reasons. U2OS NF-. kappa.B EFC reporter assay detects forced degradation at 70 ℃ for 0, 15 or 30min The efficacy of the sample was graded lost (right shift). The assay can also be used to study over time Stability of (2). It can be seen that the two batchesStored at 4 ℃ (all purchased commercially and therefore essentially equally potent at the time of shipment), one unexpired (#1047318) and one expired over 14 months (#1017235), all showed the same potency for inhibition of the NF- κ B pathway in the TNF α stimulated U2OS NF- κ B EFC reporter assay (fig. 6).

FIG. 6: NF kappa B EFC reporter assay showed that although lot #1017235 had expired over 14 months ((R))Lot #1047318 not expired), but two were detectedBatches had the same TNF α inhibitory activity and potency. In the forced degradation experiment shown in fig. 5, expired lot #1017235 also had the same stability as lot # 1047318.

FIG. 7: the NF κ B EFC reporter detects that endogenous CD40 receptor in cells responds strongly to CD40 ligand (CD40L) after 3h or 6h incubation. These same cells also endogenously expressed the TNF receptor and responded to soluble TNF α (as shown in fig. 4-6).

In general, multiple ligands acting through different cellular receptors stimulate the same signaling pathway, allowing the use of the same EFC reporter assay to study the function and functional inhibition of these multiple ligands. For example, stimulation and inhibition of NF κ B signaling and gene expression stimulated by TNF α was investigated in fig. 4-6. Using the same assay, the study in figure 7 shows that CD40L also stimulates the same transcription factor and gene expression pathway through the action of CD40 receptor.

Example 3 NFAT EFC reporter cell stimulation in Co-culture assay

Many active and inhibitory ligands of current research interest are soluble extracellular ligands such as TNF α, CD40L or soluble intracellular ligands such as PMA and ionomycin. However, EFC reporter assays based on these same cells can also be used to study cell-associated ligands (e.g., cell-cell or intercellular interactions) presented to another cell surface assay cell. Such other ligand presenting cells may be allogeneic or autologous cells with respect to the assay cells. FIG. 8 shows that OKT3 ligand presented on the surface of CHO-K1 cells was able to activate NFAT-mediated gene expression in Jurkat NFAT EFC reporter cell mixtures.

FIG. 8: in a co-culture assay, Jurkat NFAT EFC reporter cells responded to OKT3 ligand expressed and presented on the surface of CHO-K1 cells. OKT3 consists of a CD5 leader peptide fused to a single chain antibody fragment of the mouse anti-human T cell receptor CD3 subunit, said CD3 subunit being fused to leader-free human CD 14; accession number HM 208750.1. The addition of CHO OKT3 cells stimulated the expression of the carrier protein of NFAT EFC reporter gene in a hierarchical manner, and the stimulation of EC50 of OKT3 cells was-700 cells.

Example 4-IL 2-promoter-EFC reporter Gene construct

FIG. 9: plasmid map of IL 2-promoter-EFC reporter plasmid. In this reporter construct, the complete endogenous IL2 gene promoter ("IL 2 prom") comprising multiple specific transcription factor binding sites fused upstream of and driving expression of carrier protein-ePL, which ePL is fused to the carrier protein. The IL2 promoter comprises a binding site of NFAT (1), NFkB (2), OCT (3), ARRE-2(4), NFAT/AP1(6) transcription factors and IL2 minimal promoter (7).

The DNA sequence of the IL2 promoter used was

gtacCTTTTCTGAGTTACTTTTGTATCCCCACCCCCTTAAAGAAAGGAGGAAAAACTGTTTCATACAGAAGGCGTTAATTGCATGAATTAGAGCTATCACCTAAGTGTGGGCTAATGTAACAAAGAGGGATTTCACCTACATCCATTCAGTCAGTCTTTGGGGGTTTAAAGAAATTCCAAAGAGTCATCAGAAGAGGAAAAATGAAGGTAATGTTTTTTCAGACAGGTAAAGTCTTTGAAAATATGTGTAATATGTAAAACATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTGCCAgctag (SEQ ID NO:11) and comprises 8 different transcription factor binding sites and an IL-2 core promoter as described by Weaver et al (Molecular Immunology,06Mar 2007,44(11): 2813-2819). The following 8 DNA elements ACCCCCTTAAAGAAAGGAGGAA (SEQ ID NO:12), GGAGGAAAAACTGTTTCATACAGAAGGCGT (SEQ ID NO:13), AATTGCATGAA (SEQ ID NO:14), GGGATTTCACC (SEQ ID NO:15), ATGAAGGTAATGTTTTTTCAG (SEQ ID NO:16), GTCTTTGAAAATATGTGTAAT (SEQ ID NO:17), AAACATTTTG (SEQ ID NO:18) and TAATATTTTT (SEQ ID NO:19) respond to the transcription factors NFAT & AP1, NFAT, OCT, NFkB, NFAT & AP1, NFAT & AP1, OCT, NFAT, respectively, while the IL-2 core promoter is NFAT

CAGAATTAACAGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCT (SEQ ID NO:20), wherein the TATA box is underlined.

FIG. 10: the Jurkat IL2 promoter EFC reporter cell line can detect stimulation of multiple different response elements by activating different signaling pathways. 5K Jurkat IL2 promoter reporter cells were seeded into wells of 384-well plates and stimulated at 37 ℃ for 16h with OKT3 cells (squares) or OKT3 cells + CD28 antibody (circles), followed by addition of FLASH detection reagent (+5 × EA) and cell lysate (and reading of EFC luminescence). EC is expressed as the number of CHO-K1 cells presented by OKT3 per well₅₀。

CHO-K1 cells carrying OKT3 stimulated expression of the IL2 promoter reporter of Jurkat cells, which is thought to occur primarily through activation of NFAT responsive elements (FIG. 10, squares). anti-CD 28 antibodies were thought to act primarily through NF κ B response elements, enhancing OKT3 stimulation of the IL2 promoter (fig. 10, circle), such as an approximately 2-fold increase in signal and EC compared to OKT3 cells alone₅₀A reduction of about 2 fold. Comparing the response of OKT3+ against CD28 with that of OKT3 alone, demonstrated additive (or synergistic) induction of the IL2 promoter EFC reporter by stimulation of multiple response elements and signaling pathways. Thus, this more complex physiological promoter can be used to study the regulation of a more complex and integrated native IL2 promoter, which is more similar to the regulation of IL2 expression and secretion in vivo.

FIG. 11: the response of the IL2 promoter EFC reporter construct, a complex natural promoter with multiple different response elements, to the intracellular mimetics of two different signaling pathways (phorbol esters and ionomycin). 5K cells were seeded in the wells of 384-well plates and stimulated for 16h, followed by addition of FLASH detection reagent (+5 × EA) and cell lysate (and reading luminescence). S/B is calculated by RLU (with PMA/ionomycin)/RLU (without PMA/ionomycin).

Ionomycin and phorbol ester PMA increased the activation of different elements in the IL2 promoter EFC reporter construct by stimulating NFAT and AP-1 responsive elements, respectively (fig. 11). As with the example shown in fig. 10, this suggests that multiple inputs can be used to study the regulation of a more complex and integrated native IL2 promoter, which is more similar to the regulation of IL2 expression and secretion in vivo.

Example 5 promoter-EFC reporter Gene constructs for detection of antagonists

FIG. 12: in U2OS cells expressing ROR γ T transcription factor and ROR γ T EFC reporter plasmid, the inverse agonist GSK805 reduced the activity of ROR γ T transcription factor. Cells were treated with GSK805(Tocris) for 18h, then treated by addition of EA andthe FLASH detection reagent detects the expression of the carrier protein-ePL.

Specific EFC reporter constructs can be used to study the activity of inverse agonists (an inverse agonist is a ligand that binds directly to a transcription factor or receptor and reduces its activity below basal levels) on transfected transcription factors. In this case, the inverse agonist GSK805 was shown to decrease the activity of ROR γ T transcription factor to stimulate ROR γ T EFC reporter in U2OS cells (fig. 12).

Example 6-EFC-based NF-. kappa.B transcriptional reporter assay shows higher sensitivity than luciferase System

FIG. 13: the EFC-based NF κ B transcriptional reporter assay showed higher sensitivity to CD40L/CD40 receptor than the luciferase system. U2OS cells stably transfected with a plasmid encoding the NFkB transcriptional response element driven expression of either the EFC reporter (left panel) or the (firefly) luciferase-PEST, stimulated with a range of concentrations of CD40L for 6h, then lysed, added excess EA, incubated for 1h and assayed for luminescence (RLU). The detected luminescence indicates the degree of induction of the corresponding carrier protein by CD 40L.

The EFC-based NFkB transcriptional reporter assay is more than 35-fold more sensitive to CD40L than the luciferase-based assay. Note that the reporter plasmids used to prepare the stable cell lines have identical elements, the only significant difference being the type of carrier protein. Specifically, both plasmids have the same promoter elements and both have the same protein destabilizing elements (PEST sequences). Thus, the reporter assay is significantly more sensitive to detect EFC than to detect luciferase activity for CD 40L.

FIG. 14: the EFC-based NF κ B transcriptional reporter assay showed higher sensitivity than the luciferase system. U2OS cells stably transfected with a plasmid encoding the NFkB transcriptional response element driven expression of either the EFC reporter (left panel) or the (firefly) luciferase-PEST, stimulated with a range of concentrations of TNF α for 18h, then lysed, added with excess EA, incubated for 1h and assayed for luminescence (RLU). The detected luminescence indicates the extent of induction of the corresponding carrier protein by TNF α.

Figure 14 shows that EFC-based NF κ B transcription reporter assay shows higher sensitivity to TNF receptor ligand TNF α than the luciferase system. For example, ligand TNF α is 12-15 times more potent in the EFC assay (left panel) than the luciferase assay (right panel), which may be advantageous in detecting a weak response to a drug candidate in a screening assay.

For CD40L and TNF α, an increased sensitivity of EFC-based reporter assays to ligand stimulation was observed. This increased sensitivity is beneficial and important because it allows the use of EFC reporter assays to study less potent compounds that may be found early in the affinity maturation of chemical inhibitors (e.g., early in the lead optimization phase of hit discovery or drug discovery).

Example 7 cell lines for NF-. kappa.B pathway reporter assay

Reporter cell lines are engineered to express Enzyme Donor (ED) -tagged carrier proteins controlled by pathway-induced transcription responsive elements. Pathway activation leads to inducible expression of the ED marker protein. Exogenous enzyme receptor (EA) and buffer were added, the cells were lysed and ED and EA enzyme fragments were forced to complement each other. This results in the formation of a functional enzyme that hydrolyzes the substrate to produce a chemiluminescent signal.

In this example, the cell line is an NFkB (nuclear factor NF-. kappa. -B p100 subunit) pathway reporter cell line. The cell is a U2OS cell comprising a coding sequence having an amino acid sequence operably coupled to a promoter region comprising an NFkB response element

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO: 30).

Cells were seeded in 96-well plates and incubated at 37 ℃ and 5% CO₂And (d) incubating to allow cells to adhere and grow. The cells were then stimulated with a control agonist (here CD40L) using the assay conditions described below. After stimulation, use according to recommended protocolProLink^TMThe detection kit (Eurofins discovery X Corporation) detects signals.

Measurement conditions

Cell number/well	5000
		Time of cell inoculation (hours)	24
Control agonists	CD40L
		Ligand incubation time (minutes)	360
Ligand incubation temperature (. degree.C.)	37

The results are shown in FIG. 15. The reporter cell line showed EC against control agonist stimulation₅₀225.4ng/mL and agonist E_MAXThe signal of (c): the background ratio was 26.3.

The cell line proved to be stable over 10 passages without significant decrease in the assay window or EC₅₀And (4) changing.

Example 8 cell lines for NFAT pathway reporter assay

Reporter cell lines are engineered to express Enzyme Donor (ED) -tagged carrier proteins controlled by pathway-induced transcription responsive elements. Pathway activation leads to inducible expression of the ED marker protein. Exogenous enzyme receptor (EA) and buffer were added, the cells were lysed and ED and EA enzyme fragments were forced to complement each other. This results in the formation of a functional enzyme that hydrolyzes the substrate to produce a chemiluminescent signal.

In this example, the cell line is an NFAT (nuclear factor of activated T cell) pathway reporter cell line. The cell is a Jurkat cell comprising a gene encoding a polypeptide having an amino acid sequence operably coupled to a promoter region comprising NFAT response elements

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO: 30).

Cells were seeded in 96-well plates and incubated at 37 ℃ and 5% CO₂And (d) incubating to allow cells to adhere and grow. Cells were then stimulated with a control agonist (here an anti-CD 3 antibody) using the assay conditions described below. After stimulation, use according to recommended protocol /ProLink^TMDetection kit (Eurofins discovery X Corporation)And (6) measuring a signal.

Measurement conditions

Cell number/well	20000
		Time of cell inoculation (hours)	24
Control agonists	anti-CD 3 antibodies
		Ligand incubation time	Overnight
Ligand incubation temperature (. degree.C.)	37

The results are shown in FIG. 16. The reporter cell line showed EC against control agonist stimulation₅₀302.5ng/mL and agonist E_MAXThe signal of (c): the background ratio was 7.6.

For this assay, wells were pre-coated with anti-CD 3 antibody [ OKT3] by inoculating 50L of a 1:3 serial dilution prepared in PBS and incubating the plates overnight at 4 ℃. The antibody is removed from the wells prior to seeding with cells for assay.

The cell line proved to be stable over 10 passages without significant decrease in the assay window or EC₅₀And (4) changing.

Example 9 cell line for STAT3 pathway reporter assay

Reporter cell lines are engineered to express Enzyme Donor (ED) -tagged carrier proteins controlled by pathway-induced transcription responsive elements. Pathway activation leads to inducible expression of the ED marker protein. Exogenous enzyme receptor (EA) and buffer were added, the cells were lysed and ED and EA enzyme fragments were forced to complement each other. This results in the formation of a functional enzyme that hydrolyzes the substrate to produce a chemiluminescent signal.

In this example, the cell line is a STAT3 (signal transducer and activator of transcription 3) pathway reporter cell line. The cell is a HepG2 cell comprising a promoter region encoding a STAT3 response element operably coupled to a promoter region having an amino acid sequence

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO: 30).

Cells were seeded in 96-well plates and incubated at 37 ℃ and 5% CO₂And (d) incubating to allow cells to adhere and grow. The cells were then stimulated with a control agonist (here IL-6) using the assay conditions described below. After stimulation, use according to recommended protocol/ProLink^TMThe detection kit (Eurofins discovery X Corporation) detects signals.

Measurement conditions

Cell seeding reagent	CP5
		Cell number/well	5000
Plate type	96 wells
		Time of cell inoculation (hours)	4
Control agonists	IL-6
		Ligand incubation time	16h
Ligand incubation temperature (. degree.C.)	37

The results are shown in FIG. 17. The reporter cell line showed EC against control agonist stimulation₅₀0.601ng/mL and agonist E_MAXThe signal of (c): the background ratio was 21.3.

The cell line proved to be stable over 10 passages without significant decrease in the assay window or EC₅₀And (4) changing.

Cell lines used in the HepG2 STAT3 assay used endogenous IL-6 receptors in HepG2 cells to detect IL-6 signaling.

Example 10 Jurkat NFAT pathway reporter assay

The reporter cell line is engineered to express an Enzyme Donor (ED) -tagged carrier protein controlled by the NFAT pathway-induced transcription responsive element. Activation of the NFAT pathway results in activation of NFAT transcription factors that bind to NFAT through the induced transcriptional response elements and induce expression of the carrier protein with the ED tag. Exogenous enzyme receptor (EA) and buffer were added, the cells were lysed and inactive ED and EA β -galactosidase fragments were forced to complement. This results in the formation of a functional β -galactosidase enzyme that hydrolyzes the substrate to produce a chemiluminescent signal.

In this example, the cell line is an NFAT (nuclear factor of activated T cell) pathway reporter cell line. Activation of the NFAT pathway results in activation of NFAT transcription factors that bind to NFAT through the induced transcriptional response elements and induce expression of the carrier protein with the ED tag. The cell is a Jurkat cell comprising a gene encoding a polypeptide having an amino acid sequence operably coupled to a promoter region comprising NFAT response elements

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO: 30).

Cells were seeded in 96-well plates and incubated at 37 ℃ and 5% CO₂And (d) incubating to allow cells to adhere and grow. The cells were then stimulated with a control agonist (here an anti-CD 3 antibody) using the assay conditions described below. After stimulation, use according to recommended protocol ProLink^TMThe detection kit (Eurofins discovery X Corporation) detects signals.

Measurement conditions

Cell number/well	20000
		Time of cell inoculation (hours)	N/A
Control agonists	anti-CD 3 antibodies
		Ligand incubation time	Overnight
Ligand incubation temperature (. degree.C.)	37

The results are shown in FIG. 18. The reporter cell line showed EC against control agonist stimulation₅₀3.025ng/mL and agonist E_MAXThe signal of (c): the background ratio was 7.6.

For this assay, wells were pre-coated with activated T Cell Receptor (TCR) antibody CD3 antibody by inoculation at 10 μ g/ml and incubation of the plates for 20 hr. The antibody is removed from the wells prior to seeding with cells for assay.

The cell line proved to be stable over 10 passages without significant decrease in the assay window or EC₅₀And (4) changing.

Example 11-pathway reporter assays can be further modified to generate targeting to other targets (e.g., ligands and receptors) Body) measurement

Reporter cell lines are engineered to express Enzyme Donor (ED) -tagged carrier proteins controlled by pathway-induced transcription responsive elements. Pathway activation leads to inducible expression of the ED marker protein. Exogenous enzyme receptor (EA) and buffer were added, the cells were lysed and ED and EA enzyme fragments were forced to complement each other. This results in the formation of a functional enzyme that hydrolyzes the substrate to produce a chemiluminescent signal.

In this embodiment, the assay comprises co-culturing the first cell line and the second cell line. The first cell line in the co-culture assay was the Jurkat PD1 (programmed cell death-1) pathway reporter cell line, derived from expressing PD1 in the Jurkat NFAT pathway reporter cell developed above. The cells to which PD1 was added were Jurkat cells comprising a gene encoding a polypeptide having an amino acid sequence operably coupled to a promoter region comprising NFAT pathway response elements

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO: 30). The second cell line in the PD1 pathway reporter co-culture assay was the U2OS cell line, which co-expressed PD-L1 (programmed death ligand 1) and TCR (T cell receptor) activator molecules. Binding of PD1 to its ligand PDL1 (programmed death ligand 1) inhibits activation of TCR (T cell receptor), thereby inhibiting TCR-induced activation of the NFAT pathway by TCR (T cell receptor) activator molecules. This co-culture assay can be used to determine inhibitors of PDL1 binding to PD1, as these inhibitors block PD 1-mediated inhibition of TCR-induced NFAT pathway activation.

Jurkat PD-1 reporter cells were preincubated with PD-1 antagonist antibody (Ab) and then U2OS PD-L1/TCR activator cells were added to activate the TCR. PD-1Ab prevents PD-L1 activation of PD-1 and prevents PD-1 attenuation of TCR activation, with the net result that TCR activation increases with higher concentrations of PD-1 Ab.

Figure 19 shows that Jurkat NFAT pathway reporter assay cell lines can be used to produce PD-1 pathway reporter cell lines, demonstrating how the pathway reporter assay can be further modified to generate assays for other targets (other receptors and ligands).

Example 12 cell line for U2OS NF-. kappa.B pathway reporter Gene assay

Reporter cell lines are engineered to express Enzyme Donor (ED) -tagged carrier proteins controlled by pathway-induced transcription responsive elements. Pathway activation leads to inducible expression of the carrier protein, the ED marker protein. Exogenous enzyme receptor (EA) and buffer were added, the cells were lysed and ED and EA enzyme fragments were forced to complement each other. This results in the formation of a functional enzyme that hydrolyzes the substrate to produce a chemiluminescent signal.

In this example, the cell line is an NFkB (nuclear factor NF-. kappa. -B p100 subunit) pathway reporter cell line. The cell is a U2OS cell comprising a nucleic acid sequence encoding a reporter fragment operably coupled to a promoter region comprising an NFkB response element

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO: 30). The cells also endogenously express CD40 (receptor).

Cells were seeded in 96-well plates and incubated at 37 ℃ and 5% CO₂And (d) incubating to allow cells to adhere and grow. Then, control agonist (here CD40L) was used to stimulateCells were subjected to the following measurement conditions. After stimulation, use according to recommended protocol/ProLink^TMThe detection kit (Eurofins discovery X Corporation) detects signals.

Measurement conditions

Cell seeding reagent	CP3
		Cell number/well	5000
Plate type	96 wells
		Time of cell inoculation (hours)	Overnight
Control agonists	CD40L
		Ligand incubation time	6h
Ligand incubation temperature (. degree.C.)	37

The results are shown in FIG. 20. The reporter cell line showed EC against control agonist stimulation₅₀0.0886 μ g/mL andanimal agent E_MAXThe signal of (c): the background ratio was 102.8.

Other endogenous receptors and ligands that signal through the NFKB pathway have also been successfully used in this assay (e.g., TNF α and TNFR).

Example 13-U2OS RANK-NF-. kappa.B pathway reporter Gene assay

Reporter cell lines are engineered to express Enzyme Donor (ED) -tagged carrier proteins controlled by pathway-induced transcription responsive elements. Pathway activation leads to inducible expression of the carrier protein, the ED marker protein. Exogenous enzyme receptor (EA) and buffer were added, the cells were lysed and ED and EA enzyme fragments were forced to complement each other. This results in the formation of a functional enzyme that hydrolyzes the substrate to produce a chemiluminescent signal.

In this example, the cell line is an NFkB (nuclear factor NF-. kappa. -B p100 subunit) pathway reporter cell line. The cell is a U2OS cell comprising a nucleic acid sequence encoding a reporter fragment operably coupled to a promoter region comprising an NFkB response element

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO: 30). To generate the RANK-NFkB reporter assay, RANK (receptor activator of nuclear factor kb receptor) was co-expressed in U2OS NF-kb pathway reporter cells developed above.

Cells were seeded in 96-well plates and incubated at 37 ℃ and 5% CO₂And (d) incubating to allow cells to adhere and grow. Cells were then stimulated with a control agonist (here sRANKL) using the assay conditions described below. After stimulation, use according to recommended protocol/ProLink^TMThe detection kit (Eurofins discovery X Corporation) detects signals.

Measurement conditions

Cell seeding reagent	CP22
		Cell number/well	5000
Plate type	96 wells
		Time of cell inoculation (hours)	4
Control agonists	Soluble RANKL
		Ligand incubation time	16h
Ligand incubation temperature (. degree.C.)	37

The results are shown in FIG. 21. The reporter cell line showed EC against control agonist stimulation₅₀4.034ng/mL and agonist E_MAXThe signal of (c): the background ratio was 24.0.

Example 14 HEK NF-. kappa.B pathway reporter Gene assay

Reporter cell lines are engineered to express Enzyme Donor (ED) -tagged carrier proteins controlled by pathway-induced transcription responsive elements. Pathway activation leads to inducible expression of the carrier protein, the ED marker protein. Exogenous enzyme receptor (EA) and buffer were added, the cells were lysed and ED and EA enzyme fragments were forced to complement each other. This results in the formation of a functional enzyme that hydrolyzes the substrate to produce a chemiluminescent signal.

In this example, the cell line is an NF-. kappa.B (nuclear factor NF-. kappa. B p100 subunit 100) pathway reporter cell line. The cell is a HEK-293 cell (HEK) comprising a reporter fragment encoding a polypeptide operably coupled to a promoter region comprising a NFkB response element and having an amino acid sequence

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO: 30). In addition, endogenously expressed TNF α (ligand) and TNFR (receptor) in HEK were used to develop NF-. kappa.B pathway reporter assays.

Measurement conditions

Cell seeding reagent	CP3
		Cell number/well	2500
Plate type	384 wells
		Time of cell inoculation (hours)	Overnight
Control agonists	TNFα
		Ligand incubation time	6h
Ligand incubation temperature (. degree.C.)	37

The measurement results are shown in fig. 22.

Example 15 HEK CD 27-NF-. kappa.B pathway reporter assay

Reporter cell lines are engineered to express Enzyme Donor (ED) -tagged carrier proteins controlled by pathway-induced transcription responsive elements. Pathway activation leads to inducible expression of the expression vector, the ED marker protein. Exogenous enzyme receptor (EA) and buffer were added, the cells were lysed and ED and EA enzyme fragments were forced to complement each other. This results in the formation of a functional enzyme that hydrolyzes the substrate to produce a chemiluminescent signal.

In this example, the cell line is a CD 27-NF-. kappa.B pathway reporter cell line. The cell is a HEK cell comprising a reporter fragment encoding a polypeptide operably coupled to a promoter region comprising an nfkb response element and having an amino acid sequence

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO: 30). Co-expression of CD27 (receptor) in the HEK NF- κ B pathway reporter cell line developed above. The measurement results are shown in fig. 23.

Measurement conditions

Cell seeding reagent	CP7
		Cell number/well	2500
Plate type	384 wells
		Time of cell inoculation (hours)	4
Control agonists	CD27L
		Ligand incubation time	16h
Ligand incubation temperature (. degree.C.)	37

Example 16 comparison of the assay results of the NF-. kappa.B reporter cell line with the assay results of the RANK-NF-. kappa.B reporter cell line

Both reporter cell lines were engineered to express Enzyme Donor (ED) -tagged carrier proteins controlled by pathway-induced transcription response elements. Pathway activation leads to inducible expression of the carrier protein, the ED marker protein. Exogenous enzyme receptor (EA) and buffer were added, the cells were lysed and ED and EA enzyme fragments were forced to complement each other. This results in the formation of a functional enzyme that hydrolyzes the substrate to produce a chemiluminescent signal.

In this example, the first cell line is the U2 OS-NF-. kappa.B pathway reporter cell line and the second cell line is the U2OS RANK-NF-. kappa.B cell line. The cells were U2OS, which were all cell lines as prepared in this example 16. The U2 OS-NF-. kappa.B cell line comprises a reporter fragment encoding a promoter region operably coupled to a receptor comprising an NF-. kappa.B responsive element and having an amino acid sequence

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO: 30). The U2OS RANK-NF- κ B cell line comprises a nucleic acid sequence encoding a reporter fragment operably coupled to a promoter region comprising an NF κ B response element and having an amino acid sequence

NSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDR (SEQ ID NO:30), which was subsequently co-expressed with RANK-CD27 (acceptor) in the U2OS NF- κ B pathway reporter cell line developed above.

The results of the assay against the U2OS NF-. kappa.B cell line with CD40L ligand are shown in FIG. 24a, and the results of the assay against the U2OS RANK NF-. kappa.B cell line with sRANK ligand are shown in FIG. 24B. As shown in FIGS. 24a and 26B, the assay results against U2OS RANK NF-. kappa.B show lower EC₅₀And the larger signal: the background ratio.

This assay indicates that the RANK-NF-. kappa.B carrier protein shows better results than the NF-. kappa.B carrier protein. Thus, one carrier protein may show better results than another carrier protein.

Accordingly, the foregoing merely illustrates the principles of the disclosure. It will thus be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended expressly to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Further, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.

Sequence listing

<110> Otolydisca Vore products Limited liability company

<120> method for analyzing promoter region and cell for carrying out the same

<130> PBH-010-PCT

<160> 39

<170> PatentIn3.5 version

<210> 1

<211> 126

<212> DNA

<213> synthetic nucleotide

<400> 1

aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 60

aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 120

gatcgc 126

<210> 2

<211> 31

<212> DNA

<213> synthetic nucleotide

<400> 2

agagggtata taatggaagc tcgacttcca g 31

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<211> 123

<212> DNA

<213> synthetic nucleotide

<400> 3

aattctcacg gcttccctcc cgaggtggag gagcaggccg ccggcaccct gcccatgagc 60

tgcgcccagg agagcggcat ggatagacac cctgctgctt gcgccagcgc caggatcaac 120

gtc 123

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<212> DNA

<213> synthetic nucleotide

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gcttgcaaga actggttcag tagcttaagc cactttgtga tccacctt 48

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<212> DNA

<213> synthetic nucleotide

<400> 5

ggaggaaaaa ctgtttcata cagaaggcgt 30

<210> 6

<211> 52

<212> DNA

<213> synthetic nucleotide

<400> 6

gggaatttcc ggggactttc cgggaatttc cggggacttt ccgggaattt cc 52

<210> 7

<211> 14

<212> DNA

<213> synthetic nucleotide

<400> 7

ggtaagtagg tcat 14

<210> 8

<211> 25

<212> DNA

<213> synthetic nucleotide

<400> 8

agcctgattt ccccgaaatg acggc 25

<210> 9

<211> 19

<212> DNA

<213> synthetic nucleotide

<400> 9

catttcccgt aaatcgtcg 19

<210> 10

<211> 15

<212> DNA

<213> synthetic nucleotide

<400> 10

agttctgaga aaagt 15

<210> 11

<211> 372

<212> DNA

<213> synthetic nucleotide

<400> 11

cttttctgag ttacttttgt atccccaccc ccttaaagaa aggaggaaaa actgtttcat 60

acagaaggcg ttaattgcat gaattagagc tatcacctaa gtgtgggcta atgtaacaaa 120

gagggatttc acctacatcc attcagtcag tctttggggg tttaaagaaa ttccaaagag 180

tcatcagaag aggaaaaatg aaggtaatgt tttttcagac aggtaaagtc tttgaaaata 240

tgtgtaatat gtaaaacatt ttgacacccc cataatattt ttccagaatt aacagtataa 300

attgcatctc ttgttcaaga gttccctatc actctcttta atcactactc acagtaacct 360

caactcctgc ca 372

<210> 12

<211> 22

<212> DNA

<213> synthetic nucleotide

<400> 12

acccccttaa agaaaggagg aa 22

<210> 13

<211> 30

<212> DNA

<213> synthetic nucleotide

<400> 13

ggaggaaaaa ctgtttcata cagaaggcgt 30

<210> 14

<211> 11

<212> DNA

<213> synthetic nucleotide

<400> 14

aattgcatga a 11

<210> 15

<211> 11

<212> DNA

<213> synthetic nucleotide

<400> 15

gggatttcac c 11

<210> 16

<211> 21

<212> DNA

<213> synthetic nucleotide

<400> 16

atgaaggtaa tgttttttca g 21

<210> 17

<211> 21

<212> DNA

<213> synthetic nucleotide

<400> 17

gtctttgaaa atatgtgtaa t 21

<210> 18

<211> 10

<212> DNA

<213> synthetic nucleotide

<400> 18

aaacattttg 10

<210> 19

<211> 10

<212> DNA

<213> synthetic nucleotide

<400> 19

taatattttt 10

<210> 20

<211> 52

<212> DNA

<213> synthetic nucleotide

<400> 20

cagaattaac agtataaatt gcatctcttg ttcaagagtt ccctatcact ct 52

<210> 21

<211> 452

<212> DNA

<213> synthetic nucleotide

<400> 21

actcgtcctt tttcaatatt attgaagcat ttatcagggt tactagtacg tctctcaagg 60

ataagtaagt aatattaagg tacgggaggt attggacagg ccgcaataaa atatctttat 120

tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc 180

atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag 240

gtgccagaac atttctctgg cctaactggc cggtacctga gctcgctagc ggaggaaaaa 300

ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa 360

ctgtttcata cagaaggcgt agatctacta gagggtatat aatggaagct cgacttccag 420

cttggcaatc cggtactgtt ggtaaagcca cc 452

<210> 22

<211> 653

<212> DNA

<213> synthetic nucleotide

<400> 22

actcgtcctt tttcaatatt attgaagcat ttatcagggt tactagtacg tctctcaagg 60

ataagtaagt aatattaagg tacgggaggt attggacagg ccgcaataaa atatctttat 120

tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc 180

atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag 240

gtgccagaac atttctctgg cctaactggc cggtaccttt tctgagttac ttttgtatcc 300

ccaccccctt aaagaaagga ggaaaaactg tttcatacag aaggcgttaa ttgcatgaat 360

tagagctatc acctaagtgt gggctaatgt aacaaagagg gatttcacct acatccattc 420

agtcagtctt tgggggttta aagaaattcc aaagagtcat cagaagagga aaaatgaagg 480

taatgttttt tcagacaggt aaagtctttg aaaatatgtg taatatgtaa aacattttga 540

cacccccata atatttttcc agaattaaca gtataaattg catctcttgt tcaagagttc 600

cctatcactc tctttaatca ctactcacag taacctcaac tcctgccagc tag 653

<210> 23

<211> 449

<212> DNA

<213> synthetic nucleotide

<400> 23

actcgtcctt tttcaatatt attgaagcat ttatcagggt tactagtacg tctctcaagg 60

ataagtaagt aatattaagg tacgggaggt attggacagg ccgcaataaa atatctttat 120

tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc 180

atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag 240

gtgccagaac atttctctgg cctaactggc cggtaccagc ctgatttccc cgaaatgacg 300

gcagcctgat ttccccgaaa tgacggcagc ctgatttccc cgaaatgacg gcagcctgat 360

ttccccgaaa tgacggcaga tctactagag ggtatataat ggaagctcga cttccagctt 420

ggcaatccgg tactgttggt aaagccacc 449

<210> 24

<211> 494

<212> DNA

<213> synthetic nucleotide

<400> 24

actcgtcctt tttcaatatt attgaagcat ttatcagggt tactagtacg tctctcaagg 60

ataagtaagt aatattaagg tacgggaggt attggacagg ccgcaataaa atatctttat 120

tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc 180

atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag 240

gtgccagaac atttctctgg cctaactggc cggtacctga gctcagcttc atttcccgta 300

aatcgtcgaa gcttcatttc ccgtaaatcg tcgaagcttc atttcccgta aatcgtcgaa 360

gcttcatttc ccgtaaatcg tcgaagcttc atttcccgta aatcgtcgac tcgaggatat 420

caagatctac tagagggtat ataatggaag ctcgacttcc agcttggcaa tccggtactg 480

ttggtaaagc cacc 494

<210> 25

<211> 444

<212> DNA

<213> synthetic nucleotide

<400> 25

actcgtcctt tttcaatatt attgaagcat ttatcagggt tactagtacg tctctcaagg 60

ataagtaagt aatattaagg tacgggaggt attggacagg ccgcaataaa atatctttat 120

tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc 180

atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag 240

gtgccagaac atttctctgg cctaactggc cggtacctga gctcagttct gagaaaagta 300

gttctgagaa aagtagttct gagaaaagta gttctgagaa aagtagttct gagaaaagtc 360

tcgaggatat caagatctac tagagggtat ataatggaag ctcgacttcc agcttggcaa 420

tccggtactg ttggtaaagc cacc 444

<210> 26

<211> 445

<212> DNA

<213> synthetic nucleotide

<400> 26

actcgtcctt tttcaatatt attgaagcat ttatcagggt tactagtacg tctctcaagg 60

ataagtaagt aatattaagg tacgggaggt attggacagg ccgcaataaa atatctttat 120

tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc 180

atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag 240

gtgccagaac atttctctgg cctaactggc cggtacctga gctcggtaag taggtcatgg 300

taagtaggtc atggtaagta ggtcatggta agtaggtcat ggtaagtagg tcatcgtgac 360

ctcgaggata tcaagatcta ctagagggta tataatggaa gctcgacttc cagcttggca 420

atccggtact gttggtaaag ccacc 445

<210> 27

<211> 467

<212> DNA

<213> synthetic nucleotide

<400> 27

actcgtcctt tttcaatatt attgaagcat ttatcagggt tactagtacg tctctcaagg 60

ataagtaagt aatattaagg tacgggaggt attggacagg ccgcaataaa atatctttat 120

tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc 180

atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag 240

gtgccagaac atttctctgg cctaactggc cggtaccaag atttctgcag cggagtactg 300

tcctccgagc ggagtactgt cctccgagcg gagtactgtc ctccgagcgg agtactgtcc 360

tccgagcgga gtactgtcct ccgctcgagg atatcagatc tactagaggg tatataatgg 420

aagctcgact tccagcttgg caatccggta ctgttggtaa agccacc 467

<210> 28

<211> 40

<212> PRT

<213> synthetic peptide

<400> 28

Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Ala Ala Gly Thr Leu

1 5 10 15

Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala Ala

20 25 30

Cys Ala Ser Ala Arg Ile Asn Val

35 40

<210> 29

<211> 16

<212> PRT

<213> synthetic peptide

<400> 29

Ala Cys Lys Asn Trp Phe Ser Ser Leu Ser His Phe Val Ile His Leu

1 5 10 15

<210> 30

<211> 42

<212> PRT

<213> synthetic peptide

<400> 30

Asn Ser Leu Ala Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly

1 5 10 15

Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Trp

20 25 30

Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg

35 40

<210> 31

<211> 52

<212> PRT

<213> synthetic peptide

<400> 31

Met Gly Val Ile Thr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp

1 5 10 15

Trp Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro

20 25 30

Pro Phe Ala Ser Trp Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro

35 40 45

Ser Gln Gln Leu

50

<210> 32

<211> 52

<212> PRT

<213> synthetic peptide

<400> 32

Met Gly Val Ile Thr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp

1 5 10 15

Trp Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro

20 25 30

Pro Phe Ala Ser Tyr Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro

35 40 45

Ser Gln Gln Leu

50

<210> 33

<211> 4120

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide

<400> 33

aaattctcac ggcttccctc ccgaggtgga ggagcaggcc gccggcaccc tgcccatgag 60

ctgcgcccag gagagcggca tggatagaca ccctgctgct tgcgccagcg ccaggatcaa 120

cgtcttcgaa ttgggaggtg gcggtagcgg aggtggcggt agcctcgaga gctccaattc 180

actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 240

ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 300

ctagtgaggc cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac 360

cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 420

atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 480

gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg 540

tggtaaaatc gataaggatc cgtttgcgta ttgggcgctc ttccgctgat ctgcgcagca 600

ccatggcctg aaataacctc tgaaagagga acttggttag ctaccttctg aggcggaaag 660

aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc 720

agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc 780

tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg 840

cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 900

ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc 960

cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cgattcttct 1020

gacactagcg ccaccatgaa gaagcccgaa ctcaccgcta ccagcgttga aaaatttctc 1080

atcgagaagt tcgacagtgt gagcgacctg atgcagttgt cggagggcga agagagccga 1140

gccttcagct tcgatgtcgg cggacgcggc tatgtactgc gggtgaatag ctgcgctgat 1200

ggcttctaca aagaccgcta cgtgtaccgc cacttcgcca gcgctgcact acccatcccc 1260

gaagtgttgg acatcggcga gttcagcgag agcctgacat actgcatcag tagacgcgcc 1320

caaggcgtta ctctccaaga cctccccgaa acagagctgc ctgctgtgtt acagcctgtc 1380

gccgaagcta tggatgctat tgccgccgcc gacctcagtc aaaccagcgg cttcggccca 1440

ttcgggcccc aaggcatcgg ccagtacaca acctggcggg atttcatttg cgccattgct 1500

gatccccatg tctaccactg gcagaccgtg atggacgaca ccgtgtccgc cagcgtagct 1560

caagccctgg acgaactgat gctgtgggcc gaagactgtc ccgaggtgcg ccacctcgtc 1620

catgccgact tcggcagcaa caacgtcctg accgacaacg gccgcatcac cgccgtaatc 1680

gactggtccg aagctatgtt cggggacagt cagtacgagg tggccaacat cttcttctgg 1740

cggccctggc tggcttgcat ggagcagcag actcgctact tcgagcgccg gcatcccgag 1800

ctggccggca gccctcgtct gcgagcctac atgctgcgca tcggcctgga tcagctctac 1860

cagagcctcg tggacggcaa cttcgacgat gctgcctggg ctcaaggccg ctgcgatgcc 1920

atcgtccgca gcggggccgg caccgtcggt cgcacacaaa tcgctcgccg gagcgcagcc 1980

gtatggaccg acggctgcgt cgaggtgctg gccgacagcg gcaaccgccg gcccagtaca 2040

cgaccgcgcg ctaaggaggt aggtcgagtt taaactctag aaccggtcat ggccgcaata 2100

aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgttcga actagatgct 2160

gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca 2220

tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc 2280

cggcagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 2340

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 2400

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 2460

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 2520

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 2580

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 2640

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 2700

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 2760

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 2820

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 2880

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 2940

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 3000

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 3060

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 3120

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 3180

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag cggccgcaaa 3240

tgctaaacca ctgcagtggt taccagtgct tgatcagtga ggcaccgatc tcagcgatct 3300

gcctatttcg ttcgtccata gtggcctgac tccccgtcgt gtagatcact acgattcgtg 3360

agggcttacc atcaggcccc agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc 3420

ccgatttgtc agcaatgaac cagccagcag ggagggccga gcgaagaagt ggtcctgcta 3480

ctttgtccgc ctccatccag tctatgagct gctgtcgtga tgctagagta agaagttcgc 3540

cagtgagtag tttccgaaga gttgtggcca ttgctactgg catcgtggta tcacgctcgt 3600

cgttcggtat ggcttcgttc aactctggtt cccagcggtc aagccgggtc acatgatcac 3660

ccatattatg aagaaatgca gtcagctcct tagggcctcc gatcgttgtc agaagtaagt 3720

tggccgcggt gttgtcgctc atggtaatgg cagcactaca caattctctt accgtcatgc 3780

catccgtaag atgcttttcc gtgaccggcg agtactcaac caagtcgttt tgtgagtagt 3840

gtatacggcg accaagctgc tcttgcccgg cgtctatacg ggacaacacc gcgccacata 3900

gcagtacttt gaaagtgctc atcatcggga atcgttcttc ggggcggaaa gactcaagga 3960

tcttgccgct attgagatcc agttcgatat agcccactct tgcacccagt tgatcttcag 4020

catcttttac tttcaccagc gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa 4080

agaagggaat gagtgcgaca cgaaaatgtt ggatgctcat 4120

<210> 34

<211> 3990

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide

<400> 34

aagcttggag gtggcggtag cggaggtggc ggtagcctcg agagctccaa ttcactggcc 60

gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 120

gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgctagggc 180

cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga 240

atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc 300

attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt 360

cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc 420

gataaggatc cgtttgcgta ttgggcgctc ttccgctgat ctgcgcagca ccatggcctg 480

aaataacctc tgaaagagga acttggttag ctaccttctg aggcggaaag aaccagctgt 540

ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 600

aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 660

gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 720

cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 780

ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt 840

gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cgattcttct gacactagcg 900

ccaccatgaa gaagcccgaa ctcaccgcta ccagcgttga aaaatttctc atcgagaagt 960

tcgacagtgt gagcgacctg atgcagttgt cggagggcga agagagccga gccttcagct 1020

tcgatgtcgg cggacgcggc tatgtactgc gggtgaatag ctgcgctgat ggcttctaca 1080

aagaccgcta cgtgtaccgc cacttcgcca gcgctgcact acccatcccc gaagtgttgg 1140

acatcggcga gttcagcgag agcctgacat actgcatcag tagacgcgcc caaggcgtta 1200

ctctccaaga cctccccgaa acagagctgc ctgctgtgtt acagcctgtc gccgaagcta 1260

tggatgctat tgccgccgcc gacctcagtc aaaccagcgg cttcggccca ttcgggcccc 1320

aaggcatcgg ccagtacaca acctggcggg atttcatttg cgccattgct gatccccatg 1380

tctaccactg gcagaccgtg atggacgaca ccgtgtccgc cagcgtagct caagccctgg 1440

acgaactgat gctgtgggcc gaagactgtc ccgaggtgcg ccacctcgtc catgccgact 1500

tcggcagcaa caacgtcctg accgacaacg gccgcatcac cgccgtaatc gactggtccg 1560

aagctatgtt cggggacagt cagtacgagg tggccaacat cttcttctgg cggccctggc 1620

tggcttgcat ggagcagcag actcgctact tcgagcgccg gcatcccgag ctggccggca 1680

gccctcgtct gcgagcctac atgctgcgca tcggcctgga tcagctctac cagagcctcg 1740

tggacggcaa cttcgacgat gctgcctggg ctcaaggccg ctgcgatgcc atcgtccgca 1800

gcggggccgg caccgtcggt cgcacacaaa tcgctcgccg gagcgcagcc gtatggaccg 1860

acggctgcgt cgaggtgctg gccgacagcg gcaaccgccg gcccagtaca cgaccgcgcg 1920

ctaaggaggt aggtcgagtt taaactctag aaccggtcat ggccgcaata aaatatcttt 1980

attttcatta catctgtgtg ttggtttttt gtgtgttcga actagatgct gtcgaccgat 2040

gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt 2100

cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct 2160

cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 2220

cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 2280

acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 2340

ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 2400

ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 2460

gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 2520

gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 2580

ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 2640

actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 2700

gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 2760

ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 2820

ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 2880

gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 2940

tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 3000

tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 3060

aatcaatcta aagtatatat gagtaaactt ggtctgacag cggccgcaaa tgctaaacca 3120

ctgcagtggt taccagtgct tgatcagtga ggcaccgatc tcagcgatct gcctatttcg 3180

ttcgtccata gtggcctgac tccccgtcgt gtagatcact acgattcgtg agggcttacc 3240

atcaggcccc agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc ccgatttgtc 3300

agcaatgaac cagccagcag ggagggccga gcgaagaagt ggtcctgcta ctttgtccgc 3360

ctccatccag tctatgagct gctgtcgtga tgctagagta agaagttcgc cagtgagtag 3420

tttccgaaga gttgtggcca ttgctactgg catcgtggta tcacgctcgt cgttcggtat 3480

ggcttcgttc aactctggtt cccagcggtc aagccgggtc acatgatcac ccatattatg 3540

aagaaatgca gtcagctcct tagggcctcc gatcgttgtc agaagtaagt tggccgcggt 3600

gttgtcgctc atggtaatgg cagcactaca caattctctt accgtcatgc catccgtaag 3660

atgcttttcc gtgaccggcg agtactcaac caagtcgttt tgtgagtagt gtatacggcg 3720

accaagctgc tcttgcccgg cgtctatacg ggacaacacc gcgccacata gcagtacttt 3780

gaaagtgctc atcatcggga atcgttcttc ggggcggaaa gactcaagga tcttgccgct 3840

attgagatcc agttcgatat agcccactct tgcacccagt tgatcttcag catcttttac 3900

tttcaccagc gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa agaagggaat 3960

gagtgcgaca cgaaaatgtt ggatgctcat 3990

<210> 35

<211> 4119

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide

<400> 35

aattctcacg gcttccctcc cgaggtggag gagcaggccg ccggcaccct gcccatgagc 60

tgcgcccagg agagcggcat ggatagacac cctgctgctt gcgccagcgc caggatcaac 120

gtcttcgaat tgggaggtgg cggtagcgga ggtggcggta gcctcgagag ctccaattca 180

ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 240

cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 300

tagtgaggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc 360

acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 420

tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 480

tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt 540

ggtaaaatcg ataaggatcc gtttgcgtat tgggcgctct tccgctgatc tgcgcagcac 600

catggcctga aataacctct gaaagaggaa cttggttagc taccttctga ggcggaaaga 660

accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca 720

gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct 780

ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc 840

ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 900

gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc 960

agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc gattcttctg 1020

acactagcgc caccatgaag aagcccgaac tcaccgctac cagcgttgaa aaatttctca 1080

tcgagaagtt cgacagtgtg agcgacctga tgcagttgtc ggagggcgaa gagagccgag 1140

ccttcagctt cgatgtcggc ggacgcggct atgtactgcg ggtgaatagc tgcgctgatg 1200

gcttctacaa agaccgctac gtgtaccgcc acttcgccag cgctgcacta cccatccccg 1260

aagtgttgga catcggcgag ttcagcgaga gcctgacata ctgcatcagt agacgcgccc 1320

aaggcgttac tctccaagac ctccccgaaa cagagctgcc tgctgtgtta cagcctgtcg 1380

ccgaagctat ggatgctatt gccgccgccg acctcagtca aaccagcggc ttcggcccat 1440

tcgggcccca aggcatcggc cagtacacaa cctggcggga tttcatttgc gccattgctg 1500

atccccatgt ctaccactgg cagaccgtga tggacgacac cgtgtccgcc agcgtagctc 1560

aagccctgga cgaactgatg ctgtgggccg aagactgtcc cgaggtgcgc cacctcgtcc 1620

atgccgactt cggcagcaac aacgtcctga ccgacaacgg ccgcatcacc gccgtaatcg 1680

actggtccga agctatgttc ggggacagtc agtacgaggt ggccaacatc ttcttctggc 1740

ggccctggct ggcttgcatg gagcagcaga ctcgctactt cgagcgccgg catcccgagc 1800

tggccggcag ccctcgtctg cgagcctaca tgctgcgcat cggcctggat cagctctacc 1860

agagcctcgt ggacggcaac ttcgacgatg ctgcctgggc tcaaggccgc tgcgatgcca 1920

tcgtccgcag cggggccggc accgtcggtc gcacacaaat cgctcgccgg agcgcagccg 1980

tatggaccga cggctgcgtc gaggtgctgg ccgacagcgg caaccgccgg cccagtacac 2040

gaccgcgcgc taaggaggta ggtcgagttt aaactctaga accggtcatg gccgcaataa 2100

aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgttcgaa ctagatgctg 2160

tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat 2220

gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc 2280

ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 2340

gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 2400

caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 2460

tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 2520

gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 2580

ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 2640

cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 2700

tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 2760

tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 2820

cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 2880

agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 2940

agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 3000

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 3060

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 3120

ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 3180

gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagc ggccgcaaat 3240

gctaaaccac tgcagtggtt accagtgctt gatcagtgag gcaccgatct cagcgatctg 3300

cctatttcgt tcgtccatag tggcctgact ccccgtcgtg tagatcacta cgattcgtga 3360

gggcttacca tcaggcccca gcgcagcaat gatgccgcga gagccgcgtt caccggcccc 3420

cgatttgtca gcaatgaacc agccagcagg gagggccgag cgaagaagtg gtcctgctac 3480

tttgtccgcc tccatccagt ctatgagctg ctgtcgtgat gctagagtaa gaagttcgcc 3540

agtgagtagt ttccgaagag ttgtggccat tgctactggc atcgtggtat cacgctcgtc 3600

gttcggtatg gcttcgttca actctggttc ccagcggtca agccgggtca catgatcacc 3660

catattatga agaaatgcag tcagctcctt agggcctccg atcgttgtca gaagtaagtt 3720

ggccgcggtg ttgtcgctca tggtaatggc agcactacac aattctctta ccgtcatgcc 3780

atccgtaaga tgcttttccg tgaccggcga gtactcaacc aagtcgtttt gtgagtagtg 3840

tatacggcga ccaagctgct cttgcccggc gtctatacgg gacaacaccg cgccacatag 3900

cagtactttg aaagtgctca tcatcgggaa tcgttcttcg gggcggaaag actcaaggat 3960

cttgccgcta ttgagatcca gttcgatata gcccactctt gcacccagtt gatcttcagc 4020

atcttttact ttcaccagcg tttcggggtg tgcaaaaaca ggcaagcaaa atgccgcaaa 4080

gaagggaatg agtgcgacac gaaaatgttg gatgctcat 4119

<210> 36

<211> 4120

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide

<400> 36

aaattctcac ggcttccctc ccgaggtgga ggagcaggcc gccggcaccc tgcccatgag 60

ctgcgcccag gagagcggca tggatagaca ccctgctgct tgcgccagcg ccaggatcaa 120

cgtcttcgaa ttgggaggtg gcggtagcgg aggtggcggt agcctcgaga gctccaattc 180

actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 240

ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 300

ctagtgaggc cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac 360

cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 420

atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 480

gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg 540

tggtaaaatc gataaggatc cgtttgcgta ttgggcgctc ttccgctgat ctgcgcagca 600

ccatggcctg aaataacctc tgaaagagga acttggttag ctaccttctg aggcggaaag 660

aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc 720

agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc 780

tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg 840

cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 900

ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc 960

cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cgattcttct 1020

gacactagcg ccaccatgaa gaagcccgaa ctcaccgcta ccagcgttga aaaatttctc 1080

atcgagaagt tcgacagtgt gagcgacctg atgcagttgt cggagggcga agagagccga 1140

gccttcagct tcgatgtcgg cggacgcggc tatgtactgc gggtgaatag ctgcgctgat 1200

ggcttctaca aagaccgcta cgtgtaccgc cacttcgcca gcgctgcact acccatcccc 1260

gaagtgttgg acatcggcga gttcagcgag agcctgacat actgcatcag tagacgcgcc 1320

caaggcgtta ctctccaaga cctccccgaa acagagctgc ctgctgtgtt acagcctgtc 1380

gccgaagcta tggatgctat tgccgccgcc gacctcagtc aaaccagcgg cttcggccca 1440

ttcgggcccc aaggcatcgg ccagtacaca acctggcggg atttcatttg cgccattgct 1500

gatccccatg tctaccactg gcagaccgtg atggacgaca ccgtgtccgc cagcgtagct 1560

caagccctgg acgaactgat gctgtgggcc gaagactgtc ccgaggtgcg ccacctcgtc 1620

catgccgact tcggcagcaa caacgtcctg accgacaacg gccgcatcac cgccgtaatc 1680

gactggtccg aagctatgtt cggggacagt cagtacgagg tggccaacat cttcttctgg 1740

cggccctggc tggcttgcat ggagcagcag actcgctact tcgagcgccg gcatcccgag 1800

ctggccggca gccctcgtct gcgagcctac atgctgcgca tcggcctgga tcagctctac 1860

cagagcctcg tggacggcaa cttcgacgat gctgcctggg ctcaaggccg ctgcgatgcc 1920

atcgtccgca gcggggccgg caccgtcggt cgcacacaaa tcgctcgccg gagcgcagcc 1980

gtatggaccg acggctgcgt cgaggtgctg gccgacagcg gcaaccgccg gcccagtaca 2040

cgaccgcgcg ctaaggaggt aggtcgagtt taaactctag aaccggtcat ggccgcaata 2100

aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgttcga actagatgct 2160

gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca 2220

tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc 2280

cggcagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 2340

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 2400

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 2460

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 2520

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 2580

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 2640

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 2700

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 2760

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 2820

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 2880

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 2940

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 3000

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 3060

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 3120

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 3180

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag cggccgcaaa 3240

tgctaaacca ctgcagtggt taccagtgct tgatcagtga ggcaccgatc tcagcgatct 3300

gcctatttcg ttcgtccata gtggcctgac tccccgtcgt gtagatcact acgattcgtg 3360

agggcttacc atcaggcccc agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc 3420

ccgatttgtc agcaatgaac cagccagcag ggagggccga gcgaagaagt ggtcctgcta 3480

ctttgtccgc ctccatccag tctatgagct gctgtcgtga tgctagagta agaagttcgc 3540

cagtgagtag tttccgaaga gttgtggcca ttgctactgg catcgtggta tcacgctcgt 3600

cgttcggtat ggcttcgttc aactctggtt cccagcggtc aagccgggtc acatgatcac 3660

ccatattatg aagaaatgca gtcagctcct tagggcctcc gatcgttgtc agaagtaagt 3720

tggccgcggt gttgtcgctc atggtaatgg cagcactaca caattctctt accgtcatgc 3780

catccgtaag atgcttttcc gtgaccggcg agtactcaac caagtcgttt tgtgagtagt 3840

gtatacggcg accaagctgc tcttgcccgg cgtctatacg ggacaacacc gcgccacata 3900

gcagtacttt gaaagtgctc atcatcggga atcgttcttc ggggcggaaa gactcaagga 3960

tcttgccgct attgagatcc agttcgatat agcccactct tgcacccagt tgatcttcag 4020

catcttttac tttcaccagc gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa 4080

agaagggaat gagtgcgaca cgaaaatgtt ggatgctcat 4120

<210> 37

<211> 4120

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide

<400> 37

aaattctcac ggcttccctc ccgaggtgga ggagcaggcc gccggcaccc tgcccatgag 60

ctgcgcccag gagagcggca tggatagaca ccctgctgct tgcgccagcg ccaggatcaa 120

cgtcttcgaa ttgggaggtg gcggtagcgg aggtggcggt agcctcgaga gctccaattc 180

actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 240

ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 300

ctagtgaggc cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac 360

cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 420

atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 480

gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg 540

tggtaaaatc gataaggatc cgtttgcgta ttgggcgctc ttccgctgat ctgcgcagca 600

ccatggcctg aaataacctc tgaaagagga acttggttag ctaccttctg aggcggaaag 660

aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc 720

agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc 780

tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg 840

cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 900

ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc 960

cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cgattcttct 1020

gacactagcg ccaccatgaa gaagcccgaa ctcaccgcta ccagcgttga aaaatttctc 1080

atcgagaagt tcgacagtgt gagcgacctg atgcagttgt cggagggcga agagagccga 1140

gccttcagct tcgatgtcgg cggacgcggc tatgtactgc gggtgaatag ctgcgctgat 1200

ggcttctaca aagaccgcta cgtgtaccgc cacttcgcca gcgctgcact acccatcccc 1260

gaagtgttgg acatcggcga gttcagcgag agcctgacat actgcatcag tagacgcgcc 1320

caaggcgtta ctctccaaga cctccccgaa acagagctgc ctgctgtgtt acagcctgtc 1380

gccgaagcta tggatgctat tgccgccgcc gacctcagtc aaaccagcgg cttcggccca 1440

ttcgggcccc aaggcatcgg ccagtacaca acctggcggg atttcatttg cgccattgct 1500

gatccccatg tctaccactg gcagaccgtg atggacgaca ccgtgtccgc cagcgtagct 1560

caagccctgg acgaactgat gctgtgggcc gaagactgtc ccgaggtgcg ccacctcgtc 1620

catgccgact tcggcagcaa caacgtcctg accgacaacg gccgcatcac cgccgtaatc 1680

gactggtccg aagctatgtt cggggacagt cagtacgagg tggccaacat cttcttctgg 1740

cggccctggc tggcttgcat ggagcagcag actcgctact tcgagcgccg gcatcccgag 1800

ctggccggca gccctcgtct gcgagcctac atgctgcgca tcggcctgga tcagctctac 1860

cagagcctcg tggacggcaa cttcgacgat gctgcctggg ctcaaggccg ctgcgatgcc 1920

atcgtccgca gcggggccgg caccgtcggt cgcacacaaa tcgctcgccg gagcgcagcc 1980

gtatggaccg acggctgcgt cgaggtgctg gccgacagcg gcaaccgccg gcccagtaca 2040

cgaccgcgcg ctaaggaggt aggtcgagtt taaactctag aaccggtcat ggccgcaata 2100

aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgttcga actagatgct 2160

gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca 2220

tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc 2280

cggcagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 2340

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 2400

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 2460

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 2520

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 2580

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 2640

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 2700

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 2760

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 2820

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 2880

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 2940

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 3000

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 3060

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 3120

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 3180

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag cggccgcaaa 3240

tgctaaacca ctgcagtggt taccagtgct tgatcagtga ggcaccgatc tcagcgatct 3300

gcctatttcg ttcgtccata gtggcctgac tccccgtcgt gtagatcact acgattcgtg 3360

agggcttacc atcaggcccc agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc 3420

ccgatttgtc agcaatgaac cagccagcag ggagggccga gcgaagaagt ggtcctgcta 3480

ctttgtccgc ctccatccag tctatgagct gctgtcgtga tgctagagta agaagttcgc 3540

cagtgagtag tttccgaaga gttgtggcca ttgctactgg catcgtggta tcacgctcgt 3600

cgttcggtat ggcttcgttc aactctggtt cccagcggtc aagccgggtc acatgatcac 3660

ccatattatg aagaaatgca gtcagctcct tagggcctcc gatcgttgtc agaagtaagt 3720

tggccgcggt gttgtcgctc atggtaatgg cagcactaca caattctctt accgtcatgc 3780

catccgtaag atgcttttcc gtgaccggcg agtactcaac caagtcgttt tgtgagtagt 3840

gtatacggcg accaagctgc tcttgcccgg cgtctatacg ggacaacacc gcgccacata 3900

gcagtacttt gaaagtgctc atcatcggga atcgttcttc ggggcggaaa gactcaagga 3960

tcttgccgct attgagatcc agttcgatat agcccactct tgcacccagt tgatcttcag 4020

catcttttac tttcaccagc gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa 4080

agaagggaat gagtgcgaca cgaaaatgtt ggatgctcat 4120

<210> 38

<211> 4120

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide

<400> 38

aaattctcac ggcttccctc ccgaggtgga ggagcaggcc gccggcaccc tgcccatgag 60

ctgcgcccag gagagcggca tggatagaca ccctgctgct tgcgccagcg ccaggatcaa 120

cgtcttcgaa ttgggaggtg gcggtagcgg aggtggcggt agcctcgaga gctccaattc 180

actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 240

ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 300

ctagtgaggc cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac 360

cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 420

atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 480

gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg 540

tggtaaaatc gataaggatc cgtttgcgta ttgggcgctc ttccgctgat ctgcgcagca 600

ccatggcctg aaataacctc tgaaagagga acttggttag ctaccttctg aggcggaaag 660

aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc 720

agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc 780

tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg 840

cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 900

ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc 960

cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cgattcttct 1020

gacactagcg ccaccatgaa gaagcccgaa ctcaccgcta ccagcgttga aaaatttctc 1080

atcgagaagt tcgacagtgt gagcgacctg atgcagttgt cggagggcga agagagccga 1140

gccttcagct tcgatgtcgg cggacgcggc tatgtactgc gggtgaatag ctgcgctgat 1200

ggcttctaca aagaccgcta cgtgtaccgc cacttcgcca gcgctgcact acccatcccc 1260

gaagtgttgg acatcggcga gttcagcgag agcctgacat actgcatcag tagacgcgcc 1320

caaggcgtta ctctccaaga cctccccgaa acagagctgc ctgctgtgtt acagcctgtc 1380

gccgaagcta tggatgctat tgccgccgcc gacctcagtc aaaccagcgg cttcggccca 1440

ttcgggcccc aaggcatcgg ccagtacaca acctggcggg atttcatttg cgccattgct 1500

gatccccatg tctaccactg gcagaccgtg atggacgaca ccgtgtccgc cagcgtagct 1560

caagccctgg acgaactgat gctgtgggcc gaagactgtc ccgaggtgcg ccacctcgtc 1620

catgccgact tcggcagcaa caacgtcctg accgacaacg gccgcatcac cgccgtaatc 1680

gactggtccg aagctatgtt cggggacagt cagtacgagg tggccaacat cttcttctgg 1740

cggccctggc tggcttgcat ggagcagcag actcgctact tcgagcgccg gcatcccgag 1800

ctggccggca gccctcgtct gcgagcctac atgctgcgca tcggcctgga tcagctctac 1860

cagagcctcg tggacggcaa cttcgacgat gctgcctggg ctcaaggccg ctgcgatgcc 1920

atcgtccgca gcggggccgg caccgtcggt cgcacacaaa tcgctcgccg gagcgcagcc 1980

gtatggaccg acggctgcgt cgaggtgctg gccgacagcg gcaaccgccg gcccagtaca 2040

cgaccgcgcg ctaaggaggt aggtcgagtt taaactctag aaccggtcat ggccgcaata 2100

aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgttcga actagatgct 2160

gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca 2220

tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc 2280

cggcagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 2340

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 2400

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 2460

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 2520

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 2580

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 2640

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 2700

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 2760

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 2820

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 2880

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 2940

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 3000

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 3060

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 3120

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 3180

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag cggccgcaaa 3240

tgctaaacca ctgcagtggt taccagtgct tgatcagtga ggcaccgatc tcagcgatct 3300

gcctatttcg ttcgtccata gtggcctgac tccccgtcgt gtagatcact acgattcgtg 3360

agggcttacc atcaggcccc agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc 3420

ccgatttgtc agcaatgaac cagccagcag ggagggccga gcgaagaagt ggtcctgcta 3480

ctttgtccgc ctccatccag tctatgagct gctgtcgtga tgctagagta agaagttcgc 3540

cagtgagtag tttccgaaga gttgtggcca ttgctactgg catcgtggta tcacgctcgt 3600

cgttcggtat ggcttcgttc aactctggtt cccagcggtc aagccgggtc acatgatcac 3660

ccatattatg aagaaatgca gtcagctcct tagggcctcc gatcgttgtc agaagtaagt 3720

tggccgcggt gttgtcgctc atggtaatgg cagcactaca caattctctt accgtcatgc 3780

catccgtaag atgcttttcc gtgaccggcg agtactcaac caagtcgttt tgtgagtagt 3840

gtatacggcg accaagctgc tcttgcccgg cgtctatacg ggacaacacc gcgccacata 3900

gcagtacttt gaaagtgctc atcatcggga atcgttcttc ggggcggaaa gactcaagga 3960

tcttgccgct attgagatcc agttcgatat agcccactct tgcacccagt tgatcttcag 4020

catcttttac tttcaccagc gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa 4080

agaagggaat gagtgcgaca cgaaaatgtt ggatgctcat 4120

<210> 39

<211> 4120

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic nucleotide

<400> 39

aaattctcac ggcttccctc ccgaggtgga ggagcaggcc gccggcaccc tgcccatgag 60

ctgcgcccag gagagcggca tggatagaca ccctgctgct tgcgccagcg ccaggatcaa 120

cgtcttcgaa ttgggaggtg gcggtagcgg aggtggcggt agcctcgaga gctccaattc 180

actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 240

ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 300

ctagtgaggc cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac 360

cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 420

atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 480

gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg 540

tggtaaaatc gataaggatc cgtttgcgta ttgggcgctc ttccgctgat ctgcgcagca 600

ccatggcctg aaataacctc tgaaagagga acttggttag ctaccttctg aggcggaaag 660

aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc 720

agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc 780

tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg 840

cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 900

ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc 960

cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cgattcttct 1020

gacactagcg ccaccatgaa gaagcccgaa ctcaccgcta ccagcgttga aaaatttctc 1080

atcgagaagt tcgacagtgt gagcgacctg atgcagttgt cggagggcga agagagccga 1140

gccttcagct tcgatgtcgg cggacgcggc tatgtactgc gggtgaatag ctgcgctgat 1200

ggcttctaca aagaccgcta cgtgtaccgc cacttcgcca gcgctgcact acccatcccc 1260

gaagtgttgg acatcggcga gttcagcgag agcctgacat actgcatcag tagacgcgcc 1320

caaggcgtta ctctccaaga cctccccgaa acagagctgc ctgctgtgtt acagcctgtc 1380

gccgaagcta tggatgctat tgccgccgcc gacctcagtc aaaccagcgg cttcggccca 1440

ttcgggcccc aaggcatcgg ccagtacaca acctggcggg atttcatttg cgccattgct 1500

gatccccatg tctaccactg gcagaccgtg atggacgaca ccgtgtccgc cagcgtagct 1560

caagccctgg acgaactgat gctgtgggcc gaagactgtc ccgaggtgcg ccacctcgtc 1620

catgccgact tcggcagcaa caacgtcctg accgacaacg gccgcatcac cgccgtaatc 1680

gactggtccg aagctatgtt cggggacagt cagtacgagg tggccaacat cttcttctgg 1740

cggccctggc tggcttgcat ggagcagcag actcgctact tcgagcgccg gcatcccgag 1800

ctggccggca gccctcgtct gcgagcctac atgctgcgca tcggcctgga tcagctctac 1860

cagagcctcg tggacggcaa cttcgacgat gctgcctggg ctcaaggccg ctgcgatgcc 1920

atcgtccgca gcggggccgg caccgtcggt cgcacacaaa tcgctcgccg gagcgcagcc 1980

gtatggaccg acggctgcgt cgaggtgctg gccgacagcg gcaaccgccg gcccagtaca 2040

cgaccgcgcg ctaaggaggt aggtcgagtt taaactctag aaccggtcat ggccgcaata 2100

aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgttcga actagatgct 2160

gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca 2220

tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc 2280

cggcagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 2340

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 2400

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 2460

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 2520

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 2580

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 2640

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 2700

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 2760

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 2820

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 2880

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 2940

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 3000

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 3060

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 3120

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 3180

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag cggccgcaaa 3240

tgctaaacca ctgcagtggt taccagtgct tgatcagtga ggcaccgatc tcagcgatct 3300

gcctatttcg ttcgtccata gtggcctgac tccccgtcgt gtagatcact acgattcgtg 3360

agggcttacc atcaggcccc agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc 3420

ccgatttgtc agcaatgaac cagccagcag ggagggccga gcgaagaagt ggtcctgcta 3480

ctttgtccgc ctccatccag tctatgagct gctgtcgtga tgctagagta agaagttcgc 3540

cagtgagtag tttccgaaga gttgtggcca ttgctactgg catcgtggta tcacgctcgt 3600

cgttcggtat ggcttcgttc aactctggtt cccagcggtc aagccgggtc acatgatcac 3660

ccatattatg aagaaatgca gtcagctcct tagggcctcc gatcgttgtc agaagtaagt 3720

tggccgcggt gttgtcgctc atggtaatgg cagcactaca caattctctt accgtcatgc 3780

catccgtaag atgcttttcc gtgaccggcg agtactcaac caagtcgttt tgtgagtagt 3840

gtatacggcg accaagctgc tcttgcccgg cgtctatacg ggacaacacc gcgccacata 3900

gcagtacttt gaaagtgctc atcatcggga atcgttcttc ggggcggaaa gactcaagga 3960

tcttgccgct attgagatcc agttcgatat agcccactct tgcacccagt tgatcttcag 4020

catcttttac tttcaccagc gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa 4080

agaagggaat gagtgcgaca cgaaaatgtt ggatgctcat 4120

The claims (modification according to treaty clause 19)

1. A method of assessing promoter region activity comprising:

culturing a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) operably coupled to a promoter region under conditions wherein the ED is expressed when the promoter region is active;

contacting the ED with an enzyme receptor (EA) to form an ED-EA complex having enzymatic activity, if expressed;

detecting the level of the enzyme activity to assess the activity of the promoter region; and

assessing the level of activation of a transcription factor based on the detected level of the enzyme activity.

2. The method of claim 1, wherein the promoter region comprises a Transcription Factor Response Element (TFRE) for a transcription factor of interest, and wherein the activity of the promoter region is indicative of the activity of the transcription factor.

3. The method of claim 2, wherein the promoter region comprises at least one TFRE.

4. The method of claim 1, further comprising introducing an expression vector encoding the transcription factor into the cell and culturing the cell under conditions in which the transcription factor is expressed.

5. The method of claim 1, further comprising contacting the cell with an agent, and assessing the level of activity of the promoter region in response to contacting the cell with the agent based on the detected level of the enzyme activity.

6. The method of claim 5, wherein the activity of the promoter region is indicative of an activity of a target cell signaling pathway.

7. The method of claim 5, wherein contacting the cell with the agent comprises culturing the cell in the presence of the agent.

8. The method of claim 5, wherein assessing the level of activity of the promoter region in response to contacting the cell with the agent comprises comparing the level of enzyme activity detected in the absence of the agent to the level of enzyme activity detected in the presence of the agent.

9. The method of claim 5, wherein the agent is a small molecule.

10. The method of claim 5, wherein the reagent is a detection reagent.

11. The method of claim 5, wherein the cell is contacted with more than one detection reagent.

12. The method of claim 1, wherein the nucleic acid further encodes a carrier protein fused to the ED, such that an ED-carrier protein fusion is expressed when the promoter region is active.

13. The method of claim 12, wherein the carrier protein exhibits an enzymatic activity that is different from the enzymatic activity of the ED-EA complex.

14. The method of claim 1, wherein detecting the level of the enzymatic activity comprises providing a substrate for the ED-EA complex, wherein a detectable signal is generated upon hydrolysis of the substrate by the ED-EA complex.

15. The method of claim 1, wherein the ED comprises the amino acid sequence set forth in SEQ ID No.30 or a variant thereof that complexes with the EA to form an ED-EA complex.

16. A method of assessing promoter region activity comprising:

culturing a cell comprising a nucleic acid comprising a region encoding a carrier protein fused to an Enzyme Donor (ED) to form an ED-carrier protein fusion, wherein the ED-carrier protein fusion is operably coupled to a promoter region, under conditions wherein the ED-carrier protein fusion is expressed when the promoter region is active;

contacting the ED with an enzyme receptor (EA) to form an ED-EA complex having enzymatic activity, if expressed;

detecting the level of the enzyme activity to assess the activity of the promoter region; and

assessing the level of activation of a transcription factor based on the detected level of the enzyme activity.

17. The method of claim 16, wherein the promoter region comprises a Transcription Factor Response Element (TFRE) for a transcription factor of interest, and wherein the activity of the promoter region is indicative of the activity of the transcription factor.

18. The method of claim 17 wherein the carrier protein comprises a domain selected to affect the stability of the ED-carrier protein fusion.

19. The method of claim 18, wherein the domain is selected to increase the stability of an ED-carrier protein fusion as compared to the ED-carrier protein fusion lacking the domain.

20. The method of claim 18, wherein the domain is selected to destabilize the ED-carrier protein compared to an ED-carrier protein lacking the domain.

21. The method of claim 16, further comprising introducing an expression vector encoding the transcription factor into the cell and culturing the cell under conditions in which the transcription factor is expressed.

22. The method of claim 16, comprising contacting the cell with an agent, and assessing the level of activity of the promoter region in response to contacting the cell with the agent based on the detected level of the enzyme activity.

23. The method of claim 16, wherein the activity of the promoter region is indicative of an activity of a target cell signaling pathway.

24. The method of claim 22, wherein assessing the level of activity of the promoter region in response to contacting the cell with the agent comprises comparing the level of enzyme activity detected in the absence of the agent to the level of enzyme activity detected in the presence of the agent.

25. The method of claim 22, wherein the agent is a small molecule.

26. The method of claim 22, wherein the reagent is a detection reagent.

27. A kit, comprising:

a cell comprising a nucleic acid comprising a region encoding an Enzyme Donor (ED) operably coupled to a promoter region; and

instructions for using the cell to perform the method of any one of claims 1 to 26.

28. The kit of claim 27, further comprising instructions for: assessing the level of activation of the transcription factor, or a pathway that activates the transcription factor, based on the detected level of the enzyme activity; contacting the cells with an agent during the culturing; and assessing activation of the promoter region in response to contacting the cell with the agent based on the detected level of the enzyme activity.