Methods and Compositions for Diagnosing Disease

Abstract

The present invention relates to methods and compositions for diagnosing a disease or disorder in a subject by introducing into cells of the subject a diagnostic gene switch construct and monitoring expression of a reporter gene. The invention further relates to methods and compositions for monitoring the progression of a disease or disorder or the effectiveness of a treatment for a disease or disorder.

Description

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The content of the sequence listing text file (File Name: Sequence Listing.ST25.txt; Size: 107 KB bytes; and Date of Creation: Aug. 22, 2008) filed herewith with the application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to methods and compositions for diagnosing a disease or disorder in a subject by introducing into cells of the subject a diagnostic gene switch construct and monitoring expression of a reporter gene. The invention further relates to methods and compositions for monitoring the progression of a disease or disorder or monitoring the effectiveness or toxicity of a treatment for a disease or disorder.

[0004] 2. Background Art

[0005] Diagnostic tests for the presence of a disease in a subject have long been in existence, but researchers are constantly searching for improved tests exhibiting increased sensitivity (allowing earlier detection) and specificity (eliminating false positives and false negatives). Other desired characteristics for diagnostic tests include ease of use, rapid results, and the ability to constantly monitor progression of a disease or the effectiveness of ongoing treatment.

[0006] Thus, there is a need in the art for new diagnostic methods and compositions that provide these desired characteristics.

SUMMARY OF THE INVENTION

[0007] The present invention is based on a combination of the specificity and sensitivity provided by the use of disease specific promoters to detect a disease coupled with the regulatory control of a ligand-dependent gene switch system to provide diagnostic and monitoring methods. The present invention relates to methods and compositions for diagnosing a disease or disorder in a subject. The invention further relates to methods and compositions for monitoring the progression of a disease or disorder in a subject or monitoring the effectiveness or toxicity of an administered treatment for a disease or disorder in a subject.

[0008] One embodiment of the invention comprises methods of diagnosing a disease or disorder in a subject, comprising:

[0009] (a) introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0010] (2) administering ligand to said modified cells; and

[0011] (3) detecting reporter gene expression;

[0012] wherein expression of the reporter gene indicates that said subject has said disease or disorder.

[0013] In one embodiment, the diagnostic methods are carried out ex vivo in cells that have been isolated from said subject.

[0014] In one embodiment, the diagnostic methods are carried out by introducing the compositions of the invention into cells that have been isolated from said subject to produce modified cells, and the modified cells are re-introduced into said subject.

[0015] In one embodiment, the diagnostic methods are carried out in vivo.

[0016] In a further embodiment, the diagnostic methods are carried out using non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the subject, and the modified non-autologous cells are introduced into the subject. In one embodiment, the non-autologous cells are surrounded by a barrier (e.g., encapsulated) prior to being introduced into the subject.

[0017] In one aspect of the invention, the gene switch is an ecdysone receptor (EcR)-based gene switch.

[0018] In one embodiment, the gene switch comprises a first transcription factor sequence under the control of a first diagnostic switch promoter and a second transcription factor sequence under the control of a second diagnostic switch promoter,

[0019] wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor.

[0020] In another aspect of the invention, said first transcription factor sequence encodes a protein comprising a heterodimer partner and a transactivation domain and said second transcription factor sequence encodes a protein comprising a DNA binding domain and a ligand-binding domain.

[0021] An additional embodiment of the invention relates to methods of monitoring the progression of a disease or disorder in a subject, comprising:

[0022] (a) introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0023] (b) administering ligand to said modified cells; and

[0024] (c) detecting reporter gene expression at least twice;

[0025] wherein a change in the level of expression of said reporter gene indicates progression of said disease or disorder in said subject.

[0026] A further embodiment of the invention relates to methods of monitoring the effectiveness of a treatment for a disease or disorder in a subject, comprising:

[0027] (a) administering said treatment to said subject;

[0028] (b) introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0029] (c) administering ligand to said modified cells; and

[0030] (d) detecting reporter gene expression at least twice;

[0031] wherein a change in the level of expression of said reporter gene indicates the effectiveness of said treatment.

[0032] Another embodiment of the invention relates to methods of monitoring the potential toxicity of an administered treatment for a disease or disorder in a subject, comprising:

[0033] (a) administering said treatment to said subject;

[0034] (b) introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by factors found in cells that are being exposed to toxic conditions, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0035] (c) administering ligand to said modified cells; and

[0036] (d) detecting reporter gene expression at least twice;

[0037] wherein a change in the level of expression of said reporter gene indicates the toxicity of said treatment.

[0038] Another embodiment of the invention relates to methods of monitoring the level of a factor that is being administered to a subject for treatment for a disease or disorder, comprising:

[0039] (a) administering said treatment to said subject;

[0040] (b) introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by said factor that is being administered for treatment, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0041] (c) administering ligand to said modified cells; and

[0042] (d) detecting reporter gene expression;

[0043] wherein the level of expression of said reporter gene indicates the level of the factor being administered for treatment.

[0044] In a further embodiment, each of the methods may be carried out using non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the subject, and the modified non-autologous cells are administered to the subject. In one embodiment, the modified non-autologous cells are surrounded by a barrier (e.g., encapsulated) prior to being introduced into the subject.

[0045] One embodiment of the invention comprises methods of detecting transplant rejection in a subject that has received an organ or tissue transplant, comprising:

[0046] (a) introducing into cells of said organ or tissue transplant (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during transplant rejection, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0047] (b) administering ligand to said modified cells; and

[0048] (c) detecting reporter gene expression;

[0049] wherein expression of the reporter gene indicates that transplant rejection has been detected.

[0050] An additional embodiment of the invention relates to methods of monitoring the progression of transplant rejection in a subject that has received an organ or tissue transplant, comprising:

[0051] (a) introducing into cells of said organ or tissue transplant (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during transplant rejection, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0052] (b) administering ligand to said modified cells; and

[0053] (c) detecting reporter gene expression at least twice;

[0054] wherein a change in the level of expression of said reporter gene indicates progression of said transplant rejection in said subject.

[0055] In a further embodiment, the methods of detecting or monitoring transplant rejection may be carried out by introducing the polynucleotides of the invention into non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the organ or tissue being transplanted, and the modified non-autologous cells are introduced to the organ or tissue prior to transplantation. In one embodiment, the modified non-autologous cells are surrounded by a barrier (e.g., encapsulated) prior to being introduced into the organ or tissue.

[0056] In the methods described above, in one embodiment, the polynucleotide encoding the gene switch and the polynucleotide encoding the reporter gene linked to a promoter are part of one larger polynucleotide, e.g., a vector. In another embodiment, the polynucleotide encoding the gene switch and the polynucleotide encoding the reporter gene linked to a promoter are separate polynucleotides.

[0057] The invention further relates to diagnostic gene switch constructs that are useful in the disclosed methods.

[0058] The invention additionally relates to vectors comprising the diagnostic gene switch constructs of the invention.

[0059] The invention also relates to kits for carrying out the methods of the invention, comprising, e.g., gene switch constructs, vectors, ligands, etc. In one embodiment, the kits may comprise cells (e.g., autologous or non-autologous cells) that may comprise the polynucleotides of the invention. The non-autologous cells may be surrounded by a barrier (e.g., encapsulated).

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0060] FIG. 1 shows an embodiment of the diagnostic gene switch of the invention in which two transcription factor sequences encoding two separate portions of a ligand-dependent transcription factor are under the control of different promoters. "Dx-Switch Components" represents a gene switch; "AD" represents a transactivation domain; "DBD" represents a DNA binding domain; "LBD" represents a ligand binding domain; "StandardDx-Reporter" represents a reporter gene; and "P1" and "P2" represent two different disease- or disorder-responsive promoters. In an alternative embodiment of FIG. 1, "P1" is a constitutive promoter; and "P2" and "P3" are different disease- or disorder-responsive promoters.

[0061] FIG. 2 shows an embodiment of the diagnostic gene switch of the invention in which two transcription factor sequences encoding two separate portions of a ligand-dependent transcription factor are under the control of different promoters. "Dx-Switch Components" represents a gene switch; "AD" represents a transactivation domain; "DBD-A" represents a first DNA binding domain; "DBD-B" represents a second DNA binding domain; "LBD" represents a ligand binding domain; "StandardDx-Reporter-A" represents a first reporter gene; "StandardDx-Reporter-B" represents a second reporter gene; and "P1," "P2," and "P3" represent three different disease- or disorder-responsive promoters. In an alternative embodiment of FIG. 2, "P1" is a constitutive promoter; and "P2" and "P3" are different disease- or disorder-responsive promoters.

[0062] FIG. 3 shows an embodiment of the diagnostic gene switch of the invention in which two transcription factor sequences encoding two separate portions of a ligand-dependent transcription factor are under the control of different diagnostic switch promoters. "Dx-Switch Components" represents a gene switch; "AD" represents a transactivation domain; "DBD" represents a DNA binding domain; "LBD" represents a ligand binding domain; "StandardDx-Reporter" represents a reporter gene; and "P1," "P2," "P3," and "P4" represent four different disease- or disorder-responsive promoters.

[0063] FIG. 4 shows an embodiment of the diagnostic gene switch of the invention in which two transcription factor sequences encoding two separate portions of a ligand-dependent transcription factor are under the control of different promoters and a control reporter gene is present. "Dx-Switch Components" represents a gene switch; "AD" represents a transactivation domain; "DBD-A" represents a first DNA binding domain; "DBD-B" represents a second DNA binding domain; "LBD" represents a ligand binding domain; "StandardDx-Reporter-A" represents a first reporter gene; "Control-Reporter-B" represents a second reporter gene; and "P1" and "P2" represent two different disease- or disorder-responsive promoters; and "P3" and "P4" represent two different control promoters. In an alternative embodiment of FIG. 4, "P3" and "P4" are constitutive promoters.

[0064] FIG. 5 shows an embodiment of a single promoter shuttle vector (SEQ ID No.: 5), which includes the IL-24/mda-7 promoter. Adenovirus produced using this vector is used to transduce cells isolated from lymphatic samples.

[0065] FIG. 6 shows an embodiment of a dual promoter vector (SEQ ID NO.: 6), which includes TRPM4 and TRGC1/TARP promoters. This DNA vector is used to transduce a prostate biopsy using non-viral transduction systems.

[0066] FIG. 7 shows an embodiment of a single promoter vector (SEQ ID NO.: 7), which includes the ADAM-17 promoter and the CD95-ADAM8 dual reporter (SEQ ID NO.: 10).

[0067] FIG. 8 shows an embodiment of a dual promoter vector (SEQ ID NO.: 8), which includes the CXCL9 and SEMA7A promoters and the CD40-CD3 dual reporter (SEQ ID NO.: 12).

[0068] FIG. 9 shows an embodiment of a single promoter vector (SEQ ID NO.: 9), which includes the ADAM-17 promoter and the alkaline phosphatase-c terminal CD40 reporter (SEQ ID NO.: 14).

[0069] FIG. 10 shows embodiments of the serum-based reporters that are designed to exhibit no immunogenic profile when expressed within the human body. These reporters are made up of human based amino acid sequences that are present either on the cell surface or within the serum naturally. Hence, these reporters are not immunogenic nor are they subject to immune attack when expressed in the human body. In one embodiment, serum-based reporters are dual epitope reporter, for e.g., CD95-ADAM8 reporter (SEQ ID NOs.: 10-11), CD40-CD3 reporter (SEQ ID NOs.: 12-13), CD28-CD3 reporter (SEQ ID. NOs.: 16-17) and CD28-CD40 reporter (SEQ ID NOs.: 18-19) that allows ELISA based capture and detection. The designs utilize a signal peptide (Signal P) for transport into the secretory pathway, followed by epitopes from cell surface antigens with linkers (L). In alternative embodiments, different combinations of linkers and epitopes are used for each design. In another embodiment, the serum-based reporter is an alkaline phosphatase reporter, for e.g., alkaline phosphatase-c terminal CD40 reporter (SEQ ID NOs.: 14-15), that allows immunocapture followed by enzymatic detection of reporter activity. Alkaline phosphatase reporters utilize the tissue non-specific alkaline phosphatase for an enzymatic reporter that can be secreted. An epitope from a cell surface antigen is included at the carboxy terminus for immunocapture prior to measurement of alkaline phosphatase activity. In additional embodiments of FIG. 10, additional alkaline phosphatase reporters are: alkaline phosphatase--amiono terminal CD40 reporter (SEQ ID NOs.: 20-21) and alkaline phosphatase--c terminal CD28 reporter (SEQ ID. NOs.: 22-23).

DETAILED DESCRIPTION OF THE INVENTION

[0070] The invention relates to methods and compositions for using a gene switch for the diagnosis of diseases or disorders in a subject. The invention further relates to methods and compositions for monitoring the progression of diseases or disorders or the treatment thereof in a subject. The methods of the invention can be carried out either ex vivo (by introducing the gene switch into isolated cells of a subject) or in vivo (by introducing the gene switch into isolated cells of a subject and reintroducing the cells to the subject or by introducing the gene switch directly into cells of the subject). In another embodiment, the cells harboring the gene switch may be non-autologous cells (e.g., allogeneic or xenogeneic cells). The non-autologous cells may be surrounded by a barrier that prevents the non-autologous cells from raising an immune response after introduction and/or prevents the non-autologous cells from escaping from the site of introduction. The methods of the invention involve the use of a gene switch in which expression of a ligand-dependent transcription factor is under the control of one or more diagnostic switch promoters. The methods and compositions described herein provide a highly sensitive and highly specific diagnostic technique in which the timing of the diagnostic step is controlled by administration of ligand to cells comprising the gene switch, permitting optimal detection of the presence of a disease or disorder as well as continuous or intermittent monitoring of the progression of a disease or disorder or the effectiveness or toxicity of a treatment.

[0071] The following definitions are provided and should be helpful in understanding the scope and practice of the present invention.

[0072] The term "isolated" for the purposes of the present invention designates a biological material (cell, nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered "isolated."

[0073] The term "purified," as applied to biological materials does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. It is rather a relative definition.

[0074] "Nucleic acid," "nucleic acid molecule," "oligonucleotide," and "polynucleotide" are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.

[0075] The term "fragment," as applied to polynucleotide sequences, refers to a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence identical to the reference nucleic acid. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000, 3000, 4000, 5000, or more consecutive nucleotides of a nucleic acid according to the invention.

[0076] As used herein, an "isolated nucleic acid fragment" refers to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

[0077] A "gene" refers to a polynucleotide comprising nucleotides that encode a functional molecule, including functional molecules produced by transcription only (e.g., a bioactive RNA species) or by transcription and translation (e.g., a polypeptide). The term "gene" encompasses cDNA and genomic DNA nucleic acids. "Gene" also refers to a nucleic acid fragment that expresses a specific RNA, protein or polypeptide, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and/or coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A chimeric gene may comprise coding sequences derived from different sources and/or regulatory sequences derived from different sources. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene or "heterologous" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

[0078] "Heterologous DNA" refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. The heterologous DNA may include a gene foreign to the cell.

[0079] The term "genome" includes chromosomal as well as mitochondrial, chloroplast and viral DNA or RNA.

[0080] A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook et al. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.

[0081] Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T_m of 55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringency hybridization conditions correspond to a higher T_m, e.g., 40% formamide, with 5× or 6×SSC. High stringency hybridization conditions correspond to the highest T_m, e.g., 50% formamide, 5× or 6×SSC.

[0082] Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as disclosed or used herein as well as those substantially similar nucleic acid sequences.

[0083] In one embodiment of the invention, polynucleotides are detected by employing hybridization conditions comprising a hybridization step at T_m, of 55° C., and utilizing conditions as set forth above. In other embodiments, the T_m is 60° C., 63° C., or 65° C.

[0084] Post-hybridization washes also determine stringency conditions. One set of conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 minutes (min), then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS is increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

[0085] The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T_m) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T_m have been derived (see Sambrook et al., supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8).

[0086] In one embodiment of the invention, polynucleotides are detected by employing hybridization conditions comprising a hybridization step in less than 500 mM salt and at least 37° C., and a washing step in 2×SSPE at a temperature of at least 63° C. In another embodiment, the hybridization conditions comprise less than 200 mM salt and at least 37° C. for the hybridization step. In a further embodiment, the hybridization conditions comprise 2×SSPE and 63° C. for both the hybridization and washing steps.

[0087] In another embodiment, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; e.g., at least about 20 nucleotides; e.g., at least 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

[0088] The term "probe" refers to a single-stranded nucleic acid molecule that can base pair with a complementary single stranded target nucleic acid to form a double-stranded molecule.

[0089] As used herein, the term "oligonucleotide" refers to a short nucleic acid that is hybridizable to a genomic DNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule. Oligonucleotides can be labeled, e.g., with ³²P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. A labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. Oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of a nucleic acid, for DNA sequencing, or to detect the presence of a nucleic acid. An oligonucleotide can also be used to form a triple helix with a DNA molecule. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.

[0090] A "primer" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions. Such primers may be used in a polymerase chain reaction or for DNA sequencing.

[0091] "Polymerase chain reaction" is abbreviated PCR and refers to an in vitro method for enzymatically amplifying specific nucleic acid sequences. PCR involves a repetitive series of temperature cycles with each cycle comprising three stages: denaturation of the template nucleic acid to separate the strands of the target molecule, annealing a single stranded PCR oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase. PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.

[0092] "Reverse transcription-polymerase chain reaction" is abbreviated RT-PCR and refers to an in vitro method for enzymatically producing a target cDNA molecule or molecules from an RNA molecule or molecules, followed by enzymatic amplification of a specific nucleic acid sequence or sequences within the target cDNA molecule or molecules as described above. RT-PCR also provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.

[0093] A DNA "coding sequence" refers to a double-stranded DNA sequence that encodes a polypeptide and can be transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of suitable regulatory sequences. "Suitable regulatory sequences" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0094] "Open reading frame" is abbreviated ORF and refers to a length of nucleic acid sequence, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.

[0095] The term "head-to-head" is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a head-to-head orientation when the 5' end of the coding strand of one polynucleotide is adjacent to the 5' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds away from the 5' end of the other polynucleotide. The term "head-to-head" may be abbreviated (5')-to-(5') and may also be indicated by the symbols ( →) or (3'5'5'→3').

[0096] The term "tail-to-tail" is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a tail-to-tail orientation when the 3' end of the coding strand of one polynucleotide is adjacent to the 3' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds toward the other polynucleotide. The term "tail-to-tail" may be abbreviated (3')-to-(3') and may also be indicated by the symbols (→ ) or (5'→3'3'5').

[0097] The term "head-to-tail" is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a head-to-tail orientation when the 5' end of the coding strand of one polynucleotide is adjacent to the 3' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds in the same direction as that of the other polynucleotide. The term "head-to-tail" may be abbreviated (5')-to-(3') and may also be indicated by the symbols (→ →) or (5'→3'5'→3').

[0098] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In particular, downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0099] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In particular, upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0100] The terms "restriction endonuclease" and "restriction enzyme" are used interchangeably and refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.

[0101] "Homologous recombination" refers to the insertion of a foreign DNA sequence into another DNA molecule, e.g., insertion of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.

[0102] Several methods known in the art may be used to propagate a polynucleotide according to the invention. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As described herein, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.

[0103] A "vector" refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. Possible vectors include, for example, plasmids or modified viruses including, for example bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives, or the Bluescript vector. Another example of vectors that are useful in the present invention is the UltraVector® Production System (Intrexon Corp., Blacksburg, Va.) as described in WO 2007/038276, incorporated herein by reference. For example, the insertion of the DNA fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate DNA fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the DNA molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) into the DNA termini. Such vectors may be engineered to contain selectable marker genes that provide for the selection of cells that have incorporated the marker into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.

[0104] Viral vectors, and particularly retroviral vectors, have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers. In addition to a nucleic acid, a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).

[0105] The term "plasmid" refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0106] A "cloning vector" refers to a "replicon," which is a unit length of a nucleic acid, preferably DNA, that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. Cloning vectors may be capable of replication in one cell type and expression in another ("shuttle vector"). Cloning vectors may comprise one or more sequences that can be used for selection of cells comprising the vector and/or one or more multiple cloning sites for insertion of sequences of interest.

[0107] The term "expression vector" refers to a vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence following transformation into the host. The cloned gene, i.e., the inserted nucleic acid sequence, is usually placed under the control of control elements such as a promoter, a minimal promoter, an enhancer, or the like. Initiation control regions or promoters, which are useful to drive expression of a nucleic acid in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving expression of these genes can be used in an expression vector, including but not limited to, viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue specific promoters, pathogenesis or disease related promoters, developmental specific promoters, inducible promoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4, GAL10, ADH1, PGK, PHOS, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase promoters (useful for expression in Saccharomyces); AOX1 promoter (useful for expression in Pichia); β-lactamase, lac, ara, tet, tip, lP_L, lP_R, T7, tac, and trc promoters (useful for expression in Escherichia coli); light regulated-, seed specific-, pollen specific-, ovary specific-, cauliflower mosaic virus 35S, CMV 35S minimal, cassava vein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose 1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase, stress inducible, rice tungro bacilliform virus, plant super-promoter, potato leucine aminopeptidase, nitrate reductase, mannopine synthase, nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters (useful for expression in plant cells); animal and mammalian promoters known in the art including, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, a baculovirus IE1 promoter, an elongation factor 1 alpha (EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), pathogenesis or disease related-promoters, and promoters that exhibit tissue specificity and have been utilized in transgenic animals, such as the elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region active in pancreatic beta cells, immunoglobulin gene control region active in lymphoid cells, mouse mammary tumor virus control region active in testicular, breast, lymphoid and mast cells; albumin gene, Apo AI and Apo AII control regions active in liver, alpha-fetoprotein gene control region active in liver, alpha 1-antitrypsin gene control region active in the liver, beta-globin gene control region active in myeloid cells, myelin basic protein gene control region active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region active in skeletal muscle, and gonadotropic releasing hormone gene control region active in the hypothalamus, pyruvate kinase promoter, villin promoter, promoter of the fatty acid binding intestinal protein, promoter of the smooth muscle cell α-actin, and the like. In addition, these expression sequences may be modified by addition of enhancer or regulatory sequences and the like.

[0108] Vectors may be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311).

[0109] A polynucleotide according to the invention can also be introduced in vivo by lipofection. For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA. 84:7413 (1987); Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027 (1988); and Ulmer et al., Science 259:1745 (1993)). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner et al., Science 337:387 (1989)). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in WO95/18863, WO96/17823 and U.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly preferred in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey et al. 1988, supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

[0110] Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO95/21931).

[0111] It is also possible to introduce a vector in vivo as a naked DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., Hum. Gene Ther. 3:147 (1992); and Wu et al., J. Biol. Chem. 262:4429 (1987)).

[0112] The term "transfection" refers to the uptake of exogenous or heterologous RNA or DNA by a cell. A cell has been "transfected" by exogenous or heterologous RNA or DNA when such RNA or DNA has been introduced inside the cell. A cell has been "transformed" by exogenous or heterologous RNA or DNA when the transfected RNA or DNA effects a phenotypic change. The transforming RNA or DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

[0113] "Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

[0114] In addition, the recombinant vector comprising a polynucleotide according to the invention may include one or more origins for replication in the cellular hosts in which their amplification or their expression is sought, markers or selectable markers.

[0115] The term "selectable marker" refers to an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like.

[0116] The term "reporter gene" refers to a nucleic acid encoding an identifying factor that is able to be identified based upon the reporter gene's effect, wherein the effect is used to track the inheritance of a nucleic acid of interest, to identify a cell or organism that has inherited the nucleic acid of interest, and/or to measure gene expression induction or transcription. Examples of reporter genes known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like. Selectable marker genes may also be considered reporter genes.

[0117] "Promoter and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the transcription of a nucleic acid. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0118] The promoter sequence is typically bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of transcription factors that recruit RNA polymerase-mediated transcription.

[0119] "Diagnostic switch promoter" refers to a promoter the activity of which is modulated by a factor in a manner that can be used as a diagnostic in the present invention. The term encompasses promoters that increase or decrease expression of a coding sequence during a disease or disorder as a change in promoter activity in either direction will be diagnostic. The term includes, without limitation, disease-specific promoters, promoters responsive to particular physiological or pathological conditions, and promoters responsive to specific biological molecules. Diagnostic switch promoters can comprise the sequence of naturally occurring promoters, modified sequences derived from naturally occurring promoters, or synthetic sequences (e.g., insertion of a response element into a promoter sequence to alter the responsiveness of the promoter).

[0120] A "coding sequence" is a DNA sequence that encodes a polypeptide or a RNA (e.g., a functional RNA).

[0121] A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into RNA. If the coding sequence is a protein coding sequence, the primary RNA transcript is then further processed (e.g., trans-RNA spliced (if the coding sequence contains introns) and polyadenylated), exported to the cytoplasm, and translated into the protein encoded by the coding sequence. Non-protein-coding bioactive RNA species (including, but not limited to RNAi or microRNAs) can be functional in the nucleus as a primary transcript, a spliced transcript (with or without polyadenylation), and/or an excised intron; or can exert bioactivity in extra-nuclear cellular regions as any RNA form that is exported from the nucleus.

[0122] "Transcriptional and translational control sequences" refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

[0123] The term "response element" refers to one or more cis-acting DNA elements which confer responsiveness on a promoter mediated through interaction with the DNA-binding domains of a transcription factor. This DNA element may be either palindromic (perfect or imperfect) in its sequence or composed of sequence motifs or half sites separated by a variable number of nucleotides. The half sites can be similar or identical and arranged as either direct or inverted repeats or as a single half site or multimers of adjacent half sites in tandem. The response element may comprise a minimal promoter isolated from different organisms depending upon the nature of the cell or organism into which the response element will be incorporated. The DNA binding domain of the transcription factor binds, in the presence or absence of a ligand, to the DNA sequence of a response element to initiate or suppress transcription of downstream gene(s) under the regulation of this response element. Examples of DNA sequences for response elements of the natural ecdysone receptor include: RRGG/TTCANTGAC/ACYY (SEQ ID NO: 1) (see Cherbas et. al., Genes Dev. 5:120 (1991)); AGGTCAN.sub.(n)AGGTCA, where N.sub.(n) can be one or more spacer nucleotides (SEQ ID NO: 2) (see D'Avino et al., Mol. Cell. Endocrinol. 113:1 (1995)); and GGGTTGAATGAATTT (SEQ ID NO: 3) (see Antoniewski et al., Mol. Cell Biol. 14:4465 (1994)).

[0124] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0125] The term "expression" as used herein refers to the production of RNA (e.g., sense RNA, antisense RNA, microRNA, messenger RNA, heterologous nuclear RNA, ribosomal RNA, small interfering RNA, ribozymes, etc.) by transcription of a nucleic acid or polynucleotide. Expression may also include translation of mRNA into a protein or polypeptide.

[0126] The terms "cassette," "expression cassette" and "gene expression cassette" refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination. The segment of DNA comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation. "Transformation cassette" refers to a specific vector comprising a polynucleotide that encodes a polypeptide of interest and having elements in addition to the polynucleotide that facilitate transformation of a particular host cell. Cassettes, expression cassettes, gene expression cassettes and transformation cassettes of the invention may also comprise elements that allow for enhanced expression of a polynucleotide encoding a polypeptide of interest in a host cell. These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.

[0127] For purposes of this invention, the term "gene switch" refers to the combination of a response element associated with a promoter, and a ligand-dependent transcription factor-based system which, in the presence of one or more ligands, modulates the expression of a gene into which the response element and promoter are incorporated.

[0128] The term "ecdysone-based," with respect to a gene switch, refers to a gene switch comprising at least a functional part of a naturally occurring or synthetic ecdysone receptor ligand binding domain and which regulates gene expression in response to a ligand that binds to the ecdysone receptor ligand binding domain.

[0129] The terms "modulate" and "modulates" mean to induce, reduce or inhibit nucleic acid or gene expression, resulting in the respective induction, reduction or inhibition of protein or polypeptide production.

[0130] The polynucleotides or vectors according to the invention may further comprise at least one promoter suitable for driving expression of a gene in a host cell.

[0131] Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor 1 (EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.

[0132] Termination control regions, i.e., terminator or polyadenylation sequences, may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included. In a one embodiment of the invention, the termination control region may be comprised or be derived from a synthetic sequence, synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, or the like.

[0133] The terms "3' non-coding sequences" or "3' untranslated region (UTR)" refer to DNA sequences located downstream (3') of a coding sequence and may comprise polyadenylation [poly(A)] recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0134] "Regulatory region" refers to a nucleic acid sequence that regulates the expression of a second nucleic acid sequence. A regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin that are responsible for expressing different proteins or even synthetic proteins (a heterologous region). In particular, the sequences can be sequences of prokaryotic, eukaryotic, or viral genes or derived sequences that stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory regions include origins of replication, RNA splice sites, promoters, enhancers, transcriptional termination sequences, and signal sequences which direct the polypeptide into the secretory pathways of the target cell.

[0135] A regulatory region from a "heterologous source" refers to a regulatory region that is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences which do not occur in nature, but which are designed by one having ordinary skill in the art.

[0136] "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA (tnRNA)" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a double-stranded DNA that is complementary to and derived from mRNA. "Sense" RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. "Antisense RNA" refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene. The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet has an effect on cellular processes.

[0137] "Polypeptide," "peptide" and "protein" are used interchangeably and refer to a polymeric compound comprised of covalently linked amino acid residues.

[0138] An "isolated polypeptide," "isolated peptide" or "isolated protein" refer to a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.

[0139] A "substitution mutant polypeptide" or a "substitution mutant" will be understood to mean a mutant polypeptide comprising a substitution of at least one wild-type or naturally occurring amino acid with a different amino acid relative to the wild-type or naturally occurring polypeptide. A substitution mutant polypeptide may comprise only one wild-type or naturally occurring amino acid substitution and may be referred to as a "point mutant" or a "single point mutant" polypeptide. Alternatively, a substitution mutant polypeptide may comprise a substitution of two or more wild-type or naturally occurring amino acids with two or more amino acids relative to the wild-type or naturally occurring polypeptide. According to the invention, a Group H nuclear receptor ligand binding domain polypeptide comprising a substitution mutation comprises a substitution of at least one wild-type or naturally occurring amino acid with a different amino acid relative to the wild-type or naturally occurring Group H nuclear receptor ligand binding domain polypeptide.

[0140] When the substitution mutant polypeptide comprises a substitution of two or more wild-type or naturally occurring amino acids, this substitution may comprise either an equivalent number of wild-type or naturally occurring amino acids deleted for the substitution, i.e., 2 wild-type or naturally occurring amino acids replaced with 2 non-wild-type or non-naturally occurring amino acids, or a non-equivalent number of wild-type amino acids deleted for the substitution, i.e., 2 wild-type amino acids replaced with 1 non-wild-type amino acid (a substitution+deletion mutation), or 2 wild-type amino acids replaced with 3 non-wild-type amino acids (a substitution+insertion mutation).

[0141] Substitution mutants may be described using an abbreviated nomenclature system to indicate the amino acid residue and number replaced within the reference polypeptide sequence and the new substituted amino acid residue. For example, a substitution mutant in which the twentieth (20^th) amino acid residue of a polypeptide is substituted may be abbreviated as "x20z", wherein "x" is the amino acid to be replaced, "20" is the amino acid residue position or number within the polypeptide, and "z" is the new substituted amino acid. Therefore, a substitution mutant abbreviated interchangeably as "E20A" or "Glu20Ala" indicates that the mutant comprises an alanine residue (commonly abbreviated in the art as "A" or "Ala") in place of the glutamic acid (commonly abbreviated in the art as "E" or "Glu") at position 20 of the polypeptide.

[0142] A substitution mutation may be made by any technique for mutagenesis known in the art, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., J. Biol. Chem. 253:6551 (1978); Zoller et al., DNA 3:479 (1984); Oliphant et al., Gene 44:177 (1986); Hutchinson et al., Proc. Natl. Acad. Sci. USA 83:710 (1986)), use of TAB® linkers (Pharmacia), restriction endonuclease digestion/fragment deletion and substitution, PCR-mediated/oligonucleotide-directed mutagenesis, and the like. PCR-based techniques are preferred for site-directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

[0143] The term "fragment," as applied to a polypeptide, refers to a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence. Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part. Such fragments of a polypeptide according to the invention may have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200, 240, or 300 or more amino acids.

[0144] A "variant" of a polypeptide or protein refers to any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein. Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification. The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements. These variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) variants in which one or more of the amino acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art. In one embodiment, a variant polypeptide comprises at least about 14 amino acids.

[0145] The term "homology" refers to the percent of identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequence from one moiety to another can be determined by techniques known to the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s) and size determination of the digested fragments.

[0146] As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin," including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., Cell 50:667 (1987)). Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity. However, in common usage and in the present application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and not a common evolutionary origin.

[0147] Accordingly, the term "sequence similarity" in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., Cell 50:667 (1987)). In one embodiment, two DNA sequences are "substantially homologous" or "substantially similar" when at least about 50% (e.g., at least about 75%, 90%, or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art (see e.g., Sambrook et al., 1989, supra).

[0148] As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. "Substantially similar" also refers to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology. "Substantially similar" also refers to modifications of the nucleic acid fragments of the present invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

[0149] Moreover, the skilled artisan recognizes that substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), with the sequences exemplified herein. Substantially similar nucleic acid fragments of the present invention are those nucleic acid fragments whose DNA sequences are at least about 70%, 80%, 90% or 95% identical to the DNA sequence of the nucleic acid fragments reported herein.

[0150] Two amino acid sequences are "substantially homologous" or "substantially similar" when greater than about 40% of the amino acids are identical, or greater than 60% are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program.

[0151] The term "corresponding to" is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured. A nucleic acid or amino acid sequence alignment may include spaces. Thus, the term "corresponding to" refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.

[0152] A "substantial portion" of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403 (1993)); available at ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.

[0153] The term "percent identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using sequence analysis software such as the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences may be performed using the Clustal method of alignment (Higgins et al., CABIOS. 5:151 (1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method may be selected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0154] The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Typical sequence analysis software includes, but is not limited to, the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters which originally load with the software when first initialized.

[0155] "Chemically synthesized," as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

[0156] As used herein, two or more individually operable gene regulation systems are said to be "orthogonal" when; a) modulation of each of the given systems by its respective ligand, at a chosen concentration, results in a measurable change in the magnitude of expression of the gene of that system, and b) the change is statistically significantly different than the change in expression of all other systems simultaneously operable in the cell, tissue, or organism, regardless of the simultaneity or sequentially of the actual modulation. Preferably, modulation of each individually operable gene regulation system effects a change in gene expression at least 2-fold greater than all other operable systems in the cell, tissue, or organism, e.g., at least 5-fold, 10-fold, 100-fold, or 500-fold greater. Ideally, modulation of each of the given systems by its respective ligand at a chosen concentration results in a measurable change in the magnitude of expression of the gene of that system and no measurable change in expression of all other systems operable in the cell, tissue, or organism. In such cases the multiple inducible gene regulation system is said to be "fully orthogonal." The present invention is useful to search for orthogonal ligands and orthogonal receptor-based gene expression systems such as those described in US 2002/0110861 A1, which is incorporated herein by reference in its entirety.

[0157] The term "exogenous gene" means a gene foreign to the subject, that is, a gene which is introduced into the subject through a transformation process, an unmutated version of an endogenous mutated gene or a mutated version of an endogenous unmutated gene. The method of transformation is not critical to this invention and may be any method suitable for the subject known to those in the art. Exogenous genes can be either natural or synthetic genes and therapeutic genes which are introduced into the subject in the form of DNA or RNA which may function through a DNA intermediate such as by reverse transcriptase. Such genes can be introduced into target cells, directly introduced into the subject, or indirectly introduced by the transfer of transformed cells into the subject. The term "therapeutic gene" means a gene which imparts a beneficial function to the host cell in which such gene is expressed.

[0158] The term "ecdysone receptor complex" generally refers to a heterodimeric protein complex having at least two members of the nuclear receptor family, ecdysone receptor ("EcR") and ultraspiracle ("USP") proteins (see Yao et al., Nature 366:476 (1993)); Yao et al., Cell 71:63 (1992)). The functional EcR complex may also include additional protein(s) such as immunophilins. Additional members of the nuclear receptor family of proteins, known as transcriptional factors (such as DHR38, betaFTZ-1 or other insect homologs), may also be ligand dependent or independent partners for EcR and/or USP. The EcR complex can also be a heterodimer of EcR protein and the vertebrate homolog of ultraspiracle protein, retinoic acid-X-receptor ("RXR") protein. The term EcR complex also encompasses homodimer complexes of the EcR protein or USP.

[0159] An EcR complex can be activated by an active ecdysteroid or non-steroidal ligand bound to one of the proteins of the complex, inclusive of EcR, but not excluding other proteins of the complex. As used herein, the term "ligand," as applied to EcR-based gene switches, describes small and soluble molecules having the capability of activating a gene switch to stimulate expression of a polypeptide encoded therein. Examples of ligands include, without limitation, an ecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, and the like, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-diacylhydrazines such as those disclosed in U.S. Pat. Nos. 6,013,836; 5,117,057; 5,530,028; and 5,378,726 and U.S. Published Application Nos. 2005/0209283 and 2006/0020146; oxadiazolines as described in U.S. Published Application No. 2004/0171651; dibenzoylalkyl cyanohydrazines such as those disclosed in European Application No. 461,809; N-alkyl-N,N'-diaroylhydrazines such as those disclosed in U.S. Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those disclosed in European Application No. 234,994; N-aroyl-N-alkyl-N'-aroylhydrazines such as those described in U.S. Pat. No. 4,985,461; amidoketones such as those described in U.S. Published Application No. 2004/0049037; each of which is incorporated herein by reference and other similar materials including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, famesol, bile acids, 1,1-biphosphonate esters, juvenile hormone III, and the like. Examples of diacylhydrazine ligands useful in the present invention include RG-115819 (3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-(2-methyl-3-methoxy-benzoyl)-hydrazide- ), RG-115830 (3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide), and RG-115932 ((R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide). See U.S. application Ser. No. 12/155,111.

[0160] The EcR complex includes proteins which are members of the nuclear receptor superfamily wherein all members are characterized by the presence of an amino-terminal transactivation domain ("TA"), a DNA binding domain ("DBD"), and a ligand binding domain ("LBD") separated by a hinge region. Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD. The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for ecdysone response elements. These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins.

[0161] The DNA sequences making up the exogenous gene, the response element, and the EcR complex may be incorporated into archaebacteria, procaryotic cells such as Escherichia coli, Bacillus subtilis, or other enterobacteria, or eucaryotic cells such as plant or animal cells. However, because many of the proteins expressed by the gene are processed incorrectly in bacteria, eucaryotic cells are preferred. The cells may be in the form of single cells or multicellular organisms. The nucleotide sequences for the exogenous gene, the response element, and the receptor complex can also be incorporated as RNA molecules, preferably in the form of functional viral RNAs such as tobacco mosaic virus. Of the eucaryotic cells, vertebrate cells are preferred because they naturally lack the molecules which confer responses to the ligands of this invention for the EcR. As a result, they are "substantially insensitive" to the ligands of this invention. Thus, the ligands useful in this invention will have negligible physiological or other effects on transformed cells, or the whole organism. Therefore, cells can grow and express the desired product, substantially unaffected by the presence of the ligand itself.

[0162] The term "subject" means an intact insect, plant or animal. It is also anticipated that the ligands will work equally well when the subject is a fungus or yeast. When the subject is an intact animal, preferably the animal is a vertebrate, most preferably a mammal.

[0163] EcR ligands, when used with the EcR complex which in turn is bound to the response element linked to an exogenous gene (e.g., a reporter gene), provide the means for external temporal regulation of expression of the exogenous gene. The order in which the various components bind to each other, that is, ligand to receptor complex and receptor complex to response element, is not critical. Typically, modulation of expression of the exogenous gene is in response to the binding of the EcR complex to a specific control, or regulatory, DNA element. The EcR protein, like other members of the nuclear receptor family, possesses at least three domains, a transactivation domain, a DNA binding domain, and a ligand binding domain. This receptor, like a subset of the nuclear receptor family, also possesses less well-defined regions responsible for heterodimerization properties. Binding of the ligand to the ligand binding domain of EcR protein, after heterodimerization with USP or RXR protein, enables the DNA binding domains of the heterodimeric proteins to bind to the response element in an activated form, thus resulting in expression or suppression of the exogenous gene. This mechanism does not exclude the potential for ligand binding to either EcR or USP, and the resulting formation of active homodimer complexes (e.g. EcR+EcR or USP+USP). In one embodiment, one or more of the receptor domains can be varied producing a chimeric gene switch. Typically, one or more of the three domains may be chosen from a source different than the source of the other domains so that the chimeric receptor is optimized in the chosen host cell or organism for transactivating activity, complementary binding of the ligand, and recognition of a specific response element. In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein from yeast (see Sadowski et al., Nature 335:563 (1988) or LexA protein from E. coli (see Brent et al., Cell 43:729 (1985)) to accommodate chimeric EcR complexes. Another advantage of chimeric systems is that they allow choice of a promoter used to drive the exogenous gene according to a desired end result. Such double control can be particularly important in areas of gene therapy, especially when cytotoxic proteins are produced, because both the timing of expression as well as the cells wherein expression occurs can be controlled. When exogenous genes, operatively linked to a suitable promoter, are introduced into the cells of the subject, expression of the exogenous genes is controlled by the presence of the ligand of this invention. Promoters may be constitutively or inducibly regulated or may be tissue-specific (that is, expressed only in a particular type of cell) or specific to certain developmental stages of the organism.

[0164] Numerous genomic and cDNA nucleic acid sequences coding for a variety of polypeptides, such as transcription factors and reporter genes, are well known in the art. Those skilled in the art have access to nucleic acid sequence information for virtually all known genes and can either obtain the nucleic acid molecule directly from a public depository, the institution that published the sequence, or employ routine methods to prepare the molecule.

[0165] For in vivo use, the ligands described herein may be taken up in pharmaceutically acceptable carriers, such as, for example, solutions, suspensions, tablets, capsules, ointments, elixirs, and injectable compositions. Pharmaceutical compositions may contain from 0.01% to 99% by weight of the ligand. Compositions may be either in single or multiple dose forms. The amount of ligand in any particular pharmaceutical composition will depend upon the effective dose, that is, the dose required to elicit the desired gene expression or suppression.

[0166] Suitable routes of administering the pharmaceutical preparations include oral, rectal, topical (including dermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and by naso-gastric tube. It will be understood by those skilled in the art that the preferred route of administration will depend upon the condition being diagnosed and may vary with factors such as the condition of the recipient.

[0167] One embodiment of the invention comprises methods of diagnosing a disease or disorder in a subject, comprising:

[0168] (a) introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0169] (b) administering ligand to said modified cells; and

[0170] (c) detecting reporter gene expression;

[0171] wherein expression of the reporter gene indicates that said subject has said disease, disorder, or condition.

[0172] In one embodiment, the diagnostic methods are carried out ex vivo in cells that have been isolated from said subject.

[0173] In one embodiment, the diagnostic methods are carried out by introducing the compositions of the invention into cells that have been isolated from said subject to produce modified cells, and the modified cells are re-introduced into said subject.

[0174] In one embodiment, the diagnostic methods are carried out in vivo.

[0175] In a different embodiment, the diagnostic methods may be carried out using non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the subject, instead of autologous cells from the subject. The polynucleotides may be introduced into the non-autologous cells ex vivo to produce modified cells and the modified cells may then be introduced into the subject. The non-autologous cells may be any cells that are viable after transplantation into a subject, including, without limitation, stem cells (such as embryonic stem cells or hematopoietic stem cells) and fibroblasts.

[0176] One embodiment of the invention relates to methods of diagnosing a disease or disorder in a subject, comprising:

[0177] (a) introducing into non-autologous cells (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0178] (b) introducing said modified cells into said subject;

[0179] (c) administering ligand to said modified cells; and

[0180] (d) detecting reporter gene expression;

[0181] wherein expression of said reporter gene indicates that said subject has said disease or disorder.

[0182] In one aspect of this embodiment, the modified cells are surrounded by a barrier (e.g., encapsulated) prior to being introduced into the subject. The encapsulated cells will function as an implantable biosensor. In one embodiment, encapsulation of cells and methods for making them are provided, which provide improved structural characteristics and immune protection. Such encapsulated cells will withstand mechanical, chemical or immune destruction within the host, and will additionally provide for free permeability to nutrients, ions, oxygen, and other materials needed to both maintain the cell and support normal metabolic functions. In one embodiment, the encapsulated cells are impermeable to bacteria, lymphocytes, and large proteins of the type responsible for immunochemical reactions. In one embodiment, the barrier will also function to prevent the non-autologous cells from escaping from the site of introduction, e.g., rogue cells that might cause harm to the subject if allowed to circulate. In one embodiment, the barrier is a selectively permeable barrier, e.g., a barrier that is permeable to small molecules such as hormones and small peptides but impermeable to larger polypeptides such as antibodies. For example, the barrier may be impermeable to molecules with a molecular weight greater than about 100,000, about 50,000, about 25,000, about 10,000, about 5,000 or about 1,000 daltons.

[0183] Two encapsulation methods, microencapsulation and macroencapsulation, are known in the art. Typically, microencapsulated cells are sequestered in a small spherical container, whereas macroencapsulated cells are entrapped in a larger non-spherical membrane. For encapsulation, living cells and other sensitive materials are treated under sufficiently mild conditions allowing the cells or biomaterial to remain substantially unaffected by the encapsulation process, yet permitting the formation of a capsule of sufficient strength to exist over long periods of time.

[0184] In one embodiment, the cells are encapsulated within a biocompatible semi-permeable membrane. The term "biocompatible" as used herein refers collectively to both the intact capsule and its contents. Specifically, it refers to the capability of the implanted intact encapsulated cell to avoid detrimental effects of the body's various protective systems, such as immune system or foreign body fibrotic response, and remain functional for a significant period of time.

[0185] The capsules of the present invention are especially useful for the administration of cells by injection, implantation or transplantation to a subject. Living cells can be encapsulated in a variety of gels, to form implantable devices, e.g., microbeads or microspheres to physically isolate the cells once implanted into a host. To prevent entry of smaller molecular weight substances such as antibodies and complement (with a molecular weight of about 150 kDa) into these mircoparticles, they can be coated with a material such as poly-L-lysine, chitosan, or PAN-PVC, which provides an outer shell with a controlled pore size or they can be treated by e.g., cross-linking, to control their internal porosity. Additional examples of useful materials include conventional biocompatible materials made up of natural or synthetic polymers or co-polymers, such as alginate, poly-L-lysine-alginate, collagen, gelatin, laminin, methyl methacrylate, hydroxyethyl methacrylate, MATRIGEL, VIRTOGEN, polyvinylalcohol, agarose, polyethylene glycol, hydrogels, polylactic acid, polyglycolic acid, poly(lactide-co-glycolide), polyhydroxybutyrate-polyhydroxyvalerate, copolymer, poly(lactide-co-caprolactone), polyesteramides, polyorthoesters, poly 13-hydroxybutyric acid, polyanhydrides, polyethylene terephthalate, polyetrafluoroethylene, polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polysulfones (including polyether sulfones), polyphosphazenes, polyacrylonitriles, and poly(acrylonitrile/covinyl chloride).

[0186] In one embodiment, the cells are isolated and suspended in liquid medium and then encapsulated by a supporting matrix, e.g., a hydrogel matrix to form a microbead. This microbead may serve as a core of an implantable device. The core will maintain a proper cell distribution, provide strength, and enhance cell viability, longevity, and function. The core will also contribute to immunoisolation. The core will also protect the internal particle from direct cell-cell interactions that can elicit an undesirable host response.

[0187] The barrier may contain multiple layers, e.g., where each layer serves a different purpose (e.g., support, control of permeability). Barriers may also comprise contrast agents or other properties that render the barrier imageable (e.g., by x-ray, sonography, etc.) to ensure proper positioning of the implanted cells. Examples of barrier systems useful for cell implantation are described in U.S. Pat. Nos. 7,226,978, RE39,542 (agarose), 6,960,351, 6,916,640, 6,911,227 (polyethylene glycol), 6,818,018, 6,808,705, 6,783,964, 6,762,959, 6,727,322, 6,610,668 (poly-14-N-acetylglucosamine (p-GlcNAc) polysaccharide), 6,558,665, RE38,027, 6,495,161, 6,368,612, 6,365,385, 6,337,008, 6,306,454 (polyalkylene), 6,303,355, 6,287,558 (gel super matrix), 6,281,015, 6,264,941, 6,258,870, 6,180,007, 6,126,936 (polyamine acid), 6,123,700, 6,083,523, 6,020,200, 5,916,790, 5,912,005, 5,908,623, 5,902,745, 5,858,746, 5,846,530 (polysaccaharides), 5,843,743, 5,837,747, 5,837,234, 5,834,274, 5,834,001, 5,801,033, 5,800,829, 5,800,828, 5,798,113, 5,788,988, 5,786,216, 5,773,286, 5,759,578, 5,700,848, 5,656,481, 5,653,975, 5,648,099, 5,550,178, 4,806,355, 4,689,293, 4,680,174, 4,673,566, 4,409,331, 4,352,883, and U.S. Patent Application Publications 2006/0263405 (alginate/polymer) and 2004/0005302 (alignate-poly-L-lysine), each incorporated herein by references in its entirety.

[0188] In one embodiment, the polynucleotide encoding the gene switch and the polynucleotide encoding the reporter gene linked to a promoter are part of one larger polynucleotide, e.g., a vector. In another embodiment, the polynucleotide encoding the gene switch and the polynucleotide encoding the reporter gene linked to a promoter are separate polynucleotides.

[0189] The subject on which the diagnostic methods are carried out may be any subject for which a diagnosis is desired. For example, the subject may be one that is exhibiting one or more symptoms of a disease or disorder. The subject may also be one that is predisposed to a disease or disorder, e.g., due to genetics, family history, or environmental exposure. The subject may be a member of the general public, e.g., as part of a screening for the prevalence of a disease or disorder in a population.

[0190] The disease or disorder to be diagnosed by the methods of the invention may be any disease or disorder for which one or more diagnostic switch promoters are available. Examples of diseases or disorders which may be diagnosed by the methods of the invention include, without limitation, hyperproliferative diseases (e.g., cancer), cardiovascular diseases, neural diseases, autoimmune diseases, graft versus host disease, transplant rejection, bone diseases, gastrointestinal diseases, blood diseases, metabolic diseases, inflammatory diseases, and infections.

[0191] One embodiment of the invention relates to methods of preparing modified cells for diagnosing a disease or disorder in a subject, comprising introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells.

[0192] Another embodiment of the invention relates to methods of diagnosing a disease or disorder in a subject, comprising:

[0193] (a) administering ligand to modified cells of said subject; and

[0194] (b) detecting reporter gene expression;

[0195] wherein expression of said reporter gene indicates that said subject has said disease or disorder, and

[0196] wherein said modified cells of said subject comprise (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor.

[0197] The diagnostic switch promoters of the invention may be any promoter that is useful for diagnosing a specific disease or disorder, monitoring the progression of a disease, or monitoring the effectiveness or toxicity of a treatment. Examples include, without limitation, promoters of genes that exhibit increased or decreased expression only during a specific disease or disorder and promoters of genes that exhibit increased or decreased expression under specific cell conditions (e.g., proliferation, apoptosis, change in pH, oxidation state, oxygen level). In some embodiments where the gene switch comprises more than one transcription factor sequence, the specificity of the diagnostic methods can be increased by combining a disease- or condition-specific promoter with a tissue- or cell type-specific promoter to limit the tissues in which a diagnostic measurement occurs. Thus, tissue- or cell type-specific promoters are encompassed within the definition of diagnostic switch promoter.

[0198] As an example of disease-specific promoters, useful promoters for diagnosing cancer include the promoters of oncogenes. Examples of classes of oncogenes include, but are not limited to, growth factors, growth factor receptors, protein kinases, programmed cell death regulators and transcription factors. Specific examples of oncogenes include, but are not limited to, sis, erb B, erb B-2, ras, abl, myc and bcl-2 and TERT. Examples of other cancer-related genes include tumor associated antigen genes and other genes that are overexpressed in neoplastic cells (e.g., MAGE-1, carcinoembryonic antigen, tyrosinase, prostate specific antigen, prostate specific membrane antigen, p53, MUC-1, MUC-2, MUC-4, HER-2/neu, T/Tn, MART-1, gp100, GM2, Tn, sTn, and Thompson-Friedenreich antigen (TF)).

[0199] Examples of promoter sequences and other regulatory elements (e.g., enhancers) that are known in the art and are useful as diagnostic switch promoters in the present invention are disclosed in the references listed in Tables 1 and 2, along with the disease/disorder (Table 1) or tissue specificity (Table 2) associated with each promoter. The promoter sequences disclosed in these references are herein incorporated by reference in their entirety.

TABLE-US-00001 TABLE 1 Patent/ Published Application Promoter Sequence Disease/Disorder No. Her-2/neu (ERBB2/c-erbB-2) cancer 5,518,885 osteocalcin calcified tumors 5,772,993 stromelysin-1 cancer 5,824,794 prostate specific antigen prostate cancer 5,919,652 human sodium-iodide symporter thyroid carcinoma 6,015,376 H19, IF-1, IGF-2 cancer 6,306,833 thymosin β15 breast, pancreatic, 6,489,463 prostate cancer T cell factor cancer 6,608,037 cartilage-derived retinoic acid- chondrosarcoma, 6,610,509 sensitive protein mammary tumor insulin pancreatic cancer 6,716,824 PEG-3 cancer 6,737,523 telomerase reverse transcriptase cancer 6,777,203 melanoma differentiation associated cancer 6,841,362 gene-7 prostasin cancer 6,864,093 telomerase catalytic subunit; cancer 6,936,595 cyclin-A midkine; c-erbB-2 cancer 7,030,099 prostate-specific membrane antigen prostate cancer 7,037,647 p51 cancer 7,038,028 telomerase RNA cancer 7,084,267 prostatic acid phosphatase prostate cancer 7,094,533 PCA3_dd3 prostate cancer 7,138,235 DF3/MUC1 cancer 7,247,297 hex II cancer 2001/0011128 cyclooxygenase-2 cancer 2002/0107219 super PSA prostate cancer 2003/0078224 skp2 cancer 2003/0109481 PRL-3 metastatic colon 2004/0126785 cancer CA125/M17S2 ovarian cancer 2004/0126824 IAI.3B ovarian cancer 2005/0031591 CRG-L2 liver cancer 2005/0124068 TRPM4 prostate cancer 2006/0188990 RTVP glioma 2006/0216731 TARP prostate cancer, 2007/0032439 breast cancer telomere reverse transcriptase cancer 2007/0059287 A4 amyloid protein Alzheimer's 5,151,508 disease amyloid β-protein precursor Alzheimer's 5,643,726 disease precursor of the Alzheimer's Disease Alzheimer's 5,853,985 A4 amyloid protein disease neuropeptide FF CNS disorders 6,320,038 endoplasmic reticulum stress stress 7,049,132 elements urocortin II psycho- 7,087,385 pathologies tyrosine hydroxylase neurological 7,195,910 disorders complement factor 3; serum amyloid inflammation 5,851,822 A3 tissue inhibitor of metalloproteinase- rheumatism, 5,854,019 3 (TIMP-3) cancer, autoimmune disease, inflammation p75 tumor necrosis factor receptor autoimmune 5,959,094 disease tumor necrosis factor-α inflammation 6,537,784 peroxisome proliferator activated inflammation 6,870,044 receptor/IIA-1 nonpancreatic secreted phospholipase A2 SOCS-3 growth 2002/0174448 disorders, autoimmune disease, inflammation SR-BI lipid disorders 5,965,790 Ob obesity 5,698,389 site-1 protease obesity, diabetes 7,045,294 TIGR glaucoma 7,138,511 VL30 anoxia 5,681,706 excitatory amino acid transporter-2 nervous system 2004/0171108 ischemia MDTS9 renal failure 2006/0014931 LIM, pyrroline 5-carboxylate prostate disorders 2006/0134688 reductase, SIM2 Bax apoptosis 5,744,310 fas apoptosis 5,888,764 bbc3 apoptosis 7,202,024 PINK-1 PI-3 kinase/Akt 2006/0228776 pathway disorders

TABLE-US-00002 TABLE 2 Patent/Published Promoter Sequence Tissue Specificity Application No. troponin T skeletal muscle 5,266,488 myoD muscle 5,352,595 actin muscle 5,374,544 smooth muscle 22α arterial smooth 5,837,534 muscle utrophin muscle 5,972,609 myostatin muscle 6,284,882 smooth muscle myosin heavy chain smooth muscle 6,780,610 cardiac ankyrin repeat protein cardiac muscle 7,193,075 MLP muscle 2002/0042057 smoothelin smooth muscle 2003/0157494 MYBPC3 cardiomyocytes 2004/0175699 Tα1 α-tubulin neurons 5,661,032 intercellular adhesion molecule-4 neurons 5,753,502 (ICAM-4) γ-aminobutyric acid type A hippocampus 6,066,726 receptor β1 subunit neuronal nicotinic acetylcholine neurons 6,177,242 receptor β2-subunit presenilin-1 neurons 6,255,473 calcium-calmodulin-dependent forebrain 6,509,190 kinase IIα CRF₂α receptor brain 7,071,323 nerve growth factor neurons 2003/159159 GLP-2 receptor gut, brain 2002/0045173 type I transglutaminase keratinocytes 5,643,746 K14 keratinocytes 6,596,515 stearoyl-CoA desaturase skin 2002/0151018 megsin renal cells 6,790,617 prolactin pituitary 5,082,779 GDF-9 ovary, testes, 7,227,013 hypothalamus, pituitary, placenta PSP94 prostate 2003/0110522 NRL; NGAL mammary gland 5,773,290 long whey acidic protein mammary gland 5,831,141 mammary associated amyloid A mammary ductal 2005/0107315 epithelial cells endothelin-1 endothelial cells 5,288,846 serglycin hematopoietic 5,340,739 cells platelet-endothelial cell adhesion platelets, 5,668,012 molecule-1 (PECAM-1) leukocytes, endothelial cells Tie receptor tyrosine kinase endothelial cells, 5,877,020 bone marrow KDR/flk-1 endothelial cells 5,888,765 endoglin endothelial cells 6,103,527 CCR5 myeloid and 6,383,746 lymphoid cells CD11d myeloid cells 6,881,834 platelet glycoprotein IIb hematopoietic 6,884,616 cells preproendothelin-1 endothelial cells 7,067,649 interleukin-18 binding protein mononuclear cells 2006/0239984 CD34 hematopoietic 5,556,954 stem cells Tec tyrosine kinase hematopoietic 6,225,459 stem cells, liver AC133 stem cells 2005/0125849

[0200] Other genes that exhibit changes in expression levels during specific diseases or disorders and therefore are useful in the present invention include, without limitation, the genes (along with the associated disease/disorder) listed in Table 3.

TABLE-US-00003 TABLE 3 Patent/Published Gene Disease/Disorder Application No. MLH1, MSH2, MSH6, PMS1, APC Colorectal cancer 7,148,016 LEF-1 Colon cancer 2002/0169300 F₂ receptor Colon cancer 2002/0187502 TGF-β type II receptor Colon cancer 2004/0038284 EYA4 Colon cancer 2005/0003463 PCA3 Prostate cancer 7,138,235 K2 Prostate cancer 6,303,361 PROST 03 Prostate cancer metastases 2002/0009455 PCAM-1 Prostate cancer 2002/0042062 PCADM-1 Prostate cancer 2003/0100033 PCA3_dd3 Prostate cancer 2003/0165850 PCAV Prostate cancer 2006/0275747 PAcP Androgen-insensitive 2006/0294615 prostate cancer SEQ ID NO: 1 of the patent Liver cancer 5,866,329 5,866,329, incorporated by reference herein SEQ ID NOS: 1, 3 of the U.S. patent Hepatocellular cancer 2002/0115094 application publication 2002/0115094, incorporated by reference herein SEQ ID NO: 1 of the patent U.S. Hepatocellular carcinoma 2005/0037372 application publication 2005/0037372, incorporated by reference herein ATB₀ Hepatocellular carcinoma 2006/0280725 SEQ ID NOS: 1, 3 of the U.S. patent Liver cancer 2007/0042420 application publication 2007/0042420, incorporated by reference herein CSA-1 Chondrosarcoma 2001/0016649 SEQ ID NOS: 1-15 of the U.S. patent Pancreatic cancer 2001/0016651 application publication 2001/0016651, incorporated by reference herein SEQ ID NOS: 1-15 of the U.S. patent Pancreatic cancer 2003/0212264 application publication 2003/0212264, incorporated by reference herein SYG972 Breast cancer 2002/0055107 Urb-ctf Breast cancer 2003/0143546 BCU399 Breast cancer 2003/0180728 TBX2 Breast cancer 2004/0029185 Cyr61 Breast cancer 2004/0086504 DIAPH3 Breast cancer 2005/0054826 SEQ ID NOS: 1-24 of the U.S. patent Breast cancer 2007/0134669 application publication 2007/0134669, incorporated by reference herein Human aspartyl (asparaginyl) beta- CNS cancer 2002/0102263 hydroxylase BEHAB CNS cancer 2003/0068661 IL-8 Kaposi's Sarcoma 2003/0096781 SEQ ID NOS: 1-278 of the U.S. Hematological cancers 2002/0198362 patent application publication 2002/0198362, incorporated by reference herein BLSA B-cell cancer 2003/0147887 BP1 Leukemia 2003/0171273 DAP-kinase, HOXA9 Non-small cell lung cancer 2003/0224509 ARP Clear cell renal carcinoma, 2004/0010119 inflammatory disorders Nbk Renal cancer 2005/0053931 CD43 Ovarian cancer 2006/0216231 SEQ ID NOS: 1-84 of the U.S. patent Ovarian cancer 2007/0054268 application publication 2007/0054268, incorporated by reference herein β7-hcG, β6-hCG, β6e-hCG, Uterine tumors 2006/0292567 β5-hCG, β8-hcG, β3-hCG MTA1s Hormone insensitive 2006/0204957 cancer Old-35, Old-64 Tumor proliferation 2003/0099660 LAGE-1 Cancer 6,794,131 CIF150/hTAF.sub.II150 Cancer 6,174,679 P65 oncofetal protein Cancer 5,773,215 Telomerase Cancer 2002/0025518 CYP1B1 Cancer 2002/0052013 14-3-3σ Cancer 2002/0102245 NES1 Cancer 2002/0106367 CAR-1 Cancer 2002/0119541 HMGI, MAG Cancer 2002/0120120 ELL2 Cancer 2002/0132329 Ephrin B2 Cancer 2002/0136726 WAF1 Cancer 2002/0142442 CIF130 Cancer 2002/0143154 C35 Cancer 2002/0155447 BMP2 Cancer 2002/0159986 BUB3 Cancer 2002/0160403 Polymerase kappa Cancer 2003/0017573 EAG1, EAG2 Cancer 2003/0040476 SEQ ID NOS: 18, 20, 22 of the U.S. Cancer 2003/0044813 patent application publication 2003/0044813, incorporated by reference herein HMG I Cancer 2003/0051260 HLTF Cancer 2003/0082526 Barx2 Cancer 2003/0087243 SEQ ID NOS: 18, 20, 22, 32, 34, 36 Cancer 2003/0108920 of the U.S. patent application publication 2003/0108920, incorporated by reference herein Cables Cancer 2003/0109443 Pp 32r1 Cancer 2003/0129631 BMP4 Cancer 2003/0134790 TS10q23.3 Cancer 2003/0139324 Nuclear spindle-associating protein Cancer 2003/0157072 PFTAIRE Cancer 2003/0166217 SEMA3B Cancer 2003/0166557 MOGp Cancer, multiple sclerosis, 2003/0166898 inflammatory disease Fortilin Cancer 2003/0172388 SEQ ID NO: 1 of the U.S. patent Cancer 2003/0215833 application publication 2003/0215833, incorporated by reference herein IGFBP-3 Cancer 2004/0005294 Polyhomeotic 2 Cancer 2004/0006210 PNQALRE Cancer 2004/0077009 SEQ ID NOS: 1, 3 of the U.S. patent Cancer 2004/0086916 application publication 2004/0086916, incorporated by reference herein SCN5A Cancer 2004/0146877 miR15, miR16 Cancer 2004/0152112 Headpin Cancer 2004/0180371 PAOh1/SMO Cancer 2004/0229241 Hippo, Mst2 Cancer 2005/0053592 PSMA-like Cancer, neurological 2005/0064504 disorders JAB1 Cancer 2005/0069918 NF-AT Cancer 2005/0079496 P28ING5 Cancer 2005/0097626 MTG16 Cancer 2005/0107313 ErbB-2 Cancer 2005/0123538 HDAC9 Cancer 2005/0130146 GPBP Cancer 2005/0130227 MG20 Cancer 2005/0153352 KLF6 Cancer 2005/0181374 ARTS1 Cancer 2005/0266443 Dock 3 Cancer 2006/0041111 Annexin 8 Cancer 2006/0052320 MH15 Cancer 2006/0068411 DELTA-N p73 Cancer 2006/0088825 RapR6 Cancer 2006/099676 StarD10 Cancer 2006/0148032 Ciz1 Cancer 2006/0155113 HLJ1 Cancer 2006/0194235 RapR7 Cancer 2006/0240021 A34 Cancer 2006/0292154 Sef Cancer 2006/0293240 Killin Cancer 2007/0072218 SGA-1M Cancer 2007/0128593 TGFβ Type II receptor Cancer 2002/0064786 GCA-associated genes Giant cell arteritis 6,743,903 PRV-1 Polycythemia vera 6,686,153 SEQ ID NOS: 2, 4 of the U.S. Pat. No. Ischemia 5,948,637 5,948,637, incorporated by reference herein Vezf1 Vascular disorders 2002/0023277 MLP Dilatative cardiomyopathy 2002/0042057 VEGI Pathological angiogenesis 2002/0111325 PRO256 Cardiovascular disorders 2002/0123091 AOP2 Atherosclerosis 2002/0142417 Remodelin Arterial restenosis, fibrosis 2002/0161211 Phosphodiesterase 4D Stroke 2003/0054531 Prostaglandin receptor subtype EP3 Peripheral arterial 2003/0157599 occlusive disease CARP Heart disorders 2004/0014706 HOP Congenital heart disease 2004/0029158 SEQ ID NOS: 1-4 of the U.S. patent Apoplexy 2004/0087784 application publication 2004/0087784, incorporated by reference herein PLTP Atherosclerosis, vascular 2006/0252787 disease, hypercholesterolemia, Tangier's disease, familial HDL deficiency disease SEQ ID NOS: 1, 3-8, 15, 16 of the Thrombosis 2007/0160996 U.S. patent application publication 2007/0160996, incorporated by reference herein UCP-2 Stroke 2002/0172958 FLJ11011 Fanconi's Anemia 2006/0070134 Codanin-1 Anemia 2006/0154331 SEQ ID NOS: 1, 6, 8 of the U.S. Insulin-dependent diabetes 5,763,591 Pat. No. 5,763,591, incorporated by mellitus reference herein Resistin Type II diabetes 2002/0161210 Archipelin Diabetes 2003/0202976 SEQ ID NOS: 2, 7, 16, 27 of the U.S. Diabetes, hyperlipidemia 2004/0053397 patent application publication 2004/0053397, incorporated by reference herein Neuronatin Metabolic disorders 2004/0259777 Ncb5or Diabetes 2005/0031605 7B2 Endocrine disorders 2005/0086709 PTHrP, PEX Metabolic bone diseases 2005/0113303 KChIPl Type II diabetes 2005/0196784 SLIT-3 Type II diabetes 2006/0141462 CX3CR1 Type II diabetes 2006/0160076 SMAP-2 Diabetes 2006/0210974 SEQ ID NOS: 2, 8, 12, 16, 22, 26, Type II diabetes 2006/0228706 28, 32 of the U.S. patent application publication 2006/0228706, incorporated by reference herein IC-RFX Diabetes 2006/0264611 E2IG4 Diabetes, insulin 2007/0036787 resistance, obesity SEQ ID NOS: 2, 8, 10, 14, 18, 24, Diabetes 2007/0122802 26, 30, 34, 38, 44, 50, 54, 60, 62, 68, 74, 80, 86, 92, 98, 104, 110 of the U.S. patent application publication 2007/0122802, incorporated by reference herein UCP2 Body weight disorders 2002/0127600 Ob receptor Body weight disorders 2002/0182676 Ob Body weight disorders 2004/0214214 Dp1 Neurodegenerative 2001/0021771 disorders NRG-1 Schizophrenia 2002/0045577 Synapsin III Schizophrenia 2002/0064811 NRG1AG1 Schizophrenia 2002/0094954 AL-2 Neuronal disorders 2002/0142444 Proline dehydrogenase Bipolar disorder, major 2002/0193581 depressive disorder, schizophrenia, obsessive compulsive disorder MNR2 Chronic neurodegenerative 2002/0197678 disease ATM Ataxia-telangiectasia 2004/0029198 Ho-1 Dementing diseases 2004/0033563 CON202 Schizophrenia 2004/0091928 Ataxin-1 Neurodegenerative 2004/0177388 disorders NR3B Motor neuron disorders 2005/0153287 NIPA-1 Hereditary spastic 2005/0164228 paraplegia DEPP, adrenomedullin, csdA Schizophrenia 2005/0227233 Inf-20 Neurodegenerative 2006/0079675 diseases EOPA Brain development and 2007/0031830 degeneration disorders

SERT Autism 2007/0037194 FRP-1 Glaucoma 2002/0049177 Serum amyloid A Glaucoma 2005/0153927 BMP2 Osteoporosis 2002/0072066 BMPR1A Juvenile polyposis 2003/0072758 ACLP Gastroschisis 2003/0084464 Resistin-like molecule β Familial adenomatous 2003/0138826 polyposis, diabetes, insulin resistance, colon cancer, inflammatory bowel disorder Dlg5 Inflammatory bowel 2006/0100132 disease SEQ ID NOS: 1-82 of the U.S. patent Osteoarthritis 2002/0119452 application publication 2002/0119452, incorporated by reference herein TRANCE Immune system disorders 2003/0185820 Matrilin-3 Osteoarthritis 2003/0203380 Synoviolin Rheumatoid arthritis 2004/0152871 SEQ ID NOS: 9, 35 of the U.S. Osteoarthritis 2007/0028314 patent application publication 2007/0028314, incorporated by reference herein HIV LTR HIV infection 5,627,023 SHIVA HIV infection 2004/0197770 EBI 1, EBI 2, EBI 3 Epstein Barr virus infection 2002/0040133 NM23 family Skin/intestinal disorders 2002/0034741 SEQ ID NO: 1 of the U.S. patent Psoriasis 2002/0169127 application publication 2002/0169127, incorporated by reference herein Eps8 Skin disorders, wound 2003/0180302 healing Beta-10 Thyroid gland pathology 2002/0015981 SEQ ID NO: 2 of the U.S. patent Thyroid conditions 2003/0207403 application publication 2003/0207403, incorporated by reference herein SEQ ID NO: 3 of the U.S. patent Thyroid disorders 2007/0020275 application publication 2007/0020275, incorporated by reference herein Hair follicle growth factor Alopecia 2003/0036174 Corneodesmosin Alopecia 2003/0211065 GCR9 Asthma, lymphoma, 2003/0166150 leukemia SEQ ID NO: 1-71 of the U.S. patent Asthma 2004/0002084 application publication 2004/0002084, incorporated by reference herein Bg Chediak-Higashi syndrome 2002/0115144 SEQ ID NOS: 1-16 of the U.S. patent Endometriosis 2002/0127555 application publication 2002/0127555, incorporated by reference herein FGF23 Hypophosphatemic 2005/0156014 disorders BBSR Bardet-Biedl syndrome 2003/0152963 MIC-1 Fetal abnormalities, cancer, 2004/0053325 inflammatory disorders, miscarriage, premature birth MIA-2 Liver damage 2004/0076965 IL-17B Cartilage degenerative 2004/0171109 disorders Formylglycine generating enzyme Multiple sulfatase 2004/0229250 deficiency LPLA2 Pulmonary alveolar 2006/0008455 proteinosis CXCL1O Respiratory illnesses 2006/0040329 SEQ ID NOS: 1, 2 of the U.S. patent Nephropathy 2006/0140945 application publication 2006/0140945, incorporated by reference herein HFE2A Iron metabolism disease 2007/0166711

[0201] Once a gene with an expression pattern that is modulated during a disease or disorder is identified, the promoter of the gene may be used in the gene switch of the invention. The sequence of many genes, including the promoter region, is known in the art and available in public databases, e.g., GenBank. Thus, once an appropriate gene is identified, the promoter sequence can be readily identified and obtained. Another aspect of the present invention is directed towards identifying suitable genes whose promoter can be isolated and placed into a gene switch. The identity of the gene, therefore, may not be critical to specific embodiments of the present invention, provided the promoter can be isolated and used in subsequent settings or environments. The current invention thus includes the use of promoters from genes that are yet to be identified. Once suitable genes are identified, it can be a matter of routine skill or experimentation to determine the genetic sequences needed for promoter function. Indeed, several commercial protocols exist to aid in the determination of the promoter region of genes of interest. By way of example, Ding et al. recently elucidated the promoter sequence of the novel Sprouty4 gene (Am. J. Physiol. Lung Cell. Mol. Physiol. 287: L52 (2004), which is incorporated by reference) by progressively deleting the 5'-flanking sequence of the human Sprouty4 gene. Briefly, once the transcription initiation site was determined, PCR fragments were generated using common PCR primers to clone segments of the 5'-flanking segment in a unidirectional manner. The generated segments were cloned into a luciferase reporter vector and luciferase activity was measured to determine the promoter region of the human Sprouty4 gene.

[0202] Another example of a protocol for acquiring and validating gene promoters includes the following steps: (1) acquire diseased and non-diseased cell/tissue samples of similar/same tissue type; (2) isolate total RNA or mRNA from the samples; (3) perform differential microarray analysis of diseased and non-diseased RNA; (4) identify candidate disease-specific transcripts; (5) identify genomic sequences associated with the disease-specific transcripts; (6) acquire or synthesize DNA sequence upstream and downstream of the predicted transcription start site of the disease-specific transcript; (7) design and produce promoter reporter vectors using different lengths of DNA from step 6; and (8) test promoter reporter vectors in diseased and non-diseased cells/tissues, as well as in unrelated cells/tissues.

[0203] The source of the promoter that is inserted into the gene switch can be natural or synthetic, and the source of the promoter should not limit the scope of the invention described herein. In other words, the promoter may be directly cloned from cells, or the promoter may have been previously cloned from a different source, or the promoter may have been synthesized.

[0204] The gene switch may be any gene switch that regulates gene expression by addition or removal of a specific ligand. In one embodiment, the gene switch is one in which the level of gene expression is dependent on the level of ligand that is present. Examples of ligand-dependent transcription factors that may be used in the gene switches of the invention include, without limitation, members of the nuclear receptor superfamily activated by their respective ligands (e.g., glucocorticoid, estrogen, progestin, retinoid, ecdysone, and analogs and mimetics thereof) and rTTA activated by tetracycline. In one aspect of the invention, the gene switch is an EcR-based gene switch. Examples of such systems include, without limitation, the systems described in U.S. Pat. Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos. 2006/0014711, 2007/0161086, and International Published Application No. WO 01/70816. Examples of chimeric ecdysone receptor systems are described in U.S. Pat. No. 7,091,038, U.S. Published Patent Application Nos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457, and 2006/0100416, and International Published Application Nos. WO 01/70816, WO 02/066612, WO 02/066613, WO 02/066614, WO 02/066615, WO 02/29075, and WO 2005/108617, each of which is incorporated by reference in its entirety. An example of a non-steroidal ecdysone agonist-regulated system is the RheoSwitch® Mammalian Inducible Expression System (New England Biolabs, Ipswich, Mass.).

[0205] In one embodiment, the gene switch comprises a single transcription factor sequence encoding a ligand-dependent transcription factor under the control of a diagnostic switch promoter. The transcription factor sequence may encode a ligand-dependent transcription factor that is a naturally occurring or an artificial transcription factor. An artificial transcription factor is one in which the natural sequence of the transcription factor has been altered, e.g., by mutation of the sequence or by the combining of domains from different transcription factors. In one embodiment, the transcription factor comprises a Group H nuclear receptor LBD. In one embodiment, the Group H nuclear receptor LBD is from an EcR, a ubiquitous receptor, an orphan receptor 1, a NER-1, a steroid hormone nuclear receptor 1, a retinoid X receptor interacting protein-15, a liver X receptor β, a steroid hormone receptor like protein, a liver X receptor, a liver X receptor α, a farnesoid X receptor, a receptor interacting protein 14, or a farnesol receptor. In another embodiment, the Group H nuclear receptor LBD is from an ecdysone receptor.

[0206] The EcR and the other Group H nuclear receptors are members of the nuclear receptor superfamily wherein all members are generally characterized by the presence of an amino-terminal transactivation domain (TD), a DNA binding domain (DBD), and a LBD separated from the DBD by a hinge region. As used herein, the term "DNA binding domain" comprises a minimal polypeptide sequence of a DNA binding protein, up to the entire length of a DNA binding protein, so long as the DNA binding domain functions to associate with a particular response element. Members of the nuclear receptor superfamily are also characterized by the presence of four or five domains: A/B, C, D, E, and in some members F (see U.S. Pat. No. 4,981,784 and Evans, Science 240:889 (1988)). The "A/B" domain corresponds to the transactivation domain, "C" corresponds to the DNA binding domain, "D" corresponds to the hinge region, and "E" corresponds to the ligand binding domain. Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD corresponding to "F".

[0207] The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for response elements. These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins. The EcR, like a subset of the nuclear receptor family, also possesses less well-defined regions responsible for heterodimerization properties. Because the domains of nuclear receptors are modular in nature, the LBD, DBD, and TD may be interchanged.

[0208] In another embodiment, the transcription factor comprises a TD, a DBD that recognizes a response element associated with the reporter gene whose expression is to be modulated; and a Group H nuclear receptor LBD. In certain embodiments, the Group H nuclear receptor LBD comprises a substitution mutation.

[0209] In another embodiment, the gene switch comprises a first transcription factor sequence under the control of a first diagnostic switch promoter and a second transcription factor sequence under the control of a second diagnostic switch promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor, i.e., a "dual switch"- or "two-hybrid"-based gene switch. The first and second diagnostic switch promoters may be the same or different. In this embodiment, the presence of two different diagnostic switch promoters in the gene switch that are required for reporter gene expression enhances the specificity of the diagnostic method (see FIG. 3). FIG. 3 also demonstrates the ability to modify the diagnostic gene switch to detect any disease or disorder simply by inserting the appropriate diagnostic switch promoters.

[0210] In a further embodiment, the first transcription factor sequence is under the control of a diagnostic switch promoter (e.g., P2 or P3 in FIG. 1) and the second transcription factor sequence is under the control of a constitutive promoter (e.g., P1 in FIG. 1). In this embodiment, one portion of the ligand-dependent transcription factor will be constitutively present while the second portion will only be synthesized if the subject has the disease or disorder.

[0211] In another embodiment, the first transcription factor sequence is under the control of a first diagnostic switch promoter (e.g., P1 in FIG. 2) and two or more different second transcription factor sequences are under the control of different diagnostic switch promoters (e.g., P2 and P3 in FIG. 2). In this embodiment, each of the second transcription factor sequences may have a different DBD that recognizes a different inducible promoter sequence (e.g., DBD-A binds to inducible promoter A and DBD-B binds to inducible promoter B). Each of the inducible promoters may be operably linked to a different reporter gene that produces a unique signal. In this manner, multiple diagnoses may be made simultaneously or a differential diagnosis between two or more possible diseases may be made.

[0212] In one embodiment, the first transcription factor sequence encodes a polypeptide comprising a TD, a DBD that recognizes a response element associated with the reporter gene whose expression is to be modulated; and a Group H nuclear receptor LBD, and the second transcription factor sequence encodes a transcription factor comprising a nuclear receptor LBD selected from the group consisting of a vertebrate RXR LBD, an invertebrate RXR LBD, an ultraspiracle protein LBD, and a chimeric LBD comprising two polypeptide fragments, wherein the first polypeptide fragment is from a vertebrate RXR LBD, an invertebrate RXR. LBD, or an ultraspiracle protein LBD, and the second polypeptide fragment is from a different vertebrate RXR LBD, invertebrate RXR LBD, or ultraspiracle protein LBD.

[0213] In another embodiment, the gene switch comprises a first transcription factor sequence encoding a first polypeptide comprising a nuclear receptor LBD and a DBD that recognizes a response element associated with the reporter gene whose expression is to be modulated, and a second transcription factor sequence encoding a second polypeptide comprising a TD and a nuclear receptor LBD, wherein one of the nuclear receptor LBDs is a Group H nuclear receptor LBD. In a preferred embodiment, the first polypeptide is substantially free of a TD and the second polypeptide is substantially free of a DBD. For purposes of the invention, "substantially free" means that the protein in question does not contain a sufficient sequence of the domain in question to provide activation or binding activity.

[0214] In another aspect of the invention, the first transcription factor sequence encodes a protein comprising a heterodimer partner and a TD and the second transcription factor sequence encodes a protein comprising a DBD and a LBD.

[0215] When only one nuclear receptor LBD is a Group H LBD, the other nuclear receptor LBD may be from any other nuclear receptor that forms a dimer with the Group H LBD. For example, when the Group H nuclear receptor LBD is an EcR LBD, the other nuclear receptor LBD "partner" may be from an EcR, a vertebrate RXR, an invertebrate RXR, an ultraspiracle protein (USP), or a chimeric nuclear receptor comprising at least two different nuclear receptor LBD polypeptide fragments selected from the group consisting of a vertebrate RXR, an invertebrate RXR, and a USP (see WO 01/70816 A2, International Patent Application No. PCT/US02/05235 and US 2004/0096942 A1, incorporated herein by reference in their entirety). The "partner" nuclear receptor ligand binding domain may further comprise a truncation mutation, a deletion mutation, a substitution mutation, or another modification.

[0216] In one embodiment, the vertebrate RXR LBD is from a human Homo sapiens, mouse Mus musculus, rat Rattus norvegicus, chicken Gallus gallus, pig Sus scrofa domestica, frog Xenopus laevis, zebrafish Danio rerio, tunicate Polyandrocarpa misakiensis, or jellyfish Tripedalia cysophora RXR.

[0217] In one embodiment, the invertebrate RXR ligand binding domain is from a locust Locusta migratoria ultraspiracle polypeptide ("LmUSP"), an ixodid tick Amblyomma americanum RXR homolog 1 ("AmaRXR1"), an ixodid tick Amblyomma americanum RXR homolog 2 ("AmaRXR2"), a fiddler crab Celuca pugilator RXR homolog ("CpRXR"), a beetle Tenebrio molitor RXR homolog ("TmRXR"), a honeybee Apis mellifera RXR homolog ("AmRXR"), an aphid Myzus persicae RXR homolog ("MpRXR"), or a non-Dipteran/non-Lepidopteran RXR homolog.

[0218] In one embodiment, the chimeric RXR LBD comprises at least two polypeptide fragments selected from the group consisting of a vertebrate species RXR polypeptide fragment, an invertebrate species RXR polypeptide fragment, and a non-Dipteranlnon-Lepidopteran invertebrate species RXR homolog polypeptide fragment. A chimeric RXR ligand binding domain for use in the present invention may comprise at least two different species RXR polypeptide fragments, or when the species is the same, the two or more polypeptide fragments may be from two or more different isoforms of the species RXR polypeptide fragment.

[0219] In one embodiment, the chimeric RXR ligand binding domain comprises at least one vertebrate species RXR polypeptide fragment and one invertebrate species RXR polypeptide fragment.

[0220] In another embodiment, the chimeric RXR ligand binding domain comprises at least one vertebrate species RXR polypeptide fragment and one non-Dipteran/non-Lepidopteran invertebrate species RXR homolog polypeptide fragment.

[0221] The ligand, when combined with the LBD of the nuclear receptor(s), which in turn are bound to the response element linked to the reporter gene, provides external temporal regulation of expression of the reporter gene. The binding mechanism or the order in which the various components of this invention bind to each other, that is, for example, ligand to LBD, DBD to response element, TD to promoter, etc., is not critical.

[0222] In a specific example, binding of the ligand to the LBD of a Group H nuclear receptor and its nuclear receptor LBD partner enables expression of the reporter gene. This mechanism does not exclude the potential for ligand binding to the Group H nuclear receptor (GHNR) or its partner, and the resulting formation of active homodimer complexes (e.g. GHNR+GHNR or partner+partner). Preferably, one or more of the receptor domains is varied producing a hybrid gene switch. Typically, one or more of the three domains, DBD, LBD, and TD, may be chosen from a source different than the source of the other domains so that the hybrid genes and the resulting hybrid proteins are optimized in the chosen host cell or organism for transactivating activity, complementary binding of the ligand, and recognition of a specific response element. In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein from yeast (see Sadowski et al., Nature 335:563 (1988)) or LexA protein from Escherichia coli (see Brent et al., Cell 43:729 (1985)), or synthetic response elements specific for targeted interactions with proteins designed, modified, and selected for such specific interactions (see, for example, Kim et al., Proc. Natl. Acad. Sci. USA, 94:3616 (1997)) to accommodate hybrid receptors.

[0223] The functional EcR complex may also include additional protein(s) such as immunophilins. Additional members of the nuclear receptor family of proteins, known as transcriptional factors (such as DHR38 or betaFTZ-1), may also be ligand dependent or independent partners for EcR, USP, and/or RXR. Additionally, other cofactors may be required such as proteins generally known as coactivators (also termed adapters or mediators). These proteins do not bind sequence-specifically to DNA and are not involved in basal transcription. They may exert their effect on transcription activation through various mechanisms, including stimulation of DNA-binding of activators, by affecting chromatin structure, or by mediating activator-initiation complex interactions. Examples of such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the promiscuous coactivator C response element B binding protein, CBP/p300 (for review see Glass et al., Curr. Opin. Cell Biol. 9:222 (1997)). Also, protein cofactors generally known as corepressors (also known as repressors, silencers, or silencing mediators) may be required to effectively inhibit transcriptional activation in the absence of ligand. These corepressors may interact with the unliganded EcR to silence the activity at the response element. Current evidence suggests that the binding of ligand changes the conformation of the receptor, which results in release of the corepressor and recruitment of the above described coactivators, thereby abolishing their silencing activity. Examples of corepressors include N-CoR and SMRT (for review, see Horwitz et al., Mol Endocrinol. 10:1167 (1996)). These cofactors may either be endogenous within the cell or organism, or may be added exogenously as transgenes to be expressed in either a regulated or unregulated fashion.

[0224] The reporter gene may be any gene that encodes a detectable protein. The protein may be secreted or non-secreted. In one embodiment, the protein is one that can be assayed using various standard assay methods, e.g., immunoassays (such as those immunofluorescent antibodies), colorimetric assays, fluorescent assays, or luminescent assays. Examples of suitable reporter genes include, without limitation, luciferase, green fluorescent protein, β-galactosidase, β-glucuronidase, thymidine kinase, and chloramphenicol acetyltransferase.

[0225] The reporter gene is operably linked to a promoter comprising at least one response element that is recognized by the DBD of the ligand-dependent transcription factor encoded by the gene switch. In one embodiment, the promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of the response element. Promoters comprising the desired response elements may be naturally occurring promoters or artificial promoters created using techniques that are well known in the art, e.g., one or more response elements operably linked to a minimal promoter.

[0226] To introduce the polynucleotides into the cells, a vector can be used. The vector may be, for example, a plasmid vector or a single- or double-stranded RNA or DNA viral vector. Such vectors may be introduced into cells by well-known techniques for introducing DNA and RNA into cells. Viral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. As used herein, the term "host cell" or "host" is used to mean a cell of the present invention that is harboring one or more polynucleotides of the invention.

[0227] Thus, at a minimum, the vectors must include the polynucleotides of the invention. Other components of the vector may include, but are not limited to, selectable markers, chromatin modification domains, additional promoters driving expression of other polypeptides that may also be present on the vector (e.g., a lethal polypeptide), genomic integration sites, recombination sites, and molecular insertion pivots. The vectors may comprise any number of these additional elements, either within or not within the polynucleotides, such that the vector can be tailored to the specific goals of the diagnostic methods desired.

[0228] In one embodiment of the present invention, the vectors that are introduced into the cells further comprise a "selectable marker gene" which, when expressed, indicates that the diagnostic gene switch construct of the present invention has been integrated into the genome of the host cell. In this manner, the selector gene can be a positive marker for the genome integration. While not critical to the methods of the present invention, the presence of a selectable marker gene allows the practitioner to select for a population of live cells where the vector construct has been integrated into the genome of the cells. Thus, certain embodiments of the present invention comprise selecting cells where the vector has successfully been integrated. As used herein, the term "select" or variations thereof, when used in conjunction with cells, is intended to mean standard, well-known methods for choosing cells with a specific genetic make-up or phenotype. Typical methods include, but are not limited to, culturing cells in the presence of antibiotics, such as G418, neomycin and ampicillin. Other examples of selectable marker genes include, but are not limited to, genes that confer resistance to dihydrofolate reductase, hygromycin, or mycophenolic acid. Other methods of selection include, but are not limited to, a selectable marker gene that allows for the use of thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase or adenine phosphoribosyltransferase as selection agents. Cells comprising a vector construct comprising an antibiotic resistance gene or genes would then be capable of tolerating the antibiotic in culture. Likewise, cells not comprising a vector construct comprising an antibiotic resistance gene or genes would not be capable of tolerating the antibiotic in culture.

[0229] As used herein, a "chromatin modification domain" (CMD) refers to nucleotide sequences that interact with a variety of proteins associated with maintaining and/or altering chromatin structure, such as, but not limited to, DNA insulators. See Ciavatta et al., Proc. Nat'l Acad. Sci. U.S.A., 103:9958 (2006), which is incorporated by reference herein. Examples of CMDs include, but are not limited to, the chicken β-globulin insulator and the chicken hypersensitive site 4 (cHS4). The use of different CMD sequences between one or more gene programs (i.e., a promoter, coding sequence, and 3' regulatory region), for example, can facilitate the use of the differential CMD DNA sequences as "mini homology arms" in combination with various microorganism or in vitro recombineering technologies to "swap" gene programs between existing multigenic and monogenic shuttle vectors. Other examples of chromatin modification domains are known in the art or can be readily identified.

[0230] Particular vectors for use with the present invention are expression vectors that code for proteins or portions thereof. Generally, such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.

[0231] A great variety of expression vectors can be used to express proteins. Such vectors include chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as adeno-associated viruses, lentiviruses, baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. All may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain, propagate or express polynucleotides or proteins in a host may be used for expression in this regard.

[0232] The DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s) including, for instance, a promoter to direct mRNA transcription. Representatives of additional promoters include, but are not limited to, constitutive promoters and tissue specific or inducible promoters. Examples of constitutive eukaryotic promoters include, but are not limited to, the promoter of the mouse metallothionein I gene (Hamer et al., J. Mol. Appl. Gen. 1:273 (1982)); the TK promoter of Herpes virus (McKnight, Cell 31:355 (1982)); the SV40 early promoter (Benoist et al., Nature 290:304 (1981)); and the vaccinia virus promoter. All of the above listed references are incorporated by reference herein. Additional examples of the promoters that could be used to drive expression of a protein include, but are not limited to, tissue-specific promoters and other endogenous promoters for specific proteins, such as the albumin promoter (hepatocytes), a proinsulin promoter (pancreatic beta cells) and the like. In general, expression constructs will contain sites for transcription, initiation and termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating AUG at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0233] In addition, the constructs may contain control regions that regulate, as well as engender expression. Generally, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.

[0234] Examples of eukaryotic vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Amersham Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, pCMV-EGFP available from Clontech. Many other vectors are well-known and commercially available.

[0235] Particularly useful vectors, which comprise molecular insertion pivots for rapid insertion and removal of elements of gene programs, are described in United States Published Patent Application No. 2004/0185556, U.S. patent application Ser. No. 11/233,246 and International Published Application Nos. WO 2005/040336 and WO 2005/116231, all of which are incorporated by reference. An example of such vectors is the UltraVector® Production System (Intrexon Corp., Blacksburg, Va.), as described in WO 2007/038276, incorporated herein by reference. As used herein, a "gene program" is a combination of genetic elements comprising a promoter (P), an expression sequence (E) and a 3' regulatory sequence (3), such that "PE3" is a gene program. The elements within the gene program can be easily swapped between molecular pivots that flank each of the elements of the gene program. A molecular pivot, as used herein, is defined as a polynucleotide comprising at least two non-variable rare or uncommon restriction sites arranged in a linear fashion. In one embodiment, the molecular pivot comprises at least three non-variable rare or uncommon restriction sites arranged in a linear fashion. Typically any one molecular pivot would not include a rare or uncommon restriction site of any other molecular pivot within the same gene program. Cognate sequences of greater than 6 nucleotides upon which a given restriction enzyme acts are referred to as "rare" restriction sites. There are, however, restriction sites of 6 bp that occur more infrequently than would be statistically predicted, and these sites and the endonucleases that cleave them are referred to as "uncommon" restriction sites. Examples of either rare or uncommon restriction enzymes include, but are not limited to, AsiS I, Pac I, Sbf I, Fse I, Asc I, Mlu I, SnaB I, Not I, Sal I, Swa I, Rsr II, BSiW I, Sfo I, Sgr AI, AflIII, Pvu I, Ngo MIV, Ase I, Flp I, Pme I, Sda I, Sgf I, Srf I, and Sse8781 I.

[0236] The vector may also comprise restriction sites for a second class of restriction enzymes called homing endonuclease (HE) enzymes. HE enzymes have large, asymmetric restriction sites (12-40 base pairs), and their restriction sites are infrequent in nature. For example, the HE known as I-SceI has an 18 bp restriction site (5'TAGGGATAACAGGGTAAT3' (SEQ ID NO:4)), predicted to occur only once in every 7×10¹⁰ base pairs of random sequence. This rate of occurrence is equivalent to only one site in a genome that is 20 times the size of a mammalian genome. The rare nature of HE sites greatly increases the likelihood that a genetic engineer can cut a gene program without disrupting the integrity of the gene program if HE sites were included in appropriate locations in a cloning vector plasmid.

[0237] Selection of appropriate vectors and promoters for expression in a host cell is a well-known procedure, and the requisite techniques for vector construction and introduction into the host, as well as its expression in the host are routine skills in the art.

[0238] The introduction of the polynucleotides into the cells can be a transient transfection, stable transfection, or can be a locus-specific insertion of the vector. Transient and stable transfection of the vectors into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986); Keown et al., 1990, Methods Enzymol. 185: 527-37; Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N.Y., which are hereby incorporated by reference. These stable transfection methods result in random insertion of the vector into the genome of the cell. Further, the copy number and orientation of the vectors are also, generally speaking, random.

[0239] In one embodiment of the invention, the vector is inserted into a bio-neutral site in the genome. A bio-neutral site is a site in the genome where insertion of the polynucleotides interferes very little, if any, with the normal function of the cell. Bio-neutral sites may be analyzed using available bioinformatics. Many bio-neutral sites are known in the art, e.g., the ROSA-equivalent locus. Other bio-neutral sites may be identified using routine techniques well known in the art. Characterization of the genomic insertion site(s) is performed using methods known in the art. To control the location, copy number and/or orientation of the polynucleotides when introducing the vector into the cells, methods of locus-specific insertion may be used. Methods of locus-specific insertion are well-known in the art and include, but are not limited to, homologous recombination and recombinase-mediated genome insertion. Of course, if locus-specific insertion methods are to be used in the methods of the present invention, the vectors may comprise elements that aid in this locus-specific insertion, such as, but not limited to, homologous recombination. For example, the vectors may comprise one, two, three, four or more genomic integration sites (GISs). As used herein, a "genomic integration site" is defined as a portion of the vector sequence which nucleotide sequence is identical or nearly identical to portions of the genome within the cells that allows for insertion of the vector in the genome. In particular, the vector may comprise two genomic insertion sites that flank at least the polynucleotides. Of course, the GISs may flank additional elements, or even all elements present on the vector.

[0240] In another embodiment, locus-specific insertion may be carried out by recombinase-site specific gene insertion. Briefly, bacterial recombinase enzymes, such as, but not limited to, PhiC31 integrase can act on "pseudo" recombination sites within the human genome. These pseudo recombination sites can be targets for locus-specific insertion using the recombinases. Recombinase-site specific gene insertion is described in Thyagarajan et al., Mol. Cell Biol. 21:3926 (2001), which is hereby incorporated by reference. Other examples of recombinases and their respective sites that may be used for recombinase-site specific gene insertion include, but are not limited to, serine recombinases such as R4 and TP901-1 and recombinases described in WO 2006/083253, incorporated herein by reference.

[0241] In a further embodiment, the vector may comprise a chemo-resistance gene, e.g., the multidrug resistance gene mdr1, dihydrofolate reductase, or O⁶-alkylguanine-DNA alkyltransferase. The chemo-resistance gene may be under the control of a constitutive (e.g., CMV) or inducible (e.g., RheoSwitch®) promoter. In this embodiment, if it is desired to treat a disease diagnosed in a subject while maintaining the modified cells within the subject, a clinician may apply a chemotherapeutic agent to destroy diseased cells while the modified cells would be protected from the agent due to expression of a suitable chemo-resistance gene and may continue to be used for monitoring of the progression of the disease or effectiveness of the treatment. By placing the chemo-resistance gene under an inducible promoter, the unnecessary expression of the chemo-resistance gene can be avoided, yet it will still be available in case continued diagnosis is desired during treatment. If the modified cells themselves become diseased, they could still be destroyed by inducing expression of a lethal polypeptide as described below.

[0242] The methods of the invention are carried out by introducing the polynucleotides encoding the gene switch and the reporter gene into cells of a subject. Any method known for introducing a polynucleotide into a cell known in the art, such as those described above, can be used.

[0243] In an alternative embodiment, the polynucleotides encoding the gene switch and the reporter gene are introduced into non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the subject.

[0244] When the polynucleotides are to be introduced into cells ex vivo, the cells may be obtained from a subject by any technique known in the art, including, but not limited to, biopsies, scrapings, and surgical tissue removal. The isolated cells may be cultured for a sufficient amount of time to allow the polynucleotides to be introduced into the cells, e.g., 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, hours or more. Methods for culturing primary cells for short periods of time are well known in the art. For example, cells may be cultured in plates (e.g., in microwell plates) either attached or in suspension.

[0245] For ex vivo diagnosis methods, cells are isolated from a subject and cultured under conditions suitable for introducing the polynucleotides into the cells. Once the polynucleotides have been introduced into the cells, the cells are incubated for a sufficient period of time to allow the ligand-dependent transcription factor to be expressed, e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours or more. If expression of the ligand-dependent transcription factor is increased or decreased compared to control levels (i.e., if the subject has the disease or disorder and the promoters controlling expression of the transcription factor are activated or deactivated), the presence and/or level of the ligand-dependent transcription factor is detected by the addition of ligand, leading to expression of the reporter gene at a level corresponding to the level of the ligand-dependent transcription factor. The ligand may be added to the cells at any time before, during or after introduction of the polynucleotides into the cells. The optimal timing of ligand administration can be determined for each type of cell and disease or disorder using only routine techniques. In one embodiment, the ligand may be added, reporter gene expression determined, the ligand removed, and the process repeated one or more times to obtain multiple diagnostic measurements of the cells. In another embodiment, ligand is continuously present and reporter gene expression is measured periodically.

[0246] The first in vivo diagnostic embodiment of the invention (modification of isolated cells followed by reintroduction of the cells to the subject) may be used where the ex vivo method using isolated cells is insufficient, e.g., where circulating factors are necessary for diagnostic switch promoter activity to occur. In this embodiment, cells are isolated from a subject and the polynucleotides are introduced into the cells in culture as described above. At some point after the introduction of the polynucleotides into the cells, the cells are introduced back into the subject. Reintroduction may be carried out by any method known in the art, e.g., intravenous infusion or direct injection into a tissue or cavity. In one embodiment, the presence of the polynucleotides in the cells is determined prior to introducing the cells back into the subject. In another embodiment, cells containing the polynucleotides are selected (e.g., based on the presence of a selectable marker in the polynucleotides) and only those cells containing the polynucleotides are reintroduced into the subject. After the cells are reintroduced to the subject, ligand is administered to the subject and reporter gene expression is assayed. The ligand may be administered by any suitable method, either systemically (e.g., orally, intravenously) or locally (e.g., intraperitoneally, intrathecally, intraventricularly, direct injection into the tissue or organ where the cells were reintroduced). The optimal timing of ligand administration can be determined for each type of cell and disease or disorder using only routine techniques. In one embodiment, the ligand may be administered, reporter gene expression determined, the ligand removed, and the process repeated one or more times to obtain multiple diagnostic measurements of the cells. In another embodiment, ligand is continuously administered and reporter gene expression is measured periodically. The detection of reporter gene expression after ligand is administered can occur in vivo or ex vivo. For example, if the reporter gene encodes a secreted protein that circulates in the blood, detection of the protein can occur in a blood sample removed from the patient. If the reporter gene encodes a protein that produces a luminescent or fluorescent signal, the signal may be detected in vivo. In another embodiment, a sample of the modified cells can be removed and expression of the reporter gene detected ex vivo.

[0247] The second in vivo diagnostic embodiment of the invention involves direct in vivo introduction of the polynucleotides into the cells of the subject. The polynucleotides may be introduced into the subject systemically or locally (e.g., at the site of the suspected disease or disorder). Once the polynucleotides have been introduced to the subject, the ligand may be administered and reporter gene expression assayed. The ligand may be administered by any suitable method, either systemically (e.g., orally, intravenously) or locally (e.g., intraperitoneally, intrathecally, intraventricularly, direct injection into the tissue or organ where the suspected disease or disorder is occurring). The optimal timing of ligand administration can be determined for each type of cell and disease or disorder using only routine techniques. In one embodiment, the ligand may be added, reporter gene expression determined, the ligand removed, and the process repeated one or more times to obtain multiple diagnostic measurements of the cells containing the polynucleotides. In another embodiment, ligand is continuously administered and reporter gene expression is measured periodically. The detection of reporter gene expression after ligand is administered can occur in vivo or ex vivo. For example, if the reporter gene encodes a secreted protein that circulates in the blood, detection of the protein can occur in a blood sample removed from the patient. If the reporter gene encodes a protein that produces a luminescent or fluorescent signal, the signal may be detected in vivo. In another embodiment, a sample of the modified cells can be removed and expression of the reporter gene detected ex vivo.

[0248] When non-autologous cells are used in the diagnostic methods, the cells may be obtained from any source, e.g., other subjects, cell lines, or animals. The non-autologous cells may be any cells that are viable after transplantation, such as fibroblasts or stem cells (e.g., embryonic stem cells, hematopoietic stem cells). The non-autologous cells are isolated and the polynucleotides are introduced into the cells in culture as described above. At some point after the introduction of the polynucleotides into the cells, the cells are introduced into the subject. Introduction may be carried out by any method known in the art, e.g., intravenous infusion or direct injection into a tissue or cavity. In one embodiment, the presence of the polynucleotides in the cells is determined prior to introducing the cells back into the subject. In another embodiment, cells containing the polynucleotides are selected (e.g., based on the presence of a selectable marker in the polynucleotides) and only those cells containing the polynucleotides are introduced into the subject. In one embodiment, the non-autologous cells are surrounded by a barrier (e.g., encapsulated) prior to introduction into the subject. After the cells are introduced to the subject, ligand is administered to the subject and reporter gene expression is assayed. The ligand may be administered by any suitable method, either systemically (e.g., orally, intravenously) or locally (e.g., intraperitoneally, intrathecally, intraventricularly, direct injection into the tissue or organ where the cells were introduced). The optimal timing of ligand administration can be determined for each type of cell and disease or disorder using only routine techniques. In one embodiment, the ligand may be administered, reporter gene expression determined, the ligand removed, and the process repeated one or more times to obtain multiple diagnostic measurements of the cells. In another embodiment, ligand is continuously administered and reporter gene expression is measured periodically. The detection of reporter gene expression after ligand is administered can occur in vivo or ex vivo. For example, if the reporter gene encodes a secreted protein that circulates in the blood, detection of the protein can occur in a blood sample removed from the patient. If the reporter gene encodes a protein that produces a luminescent or fluorescent signal, the signal may be detected in vivo. In another embodiment, a sample of the modified cells can be removed and expression of the reporter gene detected ex vivo.

[0249] In all in vivo embodiments, the polynucleotides or vector comprising the polynucleotides may comprise a sequence encoding a lethal polypeptide that can be turned on to express a product that will kill a cell containing the polynucleotides or vector. Lethal polypeptide expression can be used to eliminate the modified cells from a subject, either because diagnostic tests are no longer needed or because of a problem with the modified cells (e.g., hyperproliferation or toxicity). A lethal polypeptide, as used herein, is a polypeptide that, when expressed, is lethal to the cell that expresses the polypeptide, either because the polypeptide itself is lethal or the polypeptide produces a compound that is lethal. As used herein, a lethal polypeptide includes polypeptides that induce cell death in any fashion, including but not limited to, necrosis, apoptosis and cytotoxicity. Examples of lethal polypeptides include, but are not limited to, apoptosis inducing tumor suppressor genes such as, but not limited to, p53, Rb and BRCA-1, toxins such as diphtheria toxin (DTA), shigella neurotoxin, botulism toxin, tetanus toxin, cholera toxin, CSE-V2 and other variants of scorpion protein toxins to name a few, suicide genes such as cytosine deaminase and thymidine kinase, and cytotoxic genes, e.g., tumor necrosis factor, interferon-alpha. The present invention is not limited by the identity of the lethal polypeptide, provided that the polypeptide is capable of being lethal to the cell in which it is expressed. If the modified cells are short-lived cells (e.g., cells with a limited lifespan (e.g., about 10 days or less, such as dendritic cells), it may not be necessary to include a lethal polypeptide in the polynucleotides or vector as the cells will be naturally removed over a short period of time.

[0250] Another aspect of the invention relates to methods of monitoring progression of a disease or disorder by administering to cells of the subject the diagnostic gene switches of the invention and measuring reporter gene expression to monitor progression of the disease or disorder. In one embodiment, the invention relates to methods of monitoring the progression of a disease or disorder in a subject, comprising:

[0251] (a) introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0252] (b) administering ligand to said modified cells; and

[0253] (c) detecting reporter gene expression at least twice;

[0254] wherein a change in the level of expression of said reporter gene indicates progression of said disease or disorder in said subject.

[0255] Progression may be indicated by increasing or decreasing reporter gene expression depending on whether the diagnostic switch promoters are responsive to factor(s) that increase or decrease during progression of the disease or disorder. These methods may be carried out using any of the variants of the diagnostic methods described above (i.e., ex vivo cells, modification of cells ex vivo followed by reintroduction of the cells in vivo, or in vivo). A disease or disorder is monitored by measuring reporter gene expression at least twice as an indication of the state of the disease or disorder and noting any change in the level of expression. In one embodiment, the monitoring can be carried out by exposing the cells to ligand continuously and measuring reporter gene expression intermittently. In another embodiment, the monitoring can be carried out by exposing cells to ligand intermittently and measuring reporter gene expression during each exposure.

[0256] One embodiment of the invention relates to methods of preparing modified cells for monitoring the progression of a disease or disorder in a subject, comprising introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells.

[0257] Another embodiment of the invention relates to methods of monitoring the progression of a disease or disorder in a subject, comprising:

[0258] (a) administering ligand to modified cells of said subject; and

[0259] (b) detecting reporter gene expression at least twice;

[0260] wherein a change in the level of expression of said reporter gene indicates progression of said disease or disorder in said subject; and

[0261] wherein said modified cells of said subject comprise (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor.

[0262] In a further embodiment, the methods of monitoring progression of a disease or disorder are carried out using non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the subject, and the modified non-autologous cells are introduced into the subject. In one embodiment, the non-autologous cells are surrounded by a barrier (e.g., encapsulated) prior to being introduced into the subject. One embodiment of the invention relates to methods of monitoring the progression of a disease or disorder in a subject, comprising:

[0263] (a) introducing into non-autologous cells (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0264] (b) introducing said modified cells into said subject;

[0265] (c) administering ligand to said modified cells; and

[0266] (d) detecting reporter gene expression at least twice;

[0267] wherein a change in the level of expression of said reporter gene indicates progression of said disease or disorder in said subject.

[0268] A further aspect of the invention relates to methods of monitoring the effectiveness of a treatment for a disease or disorder in a subject, comprising administering to the subject a treatment and carrying out the diagnostic methods of the invention at least twice to determine if reporter gene expression is increasing, decreasing, or remaining the same. In one embodiment, the invention relates to methods of monitoring the effectiveness of a treatment for a disease or disorder in a subject, comprising:

[0269] (a) administering said treatment to said subject;

[0270] (b) introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0271] (c) administering ligand to said modified cells; and

[0272] (d) detecting reporter gene expression at least twice;

[0273] wherein a change in the level of expression of said reporter gene indicates the effectiveness of said treatment.

[0274] A change in the level of expression of the reporter gene after the treatment is administered is an indication of the effectiveness of the treatment. A decrease in reporter gene expression indicates the treatment is effective if the diagnostic switch promoter(s) are responsive to factor(s) that are elevated in the disease or disorder. An increase in reporter gene expression indicates the treatment is effective if the diagnostic switch promoter(s) are responsive to factor(s) that are reduced in the disease or disorder. If reporter gene expression does not change after administration of the treatment, it may indicate that the treatment has halted the progression of the disease or disorder. These methods may be carried out using any of the variants of the diagnostic methods described above (i.e., ex vivo cells, modification of cells ex vivo followed by reintroduction of the cells in vivo, in vivo).

[0275] For the ex vivo embodiment of the invention, cells may be isolated from the subject before treatment to determine baseline levels of reporter gene expression. After the treatment is administered to the subject, cells may be isolated from the subject at various intervals to determine reporter gene expression.

[0276] For the in vivo embodiments of the invention, modified cells or the polynucleotides can be introduced into a subject before, during, or after administration of the treatment. If the cells or polynucleotides are administered prior to the treatment, a baseline level of reporter gene expression can be obtained.

[0277] The measurement of reporter gene expression may be carried out ex vivo or in vivo. In one embodiment, the monitoring can be carried out by exposing the cells to ligand continuously and measuring reporter gene expression intermittently. In another embodiment, the monitoring can be carried out by exposing cells to ligand intermittently and measuring reporter gene expression during each exposure.

[0278] In one embodiment, one or both of the polynucleotides encoding a gene switch and a reporter gene may be part of a therapeutic vector that is being administered to a subject (e.g., a vector encoding a therapeutic protein or nucleic acid for gene therapy). In this embodiment, the therapeutic treatment and the diagnostic test for monitoring effectiveness of the treatment are administered together in one unit, ensuring that all cells that receive the treatment also receive the diagnostic gene switch.

[0279] One embodiment of the invention relates to methods of preparing modified cells for monitoring the effectiveness of a treatment for a disease or disorder in a subject, comprising introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells.

[0280] Another embodiment of the invention relates to methods of monitoring the effectiveness of a treatment for a disease or disorder in a subject, comprising:

[0281] (a) administering said treatment to said subject;

[0282] (b) administering ligand to modified cells of said subject; and

[0283] (c) detecting reporter gene expression at least twice;

[0284] wherein a change in the level of expression of said reporter gene indicates the effectiveness of said treatment; and

[0285] wherein said modified cells of said subject comprise (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor.

[0286] In a further embodiment, the methods of monitoring the effectiveness of a treatment for a disease or disorder are carried out using non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the subject, and the modified non-autologous cells are introduced into the subject. In one embodiment, the non-autologous cells are surrounded by a barrier (e.g., encapsulated) prior to being introduced into the subject. In one embodiment the invention relates to methods of monitoring the effectiveness of a treatment for a disease or disorder in a subject, comprising:

[0287] (a) introducing into non-autologous cells (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0288] (b) introducing said modified cells into said subject;

[0289] (c) administering ligand to said modified cells; and

[0290] (d) detecting reporter gene expression at least twice;

[0291] wherein a change in the level of expression of said reporter gene indicates the effectiveness of said treatment.

[0292] Another aspect of the invention relates to methods of monitoring the potential toxicity of an administered treatment for a disease or disorder in a subject. In one embodiment, the invention relates to methods of monitoring the potential toxicity of an administered treatment for a disease or disorder in a subject, comprising: administering said treatment to said subject;

[0293] (a) introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by factors found in cells that are being exposed to toxic conditions, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0294] (b) administering ligand to said modified cells; and

[0295] (c) detecting reporter gene expression at least twice;

[0296] wherein a change in the level of expression of said reporter gene indicates the toxicity of said treatment.

[0297] In one embodiment, this aspect involves polynucleotides in which the transcription factor sequence(s) are under the control of promoters that are regulated by factors found in cells that are being exposed to toxic conditions, e.g., cells that are stressed or dying. Examples include, without limitation, promoters responsive to apoptosis signals, necrosis signals, hypoxia, reactive oxygen species, DNA or chromatin modification, protein degradation, oxidative/reductive state, changes in pH, etc. Suitable stress promoters include those disclosed in U.S. Published Application No. 2003/0027127 (incorporated herein by reference) and include, without limitation, promoters from the following genes: CYP1A1, GST Ya, GADD45, GRP78, JUN, FOS, XHF, HSP70, MT IIA, GADD153, ALDH 1, HMO, CRE, XRE, NFκBRE, RARE, ThRE, PPRE, TRE, ERE, and p53RE. Suitable apoptosis-responsive promoters include, without limitation, Fas/CD95, TRAMP, TNF RI, DR1, DR2, DR3, DR4, DRS, DR6, FADD, RIP, TNFα, Fas ligand, TRAILR1, TRAILR2, TRAILR3, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3 kinase, PP1, and caspase proteins. Detection of an increase in reporter gene expression following administration of a treatment is an indication that the treatment is harmful to the cells. By making the gene switch responsive to stress or death signals, it can be used to monitor the effects of a treatment and detect toxic effects on the cellular level long before the subject exhibits overt symptoms of toxicity.

[0298] These methods may be carried out using any of the variants of the diagnostic methods described above (i.e., ex vivo cells, modification of cells ex vivo followed by reintroduction of the cells in vivo, in vivo).

[0299] For the ex vivo embodiment of the invention, cells may be isolated from the subject before treatment to determine baseline levels of reporter gene expression. After the treatment is administered to the subject, cells may be isolated from the subject at various intervals to determine reporter gene expression.

[0300] For the in vivo embodiments of the invention, modified cells or the polynucleotides can be introduced into a subject before, during, or after administration of the treatment. If the cells or polynucleotides are administered prior to the treatment, a baseline level of reporter gene expression can be obtained.

[0301] The measurement of reporter gene expression may be carried out ex vivo or in vivo. In one embodiment, the monitoring can be carried out by exposing the cells to ligand continuously and measuring reporter gene expression intermittently. In another embodiment, the monitoring can be carried out by exposing cells to ligand intermittently and measuring reporter gene expression during each exposure.

[0302] One embodiment of the invention relates to methods of preparing modified cells for monitoring the potential toxicity of an administered treatment for a disease or disorder in a subject, comprising introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by factors found in cells that are being exposed to toxic conditions, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells.

[0303] Another embodiment of the invention relates to methods of monitoring the potential toxicity of an administered treatment for a disease or disorder in a subject, comprising:

[0304] (a) administering said treatment to said subject;

[0305] (b) administering ligand to modified cells of said subject; and

[0306] (c) detecting reporter gene expression at least twice;

[0307] wherein a change in the level of expression of said reporter gene indicates the toxicity of said treatment; and

[0308] wherein said modified cells of said subject comprise (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by factors found in cells that are being exposed to toxic conditions, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor.

[0309] In a further embodiment, the methods of monitoring the potential toxicity of an administered treatment for a disease or disorder are carried out using non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the subject, and the modified non-autologous cells are introduced into the subject. In one embodiment, the non-autologous cells are surrounded by a barrier (e.g., encapsulated) prior to being introduced into the subject. In one embodiment, the invention relates to methods of monitoring the potential toxicity of an administered treatment for a disease or disorder in a subject, comprising:

[0310] (a) introducing into non-autologous cells (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by factors found in cells that are being exposed to toxic conditions, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0311] (b) introducing said modified cells into said subject;

[0312] (c) administering ligand to said modified cells; and

[0313] (d) detecting reporter gene expression at least twice;

[0314] wherein a change in the level of expression of said reporter gene indicates the toxicity of said treatment.

[0315] In another embodiment of the invention, the polynucleotide comprises transcription factor sequence(s) that are under the control of promoters that are activated by the factor which is being administered as the treatment (e.g., gene therapy treatment with a therapeutic protein or nucleic acid). By making the gene switch responsive to the administered treatment, it can be used to monitor expression of the gene therapy treatment and detect undesirably high or low levels of the treatment long before the subject exhibits overt symptoms of overexpression or underexpression of the therapeutic factor. In one embodiment, the invention relates to methods of monitoring the level of a factor that is being administered to a subject for treatment for a disease or disorder, comprising: administering said treatment to said subject;

[0316] (a) introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by said factor that is being administered for treatment, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0317] (b) administering ligand to said modified cells; and

[0318] (c) detecting reporter gene expression;

[0319] wherein the level of expression of said reporter gene indicates the level of the factor being administered for treatment.

[0320] These methods may be carried out using any of the variants of the diagnostic methods described above (i.e., ex vivo cells, modification of cells ex vivo followed by reintroduction of the cells in vivo, in vivo).

[0321] For the ex vivo embodiment of the invention, cells may be isolated from the subject before treatment to determine baseline levels of reporter gene expression. After the treatment is administered to the subject, cells may be isolated from the subject at various intervals to determine reporter gene expression.

[0322] For the in vivo embodiments of the invention, modified cells or the polynucleotides can be introduced into a subject before, during, or after administration of the treatment. If the cells or polynucleotides are administered prior to the treatment, a baseline level of reporter gene expression can be obtained.

[0323] The measurement of reporter gene expression may be carried out ex vivo or in vivo. In one embodiment, the monitoring can be carried out by exposing the cells to ligand continuously and measuring reporter gene expression intermittently. In another embodiment, the monitoring can be carried out by exposing cells to ligand intermittently and measuring reporter gene expression during each exposure.

[0324] One embodiment of the invention relates to methods of preparing modified cells for monitoring the level of a factor that is being administered to a subject for treatment for a disease or disorder, comprising introducing into cells of said subject (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by said factor that is being administered for treatment, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells.

[0325] Another embodiment of the invention relates to method of monitoring the level of a factor that is being administered to a subject for treatment for a disease or disorder, comprising:

[0326] (a) administering said treatment to said subject;

[0327] (b) administering ligand to modified cells of said subject; and

[0328] (c) detecting reporter gene expression;

[0329] wherein the level of expression of said reporter gene indicates the level of the factor being administered for treatment; and

[0330] wherein said modified cells of said subject comprise (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by said factor that is being administered for treatment, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor.

[0331] In a further embodiment, the methods of monitoring the level of a factor that is being administered to a subject for treatment for a disease or disorder are carried out using non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the subject, and the modified non-autologous cells are introduced into the subject. In one embodiment, the non-autologous cells are surrounded by a barrier (e.g., encapsulated) prior to being introduced into the subject. A method of monitoring the level of a factor that is being administered to a subject for a disease or disorder in a subject, comprising:

[0332] (a) introducing into non-autologous cells (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by said factor that is being administered for treatment, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0333] (b) introducing said modified cells into said subject;

[0334] (c) administering ligand to said modified cells; and

[0335] (d) detecting reporter gene expression;

[0336] wherein the level of expression of said reporter gene indicates the level of the factor being administered for treatment.

[0337] Another aspect of the invention relates to methods of detecting transplant rejection in a subject that has received an organ or tissue transplant, comprising:

[0338] (a) introducing into cells of said organ or tissue transplant (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during transplant rejection, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0339] (b) administering ligand to said modified cells; and

[0340] (c) detecting reporter gene expression;

[0341] wherein expression of the reporter gene indicates that transplant rejection has been detected.

[0342] An additional embodiment of the invention relates to methods of monitoring the progression of transplant rejection in a subject that has received an organ or tissue transplant, comprising:

[0343] (a) introducing into cells of said organ or tissue transplant (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during transplant rejection, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells;

[0344] (b) administering ligand to said modified cells; and

[0345] (c) detecting reporter gene expression at least twice;

[0346] wherein a change in the level of expression of said reporter gene indicates progression of said transplant rejection in said subject.

[0347] The methods for detecting and monitoring transplant rejection in a transplant recipient can be used to monitor the viability of transplanted organs or tissues. The ability to detect the onset of transplant rejection will allow a medical practitioner to adjust treatment of the transplant patient accordingly, e.g., by adjusting the level of immunosuppression therapy. The sensitivity of the present methods may allow for earlier detection of rejection than is possible with the current method of taking periodic tissue biopsies to look for tissue damage. The earlier detection of the occurrence of rejection in a subject will allow for a more rapid response and avoidance of further damage to the transplanted organs or tissues. The methods may be used for any organ or tissue transplant, including, without limitation, heart, kidney, lung, liver, pancreas, small and large intestine, skin, cornea, bone marrow, bone, ligament, tendon, neural tissue, and stem cell transplants.

[0348] In the methods of the invention the gene switch comprises one or more diagnostic switch promoters that are activated during transplant rejection, i.e., rejection promoters. A rejection promoter is any promoter that is activated in a transplanted organ or tissue when the rejection process begins to occur. Examples of rejection promoters that are useful in the present invention include, without limitation, promoters from the genes listed in Table 4, along with the organs in which increased expression has been detected during rejection.

TABLE-US-00004 TABLE 4 Gene Organ/Tissue A Disintegrin And Metalloproteinase 17 (ADAM17) Kidney A Disintegrin And Metalloproteinase 19 (ADAM19) Kidney AGT Kidney Allograft Inflammatory Factor-1 (AIF-1) Liver Angiotensin II Type 1 Receptor (AGTR1) Heart APAF1 Intestine β2-Defensin Lung Brain Natriuretic Peptide Heart C-Reactive Protein (CRP) Liver C3 Kidney CCL1 Heart CCL3 Heart CCL4 Heart CCL5 Heart CCR5 Heart CD3 Kidney CD95 Liver CD95 Ligand Liver, Heart CD103 Kidney Cellular Mediator of Immune Response (MIR) Heart CFLAR Heart Chemokine (C--X--C motif) Ligand 9 (CXCL9) Heart Collagen Type IX α3 Kidney Collagenase Lung Connective Tissue Growth Factor (CTGF) Kidney CX3CR1 Heart CXCR3 Pancreas, Heart, Kidney Cyclooxygenase-2 (COX-2) Kidney, Heart Early Growth Response Gene-1 (EGR-1) Lung, Heart ENA 78 Heart Eotaxin Cornea Epidermal Growth Factor Receptor (EGFR) Kidney EPST11 Intestine Fas Heart Fas Ligand Kidney, Heart, Pancreas Fork-Head Activin Signal Transducer-1 (FAST-1) Heart FOXP3 Kidney Fractalkine Kidney, Heart Gamma 2 Kidney Granulysin Kidney Granzyme B Kidney, Pancreas, Intestine, Heart, Lung Heat Shock Protein-60 (HSP-60) Intestine Heat Shock Protein-70 (HSP-70) Intestine Hepatocyte Growth Factor (HGF) Heart IF127 Intestine Integrin-α4 (ITGA4) Heart Interferon-γ Kidney, Intestine, Heart Interferon-Inducible Protein 10 (IP-10; CXCL10) Kidney, Heart, Pancreas Interleukin-2 (IL-2) Heart Interleukin-2 Receptor (IL-2R) Intestine Interleukin-4 (IL-4) Kidney Interleukin-15 (IL-15) Heart, Lung Interleukin-18 (IL-18) Kidney Intracellular Adhesion Molecule-1 (ICAM-1) Kidney Laminin Kidney LAP3 Intestine Macrophage Inflammatory Protein-2 (MIP-2) Cornea MADCAM-1 Intestine Matrix Metalloproteinase-2 (MMP-2) Intestine, Kidney Matrix Metalloproteinase-9 (MMP-9) Kidney, Intestine Matrix Metalloproteinase-11 (MMP-11) Kidney Matrix Metalloproteinase-12 (MMP-12) Kidney Matrix Metalloproteinase-14 (MMP-14) Kidney MDK Intestine MIG Pancreas, Heart MIP-1α Cornea, Pancreas, Heart, Kidney MIP-1β Cornea, Heart Monocyte Chemotactic Protein-1 (MCP-1) Cornea, Pancreas, Heart Monocyte Chemotactic Protein-2 (MCP-2) Heart MUC2 Intestine MUC4 Intestine NKG2D Kidney p16 (INK4a) Kidney p21 (WAF/CIP1) Kidney p27 (Kip1) Kidney Perforin Kidney, Pancreas, Intestine, Heart Programmed Cell Death (PDCD1) Heart RANTES Cornea, Pancreas, Kidney, Heart RAS Homolog Gene Family, Member U (RHOU) Heart Semaphorin 7A (SEMA7A) Heart Serine Proteinase Inhibitor-9 (PI-9) Kidney SOD2 Heart STK6 Intestine Surfactant Protein-C (SP-C) Kidney TIAF-1 Kidney, Liver TIM-3 Kidney Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Kidney Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) Kidney Transforming Growth Factor-β1 (TGF-β1) Intestine, Kidney, Heart Transforming Growth Factor Type I Receptor Intestine Tumor Necrosis Factor-α (TNF-α) Intestine, Heart, Kidney Urokinase Plasminogen Activator (uPA) Kidney Urokinase Plasminogen Activator Receptor (uPAR) Kidney Vascular Cell Adhesion Molecule-1 (VCAM-1) Kidney, Lung Vasoactive Intestinal Peptide (VIP) Intestine WD Repeat Dommoain 40A (WDR40A) Heart XCL1 Heart

[0349] In one embodiment of the invention, the polynucleotides comprising the rejection promoters are administered to the organ or tissue to be transplanted prior to the transplantation process. Organs and tissues that are used for transplantation typically must be transplanted into a recipient within 24-48 hours after removal from the donor. In one embodiment, the polynucleotides of the invention are administered to the organ or tissue within 48 hours of removal from the donor, e.g., within 36, 24, 18, 12, or 6 hours of removal from the donor. In another embodiment, the polynucleotides are administered to the organ or tissue at least 48 hours prior to transplantation into the recipient, e.g., at least 36, 24, 18, 12, or 6 hours prior to transplantation. The polynucleotides may be introduced into the organ or tissue to be transplanted in one location or in more than one location within the organ or tissue, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more locations.

[0350] In another embodiment, the polynucleotides are administered to the organ or tissue after it has been transplanted into a subject, e.g., 2, 4, 6, 8, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks or more after the transplantation.

[0351] The polynucleotides may be administered to the organ or tissue by any means as discussed above, including direct injection, electroporation, viral delivery, etc. In other embodiments, the polynucleotides may be administered as part of transgenic cells, e.g., transgenic stem cells. The cells may be isolated from either the transplant donor or the transplant recipient. For example, stem cells may be isolated from a patent in need of a transplant and the polynucleotides of the invention introduced into the stem cells. The transgenic cells may then be stored (e.g., frozen) until an organ or tissue is available for transplantation. The transgenic cells may then be administered to the organ or tissue before or after transplantation.

[0352] In a further embodiment, the methods of detecting transplant rejection may be carried out by introducing the polynucleotides of the invention into non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the organ or tissue being transplanted, and the modified non-autologous cells are introduced to the organ or tissue prior to transplantation. In one embodiment, the modified non-autologous cells are surrounded by a barrier (e.g., encapsulated) prior to being introduced into the organ or tissue.

[0353] In one embodiment of the invention, the gene switch comprises a single rejection promoter operably linked to a transcription factor sequence. In another embodiment, the gene switch comprises two rejection promoters that are operably linked to two different transcription factor sequences that together encode a ligand-dependent transcription factor. The two rejection promoters may be the same or different.

[0354] In another embodiment, the gene switch may further comprise a promoter that regulates expression of a control protein that is useful for monitoring the function of the gene switch, i.e., to show that the gene switch is operating properly in its environment, e.g., has not been subjected to gene silencing. The expression of the control protein may be used to limit false negative results from the diagnostic switch. In one embodiment, the promoter linked to the control protein is a constitutive promoter so that the control protein is always expressed. In a different embodiment, the promoter linked to the control protein is a switch promoter which is different from the rejection promoter(s) present in the gene switch. The control protein may be a ligand-dependent transcription factor that binds to a promoter operably linked to a second reporter gene that is different from the first reporter gene. For example, the control protein may be a ligand-dependent transcription factor having a different DNA binding domain than the transcription factor expressed from the rejection promoter(s), and that recognizes the response elements in the promoter operably linked to the second reporter gene as shown in FIG. 4.

[0355] In one embodiment of the methods for detecting or monitoring transplant rejection, the reporter gene is any reporter gene described above. In another embodiment, the reporter gene encodes a secreted protein, e.g., one that can be readily detected in a blood or urine sample of a transplant recipient. In another embodiment, the reporter gene encodes a protein that is endogenous to the transplant recipient, e.g., a protein that is normally expressed at low levels so that an increase in reporter gene expression upon the onset of rejection can be detected.

[0356] Once the polynucleotides of the invention have been administered to the organ or tissue transplant, the methods of detecting or monitoring transplant rejection may be carried out by detecting reporter gene expression. In one embodiment, the level of reporter gene expression may be measured once. In another embodiment, the level of reporter gene expression may be measured more than once, e.g., regularly, such as once every 1, 2, 3, 4, 5, 6 days, 1, 2, 3, 4 weeks, or every 1, 2, 3, 4, 5, 6 or more months. At each time point to be measured, a measurement of reporter gene expression may be made in the absence of ligand to get a background level of expression and in the presence ligand to obtain the level of reporter gene expression due to activation of the rejection promoter(s). In one embodiment, the level of reporter gene expression in the presence of ligand is determined shortly after transplantation (e.g., within 1, 2, 3, 4, 5, 6 days or 1, 2, 3, or 4 weeks) to obtain the ligand-induced baseline level of reporter gene expression prior to the occurrence of any transplant rejection. The initial timepoint (or any subsequent timepoint) can be used to determine how much ligand must be administered to the subject and how long the ligand must be present to obtain measurable reporter gene expression. Both the dose and time may be adjusted as needed for each subject. Regular monitoring of ligand-induced reporter gene expression may then be carried out to detect any increase in reporter gene expression, which is indicative of transplant rejection.

[0357] If a polynucleotide encoding a control protein (either constitutive or inducible) is present in the gene switch, the level of the control protein or the reporter gene induced by the control protein may be measured at the same time to confirm that the gene switch is functioning properly. If the gene switch is not functioning optimally, it may be necessary to increase the ligand concentration or the amount of time between ligand administration and reporter gene detection to increase the signal from the gene switch. In another embodiment, if the gene switch is no longer functioning, additional polynucleotides may be administered to the organ or tissue transplant so that monitoring of transplant rejection can be continued.

[0358] In a further embodiment, once an increase in reporter gene expression has been detected indicating the presence of transplant rejection, the diagnosis may be confirmed using traditional means, e.g., by obtaining a biopsy of the transplanted tissue.

[0359] One embodiment of the invention relates to methods of preparing on organ or tissue transplant for detecting transplant rejection in a subject, comprising introducing into cells of said organ or tissue transplant (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during transplant rejection, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells.

[0360] Another embodiment of the invention relates to methods of detecting transplant rejection in a subject that has received an organ or tissue transplant, comprising:

[0361] (a) administering ligand to said subject; and

[0362] (b) detecting reporter gene expression;

[0363] wherein expression of the reporter gene indicates that transplant rejection has been detected; and

[0364] wherein said organ or tissue transplant comprises one or more cells comprising (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during transplant rejection, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor.

[0365] One embodiment of the invention relates to methods of preparing an organ or tissue transplant for monitoring the progression of transplant rejection in a subject, comprising introducing into cells of said organ or tissue transplant (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during transplant rejection, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells.

[0366] Another embodiment of the invention relates to methods of monitoring the progression of transplant rejection in a subject that has received an organ or tissue transplant, comprising:

[0367] (a) administering ligand to said subject; and

[0368] (b) detecting reporter gene expression at least twice;

[0369] wherein a change in the level of expression of said reporter gene indicates progression of said transplant rejection in said subject, and

[0370] wherein said organ or tissue transplant comprises one or more cells comprising (1) a polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during transplant rejection, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor.

[0371] In a further embodiment, the methods of monitoring transplant rejection may be carried out by introducing the polynucleotides of the invention into non-autologous cells, e.g., cells that are allogeneic or xenogeneic to the organ or tissue being transplanted, and the modified non-autologous cells are introduced to the organ or tissue prior to transplantation. In one embodiment, the modified non-autologous cells are surrounded by a barrier (e.g., encapsulated) prior to being introduced into the organ or tissue.

[0372] In one embodiment of the methods described above, one or both of the polynucleotides encoding a gene switch and a reporter gene may be part of a therapeutic vector that is being administered to a subject (e.g., a vector encoding a therapeutic factor (protein or nucleic acid) for gene therapy). In this embodiment, the factor and the diagnostic test for monitoring the level of the factor are administered together in one unit, ensuring that all cells that receive the treatment also receive the diagnostic gene switch.

[0373] For each of the methods described above, in one embodiment, the polynucleotide encoding the gene switch and the polynucleotide encoding the reporter gene linked to a promoter are part of one larger polynucleotide, e.g., a vector. In another embodiment, the polynucleotide encoding the gene switch and the polynucleotide encoding the reporter gene linked to a promoter are separate polynucleotides.

[0374] In one aspect, the invention relates to polynucleotides that may be used in the methods of the invention. In one embodiment, the polynucleotide encodes a gene switch, the gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during said disease or disorder. In another embodiment, the polynucleotide further encodes a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor. In one embodiment, the gene switch is an EcR-based gene switch. In another embodiment, the gene switch comprises a first transcription factor sequence under the control of a first diagnostic switch promoter and a second transcription factor sequence under the control of a second diagnostic switch promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. In one embodiment, the first diagnostic switch promoter and the second diagnostic switch promoter are different. In another embodiment, the first diagnostic switch promoter and the second diagnostic switch promoter are the same. In another embodiment, the first transcription factor sequence encodes a protein comprising a heterodimer partner and a transactivation domain and the second transcription factor sequence encodes a protein comprising a DNA binding domain and a ligand-binding domain. In a further embodiment, the polynucleotide also encodes a lethal polypeptide operably linked to an inducible promoter.

[0375] In one aspect, the invention relates to polynucleotides encoding a gene switch, the gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by factors found in cells that are being exposed to toxic conditions. In another embodiment, the polynucleotide further encodes a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor. In one embodiment, the gene switch is an EcR-based gene switch. In another embodiment, the gene switch comprises a first transcription factor sequence under the control of a first diagnostic switch promoter and a second transcription factor sequence under the control of a second diagnostic switch promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. In one embodiment, the first diagnostic switch promoter and the second diagnostic switch promoter are different. In another embodiment, the first diagnostic switch promoter and the second diagnostic switch promoter are the same. In another embodiment, the first transcription factor sequence encodes a protein comprising a heterodimer partner and a transactivation domain and the second transcription factor sequence encodes a protein comprising a DNA binding domain and a ligand-binding domain. In a further embodiment, the polynucleotide also encodes a lethal polypeptide operably linked to an inducible promoter.

[0376] In one aspect, the invention relates to polynucleotides encoding a gene switch, the gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated by said factor that is being administered for treatment. In another embodiment, the polynucleotide further encodes a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor. In one embodiment, the gene switch is an EcR-based gene switch. In another embodiment, the gene switch comprises a first transcription factor sequence under the control of a first diagnostic switch promoter and a second transcription factor sequence under the control of a second diagnostic switch promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. In one embodiment, the first diagnostic switch promoter and the second diagnostic switch promoter are different. In another embodiment, the first diagnostic switch promoter and the second diagnostic switch promoter are the same. In another embodiment, the first transcription factor sequence encodes a protein comprising a heterodimer partner and a transactivation domain and the second transcription factor sequence encodes a protein comprising a DNA binding domain and a ligand-binding domain. In a further embodiment, the polynucleotide also encodes a lethal polypeptide operably linked to an inducible promoter.

[0377] In one aspect, the invention relates to polynucleotides encoding a gene switch, the gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, linked to a diagnostic switch promoter, wherein the activity of the promoter is modulated during transplant rejection. In another embodiment, the polynucleotide further encodes a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor. In one embodiment, the gene switch is an EcR-based gene switch. In another embodiment, the gene switch comprises a first transcription factor sequence under the control of a first diagnostic switch promoter and a second transcription factor sequence under the control of a second diagnostic switch promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. In one embodiment, the first diagnostic switch promoter and the second diagnostic switch promoter are different. In another embodiment, the first diagnostic switch promoter and the second diagnostic switch promoter are the same. In another embodiment, the first transcription factor sequence encodes a protein comprising a heterodimer partner and a transactivation domain and the second transcription factor sequence encodes a protein comprising a DNA binding domain and a ligand-binding domain. In a further embodiment, the polynucleotide also encodes a lethal polypeptide operably linked to an inducible promoter.

[0378] Another aspect of the invention relates to vectors comprising any of the polynucleotides described above. In one embodiment, the vector is a plasmid vector or a viral vector. In one embodiment, the polynucleotides are present on the same vector. In a further embodiment, each of the polynucleotides is on a separate vector. The separate vectors may be the identical vector (e.g., the same plasmid), the same type of vector (e.g., both are plasmids but not the same plasmid), or different types of vectors (e.g., one vector is a plasmid, the other vector is a virus).

[0379] In another aspect, the invention provides kits that may be used in conjunction with methods the invention. Kits according to this aspect of the invention may comprise one or more containers, which may contain one or more components selected from the group consisting of one or more nucleic acid molecules, restriction enzymes and one or more cells comprising such nucleic acid molecules. Kits of the invention may further comprise one or more containers containing cell culture media suitable for culturing cells of the invention, one or more containers containing antibiotics suitable for use in culturing cells of the invention, one or more containers containing buffers, one or more containers containing transfection reagents, one or more containers containing substrates for enzymatic reactions, and/or one or more ligands for gene switch activation.

[0380] Kits of the invention may contain a wide variety of nucleic acid molecules that can be used with the invention. Examples of nucleic acid molecules that can be supplied in kits of the invention include those that contain promoters, sequences encoding gene switches, enhancers, repressors, selection markers, transcription signals, translation signals, primer hybridization sites (e.g., for sequencing or PCR), recombination sites, restriction sites and polylinkers, sites that suppress the termination of translation in the presence of a suppressor tRNA, suppressor tRNA coding sequences, sequences that encode domains and/or regions, origins of replication, telomeres, centromeres, and the like. In one embodiment, kits may comprise a polynucleotide comprising a gene switch without any diagnostic switch promoters, the polynucleotide being suitable for insertion of any diagnostic switch promoters of interest. Nucleic acid molecules of the invention may comprise any one or more of these features in addition to polynucleotides as described above.

[0381] Kits of the invention may comprise cells. The cells may comprise the polynucleotides of the invention, or the cells and the polynucleotides may be in separate containers. In one embodiment the cells may be autologous cells, e.g., as part of a kit designed for a specific subject. In another embodiment, the cells may be non-autologous cells, e.g., as part of a kit designed for any subject. In a further embodiment, the non-autologous cells may be surrounded by a barrier (e.g., encapsulated).

[0382] Kits of the invention may comprise containers containing one or more recombination proteins. Suitable recombination proteins include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, Cin, Tn3 resolvase, ΦC31, TndX, XerC, and XerD. Other suitable recombination sites and proteins are those associated with the GATEWAY® Cloning Technology available from Invitrogen Corp., Carlsbad, Calif., and described in the product literature of the GATEWAY® Cloning Technology (version E, Sep. 22, 2003), the entire disclosures of which are incorporated herein by reference.

[0383] Kits of the invention can also be supplied with primers. These primers will generally be designed to anneal to molecules having specific nucleotide sequences. For example, these primers can be designed for use in PCR to amplify a particular nucleic acid molecule. Sequencing primers may also be supplied with the kit.

[0384] One or more buffers (e.g., one, two, three, four, five, eight, ten, fifteen) may be supplied in kits of the invention. These buffers may be supplied at working concentrations or may be supplied in concentrated form and then diluted to the working concentrations. These buffers will often contain salt, metal ions, co-factors, metal ion chelating agents, etc. for the enhancement of activities or the stabilization of either the buffer itself or molecules in the buffer. Further, these buffers may be supplied in dried or aqueous forms. When buffers are supplied in a dried form, they will generally be dissolved in water prior to use.

[0385] Kits of the invention may contain virtually any combination of the components set out above or described elsewhere herein. As one skilled in the art would recognize, the components supplied with kits of the invention will vary with the intended use for the kits. Thus, kits may be designed to perform various functions set out in this application and the components of such kits will vary accordingly.

[0386] The present invention further relates to instructions for performing one or more methods of the invention. Such instructions can instruct a user of conditions suitable for performing methods of the invention. Instructions of the invention can be in a tangible form, for example, written instructions (e.g., typed on paper), or can be in an intangible form, for example, accessible via a computer disk or over the internet.

[0387] It will be recognized that a full text of instructions for performing a method of the invention or, where the instructions are included with a kit, for using the kit, need not be provided. One example of a situation in which a kit of the invention, for example, would not contain such full length instructions is where the provided directions inform a user of the kits where to obtain instructions for practicing methods for which the kit can be used. Thus, instructions for performing methods of the invention can be obtained from internet web pages, separately sold or distributed manuals or other product literature, etc. The invention thus includes kits that direct a kit user to one or more locations where instructions not directly packaged and/or distributed with the kits can be found. Such instructions can be in any form including, but not limited to, electronic or printed forms.

[0388] Having now fully described the invention, it will be understood by those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety.

EXAMPLES

Example 1

[0389] The vector shown in FIG. 5 includes IL-24/mda-7 promoter (SEQ ID NO.: 5). Adenovirus produced using the adenoviral shuttle vector is used to transduce cells isolated from lymphatic samples. The transduces cells are split into two groups and cultured either in the presence or absence of activator ligand. Both sets of cells are then disrupted, and the resulting cell lysates are used in luciferase assays.

Example 2

[0390] The vector shown in FIG. 6 includes TRPM4 and TRGC1/TARP promoters (SEQ ID NO.: 6). The DNA vector is used to transduce a prostate biopsy using non-viral transduction systems. The transduced biopsy is split into two groups and cultured either in the presence or absence of an activator ligand. Both sets of biopsied tissues are homogenized, and the resulting lysates are used in luciferase assays.

Example 3

[0391] The vector shown in FIG. 7 includes the ADAM-17 promoter (SEQ ID NO.: 7) and the CD95-ADAM8 reporter (SEQ ID NOs: 10-11). Adenovirus produced using the adenoviral shuttle vector is used to transduced a portion of a donor kidney before transplantation. Following transplantation, serum samples are assayed for the reporter gene for a period defined by the physician. The assay protocol consists of assaying reporter expression from samples not exposed to activator ligand. Immediately after blood draw of the non-activated group, ligand is administered for 24 hours and then discontinued; another blood sample is acquired within one hour before the ligand treatment is completed. Reporter assay results are compared between the ligand-treated and untreated samples. A period of 60 hours must pass before this procedure is performed again.

Example 4

[0392] The vector shown in FIG. 8 includes the CXCL9 and SEMA7A promoters (SEQ ID NO.: 8) and the CD40-CD3 reporter (SEQ ID NOs.: 12-13). This vector is used to transduce a portion of a donor heart before transplantation via direct needle injection. Following transplantation, serum samples are assayed for the reporter gene for a period defined by the physician. The assay protocol consists of assaying reporter expression from samples not exposed to an activator ligand. Immediately after the blood draw of the "non-activated" group, ligand is administered for 24 hours and then discontinued; another blood sample is acquired within one hour before the ligand treatment is completed. Reporter assay results are compared between the ligand-treated and untreated samples. A period of 60 hours must pass before this procedure can be performed again.

Example 5

[0393] The vector shown in FIG. 9 includes the ADAM-17 promoter and the alkaline phosphatase-c terminal CD40 reporter (SEQ ID NO.: 14). Adenovirus produced using this adenoviral shuttle vector is used to transduce primary porcine kidney cells. The resulting cells are encapsulated in alginate (see WO 2007/046719A2), and then implanted into a donor kidney before transplantation. Following transplantation, serum samples are assayed for the reporter gene for a period defined by the physician. The assay protocol consists of assaying reporter expression from samples not exposed to activator ligand. Immediately after the blood draw of the "non-activated" group, ligand is administered for 24 hours and then discontinued; another blood sample is acquired within one hour before the ligand treatment is completed. Reporter assay results are compared between the ligand-treated and untreated samples. A period of 60 hours must pass before this procedure can be performed again.

Sequence CWU 1

1

23115PRTArtificial SequenceSynthetic ecdysone receptor 1Arg Arg Gly Xaa Thr Cys Ala Asn Thr Gly Ala Xaa Cys Tyr Tyr 1 5 10 15 213DNAArtificial SequenceSynthetic ecdysone receptor 2aggtcanagg tca 13315DNAArtificial SequenceSynthetic ecdysone receptor 3gggttgaatg aattt 15418DNAArtificial SequenceSynthetic homing endonuclease (HE) enzyme known as I-SceI 4tagggataac agggtaat 18513028DNAArtificial SequenceSynthetic promoter 5taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac 60cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca 120agaaaattaa tcgcaccggt atctatgtcg ggtgcggaga aagaggtaat gaaatggcag 180ctagcatcat caataatata ccttattttg gattgaagcc aatatgataa tgagggggtg 240gagtttgtga cgtggcgcgg ggcgtgggaa cggggcgggt gacgtagtag tgtggcggaa 300gtgtgatgtt gcaagtgtgg cggaacacat gtaagcgacg gatgtggcaa aagtgacgtt 360tttggtgtgc gccggtgtac acaggaagtg acaattttcg cgcggtttta ggcggatgtt 420gtagtaaatt tgggcgtaac cgagtaagat ttggccattt tcgcgggaaa actgaataag 480aggaagtgaa atctgaataa ttttgtgtta ctcatagcgc gtaatagtgc gccggtgtac 540acaggaagtg acaattttcg cgcggtttta ggcggatgtt gtagtaaatt tgggcgtaac 600cgagtaagat ttggccattt tcgcgggaaa actgaataag aggaagtgaa atctgaataa 660ttttgtgtta ctcattttgt ctagggagat ccggtaccga tatcctagac aacgatgctg 720agctaactat aacggtccta aggtagcgac cgcggagact aggtgtattt atctaagcga 780tcgcttaatt aaggccggcc gccgcaataa aatatcttta ttttcattac atctgtgtgt 840tggttttttg tgtgaatcca tagtactaac atacgctctc catcaaaaca aaacgaaaca 900aaacaaacta gcaaaatagg ctgtccccag tgcaagtcca ggtgccagaa catttctcta 960tccataatgc aggggtaccg ggtgatgacg gtgaaaacct ccaattgcgg agtactgtcc 1020tccgagcgga gtactgtcct ccgagcggag tactgtcctc cgagcggagt actgtcctcc 1080gagcggagta ctgtcctccg agcggagtac tgtcctccga gcggagagtc cccggggacc 1140tagagggtat ataatgggtg ccttagctgg tgtgtgacct catcttcctg tacgcccctg 1200caggggcgcg ccacgcgtcg aagaaggtga gtaatcttaa catgctcttt tttttttttt 1260ttgctaatcc cttttgtgtg ctgatgttag gatgacattt acaacaaatg tttgttcctg 1320acaggaaaaa ccttgctggg taccttcgtt gccggacact tcttgtcctc tactttggaa 1380aaaaggaatt gagagccgct agcgccacca tggaagatgc caaaaacatt aagaagggcc 1440cagcgccatt ctacccactc gaagacggga ccgctggcga gcagctgcac aaagccatga 1500agcgctacgc cctggtgccc ggcaccatcg cctttaccga cgcacatatc gaggtggaca 1560ttacctacgc cgagtacttc gagatgagcg ttcggctggc agaagctatg aagcgctatg 1620ggctgaatac aaaccatcgg atcgtggtgt gcagcgagaa tagcttgcag ttcttcatgc 1680ccgtgttggg tgccctgttc atcggtgtgg ctgtggcccc agctaacgac atctacaacg 1740agcgcgagct gctgaacagc atgggcatca gccagcccac cgtcgtattc gtgagcaaga 1800aagggctgca aaagatcctc aacgtgcaaa agaagctacc gatcatacaa aagatcatca 1860tcatggatag caagaccgac taccagggct tccaaagcat gtacaccttc gtgacttccc 1920atttgccacc cggcttcaac gagtacgact tcgtgcccga gagcttcgac cgggacaaaa 1980ccatcgccct gatcatgaac agtagtggca gtaccggatt gcccaagggc gtagccctac 2040cgcaccgcac cgcttgtgtc cgattcagtc atgcccgcga ccccatcttc ggcaaccaga 2100tcatccccga caccgctatt ctcagcgtgg tgccatttca ccacggcttc ggcatgttca 2160ccacgctggg ctacttgatc tgcggctttc gggtcgtgct catgtaccgc ttcgaggagg 2220agctattctt gcgcagcttg caagactata agattcaatc tgccctgctg gtgcccacac 2280tatttagctt cttcgctaag agcactctca tcgacaagta cgacctaagc aacttgcacg 2340agatcgccag cggcggagcg cctctcagca aggaggtagg tgaggccgtg gccaaacgct 2400tccacctacc aggcatccgc cagggctacg gcctgacaga aacaaccagc gccattctga 2460tcacccccga aggggacgac aagcctggcg cagtaggcaa ggtggtgccc ttcttcgagg 2520ctaaggtggt ggacttggac acaggtaaga ccctgggtgt gaaccagcgc ggcgagctgt 2580gcgtccgtgg ccccatgatc atgagcggct acgtgaacaa ccccgaggct acaaacgctc 2640tcatcgacaa ggacggctgg ctgcacagcg gcgacatcgc ctactgggac gaggacgagc 2700acttcttcat cgtggaccgg ctcaagagcc tgatcaaata caagggctac caggtagccc 2760cagccgaact ggagagcatc ctgctgcaac accccaacat cttcgacgcc ggggtcgctg 2820gcctgcccga cgacgatgct ggcgagctgc ccgccgcagt cgtcgtgctg gaacacggta 2880aaaccatgac cgagaaggag atcgtggact atgtggccag ccaggttaca accgccaaga 2940agctgcgcgg tggtgttgtg ttcgtggacg aggtgcctaa aggactgacc ggcaagttgg 3000acgcccgcaa gatccgcgag attctcatta aggccaagaa gggcggcaag atcgccgtgt 3060aaatcgattg cgcaaagctt tcgcgatagg cgagaccaat gggtgtgtac gtagcggccg 3120cgtcgacgat agcttgatgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc 3180ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt 3240ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag 3300gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt 3360gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg 3420cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg 3480tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct 3540caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct 3600cccttccctg tccttctgat tttaaaataa ctataccagc aggaggacgt ccagacacag 3660cataggctac ctggccatgc ccaaccggtg ggacatttga gttgcttgct tggcactgtc 3720ctctcatgcg ttgggtccac tcagtagatg cctgttgaat tctgatttaa atcggtccgc 3780gtacggcgtg gtaggtccga acgaatccat ggattaccct gttatcccta tccggagtta 3840acctcgagga cttcggaact tctagaacca gaccgttcag tttaaacgct cttctccccc 3900tcgagccata tggtctcagt cacaactact catctctgcc tctgtagcac gaaagcaatt 3960agcaacaata tgtcaacaaa catatgtgac cccatgaaaa ctttatttat tatggatacg 4020gaaacctgaa aataatgtct ttcttttgat tttttcccca atcattaaaa aacgtaaaaa 4080ctactcttag gtcgcaaggt taagccattc tcagcttggc agtggcaggc tggatttggc 4140ttgtgaccta cagttggcca atccctgatt cccaaaatgt attcctcagg gatgtgggca 4200aatacttatg ggaagtgctg gattaaacag agttaagaag catcagacat ttccaggacg 4260ggctagcaca tgccagggct ctctaactga cctcattgga ttcatctgtt tcatggagga 4320tcttgcaaga caagaattcc tcaaacctag agtctgagga ctgtgctttg ggaaacactg 4380ctctgcttga tgccctcact gggcacatgg tagaatctgg agctgagtgc cttgctagct 4440ggagataggg tcagagctct tgactgccct ggcagtcttg acacatcacg ctgtctgtgt 4500cccctgagtg gttcagagct acacaggcca agactagccc accagagcac caggcctccc 4560agctttctgg gcttgtccat gcgtacattt ccttattctt cctggtttcc agaacctaag 4620gagaggcaca ttttggttga gtgattataa ccctagggac catgggtagc tgcatgtcag 4680gaaacactcc tcaacttcct ggccctgatg gattaaagga gaggtactta caggttattt 4740cttcgctgtg gactactgtc ccagcatgaa tagggcatca ttattgaatt attttgacag 4800gaaggagact ggtgtatgct gcacagtaat aatgtattta catgtgtaca gagtttacca 4860agcacctctg tgttgttttt gcctttgttt attacacttg ggacaaattt ttaaaattta 4920tacatgcaga gactgcagcg cagagaagct aagagacttg cccctgccca cacagccagt 4980ggtagagcct gaactcaaac ccaggtctca tctcacctca ggggctgctt tccccatcgc 5040tgtattgtcc ttaaagtgat gggtgactag gcaatgaagt aattctctag gaaagcatga 5100ccaatttccc tttctccacc tccctctttt tcctccaccc ctcccccatc agcccccata 5160tatatgccca aatctccaca aagccttgct tgcctgcaaa cctttacttc tgaaatgact 5220tccacgcatg cggggggggg ggggggcaat tggccaccat gggccccaag aagaaaagga 5280aggtggcccc ccccaccgac gtgagcctgg gcgacgagct gcacctggac ggcgaggacg 5340tggccatggc ccacgccgac gccctggacg acttcgacct ggacatgctg ggcgacggcg 5400acagccccgg ccccggcttc accccccacg acagcgcccc ctacggcgcc ctggacatgg 5460ccgacttcga gttcgagcag atgttcaccg acgccctggg catcgacgag tacggcggcc 5520atatggagat gcccgtggac aggattctgg aggccgaact cgccgtggag cagaaaagcg 5580accagggcgt ggagggcccc ggcggaaccg gcggcagcgg cagcagcccc aacgaccccg 5640tgaccaacat ctgccaggcc gccgacaagc agctgttcac cctggtggag tgggccaaga 5700ggattcccca cttcagcagc ctgcccctgg acgaccaggt gatcctgctg agggccggat 5760ggaacgagct gctgatcgcc agcttcagcc acaggagcat cgacgtgagg gacggcatcc 5820tgctggccac cggcctgcac gtccatagga acagcgccca cagcgccgga gtgggcgcca 5880tcttcgacag ggtgctgacc gagctggtga gcaagatgag ggacatgagg atggacaaga 5940ccgagctggg ctgcctgagg gccatcatcc tgttcaaccc cgaggtgagg ggcctgaaaa 6000gcgcccagga ggtggagctg ctgagggaga aggtgtacgc cgccctggag gagtacacca 6060ggaccaccca ccccgacgag cccggcagat tcgccaagct gctgctgagg ctgcccagcc 6120tgaggagcat cggcctgaag tgcctggagc acctgttctt cttcaggctg atcggcgacg 6180tgcccatcga caccttcctg atggagatgc tggagagccc cagcgacagc tgagccggca 6240actcgctgta gtaattccag cgagaggcag agggagcgag cgggcggcgg gctagggtgg 6300aggagcccgg cgagcagagc tgcgctgcgg gcgtcctggg aagggagatc cggagcgaat 6360agggggcttc gcctctggcc cagccctccc gctgatcccc cagccagcgg tgcgcaaccc 6420tagccgcatc cacgaaactt tgcccatagc agcgggcggg cactttgcac tggaacttac 6480aacacccgag caaggacgcg actctcccga cgcggggagg ctattctgcc catttgggga 6540cacttccccg ccgctgccag gacccgcttc tctgaaaggc tctccttgca gctgcttaga 6600cgctggattt ttttcgggta gtggaaaacc agcagcctcc cgcgaccaga tctgccacca 6660tgaagctgct gagcagcatc gagcaggctt gcgacatctg caggctgaag aagctgaagt 6720gcagcaagga gaagcccaag tgcgccaagt gcctgaagaa caactgggag tgcagataca 6780gccccaagac caagaggagc cccctgacca gggcccacct gaccgaggtg gagagcaggc 6840tggagaggct ggagcagctg ttcctgctga tcttccccag ggaggacctg gacatgatcc 6900tgaagatgga cagcctgcaa gacatcaagg ccctgctgac cggcctgttc gtgcaggaca 6960acgtgaacaa ggacgccgtg accgacaggc tggccagcgt ggagaccgac atgcccctga 7020ccctgaggca gcacaggatc agcgccacca gcagcagcga ggagagcagc aacaagggcc 7080agaggcagct gaccgtgagc cccgagtttc ccgggatcag gcccgagtgc gtggtgcccg 7140agacccagtg cgccatgaaa aggaaggaga agaaggccca gaaggagaag gacaagctgc 7200ccgtgagcac caccaccgtc gatgaccaca tgccccccat catgcagtgc gagccccccc 7260cccccgaggc cgccaggatt cacgaggtcg tgcccaggtt cctgagcgac aagctgctgg 7320tgaccaacag gcagaagaac atcccccagc tgaccgccaa ccagcagttc ctgatcgcca 7380ggctgatctg gtatcaggac ggctacgagc agcccagcga cgaggacctg aaaaggatca 7440cccagacctg gcagcaggcc gacgacgaga acgaggagag cgacaccccc ttcaggcaga 7500tcaccgagat gaccatcctg accgtgcagc tgatcgtgga gttcgccaag ggcctgcccg 7560gattcgccaa gatcagccag cccgaccaga tcaccctgct gaaggcttgc agcagcgagg 7620tgatgatgct gagggtggcc aggaggtacg acgccgccag cgacagcatc ctgttcgcca 7680acaaccaggc ttacaccagg gacaactaca ggaaggctgg catggccgag gtgatcgagg 7740acctcctgca cttctgcaga tgtatgtaca gcatggccct ggacaacatc cactacgccc 7800tgctgaccgc cgtggtgatc ttcagcgaca ggcccggcct ggagcagccc cagctggtgg 7860aggagatcca gaggtactac ctgaacaccc tgaggatcta catcctgaac cagctgagcg 7920gcagcgccag gagcagcgtg atctacggca agatcctgag catcctgagc gagctgagga 7980ccctgggaat gcagaacagc aatatgtgta tcagcctgaa gctgaagaac aggaagctgc 8040cccccttcct ggaggagatt tgggacgtgg ccgacatgag ccacacccag ccccccccca 8100tcctggagag ccccaccaac ctgtgaatcg attagacatg ataagataca ttgatgagtt 8160tggacaaacc acaactagaa tgcagtgaaa aaaatgctta atttgtgaaa tttgtgatgc 8220tattgcttaa tttgtaacca ttataagctg caataaacaa gttaataaaa catttgcatt 8280cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc 8340tacaaatgtg gtatctagag ctcttccaaa tagatctgga aggtgctgag gtacgatgag 8400acccgcacca ggtgcagacc ctgcgagtgt ggcggtaaac atattaggaa ccagcctgtg 8460atgctggatg tgaccgagga gctgaggccc gatcacttgg tgctggcctg cacccgcgct 8520gagtttggct ctagcgatga agatacagat tgaggtactg aaatgtgtgg gcgtggctta 8580agggtgggaa agaatatata aggtgggggt cttatgtagt tttgtatctg ttttgcagca 8640gccgccgccg ccatgagcac caactcgttt gatggaagca ttgtgagctc atatttgaca 8700acgcgcatgc ccccatgggc cggggtgcgt cagaatgtga tgggctccag cattgatggt 8760cgccccgtcc tgcccgcaaa ctctactacc ttgacctacg agaccgtgtc tggaacgccg 8820ttggagactg cagcctccgc cgccgcttca gccgctgcag ccaccgcccg cgggattgtg 8880actgactttg ctttcctgag cccgcttgca agcagtgcag cttcccgttc atccgcccgc 8940gatgacaagt tgacggctct tttggcacaa ttggattctt tgacccggga acttaatgtc 9000gtttctcagc agctgttgga tctgcgccag caggtttctg ccctgaaggc ttcctcccct 9060cccaatgcgg tttaaaacat aaataaaaaa ccagactctg tttggatttg gatcaagcaa 9120gtgtcttgct gtctttattt aggggttttg cgcgcgcggt aggcccggga ccagcggtct 9180cggtcgttga gggtcctgtg tattttttcc aggacgtggt aaaggtgact ctggatgttc 9240agatacatgg gcataagccc gtctctgggg tggaggtagc accactgcag agcttcatgc 9300tgcggggtgg tgttgtagat gatccagtcg tagcaggagc gctgggcgtg gtgcctaaaa 9360atgtctttca gtagcaagct gattgccagg ggcaggccct tggtgtaagt gtttacaaag 9420cggttaagct gggatgggtg catacgtggg gatatgagat gcatcttgga ctgtattttt 9480aggttggcta tgttcccagc catatccctc cggggattca tgttgtgcag aaccaccagc 9540acagtgtatc cggtgcactt gggaaatttg tcatgtagct tagaaggaaa tgcgtggaag 9600aacttggaga cgcccttgtg acctccaaga ttttccatgc attcgtccat aatgatggca 9660atgggcccac gggcggcggc ctgggcgaag atatttctgg gatcactaac gtcatagttg 9720tgttccagga tgagatcgtc ataggccatt tttacaaagc gcgggcggag ggtgccagac 9780tgcggtataa tggttccatc cggcccaggg gcgtagttac cctcacagat ttgcatttcc 9840cacgctttga gttcagatgg ggggatcatg tctacctgcg gggcgatgaa gaaaacggtt 9900tccggggtag gggagatcag ctgggaagaa agcaggttcc tgagcagctg cgacttaccg 9960cagccggtgg gcccgtaaat cacacctatt accgggtgca actggtagtt aagagagctg 10020cagctgccgt catccctgag caggggggcc acttcgttaa gcatgtccct gactcgcatg 10080ttttccctga ccaaatccgc cagaaggcgc tcgccgccca gcgatagcag ttcttgcaag 10140gaagcaaagt ttttcaacgg tttgagaccg tccgccgtag gcatgctttt gagcgtttga 10200ccaagcagtt ccaggcggtc ccacagctcg gtcacctgct ctacggcatc tcgatccagc 10260atatctcctc gtttcgcggg ttggggcggc tttcgctgta cggcagtagt cggtgctcgt 10320ccagacgggc cagggtcatg tctttccacg ggcgcagggt cctcgtcagc gtagtctggg 10380tcacggtgaa ggggtgcgct ccgggctgcg cgctggccag ggtgcgcttg aggctggtcc 10440tgctggtgct gaagcgctgc cggtcttcgc cctgcgcgtc ggccaggtag catttgacca 10500tggtgtcata gtccagcccc tccgcggcgt ggcccttggc gcgcagcttg cccttggagg 10560aggcgccgca cgaggggcag tgcagacttt tgagggcgta gagcttgggc gcgagaaata 10620ccgattccgg ggagtaggca tccgcgccgc aggccccgca gacggtctcg cattccacga 10680gccaggtgag ctctggccgt tcggggtcaa aaaccaggtt tcccccatgc tttttgatgc 10740gtttcttacc tctggtttcc atgagccggt gtccacgctc ggtgacgaaa aggctgtccg 10800tgtccccgta tacagacttg agaggcctgt cctcgaccga tgcccttgag agccttcaac 10860ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc 10920ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag 10980gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg 11040cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag 11100gccattatcg ccggcatggc ggccgacgcg ctgggctacg tcttgctggc gttcgcgacg 11160cgaggctgga tggccttccc cattatgatt cttctcgctt ccggcggcat cgggatgccc 11220gcgttgcagg ccatgctgtc caggcaggta gatgacgacc atcagggaca gcttcaaggc 11280cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 11340ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 11400ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 11460ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 11520agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 11580cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 11640aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 11700gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 11760agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 11820ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 11880cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 11940tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 12000aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 12060tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 12120atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 12180cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 12240gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 12300gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 12360tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc 12420tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 12480tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 12540aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 12600atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 12660tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca 12720catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 12780aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 12840tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 12900gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 12960tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 13020tagaaaaa 13028612454DNAArtificial SequenceSynthetic promoter 6taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tactgaggac 60gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtagcagac aagcccgtca 120gggcgcgtca gcgggtgttg gccctaggac gaaaggaggt cgtgaaatgg ataaaaaaat 180acagcgtttt tcatgtacaa ctatactagt tgtagtgcct aaataatgct tttaaaactt 240aaaaataatc aatgtcctca gcggggtgtc ggggatttag gtgacactat aggctgagcg 300ccgcacaggc atctagaggc tatggcaggg cctgccgccc cgacgttggc tgcgagccct 360gggccttcac ccgaacttgg ggggtggggt ggggaaaagg aagaaacgcg ggcgtattgg 420ccccaatggg gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg aaccccgcgt 480ttatgaacaa acgacccaac accgtgcgtt ttattctgtc tttttattgc cgtcatagcg 540cgggttcctt ccggtattgt ctccttccgt gtttcaatcg attcaaaaga actcgtccag 600cagacggtaa aaagcaatgc gttgagaatc cggtgcagca atgccataca gcaccagaaa 660gcgatcagcc cattcaccgc ccagttcttc agcaatgtca cgggttgcca gtgcgatgtc 720ctgatagcga tcagccacgc ccaggcgacc gcagtcgata aagccggaga aacggccgtt 780ttccaccata atgtttggca gacaagcatc gccgtgggtc acaaccaggt cctcgccatc 840tggcatacgt gctttcaggc gtgcgaacag ttctgccggt gccagaccct gatgttcctc 900gtccaggtca tcctgatcaa ccaggccagc ttccatgcga gtgcgtgcgc gctcgatacg 960gtgtttagct tggtgatcga atgggcaagt agctgggtcc agggtatgca gacggcgcat 1020agcatcagcc atgatggaaa ccttttctgc cggtgccaga tgagaggaca gcagatcctg 1080gcctggaacc tcgcccagca gcagccagtc gcggccagcc tcggtcacaa catccagcac 1140agctgcgcat ggaacgccgg tagtagccag ccaggacaga cgagctgctt catcttgcag 1200ttcgttcagt gcgccggaca gatcggtctt aacaaacagc accggacggc cttgagcgga 1260cagacggaac acagctgcgt cggagcaacc gatagtctgt tgagcccagt catagccaaa 1320cagacgttcc acccaagcag ccggagaacc agcgtgcaga ccgtcttgtt caatcatggt 1380ggcaattggg

tgtctgagcg atgtggctcg gctggcgacg caaaagaaga tgcggctgac 1440tgtcgaacag gaggagcaga gagcgaagcg ggaggctgcg ggctcaattt gcatgcttta 1500gttcctcacc ttgtcgtatt atactatgcc gatatactat gccgatgatt aattgtcaac 1560gtatacggaa tagctctgag gccgaggcag cttcggcctc tgcataaata aaaaaaatta 1620gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag 1680ttaggggcgg gactatggtt gctgactaat tgagatgctt gctttgcata cttctgcctg 1740ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc ttgctttgca 1800tacttctgcc tgctggggag cctggggact ttccacaccc taacctcgag gccatcgtgg 1860cacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgctc ttctcccccg 1920cgggaggttt tataaatccg actgtctaga ttgttgttaa atcacacaaa aaaccaacac 1980acagatgtaa tgaaaataaa gatattttat tatcgattca gctgtcgctg gggctctcca 2040gcatctccat caggaaggtg tcgatgggca cgtcgccgat cagcctgaag aagaacaggt 2100gctccaggca cttcaggccg atgctcctca ggctgggcag cctcagcagc agcttggcga 2160atctgccggg ctcgtcgggg tgggtggtcc tggtgtactc ctccagggcg gcgtacacct 2220tctccctcag cagctccacc tcctgggcgc ttttcaggcc cctcacctcg gggttgaaca 2280ggatgatggc cctcaggcag cccagctcgg tcttgtccat cctcatgtcc ctcatcttgc 2340tcaccagctc ggtcagcacc ctgtcgaaga tggcgcccac tccggcgctg tgggcgctgt 2400tcctatggac gtgcaggccg gtggccagca ggatgccgtc cctcacgtcg atgctcctgt 2460ggctgaagct ggcgatcagc agctcgttcc atccggccct cagcaggatc acctggtcgt 2520ccaggggcag gctgctgaag tggggaatcc tcttggccca ctccaccagg gtgaacagct 2580gcttgtcggc ggcctggcag atgttggtca cggggtcgtt ggggctgctg ccgctgccgc 2640cggttccgcc ggggccctcc acgccctggt cgcttttctg ctccacggcg agttcggcct 2700ccagaatcct gtccacgggc atctccatat ggccgccgta ctcgtcgatg cccagggcgt 2760cggtgaacat ctgctcgaac tcgaagtcgg ccatgtccag ggcgccgtag ggggcgctgt 2820cgtggggggt gaagccgggg ccggggctgt cgccgtcgcc cagcatgtcc aggtcgaagt 2880cgtccagggc gtcggcgtgg gccatggcca cgtcctcgcc gtccaggtgc agctcgtcgc 2940ccaggctcac gtcggtgggg ggggccacct tccttttctt cttggggccc atcaattggc 3000cacccccccc cccccccccg catgctgtgc cacctgtctt cattcttaac ctgaagattt 3060agtcttttag ggtttctttt gcacttgggg tacttaacat gccactatgc atattgtaga 3120tttatagata cttttcaact gatactgctc ccacaggcaa acatgcctgg tgaaacactt 3180gtttcttttt ggcaaacaag aaatgtttgc ataaaagact cgtgtctgga aaaatgtctt 3240gagaaattga tatgctactc taagccccaa atctcctttt cacttacctt ccccctgctg 3300caggatgaca atccatgggc ctgtgctttc tgatagtgaa agagaagaaa actcataaat 3360atagtgtcta ccatgagtca ggcactaact atgcatttat ctctttctgc tttaccctat 3420aaatggttat taccattaaa agtcatgtga ggaaacagaa ttagggagtt caaactgagc 3480caggatttca gttcaagttt tccagatcct cgagtaggcg agaccaatgg gtgcgccatg 3540ggctcttcca aaaatttagg tgacactata gggcaccgct cgcacctgcg cacaggcccg 3600cggctacaaa ctacgaacga tcattctaga taccacattt gtagaggttt tacttgcttt 3660aaaaaacctc ccacatctcc ccctgaacct gaaacataaa atgaatgcaa atgttttatt 3720aacttgttta ttgcagctta taatggttac aaattaagca atagcatcac aaatttcaca 3780aattaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 3840tatcatgtct aatcgattca caggttggtg gggctctcca ggatgggggg gggctgggtg 3900tggctcatgt cggccacgtc ccaaatctcc tccaggaagg ggggcagctt cctgttcttc 3960agcttcaggc tgatacacat attgctgttc tgcattccca gggtcctcag ctcgctcagg 4020atgctcagga tcttgccgta gatcacgctg ctcctggcgc tgccgctcag ctggttcagg 4080atgtagatcc tcagggtgtt caggtagtac ctctggatct cctccaccag ctggggctgc 4140tccaggccgg gcctgtcgct gaagatcacc acggcggtca gcagggcgta gtggatgttg 4200tccagggcca tgctgtacat acatctgcag aagtgcagga ggtcctcgat cacctcggcc 4260atgccagcct tcctgtagtt gtccctggtg taagcctggt tgttggcgaa caggatgctg 4320tcgctggcgg cgtcgtacct cctggccacc ctcagcatca tcacctcgct gctgcaagcc 4380ttcagcaggg tgatctggtc gggctggctg atcttggcga atccgggcag gcccttggcg 4440aactccacga tcagctgcac ggtcaggatg gtcatctcgg tgatctgcct gaagggggtg 4500tcgctctcct cgttctcgtc gtcggcctgc tgccaggtct gggtgatcct tttcaggtcc 4560tcgtcgctgg gctgctcgta gccgtcctga taccagatca gcctggcgat caggaactgc 4620tggttggcgg tcagctgggg gatgttcttc tgcctgttgg tcaccagcag cttgtcgctc 4680aggaacctgg gcacgacctc gtgaatcctg gcggcctcgg gggggggggg ctcgcactgc 4740atgatggggg gcatgtggtc atcgacggtg gtggtgctca cgggcagctt gtccttctcc 4800ttctgggcct tcttctcctt ccttttcatg gcgcactggg tctcgggcac cacgcactcg 4860ggcctcccga gtttcccggg atcgctcacg gtcagctgcc tctggccctt gttgctgctc 4920tcctcgctgc tgctggtggc gctgatcctg tgctgcctca gggtcagggg catgtcggtc 4980tccacgctgg ccagcctgtc ggtcacggcg tccttgttca cgttgtcctg cacgaacagg 5040ccggtcagca gggccttgat gtcttgcagg ctgtccatct tcaggatcat gtccaggtcc 5100tccctgggga agatcagcag gaacagctgc tccagcctct ccagcctgct ctccacctcg 5160gtcaggtggg ccctggtcag ggggctcctc ttggtcttgg ggctgtatct gcactcccag 5220ttgttcttca ggcacttggc gcacttgggc ttctccttgc tgcacttcag cttcttcagc 5280ctgcagatgt cgcaagcctg ctcgatgctg ctcagcagct tcatggtggc caattgcccc 5340cccccccccc cgcatgctgc ttccagaccc gcccagagca aaccggatcc tccactttcc 5400agcctagcct ggggcggggc gaggtctggg gggcggggag tcttggagac tccaaagcgg 5460gaggcgggga gagggcgggt cccaggccgc gataagggga cagagggaca gcgacctggg 5520gggtgcagag gtccgggcct gggggagaga gacacacagg ggagactcca agggcaagcg 5580cgagatggga acagaaggag ttgggggcca gtggcggaga gggaacagac tcagaaagga 5640ggatgccaga cagggagggg acaatttaac ccaaagaggg ggagacaaag acttagggga 5700cagagaatca gaaggtgggg cagcagggac ccagggatag acagagagag agggggacag 5760agacccacag agagaggggg acagagaccc agggagaggg gacagagacc cagagagagt 5820gggacagaga cccagagaga gtgggacaga gacccacaga gagaggggga cagagaccca 5880gggagaggag gacagagacc cagagagggg gggacagaga cccagagagg gggggacaga 5940gacccaggga gggggggaca gagacccagg gagaggggac agagacccac ggagaggggg 6000acagagaccc agggagaggg gacagagacc cagagagagg gggacagaga cccagagaga 6060gggggacaga gacccagaga gacagggaca gagacccaga gagaggggga cagagaccca 6120gagagagggg aacagagacc cagagagaga ggaggacaga gacccagaga gagggggaca 6180gagacccaga gagacaggga cagagaccca gagagagggg gacagagacc cagagagagg 6240ggaacagaga cccagagaga gaggaggaca gagacccaga gagaggggga cagagaccca 6300gagagggggg gacagagacc cagggatggg gggacagaga cccagggaga ggggacagag 6360acccacggag aggggtacag agacccaggg agaggggaca gagacccaga gagaggggga 6420cagagactct gagacagagg gggacagaga ccctgagaga gggggacaga gacccagaga 6480gaggggaaca gagacccaga gagagagggg gacagagacc cacagagaga ggggaacaga 6540gacccagaga gagggggaca gagatccaca gagagagggg gacagagacc cacagagaga 6600ggggaacaga gacccagaga gagggggaca gagatccaca gagagagggg gacagagacc 6660cagggagagg gggacagaga cccagagaga gggggacaga gacccaggga gaggtggaca 6720gagactctga gacagagggg gacagagact ctgagagaga gtggggacag agacccagag 6780aggtggatgc cagagatggg gaggagacac cttgactgag gcaaggggca gagggggatg 6840caaaaaccaa agaattgggg gaagggcagg aactcagaaa ggaggggcca gagatttagg 6900ggagagagag ggacacagac ttgggggagg actgacacta agataagagt gggacagaag 6960cccagagtga gagggagagg caatcccatg gaggagggac agggaccctg agagagcaga 7020tgacagaatt gaagagagag gaggacccgt gaacaggaaa ggagacccta gtgggggcag 7080tcatgaaggg tgaggagtag gaaatactga cacacagaga caggcaaggc cacttgtctc 7140ctgccagccc tctgtgatgg ctcgagtggt aatacaatgg ccggttccca tggacctgca 7200tcgtggtgta actataacgg tcctaaggta gcgaccgcgg agactaggtg tatttatcta 7260agcgatcgct taattaaggc cggccgccgc aataaaatat ctttattttc attacatctg 7320tgtgttggtt ttttgtgtga atccatagta ctaacatacg ctctccatca aaacaaaacg 7380aaacaaaaca aactagcaaa ataggctgtc cccagtgcaa gtccaggtgc cagaacattt 7440ctctatccat aatgcagggg taccgggtga tgacggtgaa aacctccaat tgcggagtac 7500tgtcctccga gcggagtact gtcctccgag cggagtactg tcctccgagc ggagtactgt 7560cctccgagcg gagtactgtc ctccgagcgg agtactgtcc tccgagcgga gagtccccgg 7620ggacctagag ggtatataat gggtgcctta gctggtgtgt gacctcatct tcctgtacgc 7680ccctgcaggg gcgcgccacg cgtcgaagaa ggtgagtaat cttaacatgc tctttttttt 7740tttttttgct aatccctttt gtgtgctgat gttaggatga catttacaac aaatgtttgt 7800tcctgacagg aaaaaccttg ctgggtacct tcgttgccgg acacttcttg tcctctactt 7860tggaaaaaag gaattgagag ccgctagcgc caccatggaa gatgccaaaa acattaagaa 7920gggcccagcg ccattctacc cactcgaaga cgggaccgct ggcgagcagc tgcacaaagc 7980catgaagcgc tacgccctgg tgcccggcac catcgccttt accgacgcac atatcgaggt 8040ggacattacc tacgccgagt acttcgagat gagcgttcgg ctggcagaag ctatgaagcg 8100ctatgggctg aatacaaacc atcggatcgt ggtgtgcagc gagaatagct tgcagttctt 8160catgcccgtg ttgggtgccc tgttcatcgg tgtggctgtg gccccagcta acgacatcta 8220caacgagcgc gagctgctga acagcatggg catcagccag cccaccgtcg tattcgtgag 8280caagaaaggg ctgcaaaaga tcctcaacgt gcaaaagaag ctaccgatca tacaaaagat 8340catcatcatg gatagcaaga ccgactacca gggcttccaa agcatgtaca ccttcgtgac 8400ttcccatttg ccacccggct tcaacgagta cgacttcgtg cccgagagct tcgaccggga 8460caaaaccatc gccctgatca tgaacagtag tggcagtacc ggattgccca agggcgtagc 8520cctaccgcac cgcaccgctt gtgtccgatt cagtcatgcc cgcgacccca tcttcggcaa 8580ccagatcatc cccgacaccg ctattctcag cgtggtgcca tttcaccacg gcttcggcat 8640gttcaccacg ctgggctact tgatctgcgg ctttcgggtc gtgctcatgt accgcttcga 8700ggaggagcta ttcttgcgca gcttgcaaga ctataagatt caatctgccc tgctggtgcc 8760cacactattt agcttcttcg ctaagagcac tctcatcgac aagtacgacc taagcaactt 8820gcacgagatc gccagcggcg gagcgcctct cagcaaggag gtaggtgagg ccgtggccaa 8880acgcttccac ctaccaggca tccgccaggg ctacggcctg acagaaacaa ccagcgccat 8940tctgatcacc cccgaagggg acgacaagcc tggcgcagta ggcaaggtgg tgcccttctt 9000cgaggctaag gtggtggact tggacacagg taagaccctg ggtgtgaacc agcgcggcga 9060gctgtgcgtc cgtggcccca tgatcatgag cggctacgtg aacaaccccg aggctacaaa 9120cgctctcatc gacaaggacg gctggctgca cagcggcgac atcgcctact gggacgagga 9180cgagcacttc ttcatcgtgg accggctcaa gagcctgatc aaatacaagg gctaccaggt 9240agccccagcc gaactggaga gcatcctgct gcaacacccc aacatcttcg acgccggggt 9300cgctggcctg cccgacgacg atgctggcga gctgcccgcc gcagtcgtcg tgctggaaca 9360cggtaaaacc atgaccgaga aggagatcgt ggactatgtg gccagccagg ttacaaccgc 9420caagaagctg cgcggtggtg ttgtgttcgt ggacgaggtg cctaaaggac tgaccggcaa 9480gttggacgcc cgcaagatcc gcgagattct cattaaggcc aagaagggcg gcaagatcgc 9540cgtgttaatc gattgcgcaa agctttcgcg ataggcgaga ccaatgggtg tgtacgtagc 9600ggccgcgtcg acgatagctt gatgggtggc atccctgtga cccctcccca gtgcctctcc 9660tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca 9720tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga 9780gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct 9840ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc 9900tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat 9960ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct 10020aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca 10080ctgctccctt ccctgtcctt ctgattttaa aataactata ccagcaggag gacgtccaga 10140cacagcatag gctacctggc catgcccaac cggtgggaca tttgagttgc ttgcttggca 10200ctgtcctctc atgcgttggg tccactcagt agatgcctgt tgaattctga tttaaatcgg 10260tccgcgtacg gcgtggtagg tccgaacgaa tccatggatt accctgttat ccctactcaa 10320ggacatcatc cctttagtga gggttaattc acgcagtggg tacggaacta aaggcagcac 10380acatcgtgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctacgtctct 10440cccccgcagt aagggctaga ttaactcgtc tcgtgaatat ccggaactcc ctttagtgag 10500ggttaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctta 10560atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctaccggaaa cgcttccttc 10620atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 10680ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 10740cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 10800tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 10860gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 10920aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 10980tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 11040aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 11100aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 11160ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 11220ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 11280atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 11340atgatctatg tcgggtgcgg agaaagaggt aatgaaatgg catacgagta aacttggtct 11400gacaccgctg catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 11460gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 11520taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 11580tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 11640tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 11700gaagcgccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaact 11760gttgccggga agctagagta agtagttcgc cagttaatag tttgcggagc gttgttgcca 11820ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 11880cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 11940tcggtcctcc gatggttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 12000cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 12060agtattcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 12120cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggga 12180agcgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 12240aacccacacg agcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 12300gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 12360gaatactcat acgcttcctt tttcaatagt attgaagcat ttatcagggt tattgtctcg 12420ggagcgaata catatttgaa tgtatttaga aaaa 12454712579DNAArtificial SequenceSynthetic promoter 7taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac 60cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca 120agaaaattaa tcgcaccggt atctatgtcg ggtgcggaga aagaggtaat gaaatggcag 180ctagcatcat caataatata ccttattttg gattgaagcc aatatgataa tgagggggtg 240gagtttgtga cgtggcgcgg ggcgtgggaa cggggcgggt gacgtagtag tgtggcggaa 300gtgtgatgtt gcaagtgtgg cggaacacat gtaagcgacg gatgtggcaa aagtgacgtt 360tttggtgtgc gccggtgtac acaggaagtg acaattttcg cgcggtttta ggcggatgtt 420gtagtaaatt tgggcgtaac cgagtaagat ttggccattt tcgcgggaaa actgaataag 480aggaagtgaa atctgaataa ttttgtgtta ctcatagcgc gtaatagtgc gccggtgtac 540acaggaagtg acaattttcg cgcggtttta ggcggatgtt gtagtaaatt tgggcgtaac 600cgagtaagat ttggccattt tcgcgggaaa actgaataag aggaagtgaa atctgaataa 660ttttgtgtta ctcattttgt ctagggagat ccggtaccga tatcctagac aacgatgctg 720agctaactat aacggtccta aggtagcgac cgcggagact aggtgtattt atctaagcga 780tcgcttaatt aaggccggcc gccgcaataa aatatcttta ttttcattac atctgtgtgt 840tggttttttg tgtgaatcca tagtactaac atacgctctc catcaaaaca aaacgaaaca 900aaacaaacta gcaaaatagg ctgtccccag tgcaagtcca ggtgccagaa catttctcta 960tccataatgc aggggtaccg ggtgatgacg gtgaaaacct ccaattgcgg agtactgtcc 1020tccgagcgga gtactgtcct ccgagcggag tactgtcctc cgagcggagt actgtcctcc 1080gagcggagta ctgtcctccg agcggagtac tgtcctccga gcggagagtc cccggggacc 1140tagagggtat ataatgggtg ccttagctgg tgtgtgacct catcttcctg tacgcccctg 1200caggggcgcg ccacgcgtcg aagaaggtga gtaatcttaa catgctcttt tttttttttt 1260ttgctaatcc cttttgtgtg ctgatgttag gatgacattt acaacaaatg tttgttcctg 1320acaggaaaaa ccttgctggg taccttcgtt gccggacact tcttgtcctc tactttggaa 1380aaaaggaatt gagagccgct agcgccacca tgattagccc tttcctcgtg ctcgccattg 1440gcacatgcct caccaatagc ctcgtgcctg agaaagagaa agacggaggc ggaggctccg 1500gcggaggcgg aagcggaggc ggaggctccg agtccctgaa actgaggaga agggtgcatg 1560agacagacaa aaactgtaga tccggcacac cccctcagac cggcctggag aaacccacag 1620gcacaggcca aagaaaacag ggagccggag cccctaccgc tcccggaccc ggaggctccc 1680ccggaggcta aatcgattgc gcaaagcttt cgcgataggc gagaccaatg ggtgtgtacg 1740tagcggccgc gtcgacgata gcttgatggg tggcatccct gtgacccctc cccagtgcct 1800ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt 1860tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta 1920tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca 1980agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg 2040attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc 2100taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac 2160tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga 2220accactgctc ccttccctgt ccttctgatt ttaaaataac tataccagca ggaggacgtc 2280cagacacagc ataggctacc tggccatgcc caaccggtgg gacatttgag ttgcttgctt 2340ggcactgtcc tctcatgcgt tgggtccact cagtagatgc ctgttgaatt ctgatttaaa 2400tcggtccgcg tacggcgtgg taggtccgaa cgaatccatg gattaccctg ttatccctat 2460ccggagttaa cctcgaggac ttcggaactt ctagaaccag accgttcagt ttaaacgctc 2520ttctccccct cgagtatgtc ctacttttat aaactttggt tgaactaaat aaaacagcaa 2580catgtaccca acgtttttag actgttttat tcagtaccat aaacattcac taaactatac 2640agagctaaaa tcatgcaaaa gattgcaaaa caaactgcct tgggaaattt ccagtctaag 2700ctaaatggac caagtatagg attgaatttt aagaagttgg tgggaagatt ccagctcttg 2760tactcacagg taaggtccta ggattctatt tgttgaagtg tctgcagacc ctctccctac 2820agcaggggcg tggagcaaat gtgcattcag gaagtgctta ttagtccccc caaaaccttt 2880ttcgtgacga cagacggatg gaggcctcag acgactccgg cagtaggacc ctgaaccaaa 2940tgcctgagct gcgctcgaaa ggctcgtctt gcaggagctg gcgagtggca cgggcttcgg 3000tggccgggct cggggctcgg ggctcggggc ccggagccgg ggggggcggg gggtccgtgc 3060ccagggcgct ctgtggctcg attcatgtcc ccgccctccc acctgagcac actgggcaga 3120gagcctgcgg catcgggcca gtcgcgcctc ctcgtccgcc ctgggcgggt cgctgggccc 3180caggccgctt tctacagctc ctttaataaa atggacagca ggggtcctaa ccagacgtgg 3240gcatcaagac aaaggaggcg gccagacgcg cttgagggcc tgctcgctag ctcccgcccc 3300cccattccgc ggtcatccgg ggacagaggc caggccggac tgggtggagt tgggactcat 3360acgtctgcgt ccaggaaggc gccgggcgag ccccagctag acgtgacggg cggggccgaa 3420cacggcagcg gaccagaggc ccgcggcgca ccggcgtggg gcggggcaag cggagccttc 3480cgggatgccg cgcggcagcc ggcttccggc tgtgggtggt gcgggggaca gcggcggccg 3540gaagctgact gagccggcct ttggtaacgc cgcctgcact tctgggggcg tcgagcctgg 3600cggtagaatc ttcccagtag gcggcgcggg agggaaaaga ggattgaggg catgcggggg 3660gggggggggg caattggcca ccatgggccc caagaagaaa aggaaggtgg ccccccccac 3720cgacgtgagc ctgggcgacg agctgcacct ggacggcgag gacgtggcca tggcccacgc 3780cgacgccctg gacgacttcg acctggacat gctgggcgac ggcgacagcc ccggccccgg 3840cttcaccccc cacgacagcg ccccctacgg cgccctggac atggccgact tcgagttcga 3900gcagatgttc

accgacgccc tgggcatcga cgagtacggc ggccatatgg agatgcccgt 3960ggacaggatt ctggaggccg aactcgccgt ggagcagaaa agcgaccagg gcgtggaggg 4020ccccggcgga accggcggca gcggcagcag ccccaacgac cccgtgacca acatctgcca 4080ggccgccgac aagcagctgt tcaccctggt ggagtgggcc aagaggattc cccacttcag 4140cagcctgccc ctggacgacc aggtgatcct gctgagggcc ggatggaacg agctgctgat 4200cgccagcttc agccacagga gcatcgacgt gagggacggc atcctgctgg ccaccggcct 4260gcacgtccat aggaacagcg cccacagcgc cggagtgggc gccatcttcg acagggtgct 4320gaccgagctg gtgagcaaga tgagggacat gaggatggac aagaccgagc tgggctgcct 4380gagggccatc atcctgttca accccgaggt gaggggcctg aaaagcgccc aggaggtgga 4440gctgctgagg gagaaggtgt acgccgccct ggaggagtac accaggacca cccaccccga 4500cgagcccggc agattcgcca agctgctgct gaggctgccc agcctgagga gcatcggcct 4560gaagtgcctg gagcacctgt tcttcttcag gctgatcggc gacgtgccca tcgacacctt 4620cctgatggag atgctggaga gccccagcga cagctgagcc ggcaactcgc tgtagtaatt 4680ccagcgagag gcagagggag cgagcgggcg gcgggctagg gtggaggagc ccggcgagca 4740gagctgcgct gcgggcgtcc tgggaaggga gatccggagc gaataggggg cttcgcctct 4800ggcccagccc tcccgctgat cccccagcca gcggtgcgca accctagccg catccacgaa 4860actttgccca tagcagcggg cgggcacttt gcactggaac ttacaacacc cgagcaagga 4920cgcgactctc ccgacgcggg gaggctattc tgcccatttg gggacacttc cccgccgctg 4980ccaggacccg cttctctgaa aggctctcct tgcagctgct tagacgctgg atttttttcg 5040ggtagtggaa aaccagcagc ctcccgcgac cagatctgcc accatgaagc tgctgagcag 5100catcgagcag gcttgcgaca tctgcaggct gaagaagctg aagtgcagca aggagaagcc 5160caagtgcgcc aagtgcctga agaacaactg ggagtgcaga tacagcccca agaccaagag 5220gagccccctg accagggccc acctgaccga ggtggagagc aggctggaga ggctggagca 5280gctgttcctg ctgatcttcc ccagggagga cctggacatg atcctgaaga tggacagcct 5340gcaagacatc aaggccctgc tgaccggcct gttcgtgcag gacaacgtga acaaggacgc 5400cgtgaccgac aggctggcca gcgtggagac cgacatgccc ctgaccctga ggcagcacag 5460gatcagcgcc accagcagca gcgaggagag cagcaacaag ggccagaggc agctgaccgt 5520gagccccgag tttcccggga tcaggcccga gtgcgtggtg cccgagaccc agtgcgccat 5580gaaaaggaag gagaagaagg cccagaagga gaaggacaag ctgcccgtga gcaccaccac 5640cgtcgatgac cacatgcccc ccatcatgca gtgcgagccc cccccccccg aggccgccag 5700gattcacgag gtcgtgccca ggttcctgag cgacaagctg ctggtgacca acaggcagaa 5760gaacatcccc cagctgaccg ccaaccagca gttcctgatc gccaggctga tctggtatca 5820ggacggctac gagcagccca gcgacgagga cctgaaaagg atcacccaga cctggcagca 5880ggccgacgac gagaacgagg agagcgacac ccccttcagg cagatcaccg agatgaccat 5940cctgaccgtg cagctgatcg tggagttcgc caagggcctg cccggattcg ccaagatcag 6000ccagcccgac cagatcaccc tgctgaaggc ttgcagcagc gaggtgatga tgctgagggt 6060ggccaggagg tacgacgccg ccagcgacag catcctgttc gccaacaacc aggcttacac 6120cagggacaac tacaggaagg ctggcatggc cgaggtgatc gaggacctcc tgcacttctg 6180cagatgtatg tacagcatgg ccctggacaa catccactac gccctgctga ccgccgtggt 6240gatcttcagc gacaggcccg gcctggagca gccccagctg gtggaggaga tccagaggta 6300ctacctgaac accctgagga tctacatcct gaaccagctg agcggcagcg ccaggagcag 6360cgtgatctac ggcaagatcc tgagcatcct gagcgagctg aggaccctgg gaatgcagaa 6420cagcaatatg tgtatcagcc tgaagctgaa gaacaggaag ctgcccccct tcctggagga 6480gatttgggac gtggccgaca tgagccacac ccagcccccc cccatcctgg agagccccac 6540caacctgtga atcgattaga catgataaga tacattgatg agtttggaca aaccacaact 6600agaatgcagt gaaaaaaatg cttaatttgt gaaatttgtg atgctattgc ttaatttgta 6660accattataa gctgcaataa acaagttaat aaaacatttg cattcatttt atgtttcagg 6720ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatct 6780agagctcttc caaatagatc tggaaggtgc tgaggtacga tgagacccgc accaggtgca 6840gaccctgcga gtgtggcggt aaacatatta ggaaccagcc tgtgatgctg gatgtgaccg 6900aggagctgag gcccgatcac ttggtgctgg cctgcacccg cgctgagttt ggctctagcg 6960atgaagatac agattgaggt actgaaatgt gtgggcgtgg cttaagggtg ggaaagaata 7020tataaggtgg gggtcttatg tagttttgta tctgttttgc agcagccgcc gccgccatga 7080gcaccaactc gtttgatgga agcattgtga gctcatattt gacaacgcgc atgcccccat 7140gggccggggt gcgtcagaat gtgatgggct ccagcattga tggtcgcccc gtcctgcccg 7200caaactctac taccttgacc tacgagaccg tgtctggaac gccgttggag actgcagcct 7260ccgccgccgc ttcagccgct gcagccaccg cccgcgggat tgtgactgac tttgctttcc 7320tgagcccgct tgcaagcagt gcagcttccc gttcatccgc ccgcgatgac aagttgacgg 7380ctcttttggc acaattggat tctttgaccc gggaacttaa tgtcgtttct cagcagctgt 7440tggatctgcg ccagcaggtt tctgccctga aggcttcctc ccctcccaat gcggtttaaa 7500acataaataa aaaaccagac tctgtttgga tttggatcaa gcaagtgtct tgctgtcttt 7560atttaggggt tttgcgcgcg cggtaggccc gggaccagcg gtctcggtcg ttgagggtcc 7620tgtgtatttt ttccaggacg tggtaaaggt gactctggat gttcagatac atgggcataa 7680gcccgtctct ggggtggagg tagcaccact gcagagcttc atgctgcggg gtggtgttgt 7740agatgatcca gtcgtagcag gagcgctggg cgtggtgcct aaaaatgtct ttcagtagca 7800agctgattgc caggggcagg cccttggtgt aagtgtttac aaagcggtta agctgggatg 7860ggtgcatacg tggggatatg agatgcatct tggactgtat ttttaggttg gctatgttcc 7920cagccatatc cctccgggga ttcatgttgt gcagaaccac cagcacagtg tatccggtgc 7980acttgggaaa tttgtcatgt agcttagaag gaaatgcgtg gaagaacttg gagacgccct 8040tgtgacctcc aagattttcc atgcattcgt ccataatgat ggcaatgggc ccacgggcgg 8100cggcctgggc gaagatattt ctgggatcac taacgtcata gttgtgttcc aggatgagat 8160cgtcataggc catttttaca aagcgcgggc ggagggtgcc agactgcggt ataatggttc 8220catccggccc aggggcgtag ttaccctcac agatttgcat ttcccacgct ttgagttcag 8280atggggggat catgtctacc tgcggggcga tgaagaaaac ggtttccggg gtaggggaga 8340tcagctggga agaaagcagg ttcctgagca gctgcgactt accgcagccg gtgggcccgt 8400aaatcacacc tattaccggg tgcaactggt agttaagaga gctgcagctg ccgtcatccc 8460tgagcagggg ggccacttcg ttaagcatgt ccctgactcg catgttttcc ctgaccaaat 8520ccgccagaag gcgctcgccg cccagcgata gcagttcttg caaggaagca aagtttttca 8580acggtttgag accgtccgcc gtaggcatgc ttttgagcgt ttgaccaagc agttccaggc 8640ggtcccacag ctcggtcacc tgctctacgg catctcgatc cagcatatct cctcgtttcg 8700cgggttgggg cggctttcgc tgtacggcag tagtcggtgc tcgtccagac gggccagggt 8760catgtctttc cacgggcgca gggtcctcgt cagcgtagtc tgggtcacgg tgaaggggtg 8820cgctccgggc tgcgcgctgg ccagggtgcg cttgaggctg gtcctgctgg tgctgaagcg 8880ctgccggtct tcgccctgcg cgtcggccag gtagcatttg accatggtgt catagtccag 8940cccctccgcg gcgtggccct tggcgcgcag cttgcccttg gaggaggcgc cgcacgaggg 9000gcagtgcaga cttttgaggg cgtagagctt gggcgcgaga aataccgatt ccggggagta 9060ggcatccgcg ccgcaggccc cgcagacggt ctcgcattcc acgagccagg tgagctctgg 9120ccgttcgggg tcaaaaacca ggtttccccc atgctttttg atgcgtttct tacctctggt 9180ttccatgagc cggtgtccac gctcggtgac gaaaaggctg tccgtgtccc cgtatacaga 9240cttgagaggc ctgtcctcga ccgatgccct tgagagcctt caacccagtc agctccttcc 9300ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac 9360tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga 9420gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag 9480ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca 9540tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct 9600tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg caggccatgc 9660tgtccaggca ggtagatgac gaccatcagg gacagcttca aggccagcaa aaggccagga 9720accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 9780acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 9840cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 9900acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 9960atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 10020agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 10080acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 10140gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 10200gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 10260gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 10320gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 10380acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 10440tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 10500ctgacagctc gagtatgtcc tacttttata aactttggtt gaactaaata aaacagcaac 10560atgtacccaa cgtttttaga ctgttttatt cagtaccata aacattcact aaactataca 10620gagctaaaat catgcaaaag attgcaaaac aaactgcctt gggaaatttc cagtctaagc 10680taaatggacc aagtatagga ttgaatttta agaagttggt gggaagattc cagctcttgt 10740actcacaggt aaggtcctag gattctattt gttgaagtgt ctgcagaccc tctccctaca 10800gcaggggcgt ggagcaaatg tgcattcagg aagtgcttat tagtcccccc aaaacctttt 10860tcgtgacgac agacggatgg aggcctcaga cgactccggc agtaggaccc tgaaccaaat 10920gcctgagctg cgctcgaaag gctcgtcttg caggagctgg cgagtggcac gggcttcggt 10980ggccgggctc ggggctcggg gctcggggcc cggagccggg gggggcgggg ggtccgtgcc 11040cagggcgctc tgtggctcga ttcatgtccc cgccctccca cctgagcaca ctgggcagag 11100agcctgcggc atcgggccag tcgcgcctcc tcgtccgccc tgggcgggtc gctgggcccc 11160aggccgcttt ctacagctcc tttaataaaa tggacagcag gggtcctaac cagacgtggg 11220catcaagaca aaggaggcgg ccagacgcgc ttgagggcct gctcgctagc tcccgccccc 11280ccattccgcg gtcatccggg gacagaggcc aggccggact gggtggagtt gggactcata 11340cgtctgcgtc caggaaggcg ccgggcgagc cccagctaga cgtgacgggc ggggccgaac 11400acggcagcgg accagaggcc cgcggcgcac cggcgtgggg cggggcaagc ggagccttcc 11460gggatgccgc gcggcagccg gcttccggct gtgggtggtg cgggggacag cggcggccgg 11520aagctgactg agccggcctt tggtaacgcc gcctgcactt ctgggggcgt cgagcctggc 11580ggtagaatct tcccagtagg cggcgcggga gggaaaagag gattgagggc atgcttacca 11640atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 11700ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 11760tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 11820agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 11880taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 11940tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 12000cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 12060ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 12120tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 12180tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 12240cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 12300tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 12360gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 12420tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 12480atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 12540tctcatgagc ggatacatat ttgaatgtat ttagaaaaa 12579811443DNAArtificial SequenceSynthetic promoter 8taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tactgaggac 60gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtagcagac aagcccgtca 120gggcgcgtca gcgggtgttg gccctaggac gaaaggaggt cgtgaaatgg ataaaaaaat 180acagcgtttt tcatgtacaa ctatactagt tgtagtgcct aaataatgct tttaaaactt 240aaaaataatc aatgtcctca gcggggtgtc ggggatttag gtgacactat aggctgagcg 300ccgcacaggc atctagaggc tatggcaggg cctgccgccc cgacgttggc tgcgagccct 360gggccttcac ccgaacttgg ggggtggggt ggggaaaagg aagaaacgcg ggcgtattgg 420ccccaatggg gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg aaccccgcgt 480ttatgaacaa acgacccaac accgtgcgtt ttattctgtc tttttattgc cgtcatagcg 540cgggttcctt ccggtattgt ctccttccgt gtttcaatcg attcaaaaga actcgtccag 600cagacggtaa aaagcaatgc gttgagaatc cggtgcagca atgccataca gcaccagaaa 660gcgatcagcc cattcaccgc ccagttcttc agcaatgtca cgggttgcca gtgcgatgtc 720ctgatagcga tcagccacgc ccaggcgacc gcagtcgata aagccggaga aacggccgtt 780ttccaccata atgtttggca gacaagcatc gccgtgggtc acaaccaggt cctcgccatc 840tggcatacgt gctttcaggc gtgcgaacag ttctgccggt gccagaccct gatgttcctc 900gtccaggtca tcctgatcaa ccaggccagc ttccatgcga gtgcgtgcgc gctcgatacg 960gtgtttagct tggtgatcga atgggcaagt agctgggtcc agggtatgca gacggcgcat 1020agcatcagcc atgatggaaa ccttttctgc cggtgccaga tgagaggaca gcagatcctg 1080gcctggaacc tcgcccagca gcagccagtc gcggccagcc tcggtcacaa catccagcac 1140agctgcgcat ggaacgccgg tagtagccag ccaggacaga cgagctgctt catcttgcag 1200ttcgttcagt gcgccggaca gatcggtctt aacaaacagc accggacggc cttgagcgga 1260cagacggaac acagctgcgt cggagcaacc gatagtctgt tgagcccagt catagccaaa 1320cagacgttcc acccaagcag ccggagaacc agcgtgcaga ccgtcttgtt caatcatggt 1380ggcaattggg tgtctgagcg atgtggctcg gctggcgacg caaaagaaga tgcggctgac 1440tgtcgaacag gaggagcaga gagcgaagcg ggaggctgcg ggctcaattt gcatgcttta 1500gttcctcacc ttgtcgtatt atactatgcc gatatactat gccgatgatt aattgtcaac 1560gtatacggaa tagctctgag gccgaggcag cttcggcctc tgcataaata aaaaaaatta 1620gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag 1680ttaggggcgg gactatggtt gctgactaat tgagatgctt gctttgcata cttctgcctg 1740ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc ttgctttgca 1800tacttctgcc tgctggggag cctggggact ttccacaccc taacctcgag gccatcgtgg 1860cacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgctc ttctcccccg 1920cgggaggttt tataaatccg actgtctaga ttgttgttaa atcacacaaa aaaccaacac 1980acagatgtaa tgaaaataaa gatattttat tatcgattca gctgtcgctg gggctctcca 2040gcatctccat caggaaggtg tcgatgggca cgtcgccgat cagcctgaag aagaacaggt 2100gctccaggca cttcaggccg atgctcctca ggctgggcag cctcagcagc agcttggcga 2160atctgccggg ctcgtcgggg tgggtggtcc tggtgtactc ctccagggcg gcgtacacct 2220tctccctcag cagctccacc tcctgggcgc ttttcaggcc cctcacctcg gggttgaaca 2280ggatgatggc cctcaggcag cccagctcgg tcttgtccat cctcatgtcc ctcatcttgc 2340tcaccagctc ggtcagcacc ctgtcgaaga tggcgcccac tccggcgctg tgggcgctgt 2400tcctatggac gtgcaggccg gtggccagca ggatgccgtc cctcacgtcg atgctcctgt 2460ggctgaagct ggcgatcagc agctcgttcc atccggccct cagcaggatc acctggtcgt 2520ccaggggcag gctgctgaag tggggaatcc tcttggccca ctccaccagg gtgaacagct 2580gcttgtcggc ggcctggcag atgttggtca cggggtcgtt ggggctgctg ccgctgccgc 2640cggttccgcc ggggccctcc acgccctggt cgcttttctg ctccacggcg agttcggcct 2700ccagaatcct gtccacgggc atctccatat ggccgccgta ctcgtcgatg cccagggcgt 2760cggtgaacat ctgctcgaac tcgaagtcgg ccatgtccag ggcgccgtag ggggcgctgt 2820cgtggggggt gaagccgggg ccggggctgt cgccgtcgcc cagcatgtcc aggtcgaagt 2880cgtccagggc gtcggcgtgg gccatggcca cgtcctcgcc gtccaggtgc agctcgtcgc 2940ccaggctcac gtcggtgggg ggggccacct tccttttctt cttggggccc atcaattggc 3000cacccccccc cccccccccg catgcccggc gggtcaggga cgcgggcgcg cggtgcgctg 3060ggggcggcac gtccgggcgg aggaggcgtc atcccgtggc cccaggagcg gcaatcagcc 3120gagactgagc cagcgcccgg ccgcaggcag acccaagcgc caggaggcgg agccagcgct 3180gaccccaccc cgcccctccc cccgcccctc gcagcctttt ctcacctcac tggctttcct 3240gagcgagagc ggaactgctg ggggaacctt cgccaccctc tcccggacaa ctctacgggg 3300aaagcccagc tgcggacaag ccagacttgt ggtcttctgc caggggcggc aggccctatc 3360ccctctctgg gcctcagttc cactccccgc accccgtaaa aagattggcg tagaagtcgt 3420ggaggacctc tctccaaggg cgcagaggag tccaacaccc aacaaccttg ctatggccag 3480ctaagtggtt cgcaccctga ggactatggt cctcggagga tgcgttcgcc taagtggaca 3540cggctccctg ccgagcctac cagcaggcac aacgatgata tgtggggagc actcagggac 3600ccgctgttgc tttgcctggg agaacactgg aaatttttca tcttggcctg gctccttccc 3660ctccctctcc tttctcctcc ctttcccagc cagcagcttt ctgggctgcc tccccccaag 3720ccagctggtg tacgctcaga aaatccaaac tcatttccat gtgagtggag ggggatataa 3780ttaggaaggc cccttcccca agtagagagg ggagcatccc ccgtgcccca ctcactgtgg 3840gggagggagg gtcaaagcag cttaagggga tctctgcccg caaagtgcct agggctcaac 3900tatctcaggc aacctgacca ttcagtgggg actcccttgg ggggtccagg cccagaccct 3960ctgcagttac ctgagcagtc cagccagctc tgccatcagc tcctcctctc agaactacgt 4020gcctgccctt tgtaaaaggg tctcctctct ctaaaccaga cagggcccag acctaccact 4080tccacttgtg ccccagggaa ccatgaatgg aaaatccacc cagctcagta agtacttatc 4140tagtgtttac tggatgtcta gctgcactga gaggagacag gccgaggaag gagtagaggt 4200gaacatccaa ccttcagaac tcacagacct agacacgatt cacgcactgc agacaccagc 4260aaaggcggtg ggctggctgc cctctgccct ctgccctgct ccatggggcc aggaccattt 4320ggttgttgtt tttacctcct ggaaccagga aagttttgct accaagaagt ttggctttac 4380cattctacat tttgcttcca gttgtcccct ccttcttccc tcagagtcct gaggcacccc 4440agcaggccag gaaggtaaac tcaaggcacc tccagagaca gtgcagagtg aggttcctgg 4500cccactggga gaagccaaca gccctcgagt aggcgagacc aatgggtgcg ccatgggctc 4560ttccaaaaat ttaggtgaca ctatagggca ccgctcgcac ctgcgcacag gcccgcggct 4620acaaactacg aacgatcatt ctagatacca catttgtaga ggttttactt gctttaaaaa 4680acctcccaca tctccccctg aacctgaaac ataaaatgaa tgcaaatgtt ttattaactt 4740gtttattgca gcttataatg gttacaaatt aagcaatagc atcacaaatt tcacaaatta 4800agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 4860tgtctaatcg attcacaggt tggtggggct ctccaggatg ggggggggct gggtgtggct 4920catgtcggcc acgtcccaaa tctcctccag gaaggggggc agcttcctgt tcttcagctt 4980caggctgata cacatattgc tgttctgcat tcccagggtc ctcagctcgc tcaggatgct 5040caggatcttg ccgtagatca cgctgctcct ggcgctgccg ctcagctggt tcaggatgta 5100gatcctcagg gtgttcaggt agtacctctg gatctcctcc accagctggg gctgctccag 5160gccgggcctg tcgctgaaga tcaccacggc ggtcagcagg gcgtagtgga tgttgtccag 5220ggccatgctg tacatacatc tgcagaagtg caggaggtcc tcgatcacct cggccatgcc 5280agccttcctg tagttgtccc tggtgtaagc ctggttgttg gcgaacagga tgctgtcgct 5340ggcggcgtcg tacctcctgg ccaccctcag catcatcacc tcgctgctgc aagccttcag 5400cagggtgatc tggtcgggct ggctgatctt ggcgaatccg ggcaggccct tggcgaactc 5460cacgatcagc tgcacggtca ggatggtcat ctcggtgatc tgcctgaagg gggtgtcgct 5520ctcctcgttc tcgtcgtcgg cctgctgcca ggtctgggtg atccttttca ggtcctcgtc 5580gctgggctgc tcgtagccgt cctgatacca gatcagcctg gcgatcagga actgctggtt 5640ggcggtcagc tgggggatgt tcttctgcct gttggtcacc agcagcttgt cgctcaggaa 5700cctgggcacg acctcgtgaa tcctggcggc ctcggggggg gggggctcgc actgcatgat 5760ggggggcatg tggtcatcga cggtggtggt gctcacgggc agcttgtcct tctccttctg 5820ggccttcttc tccttccttt tcatggcgca ctgggtctcg ggcaccacgc actcgggcct 5880cccgagtttc ccgggatcgc tcacggtcag ctgcctctgg cccttgttgc tgctctcctc 5940gctgctgctg gtggcgctga tcctgtgctg cctcagggtc aggggcatgt cggtctccac 6000gctggccagc ctgtcggtca cggcgtcctt gttcacgttg tcctgcacga acaggccggt 6060cagcagggcc ttgatgtctt gcaggctgtc catcttcagg atcatgtcca ggtcctccct 6120ggggaagatc agcaggaaca gctgctccag cctctccagc ctgctctcca cctcggtcag 6180gtgggccctg gtcagggggc tcctcttggt cttggggctg tatctgcact cccagttgtt 6240cttcaggcac ttggcgcact tgggcttctc cttgctgcac ttcagcttct tcagcctgca 6300gatgtcgcaa

gcctgctcga tgctgctcag cagcttcatg gtggccaatt gccccccccc 6360ccccccgcat gccttgcact ccaatcagaa ccagcaagat gatgcccaag aggaaaagaa 6420caccactttt cttcatagtg atagaatgga gttccaagtc actcctgtat tggattttga 6480gcctgagaaa ttctttagag aacacatttt ggggatcata tttatttagg atattcattt 6540ctctcctaaa ctctgattgg ctagtatctt tagccgagct aactaaattg accacaaact 6600tgattgtgct gggggaaacc ctagtctcag atccaaggga atttctgcat gtttttattt 6660ccatttcaca taaatgggtg gtttatagta agggatgtga aactagcaac actgatagcc 6720tttggcacat tcacagttgc tcacctgtgt gaagaatagg ttatataatg aatatactaa 6780tgttatacaa caaaggttgg agatgtggtt tccagatggg aaatattaaa gtccagataa 6840ataatatact tttcaaattc ccttactatt tatggcatat tcttctgtat aatagatggc 6900tacattgata ttttcttata ttgcccactt aacaacttaa agcaccttaa tttcctctgg 6960gaccaggtca ttttctacta gtggtaagct cattctgtat cattcctgct acagtattat 7020tttttaaact aataaattgg ttagaataag gggtcagttg catgtaagag acccaaatta 7080acagtggcta agaaatgcaa taaaaatgaa gataaatagc tgggacctaa ttaaagagct 7140tttgcacagc aaaaggaaca gtcagcagag taaacagaca acccacagag cgggagaaaa 7200tcttcacaat ctatatacct gacaaaggac taatatccag aatccagaat gaattcaaac 7260aaatcattaa gtaaaaaaca aacaatccca ttaaaaagtg ggctaaggac atcaatatac 7320aattctcaaa agaagataca cgaatagcca acaaacatat gaaaaaatgc tcatcactaa 7380tgatcaggga aatgcaaatc aaaaccacgg tgcgatacca ccttacaccc gcaagaatgg 7440ccataatcaa aaaatcaaaa aacagcagat gttggtgtgg actcgagtgg taatacaatg 7500gccggttccc atggacctgc atcgtggtgt aactataacg gtcctaaggt agcgaccgcg 7560gagactaggt gtatttatct aagcgatcgc ttaattaagg ccggccgccg caataaaata 7620tctttatttt cattacatct gtgtgttggt tttttgtgtg aatccatagt actaacatac 7680gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca 7740agtccaggtg ccagaacatt tctctatcca taatgcaggg gtaccgggtg atgacggtga 7800aaacctccaa ttgcggagta ctgtcctccg agcggagtac tgtcctccga gcggagtact 7860gtcctccgag cggagtactg tcctccgagc ggagtactgt cctccgagcg gagtactgtc 7920ctccgagcgg agagtccccg gggacctaga gggtatataa tgggtgcctt agctggtgtg 7980tgacctcatc ttcctgtacg cccctgcagg ggcgcgccac gcgtcgaaga aggtgagtaa 8040tcttaacatg ctcttttttt ttttttttgc taatcccttt tgtgtgctga tgttaggatg 8100acatttacaa caaatgtttg ttcctgacag gaaaaacctt gctgggtacc ttcgttgccg 8160gacacttctt gtcctctact ttggaaaaaa ggaattgaga gccgctagcg ccaccatgat 8220tagccctttc ctcgtgctcg ccattggcac atgcctcacc aatagcctcg tgcctggcgg 8280aggcggaagc ggaggcggag gctccggcgg aggcggaagc tgtcagcctg tgacacagga 8340ggacggaaag gaaagcagga tctccgtgca agagaggcag cctgccccta cccctagcaa 8400tggctccccc aaagacggac ccgaaatccc tcccacaggc ggaaaggcta aggccaagcc 8460cgtgacaaga ggagccggag ccaggagcgg aacccctccc caaaccggac tggaaaagcc 8520taccggaacc ggatgaatcg attgcgcaaa gctttcgcga taggcgagac caatgggtgt 8580gtacgtagcg gccgcgtcga cgatagcttg atgggtggca tccctgtgac ccctccccag 8640tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat 8700taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg 8760tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg 8820aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc 8880aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc 8940tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct 9000ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg 9060cgtgaaccac tgctcccttc cctgtccttc tgattttaaa ataactatac cagcaggagg 9120acgtccagac acagcatagg ctacctggcc atgcccaacc ggtgggacat ttgagttgct 9180tgcttggcac tgtcctctca tgcgttgggt ccactcagta gatgcctgtt gaattctgat 9240ttaaatcggt ccgcgtacgg cgtggtaggt ccgaacgaat ccatggatta ccctgttatc 9300cctactcaag gacatcatcc ctttagtgag ggttaattca cgcagtgggt acggaactaa 9360aggcagcaca catcgtgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 9420tacgtctctc ccccgcagta agggctagat taactcgtct cgtgaatatc cggaactccc 9480tttagtgagg gttaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 9540gccagcttaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc taccggaaac 9600gcttccttca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 9660ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 9720cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 9780ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 9840tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 9900gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 9960tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 10020gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 10080tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 10140ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 10200agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 10260gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 10320attttggtca tgatctatgt cgggtgcgga gaaagaggta atgaaatggc atacgagtaa 10380acttggtctg acaccgctgc atgagattat caaaaaggat cttcacctag atccttttaa 10440attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 10500accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 10560ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 10620gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 10680agccagccgg aagcgccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 10740ctattaactg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcggagcg 10800ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 10860gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 10920ttagctcctt cggtcctccg atggttgtca gaagtaagtt ggccgcagtg ttatcactca 10980tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 11040tgactggtga gtattcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 11100cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 11160tcattgggaa gcgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 11220gttcgatgta acccacacga gcacccaact gatcttcagc atcttttact ttcaccagcg 11280tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 11340ggaaatgttg aatactcata cgcttccttt ttcaatagta ttgaagcatt tatcagggtt 11400attgtctcgg gagcgaatac atatttgaat gtatttagaa aaa 11443912844DNAArtificial SequenceSynthetic promoter 9taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac 60cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca 120agaaaattaa tcgcaccggt atctatgtcg ggtgcggaga aagaggtaat gaaatggcag 180ctagcatcat caataatata ccttattttg gattgaagcc aatatgataa tgagggggtg 240gagtttgtga cgtggcgcgg ggcgtgggaa cggggcgggt gacgtagtag tgtggcggaa 300gtgtgatgtt gcaagtgtgg cggaacacat gtaagcgacg gatgtggcaa aagtgacgtt 360tttggtgtgc gccggtgtac acaggaagtg acaattttcg cgcggtttta ggcggatgtt 420gtagtaaatt tgggcgtaac cgagtaagat ttggccattt tcgcgggaaa actgaataag 480aggaagtgaa atctgaataa ttttgtgtta ctcatagcgc gtaatagtgc gccggtgtac 540acaggaagtg acaattttcg cgcggtttta ggcggatgtt gtagtaaatt tgggcgtaac 600cgagtaagat ttggccattt tcgcgggaaa actgaataag aggaagtgaa atctgaataa 660ttttgtgtta ctcattttgt ctagggagat ccggtaccga tatcctagac aacgatgctg 720agctaactat aacggtccta aggtagcgac cgcggagact aggtgtattt atctaagcga 780tcgcttaatt aaggccggcc gccgcaataa aatatcttta ttttcattac atctgtgtgt 840tggttttttg tgtgaatcca tagtactaac atacgctctc catcaaaaca aaacgaaaca 900aaacaaacta gcaaaatagg ctgtccccag tgcaagtcca ggtgccagaa catttctcta 960tccataatgc aggggtaccg ggtgatgacg gtgaaaacct ccaattgcgg agtactgtcc 1020tccgagcgga gtactgtcct ccgagcggag tactgtcctc cgagcggagt actgtcctcc 1080gagcggagta ctgtcctccg agcggagtac tgtcctccga gcggagagtc cccggggacc 1140tagagggtat ataatgggtg ccttagctgg tgtgtgacct catcttcctg tacgcccctg 1200caggggcgcg ccacgcgtcg aagaaggtga gtaatcttaa catgctcttt tttttttttt 1260ttgctaatcc cttttgtgtg ctgatgttag gatgacattt acaacaaatg tttgttcctg 1320acaggaaaaa ccttgctggg taccttcgtt gccggacact tcttgtcctc tactttggaa 1380aaaaggaatt gagagccgct agcgccacca tgattagccc tttcctcgtg ctcgccattg 1440gcacatgcct caccaatagc ctcgtgcctg agaaagagaa agaccctaag tattggaggg 1500accaagccca agagacactg aaatacgctc tggaactgca aaagctcaac acaaacgtcg 1560ccaaaaacgt catcatgttc ctcggcgatg gcatgggcgt cagcacagtg acagccgcta 1620gaatcctgaa aggccaactg catcacaatc ccggagagga aaccaggctg gaaatggata 1680agtttccctt tgtggctctg tccaagacat acaataccaa tgcccaagtg cctgactccg 1740ccggaaccgc taccgcctac ctgtgcggag tgaaagccaa tgagggaacc gtcggcgtca 1800gcgctgccac agagaggagc agatgcaata ccacacaggg aaacgaagtg acaagcattc 1860tgaggtgggc taaggatgcc ggaaagtccg tgggaatcgt caccacaacc agggtgaatc 1920acgctacccc tagcgctgcc tatgcccata gcgctgacag ggactggtac tccgacaatg 1980agatgccccc tgaggctctg tcccagggat gcaaagacat tgcctatcag ctcatgcaca 2040acattagaga catcgacgtg attatgggag gcggaagaaa atatatgtac cctaagaata 2100agacagacgt ggagtatgag tccgacgaaa aggctagagg aaccaggctg gatggcctcg 2160acctcgtgga tacctggaag tccttcaaac ccaggtacaa acactcccac tttatctgga 2220acaggaccga actgctgacc ctggaccctc acaacgtcga ttacctcctg ggactgtttg 2280agcctggcga tatgcaatac gaactgaata gaaataacgt caccgatccc tccctgtccg 2340agatggtggt cgtggctatc caaatcctca gaaaaaaccc taagggattc tttctgctcg 2400tggaaggcgg aagaattgac cacggccatc acgaaggcaa agccaagcaa gctctccacg 2460aggctgtgga aatggataga gctatcggac aggctggctc cctgacaagc tccgaggata 2520ccctcaccgt cgtgacagcc gatcactccc acgtttttac attcggaggc tataccccta 2580gaggaaactc catctttggc ctcgccccta tgctgtccga taccgataag aaacccttta 2640ccgctatcct ctacggaaac ggacccggat acaaagtggt cggcggagag agggagaatg 2700tgagcatggt ggactatgcc cataacaatt accaagccca aagcgctgtg cccctgagac 2760acgaaaccca cggcggagag gatgtggctg tgtttagcaa aggccctatg gcccacctgt 2820tgcatggcgt ccacgaacag aattacgtcc cccatgtgat ggcctatgcc gcttgcattg 2880gcgctaacct cggccattgc gctcccgcca gctccgccgg aagcctcgcc gctggccctc 2940tgctcctggc tctggctctg tatcccctga gcgtcctgtt tggcggaggc ggaagcggag 3000gcggaggctc cggcggaggc ggaagctgtc agcctgtgac acaggaggac ggaaaggaaa 3060gcaggatctc cgtgcaagag taaatcgatt gcgcaaagct ttcgcgatag gcgagaccaa 3120tgggtgtgta cgtagcggcc gcgtcgacga tagcttgatg ggtggcatcc ctgtgacccc 3180tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta 3240ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg 3300aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc 3360tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc 3420tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg 3480accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg 3540ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga 3600ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttaaaata actataccag 3660caggaggacg tccagacaca gcataggcta cctggccatg cccaaccggt gggacatttg 3720agttgcttgc ttggcactgt cctctcatgc gttgggtcca ctcagtagat gcctgttgaa 3780ttctgattta aatcggtccg cgtacggcgt ggtaggtccg aacgaatcca tggattaccc 3840tgttatccct atccggagtt aacctcgagg acttcggaac ttctagaacc agaccgttca 3900gtttaaacgc tcttctcccc ctcgagtatg tcctactttt ataaactttg gttgaactaa 3960ataaaacagc aacatgtacc caacgttttt agactgtttt attcagtacc ataaacattc 4020actaaactat acagagctaa aatcatgcaa aagattgcaa aacaaactgc cttgggaaat 4080ttccagtcta agctaaatgg accaagtata ggattgaatt ttaagaagtt ggtgggaaga 4140ttccagctct tgtactcaca ggtaaggtcc taggattcta tttgttgaag tgtctgcaga 4200ccctctccct acagcagggg cgtggagcaa atgtgcattc aggaagtgct tattagtccc 4260cccaaaacct ttttcgtgac gacagacgga tggaggcctc agacgactcc ggcagtagga 4320ccctgaacca aatgcctgag ctgcgctcga aaggctcgtc ttgcaggagc tggcgagtgg 4380cacgggcttc ggtggccggg ctcggggctc ggggctcggg gcccggagcc ggggggggcg 4440gggggtccgt gcccagggcg ctctgtggct cgattcatgt ccccgccctc ccacctgagc 4500acactgggca gagagcctgc ggcatcgggc cagtcgcgcc tcctcgtccg ccctgggcgg 4560gtcgctgggc cccaggccgc tttctacagc tcctttaata aaatggacag caggggtcct 4620aaccagacgt gggcatcaag acaaaggagg cggccagacg cgcttgaggg cctgctcgct 4680agctcccgcc cccccattcc gcggtcatcc ggggacagag gccaggccgg actgggtgga 4740gttgggactc atacgtctgc gtccaggaag gcgccgggcg agccccagct agacgtgacg 4800ggcggggccg aacacggcag cggaccagag gcccgcggcg caccggcgtg gggcggggca 4860agcggagcct tccgggatgc cgcgcggcag ccggcttccg gctgtgggtg gtgcggggga 4920cagcggcggc cggaagctga ctgagccggc ctttggtaac gccgcctgca cttctggggg 4980cgtcgagcct ggcggtagaa tcttcccagt aggcggcgcg ggagggaaaa gaggattgag 5040ggcatgcggg gggggggggg ggcaattggc caccatgggc cccaagaaga aaaggaaggt 5100ggcccccccc accgacgtga gcctgggcga cgagctgcac ctggacggcg aggacgtggc 5160catggcccac gccgacgccc tggacgactt cgacctggac atgctgggcg acggcgacag 5220ccccggcccc ggcttcaccc cccacgacag cgccccctac ggcgccctgg acatggccga 5280cttcgagttc gagcagatgt tcaccgacgc cctgggcatc gacgagtacg gcggccatat 5340ggagatgccc gtggacagga ttctggaggc cgaactcgcc gtggagcaga aaagcgacca 5400gggcgtggag ggccccggcg gaaccggcgg cagcggcagc agccccaacg accccgtgac 5460caacatctgc caggccgccg acaagcagct gttcaccctg gtggagtggg ccaagaggat 5520tccccacttc agcagcctgc ccctggacga ccaggtgatc ctgctgaggg ccggatggaa 5580cgagctgctg atcgccagct tcagccacag gagcatcgac gtgagggacg gcatcctgct 5640ggccaccggc ctgcacgtcc ataggaacag cgcccacagc gccggagtgg gcgccatctt 5700cgacagggtg ctgaccgagc tggtgagcaa gatgagggac atgaggatgg acaagaccga 5760gctgggctgc ctgagggcca tcatcctgtt caaccccgag gtgaggggcc tgaaaagcgc 5820ccaggaggtg gagctgctga gggagaaggt gtacgccgcc ctggaggagt acaccaggac 5880cacccacccc gacgagcccg gcagattcgc caagctgctg ctgaggctgc ccagcctgag 5940gagcatcggc ctgaagtgcc tggagcacct gttcttcttc aggctgatcg gcgacgtgcc 6000catcgacacc ttcctgatgg agatgctgga gagccccagc gacagctgag ccggcaactc 6060gctgtagtaa ttccagcgag aggcagaggg agcgagcggg cggcgggcta gggtggagga 6120gcccggcgag cagagctgcg ctgcgggcgt cctgggaagg gagatccgga gcgaataggg 6180ggcttcgcct ctggcccagc cctcccgctg atcccccagc cagcggtgcg caaccctagc 6240cgcatccacg aaactttgcc catagcagcg ggcgggcact ttgcactgga acttacaaca 6300cccgagcaag gacgcgactc tcccgacgcg gggaggctat tctgcccatt tggggacact 6360tccccgccgc tgccaggacc cgcttctctg aaaggctctc cttgcagctg cttagacgct 6420ggattttttt cgggtagtgg aaaaccagca gcctcccgcg accagatctg ccaccatgaa 6480gctgctgagc agcatcgagc aggcttgcga catctgcagg ctgaagaagc tgaagtgcag 6540caaggagaag cccaagtgcg ccaagtgcct gaagaacaac tgggagtgca gatacagccc 6600caagaccaag aggagccccc tgaccagggc ccacctgacc gaggtggaga gcaggctgga 6660gaggctggag cagctgttcc tgctgatctt ccccagggag gacctggaca tgatcctgaa 6720gatggacagc ctgcaagaca tcaaggccct gctgaccggc ctgttcgtgc aggacaacgt 6780gaacaaggac gccgtgaccg acaggctggc cagcgtggag accgacatgc ccctgaccct 6840gaggcagcac aggatcagcg ccaccagcag cagcgaggag agcagcaaca agggccagag 6900gcagctgacc gtgagccccg agtttcccgg gatcaggccc gagtgcgtgg tgcccgagac 6960ccagtgcgcc atgaaaagga aggagaagaa ggcccagaag gagaaggaca agctgcccgt 7020gagcaccacc accgtcgatg accacatgcc ccccatcatg cagtgcgagc cccccccccc 7080cgaggccgcc aggattcacg aggtcgtgcc caggttcctg agcgacaagc tgctggtgac 7140caacaggcag aagaacatcc cccagctgac cgccaaccag cagttcctga tcgccaggct 7200gatctggtat caggacggct acgagcagcc cagcgacgag gacctgaaaa ggatcaccca 7260gacctggcag caggccgacg acgagaacga ggagagcgac acccccttca ggcagatcac 7320cgagatgacc atcctgaccg tgcagctgat cgtggagttc gccaagggcc tgcccggatt 7380cgccaagatc agccagcccg accagatcac cctgctgaag gcttgcagca gcgaggtgat 7440gatgctgagg gtggccagga ggtacgacgc cgccagcgac agcatcctgt tcgccaacaa 7500ccaggcttac accagggaca actacaggaa ggctggcatg gccgaggtga tcgaggacct 7560cctgcacttc tgcagatgta tgtacagcat ggccctggac aacatccact acgccctgct 7620gaccgccgtg gtgatcttca gcgacaggcc cggcctggag cagccccagc tggtggagga 7680gatccagagg tactacctga acaccctgag gatctacatc ctgaaccagc tgagcggcag 7740cgccaggagc agcgtgatct acggcaagat cctgagcatc ctgagcgagc tgaggaccct 7800gggaatgcag aacagcaata tgtgtatcag cctgaagctg aagaacagga agctgccccc 7860cttcctggag gagatttggg acgtggccga catgagccac acccagcccc cccccatcct 7920ggagagcccc accaacctgt gaatcgatta gacatgataa gatacattga tgagtttgga 7980caaaccacaa ctagaatgca gtgaaaaaaa tgcttaattt gtgaaatttg tgatgctatt 8040gcttaatttg taaccattat aagctgcaat aaacaagtta ataaaacatt tgcattcatt 8100ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa aacctctaca 8160aatgtggtat ctagagctct tccaaataga tctggaaggt gctgaggtac gatgagaccc 8220gcaccaggtg cagaccctgc gagtgtggcg gtaaacatat taggaaccag cctgtgatgc 8280tggatgtgac cgaggagctg aggcccgatc acttggtgct ggcctgcacc cgcgctgagt 8340ttggctctag cgatgaagat acagattgag gtactgaaat gtgtgggcgt ggcttaaggg 8400tgggaaagaa tatataaggt gggggtctta tgtagttttg tatctgtttt gcagcagccg 8460ccgccgccat gagcaccaac tcgtttgatg gaagcattgt gagctcatat ttgacaacgc 8520gcatgccccc atgggccggg gtgcgtcaga atgtgatggg ctccagcatt gatggtcgcc 8580ccgtcctgcc cgcaaactct actaccttga cctacgagac cgtgtctgga acgccgttgg 8640agactgcagc ctccgccgcc gcttcagccg ctgcagccac cgcccgcggg attgtgactg 8700actttgcttt cctgagcccg cttgcaagca gtgcagcttc ccgttcatcc gcccgcgatg 8760acaagttgac ggctcttttg gcacaattgg attctttgac ccgggaactt aatgtcgttt 8820ctcagcagct gttggatctg cgccagcagg tttctgccct gaaggcttcc tcccctccca 8880atgcggttta aaacataaat aaaaaaccag actctgtttg gatttggatc aagcaagtgt 8940cttgctgtct ttatttaggg gttttgcgcg cgcggtaggc ccgggaccag cggtctcggt 9000cgttgagggt cctgtgtatt ttttccagga cgtggtaaag gtgactctgg atgttcagat 9060acatgggcat aagcccgtct ctggggtgga ggtagcacca ctgcagagct tcatgctgcg 9120gggtggtgtt gtagatgatc cagtcgtagc aggagcgctg ggcgtggtgc ctaaaaatgt 9180ctttcagtag caagctgatt gccaggggca ggcccttggt gtaagtgttt acaaagcggt 9240taagctggga tgggtgcata cgtggggata tgagatgcat cttggactgt atttttaggt 9300tggctatgtt cccagccata tccctccggg gattcatgtt gtgcagaacc accagcacag 9360tgtatccggt gcacttggga aatttgtcat gtagcttaga aggaaatgcg tggaagaact 9420tggagacgcc cttgtgacct ccaagatttt ccatgcattc gtccataatg atggcaatgg 9480gcccacgggc ggcggcctgg gcgaagatat ttctgggatc actaacgtca tagttgtgtt 9540ccaggatgag atcgtcatag gccattttta caaagcgcgg gcggagggtg ccagactgcg 9600gtataatggt tccatccggc ccaggggcgt agttaccctc acagatttgc atttcccacg 9660ctttgagttc agatgggggg atcatgtcta cctgcggggc gatgaagaaa acggtttccg 9720gggtagggga gatcagctgg gaagaaagca ggttcctgag cagctgcgac ttaccgcagc 9780cggtgggccc gtaaatcaca cctattaccg ggtgcaactg gtagttaaga gagctgcagc 9840tgccgtcatc

cctgagcagg ggggccactt cgttaagcat gtccctgact cgcatgtttt 9900ccctgaccaa atccgccaga aggcgctcgc cgcccagcga tagcagttct tgcaaggaag 9960caaagttttt caacggtttg agaccgtccg ccgtaggcat gcttttgagc gtttgaccaa 10020gcagttccag gcggtcccac agctcggtca cctgctctac ggcatctcga tccagcatat 10080ctcctcgttt cgcgggttgg ggcggctttc gctgtacggc agtagtcggt gctcgtccag 10140acgggccagg gtcatgtctt tccacgggcg cagggtcctc gtcagcgtag tctgggtcac 10200ggtgaagggg tgcgctccgg gctgcgcgct ggccagggtg cgcttgaggc tggtcctgct 10260ggtgctgaag cgctgccggt cttcgccctg cgcgtcggcc aggtagcatt tgaccatggt 10320gtcatagtcc agcccctccg cggcgtggcc cttggcgcgc agcttgccct tggaggaggc 10380gccgcacgag gggcagtgca gacttttgag ggcgtagagc ttgggcgcga gaaataccga 10440ttccggggag taggcatccg cgccgcaggc cccgcagacg gtctcgcatt ccacgagcca 10500ggtgagctct ggccgttcgg ggtcaaaaac caggtttccc ccatgctttt tgatgcgttt 10560cttacctctg gtttccatga gccggtgtcc acgctcggtg acgaaaaggc tgtccgtgtc 10620cccgtataca gacttgagag gcctgtcctc gaccgatgcc cttgagagcc ttcaacccag 10680tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct 10740ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc 10800gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg 10860ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca 10920ttatcgccgg catggcggcc gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag 10980gctggatggc cttccccatt atgattcttc tcgcttccgg cggcatcggg atgcccgcgt 11040tgcaggccat gctgtccagg caggtagatg acgaccatca gggacagctt caaggccagc 11100aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 11160ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 11220aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 11280cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 11340cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 11400aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 11460cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 11520ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 11580ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 11640gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 11700agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 11760acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 11820tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 11880agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 11940gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 12000agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 12060cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 12120ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 12180cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt 12240cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 12300ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 12360tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 12420catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 12480gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata 12540gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 12600tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 12660catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 12720aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 12780attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 12840aaaa 1284410300DNAArtificial SequenceSynthetic CD95-ADAM8 reporter 10gctagcgcca ccatgattag ccctttcctc gtgctcgcca ttggcacatg cctcaccaat 60agcctcgtgc ctgagaaaga gaaagacgga ggcggaggct ccggcggagg cggaagcgga 120ggcggaggct ccgagtccct gaaactgagg agaagggtgc atgagacaga caaaaactgt 180agatccggca caccccctca gaccggcctg gagaaaccca caggcacagg ccaaagaaaa 240cagggagccg gagcccctac cgctcccgga cccggaggct cccccggagg ctaaatcgat 3001193PRTArtificial SequenceSynthetic CD95-ADAM8 reporter 11Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn 1 5 10 15 Ser Leu Val Pro Glu Lys Glu Lys Asp Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Leu Lys Leu Arg Arg Arg 35 40 45 Val His Glu Thr Asp Lys Asn Cys Arg Ser Gly Thr Pro Pro Gln Thr 50 55 60 Gly Leu Glu Lys Pro Thr Gly Thr Gly Gln Arg Lys Gln Gly Ala Gly 65 70 75 80 Ala Pro Thr Ala Pro Gly Pro Gly Gly Ser Pro Gly Gly 85 90 12339DNAArtificial SequenceSynthetic CD40-CD3 reporter 12gctagcgcca ccatgattag ccctttcctc gtgctcgcca ttggcacatg cctcaccaat 60agcctcgtgc ctggcggagg cggaagcgga ggcggaggct ccggcggagg cggaagctgt 120cagcctgtga cacaggagga cggaaaggaa agcaggatct ccgtgcaaga gaggcagcct 180gcccctaccc ctagcaatgg ctcccccaaa gacggacccg aaatccctcc cacaggcgga 240aaggctaagg ccaagcccgt gacaagagga gccggagcca ggagcggaac ccctccccaa 300accggactgg aaaagcctac cggaaccgga tgaatcgat 33913106PRTArtificial SequenceSynthetic CD40-CD3 reporter 13Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn 1 5 10 15 Ser Leu Val Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg 35 40 45 Ile Ser Val Gln Glu Arg Gln Pro Ala Pro Thr Pro Ser Asn Gly Ser 50 55 60 Pro Lys Asp Gly Pro Glu Ile Pro Pro Thr Gly Gly Lys Ala Lys Ala 65 70 75 80 Lys Pro Val Thr Arg Gly Ala Gly Ala Arg Ser Gly Thr Pro Pro Gln 85 90 95 Thr Gly Leu Glu Lys Pro Thr Gly Thr Gly 100 105 141692DNAArtificial SequenceSynthetic alkaline phosphatase - c terminal 14gctagcgcca ccatgattag ccctttcctc gtgctcgcca ttggcacatg cctcaccaat 60agcctcgtgc ctgagaaaga gaaagaccct aagtattgga gggaccaagc ccaagagaca 120ctgaaatacg ctctggaact gcaaaagctc aacacaaacg tcgccaaaaa cgtcatcatg 180ttcctcggcg atggcatggg cgtcagcaca gtgacagccg ctagaatcct gaaaggccaa 240ctgcatcaca atcccggaga ggaaaccagg ctggaaatgg ataagtttcc ctttgtggct 300ctgtccaaga catacaatac caatgcccaa gtgcctgact ccgccggaac cgctaccgcc 360tacctgtgcg gagtgaaagc caatgaggga accgtcggcg tcagcgctgc cacagagagg 420agcagatgca ataccacaca gggaaacgaa gtgacaagca ttctgaggtg ggctaaggat 480gccggaaagt ccgtgggaat cgtcaccaca accagggtga atcacgctac ccctagcgct 540gcctatgccc atagcgctga cagggactgg tactccgaca atgagatgcc ccctgaggct 600ctgtcccagg gatgcaaaga cattgcctat cagctcatgc acaacattag agacatcgac 660gtgattatgg gaggcggaag aaaatatatg taccctaaga ataagacaga cgtggagtat 720gagtccgacg aaaaggctag aggaaccagg ctggatggcc tcgacctcgt ggatacctgg 780aagtccttca aacccaggta caaacactcc cactttatct ggaacaggac cgaactgctg 840accctggacc ctcacaacgt cgattacctc ctgggactgt ttgagcctgg cgatatgcaa 900tacgaactga atagaaataa cgtcaccgat ccctccctgt ccgagatggt ggtcgtggct 960atccaaatcc tcagaaaaaa ccctaaggga ttctttctgc tcgtggaagg cggaagaatt 1020gaccacggcc atcacgaagg caaagccaag caagctctcc acgaggctgt ggaaatggat 1080agagctatcg gacaggctgg ctccctgaca agctccgagg ataccctcac cgtcgtgaca 1140gccgatcact cccacgtttt tacattcgga ggctataccc ctagaggaaa ctccatcttt 1200ggcctcgccc ctatgctgtc cgataccgat aagaaaccct ttaccgctat cctctacgga 1260aacggacccg gatacaaagt ggtcggcgga gagagggaga atgtgagcat ggtggactat 1320gcccataaca attaccaagc ccaaagcgct gtgcccctga gacacgaaac ccacggcgga 1380gaggatgtgg ctgtgtttag caaaggccct atggcccacc tgttgcatgg cgtccacgaa 1440cagaattacg tcccccatgt gatggcctat gccgcttgca ttggcgctaa cctcggccat 1500tgcgctcccg ccagctccgc cggaagcctc gccgctggcc ctctgctcct ggctctggct 1560ctgtatcccc tgagcgtcct gtttggcgga ggcggaagcg gaggcggagg ctccggcgga 1620ggcggaagct gtcagcctgt gacacaggag gacggaaagg aaagcaggat ctccgtgcaa 1680gagtaaatcg at 169215557PRTArtificial SequenceSynthetic alkaline phosphatase - c terminal CD40 15Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn 1 5 10 15 Ser Leu Val Pro Glu Lys Glu Lys Asp Pro Lys Tyr Trp Arg Asp Gln 20 25 30 Ala Gln Glu Thr Leu Lys Tyr Ala Leu Glu Leu Gln Lys Leu Asn Thr 35 40 45 Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val 50 55 60 Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His Asn 65 70 75 80 Pro Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Phe Val Ala 85 90 95 Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly 100 105 110 Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val 115 120 125 Gly Val Ser Ala Ala Thr Glu Arg Ser Arg Cys Asn Thr Thr Gln Gly 130 135 140 Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser 145 150 155 160 Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala 165 170 175 Ala Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met 180 185 190 Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu 195 200 205 Met His Asn Ile Arg Asp Ile Asp Val Ile Met Gly Gly Gly Arg Lys 210 215 220 Tyr Met Tyr Pro Lys Asn Lys Thr Asp Val Glu Tyr Glu Ser Asp Glu 225 230 235 240 Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asp Leu Val Asp Thr Trp 245 250 255 Lys Ser Phe Lys Pro Arg Tyr Lys His Ser His Phe Ile Trp Asn Arg 260 265 270 Thr Glu Leu Leu Thr Leu Asp Pro His Asn Val Asp Tyr Leu Leu Gly 275 280 285 Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn Val 290 295 300 Thr Asp Pro Ser Leu Ser Glu Met Val Val Val Ala Ile Gln Ile Leu 305 310 315 320 Arg Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile 325 330 335 Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala 340 345 350 Val Glu Met Asp Arg Ala Ile Gly Gln Ala Gly Ser Leu Thr Ser Ser 355 360 365 Glu Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr 370 375 380 Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro 385 390 395 400 Met Leu Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly 405 410 415 Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val Ser 420 425 430 Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro 435 440 445 Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ser Lys 450 455 460 Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Val 465 470 475 480 Pro His Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Leu Gly His 485 490 495 Cys Ala Pro Ala Ser Ser Ala Gly Ser Leu Ala Ala Gly Pro Leu Leu 500 505 510 Leu Ala Leu Ala Leu Tyr Pro Leu Ser Val Leu Phe Gly Gly Gly Gly 515 520 525 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Gln Pro Val Thr 530 535 540 Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu 545 550 555 16354DNAArtificial SequenceSynthetic CD28-CD3 reporter 16gctagcgcca ccatgattag ccctttcctc gtgctcgcca ttggcacatg cctcaccaat 60agcctcgtgc ctagatccga cgtgaactcc agaacaggcc ctagcggagc cacaccccct 120agcggaaacc cttacacaat cacaggctcc cagcaactgc aagtgtatag caaaaccgga 180ttcaatcccg ctcccacacc ctccaacgga agccctaagg atggccctga gattccccct 240accggaggct ccggcggagg cggaagcgga ggcggaggct ccaaggctaa ggccaagccc 300gtgacaagag gagccggagc cggacccgga ggctcccccg gaggctaaat cgat 35417111PRTArtificial SequenceSynthetic CD28-CD3 reporter 17Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn 1 5 10 15 Ser Leu Val Pro Arg Ser Asp Val Asn Ser Arg Thr Gly Pro Ser Gly 20 25 30 Ala Thr Pro Pro Ser Gly Asn Pro Tyr Thr Ile Thr Gly Ser Gln Gln 35 40 45 Leu Gln Val Tyr Ser Lys Thr Gly Phe Asn Pro Ala Pro Thr Pro Ser 50 55 60 Asn Gly Ser Pro Lys Asp Gly Pro Glu Ile Pro Pro Thr Gly Gly Ser 65 70 75 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Ala Lys Ala Lys Pro 85 90 95 Val Thr Arg Gly Ala Gly Ala Gly Pro Gly Gly Ser Pro Gly Gly 100 105 110 18375DNAArtificial SequenceSynthetic CD28-CD40 reporter 18gctagcgcca ccatgattag ccctttcctc gtgctcgcca ttggcacatg cctcaccaat 60agcctcgtgc ctagatccga cgtgaactcc agaacaggcc ctagcggagc cacaccccct 120agcggaaacc cttacacaat cacaggctcc cagcaactgc aagtgtatag caaaaccgga 180ttcaatcccg ctcccacacc ctccaacgga agccctaagg atggccctga gattccccct 240accggaggct ccggcggagg cggaagcgga ggcggaggct cctgccaacc cgtcacccag 300gaggacggca aagagtccag aattagcgtc caggaaagac aaggccctgg cggaagccct 360ggcggatgaa tcgat 37519118PRTArtificial SequenceSynthetic CD28-CD40 reporter 19Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn 1 5 10 15 Ser Leu Val Pro Arg Ser Asp Val Asn Ser Arg Thr Gly Pro Ser Gly 20 25 30 Ala Thr Pro Pro Ser Gly Asn Pro Tyr Thr Ile Thr Gly Ser Gln Gln 35 40 45 Leu Gln Val Tyr Ser Lys Thr Gly Phe Asn Pro Ala Pro Thr Pro Ser 50 55 60 Asn Gly Ser Pro Lys Asp Gly Pro Glu Ile Pro Pro Thr Gly Gly Ser 65 70 75 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Gln Pro Val Thr Gln 85 90 95 Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln Gly Pro 100 105 110 Gly Gly Ser Pro Gly Gly 115 201737DNAArtificial SequenceSynthetic alkaline phosphatase - amino terminal CD40 20gctagcgcca ccatgattag ccctttcctc gtgctcgcca ttggcacatg cctcaccaat 60agcctcgtgc cttgccaacc cgtcacccag gaggacggca aagagtccag aattagcgtc 120caggaaggcg gaggcggaag cggaggcgga ggctccggcg gaggcggaag cgaaaaggaa 180aaggacccca aatactggag agatcaggct caggaaaccc tcaagtatgc cctggaactc 240cagaaactga ataccaatgt ggctaagaat gtgattatgt ttctgggcga cggcatggga 300gtgtccaccg tcaccgctgc cagaatcctg aagggacagc tccaccacaa ccccggcgag 360gagacaagac tggagatgga caaattccct ttcgtcgccc tgtccaaaac ctataacaca 420aacgctcagg tccccgatag cgctggcaca gccacagcct atctgtgtgg cgtcaaggct 480aacgaaggca cagtgggagt gtccgccgct accgaaagat ccagatgtaa cacaacccaa 540ggcaatgagg tcacctccat cctcagatgg gccaaagacg ctggcaaaag cgtcggcatt 600gtgacaacca caagagtcaa ccatgccaca ccctccgccg cttacgctca ctccgccgat 660agagattggt atagcgataa cgaaatgcct cccgaagccc tgagccaagg ctgtaaggac 720atcgcttacc aactgatgca caatatcagg gacattgacg ttatcatggg cggaggcagg 780aagtatatgt atcccaaaaa caaaaccgat gtggaatacg aaagcgatga gaaagccagg 840ggcacaagac tcgacggact ggatctggtg gatacatgga agtccttcaa gcctagatat 900aagcatagcc atttcatttg gaatagaaca gagcttctga cactggaccc ccataacgtg 960gactatctgc tcggcctgtt cgagcccgga gacatgcagt atgagctgaa caggaacaat 1020gtgacagacc ctagcctgag cgaaatggtc gtggtcgcca ttcagattct gaggaagaat 1080cccaaaggct ttttcctcct ggtcgaggga ggcagaatcg accacggcca ccacgaggga 1140aaggccaagc aagccctcca cgaagccgtc gagatggaca gggccattgg ccaagccgga 1200agcctcacct ccagcgagga tacactgaca gtggtcaccg ctgaccatag ccatgtgttt 1260acctttggcg gatacacacc caggggcaat agcattttcg gactggctcc catgctgtcc 1320gacacagaca aaaagccttt cacagccatt ctgtatggca atggccctgg ctataaggtc 1380gtgggaggcg aaagagaaaa cgtcagcatg gtggattacg ctcacaataa ctatcaggct 1440cagtccgccg tccccctcag acatgagaca cacggaggcg aggacgtggc cgtgttctcc 1500aagggaccta tggcccatct gctccacggc gtccatgagc aaaactatgt gcctcacgtc 1560atggcttacg ctgcctgtat cggagccaat ctgggacact gtgcccctgc ctccagcgct 1620ggctccctgg ctgccggacc cctcctgctc gccctcgccc tctaccctct gtccgtgctg 1680ttcggaggcg gaggctccgg cggaggcgga agcggaggcg gaggctcctg aatcgat

173721572PRTArtificial SequenceSynthetic alkaline phosphatase - amino terminal CD40 21Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn 1 5 10 15 Ser Leu Val Pro Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser 20 25 30 Arg Ile Ser Val Gln Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 45 Gly Gly Gly Gly Ser Glu Lys Glu Lys Asp Pro Lys Tyr Trp Arg Asp 50 55 60 Gln Ala Gln Glu Thr Leu Lys Tyr Ala Leu Glu Leu Gln Lys Leu Asn 65 70 75 80 Thr Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly 85 90 95 Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His 100 105 110 Asn Pro Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Phe Val 115 120 125 Ala Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala 130 135 140 Gly Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr 145 150 155 160 Val Gly Val Ser Ala Ala Thr Glu Arg Ser Arg Cys Asn Thr Thr Gln 165 170 175 Gly Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys 180 185 190 Ser Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser 195 200 205 Ala Ala Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu 210 215 220 Met Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln 225 230 235 240 Leu Met His Asn Ile Arg Asp Ile Asp Val Ile Met Gly Gly Gly Arg 245 250 255 Lys Tyr Met Tyr Pro Lys Asn Lys Thr Asp Val Glu Tyr Glu Ser Asp 260 265 270 Glu Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asp Leu Val Asp Thr 275 280 285 Trp Lys Ser Phe Lys Pro Arg Tyr Lys His Ser His Phe Ile Trp Asn 290 295 300 Arg Thr Glu Leu Leu Thr Leu Asp Pro His Asn Val Asp Tyr Leu Leu 305 310 315 320 Gly Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn 325 330 335 Val Thr Asp Pro Ser Leu Ser Glu Met Val Val Val Ala Ile Gln Ile 340 345 350 Leu Arg Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg 355 360 365 Ile Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu 370 375 380 Ala Val Glu Met Asp Arg Ala Ile Gly Gln Ala Gly Ser Leu Thr Ser 385 390 395 400 Ser Glu Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe 405 410 415 Thr Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala 420 425 430 Pro Met Leu Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr 435 440 445 Gly Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val 450 455 460 Ser Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val 465 470 475 480 Pro Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ser 485 490 495 Lys Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr 500 505 510 Val Pro His Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Leu Gly 515 520 525 His Cys Ala Pro Ala Ser Ser Ala Gly Ser Leu Ala Ala Gly Pro Leu 530 535 540 Leu Leu Ala Leu Ala Leu Tyr Pro Leu Ser Val Leu Phe Gly Gly Gly 545 550 555 560 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 565 570 221677DNAArtificial SequenceSynthetic alkaline phosphatase - c terminal CD28 22gctagcgcca ccatgattag ccctttcctc gtgctcgcca ttggcacatg cctcaccaat 60agcctcgtgc ctgagaaaga gaaagaccct aagtattgga gggaccaagc ccaagagaca 120ctgaaatacg ctctggaact gcaaaagctc aacacaaacg tcgccaaaaa cgtcatcatg 180ttcctcggcg atggcatggg cgtcagcaca gtgacagccg ctagaatcct gaaaggccaa 240ctgcatcaca atcccggaga ggaaaccagg ctggaaatgg ataagtttcc ctttgtggct 300ctgtccaaga catacaatac caatgcccaa gtgcctgact ccgccggaac cgctaccgcc 360tacctgtgcg gagtgaaagc caatgaggga accgtcggcg tcagcgctgc cacagagagg 420agcagatgca ataccacaca gggaaacgaa gtgacaagca ttctgaggtg ggctaaggat 480gccggaaagt ccgtgggaat cgtcaccaca accagggtga atcacgctac ccctagcgct 540gcctatgccc atagcgctga cagggactgg tactccgaca atgagatgcc ccctgaggct 600ctgtcccagg gatgcaaaga cattgcctat cagctcatgc acaacattag agacatcgac 660gtgattatgg gaggcggaag aaaatatatg taccctaaga ataagacaga cgtggagtat 720gagtccgacg aaaaggctag aggaaccagg ctggatggcc tcgacctcgt ggatacctgg 780aagtccttca aacccaggta caaacactcc cactttatct ggaacaggac cgaactgctg 840accctggacc ctcacaacgt cgattacctc ctgggactgt ttgagcctgg cgatatgcaa 900tacgaactga atagaaataa cgtcaccgat ccctccctgt ccgagatggt ggtcgtggct 960atccaaatcc tcagaaaaaa ccctaaggga ttctttctgc tcgtggaagg cggaagaatt 1020gaccacggcc atcacgaagg caaagccaag caagctctcc acgaggctgt ggaaatggat 1080agagctatcg gacaggctgg ctccctgaca agctccgagg ataccctcac cgtcgtgaca 1140gccgatcact cccacgtttt tacattcgga ggctataccc ctagaggaaa ctccatcttt 1200ggcctcgccc ctatgctgtc cgataccgat aagaaaccct ttaccgctat cctctacgga 1260aacggacccg gatacaaagt ggtcggcgga gagagggaga atgtgagcat ggtggactat 1320gcccataaca attaccaagc ccaaagcgct gtgcccctga gacacgaaac ccacggcgga 1380gaggatgtgg ctgtgtttag caaaggccct atggcccacc tgttgcatgg cgtccacgaa 1440cagaattacg tcccccatgt gatggcctat gccgcttgca ttggcgctaa cctcggccat 1500tgcgctcccg ccagctccgc cggaagcctc gccgctggcc ctctgctcct ggctctggct 1560ctgtatcccc tgagcgtcct gtttggcgga ggcggaagcg gaggcggagg ctccggcgga 1620ggcggaagct cccagcaact gcaagtgtat agcaaaaccg gattcaactg aatcgat 167723552PRTArtificial SequenceSynthetic alkaline phosphatase - c terminal CD28 23Met Ile Ser Pro Phe Leu Val Leu Ala Ile Gly Thr Cys Leu Thr Asn 1 5 10 15 Ser Leu Val Pro Glu Lys Glu Lys Asp Pro Lys Tyr Trp Arg Asp Gln 20 25 30 Ala Gln Glu Thr Leu Lys Tyr Ala Leu Glu Leu Gln Lys Leu Asn Thr 35 40 45 Asn Val Ala Lys Asn Val Ile Met Phe Leu Gly Asp Gly Met Gly Val 50 55 60 Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Leu His His Asn 65 70 75 80 Pro Gly Glu Glu Thr Arg Leu Glu Met Asp Lys Phe Pro Phe Val Ala 85 90 95 Leu Ser Lys Thr Tyr Asn Thr Asn Ala Gln Val Pro Asp Ser Ala Gly 100 105 110 Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Ala Asn Glu Gly Thr Val 115 120 125 Gly Val Ser Ala Ala Thr Glu Arg Ser Arg Cys Asn Thr Thr Gln Gly 130 135 140 Asn Glu Val Thr Ser Ile Leu Arg Trp Ala Lys Asp Ala Gly Lys Ser 145 150 155 160 Val Gly Ile Val Thr Thr Thr Arg Val Asn His Ala Thr Pro Ser Ala 165 170 175 Ala Tyr Ala His Ser Ala Asp Arg Asp Trp Tyr Ser Asp Asn Glu Met 180 185 190 Pro Pro Glu Ala Leu Ser Gln Gly Cys Lys Asp Ile Ala Tyr Gln Leu 195 200 205 Met His Asn Ile Arg Asp Ile Asp Val Ile Met Gly Gly Gly Arg Lys 210 215 220 Tyr Met Tyr Pro Lys Asn Lys Thr Asp Val Glu Tyr Glu Ser Asp Glu 225 230 235 240 Lys Ala Arg Gly Thr Arg Leu Asp Gly Leu Asp Leu Val Asp Thr Trp 245 250 255 Lys Ser Phe Lys Pro Arg Tyr Lys His Ser His Phe Ile Trp Asn Arg 260 265 270 Thr Glu Leu Leu Thr Leu Asp Pro His Asn Val Asp Tyr Leu Leu Gly 275 280 285 Leu Phe Glu Pro Gly Asp Met Gln Tyr Glu Leu Asn Arg Asn Asn Val 290 295 300 Thr Asp Pro Ser Leu Ser Glu Met Val Val Val Ala Ile Gln Ile Leu 305 310 315 320 Arg Lys Asn Pro Lys Gly Phe Phe Leu Leu Val Glu Gly Gly Arg Ile 325 330 335 Asp His Gly His His Glu Gly Lys Ala Lys Gln Ala Leu His Glu Ala 340 345 350 Val Glu Met Asp Arg Ala Ile Gly Gln Ala Gly Ser Leu Thr Ser Ser 355 360 365 Glu Asp Thr Leu Thr Val Val Thr Ala Asp His Ser His Val Phe Thr 370 375 380 Phe Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Leu Ala Pro 385 390 395 400 Met Leu Ser Asp Thr Asp Lys Lys Pro Phe Thr Ala Ile Leu Tyr Gly 405 410 415 Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg Glu Asn Val Ser 420 425 430 Met Val Asp Tyr Ala His Asn Asn Tyr Gln Ala Gln Ser Ala Val Pro 435 440 445 Leu Arg His Glu Thr His Gly Gly Glu Asp Val Ala Val Phe Ser Lys 450 455 460 Gly Pro Met Ala His Leu Leu His Gly Val His Glu Gln Asn Tyr Val 465 470 475 480 Pro His Val Met Ala Tyr Ala Ala Cys Ile Gly Ala Asn Leu Gly His 485 490 495 Cys Ala Pro Ala Ser Ser Ala Gly Ser Leu Ala Ala Gly Pro Leu Leu 500 505 510 Leu Ala Leu Ala Leu Tyr Pro Leu Ser Val Leu Phe Gly Gly Gly Gly 515 520 525 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gln Gln Leu Gln 530 535 540 Val Tyr Ser Lys Thr Gly Phe Asn 545 550

Claims

1-38. (canceled)

39. A method of monitoring the effectiveness of a treatment for a disease or disorder in a subject, comprising: (a) administering said treatment to said subject; (b) introducing into cells of said subject a polynucleotide, wherein said polynucleotide encodes a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence, which is operably linked to a diagnostic switch promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, wherein the activity of said promoter is modulated during said disease or disorder of said subject, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells; (c) administering ligand to said modified cells; and (d) detecting reporter gene expression at least twice; wherein a change in the level of expression of said reporter gene indicates the effectiveness of said treatment.

40. The method of claim 39, wherein step (b) is carried out before step (a).

41. The method of claim 40, wherein a baseline level of reporter gene expression is determined prior to step (a).

42. The method of claim 39, wherein said method is carried out ex vivo in cells that have been isolated from said subject.

43. The method of claim 39, wherein said polynucleotides are introduced into cells that have been isolated from said subject to produce modified cells, and the modified cells are re-introduced into said subject.

44. The method of claim 39, wherein said method is carried out in vivo.

45. The method of claim 39, wherein said gene switch is an EcR-based gene switch.

46. The method of claim 45, wherein said ligand binds to the EcR ligand binding domain.

47. The method of claim 46, wherein said ligand is a diacylhydrazine.

48. The method of claim 47, wherein said ligand is selected from the group consisting of RG-115819, RG-115830, and RG-115932.

49. The method of claim 46, wherein said ligand is an amidoketone or oxadiazoline.

50. The method of claim 39, wherein said gene switch comprises a first transcription factor sequence under the control of a first diagnostic switch promoter and a second transcription factor sequence under the control of a second diagnostic switch promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor.

51. The method of claim 50, wherein said first diagnostic switch promoter and said second diagnostic switch promoter are different.

52. The method of claim 50, wherein said first diagnostic switch promoter and said second diagnostic switch promoter are the same.

53. The method of claim 50, wherein said first transcription factor sequence encodes a protein comprising a heterodimer partner and a transactivation domain.

54. The method of claim 50, wherein said second transcription factor sequence encodes a protein comprising a DNA binding domain and a ligand-binding domain.

55. The method of claim 39, wherein one of said polynucleotides further encodes a lethal polypeptide operably linked to an inducible promoter.

56-59. (canceled)

60. A method of monitoring the potential toxicity of an administered treatment for a disease or disorder in a subject, comprising: (a) administering said treatment to said subject; (b) introducing into cells of said subject a polynucleotide, wherein said polynucleotide encodes a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence, which is operably linked to a diagnostic switch promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, wherein the activity of said promoter is modulated by factors found in cells that are being exposed to toxic conditions, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells; (c) administering ligand to said modified cells; and (d) detecting reporter gene expression at least twice; wherein a change in the level of expression of said reporter gene indicates the toxicity of said treatment.

61-80. (canceled)

81. A method of monitoring the level of a factor that is being administered to a subject for treatment for a disease or disorder, comprising: (a) administering said treatment to said subject; (b) introducing into cells of said subject a polynucleotide, wherein said polynucleotide encodes a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence, which is operably linked to a diagnostic switch promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, wherein the activity of said promoter is modulated by said factor that is being administered for treatment, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells; (c) administering ligand to said modified cells; and (d) detecting reporter gene expression; wherein the level of expression of said reporter gene indicates the level of the factor being administered for treatment.

82-102. (canceled)

103. A method of detecting transplant rejection in a subject that has received an organ or tissue transplant, comprising: (a) introducing into cells of said organ or tissue transplant a polynucleotide, wherein said polynucleotide encodes a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence, which is operably linked to a diagnostic switch promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, wherein the activity of said promoter is modulated during transplant rejection, and (2) a polynucleotide encoding a reporter gene linked to a promoter which is activated by said ligand-dependent transcription factor, to produce modified cells; (b) administering ligand to said modified cells; and (c) detecting reporter gene expression; wherein expression of the reporter gene indicates that transplant rejection has been detected.

104-175. (canceled)

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