Methods of Protein Destabilization and Uses Thereof

Abstract

This invention is directed towards methods of destabilizing proteins in living cells, and their use for the development of novel assays. In one embodiment, the invention comprises the use of non-cleavable multimerized ubiquitin fusion proteins to destabilize a target protein, such as a reporter moiety. In one aspect of this method the constructs also comprises a linker that operatively couples the reporter moiety to the multimerized ubiquitin fusion protein. In this embodiment, enzymatic modification of the linker results in a modulation of the coupling of the reporter protein to the multimerized ubiquitin domains resulting in a change in the stability of the reporter moiety. The level of the reporter moiety in the cell can then be used as a measure of the enzymatic activity in the cell. In another embodiment the invention provides for a generalized way of coordinately regulating the cellular concentration of a plurality of target proteins. In one aspect of this method, the target proteins are operatively coupled to a ubiquitin fusion protein via a linker containing a protease cleavage site. Cleavage of the linker by a protease results in uncoupling of the target protein from the multimerized ubiquitin construct, and results in an increase in the stability and concentration of the target protein.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of U.S. application Ser. No. 11/821,562 filed Jun. 22, 2007, now issued as U.S. Pat. No. 7,824,850; which is a continuation application of U.S. application Ser. No. 09/498,098 filed Feb. 4, 2000, now issued as U.S. Pat. No. 7,262,005. The disclosure of the prior application is considered part of and is incorporated by reference in the disclosure of this application.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention is in the field of protein analysis and more particularly methods of destabilizing proteins and using the destabilized proteins for novel cell based assays.

[0004] 2. Background of the Invention

[0005] While genomic programs provide ever more sophisticated information on the sequence and patterns of expression of mammalian genes, it is increasingly recognized that integrating this information into a functional model of how a cell works requires an understanding of how the protein products of expressed genes interact within the cell. Although we have made significant improvements in our ability to clone, sequence and analyze DNA sequences, our reciprocal abilities for studying RNA and protein molecules are significantly less facile or advanced. Furthermore, proteins themselves represent significantly more complex molecules in terms of composition, shape and activity compared to double stranded DNA. A central challenge facing workers in the field today is to understand out how a protein's activity and function within a cell are regulated and coordinated within the native physiological context.

[0006] Traditionally, genetic analysis has been used for determining the function of gene products and how they interact with other proteins within a common pathway. Unfortunately genetic analysis in vertebrate organisms is extremely time consuming and expensive. An alternative approach is to devise an assay system for a given protein and then screen for compounds that activate or inhibit its function. These compounds can be used to dissect the cellular pathways the protein functions in, as well as serving as potential compounds of therapeutic value.

[0007] Although there is tremendous interest in understanding the regulation and interactions of proteins within cells there are relatively few methods that are robust, simple to use, amenable to high throughput screening or can be used effectively within Jiving ceils. Furthermore in many cases where specific assays do exist these are restricted in scope to individual enzymatic steps or to one or two defined pathways.

[0008] A need thus exists for sensitive methods of interfacing the functional modifications of proteins with optical signals that can be used detect and monitor these changes, for example for use in high throughput screening. In drug screening applications these assays can be applied to find useful compounds that are specific and selective for a particular protein or signal transduction or metabolic pathway.

[0009] Proteins may undergo a huge variety of post-translational modifications subsequent to their synthesis in the cell. In many cases these modifications can play critical roles in the functioning and stability of the modified proteins. For example, proteolysis, phosphorylation, covalent attachment of a lipid or lipid derivative, disulfide bond formation, glycosylation and oxidation all can have important functional effects. Many other examples also exist and may play important functional roles within a cell for defined proteins.

[0010] One approach to developing a generic assay capable of detecting these myriad post-translational modifications is to operatively couple these activities through a central pathway of protein modification that can be sensitively measured with a common reporter system. In the present invention, the inventors have recognized that by coupling post-translational activities to the stability of a high sensitivity reporter moiety it is possible to develop uniform cell based assays for a range of activities. Importantly these measurements are robust enough for high throughput screening applications, readily adaptable to a range of activities and provide cellular assays that provide information within a living cell.

[0011] In the present invention, post-translational activities can be measured by providing one or more constructs in which the activity to be measured influences the stability of a reporter moiety. In one embodiment, this may be achieved by providing a reporter moiety that is operatively coupled to a multimerized destabilization domain via a linking moiety. The linking moiety comprises a recognition motif for the target activity such that modification of the linker by the activity results in altered stability of the reporter moiety. If the reporter moiety is an enzymatic reporter gene the method provides a high sensitivity readout that is generally applicable to a range of activities which are otherwise difficult to measure within living cells. The multimerized destabilization domain described herein provides a key advantage in the method because it enables the degree of destabilization to be predictably tuned to any activity level or intrinsic stability of the target protein or reporter moiety.

[0012] The regulation of protein stability is an area of particular interest because of its increased recognition as a key regulator of a protein's concentration and function in the cell. Although our knowledge of the factors that control protein stability have grown dramatically in recent years, it is clear that a variety of cellular pathways and environmental cues participate in and control a protein's fate. For example, mis-folding, proteolysis, oxidation and some conformational changes that expose significant surface hydrophobicity readily contribute to the recognition of a protein by the cellular machinery for protein degradation. The majority of cytoplasmic protein degradation involves the ubiquitination of the target protein followed by binding and degradation by the proteasome. (For review see Hershko and Ciechanover (1998) Annu. Rev. Biochem. 67 425-79)

[0013] A key step in protein ubiquitination, and degradation, is recognition of the target protein by ubiquitin protein ligase or E3 enzyme. This class of enzymes is responsible for the covalent attachment of ubiquitin to the target protein via an amide isopeptide linkage to an e-amino group of one of the substrate protein's lysine residues. There are currently believed to be multiple families of E3 enzymes, additionally there is increasing evidence that some E3 proteins exist as multi-subunit protein complexes (Laney and Hochstrasser (1999) Cell 97 427-430). E3 proteins and their associated complexes are believed to be largely responsible for recognizing and ubiquitinating damaged proteins as well as specific destabilization domains present in target proteins. Once recognized, a protein target that has been modified by the addition of a single ubiquitin domain, becomes a substrate for further ubiquitination, either at different sites in the substrate protein, or through extension of the conjugated ubiquitin. This process can thus lead to a poly-ubiquitinated protein with numerous branched ubiquitin domains attached. Once poly-ubiquitinated, the protein is recognized with high affinity by the proteasome where it is degraded.

[0014] The addition of specific destabilization domains to a target protein has in some cases been demonstrated to destabilize that target protein. A key challenge in this area has been to provide a predictable way of creating graded levels of destabilization for a given protein that that can be utilized in manipulating the steady state levels or dynamic temporal regulation of that protein. The present inventors have discovered for the first time that by providing stable multimerized linear chains of individual destabilization domains, such as ubiquitin, it is possible to create a generic method of protein destabilization that is widely applicable to virtually any protein. Importantly, this approach has the advantage that the degree of destabilization can be accurately controlled by varying the number of destabilization domains added to the target protein. As a result, the actual cellular concentration and half-life of an exogenously expressed protein in a cell or living organism can be accurately and reproducibly controlled. By coupling 1, 2, 3, 4 or more copies of ubiquitin to the reporter gene β-lactamase it has been possible to regulate the protein concentration of this protein in the cell over a 10-fold range compared to the native protein. The present inventors have applied this discovery to create an assay technology that is broadly capable of measuring a wide range of post-translational activities.

SUMMARY OF THE INVENTION

[0015] This invention provides a fluorescent, bioluminescent or enzymatic substrate useful as an optical probe or sensor of post-translational modifications, such as proteolysis. In one embodiment, the invention comprises a reporter moiety that is functionally coupled to one or more destabilizing domains via a linker. The linker typically contains a recognition motif for an activity. Modification of the linker by the activity results in uncoupling of the reporter moiety from the destabilizing domain(s) with a corresponding change in the stability of the reporter moiety. The level of activity within a sample is sensed by a measurable change in the level of the reporter moiety, for example by detecting at least one optical property of the reporter moiety, or by detecting at least one optical property of detectable product of the reporter moiety. FIG. 1.

[0016] In one embodiment the reporter moiety is an enzymatic reporter such as alkaline phosphatase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase, peroxidase, β-lactamase, bioluminescent proteins, luciferases and catalytic antibodies. In another embodiment the reporter moiety is a naturally fluorescent protein, epitope or structural protein.

[0017] In one aspect the linker moiety is an amino acid sequence that covalently couples the reporter moiety to the multimerized destabilization domain. In another aspect, the linker moiety comprises two separate amino acid sequences, one of which is covalently coupled to the reporter moiety, and one of which is coupled to the multimerized destabilization domain. Coupling of the reporter moiety to the destabilization domains occurs through the non-covalent interaction or binding of the two ammo acid sequences of the linker together. In either case, modification of the linker by the activity results in a modulation of the coupling of the reporter moiety to the multimerized destabilization domains. In one aspect of this method the activity is selected from the group consisting of a protease activity, a protein kinase activity and a phosphoprotein phosphatase activity.

[0018] In one aspect the multimerized destabilization domain comprises two, three, four, or more copies of the destabilization domain covalently coupled together in a linear chain. In one embodiment, the destabilization domains comprise ubiquitin, or a homolog thereof. In a preferred embodiment the multimerized copies of ubiquitin are not cleavable by α-NH-ubiquitin protein endoproteases. In one embodiment the ubiquitin domains comprise a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases. In one aspect of this embodiment the mutation represents the mutation of glycine 76 to a larger or more bulky amino acid.

[0019] In another aspect the invention comprises a method of regulating the concentration of one or more target proteins in a cell. The method involves the creation of a fusion protein containing the protein of interest coupled to one or more destabilization domains. In different embodiments the protein of interest may be coupled to a multimerized destabilization domain comprising two or more copies of the destabilization domain. In one embodiment, the destabilization domains comprise ubiquitin, or a homolog thereof. In a preferred embodiment the multimerized copies of ubiquitin are not cleavable by oc-NH-ubiquitin protein endoproteases. In one embodiment the ubiquitin domains comprise a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases. In one aspect of this embodiment the mutation represents the mutation of glycine 76 to a larger or more bulky amino acid.

[0020] In one aspect of this method, the fusion protein may additionally comprise a linker that couples the protein of interest to one or more destabilization domains. The linker typically comprises a protease cleavage site for a protease. Cleavage of the linker by the protease modulates the coupling of the multimerized destabilization domain to the protein of interest, thereby providing a method of rapidly modulating the stability of one or more proteins of interest in the cell simultaneously. The protease may be introduced into the cell, or its activity regulated by the presence of a membrane permeant small molecule inhibitor. In one embodiment of this method, the protease does not naturally occur in the target cell.

[0021] In another aspect the invention includes a recombinant DNA molecule, comprising a nucleic acid sequence encoding for one or more destabilization domains, a target protein, and a linker moiety that operatively couples the destabilization domain(s) to the target protein. In different embodiments the protein of interest may be coupled to one, two, three, four or more copies of the destabilization domain. In one embodiment, the destabilization domains comprise ubiquitin, or a homolog thereof. In a preferred embodiment the multimerized copies of ubiquitin are not cleavable by α-NH-ubiquitin protein endoproteases. In one embodiment the ubiquitin domains comprise a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases. In one aspect of this embodiment the mutation represents the mutation of glycine 76 to a larger or more bulky amino acid.

[0022] In another embodiment the invention includes a recombinant protein molecule, comprising an amino acid sequence encoding for one or more destabilization domains, a target protein, and a linker moiety that operatively couples the multimerized destabilization domain to the target protein.

[0023] In another aspect the invention includes a cell or transgenic organism comprising a nucleic acid sequence encoding for a one or more destabilization domains, a target protein, and a linker moiety that operatively couples the destabilization domain(s) to the target protein. In different embodiments the protein of interest may be coupled to one, two, three, four or more copies of the destabilization domain. In one embodiment, the destabilization domains comprise ubiquitin, or a homolog thereof. In a preferred embodiment the multimerized copies of ubiquitin are not cleavable by ot-NH-ubiquitin protein endoproteases. In one embodiment the ubiquitin domains comprise a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases. In one aspect of this embodiment the mutation represents the mutation of glycine 76 to a larger or more bulky amino acid.

[0024] In another embodiment the invention includes a method for identifying a modulator of an activity, comprising the use of the inventions cells or transgenic organisms. The method includes contacting the cells with a test chemical and detecting the activity of the reporter moiety. Additional claims involve the steps of contacting the cell with an activator of the activity prior to the addition said test chemical, and of in parallel determining the cell viability of the cell in the presence of the test chemical.

[0025] In another embodiment the invention is directed to the test chemical and a pharmaceutical composition comprising a test chemical identified by the methods of the present invention.

[0026] The accompanying drawings, which are incorporated in and form part of the specification, merely illustrate embodiments of the present invention. Together with the remainder of the specification, they are meant to serve to explain certain principles of the invention to those of skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 General schematic overview of parent construct pcDNA3-UbiquitinG76V-Bla. Shown are important coding regions including the ubiquitin-β-lactamase fusion coding region, various promoters and important restriction sites used in the cloning of derivative constructs.

[0028] FIG. 2 TNT in vitro synthesis and degradation experiments with Met, 1, 2, 3 or 4 copies of ubiquitinG76V fused to β-lactamase. The kinetics of turnover in vitro in (A) were determined by chase reactions at 37° C. and products analyzed by SDS-PAGE. The effect of the proteasome inhibitor MG132 at 50 U.M in the TNT synthesis reaction is shown in (B).

[0029] FIG. 3 Turnover in vitro of labeled fusion proteins of uncleavable ubiquitinG76V fused to GFP. TNT synthesis reactions were incubated in chase lysate at 37° C. and products analyzed by SDS-PAGE.

[0030] FIG. 4 Turnover reactions in vitro of labeled uncleavable ubiquitin caspase-3 fusions. TNT reactions were incubated in chase lysate at 37° C. and products analyzed by SDS-PAGE.

[0031] FIG. 5 FACS® analysis of uncleavable ubiquitin β-lactamase fusions. Jurkat cells expressing ubiquitinG76V-Bla fusion proteins were analyzed for β-lactamase expression by flow cytometry. The R5+R6+R7 region was designated as Bla.sup.+ and the percentage of cells in that region is shown in the bar graph.

[0032] FIG. 6 Kinetics of degradation in vivo of ubiquitinG76V-β-lactamase fusion proteins. Jurkat cells expressing the various ubiquitinG76V-Bla fusions were treated with cycloheximide to initiate a chase and aliquots of cells were removed at the indicated times. The cells were lysed and the β-lactamase activity in the lysates was determined by an in vitro reaction using the fluorescent substrate CCF2. The β-lactamase activity was measured by cleavage of CCF2 and represented as emission at 460 nm.

[0033] FIG. 7 Caspase cleavage of 2XUb-DEVD-Bla results in the stabilization of β-lactamase. TNT synthesis reactions were performed to generate labeled fusion proteins of the caspase substrate 2XUb-DEVD-Bla and control 2XUb-DEVA-Bla. In (A), the labeled proteins were incubated with purified caspase-3 to verify that 2XUb-DEVD-Bla can be cleaved by caspase-3 and 2XUb-DEVA-Bla cannot. In (B), the products of the caspase-3 cleavage reactions were incubated with chase extract and samples analyzed by SDS-PAGE.

[0034] FIG. 8 Dose-response curves for an inducer and an inhibitor of caspase activation with Jurkat cells expressing 2XUb-DEVD-Bla. Varying concentrations of antiFas IgM were incubated with 2XUb-DEVD-Bla-expressing Jurkat cells for 6 hours at 37° C. and caspase activity was measured following a cycloheximide chase to clear uncleaved reporters. The cells were loaded with CCF2-AM and β-lactamase activity measured and expressed as a 460/530 nm ratio. Jurkat cells expressing 2XUb-DEVD-Bla were treated with varying concentrations of the caspase inhibitor ZVAD-fmk and then treated with 75 ng/ml antiFas IgM. The cells were incubated for 6 hours at 37° C., cycloheximide for 1 hour at 37° C. and β-lactamase activity measured using CCF2-AM as described above.

[0035] FIG. 9 In vitro cis-cleavage activity of UbG76V-HRV 2A-Bla fusions. Labeled UbiquitinG76V-HRV 2A protease β-lactamase fusions were produced in TNT reactions and then analyzed by SDS-PAGE. (A) shows that the cis-cleavage of HRV-Bla fusions is blocked by mutation of putative catalytic residues (C106 and D35). (B) The TNT reactions were incubated in chase extract to show the selective stabilization of the cleavage product.

[0036] FIG. 10 Rapid degradation of 2XUb-Bla in vitro requires polyubiquitination and proteasome activity. TNT synthesis reactions were incubated in chase extract containing the indicated inhibitors for 20 minutes at 37° C. MG132 and ALLN were present at 50 μM, lactacystin at 10 mM and MeUb at 200 μg/ml.

[0037] FIG. 11 Dose-response curves for proteasome inhibitors on Jurkat cells expressing 2XUb-Bla reporter. Cells were treated with varying concentrations of MG132 or ALLN for 30 minutes and then cycloheximide was added and the cells incubated at 37° C. for one hour. The cells were loaded with CCF2-AM to measure β-lactamase activity as described above.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0038] The techniques and procedures are generally performed according to conventional methods in the art and various general references. (Lakowicz, J. R. Topics in Fluorescence Spectroscopy, (3 volumes) New York: Plenum Press (1991), and Lakowicz, J. R. (1996) Scanning Microsc Suppl. 10 213-24, for fluorescence techniques; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., for molecular biology methods; Cells: A Laboratory Manual, 1st edition (1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., for cell biology methods; Optics Guide 5 Melles Griot® Irvine Calif., and Optical Waveguide Theory, Snyder & Love published by Chapman & Hall for general optical methods, which are incorporated herein by reference which are provided throughout this document).

[0039] "Activity" refers to the enzymatic or non-enzymatic activity capable of modifying an amino acid residue or peptide bond (preferably enzymatic). Such covalent modifications include proteolysis, phosphorylation, dephosphorylation, glycosylation, methylation, sulfation, prenylation and ADP-ribsoylation. The term includes non-covalent modifications including protein-protein interactions, and the binding of allosteric, or other modulators or second messengers such as calcium, or cAMP or inositol phosphates to a polypeptide.

[0040] Amino acid "substitutions" are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

[0041] Ammo acid "insertions" or "deletions" are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. The variation allowed in a particular amino acid sequence may be experimentally determined by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in the gene sequence using recombinant DNA techniques.

[0042] "Animal" as used herein may be defined to include human, domestic (cats, dogs, etc), agricultural (cows, horses, sheep, goats, chicken, fish, etc) or test species (frogs, mice, rats, rabbits, simians, etc).

[0043] "Chimeric" molecules are polynucleotides or polypeptides which are created by combining one or more of nucleotide sequences of this invention (or their parts) with additional nucleic acid sequence(s). Such combined sequences may be introduced into an appropriate vector and expressed to give rise to a chimeric polypeptide which may be expected to be different from the native molecule in one or more of the following characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.

[0044] The terms "cleavage site" or "protease site" refers to the bond cleaved by the protease (e.g. a scissile bond) and typically the surrounding three to four amino acids of either side of the bond.

[0045] "Control elements" or "regulatory sequences" are those non-translated regions of the gene or DNA such as enhancers, promoters, introns and 3' untranslated regions which interact with cellular proteins to carry out replication, transcription, and translation. They may occur as boundary sequences or even split the gene. They function at the molecular level and along with regulatory genes are very important in development, growth, differentiation and aging processes.

[0046] "Corresponds to" refers to a polynucleotide sequence that is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to all or a portion of a reference polypeptide sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA".

[0047] "Derivative" refers to those polypeptides which have been chemically modified by such techniques as ubiquitination, labeling, pegylation (denvatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.

[0048] A "destabilization domain" refers to a protein, polypeptide or amino acid sequence that is capable of modulating the stability of a protein of interest when functionally coupled to the protein of interest. Examples of destabilizing domains include ubiquitin, PEST sequences, cyclin destruction boxes and hydrophobic stretches of amino acids. Preferred destabilization domains include ubiquitin and homologs thereof, particularly those comprising mutations that prevent, or significantly reduce, the cleavage of ubiquitin multimers by α-NH-ubiquitin protein endoproteases. Examples of such mutations include the mutation of glycine 76 to another amino acid, particularly an amino acid selected from the group consisting of Ala, Leu, Ile, Phe, Tyr, Val, Met, Cys, His, Trp, Pro, Arg, Lys, Thr and Ser. Preferred is UbiquitinG76V.

[0049] A "detectable product" is a chemical moiety used for detecting a reporter moiety. They include, but are not limited to, radionuclides, enzymes, fluorescent, chemi-luminescent, or chromogenic agents. Detectable products associate with, establish the presence of, and may allow quantification of a particular nucleic sequence, amino acid sequence or reporter moiety. Preferred detectable products are retained within living cells and provide a fluorescence readout that is compatible with fluorescent activated cell sorting (FACS) analysis.

[0050] The term "engineered protease site" refers to a protease site that has been modified from the naturally existing sequence by at least one amino acid substitution.

[0051] The term "homolog" refers to two sequences or parts thereof, that are greater than, or equal to 85% identical when optimally aligned using the ALIGN program. Homology or sequence identity refers to the following. Two amino acid sequences arc homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 ammo acids in length) are homologous, as this term is used herein, if they have an alignment score of more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., (1972) in Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, 101-110, and Supplement 2 to this volume, pp. 1-10.

[0052] An "inhibitor" is a substance that retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, antagonists and their derivatives.

[0053] "Isolated" refers to material removed from its original environment (e.g. the natural environment if it is naturally occurring), and thus is altered from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be "isolated" because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.

[0054] The term "linker" or "linker moiety" refers to an amino acid, polypeptide or protein sequence that serves to operatively couple a reporter moiety to one or more destabilization domains. Linkers may comprise a single polypeptide chain that covalently couples the reporter moiety to the multimerized destabilization domain. Alternatively the linker may comprise two separate polypeptides. Typically the first polypeptide is covalently coupled to the reporter moiety, and the second polypeptide is covalently coupled to the multimerized destabilization domain. Generally the first and second polypeptides comprising the linker moiety in this embodiment are capable of interacting or associating such that the interaction or association operatively couples the reporter moiety to the multimerized destabilization domain. Preferably the linker moiety is non-cleavable by α-NH-ubiquitin protein endoproteases. Linkers may be of any size.

[0055] The term "modulates" refers to, either the partial or complete, enhancement or inhibition (e.g. attenuation of the rate or efficiency) of an activity or process.

[0056] The term "modulator" refers to a chemical compound (naturally occurring or non-naturally occurring), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian, including human) cells or tissues. Modulators are evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (e.g., agonist, partial antagonist, partial agonist, inverse agonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation-promoting agents, and the like) by inclusion in screening assays described herein. The activity of a modulator may be known, unknown or partially known.

[0057] The term "multimerized destabilization domain" refers to at least two destabilization domains that are linearly coupled together. Preferred multimerized domains are non-cleavable by α-NH-ubiquitin protein endoproteases. The term does not include naturally occurring poly-ubiquitin chains in which the ubiquitin monomers are coupled together via isopeptide bonds attached to the ε-amino group of lysine. The term also does not include naturally occurring multi-ubiquitin genes, are cleavable by α-NH-ubiquitin protein endoproteases to create ubiquitin monomers. The destabilization domains present in the multimerized destabilization domain are typically the same, but need not necessarily be identical.

[0058] "Naturally fluorescent protein" refers to proteins capable of forming a highly fluorescent, intrinsic chromophore either through the cyclization and oxidation of internal amino acids within the protein or via the enzymatic addition of a fluorescent co-factor. Typically such chromophores can be spectrally resolved from weakly fluorescent amino acids such as tryptophan and tyrosine.

[0059] "Naturally occurring" refers to a polypeptide produced by cells which have not been genetically engineered or which have been genetically engineered to produce the same, sequence as that naturally produced. Specifically contemplated are various polypeptides that arise from post-transnational modifications. Such modifications of the polypeptide include but are not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation, proteolytic cleavage and acylation.

[0060] An "oligonucleotide" or "oligomer" is a stretch of nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR), a site directed mutagenesis reaction or a cassette to create a desired sequence element. These short sequences are based on (or designed from) genomic or cDNA sequences and are used to amplify, mutate or create particular sequence elements. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthesized and may also be used as probes.

[0061] An "oligopeptide" is a short stretch of amino acid residues and may be expressed from an oligonucleotide. It may be functionally equivalent to and either the same length as or considerably shorter than a "fragment", "portion", or "segment" of a polypeptide. Such sequences comprise a stretch of amino acid residues of at least about 5 amino acids and often about 17 or more amino acids, typically at least about 9 to 13 amino acids, and of sufficient length to display biologic and/or immunogenic activity.

[0062] The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0063] The term "operably coupled" refers to a juxtaposition wherein the components so described are either directly or indirectly coupled. Examples of directly coupled components include proteins that are translationally fused together. Examples of indirectly coupled components include proteins that can functionally associate either transiently, or persistently, through a binding interaction.

[0064] The term "polynucleotide" refers to a polymeric form of nucleotides of at least 10 bases m length, either ribonucleotides or deoxynucleotides. Modified forms and analogs of either type of nucleotide are also included, as are ribonucleotides or deoxynucleotides linked via novel bonds such as those described in U.S. Pat. No. 5,532,130, European Patent Applications EP 0 839 830, EP 0 742 287, EP 0 285 057 and HP 0 694 559. The term includes single and double stranded forms of nucleotides, or a mixture of single and double stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine, as well as other chemical or enzymatic modifications.

[0065] The term "polypeptide" refers to a amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modification include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins-Structure and Molecular Properties 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Pres. New York, pp. 1-12 (1983).

[0066] A "portion" or "fragment" of a polynucleotide or nucleic acid comprises all or any part of the nucleotide sequence having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb which can be used as a probe. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR of other methods well known in the art. After pretesting to optimize reaction conditions and to eliminate false positives, nucleic acid probes may be used in Southern, northern or in situ hybridizations to determine whether DNA or RNA encoding the protein is present in a biological sample, cell type, tissue, organ or organism.

[0067] "Probes" are nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used m the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA-like technologies.

[0068] The term "recognition motif refers to all or part of a polypeptide sequence recognized by a post-translational modification activity to enable a polypeptide to become modified by that post-translational modification activity. Typically, the affinity of a protein, e.g. enzyme, for the recognition motif is about 1 mM (apparent K_d), preferably a greater affinity of about 10 μM, more preferably, 1 μM or most preferably has an apparent K_d of about 0.1 μM. The term is not meant to be limited to optimal or preferred recognition motifs, but encompasses all sequences that can specifically confer substrate recognition to a peptide. In some embodiments the recognition motif is a phosphorylated recognition motif (e.g., includes a phosphate group), or comprises other post-translationally modified residues.

[0069] "Recombinant nucleotide variants" are polynucleotides that encode a protein. They may be synthesized by making use of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce specific restriction sites or codon usage-specific mutations, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic host system, respectively.

[0070] "Recombinant polypeptide variant" refers to any polypeptide which differs from a naturally occurring polypeptide by amino acid insertions, deletions and/or substitutions, created using recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing characteristics of interest may be found by comparing the sequence of a polypeptide with that of related polypeptides and minimizing the number of amino acid sequence changes made in highly conserved regions.

[0071] A "reporter moiety" includes any protein that directly or indirectly produces a specific detectable product, or cellular phenotype, such as drug resistance that can be used to monitor transcription of a gene. Preferred reporter moieties include proteins with an enzymatic activity that provides enzymatic amplification of gene expression such as alkaline phosphatase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase, peroxidase, β-lactamase, bioluminescent proteins, luciferases and catalytic antibodies. Other reporter moieties include proteins such as naturally fluorescent proteins or homologs thereof, cell surface proteins or the native or modified forms of an endogenous protein to which a specific assay exists or can be developed in the future. Preferred reporter moieties for use in the present invention provide for a fluorescent readout that is compatible with fluorescent activated cell sorting (FACS) analysis.

[0072] A "signal or leader sequence" is a short amino acid sequence which is or can be used, when desired, to direct the polypeptide through a membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.

[0073] A "standard" is a quantitative or qualitative measurement for comparison. Preferably, it is based on a statistically appropriate number of samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles. The samples of a particular standard may be normal or similarly abnormal.

[0074] The term "stringent hybridization conditions", refers to an overnight incubation at 42° C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate and 20 μg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lower stringency); salt conditions, or temperature. For example, lower stringencyconditions include an overnight incubation at 37° C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2P04; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC). Variation in the above conditions may be accomplished through the inclusion and/or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. A polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues would not be included in the definition of a "polynucleotide" since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch, or the complement thereof.

[0075] The term "target" refers to a biochemical entity involved a biological process. Targets are typically proteins that play a useful role in the physiology or biology of an organism. A therapeutic chemical binds to target to alter or modulate its function. As used herein, targets can include cell surface receptors, G-proteins, kinases, ion channels, phopholipases, proteases and other proteins mentioned herein.

[0076] The term "test chemical" refers to a chemical to be tested by one or more screening method(s) of the invention as a putative modulator. A test chemical can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate, a lipid, or a combination thereof. Usually, various predetermined concentrations of test chemicals are used for screening, such as 0.01 micromolar, 1 micromolar and 10 micromolar. Test chemical controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.

[0077] The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage identical to a sequence", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may he conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods selected. The term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage identical to a sequence" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 30 percent sequence identity, preferably at least 50 to 60 percent sequence identity, more usually at least 60 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.

[0078] As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 30 percent sequence identity, preferably at least 40 percent sequence identity, more preferably at least 50 percent sequence identity, and most preferably at least 60 percent sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of ammo acids having aliphatic-hydroxyl side chains is serine and threonine; a group of ammo acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.

[0079] Since the list of technical and scientific terms cannot be all encompassing, any undefined terms shall be construed to have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. Furthermore, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "restriction enzyme" or a "high fidelity enzyme" may include mixtures of such enzymes and any other enzymes fitting the stated criteria, or reference to the method includes reference to one or more methods for obtaining cDNA sequences which will be known to those skilled in the art or will become known to them upon reading this specification.

[0080] Before the present sequences, variants, formulations and methods for making and using the invention are described, it is to be understood that the invention is not to be limited only to the particular sequences, variants, formulations or methods described. The sequences, variants, formulations and methodologies may vary, and the terminology used herein is for the purpose of describing particular embodiments. The terminology and definitions are not intended to be limiting since the scope of protection will ultimately depend upon the claims.

I. Multimerized Destabilization Domains

[0081] Destabilization domains include proteins, protein domains and amino acid sequences that when functionally coupled to a target protein effect a change in the half-life of that protein when expressed in a cell. Examples include PEST domains, stretches of hydrophobic amino acids, phosphorylation dependent degradation signals, cyclin destruction boxes and the addition of ubiquitin domains. Preferred as a destabilization domain is ubiquitin and homologs thereof, particularly mutants or homologs comprising mutations that prevent, or significantly reduce, the cleavage of ubiquitin multimers by α-NH-ubiquitin protein endoproteases. In general, destabilization domains function by causing the target protein to be recognized by one or more elements of the cellular protein degradation apparatus. Once marked for destruction, the protein is actively recruited into the 28S proteasome where the protein is degraded. Within the cell a variety of signals may target a protein for degradation. In some cases a destabilization feature may be revealed in a protein as a result of oxidation, mis-folding or proteolysis. For example, stretches of hydrophobic amino acids are often exposed in denatured or improperly folded proteins thereby targeting them for degradation. Short stretches of hydrophobic amino acids, or hydrophobic domains, also occur in correctly folded proteins and have been identified in proteins with short half lives.

[0082] For example, the Deg 1 domain of yeast mating type transcription factor α2 is a 19 residue element that forms an amphipathic helix with an exposed hydrophobic face, and is responsible for the rapid degradation of this protein (Johnson et al, (1998) Cell 94 217-227). These elements are believed to be recognized by E3 ubiquitin ligases and target the protein to degradation through the ubiquitin system described below.

[0083] PEST domains (regions rich in the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T)) are often located at the C-terminal domains of relatively unstable proteins. (Rogers, et al., (1986) Science 234 (4774) 364-8). A well characterized PEST domain is located in residues 422 to 461 of ornithine decarboxylase, and has been used to successfully destabilize a number of proteins including the green fluorescent protein from Aequorea green fluorescent protein (Li et al. J. Biol. Chem. (1998) 273 (52) 34970-5). Certain PEST sequences are believed to be recognized by the 26S proteasome subunit directly and do not require ubiquitination.

[0084] PEST sequences may also be regulated by phosphorylation, for example multiple phosphorylation within the PEST sequences of the yeast G1 cyclins Cln3 and Cln2 are required for degradation.

[0085] Phosphorylation dependent degradation signals have also been identified in the transcription factors NF-κB and β-catenin, in addition to many cell cycle regulatory proteins such as cyclins. (Ghosh et al., (1998) Ann. Rev. Immunol. 16 225-260; Aberle et al., (1997) EMBO J. 16 3797-3804; Koepp et al., (1999) 97 431-434). These proteins include phosphorylation dependent recognition sequences that bind to one of the growing family of E3 ubiquitin ligases only when the site is phosphorylated. In NF-κB, the binding domain for the E3 ubiquitin ligase comprises the relatively short sequence DS*GLDS*, (SEQ. ID. NO.: 1) where S* denotes phosphoserine. Binding to the E3 ubiquitin ligase does not require a ubiquitination conjugation site in this case.

[0086] The cell-cycle destruction box is a partially conserved 9 amino acid sequence motif usually located approximately 40-50 amino acid residues from the N-terminus of the protein first described for the A and B type cyclins. The consensus destruction box sequence has the general structure as shown in Table 1 below.

TABLE-US-00001 TABLE 1 Consensus destruction box sequence R (A/T) (A) L (G) x (I/V) (G/T) (N) 1 2 3 4 5 6 7 8 9

[0087] Amino acid residues, or combinations of two residues, that appear in parentheses in the above structure occur in more than 50% of known destruction sequences. The residues at positions 1 and 4 are conserved in all destruction boxes. Ubiquitin (SEQ. ED. NO.: 2), a 76 amino acid polypeptide found in all eukaryotic cells, is centrally involved in the mechanism of targeting a protein for degradation by the cell. In general, the covalent attachment of a ubiquitin domain (SEQ. ID. NO.: 2), to a protein represents a primary recognition motif for binding of that protein to the proteasome. The attachment of ubiquitin (SEQ. ID. NO.: 2) to the protein typically occurs after recognition of one or more of the destabilization domains discussed above, or some other destabilizing feature of a protein. Attachment of ubiquitin (SEQ. ED. NO.: 2) occurs via the reversible isopeptide linkage of the carboxy-terminus of ubiquitin (SEQ. ED. NO.: 2) to lysine residues in the target protein. After the addition of the first ubiquitin domain (SEQ. ID. NO.: 2), further ubiquitin moieties (SEQ. ED. NO.: 2) may subsequently be added via free lysine residues in ubiquitin (SEQ. ED. NO.: 2) to create branched poly-ubiquitin chains on the substrate protein. These reactions are catalyzed by a family of enzymes that are often referred to as the ubiquitination complex. Once the target protein comprises one or more copies of ubiquitin (SEQ. ED. NO.: 2) it binds with high affinity to the proteasome where it is degraded. (See generally, Hershko et al., (1998) Annu. Rev. Biochem. 76 425-79; Laney et al, (1999) Cell 97 427-430).

[0088] The ubiquitin gene typically comprises multiple copies of the ubiquitin coding sequence (SEQ. ED. NO.: 2). Individual ubiquitin domains (SEQ. ID. NO.: 2) are post-translationally formed from the poly-ubiquitin gene by cleavage of the expressed protein by specific α-NH-ubiquitin protein endoproteases that are present within all eukaryotic cells. (Jonnalagadda et al, (1989) J. Biol. Chem. 264 10637-10642. The endoproteases will cleave either multiple ubiquitin--ubiquitin chains, or ubiquitin--fusion protein constructs, provided that the last amino acid of the ubiquitin moiety (SEQ. ED. NO.: 2) is glycine. If this last amino acid is mutated to a more bulky amino acid the ubiquitin fusion protein is not cleavable by α-NH-ubiquitin protein endoproteases.

[0089] The present inventors have recognized for the first time that the creation of multiple ubiquitin fusion proteins that are not cleavable by the α-NH-ubiquitin protein endoproteases provides for a facile and tunable method of regulating protein stability. This invention has many important applications for developing novel assays for intracellular activities, and as a regulatable method of coordinately controlling protein concentrations within the cell.

II. Reporter Moieties

[0090] Enzymatic reporter moieties include any protein capable of catalyzing the creation of a detectable product. Specific examples include alkaline phosphatase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase, peroxidase, β-lactamase, catalytic antibodies, luciferases and other bioluminescent proteins.

[0091] Alkaline phosphatase, including human placental and calf intestinal alkaline phosphatase (for example, GenBank Accession # U89937), can be measured using colonmetric, fluorescent and chemiluminescent substrates. (Berger, J., et al. (1988) Gene 66 1-10; Kain, S. R. (1997) Methods. Mol. Biol. 63 49-60) Alkaline phosphatase is widely used in transcriptional assays, typically by measuring secreted alkaline phosphatase (SEAP).

[0092] β-galactosidase (β-Gal) the gene product of the bacterial gene LacZ, is also widely used as a reporter gene for transcriptional analysis and may be assayed via histochemical, fluorescent or chemiluminescent substrates, either within intact, or permeabilized cells. (See, U.S. Pat. No. 5,070,012, issued Dec. 3, 1991 to Nolan et al. and Bronstein, I., et al, (1989) J. Chemilum. Biolum. 99-111).

[0093] β-glucuronidase (GUS) is widely used for transcriptional analysis in higher plants and may also be assayed using a variety of histochemical and fluorescent substrates. (See generally U.S. Pat. No. 5,599,670, issued Feb. 4, 1997 to Jefferson).

[0094] Chloramphenicol acetyltransferase (CAT), encoded by the bacterial Tn9 gene, is widely used for transcriptional assays and is traditionally measured using a radioisotopic assay in cell extracts (See Gorman et al, (1982) 2 1044-51).

[0095] Catalytic antibodies are also amenable for use as reporter genes, if the reaction catalyzed by the antibody results in the formation of a detectable product. Examples include the aldolase specific antibodies 38C2 and 33F12 that catalyze the synthesis of novel fluorogenic retro-aldol reactions (List et al., (1998) Proc. Natl. Acad. Sci USA 95 15351-15355). Typical antibody substrates are cell permeant nonpolar organic molecules that are not substrates for the natural enzymes and are thus good markers of enzyme activity.

β-Lactamases

[0096] A large number of β-lactamases have been isolated and characterized, all of which would be suitable for use in accordance with the present method. Initially, β-lactamases were divided into different classes (I through V) on the basis of their substrate and inhibitor profiles and their molecular weight (Richmond, M. H. and Sykes, R. B., (1973) Adv. Microb. Physiol. 9 31-88). More recently, a classification system based on amino acid and nucleotide sequence has been introduced (Ambler, R. P., (1980) Phil. Trans. R. Soc. Lond. [Ser. B.] 289 321-331). Class A β-lactamases possess a serine in the active site and have an approximate weight of 29 kd. This class contains the plasmid mediated TEM β-lactamases such as the RTEM enzyme of pBR322. Class B β-lactamases have an active-site zinc bound to a cysteine residue. Class C enzymes have an active site serine and a molecular weight of approximately 39 kd, but have no amino acid homology to the class A enzymes.

[0097] The coding regions of an exemplary β-lactamase employed in the methods described herein include SEQ. ED. NOs: 3 through 7. Nucleic acids encoding proteins with β-lactamase activity can be obtained by methods known in the art, for example, by polymerase chain reaction of cDNA using primers based on a DNA sequence in SEQ. ID. NO.: 3. PCR methods are described in, for example, U.S. Pat. No. 4,683,195; Mullis et al. (1987) Cold Spring Harbor Symp. Quant. Biol. 51 263; and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).

[0098] Preferably, beta-lactamase polynucleotides encode an intracellular-form of a protein with beta-lactamase activity that lacks a functional signal sequence. This provides the advantage of trapping the normally secreted beta-lactamase protein within the cell, which enhances the signal to noise ratio of the signal associated with beta-lactamase activity, and enables the individual cells to be FACS® sorted. For example, in any of the polypeptides of SEQ. ID. NO.: 3-7, the signal sequence has been replaced with the amino acids Met-Ser. Accordingly, upon expression, beta-lactamase activity remains within the cell. For expression in mammalian cells it is preferable to use beta-lactamase polynucleotides with nucleotide sequences preferred by mammalian cells. In some applications secreted forms of beta-lactamase can be used with the methods described herein.

[0099] A variety of colonmetric and fluorescent substrates of β-lactamase are available. Fluorescent substrates include those capable of changes, either individually or in combination, of total fluorescence, excitation or emission spectra or fluorescence resonance energy transfer (FRET), for example those described in U.S. Pat. No. 5,741,657, issued Apr. 21, 1998, and U.S. Pat. No. 5,955,604, issued Sep. 22, 1999. Any membrane permanent β-lactamase substrate capable of being measured inside the cell after cleavage can be used in the methods and compositions of the invention. Membrane permanent β-lactamase substrates will not require permeablizing eukaryotic cells either by hypotonic shock or by electroporation. Generally, such non-specific pore forming methods are not desirable to use in eukaryotic cells because such methods injure the cells, thereby decreasing viability and introducing additional variables into the screening assay (such as loss of ionic and biological contents of the shocked or electroplated cells). Such methods can be used in cells with cell walls or membranes that significantly prevent or retard the diffusion of such substrates. Preferably, the membrane permeant β-lactamase substrates are transformed in the cell into a β-lactamase substrate of reduced membrane permeability (usually at least five-fold less permeable) or that is membrane impermeant. Transformation inside the cell can occur via intracellular enzymes (e.g. esterases) or intracellular metabolites or organic molecules (e.g. sulfhydryl groups).

Bioluminescent Proteins

[0100] Preferred bioluminescent proteins include firefly, bacterial or click beetle luciferases, aequorins and other photoproteins, for example as described in U.S. Pat. No. 5,221,623, issued Jun. 22, 1989 to Thompson et al., U.S. Pat. No. 5,683,888 issued Nov. 4, 1997 to Campbell; U.S. Pat. No. 5,674,713 issued Sep. 7, 1997 to DeLuca et al, U.S. Pat. No. 5,650,289 issued Jul. 22, 1997 to Wood and U.S. Pat. No. 5,843,746 issued Dec. 1, 1998 to Tatsumi et al. Particularly preferred are bioluminescent proteins isolated from the ostracod Cypridina (or Vargula) hilgendorfii. (Johnson and Shimomura, (1978) Methods Hn/.ymol 57 331-364; Thompson, Nagata & Tsuji (1989) Proc. Natl. Acad. Sci. USA 86, 6567-6571).

[0101] Beyond the availability of bioluminescent proteins (luciferases) isolated directly from the light organs of beetles, cDNAs encoding luciferases of several beetle species (including, among others, the luciferase of P. pyralis (firefly), the four luciferase isozymes of P. plagiophthalamus (click beetle), the luciferase of L. cruciata (firefly) and the luciferase of L. lateralis) (deWet et al., (1987) Molec. Cell. Biol. 7, 725-737; Masuda et al, (1989) Gene 77, 265-270; Wood et al, (1989) Science 244, 700-702; European Patent Application Publication No. 0 353 464) are available. Further, the cDNAs encoding luciferases of any other beetle species, which make bioluminescent proteins, are readily obtainable by the skilled using known techniques (de Wet et al. (1986) Meth. Enzymol. B3, 3-14; Wood et al, (1989) Science 244, 700-702).

[0102] Most firefly and click beetle luciferases are ATP- and magnesium dependent and require oxygen for light production. Typically light emission from these enzymes exhibits a rapid burst in intensity followed by a rapid decrease in the first few seconds, followed by a significantly slower sustained light emission. Relatively sustained light output at high rates has been accomplished in these systems by inclusion of coenzyme A, dithiothreitol and other reducing agents that reduce product inhibition and slows inactivation of the luciferase that occurs during catalysis of the light producing reaction, as described in U.S. Pat. No. 5,641,641, issued Jun. 24, 1997, and U.S. Pat. No. 5,650,289, issued Jul. 22, 1997. Such stable light emitting systems are preferred for use in the present invention.

[0103] Particularly preferred bioluminescent proteins are those derived from the ostracod Cypridina (or Vargula) hilgendorfii. The Cypridina luciferase (GenBank accession no. U89490) uses no cofactors other than water and oxygen, and its luminescent reaction proceeds optimally at pH 7.2 and physiological salt concentrations, (Shimomura, O., Johnson, F. H. and Saiga, Y. (1961) J. Cell. Comp. Physiol. 58 113-124). By comparison, firefly luciferase has optimal activity at low ionic strength, alkaline pH and reducing conditions, that are typically quite different to those usually found within mammalian cells. Because Cypridina luciferase has a turnover number of 1600 min^-1 and a quantum yield of 0.29, (Shimomura, O. & Johnson, F. H. and Masugi, T. (1969) Science 164 1299-1300; Shimomura, O. & Johnson, F. H. (1970) Photochem. Photobiol. 12 291-295), the Cypridina luciferase produces a specific photon flux exceeding that of the optimized firefly system by a factor of at least 50 (Miesenbock and Rothman, (1997) Proc. Natl. Acad. Sci. USA 94 3402-3407).

Naturally Fluorescent Proteins

[0104] Another preferred class of embodiments of the reporter moiety includes naturally fluorescent proteins such as the Green Fluorescent Protein (GFP) of Aequorea victoria (Tsien, R. Y. (1998) Annu. Rev. Biochem. 67 509-44). Because the entire fluorophore and peptide of a naturally fluorescent protein can be expressed within intact living cells without the addition of other co-factors or fluorophores, optical probes comprising such proteins as the reporter moiety provide the ability to monitor activities, within defined cell populations, tissues or in an entire transgenic organism. For example, by the use of cell type specific promoters and subcellular targeting motifs, it is possible to selectively target the probe to a discrete location to enable highly spatially defined measurements.

[0105] Naturally fluorescent proteins have been isolated and cloned from a number of marine species including the sea pansies Renilla reniformis, R. kollikeri and R. mullerei and from the sea pens Ptilosarcus, Stylatula and Acanthoptilum, as well as from the Pacific Northwest jellyfish, Aequorea victoria; Szent-Gyorgyi et al. (SPIE conference 1999); D. C. Prasher et al, (1992) Gene, 111:229-233 and several species of coral (Matz et al. (1999). Nature Biotechnology 17 969-973. These proteins are capable of forming a highly fluorescent, intrinsic chromophore through the cyclization and oxidation of internal amino acids within the protein that can be spectrally resolved from weakly fluorescent amino acids such as tryptophan and tyrosine.

[0106] Additionally naturally fluorescent proteins have also been observed in other organisms, although in most cases these require the addition of some exogenous t actor to enable fluorescence development. For example, the cloning and expression of yellow fluorescent protein from Vibrio fischeri strain Y-1 has been described by T. O. Baldwin et al., Biochemistry (1990) 29 5509-15. This protein requires flavins as fluorescent co-factors. The cloning of Peridinin-chlorophyll a binding protein from the dinoflagellate Symbiodinium sp. was described by B. J. Morris et al., (1994) Plant Molecular Biology, 24 613:11. One useful aspect of this protein is that it fluoresces in red. The cloning of phycobiliproteins from marine cyanobacteria such as Synechococcus, e.g., phycoerythrin and phycocyanin, is described in S. M. Wilbanks et al. (1993) J. Biol. Chem. 268 1226-35. These proteins require phycobilins as fluorescent co-factors, whose insertion into the proteins involves auxiliary enzymes. The proteins fluoresce at yellow to red wavelengths.

[0107] A variety of mutants of the GFP from Aequorea victoria have been created that have distinct spectral properties, improved brightness and enhanced expression and folding in mammalian cells compared to the native GFP, (SEQ. ID. NO.: 8), Table 2. (Green Fluorescent Proteins, Chapter 2, pages 19 to 47, edited Sullivan and Kay, Academic Press, U.S. Pat. No. 5,625,048 to Tsien et al., issued Apr. 29, 1997; U.S. Pat. No. 5,777,079 to Tsien et al, issued Jul. 7, 1998; and U.S. Pat. No. 5,804,387 to Cormack et al., issued Sep. 8, 1998). In many cases these functional engineered fluorescent proteins have superior spectral properties to wild-type Aequorea GFP and are preferred for use as reporter moieties in the present invention.

TABLE-US-00002 TABLE 2 Aequorea Fluorescent Proteins Quantum Yield (Φ) & Relative Sensitivity To Low Common Molar Excitation & Fluorescence At _PH Mutations Name Extinction (ε) Emission Max 37° C. % max F at pH 6 S65T type S65T. S72A, Emerald Φ = 0.68 487 100 91 N149K, (SEQ. ID. ε = 57,500 509 M153T, I167T NO.: 28) F64L, S65T, Φ = 0.58 488 54 43 VI63 A ε = 42,000 511 F64L.S65T EGFP Φ = 0.60 488 20 57 ε = 55,900 507 S65T Φ = 0.64 489 12 56 ε = 52,000 511 Y66H type F64L, Y66H, P4-3E Φ = 0.27 384 100 N.D. Y145F, V163A ε = 22,000 448 F64L, Y66H, Φ = 0.26 383 82 57 Y145F ε = 26,300 447 Y66H. Y145F P4-3 Φ = 0.3 382 51 64 ε = 22,300 446 Y66H BFP Φ = 0.24 384448 15 59 ε = 21,000 Y66W type S65A. Y66W, W1C Φ = 0.39 435 100 82 S72A, N146I, ε = 21,200 495 M153T, V163A F64L, S65T, W1B Φ = 0.4 434 452 80 71 Y66W, N146I, ε = 32,500 476 (505) M153T, V163A Y66W, N146I, hW7 Φ = 0.42 434 452 61 88 M153T, V163A ε = 23,900 476 (505) Y66W 436 N.D. N.D. 485 T203Y type S65G, S72A, Topaz Φ = 0.60 514 100 14 K79R, T203Y ε = 94,500 527 S65G, V68L, IOC Φ = 0.61 514 58 21 S72A, T203Y ε = 83,400 527 S65G, V68L, hlOC+ Φ = 0.71 516 50 54 Q69K, S72A, ε = 62,000 529 T203Y S65G, S72A, Φ = 0.78 508 12 30 T203H ε = 48,500 518 S65G, S72A Φ = 0.70 512 6 28 T203F ε = 65,500 522 T203I type T203I, S72A, Sapphire Φ = 0.64 395 100 90 Y145F ε = 29,000 511 T203I T202F H9 Φ = 0.6 395 13 80 ε = 20,000 511

[0108] Non Aequorea, naturally fluorescent proteins, for example Anthozoan fluorescent proteins, and functional engineered homologs thereof, are also suitable for use in the present invention including those shown in Table 3 below.

TABLE-US-00003 TABLE 3 Anthozoa Fluorescent Proteins Quantum Yield (Φ) & Excitation & Protein Molar Emission Relative Species Name Extinction (ε) Max Brightness SEQ. ID. NO.: Anemonia amFP486 Φ = 0.24 458 0.43 SEQ. ID. NO.: 9 majano ε = 40,000 486 Zoanthus sp zFP506 Φ = 0.63 496,506 1.02 SEQ. ID. NO.: 10 ε = 35,600 zFP538 Φ = 0.42 528,538 0.38 SEQ. ID. NO.: 11 ε = 20,200 Discosoma dsFP483 Φ = 0.46 443 0.5 SEQ. ID. NO.: 12 striata ε = 23,900 483 Discosoma sp drFP583 Φ = 0.23 558 0.24 SEQ. ID. NO.: 13 red" ε = 22,500 583 Clavularia sp CFP484 Φ = 0.48 456 0.77 SEQ. ID. NO.: 14 ε = 35,300 484

III Linker Moieties

[0109] Generally linker moieties for measuring a post-translational activity encompass a post-translational recognition motif that contains a residue that, when modified, modulates the coupling of the reporter moiety to the multimerized destabilization domain, thus effecting a change in the stability of the reporter moiety. Typically, for measuring proteases, such linkers contain a single scissile bond (bond that is cleaved within the substrate) for a specific protease and preserve "the native function and activity of the reporter moiety and destabilization domains in the intact fusion protein. The design and size of peptide sequences for specific constructs, is dependent upon the application for which the optical probe is to be used. For example, for most applications, the peptide linker separating the reporter moiety and the multimerized destabilization domains will typically be in the range of 5 to 50 amino acids in length, preferably 10 to 25 amino acids in length, or more preferably 10 to 15 amino acids in length. For certain applications, the peptide may be significantly larger, up to and including entire protein domains, for example 50 to 100 ammo acids in length. Smaller peptides, in the range of 5 to 50 amino acids may also be used. Typically the protease site may be located at any position within the linker with respect to the reporter moiety and destabilization domains.

[0110] In one embodiment the linker comprises a single polypeptide chain that covalently couples the destabilization domains to the reporter moiety. Typically in this embodiment, the linker will comprise a post-translational recognition motif such as a protease recognition motif. Cleavage of the linker by the protease at the cleavage site results in uncoupling of the multimerized destabilization domains from the reporter moiety resulting in a modulation in the stability of the reporter moiety. An important feature of the linker is that it does not contain a protease recognition site for α-NH-ubiquitin protein endoproteases that would otherwise result in the post-translational processing of the construct irrespective of the presence or absence of the target post-translational activity. Any cleavage activity capable of hydrolyzing the linker moiety may be assayed with this embodiment of the present invention, provided it does not also cleave the reporter moiety thereby directly modulating its function.

[0111] In another aspect of this method, the linker may comprise distinct post-translational recognition motifs and cleavage sites for example, a phosphorylation site and a protease cleavage site, as described in commonly owned U.S. patent application Ser. No. 09/306,542 filed May 5, 1999. In this case, post-translational modification of the linker results in the modulation of the rate and efficiency of cleavage of the modified linker compared to the non-modified linker. This approach enables the present method to be used to detect a broad range of post translational activities.

[0112] In some embodiments, the linker functions to couple a target protein to one or more destabilization domains for the purpose of regulating the concentration of the target protein in the cell. In this case the linker need not contain a protease cleavage site, and may be significantly smaller, in the order of about 1 to 10 amino acids in length.

[0113] In another aspect, the linker may comprise two separate polypeptide chains that are capable of interacting with each other to functionally couple the multimerized destabilization domains to the reporter gene. This approach enables an additional range of post-translational activities to be assayed. In this embodiment, one polypeptide chain is typically covalently coupled to the multimerized destabilization domain, and a separate polypeptide chain is covalently coupled to the reporter moiety. (FIG. 1) Binding of the first polypeptide chain to the second polypeptide chain results in coupling of the destabilization domain to reporter moiety resulting in a modulation of the stability of the reporter moiety. This approach thus enables the identification and detection of protein-protein interactions between defined proteins as well as the ability to detect post-translational modifications that influence these protein-protein interactions.

[0114] Examples of suitable interaction domains include protein-protein interaction domains such as SH2, SH3, PDZ, 14-3-3, WW and PTB domains. Other interaction domains are described in for example, the database of interacting proteins available on the web at http://www.doe-mbi.ucla.edu.

[0115] To identify and characterize the interaction of two test proteins, the method would typically involve 1) the creation of a first fusion protein comprising the first test protein coupled to the reporter moiety, and a second fusion protein comprising the second test protein coupled to the multimerized destabilization domain construct. 2) The introduction of the test protein fusion proteins alone in to control cells, and in combination into test cells. 3) The measurement of the stability of the reporter moiety in the control cells and test cells. 4) Comparison of the stability of the reporter moiety in the control cells, compared to the stability of the reporter moiety in the test cells. If the cell expressing both test fusion proteins exhibits a reporter moiety with a significantly altered stability (or level of expression) compared to the control cells, then the results indicate that the two proteins do interact under the experimental conditions chosen. Conversely if the stability's of the reporter moieties in the control cells, and in the test cells are the same, then the results indicate that the proteins probably don't interact strongly under the test conditions.

[0116] The method also enables the detection and characterization of stimuli (such as receptor stimulation) that cause two proteins to alter their degree of interaction. In this case, a cell line is created that expresses the first and second fusion proteins, as described above, comprising interaction domains that exhibit, or are believed to exhibit post-translational regulated interactions. For example, post-translational modification by phosphorylation of serine or threonine residues can modulate 14-3-3 domain interactions, tyrosine phosphorylation can influence SH2 domain interactions, the redox state can influence disulfide bond formation. The cell line is then exposed to a test stimulus to determine whether the stimulus regulates the interaction of the two proteins. If the stimulus does regulate the interaction of the two proteins, then this will result in the coupling of the multimerized destabilization domain fusion protein to the reporter moiety fusion protein, subsequently resulting in a modulation of the stability of the reporter moiety in the treated cells, compared to the non-treated cells.

[0117] The invention is also readily amenable to identifying new protein-protein interactions. For example, where a first protein is known, but the protein(s) with which it interacts are unknown. In this case, a first fusion protein is made between the first protein and the reporter moiety (or destabilization domain) and cloned into a suitable expression vector. Second, a library of test proteins, for example isolated from a cDNA expression library, is fused in frame to the multimerized destabilization domains (or reporter moiety) and subcloned into a second expression vector. Typically the first fusion protein would be then be introduced into a population of test cells and single clones identified that stably expressed the reporter moiety. The library of test proteins (typically in the form of expression vectors) would be introduced into the clonal cells, stably expressing the first fusion protein. The resulting transformed cells would then be screened to identify cells with altered expression of the reporter moiety fusion compared to the control cells. Suitable clones expressing the reporter moieties with modulated stability, (i.e., reduced levels of the reporter moiety) may then be identified, isolated and characterized, for example by fluorescence activated cell sorting (FACS®). Those library members that display reporter moieties with larger relative changes in expression level may then be identified by the degree to which the stability of the reporter moiety is altered for each library member after exposure to the library of test fusion proteins.

IV Methods of Use

[0118] Introduction of Constructs into Cells

[0119] Typically the constructs of the present invention will be introduced and expressed in target cells via the use of standard molecular biology techniques known in the art. Another approach involves the use of membrane translocating sequences, as described in U.S. Pat. No. 5,807,746, issued Sep. 15, 1998 to Lin et al. to introduce the protein constructs into cells.

[0120] Nucleic acids may also be used to transfect cells with sequences coding for expression of the multimerized destabilization domain, linker and reporter moiety. Generally these will be in the form of an expression vector including expression control sequences operatively linked to a nucleotide sequence coding for expression of the polypeptide. As used, the term "nucleotide sequence coding for expression of a polypeptide refers to a sequence that, upon transcription and translation of mRNA, produces the polypeptide. This can include sequences containing, e.g., introns. As used herein, the term "expression control sequences" refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, ERES sequences (internal ribosome entry site) maintenance of the correct reading frame of that gene to permit proper translation of the mRNA, and stop codons.

[0121] Methods that are well known to those skilled in the art can be used to construct expression vectors containing the multimerized destabilization domain, linker, reporter moiety construct. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. (See, for example, the techniques described in Maniatis, et al., (1989) Cold Spring Harbor Laboratory, N.Y.). Many commercially available expression vectors are available from a variety of sources including Clontech (Palo Alto, Calif.), Stratagene (San Diego, Calif.) and Invitrogen (San Diego, Calif.) as well as and many other commercial sources.

[0122] A contemplated version of the method is to use inducible controlling nucleotide sequences to produce a sudden increase in the expression of the reporter moiety, linker and multimerized destabilization domain construct e.g., by inducing expression of the construct. Example inducible systems include the tetracycline inducible system first described by Bujard and colleagues (Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89 5547-5551, Gossen et al. (1995) Science 268 1766-1769) and described in U.S. Pat. No. 5,464,758.

[0123] Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells that are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaC₂ method by procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.

[0124] When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be co-transfected with DNA sequences encoding the fusion polypeptide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use an eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Preferably, an eukaryotic host is utilized as the host cell as described herein.

[0125] The construction of expression vectors and the expression of genes in transfected cells involve the use of molecular cloning techniques also well known in the art. Sambrook et al., (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., (Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (most recent Supplement). Nucleic acids used to transfect cells with sequences coding for expression of the polypeptide of interest generally will be in the form of an expression vector including expression control sequences operatively linked to a nucleotide sequence coding for expression of the polypeptide comprising the optical probe.

Assays for Post-Translational Activities

[0126] In one class of embodiments, the present invention can be used to measure post-translational activities, such as proteolysis, phosphorylation, dephosphorylation, glycosylation, methylation, sulfation, prenylation, disulfide bond formation and ADP-ribsoylation within cells.

[0127] The method generally involves the expression within, or introduction into a cell of a reporter moiety that is functionally coupled to one or more destabilizing domains via a linker. The linker typically contains a recognition motif that is specific for the post-translational activity to be assayed. Modification of the linker by the post-translational activity, results in uncoupling of the reporter moiety from the destabilizing domain resulting in a modulation in the stability of the reporter moiety. The level of activity within a sample is sensed by a measurable change in the level of the reporter moiety, for example by detecting at least one optical property of the reporter moiety, or by detecting at least one optical property of detectable product of the reporter moiety.

[0128] To measure protease activity, it is typically desirable to provide an expression vector in which the expressed fusion gene product comprises a reporter moiety covalently linked to the multimerized destabilization moieties via a single amino acid chain. Thus under these conditions the expressed construct is destabilized until acted upon by the target protease. Upon proteolysis, the cleaved reporter moiety exhibits significantly increased stability, resulting in its steady state accumulation within the cell to a higher level.

[0129] The choice of reporter moiety depends in part on the cellular system in which the assays are conducted, and the sensitivity and detection means at hand. For mammalian cells, the β-lactamase, β-galactosidase, and naturally fluorescent protein based reporter genes provide for intracellular fluorescent measurements, which are preferred. Preferred reporter moieties for luminescent readouts include luciferase and other bioluminescent protein based reporters. In plant studies, preferred reporters include β-glucuronidase and luciferase. For transgenic applications in whole animals or intact tissue samples, naturally fluorescent proteins are preferred because the reporter does not require the addition of any substrates or co-factors in order to produce a detectable product. For applications were high sensitivity is required, for example because the target activity has a low turnover number, enzymatic reporter moieties are preferred because they provide enzymatic amplification. That is, each reporter moiety is capable of generating hundreds or thousands of detectable products per minute. By comparison a non enzymatic reporter, such as a naturally fluorescent protein, provides for little signal amplification.

[0130] The choice of the multimerized destabilization domain, and the number of copies of the destabilization domain to use are also dependent on the reporter moiety and type of activity being measured. Preferred destabilization domains include, those based on ubiquitin (SEQ. ID. NO.: 2) and mutants and homologs thereof. Particularly preferred are mutants or homologs of ubiquitin (SEQ. ID. NO.: 2) comprising mutations that prevent, or significantly reduce, the cleavage of ubiquitin multimers by α-NH-ubiquitin protein endoproteases.

[0131] To establish the optimal number of destabilization domains one would generally start by evaluating a construct containing three copies of the destabilization domain. Depending upon the results, one would either increase or decrease the number of copies of destabilization domains. Generally one would increase the number of copies of the destabilization domain if the steady state levels of the non-protease treated samples were too high (too little degradation), and decrease the number of copies of the destabilization domain if the steady state level of the non-protease treated samples were too low (too much degradation). If the target protein was subject to excessive degradation, the steady state level of the target protein may be too low to provide for effective cleavage by the protease, particularly if that protease exhibits a relatively low affinity for that substrate.

[0132] An important advantage of the present invention is the ability to titrate the degree of destabilization, and therefore the steady state concentration, of the target protein in the cell. Since the destabilized, unmodified sensor represents the substrate for the target activity, it is preferable to provide the substrate at a physiologically relevant concentration within the cell while retaining the appropriate turnover characteristics for each individual reporter molecule.

[0133] For assays measuring protease activity, the linker generally comprises a protease recognition motif within its sequence. The protease recognition motif may be placed anywhere within the linker moiety, but is conveniently placed close to the center of the linker unless there are steric, or other reasons, to position the recognition motif at a specific location. Typically, the recognition motif will provide for relatively specific recognition of the sequence by the target protease. In some cases it may be preferable for the linker to contain a second "control" protease site for a known protease for use as a positive control.

[0134] The expression vector will normally direct expression of the sensor to the cytosol of the cell, although other cellular compartments, such as the plasma membrane are also practical. Once the expression vector is introduced in a population of cells, the cells are typically screened for reporter moiety expression level in the absence of the target protease. This can be achieved by FACS®, after addition of appropriate substrates for the reporter moieties (if required). While cells may be selected for varying levels of expression of the reporter moiety within the population of cells, observations to date suggest that cells exhibiting somewhat lower levels of reporter moiety are superior to those that initially exhibit high levels of reporter moieties under these conditions. Cells may also be selected via antibiotic resistance to provide for stable cell lines.

[0135] Once isolated and characterized, the resulting cell line represents a living sensor for the activation or expression of the target protease that enables the identification and screening of compounds that modulate the activation of the target protease. Importantly these determinations can be completed within the living cell where other issues such as membrane permeability, specificity and toxicity may be directly assessed.

[0136] In most cases, it will be preferable to start with a cell line that does not normally express high levels of the active target protease. However if this is not possible, then the initial evaluation of the cell lines may be modified in order to screen for cells initially exhibiting high levels of reporter moiety expression. For example, by using an inhibitor of the reporter moiety to inhibit basal reporter gene activity, (as discussed below). In general any types of cells may be used with the present invention, including animal, plant, insect, yeast and other eukaryotic cells or prokaryotic cells.

[0137] In whole cell studies it may be desirable to add an inhibitor of protein synthesis such as cycloheximide in order to reduce the steady state level of the destabilized reporter moiety in the cell immediately prior to the measurement of reporter activity. This approach has the advantage of improving the dynamic range of the assay because in the absence of new protein synthesis, uncleaned and therefore destabilized reporter moieties are destroyed by targeting to the proteasome leaving the cleaved and stabilized reporters intact within the cell. (i.e. the background is reduced). This results in a larger net difference in reporter moiety activity in cells containing a suitable protease compared with those lacking a suitable protease. Typically for such uses, cycloheximide is added to cell in the range of 10 to 150 μg/ml cycloheximide, preferably 50 to 100 μg/ml. Generally cells are pretreated with an appropriate stimulus to activate the target protease, and then cycloheximide is added one to two hours prior to the addition of suitable substrates for the reporter moiety.

[0138] In another aspect of this method, it sometimes may also be desirable to add an inhibitor of the enzymatic reporter moiety to reduce the activity of the reporter moiety prior to compound addition in screening applications. For example, in order to screen for inhibitors of a constitutively active protease, such inhibitors of reporter activity can be used to eliminate the pool of cleaved and stabilized reporter prior to adding compound, in effect zeroing out the cells to begin the experiment. This approach also has the advantage that the actual concentration of destabilized substrate molecules is not reduced in the cell, so that the protein substrate can be effectively degraded by the target protease. Example inhibitors include clavulanic acid for the β-lactamase reporter gene (see commonly owned U.S. patent application Ser. No. 09/067,612 filed Apr. 28, 1999) and phenylethyl-β-D-thiogalactoside for β-galactosidase (see Fiering et al., (1991) Cytometry 12 291-301). These membrane permeable inhibitors may be added prior to, simultaneously with, or after exposure of the cells to an inhibitor of protein synthesis.

[0139] To measure the degree of protein-protein interaction between two defined test proteins, it is typically desirable to separately couple one protein to one or more destabilization domains, and the second protein to the reporter moiety, and then express both fusion proteins in a test cell. This could be achieved for example by transfecting a cell with two compatible expression vectors. In one expression vector, the expressed fusion protein typically comprises a reporter moiety coupled to the first test protein, and in the second expression vector, the expressed fusion protein typically comprises the second test protein, coupled to one or more destabilization domains.

[0140] If the first polypeptide fusion protein binds to the second polypeptide fusion protein then the destabilization domain(s) are effectively coupled to the reporter moiety resulting in a modulation of its stability. Thus the relative degree of destabilization of the reporter moiety is a direct indicator of the extent to which the proteins physically interact. Typically this can be accomplished by determining the stability of the reporter moiety in a cell expressing both proteins compared to a control cell, expressing the reporter moiety fusion protein alone. If the cell expressing both constructs exhibits a reporter moiety with a significantly altered stability compared to the control cell, the results indicate that the two proteins are interacting when co-expressed within the cell.

[0141] The choice and selection of the appropriate reporter moiety and destabilization domain are determined by the same issues of sensitivity and ease of detection discussed above. Preferred reporter moieties include β-lactamase and naturally fluorescent proteins. Preferred destabilization domains include those based on ubiquitin (SEQ. ID. NO.: 2), and mutants and functional homologs thereof. Particularly preferred are mutants or homologs of ubiquitin (SEQ. ID. NO.: 2) comprising mutations that prevent, or significantly reduce, the cleavage of ubiquitin multimers by α-NH-ubiquitin protein endoproteases.

[0142] The choice of the number of copies of the destabilization domain is dependent on the affinity of the target interaction to be measured, and the degree of destabilization exerted on the reporter moiety when the proteins are associated. In many cases, the affinity of the interaction will not be known and it will be necessary to evaluate a range of multimerized constructs in order to identify the optimal assay characteristics. Ideally a multimerized construct will be selected in which both the first test protein and the second test protein are present at physiologically relevant concentrations. One way to achieve this result may be to couple both the first test protein and the second test protein with at least one ubiquitin (SEQ. ID. NO.: 2) domain. Under these circumstances both proteins are slowly degraded when separated, but more rapidly degraded when complexed together.

Induction and Regulation of Expression Levels of Target Proteins

[0143] In another embodiment, the invention provides for a generalized way of coordinately regulating the cellular concentration of a plurality of target proteins in a cell, or transgenic organism. In this method, the target proteins are operatively coupled to a multimerized destabilization domain via a linker. By varying the number of destabilization domains present in the multimerized destabilization domain, it is possible to titrate the degree of destabilization, and therefore the steady state concentration of the target protein within the cell or transgenic organism. Thus using this approach it is possible to reproducibly vary the relative stoichometery, as well as, the level of expression, of one or more target proteins.

[0144] In some embodiments the linker may comprise about 1 to 10 amino acids. Typically the linker is non-cleavable by α-NH-ubiquitin protein endoproteases.

[0145] In one embodiment the linker may contain a non-naturally occurring protease cleavage site (in that cell type), such that cleavage of the linker by the protease results in uncoupling of the target protein from the multimerized destabilization domain hence creating an increase in the stability and concentration of the target protein after protease digestion. In one aspect of this method, regulation of the activity of the protease can be achieved via regulating the concentration and exposure of the cell to an inhibitor of the protease.

[0146] This approach enables the coordinate regulation of the intracellular concentration of a number of target proteins that contain the same protease recognition sites in their linker moieties, simultaneously within a cell. The approach is particularly well suited for the engineering of organisms or cells where multiple proteins need to be induced and expressed in order to create the desired effect, for example for regulating a multi-step metabolic or signal transduction pathway.

[0147] In one embodiment the protease is a non-naturally occurring protease in the host cell, which recognizes a relatively rare recognition motif in the linker moiety, for example, including proteases such as Factor Xa (EC 3.4.21.6), Entrokinase (EC 3.4.21.9) and IgA protease (EC 3.4.21.72). Proteases that recognize defined sequences of at least 4, or preferably at least 5 or more preferably about 6 amino acid residues, are generally preferred. Viral proteases, such as a CMV protease or other non-naturally occurring proteases (for that particular cell or organism) are also preferred. If this is the case, then expression of the protease should not significantly impact the cell, and the fusion proteins should not suffer non-specific degradation via the host cells endogenous proteases. Induction or activation of the protease in the cell results in a rapid increase in protease activity within the cell that can cleave the target fusion proteins thereby increasing their stability and steady state concentration in the cell.

V. Screening Applications

[0148] The present invention is suited for use with systems and methods that utilize automated and integratable workstations for identifying modulators, and chemicals having useful activity. Such systems are described generally in the art (see, U.S. Pat. No. 4,000,976 to Kramer et al. (issued Jan. 4, 1977), U.S. Pat. No. 5,104,621 to Pfost et al. (issued Apr. 14, 1992), U.S. Pat. No. 5,125,748 to Bjornson et al. (issued Jun. 30, 1992), U.S. Pat. No. 5,139,744 to Kowalski (issued Aug. 18, 1992), U.S. Pat. No. 5,206,568 Bjornson et al. (issued Apr. 27, 1993), U.S. Pat. No. 5,350,564 to Mazza et al. (Sep. 27, 1994), U.S. Pat. No. 5,589,351 to Harootunian (issued Dec. 31, 1996), and PCT Application Nos: WO 93/20612 to Baxter Deutschland GMBH (published Oct. 14, 1993), WO 96/05488 to McNeil et al. (published Feb. 22, 1996), WO 93/13423 to Agong et al. (published Jul. 8, 1993) and U.S. Pat. No. 5,985,214, issued Nov. 16, 1999.

[0149] Typically, such a system includes: A) a storage and retrieval module comprising storage locations for storing a plurality of chemicals in solution in addressable chemical wells, a chemical well retriever and having programmable selection and retrieval of the addressable chemical wells and having a storage capacity for at least 100,000 addressable wells, B) a sample distribution module comprising a liquid handler to aspirate or dispense solutions from selected addressable chemical wells, the chemical distribution module having programmable selection of, and aspiration from, the selected addressable chemical wells and programmable dispensation into selected addressable sample wells (including dispensation into arrays of addressable wells with different densities of addressable wells per centimeter squared) or at locations, preferably pre-selected, on a plate, C) a sample transporter to transport the selected addressable chemical wells to the sample distribution module and optionally having programmable control of transport of the selected addressable chemical wells or locations on a plate (including adaptive routing and parallel processing), D) a reaction module comprising either a reagent dispenser to dispense reagents into the selected addressable sample wells or locations on a plate or a fluorescent detector to detect chemical reactions in the selected addressable sample wells or locations on a plate, and a data processing and integration module.

[0150] The storage and retrieval module, the sample distribution module, and the reaction module are integrated and programmably controlled by the data processing and integration module. The storage and retrieval module, the sample distribution module, the sample transporter, the reaction module and the data processing and integration module are operably linked to facilitate rapid processing of the addressable sample wells or locations on a plate. Typically, devices of the invention can process at least 100,000 addressable wells or locations on a plate in 24 hours. This type of system is described in commonly owned U.S. Pat. No. 5,985,214, issued Nov. 16, 1999. If desired, each separate module is integrated and programmably controlled to facilitate the rapid processing of liquid samples, as well as being operably linked to facilitate the rapid processing of liquid samples. In one embodiment the system provides for a reaction module that is a fluorescence detector to monitor fluorescence. The fluorescence detector is integrated to other workstations with the data processing and integration module and operably linked with the sample transporter. Preferably, the fluorescence detector is of the type described herein and can be used for epi-fluorescence. Other fluorescence detectors that are compatible with the data processing and integration module and the sample transporter, if operable linkage to the sample transporter is desired can be used as known in the art or developed in the future. For some embodiments of the invention, particularly for plates with 96, 192, 384 and 864 wells per plate, detectors are available for integration into the system. Such detectors are described in U.S. Pat. No. 5,589,351 (Harootunian), U.S. Pat. No. 5,355,215 (Schroeder), and PCT patent application WO 93/13423 (Akong). Alternatively, an entire plate may be "read" using an imager, such as a Molecular Dynamics Fluor-Imager 595 (Sunnyvale, Calif.). Multi-well platforms having greater than 864 wells, including 3,456 wells, can also be used in the present invention (see, for example, the PCT Application PCT/US98/11061, filed Jun. 2, 1998. These higher density well plates require miniaturized assay volumes that necessitate the use of highly sensitivity assays that do not require washing. The present invention provides such assays as described herein.

[0151] The screening methods described herein can be made on cells growing in or deposited on solid surfaces. A common technique is to use a microtiter plate well wherein the fluorescence measurements are made by commercially available fluorescent plate readers. One such method is to use cells in Costar 96 well microtiter plates (flat with a clear bottom) and measure fluorescent signal with CytoFluor multiwell plate reader (Perseptive Biosystems, Inc., MA) using two emission wavelengths to record fluorescent emission ratios. In another embodiment, the system comprises a microvolume liquid handling system that uses electrokinetic forces to control the movement of fluids through channels of the system, for example as described in U.S. Pat. No. 5,800,690 issued Sep. 1, 1998 to Chow et al, European patent application EP 0 810 438 A2 filed May 5, 1997, by Pelc et al. and PCT application WO 98/00231 filed 24 Jun. 1997 by Parce et al. These systems use "chip" based analysis systems to provide massively parallel miniaturized analysis. Such systems are preferred systems of spectroscopic measurements in some instances that require miniaturized analysis.

A Method for Identifying a Chemical, Modulator or a Therapeutic

[0152] The present invention can also be used for testing a therapeutic for useful therapeutic activity. A therapeutic is identified by contacting a test chemical suspected of having a modulating activity of a biological process or target with a test cell comprising the constructs of the present invention. Typically the cells are located within at least one well of a multi-well platform. The test chemical can be part of a library of test chemicals that is screened for activity, such as biological activity. The library can have individual members that are tested individually or in combination, or the library can be a combination of individual members. Such libraries can have at least two members, preferably greater than about 100 members or greater than about 1,000 members, more preferably greater than about 10,000 members, and most preferably greater than about 100,000 or 1,000,000 members. After appropriate incubation of the sample with the test cell, an inhibitor of protein synthesis may be added and a substrate for the reporter moiety added. At least one optical property (such as fluorescence or absorbance) of the sample is determined and compared to a non-treated control to determine the level of reporter gene expression or activity. If the sample having the test chemical exhibits increased or decreased reporter moiety expression or activity relative to that of the control cell then a candidate modulator has been identified.

[0153] The candidate modulator can be further characterized and monitored for structure, potency, toxicology, and pharmacology using well-known methods. The structure of a candidate modulator identified by the invention can be determined or confirmed by methods known in the art, such as mass spectroscopy. For putative modulators stored for extended periods of time, the structure, activity, and potency of the putative modulator can be confirmed.

[0154] Depending on the system used to identify a candidate modulator, the candidate modulator will have putative pharmacological activity. For example, if the candidate modulator is found to inhibit a protein tyrosine phosphatase involved, for example in T-cell proliferation in vitro, then the candidate modulator would have presumptive pharmacological properties as an immunosuppressant or anti-inflammatory (see, Suthanthiran et al, (1996) Am. J. Kidney Disease, 28 159-172). Such nexuses are known in the art for several disease states, and more are expected to be discovered over time. Based on such nexuses, appropriate confirmatory in vitro and in vivo models of pharmacological activity, as well as toxicology, can be selected. The assays, and methods of use described herein, enable rapid pharmacological profiling to assess selectivity and specificity, and toxicity. This data can subsequently be used to develop new candidates with improved characteristics.

Bioavailability and Toxicology of Candidate Modulators

[0155] Once identified, candidate modulators can be evaluated for bioavailability and toxicological effects using known methods (see, Lu, Basic Toxicology, Fundamentals, Target Organs, and Risk Assessment, Hemisphere Publishing Corp., Washington (1985); U.S. Pat. Nos. 5,196,313 to Culbreth (issued Mar. 23, 1993) and U.S. Pat. No. 5,567,952 to Benet (issued Oct. 22, 1996). For example, toxicology of a candidate modulator can be established by determining in vitro toxicity towards a cell line, such as a mammalian i.e. human, cell line. Candidate modulators can be treated with, for example, tissue extracts, such as preparations of liver, such as microsomal preparations, to determine increased or decreased toxicological properties of the chemical after being metabolized by a whole organism. The results of these types of studies are often predictive of toxicological properties of chemicals in animals, such as mammals, including humans.

[0156] The toxicological activity can be measured using reporter genes that are activated during toxicological activity or by cell lysis (see WO 98/13353, published Apr. 2, 1998). Preferred reporter genes produce a fluorescent or luminescent translational product (such as, for example, a Green Fluorescent Protein (see, for example, U.S. Pat. No. 5,625,048 to Tsien et al, issued Apr. 29, 1998; U.S. Pat. No. 5,777,079 to Tsien et al, issued Jul. 7, 1998; WO 96/23810 to Tsien, published Aug. 8, 1996; WO 97/28261, published Aug. 7, 1997; PCT/US97/12410, filed Jul. 16, 1997; PCT/US97/14595, filed Aug. 15, 1997)) or a translational product that can produce a fluorescent or luminescent product (such as, for example, beta-lactamase (see, for example, U.S. Pat. No. 5,741,657 to Tsien, issued Apr. 21, 1998, and WO 96/30540, published Oct. 3, 1996)), such as an enzymatic degradation product. Cell lysis can be detected in the present invention as a reduction in a fluorescence signal from at least one photon-producing agent within a cell in the presence of at least one photon reducing agent. Such toxicological determinations can be made using prokaryotic or eukaryotic cells, optionally using toxicological profiling, such as described in PCT/US94/00583, filed Jan. 21, 1994 (WO 94/17208), German Patent No 69406772.5-08, issued Nov. 25, 1997; EPC 0680517, issued Nov. 12, 1994; U.S. Pat. No. 5,589,337, issued Dec. 31, 1996; EPO 651825, issued Jan. 14, 1998; and U.S. Pat. No. 5,585,232, issued Dec. 17, 1996).

[0157] Alternatively, or in addition to these in vitro studies, the bioavailability and toxicological properties of a candidate modulator in an animal model, such as mice, rats, rabbits, or monkeys, can be determined using established methods (see, Lu, supra (1985); and Creasey, Drug Disposition in Humans, The Basis of Clinical Pharmacology, Oxford University Press, Oxford (1979), Osweiler, Toxicology, Williams and Wilkins, Baltimore, Md. (1995), Yang, Toxicology of Chemical Mixtures; Case Studies, Mechanisms, and Novel Approaches, Academic Press, Inc., San Diego, Calif. (1994), Burrell et al., Toxicology of the Immune System; A Human Approach, Van Nostrand Reinhld, Co. (1997), Niesink et al., Toxicology; Principles and Applications, CRC Press, Boca Raton, Fla. (1996)). Depending on the toxicity, target organ, tissue, locus, and presumptive mechanism of the candidate modulator, the skilled artisan would not be burdened to determine appropriate doses, LD₅₀ values, routes of administration, and regimes that would be appropriate to determine the toxicological properties of the candidate modulator. In addition to animal models, human clinical trials can be performed following established procedures, such as those set forth by the United States Food and Drug Administration (USFDA) or equivalents of other governments. These toxicity studies provide the basis for determining the therapeutic utility of a candidate modulator in vivo.

Efficacy of Candidate Modulators

[0158] Efficacy of a candidate modulator can be established using several art-recognized methods, such as in vitro methods, animal models, or human clinical trials (see, Creasey, supra (1979)). Recognized in vitro models exist for several diseases or conditions. For example, the ability of a chemical to extend the life-span of HIV-infected cells in vitro is recognized as an acceptable model to identify chemicals expected to be efficacious to treat HIV infection or AIDS (see, Daluge et al., (1995) Antimicro. Agents Chemother. 41 1082-1093). Furthermore, the ability of cyclosporin A (CsA) to prevent proliferation of T-cells in vitro has been established as an acceptable model to identify chemicals expected to be efficacious as immunosuppressants (see, Suthanthiran et al., supra, (1996)). For nearly every class of therapeutic, disease, or condition, an acceptable in vitro or animal model is available. Such models exist, for example, for gastro-intestinal disorders, cancers, cardiology, neurobiology, and immunology. In addition, these in vitro methods can use tissue extracts, such as preparations of liver, such as microsomal preparations, to provide a reliable indication of the effects of metabolism on the candidate modulator. Similarly, acceptable animal models may be used to establish efficacy of chemicals to treat various diseases or conditions. For example, the rabbit knee is an accepted model for testing chemicals for efficacy in treating arthritis (see, Shaw and Lacy, J. (1973) Bone Joint Surg. (Br) 55 197-205. Hydrocortisone, which is approved for use in humans to treat arthritis, is efficacious in this model which confirms the validity of this model (see, McDonough, (1982) Phys. Ther. 62 835-839). When choosing an appropriate model to determine efficacy of a candidate modulator, the skilled artisan can he guided by the state of the art to choose an appropriate model, dose, and route of administration, regime, and endpoint and as such would not be unduly burdened.

[0159] In addition to animal models, human clinical trials can be used to determine the efficacy of a candidate modulator in humans. The USFDA, or equivalent governmental agencies, have established procedures for such studies (see, www.fda.gov).

Selectivity of Candidate Modulators

[0160] The in vitro and in vivo methods described above also establish the selectivity of a candidate modulator. It is recognized that chemicals can modulate a wide variety of biological processes or be selective. Panels of cells, each containing constructs with varying specificity, based on the present invention, can be used to determine the specificity of the candidate modulator. Selective modulators are preferable because they have fewer side effects in the clinical setting. The selectivity of a candidate modulator can be established in vitro by testing the toxicity and effect of a candidate modulator on a plurality of cell lines that exhibit a variety of cellular pathways and sensitivities. The data obtained from these in vitro toxicity studies can be extended into in vivo animal model studies, including human clinical trials, to determine toxicity, efficacy, and selectivity of the candidate modulator suing art-recognized methods.

An Identified Chemical, Modulator, or Therapeutic and Compositions

[0161] The invention includes compositions, such as novel chemicals, and therapeutics identified by at least one method of the present invention as having activity by the operation of methods, systems or components described herein. Novel chemicals, as used herein, do not include chemicals already publicly known in the art as of the filing date of this application. Typically, a chemical would be identified as having activity from using the invention and then its structure revealed from a proprietary database of chemical structures or determined using analytical techniques such as mass spectroscopy.

[0162] One embodiment of the invention is a chemical with useful activity, comprising a chemical identified by the method described above. Such compositions include small organic molecules, nucleic acids, peptides and other molecules readily synthesized by techniques available in the art and developed in the future. For example, the following combinatorial compounds are suitable for screening: peptoids (PCT Publication No. WO 91/19735, 26 Dec. 1991), encoded peptides (PCT Publication No. WO 93/20242, 14 Oct. 1993), random bio-oligomers (PCT Publication. WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomeres such as hydantoins, benzodiazepines and dipeptides (Hobbs DeWitt, S. et al., (1993) Proc. Nat. Acad. Sci. USA 90 6909-6913), vinylogous polypeptides (Hagihara et al., (1992) J. Amer. Chem. Soc. 114 6568), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann, R. et al, (1992) J. Amer. Chem. Soc. 114 9217-9218), analogous organic syntheses of small compound libraries (Chen, C. et al., (1994) J. Amer. Chem. Soc. 116 2661), oligocarbamates (Cho, C. Y. et al., (1993) Science 261: 1303), and/or peptidyl phosphonates (Campbell, D. A. et al, (1994) J. Org. Chem. 59 658). See, generally, Gordon, E. M. et al. (1994). J. Med Chem. 37 1385. The contents of all of the aforementioned publications are incorporated herein by reference.

[0163] The present invention also encompasses the identified compositions in a pharmaceutical composition comprising a pharmaceutically acceptable carrier prepared for storage and subsequent administration, which have a pharmaceutically effective amount of the products disclosed above in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, acsorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used.

[0164] The compositions of the present invention may be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions for injectable administration; and the like. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (e.g., liposomes) may be utilized.

[0165] The pharmaceutically effective amount of the composition required as a dose will depend on the route of administration, the type of animal being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize. In practicing the methods of the invention, the products or compositions can be used alone or in combination with one another or in combination with other therapeutic or diagnostic agents. These products can be utilized in vivo, ordinarily in a mammal, preferably in a human, or in vitro. In employing them in vivo, the products or compositions can be administered to the mammal in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms. Such methods may also be applied to testing chemical activity in vivo.

[0166] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.

[0167] In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear. The dosage for the products of the present invention can range broadly depending upon the desired affects and the therapeutic indication. Typically, dosages may be between about 10 mg/kg and 100 mg/kg body weight, and preferably between about 100 μg/kg and 10 mg/kg body weight. Administration is preferably oral on a daily basis.

[0168] The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al., in The Pharmacological Basis of Therapeutics, 1975). It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0169] Depending on the specific conditions being treated, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0170] For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Use of pharmaceutically acceptable earners to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

[0171] Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external micro-environment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.

[0172] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0173] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0174] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Such formulations can be made using methods known in the art (see, for example, U.S. Pat. No. 5,733,888 (injectable compositions); U.S. Pat. No. 5,726,181 (poorly water soluble compounds); U.S. Pat. No. 5,707,6411 therapeutically active proteins or peptides); U.S. Pat. No. 5,667,809 (lipophilic agents); U.S. Pat. No. 5,576,012 (solubilizing polymeric agents); U.S. Pat. No. 5,707,615 (anti-viral formulations); U.S. Pat. No. 5,683,676 (particulate medicaments); U.S. Pat. No. 5,654,286 (topical formulations); U.S. Pat. No. 5,688,529 (oral suspensions); U.S. Pat. No. 5,445,829 (extended release formulations); U.S. Pat. No. 5,653,987 (liquid formulations); U.S. Pat. No. 5,641,515 (controlled release formulations) and U.S. Pat. No. 5,601,845 (spheroid formulations).

VII. Transgenic Animals

[0175] In another embodiment, the invention provides a transgenic non-human organism that expresses a nucleic acid sequence that encodes a target protein, (such as a reporter moiety, enzyme or structural protein) functionally coupled to one or more destabilization domains by a linker. Because such constructs can be expressed within intact living cells, with preset degrees of stability, the invention provides the ability to regulate the expression level of the target protein, or to monitor post translational activities within defined cell populations, tissues or in an entire transgenic organism.

[0176] In one embodiment the approach may be used to regulate the expression level of an enzyme or group of enzymes involved in a particular signal transduction, disease, or metabolic pathway. Such methods may be useful, for example, for creating transgenic model animals for certain disease states, or for modulating the intracellular concentration of enzymatic intermediates though the manipulation of the expression levels of the enzymes involved. For example, to increase the intracellular concentration of an intermediate one could increase the concentration of the enzyme(s) involved in the synthesis of the intermediate, and/or decrease the concentration of the enzyme(s) involved in degradation of the intermediate. Typically the approach would require the replacement of the native enzymes with fusion proteins of the enzymes with the multimerized destabilization domains of the present invention. For target proteins in which the desired concentration was relatively high, one would select fusion proteins with relatively few (i.e. one or two), or even no, (zero) copies of the destabilization domain. For target proteins for which a relatively low intracellular concentration was desired, one would select fusion proteins with relatively more copies of the destabilization domain (i.e. three or more).

[0177] In another embodiment, the approach can be used to identify where in specific tissues a particular activity is located, for example, by expression of a reporter moiety coupled to the multimerized destabilization domain via a linker comprising recognition and cleavage motifs for that activity, in the organism. Typically the linker would comprise a single polypeptide chain that covalently couples the destabilization domains to the reporter moiety. Typically in this embodiment, the linker will comprise a post-translational recognition motif such as a protease recognition motif. Cleavage of the linker by the protease at the cleavage site results in uncoupling of the multimerized destabilization domains from the reporter moiety resulting in a modulation in the stability of the reporter moiety, thereby resulting in an accumulation of reporter moiety in cells or tissues that exhibit protease activity.

[0178] Such non-human organisms include vertebrates such as rodents, fish such as Zebrafish, non-human primates and reptiles as well as invertebrates. Preferred non-human organisms are selected from the rodent family including rat and mouse, most preferably mouse. The transgenic non-human organisms of the invention are produced by introducing transgenes into the germline of the non-human organism. Embryonic target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the organism and stage of development of the embryonic target cell. In vertebrates, the zygote is the best target for microinjection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter, which allows reproducible injection of 1-2 pi of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al, (1985) Proc. Natl. Acad. Sci. USA 82 4438-4442.). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.

[0179] A transgenic organism can be produced by cross-breeding two chimeric organisms which include exogenous genetic material within cells used in reproduction. Twenty-five percent of the resulting offspring will be transgenic i.e., organisms that include the exogenous genetic material within all of their cells in both alleles 50% of the resulting organisms will include the exogenous genetic material within one allele and 25% will include no exogenous genetic material.

[0180] Retroviral infection can also be used to introduce transgene into a non-human organism. In vertebrates, the developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retro viral infection (Jaenich, R., (1976) Proc. Natl. Acad. Sci USA 73 1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viral vector system used to introduce the transgene is typically a replication-defective retro virus carrying the transgene (Jahner, et al, (1985) Proc. Natl. Acad. Sci. USA 82 6927-6931; Van der Putten, et al, (1985) Proc. Natl. Acad. Sci USA 82 6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al, (1987) EMBO J. 6 383-388).

[0181] Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (D. Jahner et al, (1982) Nature 298 623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells that formed the transgenic nonhuman animal. Further, the founder may contain various retro viral insertions of the transgene at different positions in the genome that generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retro viral infection of the midgestation embryo (D. Jahner et al., supra). A third type of target cell for transgene introduction for vertebrates is the embryonic stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al. (1981) Nature 292 154-156; M. O. Bradley et al., (1984) Nature 309 255-258; Gossler, et al, (1986) Proc. Natl. Acad. Sci USA 83 9065-9069; and Robertson et al, (1986) Nature 322 445-448). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. (For review see Jaenisch, R., (1988) Science 240 1468-1474).

VIII Transgenic Plants

[0182] In another embodiment, the invention provides a transgenic plant that expresses a nucleic acid sequence that encodes a target protein, (such as a reporter moiety, enzyme or structural protein) functionally coupled to a multimerized destabilization domain by a linker. Because such constructs can be specifically expressed, both spatially and temporally, within intact living cells, the invention provides the ability to regulate the expression level of the target protein, within defined cell populations, tissues, or in the entire transgenic plant.

[0183] In one embodiment the approach may be used to regulate the expression level of an enzyme or group of enzymes involved in a particular signal transduction, developmental or metabolic pathway. Such methods may be useful for creating transgenic plants with improved disease resistance or other favorable traits. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest, for example by allowing for the regulated expression of agronomically important genes. Given potential concerns about the safety of transgenic plants, the ability to reduce or eliminate the expression of certain resistance genes prior to harvesting and human consumption is of particular interest. Examples of the types of genes that could be manipulated using the methods described herein, include disease resistance genes, herbicide resistance genes and genes that improve plant traits, including those shown in Table 4, below.

TABLE-US-00004 TABLE 4 Gene or Gene Product Function Reference I. Disease Resistance Genes Tomato Cf-9 gene Resistance to Cladosporium Jones et al., Science fulvum 266 789 (1994) Tomato Pto gene Resistance to Martin et al., Science Pseudomonassy.ringae 262: 1432 (1993) Arabidopsis RSP2 gene Resistance to Pseudomonas Mindrinos et al., Cell syringae 78: 1089 (1994) Bacillus thuringiensis protein Insect resistance Geiser et al., Gene 48: 109 (1986), Streptomyces nitrospoeus a- Inhibition of amylase activity. Sumitani et al., Biosci. amylase inhibitor Biotech. Biochem. 57 1243 (1993) Expression of insect-specific Disruption of insect Hammock et al., Nature hormones or pheromones development 344: 458 (1990) such as an ecdysteroid and juvenile hormone Expression insect-specific Insect resistance Pang et al., Gene 116: scorpion venom 165 (1992) Altered expression of Expression of enzymes metabolic enzymes responsible for the formation of non protein molecules with insecticidal activity Altered expression of signal Expression of enzymes See PCT application transduction enzymes responsible for the post- WO 93/02197, Botella translational modification of et al., Plant Molec. biologically active molecules Biol. 24: 757 (1994), Expression of synthetic Improved disease resistance antimicrobial peptides, such as peptide derivatives of Tachyplesin Altered expression of Ion Improved resistance to Jaynes et al., Plant Sci. channels, blockers or Pseudomonas solanacearum. 89: 43 (1993), permeases such as cecropin-3 lytic peptide Expression of viral coat Improved viral resistance to See Beachy et al., Ann. proteins or viral-invasive alfalfa mosaic virus, cucumber Rev. Phytopathol. proteins or toxins. mosaic virus, tobacco streak 28: 451 (1990). virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus Expression of insect-specific Improved resistance to insects. Taylor et al., Abstract antibody or immunotoxins #497, SEVENTH INTL SYMPOSIUM ON MOLECULAR PLANT-MICROBE INTERACTIONS (1994) Expression of virus-specific Improved resistance to viruses Tavladoraki et al., antibodies. Nature 366: 469 (1993) Expression of developmental- Increased resistance to See Lamb et al., Biol arrestive proteins or gene pathogens or parasites Technology; Q: 1436 products, as endo al, 4-D- (1992). Logemann et polygalacturonase, or al., BiolTechnology.10: expression of barley 30 (1992) ribosome-inactivating gene II. Herbicide Resistance Genes Expression of mutant ALS Inhibition of the growing Lee et al., EMBO J. 7: and AHAS enzymes point or meristem, increasing 1241 (1988), and Miki resistance to herbicides et al., Theor. Appl. Genet. 8: 449 (1990), Expression of mutant EPSP Resistance to glyphosate and U.S. Pat. No. synthase and aroA genes, other phosphono compounds 4,940,835 to Shah et such as glufosinate al., U.S. Pat. No. 4,769,061 to Comai. European patent application No. 0 333 033 to Kumada et al. and U.S. Pat. No. 4,975,374 to Goodman et al. III. Genes That Confer Or Contribute To A Value-added Trait Expression of antisense gene Improved fatty acid Knultzon et al., Proc. of stearoyl-ACT desaturase composition Natl. Acad. Sci. USA 89: 2624 (1992). Expression of phytic acid Improved free phosphate Van Hartingsveldt et degrading enzymes composition al.. Gene 127: 87 (1993) Expression of Improved carbohydrate See Shiroza et al., J. tructosyltransferase. composition Bacteriol. 170: levansucrase, or invertase 810 (1988), Steinmetz et genes al., Mol. Gen. Genet. 200 220 (1985), Elliot etal., Plant Molec. Biol. 21 515 (1993)

[0184] In another embodiment, the approach can be used to specifically identify where in specific tissues a particular activity is expressed, for example by expression of the protease sensor in specific plant tissues.

[0185] Transgenic plants may be produced by any one of a number of methods of plant transformation and regeneration. Numerous methods for plant transformation have been developed, including biological and physical, plant transformation protocols. See, for example, Miki et al., "Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al., "Vectors for Plant Transformation" in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

[0186] The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al., (1985) Science 227 1229. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I., Crit. Rev. Plant. Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber et al., supra, Miki et al., supra, and Moloney et al., (1989) Plant Cell Reports 8 238.

[0187] Despite the fact the host range for Agrobacterium mediated transformation is broad, some major cereal crop species and gymnosperms have generally been recalcitrant to this mode of gene transfer, even though some success has recently been achieved in rice. Hiei et al., (1994) The Plant Journal 6 271-282. Several methods of plant transformation, collectively referred to as direct gene transfer, have been developed as an alternative to Agrobactenum-mediated transformation.

[0188] A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 to 4 Am. The expression vector is introduced into plant tissues with a biohstic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes. Sanford et al., (1987), Part. Sci. Technol. 5 27, Sanford, J. C., (1988) Trends Biotech. 6 299, Sanford, J. C., (1990) Physiol. Plant 79 206, Klein et al., (1992) Biotechnology 10 268.

[0189] Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., (1991) BioTechnology 9 996. Alternatively, liposome or spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al., (1985) EMBO J., 4 2731, Christou et al., (1987) Proc Natl. Acad. Sci. U.S.A. 84 3962. Direct uptake-of DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol or poly-Lornithine have also been reported. Hain et al., (1985) Mol. Gen. Genet. 199 161 and Draper et al., (1982) Plant Cell Physiol. 23 451. Electroporation of protoplasts and whole cells and tissues have also been described. Donn et al., In Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin et al., (1992) Plant Cell 4 1495-1505 and Spencer et al, (1994) Plant Mol. Biol. 24 51-61.

[0190] A preferred method is microprojectile-mediated bombardment of immature embryos. The embryos can be bombarded on the embryo axis side to target the meristem at a very early stage of development or bombarded on the scutellar side to target cells that typically form callus and somatic embryos. Targeting of the scutellum using projectile bombardment is well known to those in the art of cereal tissue culture. Klein et al., (1988) BioTechnol., 6 559-563; Sautter et al., Bio/Technol., 9 1080-1085 (1991); Chibbar et al., (1991) Genome, 34 435-460. The scutellar origin of regenerable callus from cereals is well known. Green et al., (1975) Crop Sci., 15 417-421; Lu et al., (1982) TAG 62 109-112; and Thomas and Scott, (1985) J. Plant Physiol. 121 159-169--Targeting the scutellum and then using chemical selection to recover transgenic plants is well established in cereals. D/Halluin et al., Plant Cell 4: 1495-1505 (1992); Perl et al., MGG 235: 279-284 (1992); Cristou et al., Bio/Technol. 9: 957-962 (1991). This literature reports DNA targeting of the scutellum and recovery of transgenic callus, plants and progeny based on a chemical selection regime. None of these references teach successful plant transformation wherein transformed cells are visualized with a screenable marker such as GUS.

[0191] A preferred transformation method involves bombardment of the scutellar surface of immature embryos to introduce the expression cassette with the gene for a bioluminescent protein, such as Aequorea victoria GFP (See PCT publication WO 97/41228 to Gordon-Kamm et al., incorporated herein by reference). Embryos can be pretreated for 1 to 48 hours with a high osmoticum medium or left on a highosmoticum medium for 24-48 hours after bombardment to improve cell survival and transformation frequencies. Immature embryos are then cultured on typical callusinducing medium with no selective agent. At each subculture transfer, i.e., every two weeks, the culture is monitored using UV-blue light for GFP fluorescence. Fluorescing calli are separated from non-fluorescing callus, and grown to the point where plants can be regenerated through standard media progressions.

[0192] Plants can be manipulated, for example, by removal of the apical meristem, to stimulate axillary or secondary buds which can exhibit larger transgenic sectors relative to the primary shoot. Flowers above transgenic shoots are pollinated and the progeny are analyzed for transgene presence and expression. A variety of starting explants can regenerate shoots in sunflower, and thus represent alternative targets for GFP-encoding DNA delivery and transmission to progeny. These include the seedling meristem (as above), also the seedling hypocotyl, the mature cotyledon, the immature cotyledon, zygotic immature embryos, somaticembryos, and primary leaflets. See for example, respectively, Greco et al., (1984) Plant Sci. Lett. 36 73-77; Krauter et al., (1991) Helia 14 117-122; Power (1987) Am. J. Bot. 74 497503; Krauter et al., (1991) Theor. Appl. Genet. a2: 521525; Finer, (1987) Plant Cell Rep. J: 372-374, and Greco et al., (1984) Plant Sci. Lett. 36 73-77.

Example 1

Generation of Multimerized Destabilization Domains

[0193] The cDNA encoding human ubiquitin was isolated from a human genomic DNA preparation obtained from Jurkat cells by polymerase chain reaction (PCR) using the PCR primers Ubi5 (SEQ. ID. NO. 15) and Ubi3 (SEQ. ID. NO. 16) and cloned into pBluescript II vector (Stratagene). The C-terminal residue of ubiquitin was altered from glycine to valine by site-directed mutagenesis (Kunkel) in order to generate a mutant form of ubiquitin that cannot be cleaved by cellular α-NH-ubiquitin endopeptidases. This mutant is hereafter referred to as ubiquitinG76V (SEQ. ID. NO. 17). The ubiquitinG76V (SEQ. ID. NO. 17) mutant was then amplified by PCR using the oligonucleotide primers Ub5' (SEQ. ID. NO. 18) and Ub3\ (SEQ. ID. NO. 19). These primers introduce a Bgl II restriction site at the 5' end of the coding sequence and a BamH I site at the 3' end of the coding sequence. The PCR fragment from the reaction was digested with Bgl II and BamH I and ligated into BamH I-digested pBluescript II vector. This plasmid was then digested with Bgl II and BamH I and the ubiquitinG76V (SEQ. ID. NO. 17) containing fragment was isolated and ligated to generate multimerized ubiquitinG76V domains. The ubiquitinG76V multimers were digested with Bgl II and BamH I to ensure that the individual ubiquitinG76V domains (SEQ. ID. NO. 17) were in the correct orientation. The digested ubiquitinG76V multimers were separated by agarose gel electrophoresis and multimers of the appropriate sizes were isolated and cloned into BamH I-digested pBluescript II. The ubiquitinG76V multimers were then excised using BamH I and Hind III and subcloned to generate a series of plasmids containing in frame fusions of from one to four copies of ubiquitinG76V (SEQ. ED. NO. 17) fused to the reporter moiety or protein of interest. These constructs are referred to as 1XUb (one copy of ubiquitinG76V (SEQ. ED. NO. 17)), 2XUb (two copies of ubiquitinG76V (SEQ. ED. NO. 17)), 3XUb (three copies of ubiquitinG76V (SEQ. ED. NO. 17)) and 4XUb (four copies of ubiquitinG76V (SEQ. ED. NO. 17)).

Example 2

Creation of Multimerized Destabilization Domain-β-Lactamase Fusion Proteins

[0194] The gene encoding the E. coli TEM-1 β-lactamase was isolated from the plasmid pBluescnpt (Stratagene) by polymerase chain reaction (PCR) amplification using the PCR primers BLAS (SEQ. ID. NO. 20) and ABSC107, (SEQ. ID. NO. 21) resulting in the deletion of the signal sequence and introduction of a BamH I restriction site and the amino acids below at the 5' end of the coding sequence.

TABLE-US-00005 BamH1 H G S G A W L H P E T L V K V K

[0195] Amino acids in bold represent original β-lactamase coding sequence, underlined amino acids represent the BamH I restriction site. An Xba I site was inserted at the 3' end of the coding sequence. The PCR fragments from these reactions were digested with BamH I and Xba I and ligated into pcDNA3 (Invitrogen) via the same sites. The resulting construct, pcDNA3-Bla (SEQ. ID. NO. 22), was then used to create in-frame fusions with the multimerized ubiquitinG76V constructs above. This was achieved by digesting the multimerized ubiquitinG76V constructs with the restriction enzymes BamH I and Hind III, and then ligating them via the same sites into the pcDNA3-Bla construct. These constructs were named pcDNA3-1XUb-Bla (SEQ. ID. NO. 23), pcDNA3-2XUb-Bla (SEQ. ID. NO. 24), pcDNA3-3XUb-Bla (SEQ. ID. NO. 25), pcDNA3-4XUb-Bla (SEQ. ID. NO. 26). To produce the wild-type β-lactamase protein, we used a construct that contains one copy of wild-type (cleavable) ubiquitin (SEQ. ID. NO. 2) fused to the β-lactamase coding region in the pcDNA3 vector; this plasmid is referred to as pcDNA3-Ub-Met-Bla (SEQ. ID. NO. 27). Upon synthesis of the Ub-Met-Bla fusion protein, ubiquitin isopeptidases efficiently cleave off the N-terminal ubiquitin (SEQ. ID. NO. 2) precisely after glycine-76, generating the wild-type β-lactamase protein with methionine at its N-terminus.

Example 3

Creation of Multimerized Destabilization Domain-Naturally Fluorescent Protein Fusions

[0196] The gene encoding the GFP mutant Emerald (S65T, S72A, N149K, M153T, 1167T) (SEQ. ID. NO. 28) was amplified by PCR using the oligonucleotides GFP5' (SEQ. ID. NO. 29) and GFP3', (SEQ. ID. NO. 30). The resulting PCR product had a BamH I restriction site at the 5' end of the coding sequence and a Xba I site at the 3' end of the coding sequence. The PCR fragment from this reaction was digested with BamH I and Xba I and ligated into pcDNA3 via the same sites. The resulting construct, pcDNA3-GFP was then used to create in-frame fusions with the multimenzed ubiquitinG76V constructs described above. This was achieved by digesting the pcDNA3-1-4XUb-Bla constructs (SEQ. ID. NOs. 23 to 26) with the restriction enzymes BamH I and Hind III, and then ligating the fragment encoding the various multiUb destabilization domains via the same sites into the pcDNA3-GFP construct. These constructs were named pcDNA3-1XUb-GFP (SEQ. ID. NO. 31), pcDNA3-2XUb-GFP (SEQ. ID. NO. 32), pcDNA3-3XUb-GFP (SEQ. ID. NO. 33), pcDNA3-4XUb-GFP (SEQ. ID. NO. 34).

Example 4

Creation of Multimerized Destabilization Domain-Naturally Occurring Mammalian Protein Fusions

[0197] Fusions between multimerized uncleavable ubiquitinG76V (SEQ. ED. NO. 17) and caspase-3 were constructed to further investigate the relationship between the degree of destabilization exerted by varying the number of copies of the destabilization domain with different target proteins.

[0198] The caspase-3 cDNA (SEQ. ED. NO. 35) was amplified by PCR using the pnmers C35' (SEQ. ID. NO. 36) and C33' (SEQ. ID. NO. 37) to add BamH I sites at the ends of the caspase-3 cDNA. The amplified caspase-3 cDNA was digested with BamH I then cloned into BamH I-digested pcDNA3-1-4XUb-Bla plasmids (SEQ. ED. NOs. 23 to 26), to create fusions of the different multiubiquitin destabilization domains to a caspase-3-β-lactamase fusion. The β-lactamase coding region was then removed from these plasmids by digesting to completion with Xba I followed by a partial digest with BamH I. The digests were separated by agarose gel electrophoresis and the correct size DNA band was purified from the gel. The ends of the digested plasmid were blunted with the Klenow fragment of DNA polymerase and the plasmid recircularized by ligation. The resulting plasmids contained an in-frame fusion of the uhiquitinG76V destabilization domain (with from one to four copies of ubiquitinG76V (SEQ. ID. NO. 17)) to the caspase-3 coding region. These plasmids were designated pcDNA3-1-4XUb-C3 (SEQ. ID. NO. 38 to 41). To produce the wild-type caspase-3 protein, the caspase-3 cDNA was amplified by PCR with primers C35Met (SEQ. ID. NO. 42) and C33' (SEQ. ID. NO. 43) and cloned directly into pcDNA3-Ub-Met-Bla (SEQ. ID. NO. 27). The resulting plasmid was then digested with BamH I and Xba I and recircularized as described above to create the wild-type caspase-3 control construct; this plasmid was designated as pcDNA3-Ub-Met-C3 (SEQ. ID. NO. 44). Upon synthesis of the Ub-Met-caspase-3 fusion protein, ubiquitin isopeptidases efficiently cleave off the N-terminal ubiquitin precisely after glycine-76, generating the wild-type caspase-3 protein with methionine at its N-terminus (data not shown).

Example 5

Characterization of Multimerized Destabilization Domain-β-Lactamase Fusion Proteins In Vitro

[0199] S-Labeled multimerized destabilization domain-β-lactamase fusion protein molecules were produced using a coupled in vitro transcription/translation system based on a rabbit reticulocyte lysate (TNT T7 Quick; Promega). Constructs containing from one to four copies of the destabilization domain (pcDNA3-1-4XUb-Bla (SEQ. ID. NOs. 23 to 26) from Example 2) were incubated in the TNT lysate essentially as described in the manufacturer's directions in the presence of 0.25 mCi/ml ³⁵S-methionine (10 mCi/ml, 1175 Ci/mmol; New England Nuclear) to generate ³⁵S-labeled fusion proteins.

[0200] To determine the half life of the constructs, 1 μl samples of the synthesis reactions were incubated at 37° C. in 9 μl of chase extract (crude rabbit reticulocyte lysate (Promega) supplemented with 100 μg/ml cycloheximide, 1 mM ATP, 20 mM phosphocreatine, 2.5 mM MgCb, 5 Hg/ml creatine kinase, 200 μg/ml ubiquitin, and 50 μM methionine). The rabbit reticulocyte lysate system contains all of the components necessary for efficient recognition and degradation of proteins by the ubiquitin-proteasome pathway. Samples were removed at 0, 5, 10, 20, 30, 45 and 60 minutes of reaction and analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). The gels were treated with Amplify (Amersham) and the labeled species detected by autoradiography. This analysis showed that wild-type β-lactamase was stable over the 1 hour chase period while the ubiquitinG76V-β-lactamase fusions were considerably less stable (FIG. 2A). In particular, the 1XUb-Bla fusions were modestly destabilized (t₁/2 ˜20 min) and β-lactamase fusions containing 2, 3 or 4 copies of ubiquitinG76V (SEQ. ED. NO. 17) were strongly destabilized (t₁/2<5 min). In addition, the degradation of the 2XUb-Bla fusion was slightly slower than the degradation of β-lactamase fusions containing 3 or 4 copies of ubiquitinG76V (SEQ. ID. NO. 17) (FIG. 2A).

[0201] In order to test whether the degradation of multiUb-Bla fusions in vitro is dependent on the proteasome, TNT synthesis reactions were performed in the absence or presence of the proteasome inhibitor MG132 (Calbiochem) at 50 μM and analyzed by SDS-PAGE as described above. These experiments showed that inhibition of the proteasome resulted in a dramatic increase in the amount of fusion protein synthesized for β-lactamase fusions containing 2, 3 or 4 copies of ubiquitinG76V (SEQ. ID. NO. 17) while MG132 had very little or no significant effect on the synthesis of wild-type B-lactamase or 1XUb-Bla (FIG. 2B). Use of MG132 in these in vitro reactions also revealed the presence of labeled high molecular weight species that represent extended ubiquitin chains conjugated to the ubiquitinG76V-β-lactamase fusions (also see Example 16). Therefore, the uncleavable ubiquitinG76V domains (SEQ. ID. NO. 17) in the multiubiquitin destabilization domain may be acting as high affinity conjugation sites for further ubiquitination by E2/E3 ubiquitin ligases. The relative lack of these high molecular weight species in the absence of MG132 reflects the highly efficient recognition and degradation by the proteasome of proteins tagged with extended polyubiquitin chains.

Example 6

Characterization of Multimerized Destabilization Domain-Naturally Fluorescent Protein Fusions In Vitro

[0202] Characterization of the turnover of multiubiquitin-GFP fusion proteins in vitro was similar to the multiubiquitin-β-lactamase analyses described in Example 5, except that time points were taken at 0, 30, 60, 90 and 120 min. These experiments showed that Emerald GFP (SEQ. ID. NO. 28) is extremely stable under these conditions, and that the multiubiquitin destabilization domain was able to impart a short half-life upon the multiUb-GFP fusion proteins (FIG. 3). A striking feature of this analysis was that significant destabilization of GFP required higher numbers of ubiquitinG76V (SEQ. ID. NO. 17) domains than was the case for β-lactamase; β-lactamase could be strongly destabilized in vitro by fusion with as few as two ubiquitinG76V domains (SEQ. ID. NO. 17) (FIG. 2A) whereas GFP required at least three ubiquitinG76V domains (SEQ. ID. NO. 17) to be strongly destabilized (FIG. 3). This relationship between the destabilization domain, and the protein to be destabilized, emphasizes the utility of the multiubiquitin destabilization system, in that the extent of destabilization can be manipulated to give the desired properties by altering the number of ubiquitinG76V (SEQ. ID. NO. 17) domains that are present in the destabilization domain.

Example 7

Characterization of Multimerized Destabilization Domain-Endogenous Mammalian Protein Fusions In Vitro

[0203] Characterization of the turnover of multiubiquitin-caspase-3 fusion proteins in vitro was performed as described in Example 5. The TNT synthesis reactions were diluted into chase lysate in the presence of cycloheximide and chase time points were taken and analyzed by SDS-PAGE and autoradiography. FIG. 4 shows that wild-type caspase-3 is stable over a 60 minute chase in vitro, and that fusion to the multiubiquitin destabilization domain results in rapid degradation. In particular, the ubiquitinG76V-caspase-3 fusions are degraded in a very similar manner to the ubiquitinG76V-β-lactamase fusions although the Ub-caspase-3 fusions appear to be degraded slightly slower in vitro than the Ub-β-lactamase fusions. Altogether, these data demonstrate the generalized applicability of the multiubiquitin destabilization domain approach to provide for predictable destabilized of any given chosen target protein using this system.

Example 8

Characterization of the Half-Life of Multimerized Destabilization Domain-β-Lactamase Fusion Proteins within Cells

[0204] UbiquitinG76V-pMactamase constructs in pcDNA3 (SEQ. ID. NOs. 23 to 26) were introduced into Jurkat T-lymphocytes by electroporation. Stable transfectants were selected in RPMI 1640 media containing 10% fetal bovine serum (Gibco) and 0 8 mg/ml G418 (Geneticin, Gibco). Analysis of β-lactamase activity in intact Jurkat cells stably transfected with the pcDNA3-1-4XUb-Bla (SEQ. ID. NOs. 23 to 26) constructs was accomplished by loading the cells with the fluorescent β-lactamase substrate CCF2/AM as described in Zlokarnik et al. (1998) (Science 279, 1848) followed by analysis by fluorescence activated cell sorter (Becton Dickinson FACS® Vantage®) or CytoFluor microtiter plate fluorimeter (Perseptive Biosystems). For kinetic measurements, to determine the half-life of the fusion protein in vivo, direct measurements were made of B-lactamase activity in lysates prepared from cells expressing the various ubiquitinG76V-Bla fusions.

[0205] Flow cytometry and cell sorting were conducted using a Becton Dickinson FACS® Vantage® with a Coherent Enterprise II® argon laser producing 60 mW of 351-364 nm multi-line UV excitation. The flow cytometer was equipped with pulse processing and the Macrosort® flow cell. Cells were loaded with 1 μM CCF2/AM for 1-2 hours at room temperature prior to sorting, and fluorescence emission was detected via 460/50 nm (blue) and 535/40 nm (green) emission filters, separated by a 490 nm long-pass dichroic mirror. The results from one such experiment are shown in FIG. 5, where the abundance of cells expressing relatively high levels of β-lactamase (regions R5+R6+R7) was determined. This analysis showed that the relative abundance of cells expressing high steady state levels of β-lactamase was inversely proportional to the number of copies of ubiquitinG76V (SEQ. ID. NO. 17) fused to β-lactamase, the lowest levels of β-lactamase expression were found in cells expressing β-lactamase fusions containing the most copies of ubiquitinG76V (SEQ. ID. NO. 17).

[0206] Similar cytometric analysis experiments were used to investigate the degradation properties of multiUb-Bla fusions in vivo. Jurkat cells expressing multiUb-Bla fusions were treated with 50 μM MG132 to investigate whether the low β-lactamase activity found in cells expressing 3-4XUb-Bla requires proteasome activity. The results, shown in Table 5, below show that the addition of inhibitor (+inh/-chx samples) results in a significant increase in the percentage of positive BLA expressing cells for the 2×, 3× and 4× ubiquitinG76V fusion protein constructs compared to the untreated controls (-inh/-chx samples.)

TABLE-US-00006 TABLE 5 -inh/-chx +inh/-chx -inh/+chx +inh/+chx % Bla.sup.+ cells % Bla.sup.+ cells % Bla.sup.+ cells % Bla.sup.+ cells WTBla 22.5 22.7 17.6 19.0 1XUb-Bla 17.4 18.8 8.5 16.2 2XUb-Bla 12.0 17.1 2.1 12.2 3XUb-Bla 8.3 14.6 1.5 9.8 4XUb-Bla 4.1 12.1 0.5 5.0

[0207] Furthermore, treating these cells with 100 M-g/ml cycloheximide (to block protein synthesis) for one hour prior to CCF2 loading and cytometric analysis (compare columns [-inh/+chx] and [-inh/-chx]) resulted in a strong decrease in β-lactamase activity only in cells expressing 2-4XUb-Bla and this decrease could largely be blocked by preincubating the cells with 50 μM MG132 prior to cycloheximide addition (column +inh/+chx, in Table 5).

[0208] These data are strong evidence that the multiubiquitin domain in ubiquitinG76V-Bla fusions is acting as a destabilization motif that directs the rapid degradation of the fusions in a proteasome-dependent manner that is controlled by the number of ubiquitinG76V (SEQ, ID. NO. 17) domains within the multiubiquitin destabilization domain.

[0209] In order to determine accurate quantitative measurements of the kinetic characteristics of the degradation of UbG76V-B-lactamase fusions in vivo, β-lactamase activity was determined in cellular lysates. To do this, Jurkat cells expressing the various forms of multiUb-Bla fusion proteins were sorted by flow cytometry to obtain a pool of cells representative of the Bla+ population seen in FIG. 5 (Region R5+R6+R7). These cells were treated with 100 μg/ml cycloheximide to inhibit new protein synthesis, and aliquots of cells were taken at appropriate intervals, to measure the β-lactamase activity remaining. This approach enabled a determination of the rate of destruction of the cellular pool of β-lactamase fusion proteins within the cell, β-lactamase activity was determined in these cell samples by transferring them to ice to terminate further metabolism, and then pelleted by centrifugation. The cell pellets were converted to lysates and β-lactamase activity was measured in vitro using the free acid form of the β-lactamase substrate CCF2. Aliquots of the lysates were assayed using 10 U.M CCF2 in PBS at room temperature. Hydrolysis of the fluorescent substrate was monitored in a Perseptive Biosystems CytoFluor plate reader using a 395/25 nm excitation filter and 460/40 nm emission filter.

[0210] In agreement with the cell analyses by flow cytometry, cells expressing wild-type β-lactamase had high levels of β-lactamase activity, that was relatively resistant to proteolytic degradation over a 90 minute incubation period with cycloheximide; wild-type β-lactamase activity decayed with a half-life >2 hours (FIG. 6). Cells expressing 1XUb-Bla fusions also contained relatively high levels of β-lactamase activity that decayed with a half-life of about 20-30 minutes. Cells expressing β-lactamase fused to 2 or more copies of ubiquitinG76V (SEQ. ID. NO. 17) had significantly less β-lactamase activity at steady state (compare 0 minute time points) and the half-lives of these pools of fusion proteins were strikingly short, with all three fusion proteins decaying with in vivo half-lives of less than 10 minutes.

[0211] The β-lactamase measurements from the Jurkat cell lysates allows a calculation of the intracellular concentration and copy number of β-lactamase fusion proteins in the respective cell lines. A standard curve created of the hydrolysis of CCF2 by purified β-lactamase enzyme was generated and used to calculate the steady state concentration of β-lactamase fusion protein for each cell line. This analysis showed that there was a ten-fold difference in intracellular concentration between wild-type B-lactamase and 4XUb-β-lactamase at steady state (Table 6). The calculated concentration of wild-type β-lactamase corresponds to 21,000 molecules per cell, in very good agreement with the values reported by Zlokarnik et al. (1998) (Science 279, 1848) for cells expressing high levels of wild-type β-lactamase.

TABLE-US-00007 TABLE 6 Intracellular Construct Half-life Concentration WTBla >120 min 35 nM 1XUb-Bla 20-30 min 30 nM 2XUb-Bla <10 min 7 nM 3XUb-Bla <10 min 5 nM 4XUb-Bla <10 min 3.5 nM

[0212] The kinetic data on fusion protein turnover, together with the steady state concentration measurements, demonstrate that the fusion of a multiubiquitin destabilization domain to a target protein allows for the manipulation of both the intracellular concentration, as well as, the turnover kinetics of the resulting fusion proteins. The present invention provides for a method of regulating the intracellular concentration of any target protein within a cell, independently of the rate of transcription of that protein. Unlike other systems of regulating the intracellular concentrations of target proteins, the present invention provides for the ability to "preset" the final concentration of the target protein within a ten-fold range of expression.

[0213] The data with multiubiquitinG76V-β-lactamase fusions demonstrate that fusions containing one to four copies of ubiquitinG76V fused to β-lactamase results in chimeric proteins with half-lives in vivo of from 5 to 30 minutes. There are likely to be applications that require proteins that have a half-life longer than that obtained with fusion to one copy of ubiquitinG76V. For such instances, it would be useful to have a form of uncleavable ubiquitin that is recognized by E2/E3 ubiquitin ligases with lower affinity and therefore result in less destabilization than with fusions to ubiquitinG76V. The efficient recognition and degradation of proteins by the proteasome requires the formation of extended polyubiquitin chains that are extended in isopeptide linkage between a critical lysine residue on ubiquitin to the C-terminus of the incoming ubiquitin. The internal lysine in ubiquitin most often used in such polyubiquitin chains is lysine-48. In order to create a longer half-life protein, it is recognized that it is possible to mutagenize the ubiquitin homolog fused to the protein of interest such that it is not recognized by E2/E3 ubiquitin ligases as efficiently as wild-type ubiquitin. It is likely that mutagenesis of lysine-48, (to Arg, His, Gin or Asn for example) and/or the residues surrounding it will yield a form of ubiquitin that is recognized and extended with lower affinity, than the non-mutant forms. The non extandable homologs would thus serve to create fusion proteins with longer half lives than is otherwise possible with wild-type ubiquitin. Typically such constructs would contain between one and five copies of the non-extendable, non-cleavable ubiquitin homologs to provide for a wide range of destabilization.

[0214] Alternatively, random mutagenesis of the ubiquitin or mutation of other lysines in ubiquitin may result in a form of ubiquitin with the desired properties.

Example 9

Characterization of the Stability of Multimerized Destabilization Domain-Naturally Fluorescent Protein Fusions within Cells

[0215] UbiquitinG76V-GFP constructs in pcDNA3 (SEQ. ID. NOs. 31 to 34) were introduced into CHO cells by Lipofectamine (Life Technologies) transfection. Stable transfectants were selected in RPMI 1640 media containing 10% fetal bovine serum (Gibco) and 0.8 mg/ml G418 (Geneticin, Gibco). Analysis of GFP fluorescence in CHO cells stably transfected with various ubiquitinG76V-GFP constructs was analyzed by flow cytometry on a Becton Dickinson FACS® Vantage® with a Coherent Enterprise II® argon laser producing 60 mW of 488 nm UV excitation. The flow cytometer was equipped with pulse processing and the Macrosort® flow cell. Fluorescence emission was detected via 530/30 nm emission filter. The FACS analyses of stable populations determined that the steady state percentage of bright green GFP+ cells varied depending on the presence of the multiubiquitin destabilization domain. The relative percentages of GFP+ cells are shown in the Table 7.

TABLE-US-00008 TABLE 7 Stable CHO cell line % GFP.sup.+ cells Wild-type GFP 39.13 1XUb-GFP 5.74 2XUb-GFP 3.06 3XUb-GFP 2.2 4XUb-GFP 1.93

[0216] This analysis showed that the relative abundance of cells expressing high steady state levels of GFP fluorescence was inversely proportional to the number of copies of ubiquitinG76V (SEQ. ID. NO. 17) fused to the protein, i.e., the lowest levels of GFP-expressing cells were found in the fusions containing the most copies of ubiquitinG76V (SEQ. ED. NO. 17). The steady state concentration measurements demonstrate that fusions of a multiubiquitin destabilization domain to the highly stable GFP mutant Emerald (SEQ. ID. NO. 28) allows for the predictable and controllable manipulation of the intracellular concentrations of naturally fluorescent proteins.

Example 10

Construction of Destabilization Domain-Linker-Reporter Moiety Fusion Proteins

[0217] Ubiquitin-β-lactamase fusion proteins containing a specific protease cleavage site were constructed by annealing the complementary oligonucleotides DEVD-1 (SEQ. ID. NO. 45) and DEVD-2 (SEQ. ID. NO. 46) that encode a caspase-3-type cleavage site and produce BamH I compatible ends. This oligonucleotide cassette was ligated into BamH I-digested pcDNA3-1-4XUb-Bla plasmid constructs (SEQ. ID. NOs. 23 to 26) described in Example 2. The resulting constructs encode an in-frame fusion protein consisting of from one, to four, copies of ubiquitinG76V (SEQ. ID. NO. 17) separated from β-lactamase by linker containing a caspase-3 cleavage site; the plasmids were designated as pcDNA3-1-4XUb-DEVD-Bla (SEQ. ID. NOs. 47-50). A control linker containing a DEVA site that should not serve as a cleavage site for caspase-3-like proteases was constructed in an identical manner using DEVA1 (SEQ. ED. NO. 51) and DEVA2 primers (SEQ. ED. NO. 52) and the resulting plasmids were designated as pcDNA3-1-4XUb-DEVA-Bla (SEQ. ED. Nos. 53-56).

##STR00001##

Example 11

Detection of Caspase Activity Using Destabilized Reporter Moieties In Vitro

[0218] ³⁵S-labeled ubiquitin-B-lactamase fusion proteins containing a cleavage site for the group II effector caspase-3 were produced by in vitro transcription/translation reactions as described in Example 2 except that plasmids pcDNA3-1-4XUb-DEVD-Bla (SEQ. ID. NOs. 47-50) or control plasmids pcDNA3-1-4XUb-DEVA-Bla (SEQ. ID NOs. 53-56) were used as templates. The ³⁵S-labeled proteins were then used as substrates for purified caspase-3 in an in vitro cleavage reaction. The 12 μl reaction consisted of 4 μl of ³⁵S-labeled ubiquitin-DEVD/A-Bla fusion proteins, 100 mM HEPES pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mM DTT and 25 nM purified recombinant caspase-3. The reactions were incubated at 30° C. and samples taken at 0, 5, 10, 20, 30, 45, and 60 minutes and analyzed by SDS-PAGE and autoradiography. The results from 2XUb-DEVD-Bla and 2XUb-DEVA-Bla fusion proteins are shown in FIG. 7A. The 2XUb-DEVD-Bla fusion served as a very good substrate for caspase-3 with over 90% cleavage within 5 minutes. In contrast, the 2XUb-DEVA-Bla fusion was not cleaved by caspase-3 in vitro, even at extended incubation times. The 2XUb-DEVD-Bla cleavage product seen in FIG. 7A co-migrates on SDS-PAGE gels with β-lactamase fused to the short DEVD linker region (data not shown) and verifies the position of the cleavage site and identifies the labeled cleavage product as the β-lactamase portion of the cleaved fusion. The liberated destabilization domain is much smaller and has run off the gel in this experiment. These data demonstrate that the DEVD fusion serves as an efficient substrate for caspase-3 and the lack of cleavage with the DEVA fusion confirms that the cleavage is occurring ate the DEVD site.

[0219] The protease assay outlined above requires that the protease cleavage result in a stabilization of the catalytic domain of the reporter. To test whether this is the case, we mixed approximately equal portions of cleaved and uncleaved ³⁵S-labeled reporters from in vitro cleavage reactions identical to those in FIG. 7A and then diluted the fragments into crude chase lysate containing cycloheximide to perform a chase experiment. The reactions were incubated at 37° C. and samples were taken at 0, 5, 10, 20, 30 and 60 minutes and analyzed by SDS-PAGE and autoradiography. FIG. 7B shows that the uncleaved intact 2XUb-DEVD-Bla or 2XUb-DEVA-Bla reporters were degraded very rapidly in vitro with a half-life of less than 5 minutes. In contrast, the cleavage product from the 2XUb-DEVD-Bla reporter lacks the destabilization domain and as a result is very stable in vitro. These data confirm that the intact and cleaved versions of the β-lactamase reporters have dramatically different half-lives and provide evidence that this difference in stability may provide a format for assaying endoprotease activity in vivo.

Example 12

Detection of Effector Caspase Protease Activity Using Destabilized Reporter Moieties within Cells

[0220] Plasmids pcDNA3-1-4XUb-DEVD-Bla (SEQ. ED. NOs. 47-50) and pcDNA3-1-4XUb-DEVA-Bla (SEQ. ID. NOs. 53-56) were transfected into Jurkat cells and selected for stable transfectants as described in Example 8. The stable transfectants were sorted by flow cytometry using Becton Dickinson FACS® Vantage® SE and FACS® Vantage® flow cytometers. The FACS® Vantage® SE was equipped with Turbosort Option, pulse processing, ACDU, and Coherent Innova 302C krypton and Coherent Innova 70 Spectrum mixed-gas krypton-argon lasers. The FACS® Vantage® was equipped with pulse processing, ACDU, and Coherent Enterprise II and Coherent Innova 70 Spectrum mixed-gas krypton-argon (with violet option) lasers. For β-lactamase experiments, 60 mW of 413 nm laser emission was used for CCF2 excitation, with a 500 nm dichroic filter separating a 460/50 nm (CCF2 blue fluorescence) and a 535/40 nm bandpass filter (green fluorescence). Single cells with the desired level of B-lactamase expression were sorted into individual wells of 96-well plates using the Automatic Cell Deposition Unit (ACDU) on the FACS® Vantage® and expanded for analysis of homogeneous clonal populations. All results in this Example utilized clonal lines.

[0221] The clonal cell lines were initially screened for expression of β-lactamase and the ability to degrade the Ub-DEVD-Bla or Ub-DEVA-Bla fusion rapidly. This initial screen was accomplished by treating an aliquot of cells with 100 mg/ml cycloheximide followed by incubation at 37° C. for 1 hour (chase period). Treated and untreated cells were loaded with 1 μM CCF2-AM for 1 hour at room temperature and β-lactamase levels were quantified using a CytoFluor microtiter plate fluorimeter (Perseptive Biosystems) using 395/25 nm excitation and 460/40 (blue) nm and 530/30 (green) nm emission filters. Emission ratios were calculated from background-subtracted values (background=media+CCF2 alone) and expressed as a 460/530 nm ratio where a high ratio indicates high β-lactamase activity. This analysis showed that Ub-DEVD-Bla fusions with two or more copies of ubiquitinG76V (SEQ. ID. NO. 17) gave satisfactory chase characteristics, with fusions to two copies of UbiquitinG76V (SEQ. ID. NO. 17) giving the highest steady state levels (no chase) of fusion protein (data not shown). In contrast, 1XUb-DEVD-Bla fusions were not sufficiently destabilized to be usable with this assay format as cells expressing the fusion required extended cycloheximide treatments (data not shown). As the 2-4XUb-DEVD-Bla fusions all exhibited satisfactory rates of proteolytic turnover in cells, the 2× ubiquitinG76V destabilization domain was used with the DEVD-Bla fusions because it gave the best performance (expression levels vs. turnover kinetics) in this particular application. It is worth noting here that due to the variability in the intrinsic stability of different proteins fused to the ubiquitinG76V (SEQ. ED. NO. 17) destabilization domain; fusions of other cellular proteins with multimerized destabilization constructs would be expected to require a dissimilar number of copies of ubiquitinG76V (SEQ. ID. NO. 17) to impart sufficiently rapid turnover kinetics (data not shown). A key advantage of the present invention is the ability to meet this need by varying the number of destabilization domains present within the multimerized destabilization domain construct.

[0222] One clonal cell line from each of 2XUb-DEVD-Bla and 2XUb-DEVA-Bla cell populations was characterized in detail. To establish the background (no β-lactamase) control value, wild-type Jurkat cells containing no β-lactamase activity were loaded with CCF2-AM and the 460/530 fluorescence ratio measured. The value obtained, about 0.05, establishes the background ratio exhibited by cells in the absence of β-lactamase activity. When the 2XUb-DEVD-Bla and 2XUb-DEVA-Bla clones were treated with cycloheximide (chx) for 1 hour at 37° C. prior to CCF2-AM loading, they both exhibited 460/530 ratios very near the background ratio of 0.05, demonstrating that the cells retained the ability to degrade the 2XUb-Bla fusion very efficiently (Table 8).

TABLE-US-00009 TABLE 8 2XUb-DEVD-Bla 2XUb-DEVA-Bla 460/530 emission ratio 460/530 emission ratio no chx 1.80 1.60 + chx 0.07 0.07 +ocFas/-chx 1.25 1.10 +aFas/+chx 0.67 0.12 +aFas/+inh/+chx 0.08 0.09

[0223] The fact that there is a significant difference in stability between the uncleaved reporter and the cleavage product in vitro (FIG. 7B) forms the basis for an assay for protease activity in intact cells. As shown in Table 8, in the absence of caspase activity, both 2XUb-DEVD-Bla and 2X-Ub-DEVA-Bla fusions are rapidly degraded to very low levels in the presence of cycloheximide to inhibit new protein synthesis. Treatment of Jurkat cells with Fas ligand will result in the activation of Fas receptor--an apoptosis signaling receptor found on the surface of a number of cell types that belongs to the tumor necrosis factor (TNF)/nerve growth factor family. Fas activation ultimately leads to the activation of the group II caspases that efficiently cleave substrates containing DEVD recognition motifs. In order to activate this pathway and measure the activity of group II caspases using the DEVD-Bla reporter in intact cells, an anti-Fas antibody (CH-11 anti-Fas IgM; Kamiya Biomedical Co., Seattle, Wash.) was used to cross-link the receptor and stimulate the activation of group II caspases. Western blot analysis of the anti-Fas-treated cells confirmed the proteolytic activation of caspase-3 (data not shown), the major group II caspase activity in Jurkat cells. Treatment of Jurkat cells expressing 2XUb-DEVD-Bla or 2XUb-DEVA-Bla reporter with 50 ng/ml anti-Fas IgM for 6 hours at 37° C. resulted in a modest decrease in the steady-state levels of the reporter (Table 8), most likely due to the inhibition of protein synthesis that is known to accompany apoptosis. At this point, the activation of group II caspases will result in the cleavage and stabilization of some proportion of the DEVD-Bla (but not the control DEVA-Bla) reporters. Treatment of such cells with cycloheximide would then allow for the clearing of the uncleaned, short half-life reporters while leaving the stable cleaved reporters as the sole forms of β-lactamase activity in the cells. Table 8 shows that cycloheximide addition to anti-Fas treated cells (+αFas/+chx) resulted in the stabilization of a significant fraction of the DEVD-Bla reporters while the DEVA-Bla reporters cannot be cleaved and stabilized. To show that the stabilization of the DEVD-Bla reporters is due to caspase activation, we used the peptide inhibitor Z-VAD-fmk (Enzyme Systems Products, Livermore, Calif.) that is a potent broad inhibitor of caspases. Treatment of the cells with 10 μM Z-VAD-fmk coincident with anti-Fas addition blocked the stabilization of DEVD-Bla reporters. Treatment of the cells with cycloheximide resulted in the degradation of the non cleaved constructs to background levels of β-lactamase activity (+αFas/+Inh/+chx). Comparison of β-lactamase levels in antiFas-treated DEVD-Bla-expressing cells in the presence or absence of Z-VAD-fmk inhibitor determines the dynamic range of the assay; in this particular experiment the dynamic range is approximately 8-fold. These data demonstrate that the cleavage and stabilization of short half-life β-lactamase protease reporters provides a sensitive and specific assay for measuring the activation of caspases in intact cells.

[0224] It is of note that this assay format would permit the identification of compounds that stimulate group II caspases and subsequent apoptosis (agonist/inducer format) as well as compounds that inhibit caspase activity stimulated by a known reagent such as anti-Fas IgM (antagonist/inhibitor format). As evidence for this assay being useful for both inducer and inhibitor applications, we generated dose-response curves for both an inducer of caspases and apoptosis (anti-Fas IgM) and an inhibitor of anti-Fas induced apoptosis (Z-VAD-fnk). FIG. 8 shows that the assay in Jurkat cells expressing 2XUb-DEVD-Bla generates sufficient dynamic range to detect low concentrations of the inducer anti-Fas IgM (EC₅₀=11 ng/ml). In addition, treatment of Jurkat cells expressing 2XUb-DEVD-Bla with 50 ng/ml anti-Fas IgM allows sensitive detection of inhibition by Z-VAD-fink with IC₅₀=5 μM (FIG. 8).

Example 13

Creation of Reporters for Viral Self-Cleaving Proteases Using Multimerized Destabilization Domain-B-Lactamase-Rhinovirus 2A Protease Fusions

[0225] The gene encoding the human rhinovirus 14 2A protease (SEQ. ED. NO. 57) was isolated by PCR amplification from genomic RNA by RT-PCR using oligonucleotides HRV145' (SEQ. ID. NO. 58) and HRV143', (SEQ. ID. NO. 59). The resulting PCR product had BamH I sites at both ends of the HRV14 2A protease sequence and could be inserted in frame into the pcDNA3-1-4XUb-Bla vectors (SEQ. ID. Nos. 23-26) from example 2. The PCR fragment from this reaction was digested with BamH I and Hgated into pcDNA3-3XUb-Bla (SEQ. ID. NO. 25). The resulting construct, pcDNA3-3XUb-Bla HRV14 (SEQ. ID. NO. 60) was further characterized in vitro and within cells.

[0226] In addition to the HRV14 2A protease constructs, two additional constructs were made for the HRV16 2A protease. The gene for the human rhinovirus 16 sequence 2A protease (SEQ. ED. NO. 61) was isolated by polymerase chain reaction (PCR) amplification of a plasmid template. The PCR template was a plasmid construct containing the entire HRV 16 genome (a gift from Dr. Wai Ming Ixe at the University of Wisconsin). Oligonucleotides HRV 165' (SEQ. ED. NO. 62) and HRV163', (SEQ. ID. NO. 63) were used in a PCR reaction with the HRV16 plasmid resulting in a PCR product that had BamH I sites at both ends of the HRV16 2A protease sequence. The PCR fragment from this reaction was digested with BamH I and ligated into pcDNA3-3XUb-Bla (SEQ. ED. NO. 25) and pcDNA3-Ub-Met Bla (SEQ. ED. NO. 27) via the BamH I site. The resulting constructs were pcDNA3-3XUb-Bla HRV16 (SEQ. ID. NO. 64) and pcDNA3-Ub-Met-Bla HRV16 (SEQ. ED. NO. 65). In addition, two mutant constructs were made for the HRV16 2A protease. These mutants corresponded to mutations at two residues of the putative catalytic triad for the 2A protease and should result in a catalytically inactive mutant, specifically, aspartate 35 was mutated to alanine (D35A) and cysteine 106 was mutated to alanine (C106A). These derivatives were generated by mutagenesis of the HRV16 2A protease using oligonucleotide HRV16 D35A (SEQ. ID. NO. 66) and oligonucleotide HRV16 C106A (SEQ. ID. NO. 67). The resulting plasmids were designated as pcDNA3-3XUb-Bla HRV16(C106A) (SEQ. ID. NO. 68), pcDNA3-3XUb-Bla HRV16(D35A) (SEQ. ID. NO. 69), pcDNA3-Ub-Met-Bla HRV16(C106A) (SEQ. ID. NO. 70) and pcDNA3-Ub-Met-Bla HRV16(D35A) (SEQ. ID NO. 71).

Example 14

Detection of Rhinovirus Protease Activity Using Destabilized Reporter Moieties In Vitro

[0227] ³⁵S-labeled ubiquitin-β-lactamase fusion proteins containing the HRV14 and HRV16 2A proteases, as well as the mutants above, were produced by in vitro transcription/translation reactions as described in Example 5. The plasmids pcDNA3-3XUb-Bla HRV16 (SEQ. ID. NO. 64), pcDNA3-3XUb-Bla HRV16(C106A) (SEQ. ID. NO. 68), pcDNA3-3XUb-Bla HRV16(D35A) (SEQ. ID. NO. 69), pcDNA3-Met Ub-Bla HRV16 (SEQ. ID. NO. 65), pcDNA3-3XUb-Bla HRV14 (SEQ. ID. NO. 60), and pcDNA3-MetUb-Bla HRV14 (SEQ. ID. NO. 72) were used as templates. The reactions were incubated at 30° C. for 45 min and analyzed by SDS-PAGE and autoradiography. FIG. 9A shows the results of TNT synthesis reactions for the wild-type HRV16 2A and the two mutant HRV16 2A constructs. Shown are the levels of expression for the stable (Met) and destabilized 3× ubiquitinG76V HRV16 2A-Bla fusions. As expected, the level of expression is higher in the stable methionine containing constructs than the destabilized 3XUb constructs (FIG. 9A). The wild-type HRV16 2A fusions also show significant accumulation of the lower molecular weight stable cleavage product indicating that the fusions exhibit robust autocatalytic cleavage activity in these in vitro reactions. In contrast, mutation of residues in the putative catalytic triad (aspartate 35 and cysteine 106) blocked formation of the stable cleavage product, indicating that these mutants are indeed catalytically inactive.

[0228] The protease assay outlined in Example 10 requires that protease cleavage results in a stabilization of the catalytic domain of the reporter. To test for this requirement the pcDNA3-3XUb-Bla HRV14 TNT reaction was diluted into chase lysate containing cycloheximide to perform a chase experiment. The reactions were incubated at 37° C. for 60 minutes and analyzed by SDS-PAGE and autoradiography. FIG. 9B shows that the uncleaved intact 3XUb-HRV14-Bla reporter was completely degraded during the 60 minute chase. In contrast, the cleavage product from the 3XUb-HRV14-Bla reporter lacks the destabilization domain, and as a result, is stable in vitro. These data confirm that the intact and cleaved versions of the HRV 2A-β-lactamase fusion reporters have dramatically different half-lives and provide evidence that this difference in stability can provide the basis for assaying self-cleaving protease activity in side intact cells.

Example 15

Detection of Rhinovirus Protease Activity Using Destabilized Reporter Moieties In Vivo

[0229] The biochemical properties of self-cleaving cis proteases such as rhinovirus 2A pose several technical challenges that have hampered the development of a screening format to allow for the identification of inhibitors or activators in cell based assays. First, the activity of the protease is directed toward cleavage of itself. This rules out the use of separate reporters that are cleaved in trans and limit the catalytic output of the assay, i.e., a single protease molecule generates a single cleavage product and this fact eliminates the catalytic amplification used in traditional assays for trans-cleaving proteases. In order to address these limitations, the β-lactamase reporters are incorporated into the 2A protease itself, thereby measuring the cis cleavage reaction directly and gaining the advantage of a catalytic reporter that can cleave many CCF2 substrate molecules per reporter. Since the HRV 2A protease undergoes the self-cleavage reaction immediately upon synthesis, the screening assay must be performed on newly synthesized HRV 2A-β-lactamase reporters. A screen to identify inhibitors of the protease must incorporate a step where test compounds are added and their effect then measured. As cleaved stable β-lactamase reporters will accumulate in the cell as the HRV 2A-Bla reporters are being constitutively expressed, it is essential to eliminate the readout due to such cleavage products that are generated before the test compound is added. To do this, the β-lactamase inhibitor clavulanate was used. Clavulanate is a non-cytotoxic irreversible inhibitor of fi-lactamase and overnight treatment of Jurkat cells reduces β-lactamase levels to background (See commonly owned U.S. patent application Ser. No. 09/067,612 filed Apr. 28, 1998). Therefore, clavulanate treatment of Jurkat cells expressing HRV 2A-Bla fusions eliminates the β-lactamase activity that is present in the cell resulting from both uncleaved and cleaved β-lactamase reporters. In essence, this has the effect of "zeroing out" the β-lactamase activity in the cells and bringing the cells back down to baseline activity. The clavulanate can then be washed out and test compound added. New synthesis of HRV 2A-Bla reporters will result in the accumulation of the fusion protein reporter in the cells and the self-cleavage reaction will now be subject to inhibition by the test compound. After an appropriate interval to allow for the cleavage of newly synthesized reporters has passed, the cells can be treated with cycloheximide to clear out the unstable uncleaved reporters and the resulting β-lactamase activity will be due exclusively to cleaved, stabilized reporters.

[0230] Plasmids pcDNA3-3XUb-Bla HRV 16 (SEQ. ID. NO. 64) and pcDNA3-3XUb-Bla HRV 14 (SEQ. ID. NO. 60) were transfected into Jurkat cells and selected for stable transfectants as described in Example 8. The stable transfectants were sorted by flow cytometry using Becton Dickinson FACS® Vantage® SE and FACS® Vantage® flow cytometers. The FACS® Vantage® SE was equipped with Turbosort Option, pulse processing, and Coherent Innova 302C krypton and Coherent Innova 70 Spectrum mixed-gas krypton-argon lasers. The FACS® Vantage® was equipped with pulse processing, and Coherent Enterprise II and Coherent Innova 70 Spectrum mixed-gas krypton-argon (with violet option) lasers. For β-lactamase experiments, 60 mW of 413 nm laser emission was used for CCF2 excitation, with a 500 nm dichroic filter separating a 460/50 nm (CCF2 blue fluorescence) and a 535/40 nm bandpass filter (CCF2 green fluorescence). Single cells with the desired level of β-lactamase expression were sorted into individual wells of 96-well plates using the Automatic Cell Deposition Unit (ACDU) on the FACS® Vantage® and expanded for analysis as homogeneous clonal populations. All results in Example 15 utilized clonal lines.

[0231] Selected clones (25-50 for each construct) were then expanded further for analysis. Clones were treated for 16 hours with 300 μM clavulanate, washed twice with phosphate buffered saline (PBS), incubated for 2 hours at 37° C., treated for 1 hour at 37° C. with 100 μg/ml cycloheximide, and then loaded with CCF2-AM for 2 hours at room temperature. The individual clones were then screened visually by fluorescence microscopy. At least 24 individual clones were tested in this manner for each construct and one clone chosen for each construct.

[0232] To assay HRV 2A protease activity, the selected Jurkat stable cell clones were treated for 16 hours with 300 fxM clavulanate to inactivate pre-existing cleaved and uncleaved HRV 2A-Bla fusion protein. Cells were then washed twice with PBS, resuspended at 100,000 cells/well in 100 μl RPMI+10% FBS in 96-well plates. The cells were incubated at 37° C. for 4 hours in the presence or absence of an inhibitor of the 2A protease. Cells were treated with 100 μg/ml cycloheximide for 30 minutes at 37° C., loaded with CCF2-AM for 2 hours at room temperature and read on the CytoFluor plate reader as described in Example 8. Inhibitor compounds, radicicol and geldanamycin, were used for the validation of the HRV protease cell-based assay. These compounds are known inhibitors of the Hsp90 heat shock protein (see Roe et al., (1999) J. Med. Chem. 42 260-266), which is required for the folding and regulation of a number of cellular proteins and can inhibit HRV 2A protease activity in vitro (data not shown). Compounds were tested at 1 μM for their ability to inhibit the HRV 2A protease cell-based assay using clones expressing HRV16 and HRV14 2A protease reporters. Jurkat cells expressing 3XUb-Bla-HRV14 or HRV16 2A protease fusion proteins contained significant β-lactamase activity in the absence of the inhibitors (Table 9). Both radicicol and geldanamycin showed strong inhibition of cellular β-lactamase activity remaining after the cycloheximide chase. The inhibitors are not simply inhibiting β-lactamase enzyme activity because control experiments showed that radicicol and geldanamycin did not inhibit β-lactamase activity in Jurkat cells expressing wild-type β-lactamase (data not shown). These data demonstrate that the β-lactamase activity present after a cycloheximide chase is due to HRV 2A protease activity and that this β-lactamase activity can be blocked using inhibitors of HRV 2A protease. These results further demonstrate that Jurkat cells expressing 3XUb-Bla HRV 2A fusion proteins constitute a robust cell-based assay for HRV 2A cis-protease activity. The difference in β-lactamase activity between untreated and inhibitor-treated cells determines the dynamic range of this assay; in this particular experiment, the assay dynamic range is approximately 6-fold.

TABLE-US-00010 TABLE 9 3XUb-HRV14-Bla 3XUb-HRV16-Bla 460/530 nm ratio 460/530 nm ratio no inhibitor 1.022 0.895 + radicicol 0.152 0.229 + geldanamycin 0.153 0.239

Example 16

Detection of Proteasome Activity within Cells Using Destabilized Reporter Moieties and Use in the Identification of Proteasome Inhibitors

[0233] A direct application of the destabilized reporter fusions is in the measurement of the activity of the proteolytic activity that responsible for the constitutive degradation of the reporter in cells. Ubiquitinated proteins are known to be degraded by the multi-subunit proteasome. In addition, the proteasome is responsible for the degradation of the large majority of cellular proteins see Lee and Goldberg, (1998) Trends Cell Biol., 8 397-403). The proteasome itself has been implicated in a number of pathological conditions resulting from either increased or decreased proteasome activity (see Ciechanover, (1998) EMBO J. 17 7151-7160). As such, the proteasome represents an attractive target for intervention in pathological conditions using small molecule inhibitors or activators.

[0234] Inhibitors of the proteasome were initially tested in vitro for inhibition of degradation of 2XUb-Bla. Transcription/translation reactions on the pcDNA3-2XUb-Bla (SEQ. ID. NO. 24) construct were preformed as described in Example 5. The ³⁵S-labeled synthesis reactions were diluted into crude chase lysates in the presence of cycloheximide and inhibitor and incubated at 37° C. for 20 minutes. Samples were then analyzed by SDS-PAGE and autoradiography. FIG. 10 shows that >90% of the starting ³⁵S-labeled fusion protein is degraded by the 20 minute time point in the absence of proteasome inhibitors. Addition of the inhibitor MG132 (Calbiochem) at 50 μM resulted in a significant increase in the intact, un-conjugated fusion protein as well as the appearance of high molecular weight labeled species that represent extensive further ubiquitination of the fusion protein. The high molecular weight ubiquitin conjugates accumulate prominently in the presence of MG132 because they are recognized so efficiently by the proteasome that they are barely visible without inhibiting their degradation. Additional proteasome inhibitors gave very similar results: 10 μM lactacystin (3-lactone (Calbiochem) and 50 μM Ac-LLN (Sigma) stabilized the 2XUb-Bla fusion protein and caused the accumulation of high molecular weight ubiquitin conjugates.

[0235] Proteins destined to be degraded by the proteasome are initially modified by the covalent addition of ubiquitin to lysines within the targeted protein through an isopeptide linkage between the C-terminal residue of ubiquitin and the e-amino groups of the substrate protein. The conjugated ubiquitin(s) acts as a high affinity conjugation site for the addition of additional ubiquitin polypeptides in isopeptide linkage between the C-terminus of the incoming ubiquitin to a lysine residue within the conjugated ubiquitin. When the ubiquitin chains reach a critical size four or more ubiquitin residues long (see Thrower et al., (2000) EMBO J. 19 94-102)), the ubiquitin-protein conjugate is recognized by the proteasome with high affinity, the substrate protein is degraded and the ubiquitin residues are recycled for further rounds of ubiquitination. To test whether poly-ubiquitination is required for the degradation of 2XUb-Bla, we used a form of ubiquitin where all amines had been reductively methylated, thereby producing a form of ubiquitin that can be conjugated but not extended. When methylated ubiquitin (MeUb) was added to the in vitro degradation system at 1 mg/ml, it significantly stabilized 2XUb-Bla and resulted in the appearance of ladders of labeled species that contain low (1-5 copies) numbers of conjugated ubiquitin polypeptides. (FIG. 10) It also inhibited the formation of the very high molecular weight ubiquitin-substrate conjugates observed with the proteasome inhibitors. Collectively, the in vitro inhibitor data demonstrate that the multiubiquitin destabilization domain targets degradation of the protein it is fused to in a proteasome-dependent manner that requires poly-ubiquitination of the substrate for high efficiency recognition/degradation.

[0236] Jurkat cells expressing 2XUb-Bla fusion protein were used to test several inhibitors of proteasome function that were active in the in vitro system to determine if they were also active within living cells. Cells were treated with various concentrations of the proteasome inhibitors MG132 or Ac-LLN for 30 minutes at 37° C. and then cycloheximide was added to 100 μg/ml to initiate a chase period. After 1 hour at 37° C., the cells were cooled to room temperature and then loaded with 1 μM CCF2-AM and β-lactamase activity quantified using a CytoFluor plate reader. The background-subtracted emission values at 460 nm and 530 nm were expressed as a 460/530 ratio and dose-response curves were plotted. FIG. 11 shows that both MG132 and Ac-LLN exhibited a dose-dependent inhibition of the decay of β-lactamase activity indicating that they had inhibited the intracellular degradation of the ubiquitin-β-lactamase fusion protein. IC₅₀ values calculated from linear regression analysis were found to be 13 μM for Ac-LLN and 2.1 μM for MG132 and are within the characteristic range for inhibition of substrates degraded by the proteasome (see Lee and Goldberg, (1998) Trends Cell Biol., 8 397-403). These data demonstrate that the multiubiquitin destabilization domain fused to β-lactamase can serve as a robust cell-based 96-well format screening assay for inhibitors of the proteasome.

[0237] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

Sequence CWU 1

7416PRTArtificial SequenceSynthetic construct 1Asp Ser Gly Leu Asp Ser1 52228DNAArtificial SequenceSynthetic construct 2atggagatct tcgtgaagac tctgactggt aagaccatca ccctcgaagt ggagccgagt 60gacaccattg agaatgtcaa ggcaaagatc caagacaagg aaggcatccc tcctgaccag 120cagaggttga tctttgctgg gaaacagctg gaagatggac gcaccctgtc tgactacaac 180atccagaaag agtccaccct gcacctggta ctccgtctca gaggtggg 2283795DNAArtificial SequenceCDS(1)...(795)Synthetic construct cloning vector 3atg agt cac cca gaa acg ctg gtg aaa gta aaa gat gct gaa gat cag 48Met Ser His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln1 5 10 15ttg ggt gca cga gtg ggt tac atc gaa ctg gat ctc aac agc ggt aag 96Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys 20 25 30atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc act 144Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr 35 40 45ttt aaa gtt ctg cta tgt ggc gcg gta tta tcc cgt gtt gac gcc ggg 192Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Val Asp Ala Gly 50 55 60caa gag caa ctc ggt cgc cgc ata cac tat tct cag aat gac ttg gtt 240Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val65 70 75 80gag tac tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta 288Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val 85 90 95aga gaa tta tgc agt gct gcc ata acc atg agt gat aac act gcg gcc 336Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala 100 105 110aac tta ctt ctg aca acg atc gga gga ccg aag gag cta acc gct ttt 384Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe 115 120 125ttg cac aac atg ggg gat cat gta act cgc ctt gat cgt tgg gaa ccg 432Leu His Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro 130 135 140gag ctg aat gaa gcc ata cca aac gac gag cgt gac acc acg atg cct 480Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro145 150 155 160gca gca atg gca aca acg ttg cgc aaa cta tta act ggc gaa cta ctt 528Ala Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu 165 170 175act cta gct tcc cgg caa caa tta ata gac tgg atg gag gcg gat aaa 576Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys 180 185 190gtt gca gga cca ctt ctg cgc tcg gcc ctt ccg gct ggc tgg ttt att 624Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile 195 200 205gct gat aaa tct gga gcc ggt gag cgt ggg tct cgc ggt atc att gca 672Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala 210 215 220gca ctg ggg cca gat ggt aag ccc tcc cgt atc gta gtt atc tac acg 720Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr225 230 235 240acg ggg agt cag gca act atg gat gaa cga aat aga cag atc gct gag 768Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu 245 250 255ata ggt gcc tca ctg att aag cat tgg 795Ile Gly Ala Ser Leu Ile Lys His Trp 260 2654858DNAArtificial SequenceCDS(1)...(858)Synthetic construct cloning vector 4atg aga att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca 48Met Arg Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala1 5 10 15ttt tgc ctt cct gtt ttt ggt cac cca gaa acg ctg gtg aaa gta aaa 96Phe Cys Leu Pro Val Phe Gly His Pro Glu Thr Leu Val Lys Val Lys 20 25 30gat gct gaa gat cag ttg ggt gca cga gtg ggt tac atc gaa ctg gat 144Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp 35 40 45ctc aac agc ggt aag atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt 192Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 50 55 60cca atg atg agc act ttt aaa gtt ctg cta tgt ggc gcg gta tta tcc 240Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser65 70 75 80cgt gtt gac gcc ggg caa gag caa ctc ggt cgc cgc ata cac tat tct 288Arg Val Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 85 90 95cag aat gac ttg gtt gag tac tca cca gtc aca gaa aag cat ctt acg 336Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 100 105 110gat ggc atg aca gta aga gaa tta tgc agt gct gcc ata acc atg agt 384Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser 115 120 125gat aac act gcg gcc aac tta ctt ctg aca acg atc gga gga ccg aag 432Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys 130 135 140gag cta acc gct ttt ttg cac aac atg ggg gat cat gta act cgc ctt 480Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu145 150 155 160gat cgt tgg gaa ccg gag ctg aat gaa gcc ata cca aac gac gag cgt 528Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg 165 170 175gac acc acg atg cct gca gca atg gca aca acg ttg cgc aaa cta tta 576Asp Thr Thr Met Pro Ala Ala Met Ala Thr Thr Leu Arg Lys Leu Leu 180 185 190act ggc gaa cta ctt act cta gct tcc cgg caa caa tta ata gac tgg 624Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp 195 200 205atg gag gcg gat aaa gtt gca gga cca ctt ctg cgc tcg gcc ctt ccg 672Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 210 215 220gct ggc tgg ttt att gct gat aaa tct gga gcc ggt gag cgt ggg tct 720Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser225 230 235 240cgc ggt atc att gca gca ctg ggg cca gat ggt aag ccc tcc cgt atc 768Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile 245 250 255gta gtt atc tac acg acg ggg agt cag gca act atg gat gaa cga aat 816Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn 260 265 270aga cag atc gct gag ata ggt gcc tca ctg att aag cat tgg 858Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp 275 280 2855795DNAArtificial SequenceCDS(1)...(795)Synthetic construct cloning vector 5atg ggg cac cca gaa acg ctg gtg aaa gta aaa gat gct gaa gat cag 48Met Gly His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln1 5 10 15ttg ggt gca cga gtg ggt tac atc gaa ctg gat ctc aac agc ggt aag 96Leu Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys 20 25 30atc ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc act 144Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr 35 40 45ttt aaa gtt ctg cta tgt ggc gcg gta tta tcc cgt gat gac gcc ggg 192Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Asp Asp Ala Gly 50 55 60caa gag caa ctc ggt cgc cgc ata cac tat tct cag aat gac ttg gtt 240Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val65 70 75 80gag tac tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta 288Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val 85 90 95aga gaa tta tgc agt gct gcc ata acc atg agt gat aac act gcg gcc 336Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala 100 105 110aac tta ctt ctg aca acg atc gga gga ccg aag gag cta acc gct ttt 384Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe 115 120 125ttg cac aac atg ggg gat cat gta act cgc ctt gat cat tgg gaa ccg 432Leu His Asn Met Gly Asp His Val Thr Arg Leu Asp His Trp Glu Pro 130 135 140gag ctg aat gaa gcc ata cca aac gac gag cgt gac acc acg atg cct 480Glu Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro145 150 155 160gta gca atg gca aca acg ttg cgc aaa cta tta act ggc gaa cta ctt 528Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu 165 170 175act cta gct tcc cgg caa caa tta ata gac tgg atg gag gcg gat aaa 576Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys 180 185 190gtt gca gga cca ctt ctg cgc tcg gcc ctt ccg gct ggc tgg ttt att 624Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile 195 200 205gct gat aaa tct gga gcc ggt gag cgt ggg tct cgc ggt atc att gca 672Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala 210 215 220gca ctg ggg cca gat ggt aag ccc tcc cgt atc gta gtt atc tac acg 720Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr225 230 235 240acg ggg agt cag gca act atg gat gaa cga aat aga cag atc gct gag 768Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu 245 250 255ata ggt gcc tca ctg att aag cat tgg 795Ile Gly Ala Ser Leu Ile Lys His Trp 260 2656792DNAArtificial SequenceCDS(1)...(792)Synthetic construct cloning vector 6atg gac cca gaa acg ctg gtg aaa gta aaa gat gct gaa gat cag ttg 48Met Asp Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu1 5 10 15ggt gca cga gtg ggt tac atc gaa ctg gat ctc aac agc ggt aag atc 96Gly Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile 20 25 30ctt gag agt ttt cgc ccc gaa gaa cgt ttt cca atg atg agc act ttt 144Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe 35 40 45aaa gtt ctg cta tgt ggc gcg gta tta tcc cgt att gac gcc ggg caa 192Lys Val Leu Leu Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln 50 55 60gag caa ctc ggt cgc cgc ata cac tat tct cag aat gac ttg gtt gag 240Glu Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu65 70 75 80tac tca cca gtc aca gaa aag cat ctt acg gat ggc atg aca gta aga 288Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg 85 90 95gaa tta tgc agt gct gcc ata acc atg agt gat aac act gcg gcc aac 336Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn 100 105 110tta ctt ctg aca acg atc gga gga ccg aag gag cta acc gct ttt ttg 384Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu 115 120 125cac aac atg ggg gat cat gta act cgc ctt gat cat tgg gaa ccg gag 432His Asn Met Gly Asp His Val Thr Arg Leu Asp His Trp Glu Pro Glu 130 135 140ctg aat gaa gcc ata cca aac gac gag cgt gac acc acg atg cct gta 480Leu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val145 150 155 160gca atg gca aca acg ttg cgc aaa cta tta act ggc gaa cta ctt act 528Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr 165 170 175cta gct tcc cgg caa caa tta ata gac tgg atg gag gcg gat aaa gtt 576Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val 180 185 190gca gga cca ctt ctg cgc tcg gcc ctt ccg gct ggc tgg ttt att gct 624Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala 195 200 205gat aaa tct gga gcc ggt gag cgt ggg tct cgc ggt atc att gca gca 672Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala 210 215 220ctg ggg cca gat ggt aag ccc tcc cgt atc gta gtt atc tac acg acg 720Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr225 230 235 240ggg agt cag gca act atg gat gaa cga aat aga cag atc gct gag ata 768Gly Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile 245 250 255ggt gcc tca ctg att aag cat tgg 792Gly Ala Ser Leu Ile Lys His Trp 2607786DNAArtificial SequenceCDS(1)...(786)Synthetic construct cloning vector 7atg aaa gat gat ttt gca aaa ctt gag gaa caa ttt gat gca aaa ctc 48Met Lys Asp Asp Phe Ala Lys Leu Glu Glu Gln Phe Asp Ala Lys Leu1 5 10 15ggg atc ttt gca ttg gat aca ggt aca aac cgg acg gta gcg tat cgg 96Gly Ile Phe Ala Leu Asp Thr Gly Thr Asn Arg Thr Val Ala Tyr Arg 20 25 30ccg gat gag cgt ttt gct ttt gct tcg acg att aag gct tta act gta 144Pro Asp Glu Arg Phe Ala Phe Ala Ser Thr Ile Lys Ala Leu Thr Val 35 40 45ggc gtg ctt ttg caa cag aaa tca ata gaa gat ctg aac cag aga ata 192Gly Val Leu Leu Gln Gln Lys Ser Ile Glu Asp Leu Asn Gln Arg Ile 50 55 60aca tat aca cgt gat gat ctt gta aac tac aac ccg att acg gaa aag 240Thr Tyr Thr Arg Asp Asp Leu Val Asn Tyr Asn Pro Ile Thr Glu Lys65 70 75 80cac gtt gat acg gga atg acg ctc aaa gag ctt gcg gat gct tcg ctt 288His Val Asp Thr Gly Met Thr Leu Lys Glu Leu Ala Asp Ala Ser Leu 85 90 95cga tat agt gac aat gcg gca cag aat ctc att ctt aaa caa att ggc 336Arg Tyr Ser Asp Asn Ala Ala Gln Asn Leu Ile Leu Lys Gln Ile Gly 100 105 110gga cct gaa agt ttg aaa aag gaa ctg agg aag att ggt gat gag gtt 384Gly Pro Glu Ser Leu Lys Lys Glu Leu Arg Lys Ile Gly Asp Glu Val 115 120 125aca aat ccc gaa cga ttc gaa cca gag tta aat gaa gtg aat ccg ggt 432Thr Asn Pro Glu Arg Phe Glu Pro Glu Leu Asn Glu Val Asn Pro Gly 130 135 140gaa act cag gat acc agt aca gca aga gca ctt gtc aca agc ctt cga 480Glu Thr Gln Asp Thr Ser Thr Ala Arg Ala Leu Val Thr Ser Leu Arg145 150 155 160gcc ttt gct ctt gaa gat aaa ctt cca agt gaa aaa cgc gag ctt tta 528Ala Phe Ala Leu Glu Asp Lys Leu Pro Ser Glu Lys Arg Glu Leu Leu 165 170 175atc gat tgg atg aaa cga aat acc act gga gac gcc tta atc cgt gcc 576Ile Asp Trp Met Lys Arg Asn Thr Thr Gly Asp Ala Leu Ile Arg Ala 180 185 190gga gcg gca tca tat gga acc cgg aat gac att gcc atc att tgg ccg 624Gly Ala Ala Ser Tyr Gly Thr Arg Asn Asp Ile Ala Ile Ile Trp Pro 195 200 205cca aaa gga gat cct gtc ggt gtg ccg gac ggt tgg gaa gtg gct gat 672Pro Lys Gly Asp Pro Val Gly Val Pro Asp Gly Trp Glu Val Ala Asp 210 215 220aaa act gtt ctt gca gta tta tcc agc agg gat aaa aag gac gcc aag 720Lys Thr Val Leu Ala Val Leu Ser Ser Arg Asp Lys Lys Asp Ala Lys225 230 235 240tat gat gat aaa ctt att gca gag gca aca aag gtg gta atg aaa gcc 768Tyr Asp Asp Lys Leu Ile Ala Glu Ala Thr Lys Val Val Met Lys Ala 245 250 255tta aac atg aac ggc aaa 786Leu Asn Met Asn Gly Lys 2608720DNAAequorea victoria 8atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180ctcgtgacca ccttctccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420aacctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 7209690DNAAnemonia majano 9atggctcttt caaacaagtt tatcggagat gacatgaaaa tgacctacca tatggatggc 60tgtgtcaatg ggcattactt taccgtcaaa ggtgaaggca acgggaagcc atacgaaggg 120acgcagactt cgacttttaa agtcaccatg gccaacggtg ggccccttgc attctccttt 180gacatactat ctacagtgtt caaatatgga aatcgatgct ttactgcgta tcctaccagt 240atgcccgact atttcaaaca agcatttcct gacggaatgt catatgaaag gacttttacc 300tatgaagatg gaggagttgc tacagccagt tgggaaataa gccttaaagg caactgcttt

360gagcacaaat ccacgtttca tggagtgaac tttcctgctg atggacctgt gatggcgaag 420aagacaactg gttgggaccc atcttttgag aaaatgactg tctgcgatgg aatattgaag 480ggtgatgtca ccgcgttcct catgctgcaa ggaggtggca attacagatg ccaattccac 540acttcttaca agacaaaaaa accggtgacg atgccaccaa accatgtggt ggaacatcgc 600attgcgagga ccgaccttga caaaggtggc aacagtgttc agctgacgga gcacgctgtt 660gcacatataa cctctgttgt ccctttctga 69010696DNAZoanthus sp. 10atggctcagt caaagcacgg tctaacaaaa gaaatgacaa tgaaataccg tatggaaggg 60tgcgtcgatg gacataaatt tgtgatcacg ggagagggca ttggatatcc gttcaaaggg 120aaacaggcta ttaatctgtg tgtggtcgaa ggtggaccat tgccatttgc cgaagacata 180ttgtcagctg cctttaacta cggaaacagg gttttcactg aatatcctca agacatagtt 240gactatttca agaactcgtg tcctgctgga tatacatggg acaggtcttt tctctttgag 300gatggagcag tttgcatatg taatgcagat ataacagtga gtgttgaaga aaactgcatg 360tatcatgagt ccaaatttta tggagtgaat tttcctgctg atggacctgt gatgaaaaag 420atgacagata actgggagcc atcctgcgag aagatcatac cagtacctaa gcaggggata 480ttgaaagggg atgtctccat gtacctcctt ctgaaggatg gtgggcgttt acggtgccaa 540ttcgacacag tttacaaagc aaagtctgtg ccaagaaaga tgccggactg gcacttcatc 600cagcataagc tcacccgtga agaccgcagc gatgctaaga atcagaaatg gcatctgaca 660gaacatgcta ttgcatccgg atctgcattg ccctga 69611696DNAZoanthus sp. 11atggctcatt caaagcacgg tctaaaagaa gaaatgacaa tgaaatacca catggaaggg 60tgcgtcaacg gacataaatt tgtgatcacg ggcgaaggca ttggatatcc gttcaaaggg 120aaacagacta ttaatctgtg tgtgatcgaa gggggaccat tgccattttc cgaagacata 180ttgtcagctg gctttaagta cggagacagg attttcactg aatatcctca agacatagta 240gactatttca agaactcgtg tcctgctgga tatacatggg gcaggtcttt tctctttgag 300gatggagcag tctgcatatg caatgtagat ataacagtga gtgtcaaaga aaactgcatt 360tatcataaga gcatatttaa tggaatgaat tttcctgctg atggacctgt gatgaaaaag 420atgacaacta actgggaagc atcctgcgag aagatcatgc cagtacctaa gcaggggata 480ctgaaagggg atgtctccat gtacctcctt ctgaaggatg gtgggcgtta ccggtgccag 540ttcgacacag tttacaaagc aaagtctgtg ccaagtaaga tgccggagtg gcacttcatc 600cagcataagc tcctccgtga agaccgcagc gatgctaaga atcagaagtg gcagctgaca 660gagcatgcta ttgcattccc ttctgccttg gcctga 69612699DNADiscosoma striata 12atgagttgtt ccaagagtgt gatcaaggaa gaaatgttga tcgatcttca tctggaagga 60acgttcaatg ggcactactt tgaaataaaa ggcaaaggaa aaggacagcc taatgaaggc 120accaataccg tcacgctcga ggttaccaag ggtggacctc tgccatttgg ttggcatatt 180ttgtgcccac aatttcagta tggaaacaag gcatttgtcc accaccctga caacatacat 240gattatctaa agctgtcatt tccggaggga tatacatggg aacggtccat gcactttgaa 300gacggtggct tgtgttgtat caccaatgat atcagtttga caggcaactg tttctactac 360gacatcaagt tcactggctt gaactttcct ccaaatggac ccgttgtgca gaagaagaca 420actggctggg aaccgagcac tgagcgtttg tatcctcgtg atggtgtgtt gataggagac 480atccatcatg ctctgacagt tgaaggaggt ggtcattacg catgtgacat taaaactgtt 540tacagggcca agaaggccgc cttgaagatg ccagggtatc actatgttga caccaaactg 600gttatatgga acaacgacaa agaattcatg aaagttgagg agcatgaaat cgccgttgca 660cgccaccatc cgttctatga gccaaagaag gataagtaa 69913678DNADiscosoma sp. 13atgaggtctt ccaagaatgt tatcaaggag ttcatgaggt ttaaggttcg catggaagga 60acggtcaatg ggcacgagtt tgaaatagaa ggcgaaggag aggggaggcc atacgaaggc 120cacaataccg taaagcttaa ggtaaccaag gggggacctt tgccatttgc ttgggatatt 180ttgtcaccac aatttcagta tggaagcaag gtatatgtca agcaccctgc cgacatacca 240gactataaaa agctgtcatt tcctgaagga tttaaatggg aaagggtcat gaactttgaa 300gacggtggcg tcgttactgt aacccaggat tccagtttgc aggatggctg tttcatctac 360aaggtcaagt tcattggcgt gaactttcct tccgatggac ctgttatgca aaagaagaca 420atgggctggg aagccagcac tgagcgtttg tatcctcgtg atggcgtgtt gaaaggagag 480attcataagg ctctgaagct gaaagacggt ggtcattacc tagttgaatt caaaagtatt 540tacatggcaa agaagcctgt gcagctacca gggtactact atgttgactc caaactggat 600ataacaagcc acaacgaaga ctatacaatc gttgagcagt atgaaagaac cgagggacgc 660caccatctgt tcctttaa 67814801DNAClavularia sp. 14atgaagtgta aatttgtgtt ctgcctgtcc ttcttggtcc tcgccatcac aaacgcgaac 60atttttttga gaaacgaggc tgacttagaa gagaagacat tgagaatacc aaaagctcta 120accaccatgg gtgtgattaa accagacatg aagattaagc tgaagatgga aggaaatgta 180aacgggcatg cttttgtgat cgaaggagaa ggagaaggaa agccttacga tgggacacac 240actttaaacc tggaagtgaa ggaaggtgcg cctctgcctt tttcttacga tatcttgtca 300aacgcgttcc agtacggaaa cagagcattg acaaaatacc cagacgatat agcagactat 360ttcaagcagt cgtttcccga gggatattcc tgggaaagaa ccatgacttt tgaagacaaa 420ggcattgtca aagtgaaaag tgacataagc atggaggaag actcctttat ctatgaaatt 480cgttttgatg ggatgaactt tcctcccaat ggtccggtta tgcagaaaaa aactttgaag 540tgggaaccat ccactgagat tatgtacgtg cgtgatggag tgctggtcgg agatattagc 600cattctctgt tgctggaggg aggtggccat taccgatgtg acttcaaaag tatttacaaa 660gcaaaaaaag ttgtcaaatt gccagactat cactttgtgg accatcgcat tgagatcttg 720aaccatgaca aggattacaa caaagtaacg ctgtatgaga atgcagttgc tcgctattct 780ttgctgccaa gtcaggccta g 8011537DNAArtificial Sequenceprimer for PCR 15gatcggtacc accatggaga tcttcgtgaa gactctg 371640DNAArtificial Sequenceprimer for PCR 16tgcaggatcc gtgcatccca cctctgagac ggagtaccag 4017228DNAArtificial SequenceSynthetic construct UbquitinG76V mutant 17atggagatct tcgtgaagac tctgactggt aagaccatca ccctcgaagt ggagccgagt 60gacaccattg agaatgtcaa ggcaaagatc caagacaagg aaggcatccc tcctgaccag 120cagaggttga tctttgctgg gaaacagctg gaagatggac gcaccctgtc tgactacaac 180atccagaaag agtccaccct gcacctggta ctccgtctca gaggtgtg 2281832DNAArtificial Sequenceprimer for PCR 18cgagatctac catggaaatc ttcgtgaaga ct 321922DNAArtificial Sequenceprimer for PCR 19ggatccgtgg tgcacacctc tg 222049DNAArtificial Sequenceprimer for PCR 20gataggatcc ggggcgtggc tgcacccaga aacgctggtg aaagtaaaa 492128DNAArtificial Sequenceprimer for PCR 21gaactctaga ttaccaatgc ttaatcag 28226180DNAArtificial SequenceSynthetic construct pcDNA3-Bla 22gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900gagctcggat ccggggcgtg gctgcaccca gaaacgctgg tgaaagtaaa agatgctgaa 960gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 1020gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 1080ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 1140tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 1200acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 1260cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 1320catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 1380cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa 1440ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 1500ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 1560ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 1620atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 1680gctgagatag gtgcctcact gattaagcat tggtaatcta gagggcccta ttctatagtg 1740tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 1800tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 1860ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 1920gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg 1980ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta 2040tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 2100gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 2160cgccacgttc gccggctttc cccgtcaagc tctaaatcgg ggcatccctt tagggttccg 2220atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 2280tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 2340tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 2400tttataaggg attttgggga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 2460atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc 2520tccccaggca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg 2580aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 2640aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca 2700ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc 2760ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa 2820gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt 2880tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 2940attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 3000gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 3060actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 3120tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 3180gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 3240aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 3300tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 3360cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc 3420cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 3480aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 3540ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 3600cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 3660tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc 3720aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga 3780atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc 3840ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 3900acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 3960atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca 4020tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 4080gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 4140gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 4200atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 4260actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 4320gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 4380cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 4440ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 4500ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 4560ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa 4620tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 4680cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 4740aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 4800gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 4860agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 4920ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 4980cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 5040tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 5100aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 5160tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 5220atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 5280cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 5340gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 5400gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 5460tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 5520tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 5580tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 5640aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 5700atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 5760tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 5820catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 5880aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 5940tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 6000gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 6060tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 6120tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 6180236411DNAArtificial SequenceSynthetic construct pcDNA3-1XUb-Bla 23gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgt gcaccacgga 1140tccggggcgt ggctgcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 1200ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 1260cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 1320ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 1380gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 1440gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 1500acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 1560cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 1620acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 1680ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 1740ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 1800gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 1860atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 1920ggtgcctcac tgattaagca ttggtaatct agagggccct attctatagt gtcacctaaa 1980tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 2040cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 2100aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 2160ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 2220gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc 2280gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 2340acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 2400cgccggcttt ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc 2460tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 2520gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 2580cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 2640gattttgggg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 2700gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc 2760aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc 2820aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt 2880cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc 2940ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct 3000attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg 3060agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat 3120tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta 3180tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca 3240ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga 3300cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga 3360cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct 3420cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg 3480gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga 3540gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca 3600tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga

3660ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg 3720cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc 3780gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt 3840gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga 3900gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca 3960tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc 4020cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac 4080cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc 4140acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta 4200tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag 4260ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 4320ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 4380tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 4440cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 4500ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 4560ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 4620gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 4680gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 4740taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 4800accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 4860tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 4920cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 4980agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 5040gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 5100gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 5160tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 5220acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 5280cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 5340acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 5400acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 5460tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 5520ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 5580ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 5640tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 5700aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 5760ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 5820ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 5880gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 5940gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 6000cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 6060actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 6120ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 6180tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 6240ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 6300agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 6360aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c 6411246678DNAArtificial SequenceSynthetic construct pcDNA3-2XUb-Bla 24gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatcc ggggcgtggc tgcacccaga aacgctggtg 1440aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 1500aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 1560tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 1620ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 1680catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 1740aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 1800ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 1860gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 1920aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 1980gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 2040gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 2100gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 2160gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaatctaga 2220gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc 2280ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 2340tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 2400gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 2460caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag 2520ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 2580ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 2640tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 2700catcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 2760gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 2820ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 2880ctcggtctat tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa 2940tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg 3000tgtggaaagt ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta 3060gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 3120gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 3180tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 3240ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 3300cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga 3360gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc 3420cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 3480tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 3540gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac 3600gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 3660attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 3720atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 3780cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 3840cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 3900gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 3960gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 4020tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 4080cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 4140catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg 4200accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat 4260gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 4320gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 4380aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 4440tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 4500tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 4560attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 4620agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 4680tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 4740tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 4800tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 4860aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 4920tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 4980tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 5040cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 5100agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 5160tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 5220aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 5280ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 5340cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 5400accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 5460ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 5520ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 5580gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 5640aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 5700gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 5760gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 5820cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 5880gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 5940gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 6000ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 6060tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 6120ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 6180cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 6240accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 6300cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 6360tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 6420cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 6480acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 6540atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 6600tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 6660aaagtgccac ctgacgtc 6678256921DNAArtificial SequenceSynthetic construct pcDNA3-3XUb-Bla 25gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tccggggcgt ggctgcaccc agaaacgctg 1680gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat 1740ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 1800acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa 1860ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa 1920aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt 1980gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 2040tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 2100gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg 2160cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 2220atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 2280attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg 2340ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg 2400gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaatct 2460agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt 2520gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 2580aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 2640taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 2700agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 2760cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg 2820tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 2880cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 2940gggcatccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 3000ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 3060gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 3120tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct attggttaaa 3180aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta 3240gggtgtggaa agtccccagg ctccccaggc aggcagaagt atgcaaagca tgcatctcaa 3300ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 3360catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 3420aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 3480agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 3540aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 3600agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 3660ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 3720tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 3780cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac 3840gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 3900gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 3960agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 4020attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 4080tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 4140caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 4200cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 4260gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 4320tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 4380gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa 4440atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc 4500tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 4560ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 4620tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 4680agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 4740agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 4800acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 4860gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 4920tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4980cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 5040gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 5100aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 5160gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 5220aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 5280gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 5340ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 5400cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc

5460ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 5520actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 5580tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 5640gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 5700ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 5760cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 5820ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 5880tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 5940agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 6000gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 6060ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 6120gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 6180cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 6240acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 6300cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 6360cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 6420ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 6480tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 6540atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 6600tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 6660actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 6720aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 6780ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 6840ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 6900cgaaaagtgc cacctgacgt c 6921267164DNAArtificial SequenceSynthetic construct pcDNA3-4XUb-Bla 26gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tctaccatgg aaatcttcgt gaagactctg 1680actggtaaga ccatcactct cgaagtggag ccgagtgaca ccattgagaa tgtcaaggca 1740aagatccaag acaaggaagg catccctcct gaccagcaga ggttgatctt tgctgggaaa 1800cagctggaag atggacgcac cctgtctgac tacaacatcc agaaagagtc caccctgcac 1860ctggtactcc gtctcagagg tgtgcaccac ggatccgggg cgtggctgca cccagaaacg 1920ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 1980gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 2040agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag 2100caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 2160gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 2220agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 2280gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 2340aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 2400ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 2460tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 2520tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 2580gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 2640atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 2700tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac 2760tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 2820ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 2880gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 2940ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 3000aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc 3060gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 3120tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 3180tcggggcatc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 3240tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 3300gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 3360ccctatctcg gtctattctt ttgatttata agggattttg gggatttcgg cctattggtt 3420aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag 3480ttagggtgtg gaaagtcccc aggctcccca ggcaggcaga agtatgcaaa gcatgcatct 3540caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 3600aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc 3660cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 3720tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt 3780tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga 3840tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc 3900tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg 3960ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac 4020cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc 4080cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg 4140gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga 4200gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg 4260cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg 4320tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt 4380cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc 4440ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg 4500gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga 4560gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc 4620gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc 4680gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc 4740ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag 4800cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat 4860ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 4920tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc 4980tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 5040ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 5100tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 5160ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 5220gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 5280gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 5340ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 5400ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 5460cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 5520ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 5580tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 5640gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 5700tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 5760gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 5820tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 5880ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 5940agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 6000gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 6060attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 6120agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 6180atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 6240cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 6300ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 6360agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 6420tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 6480gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 6540caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 6600ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 6660gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 6720tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 6780tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 6840cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 6900cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 6960gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 7020atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 7080agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 7140ccccgaaaag tgccacctga cgtc 7164276411DNAArtificial SequenceSynthetic construct pcDNA3-Ub-Met-Bla 27gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgg gatgcacgga 1140tccggggcgt ggctgcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 1200ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 1260cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 1320ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 1380gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 1440gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 1500acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 1560cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 1620acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 1680ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 1740ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 1800gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 1860atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 1920ggtgcctcac tgattaagca ttggtaatct agagggccct attctatagt gtcacctaaa 1980tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 2040cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 2100aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 2160ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 2220gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc 2280gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 2340acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 2400cgccggcttt ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc 2460tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 2520gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 2580cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 2640gattttgggg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 2700gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc 2760aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc 2820aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt 2880cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc 2940ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct 3000attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg 3060agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat 3120tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta 3180tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca 3240ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga 3300cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga 3360cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct 3420cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg 3480gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga 3540gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca 3600tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga 3660ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg 3720cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc 3780gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt 3840gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga 3900gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca 3960tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc 4020cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac 4080cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc 4140acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta 4200tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag 4260ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 4320ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 4380tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 4440cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 4500ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 4560ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 4620gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 4680gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 4740taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 4800accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 4860tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 4920cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 4980agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 5040gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 5100gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 5160tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 5220acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 5280cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 5340acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 5400acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 5460tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 5520ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 5580ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 5640tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 5700aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 5760ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 5820ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 5880gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 5940gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 6000cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 6060actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 6120ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 6180tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag

6240ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 6300agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 6360aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c 641128720DNAArtificial SequenceSynthetic construct green fluorescent protein mutant Emerald 28atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180ctcgtgacca ccttcaccta cggcgtgcag tgcttcgccc gctaccccga ccacatgaag 240cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420aagctggagt acaactacaa cagccacaag gtctatatca ccgccgacaa gcagaagaac 480ggcatcaagg tgaacttcaa gacccgccac aacatcgagg acggcagcgt gcagctcgcc 540gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 7202924DNAArtificial SequenceSynthetic construct oligonucleotide for PCR 29ggatccgaat tcgccaccat ggtg 243030DNAArtificial SequenceSynthetic construct oligonucleotide for PCR 30ccggaatcaa agcgcttctc agacttactt 30316340DNAArtificial SequenceSynthetic construct pcDNA3-1XUb-GFP 31gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgt gcaccacgga 1140tccgaattcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 1200ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 1260ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 1320gtgccctggc ccaccctcgt gaccaccttc acctacggcg tgcagtgctt cgcccgctac 1380cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 1440gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 1500gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 1560aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaaggtcta tatcaccgcc 1620gacaagcaga agaacggcat caaggtgaac ttcaagaccc gccacaacat cgaggacggc 1680agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 1740ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 1800cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 1860gagctgtaca agtaagtcta gagggcccta ttctatagtg tcacctaaat gctagagctc 1920gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg 1980tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa 2040ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca 2100gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg 2160cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg 2220gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 2280ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 2340cccgtcaagc tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc 2400tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 2460cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 2520ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgggga 2580tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct 2640gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccaggca ggcagaagta 2700tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag 2760caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa 2820ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac 2880taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt 2940agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat 3000ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 3060gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 3120aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 3180ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc 3240ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 3300aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 3360accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 3420ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 3480ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 3540cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg 3600tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 3660tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 3720gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 3780tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag 3840cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt 3900cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg 3960ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt 4020tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 4080atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 4140ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt 4200gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 4260agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 4320tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 4380aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 4440cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4500atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4560taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 4620aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 4680tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 4740gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct 4800cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 4860cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 4920atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 4980tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 5040ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 5100acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 5160aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 5220aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 5280tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 5340cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 5400catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 5460ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5520aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5580ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 5640caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 5700attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 5760agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 5820actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 5880ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 5940ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 6000gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 6060atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 6120cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 6180gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 6240gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 6300ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 6340326607DNAArtificial SequenceSynthetic construct pcDNA3-2XUb-GFP 32gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatcc gaattcgcca ccatggtgag caagggcgag 1440gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 1500aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag 1560ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcacc 1620tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag 1680tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac 1740tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 1800aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac 1860aacagccaca aggtctatat caccgccgac aagcagaaga acggcatcaa ggtgaacttc 1920aagacccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac 1980acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc 2040gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc 2100gccgccggga tcactctcgg catggacgag ctgtacaagt aagtctagag ggccctattc 2160tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc 2220agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 2280ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 2340ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 2400atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta 2460gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 2520gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 2580cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggc atccctttag 2640ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 2700cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 2760tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 2820cttttgattt ataagggatt ttggggattt cggcctattg gttaaaaaat gagctgattt 2880aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc 2940cccaggctcc ccaggcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 3000ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 3060agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 3120ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 3180cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 3240gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag 3300gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg 3360agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt 3420tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc 3480tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt 3540gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag 3600tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg 3660ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag 3720cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg 3780atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc 3840gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca 3900tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc 3960gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg 4020ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct 4080atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc 4140gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg 4200cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct 4260ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa 4320tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 4380caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc 4440gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 4500catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 4560attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 4620ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 4680ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 4740aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 4800aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 4860gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 4920gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 4980tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 5040ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 5100ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 5160tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 5220tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 5280ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 5340aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 5400ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 5460tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 5520atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 5580aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 5640ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 5700tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 5760ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 5820tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 5880aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 5940gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 6000tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 6060cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 6120tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 6180ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 6240cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 6300actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 6360ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 6420aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 6480ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 6540atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 6600tgacgtc 6607336850DNAArtificial SequenceSynthetic construct pcDNA3-3XUb-GFP 33gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga

tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tccgaattcg ccaccatggt gagcaagggc 1680gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 1740cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 1800aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc 1860acctacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc 1920aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 1980aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 2040ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 2100tacaacagcc acaaggtcta tatcaccgcc gacaagcaga agaacggcat caaggtgaac 2160ttcaagaccc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 2220aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 2280tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 2340accgccgccg ggatcactct cggcatggac gagctgtaca agtaagtcta gagggcccta 2400ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt 2460gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 2520ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 2580ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 2640ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 2700ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 2760cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 2820cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg ggcatccctt 2880tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 2940gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 3000cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 3060attcttttga tttataaggg attttgggga tttcggccta ttggttaaaa aatgagctga 3120tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa 3180gtccccaggc tccccaggca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 3240ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 3300attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 3360gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 3420ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 3480tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat 3540gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg 3600tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg 3660tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 3720ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc 3780cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg 3840aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca 3900tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc 3960aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg 4020atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg 4080cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 4140tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg 4200accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 4260gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 4320tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca 4380agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt 4440gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 4500gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag 4560caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 4620gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt 4680ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 4740caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 4800cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 4860gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 4920ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 4980ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 5040agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 5100taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 5160cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 5220tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 5280gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 5340gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 5400tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 5460gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 5520cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 5580aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 5640tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 5700ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5760attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 5820ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 5880tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 5940aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 6000acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 6060aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 6120agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 6180ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 6240agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 6300tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 6360tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 6420attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 6480taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 6540aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 6600caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 6660gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 6720cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 6780tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 6840acctgacgtc 6850347093DNAArtificial SequenceSynthetic construct pcDNA3-4XUb-GFP 34gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tctaccatgg aaatcttcgt gaagactctg 1680actggtaaga ccatcactct cgaagtggag ccgagtgaca ccattgagaa tgtcaaggca 1740aagatccaag acaaggaagg catccctcct gaccagcaga ggttgatctt tgctgggaaa 1800cagctggaag atggacgcac cctgtctgac tacaacatcc agaaagagtc caccctgcac 1860ctggtactcc gtctcagagg tgtgcaccac ggatccgaat tcgccaccat ggtgagcaag 1920ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 1980ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 2040ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 2100ttcacctacg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc 2160ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 2220ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 2280gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 2340aactacaaca gccacaaggt ctatatcacc gccgacaagc agaagaacgg catcaaggtg 2400aacttcaaga cccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 2460cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 2520cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 2580gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaagt ctagagggcc 2640ctattctata gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta 2700gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 2760ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 2820attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 2880gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg 2940gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 3000ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 3060tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc 3120ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 3180atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 3240ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 3300tctattcttt tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc 3360tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg 3420aaagtcccca ggctccccag gcaggcagaa gtatgcaaag catgcatctc aattagtcag 3480caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 3540tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc 3600ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg 3660aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag 3720gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag 3780gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt 3840gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg 3900ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg 3960gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg 4020ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg 4080gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca 4140tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc 4200accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc 4260aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca 4320aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga 4380atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg 4440cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg 4500aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg 4560ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga 4620ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag 4680gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct 4740catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata 4800aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 4860tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag 4920cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 4980acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 5040actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 5100gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 5160cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 5220tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 5280gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 5340ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 5400aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 5460tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 5520ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 5580gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 5640tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 5700caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 5760ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 5820cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 5880ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 5940cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 6000gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 6060aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 6120acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 6180gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 6240cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 6300cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 6360tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 6420cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 6480gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 6540cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 6600ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 6660gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 6720taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 6780gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 6840acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 6900aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 6960cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 7020atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 7080gccacctgac gtc 709335834DNAHomo sapiens 35atggagaaca ctgaaaactc agtggattca aaatccatta aaaatttgga accaaagatc 60atacatggaa gcgaatcaat ggactctgga atatccctgg acaacagtta taaaatggat 120tatcctgaga tgggtttatg tataataatt aataataaga attttcataa aagcactgga 180atgacatctc ggtctggtac agatgtcgat gcagcaaacc tcagggaaac attcagaaac 240ttgaaatatg aagtcaggaa taaaaatgat cttacacgtg aagaaattgt ggaattgatg 300cgtgatgttt ctaaagaaga tcacagcaaa aggagcagtt ttgtttgtgt gcttctgagc 360catggtgaag aaggaataat ttttggaaca aatggacctg ttgacctgaa aaaaataaca 420aactttttca gaggggatcg ttgtagaagt ctaactggaa aacccaaact tttcattatt 480caggcctgcc gtggtacaga actggactgt ggcattgaga cagacagtgg tgttgatgat 540gacatggcgt gtcataaaat accagtggag gccgacttct tgtatgcata ctccacagca 600cctggttatt attcttggcg aaattcaaag gatggctcct ggttcatcca gtcgctttgt 660gccatgctga aacagtatgc cgacaagctt gaatttatgc acattcttac ccgggttaac 720cgaaaggtgg caacagaatt tgagtccttt tcctttgacg ctacttttca tgcaaagaaa 780cagattccat gtattgtttc catgctcaca aaagaactct atttttatca ctaa 8343650DNAArtificial Sequenceprimer for PCR 36cggatccaac actgaaaact cagtggattc aaaatccatt aaaaatttgg 503748DNAArtificial Sequenceprimer for PCR 37cggatccgtg ataaaaatag agttcttttg tgagcatgga aacaatac 48386436DNAArtificial SequenceSynthetic construct pcDNA3-1XUb-C3 38gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc

cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgt gcaccacgga 1140tccaacactg aaaactcagt ggattcaaaa tccattaaaa atttggaacc aaagatcata 1200catggaagcg aatcaatgga ctctggaata tccctggaca acagttataa aatggattat 1260cctgagatgg gtttatgtat aataattaat aataagaatt ttcataaaag cactggaatg 1320acatctcggt ctggtacaga tgtcgatgca gcaaacctca gggaaacatt cagaaacttg 1380aaatatgaag tcaggaataa aaatgatctt acacgtgaag aaattgtgga attgatgcgt 1440gatgtttcta aagaagatca cagcaaaagg agcagttttg tttgtgtgct tctgagccat 1500ggtgaagaag gaataatttt tggaacaaat ggacctgttg acctgaaaaa aataacaaac 1560tttttcagag gggatcgttg tagaagtcta actggaaaac ccaaactttt cattattcag 1620gcctgccgtg gtacagaact ggactgtggc attgagacag acagtggtgt tgatgatgac 1680atggcgtgtc ataaaatacc agtggaggcc gacttcttgt atgcatactc cacagcacct 1740ggttattatt cttggcgaaa ttcaaaggat ggctcctggt tcatccagtc gctttgtgcc 1800atgctgaaac agtatgccga caagcttgaa tttatgcaca ttcttacccg ggttaaccga 1860aaggtggcaa cagaatttga gtccttttcc tttgacgcta cttttcatgc aaagaaacag 1920attccatgta ttgtttccat gctcacaaaa gaactctatt tttatcacgg atcctagagg 1980gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt 2040ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 2100ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 2160gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 2220atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 2280ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 2340tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 2400tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcggggca 2460tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 2520gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 2580agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 2640cggtctattc ttttgattta taagggattt tggggatttc ggcctattgg ttaaaaaatg 2700agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg 2760tggaaagtcc ccaggctccc caggcaggca gaagtatgca aagcatgcat ctcaattagt 2820cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 2880atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc 2940cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg 3000ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc 3060taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga 3120caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg 3180cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg 3240ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt 3300ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg 3360gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat 3420tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat 3480ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg 3540accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg 3600atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc 3660tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc 3720cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg 3780tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg 3840gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca 3900tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac 3960cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga 4020aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga 4080tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa 4140ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 4200tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta 4260gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 4320tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 4380ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 4440ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 4500ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 4560agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 4620catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 4680tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 4740gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 4800ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 4860cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 4920caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 4980ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 5040taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 5100taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 5160cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 5220tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 5280gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 5340catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 5400atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 5460ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 5520gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 5580agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 5640gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 5700agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 5760catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 5820aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 5880gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 5940taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 6000caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 6060ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 6120ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 6180tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 6240aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 6300actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 6360catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 6420agtgccacct gacgtc 6436396703DNAArtificial SequenceSynthetic construct pcDNA3-2XUb-C3 39gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatcc aacactgaaa actcagtgga ttcaaaatcc 1440attaaaaatt tggaaccaaa gatcatacat ggaagcgaat caatggactc tggaatatcc 1500ctggacaaca gttataaaat ggattatcct gagatgggtt tatgtataat aattaataat 1560aagaattttc ataaaagcac tggaatgaca tctcggtctg gtacagatgt cgatgcagca 1620aacctcaggg aaacattcag aaacttgaaa tatgaagtca ggaataaaaa tgatcttaca 1680cgtgaagaaa ttgtggaatt gatgcgtgat gtttctaaag aagatcacag caaaaggagc 1740agttttgttt gtgtgcttct gagccatggt gaagaaggaa taatttttgg aacaaatgga 1800cctgttgacc tgaaaaaaat aacaaacttt ttcagagggg atcgttgtag aagtctaact 1860ggaaaaccca aacttttcat tattcaggcc tgccgtggta cagaactgga ctgtggcatt 1920gagacagaca gtggtgttga tgatgacatg gcgtgtcata aaataccagt ggaggccgac 1980ttcttgtatg catactccac agcacctggt tattattctt ggcgaaattc aaaggatggc 2040tcctggttca tccagtcgct ttgtgccatg ctgaaacagt atgccgacaa gcttgaattt 2100atgcacattc ttacccgggt taaccgaaag gtggcaacag aatttgagtc cttttccttt 2160gacgctactt ttcatgcaaa gaaacagatt ccatgtattg tttccatgct cacaaaagaa 2220ctctattttt atcacggatc ctagagggcc ctattctata gtgtcaccta aatgctagag 2280ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 2340ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 2400aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 2460acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 2520tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta 2580gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 2640gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 2700ttccccgtca agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc 2760acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 2820agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 2880aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg 2940ggatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat 3000tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag gcaggcagaa 3060gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 3120cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 3180taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 3240gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga 3300agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta 3360tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag 3420atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg 3480cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc 3540cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag 3600cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca 3660ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat 3720ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata 3780cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac 3840gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc 3900tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg 3960tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg 4020gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta 4080cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg 4140gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct 4200gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga 4260tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc 4320cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt 4380gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 4440agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 4500tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc 4560tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 4620taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 4680cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 4740gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 4800ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 4860agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 4920ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 4980caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 5040gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 5100cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 5160tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 5220gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 5280cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 5340tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 5400tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 5460caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 5520aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 5580cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 5640ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 5700tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 5760atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 5820tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 5880aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 5940catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 6000gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 6060ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 6120aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 6180atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 6240cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 6300gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 6360agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 6420gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 6480caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 6540ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 6600tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 6660aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc 6703406946DNAArtificial SequenceSynthetic construct pcDNA3-3XUb-C3 40gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tccaacactg aaaactcagt ggattcaaaa 1680tccattaaaa atttggaacc aaagatcata catggaagcg aatcaatgga ctctggaata 1740tccctggaca acagttataa aatggattat cctgagatgg gtttatgtat aataattaat 1800aataagaatt ttcataaaag cactggaatg acatctcggt ctggtacaga tgtcgatgca 1860gcaaacctca gggaaacatt cagaaacttg aaatatgaag tcaggaataa aaatgatctt 1920acacgtgaag aaattgtgga attgatgcgt gatgtttcta aagaagatca cagcaaaagg 1980agcagttttg tttgtgtgct tctgagccat ggtgaagaag gaataatttt tggaacaaat 2040ggacctgttg acctgaaaaa aataacaaac tttttcagag gggatcgttg tagaagtcta 2100actggaaaac ccaaactttt cattattcag gcctgccgtg gtacagaact ggactgtggc 2160attgagacag acagtggtgt tgatgatgac atggcgtgtc ataaaatacc agtggaggcc 2220gacttcttgt atgcatactc

cacagcacct ggttattatt cttggcgaaa ttcaaaggat 2280ggctcctggt tcatccagtc gctttgtgcc atgctgaaac agtatgccga caagcttgaa 2340tttatgcaca ttcttacccg ggttaaccga aaggtggcaa cagaatttga gtccttttcc 2400tttgacgcta cttttcatgc aaagaaacag attccatgta ttgtttccat gctcacaaaa 2460gaactctatt tttatcacgg atcctagagg gccctattct atagtgtcac ctaaatgcta 2520gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 2580cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 2640aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 2700aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 2760ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct 2820gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 2880ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 2940gctttccccg tcaagctcta aatcggggca tccctttagg gttccgattt agtgctttac 3000ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct 3060gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 3120tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt 3180tggggatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 3240aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc caggcaggca 3300gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct 3360ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc 3420ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 3480gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc 3540agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt 3600gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac 3660aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact 3720gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc 3780gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg 3840cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg 3900tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt 3960catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc 4020atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag 4080cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg 4140ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc 4200tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt 4260ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg 4320ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt 4380acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct 4440tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg 4500agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga 4560cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa 4620cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa 4680taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta 4740tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt 4800tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 4860gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 4920gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 4980ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 5040ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 5100cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 5160gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 5220tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 5280ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 5340atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 5400gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 5460tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 5520cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 5580cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 5640tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 5700cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 5760cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 5820gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 5880gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 5940gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 6000ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 6060atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 6120agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 6180ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 6240tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 6300ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 6360caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 6420gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 6480atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 6540accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 6600aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 6660gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 6720tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 6780aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 6840ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 6900aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc 6946417189DNAArtificial SequenceSynthetic construct pcDNA3-4XUb-C3 41gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tctaccatgg aaatcttcgt gaagactctg 1680actggtaaga ccatcactct cgaagtggag ccgagtgaca ccattgagaa tgtcaaggca 1740aagatccaag acaaggaagg catccctcct gaccagcaga ggttgatctt tgctgggaaa 1800cagctggaag atggacgcac cctgtctgac tacaacatcc agaaagagtc caccctgcac 1860ctggtactcc gtctcagagg tgtgcaccac ggatccaaca ctgaaaactc agtggattca 1920aaatccatta aaaatttgga accaaagatc atacatggaa gcgaatcaat ggactctgga 1980atatccctgg acaacagtta taaaatggat tatcctgaga tgggtttatg tataataatt 2040aataataaga attttcataa aagcactgga atgacatctc ggtctggtac agatgtcgat 2100gcagcaaacc tcagggaaac attcagaaac ttgaaatatg aagtcaggaa taaaaatgat 2160cttacacgtg aagaaattgt ggaattgatg cgtgatgttt ctaaagaaga tcacagcaaa 2220aggagcagtt ttgtttgtgt gcttctgagc catggtgaag aaggaataat ttttggaaca 2280aatggacctg ttgacctgaa aaaaataaca aactttttca gaggggatcg ttgtagaagt 2340ctaactggaa aacccaaact tttcattatt caggcctgcc gtggtacaga actggactgt 2400ggcattgaga cagacagtgg tgttgatgat gacatggcgt gtcataaaat accagtggag 2460gccgacttct tgtatgcata ctccacagca cctggttatt attcttggcg aaattcaaag 2520gatggctcct ggttcatcca gtcgctttgt gccatgctga aacagtatgc cgacaagctt 2580gaatttatgc acattcttac ccgggttaac cgaaaggtgg caacagaatt tgagtccttt 2640tcctttgacg ctacttttca tgcaaagaaa cagattccat gtattgtttc catgctcaca 2700aaagaactct atttttatca cggatcctag agggccctat tctatagtgt cacctaaatg 2760ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 2820cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 2880atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 2940ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 3000gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc 3060cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 3120ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 3180ccggctttcc ccgtcaagct ctaaatcggg gcatcccttt agggttccga tttagtgctt 3240tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 3300cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 3360tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 3420ttttggggat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 3480attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccaggcag 3540gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag 3600gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc 3660cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc 3720atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat 3780tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag 3840cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg 3900aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 3960actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 4020ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 4080aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 4140ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 4200tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 4260tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 4320gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 4380aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 4440atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 4500tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 4560tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 4620tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 4680tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc 4740acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg 4800ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc 4860caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 4920aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc 4980ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct 5040gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 5100aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 5160actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 5220cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 5280gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 5340atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 5400caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 5460gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 5520ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 5580cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg 5640taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 5700cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 5760acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 5820aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 5880atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 5940atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 6000gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 6060gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 6120ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 6180ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 6240tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 6300accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 6360atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 6420cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 6480tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 6540tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 6600gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 6660agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 6720aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 6780gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 6840tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 6900gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 6960tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 7020aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 7080catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 7140acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc 71894253DNAArtificial Sequenceprimer for PCR 42cggatccatg aacactgaaa actcagtgga ttcaaaatcc attaaaaatt tgg 534348DNAArtificial Sequenceprimer for PCR 43cggatccgtg ataaaaatag agttcttttg tgagcatgga aacaatac 48447248DNAArtificial SequenceSynthetic construct pcDNA3-Ub-Met-C3 44gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgg gatgcacgga 1140tcccacatca acactgaaaa ctcagtggcc tcaaaatcca ttaaaaattt ggaaccaaag 1200atcatacatg gaagcgaatc aatggactct ggaatatccc tggacaacag ttataaaatg 1260gattatcctg agatgggttt atgtataata attaataata agaattttca taaaagcact 1320ggaatgacat ctcggtctgg tacagatgtc gatgcagcaa acctcaggga aacattcaga 1380aacttgaaat atgaagtcag gaataaaaat gatcttacac gtgaagaaat tgtggaattg 1440atgcgtgatg tttctaaaga agatcacagc aaaaggagca gttttgtttg tgtgcttctg 1500agccatggtg aagaaggaat aatttttgga acaaatggac ctgttgacct gaaaaaaata 1560acaaactttt tcagagggga tcgttgtaga agtctaactg gaaaacccaa acttttcatt 1620attcaggcct gccgtggtac agaactggac tgtggcattg agacagacag tggtgttgat 1680gatgacatgg cgtgtcataa aataccagtg gatgccgact tcttgtatgc atactccaca 1740gcacctggtt attattcttg gcgaaattca aaggatggct cctggttcat ccagtcgctt 1800tgtgccatgc tgaaacagta tgccgacaag cttgaattta tgcacattct tacccgggtt 1860aaccgaaagg tggcaacaga atttgagtcc ttttcctttg acgctacttt tcatgcaaag 1920aaacagattc catgtattgt ttccatgctc acaaaagaac tctattttta tcacggatcc 1980ggggcgtggc tgcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 2040gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 2100cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 2160tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 2220ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 2280ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 2340atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 2400cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 2460atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 2520gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 2580cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 2640tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 2700tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 2760gcctcactga ttaagcattg gtaatctaga gggccctatt ctatagtgtc acctaaatgc

2820tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc 2880ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 2940tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3000gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 3060ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc 3120ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 3180tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 3240cggctttccc cgtcaagctc taaatcgggg catcccttta gggttccgat ttagtgcttt 3300acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 3360ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 3420gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 3480tttggggatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 3540ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccaggcagg 3600cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 3660ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 3720gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 3780tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt 3840ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc 3900ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga 3960acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga 4020ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg 4080gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga 4140ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt 4200tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct 4260gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct 4320gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg 4380agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca 4440ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga 4500tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt 4560ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt 4620ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct 4680ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt 4740cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca 4800cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg 4860gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc 4920aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 4980aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 5040tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg 5100tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 5160aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 5220ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 5280gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 5340cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 5400tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 5460aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 5520catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 5580caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 5640ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt 5700aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 5760gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 5820cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 5880ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 5940tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 6000tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 6060cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 6120tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 6180tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 6240tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 6300cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 6360ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 6420tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 6480gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 6540agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 6600atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 6660tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 6720gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 6780agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 6840cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 6900ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 6960ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 7020actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 7080ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 7140atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 7200caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc 72484548DNAArtificial SequenceSynthetic construct oligonucleotide cassette 45gatccgtcgg cgctgtcggc agcgtcggcg acgaggtcga cggcgtcg 484648DNAArtificial SequenceSynthetic construct oligonucleotide cassette 46gatccgacgc cgtcgacctc gtcgccgacg ctgccgacag cgccgacg 48476459DNAArtificial SequenceSynthetic construct pcDNA3-1XUb-DEVD-Bla 47gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgt gcaccacgga 1140tccgtcggcg ctgtcggcag cgtcggcgac gaggtcgacg gcgtcggatc cggggcgtgg 1200ctgcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 1260ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 1320cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 1380gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1440tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1500gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1560ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 1620tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 1680gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 1740caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 1800cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 1860atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 1920gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 1980attaagcatt ggtaatctag agggccctat tctatagtgt cacctaaatg ctagagctcg 2040ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt 2100gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 2160tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 2220caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 2280ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg 2340cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 2400cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 2460ccgtcaagct ctaaatcggg gcatcccttt agggttccga tttagtgctt tacggcacct 2520cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 2580ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 2640tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttggggat 2700ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg 2760tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccaggcag gcagaagtat 2820gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 2880aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 2940tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 3000aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta 3060gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc 3120cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg 3180attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 3240acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 3300tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg 3360gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 3420agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 3480ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 3540tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 3600tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 3660gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt 3720gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 3780catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 3840tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 3900cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc 3960gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc 4020gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 4080tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt 4140attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 4200tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 4260tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg 4320tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 4380gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 4440ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 4500ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 4560gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 4620tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 4680aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 4740aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4800ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 4860tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc 4920agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 4980gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 5040tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 5100acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 5160tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 5220caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 5280aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 5340aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 5400ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 5460agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 5520atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 5580cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 5640aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 5700cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 5760aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 5820ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 5880gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 5940ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 6000tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 6060tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 6120ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 6180tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 6240agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 6300acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 6360ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 6420gttccgcgca catttccccg aaaagtgcca cctgacgtc 6459486726DNAArtificial SequenceSynthetic construct pcDNA3-2XUb-DEVD-Bla 48gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatcc gtcggcgctg tcggcagcgt cggcgacgag 1440gtcgacggcg tcggatccgg ggcgtggctg cacccagaaa cgctggtgaa agtaaaagat 1500gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 1560atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 1620ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 1680cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 1740ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 1800aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 1860ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 1920gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 1980ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 2040gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 2100ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 2160tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 2220cagatcgctg agataggtgc ctcactgatt aagcattggt aatctagagg gccctattct 2280atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 2340gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 2400tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 2460tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 2520tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag 2580ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 2640cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 2700ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcggggca tccctttagg 2760gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 2820acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 2880ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 2940ttttgattta taagggattt tggggatttc ggcctattgg ttaaaaaatg agctgattta 3000acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc 3060ccaggctccc caggcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag 3120gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 3180gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 3240cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 3300ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 3360caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg 3420atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga 3480gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt 3540ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct 3600gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg 3660cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt 3720gccggggcag gatctcctgt catctcacct tgctcctgcc

gagaaagtat ccatcatggc 3780tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc 3840gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga 3900tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg 3960catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat 4020ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg 4080ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc 4140tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta 4200tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg 4260acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc 4320ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg 4380gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat 4440agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 4500aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg 4560taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 4620atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 4680ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 4740taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 4800tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 4860aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 4920aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 4980ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 5040acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 5100ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 5160tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 5220tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 5280gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 5340agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 5400tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 5460agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 5520tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 5580acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 5640tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 5700agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 5760tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 5820acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 5880tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 5940ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 6000agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 6060tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 6120acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 6180agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 6240actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 6300tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 6360gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 6420ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 6480tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 6540aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 6600tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 6660tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 6720gacgtc 6726496969DNAArtificial SequenceSynthetic construct pcDNA3-3XUb-DEVD-Bla 49gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tccgtcggcg ctgtcggcag cgtcggcgac 1680gaggtcgacg gcgtcggatc cggggcgtgg ctgcacccag aaacgctggt gaaagtaaaa 1740gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 1800aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 1860ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 1920atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 1980gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 2040gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 2100atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 2160aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 2220actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 2280aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 2340tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 2400ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 2460agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaatctag agggccctat 2520tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg 2580ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 2640cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 2700tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 2760gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 2820tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 2880gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 2940ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg gcatcccttt 3000agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 3060ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 3120gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 3180ttcttttgat ttataaggga ttttggggat ttcggcctat tggttaaaaa atgagctgat 3240ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag 3300tccccaggct ccccaggcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 3360caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 3420ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 3480ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 3540cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 3600ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg 3660aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt 3720ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt 3780gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc 3840cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc 3900ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga 3960agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat 4020ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca 4080agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga 4140tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc 4200gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat 4260catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga 4320ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg 4380ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt 4440ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa 4500gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg 4560ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 4620ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc 4680aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 4740tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg 4800gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 4860aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 4920acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 4980cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 5040tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 5100tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 5160gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 5220aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 5280ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 5340gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 5400ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 5460ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 5520cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 5580attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 5640ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 5700aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 5760gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 5820tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 5880ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 5940taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 6000atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 6060actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 6120cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 6180agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 6240gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 6300gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 6360gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 6420gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 6480cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 6540ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 6600accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 6660aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 6720aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 6780caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 6840ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 6900gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 6960cctgacgtc 6969507212DNAArtificial SequenceSynthetic construct pcDNA3-4XUb-DEVD-Bla 50gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tctaccatgg aaatcttcgt gaagactctg 1680actggtaaga ccatcactct cgaagtggag ccgagtgaca ccattgagaa tgtcaaggca 1740aagatccaag acaaggaagg catccctcct gaccagcaga ggttgatctt tgctgggaaa 1800cagctggaag atggacgcac cctgtctgac tacaacatcc agaaagagtc caccctgcac 1860ctggtactcc gtctcagagg tgtgcaccac ggatccgtcg gcgctgtcgg cagcgtcggc 1920gacgaggtcg acggcgtcgg atccggggcg tggctgcacc cagaaacgct ggtgaaagta 1980aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 2040ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 2100gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 2160cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 2220acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 2280gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 2340aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 2400ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 2460ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 2520gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 2580aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 2640aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 2700aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaatc tagagggccc 2760tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag 2820ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 2880tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 2940ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 3000caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg 3060ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 3120tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 3180cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc ggggcatccc 3240tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 3300tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 3360cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 3420ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa aaaatgagct 3480gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga 3540aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca attagtcagc 3600aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 3660caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 3720cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 3780ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 3840cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg 3900atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg 3960ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc 4020cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg 4080tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt 4140tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg 4200cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat 4260catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca 4320ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca 4380ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa 4440ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 4500tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc 4560ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga 4620atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc 4680cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac 4740caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg 4800ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc 4860atgctggagt tcttcgccca ccccaacttg tttattgcag

cttataatgg ttacaaataa 4920agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 4980ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc 5040ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 5100cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 5160ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 5220ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 5280gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 5340cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 5400tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 5460cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 5520aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 5580cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 5640gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 5700ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 5760cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 5820aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 5880tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 5940ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 6000tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 6060ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 6120agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 6180atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 6240cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 6300ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 6360ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 6420agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 6480agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 6540gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 6600cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 6660gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 6720tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 6780tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 6840aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 6900cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 6960cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 7020aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 7080ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 7140tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 7200ccacctgacg tc 72125148DNAArtificial SequenceSynthetic construct oligonucleotide cassette 51gatccgtcgg cgctgtcggc agcgtcggcg acgaggtcgc tggcgtcg 485248DNAArtificial SequenceSynthetic construct oligonucleotide cassette 52gatccgacgc cagcgacctc gtcgccgacg ctgccgacag cgccgacg 48536459DNAArtificial SequenceSynthetic construct pcDNA3-1XUb-DEVA-Bla 53gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgt gcaccacgga 1140tccgtcggcg ctgtcggcag cgtcggcgac gaggtcgctg gcgtcggatc cggggcgtgg 1200ctgcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 1260ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 1320cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 1380gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1440tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1500gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1560ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 1620tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 1680gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 1740caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 1800cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 1860atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 1920gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 1980attaagcatt ggtaatctag agggccctat tctatagtgt cacctaaatg ctagagctcg 2040ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt 2100gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 2160tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 2220caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 2280ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg 2340cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 2400cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 2460ccgtcaagct ctaaatcggg gcatcccttt agggttccga tttagtgctt tacggcacct 2520cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 2580ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 2640tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttggggat 2700ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg 2760tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccaggcag gcagaagtat 2820gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 2880aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 2940tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 3000aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta 3060gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc 3120cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg 3180attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 3240acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 3300tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg 3360gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 3420agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 3480ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 3540tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 3600tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 3660gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt 3720gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 3780catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 3840tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 3900cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc 3960gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc 4020gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 4080tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt 4140attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 4200tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 4260tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg 4320tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 4380gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 4440ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 4500ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 4560gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 4620tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 4680aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 4740aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4800ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 4860tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc 4920agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 4980gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 5040tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 5100acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 5160tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 5220caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 5280aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 5340aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 5400ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 5460agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 5520atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 5580cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 5640aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 5700cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 5760aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 5820ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 5880gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 5940ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 6000tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 6060tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 6120ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 6180tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 6240agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 6300acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 6360ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 6420gttccgcgca catttccccg aaaagtgcca cctgacgtc 6459546726DNAArtificial SequenceSynthetic construct pcDNA3-2XUb-DEVA-Bla 54gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatcc gtcggcgctg tcggcagcgt cggcgacgag 1440gtcgctggcg tcggatccgg ggcgtggctg cacccagaaa cgctggtgaa agtaaaagat 1500gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 1560atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 1620ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 1680cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 1740ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 1800aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 1860ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 1920gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 1980ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 2040gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 2100ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 2160tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 2220cagatcgctg agataggtgc ctcactgatt aagcattggt aatctagagg gccctattct 2280atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 2340gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 2400tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 2460tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 2520tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag 2580ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 2640cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 2700ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcggggca tccctttagg 2760gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 2820acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 2880ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 2940ttttgattta taagggattt tggggatttc ggcctattgg ttaaaaaatg agctgattta 3000acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc 3060ccaggctccc caggcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag 3120gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 3180gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 3240cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 3300ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 3360caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg 3420atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga 3480gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt 3540ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct 3600gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg 3660cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt 3720gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc 3780tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc 3840gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga 3900tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg 3960catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat 4020ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg 4080ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc 4140tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta 4200tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg 4260acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc 4320ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg 4380gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat 4440agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 4500aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg 4560taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 4620atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 4680ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 4740taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 4800tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 4860aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 4920aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 4980ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 5040acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 5100ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 5160tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 5220tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 5280gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 5340agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 5400tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 5460agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 5520tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 5580acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 5640tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 5700agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 5760tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 5820acgatacggg agggcttacc

atctggcccc agtgctgcaa tgataccgcg agacccacgc 5880tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 5940ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 6000agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 6060tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 6120acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 6180agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 6240actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 6300tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 6360gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 6420ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 6480tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 6540aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 6600tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 6660tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 6720gacgtc 6726556969DNAArtificial SequenceSynthetic construct pcDNA3-3XUb-DEVA-Bla 55gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tccgtcggcg ctgtcggcag cgtcggcgac 1680gaggtcgctg gcgtcggatc cggggcgtgg ctgcacccag aaacgctggt gaaagtaaaa 1740gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 1800aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 1860ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 1920atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 1980gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 2040gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 2100atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 2160aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 2220actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 2280aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 2340tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 2400ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 2460agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaatctag agggccctat 2520tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg 2580ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 2640cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 2700tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 2760gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 2820tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 2880gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 2940ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg gcatcccttt 3000agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 3060ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 3120gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 3180ttcttttgat ttataaggga ttttggggat ttcggcctat tggttaaaaa atgagctgat 3240ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag 3300tccccaggct ccccaggcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 3360caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 3420ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 3480ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 3540cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 3600ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg 3660aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt 3720ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt 3780gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc 3840cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc 3900ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga 3960agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat 4020ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca 4080agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga 4140tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc 4200gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat 4260catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga 4320ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg 4380ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt 4440ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa 4500gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg 4560ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 4620ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc 4680aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 4740tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg 4800gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 4860aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 4920acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 4980cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 5040tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 5100tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 5160gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 5220aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 5280ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 5340gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 5400ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 5460ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 5520cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 5580attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 5640ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 5700aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 5760gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 5820tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 5880ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 5940taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 6000atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 6060actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 6120cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 6180agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 6240gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 6300gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 6360gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 6420gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 6480cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 6540ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 6600accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 6660aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 6720aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 6780caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 6840ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 6900gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 6960cctgacgtc 6969567212DNAArtificial SequenceSynthetic construct pcDNA3-4XUb-DEVA-Bla 56gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tctaccatgg aaatcttcgt gaagactctg 1680actggtaaga ccatcactct cgaagtggag ccgagtgaca ccattgagaa tgtcaaggca 1740aagatccaag acaaggaagg catccctcct gaccagcaga ggttgatctt tgctgggaaa 1800cagctggaag atggacgcac cctgtctgac tacaacatcc agaaagagtc caccctgcac 1860ctggtactcc gtctcagagg tgtgcaccac ggatccgtcg gcgctgtcgg cagcgtcggc 1920gacgaggtcg ctggcgtcgg atccggggcg tggctgcacc cagaaacgct ggtgaaagta 1980aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 2040ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 2100gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 2160cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 2220acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 2280gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 2340aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 2400ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 2460ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 2520gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 2580aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 2640aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 2700aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaatc tagagggccc 2760tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag 2820ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 2880tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 2940ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 3000caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg 3060ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 3120tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 3180cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc ggggcatccc 3240tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 3300tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 3360cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 3420ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa aaaatgagct 3480gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga 3540aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca attagtcagc 3600aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 3660caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 3720cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 3780ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 3840cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg 3900atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg 3960ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc 4020cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg 4080tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt 4140tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg 4200cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat 4260catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca 4320ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca 4380ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa 4440ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 4500tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc 4560ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga 4620atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc 4680cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac 4740caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg 4800ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc 4860atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa 4920agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 4980ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc 5040ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 5100cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 5160ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 5220ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 5280gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 5340cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 5400tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 5460cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 5520aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 5580cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 5640gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 5700ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 5760cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 5820aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 5880tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 5940ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 6000tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 6060ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 6120agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 6180atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 6240cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 6300ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 6360ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 6420agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 6480agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 6540gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 6600cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 6660gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 6720tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 6780tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 6840aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 6900cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 6960cccaactgat cttcagcatc

ttttactttc accagcgttt ctgggtgagc aaaaacagga 7020aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 7080ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 7140tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 7200ccacctgacg tc 7212571095DNAHuman rhinovirus 14 57ttgggtcgtg cagcttgtgt gcatgtaact gaaatacaaa acaaagatgc tactggaata 60gataatcaca gagaagcaaa attgttcaat gattggaaaa tcaacctgtc cagccttgtc 120caacttagaa agaaactgga actcttcact tatgttaggt ttgattctga gtataccata 180ctggccactg catctcaacc tgattcagca aactattcaa gcaatttggt ggtccaagcc 240atgtatgttc cacatggtgc cccgaaatcc aaaagagtgg gcgattacac atggcaaagt 300gcttcaaacc ccagtgtatt cttcaaggtg ggggatacat caaggtttag tgtgccttat 360gtaggattgg catcagcata taattgtttt tatgatggtt actcacatga tgatgcagaa 420actcagtatg gcataactgt tctaaaccat atgggtagta tggcattcag aatagtaaat 480gaacatgatg aacacaaaac tcttgtcaag atcagagttt atcacagggc aaagctcgtt 540gaagcatgga ttccaagagc acccagagca ctaccctaca catcaatagg gcgcacaaat 600tatcctaaga atacagaacc agtaattaag aagaggaaag gtgacattaa atcctatggt 660ttaggaccta ggtacggtgg gatttataca tcaaatgtta aaataatgaa ttaccacttg 720atgacaccag aagaccacca taatctgata gcaccctatc caaatagaga tttagcaata 780gtctcaacag gaggacatgg tgcagaaaca ataccacact gtaaccgtac atcaggtgtt 840tactattcca catattacag aaagtattac cccataattt gcgaaaagcc caccaacatc 900tggattgaag gaagccctta ttacccaagt agatttcaag caggagtgat gaaaggggtt 960gggccggcag agctaggaga ctgcggtggg attttgagat gcatacatgg tcccattgga 1020ttgttaacag ctgaaggtag tggatatgtt tgttttgctg acatacgaca gttggagtgt 1080atcgcagagg aacag 10955829DNAArtificial SequenceSynthetic construct oligonucleotide for RT-PCR 58taggatcctt gggtcgtgca gcttgtgtg 295929DNAArtificial SequenceSynthetic construct oligonucleotide for RT-PCR 59aaggatccct gttcctctgc catacactc 29608022DNAArtificial SequenceSynthetic construct pcDNA3-3XUb-Bla HRV 14 60gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tccttgggtc gtgcagcttg tgtgcatgta 1680actgaaatac aaaacaaaga tgctactgga atagataatc acagagaagc aaaattgttc 1740aatgattgga aaatcaacct gtccagcctt gtccaactta gaaagaaact ggaactcttc 1800acttatgtta ggtttgattc tgagtatacc atactggcca ctgcatctca acctgattca 1860gcaaactatt caagcaattt ggtggtccaa gccatgtatg ttccacatgg tgccccgaaa 1920tccaaaagag tgggcgatta cacatggcaa agtgcttcaa accccagtgt attcttcaag 1980gtgggggata catcaaggtt tagtgtgcct tatgtaggat tggcatcagc atataattgt 2040ttttatgatg gttactcaca tgatgatgca gaaactcagt atggcataac tgttctaaac 2100catatgggta gtatggcatt cagaatagta aatgaacatg atgaacacaa aactcttgtc 2160aagatcagag tttatcacag ggcaaagctc gttgaagcat ggattccaag agcacccaga 2220gcactaccct acacatcaat agggcgcaca aattatccta agaatacaga accagtaatt 2280aagaagagga aaggtgacat taaatcctat ggtttaggac ctaggtacgg tgggatttat 2340acatcaaatg ttaaaataat gaattaccac ttgatgacac cagaagacca ccataatctg 2400atagcaccct atccaaatag agatttagca atagtctcaa caggaggaca tggtgcagaa 2460acaataccac actgtaaccg tacatcaggt gtttactatt ccacatatta cagaaagtat 2520taccccataa tttgcgaaaa gcccaccaac atctggattg aaggaagccc ttattaccca 2580agtagatttc aagcaggagt gatgaaaggg gttgggccgg cagagctagg agactgcggt 2640gggattttga gatgcataca tggtcccatt ggattgttaa cagctgaagg tagtggatat 2700gtttgttttg ctgacatacg acagttggag tgtatcgcag aggaacaggg atccggggcg 2760tggctgcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 2820gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 2880gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 2940attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 3000gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 3060agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 3120ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 3180cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 3240gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 3300cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 3360gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 3420ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 3480acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 3540ctgattaagc attggtaatc tagagggccc tattctatag tgtcacctaa atgctagagc 3600tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 3660cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 3720aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 3780cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 3840ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag 3900cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 3960cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 4020tccccgtcaa gctctaaatc ggggcatccc tttagggttc cgatttagtg ctttacggca 4080cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 4140gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 4200aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttggg 4260gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt 4320ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagg caggcagaag 4380tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc 4440agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct 4500aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 4560actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa 4620gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat 4680atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 4740tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 4800acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 4860ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc 4920gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 4980tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 5040tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 5100gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 5160tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 5220cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt 5280cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 5340attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 5400ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 5460tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 5520agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat 5580ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 5640ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 5700tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 5760gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 5820gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 5880gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 5940aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 6000gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 6060agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 6120gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 6180gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 6240cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 6300aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 6360tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 6420ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 6480ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 6540cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 6600ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 6660gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 6720atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 6780aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 6840aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 6900gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 6960cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 7020gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 7080tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 7140ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 7200ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 7260atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 7320cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 7380tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 7440aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 7500tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 7560ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 7620agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 7680gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 7740agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 7800accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 7860gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 7920cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 7980ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc 802261636DNAHuman rhinovirus 16 61atgggaactt tgtgttcgcg tattgtgacc agtgagcaat tacacaaagt caaagtggta 60acaaggatat atcacaaagc caaacacacc aaagcttggt gcccaagacc acccagagct 120gttcaatact cacatacaca taccaccaac tacaaattga gttcagaagt acacaatgat 180gtggctataa gacctagaac aaatctaaca actgttgggc ctagtgacat gtatgtgcat 240gttggtaatc taatatacag aaatctacat ttatttaact ctgacataca tgattccatt 300ttagtgtctt attcatcaga tttaatcata taccgaacaa gcacacaagg tgatggttat 360attccaacat gtaattgcac tgaagctaca tattactgca aacacaaaaa caggtactac 420ccaattaatg tcacacctca tgactggtat gagatacaag agagtgaata ttatccaaaa 480catatccagt acaatttact aataggtgaa ggaccatgtg aaccaggtga ttgtggtggg 540aaattattat gcaaacatgg agtgataggt attattacag caggtggtga gggccatgtt 600gcattcatag atcttagaca ctttcactgt gctgaa 6366229DNAArtificial SequenceSynthetic construct oligonucleotide for PCR 62aaggatccat gggaactttg tgttcgcgt 296331DNAArtificial SequenceSynthetic construct oligonucleotide for PCR 63ttggatcctt cttcagcaca gtgaaagtgt c 31647563DNAArtificial SequenceSynthetic construct pcDNA3-3XUb-Bla HRV16 64gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tccatgggaa ctttgtgttc gcgtattgtg 1680accagtgagc aattacacaa agtcaaagtg gtaacaagga tatatcacaa agccaaacac 1740accaaagctt ggtgcccaag accacccaga gctgttcaat actcacatac acataccacc 1800aactacaaat tgagttcaga agtacacaat gatgtggcta taagacctag aacaaatcta 1860acaactgttg ggcctagtga catgtatgtg catgttggta atctaatata cagaaatcta 1920catttattta actctgacat acatgattcc attttagtgt cttattcatc agatttaatc 1980atataccgaa caagcacaca aggtgatggt tatattccaa catgtaattg cactgaagct 2040acatattact gcaaacacaa aaacaggtac tacccaatta atgtcacacc tcatgactgg 2100tatgagatac aagagagtga atattatcca aaacatatcc agtacaattt actaataggt 2160gaaggaccat gtgaaccagg tgattgtggt gggaaattat tatgcaaaca tggagtgata 2220ggtattatta cagcaggtgg tgagggccat gttgcattca tagatcttag acactttcac 2280tgtgctgaag gatccggggc gtggctgcac ccagaaacgc tggtgaaagt aaaagatgct 2340gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 2400cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 2460tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 2520tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 2580atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 2640ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 2700gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 2760gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 2820gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 2880gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 2940gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 3000cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 3060atcgctgaga taggtgcctc actgattaag cattggtaat ctagagggcc ctattctata 3120gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 3180atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 3240cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 3300ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 3360tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg 3420gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 3480cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 3540tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt 3600ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 3660tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 3720taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 3780tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca 3840aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca 3900ggctccccag gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 3960tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 4020agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 4080ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 4140tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 4200aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc

4260gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 4320gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 4380gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 4440tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 4500agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 4560ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 4620tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 4680acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 4740ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 4800gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 4860ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 4920tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 4980ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 5040ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 5100cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 5160ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 5220ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 5280atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 5340ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 5400tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 5460cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 5520attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 5580tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5640ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5700gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 5760ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5820cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 5880ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 5940accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6000caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6060gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6120tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6180agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6240actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6300gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6360aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6420gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6480aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6540atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6600gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6660atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6720ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6780cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6840agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 6900cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 6960tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7020agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7080gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7140gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 7200ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7260tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 7320tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7380gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7440caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7500atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 7560gtc 7563657053DNAArtificial SequenceSynthetic construct pcDNA3-Ub-Met-Bla HRV16 65gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgg gatgcacgga 1140tccatgggaa ctttgtgttc gcgtattgtg accagtgagc aattacacaa agtcaaagtg 1200gtaacaagga tatatcacaa agccaaacac accaaagctt ggtgcccaag accacccaga 1260gctgttcaat actcacatac acataccacc aactacaaat tgagttcaga agtacacaat 1320gatgtggcta taagacctag aacaaatcta acaactgttg ggcctagtga catgtatgtg 1380catgttggta atctaatata cagaaatcta catttattta actctgacat acatgattcc 1440attttagtgt cttattcatc agatttaatc atataccgaa caagcacaca aggtgatggt 1500tatattccaa catgtaattg cactgaagct acatattact gcaaacacaa aaacaggtac 1560tacccaatta atgtcacacc tcatgactgg tatgagatac aagagagtga atattatcca 1620aaacatatcc agtacaattt actaataggt gaaggaccat gtgaaccagg tgattgtggt 1680gggaaattat tatgcaaaca tggagtgata ggtattatta cagcaggtgg tgagggccat 1740gttgcattca tagatcttag acactttcac tgtgctgaag gatccggggc gtggctgcac 1800ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 1860atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 1920ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 1980gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 2040ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 2100ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 2160gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 2220ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 2280gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 2340ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 2400gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 2460gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 2520caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 2580cattggtaat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc 2640agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 2700cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 2760gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 2820ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga 2880ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt 2940aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 3000gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 3060agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 3120caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 3180tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 3240aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc 3300ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat 3360gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag gcaggcagaa gtatgcaaag 3420catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 3480aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 3540catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 3600ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 3660aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 3720cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 3780cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 3840aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 3900tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 3960gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 4020aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 4080tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 4140ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 4200ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 4260cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 4320tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 4380ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 4440tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 4500tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 4560ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 4620accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 4680atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 4740gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 4800tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 4860ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 4920tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 4980ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 5040tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 5100ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 5160ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 5220gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 5280gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 5340cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 5400ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 5460tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 5520gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 5580tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 5640ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 5700ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 5760ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 5820accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 5880tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 5940cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 6000taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 6060caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 6120gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 6180gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 6240ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 6300attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 6360gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 6420tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 6480agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 6540gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 6600actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 6660tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 6720attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 6780tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 6840tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 6900aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 6960tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 7020cgcacatttc cccgaaaagt gccacctgac gtc 70536632DNAArtificial SequenceSynthetic construct oligonucleotide for mutagenesis 66gtgtcttatt catcagcttt aatcatatac cg 326734DNAArtificial SequenceSynthetic construct oligonucleotide for mutagenesis 67gtgaaccagg tgatgctggt gggaaattat tatg 34687563DNAArtificial SequenceSynthetic construct pcDNA3-3XUb-Bla HRV16 (C106A) 68gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tccatgggaa ctttgtgttc gcgtattgtg 1680accagtgagc aattacacaa agtcaaagtg gtaacaagga tatatcacaa agccaaacac 1740accaaagctt ggtgcccaag accacccaga gctgttcaat actcacatac acataccacc 1800aactacaaat tgagttcaga agtacacaat gatgtggcta taagacctag aacaaatcta 1860acaactgttg ggcctagtga catgtatgtg catgttggta atctaatata cagaaatcta 1920catttattta actctgacat acatgattcc attttagtgt cttattcatc agatttaatc 1980atataccgaa caagcacaca aggtgatggt tatattccaa catgtaattg cactgaagct 2040acatattact gcaaacacaa aaacaggtac tacccaatta atgtcacacc tcatgactgg 2100tatgagatac aagagagtga atattatcca aaacatatcc agtacaattt actaataggt 2160gaaggaccat gtgaaccagg tgatgctggt gggaaattat tatgcaaaca tggagtgata 2220ggtattatta cagcaggtgg tgagggccat gttgcattca tagatcttag acactttcac 2280tgtgctgaag gatccggggc gtggctgcac ccagaaacgc tggtgaaagt aaaagatgct 2340gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 2400cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 2460tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 2520tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 2580atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 2640ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 2700gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 2760gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 2820gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 2880gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 2940gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 3000cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 3060atcgctgaga taggtgcctc actgattaag cattggtaat ctagagggcc ctattctata 3120gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 3180atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 3240cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 3300ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 3360tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg 3420gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 3480cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 3540tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt 3600ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 3660tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 3720taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 3780tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca 3840aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca 3900ggctccccag gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 3960tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 4020agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 4080ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 4140tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa

4200aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc 4260gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 4320gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 4380gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 4440tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 4500agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 4560ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 4620tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 4680acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 4740ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 4800gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 4860ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 4920tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 4980ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 5040ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 5100cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 5160ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 5220ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 5280atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 5340ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 5400tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 5460cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 5520attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 5580tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5640ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5700gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 5760ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5820cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 5880ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 5940accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6000caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6060gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6120tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6180agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6240actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6300gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6360aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6420gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6480aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6540atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6600gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6660atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6720ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6780cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6840agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 6900cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 6960tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7020agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7080gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7140gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 7200ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7260tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 7320tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7380gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7440caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7500atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 7560gtc 7563697563DNAArtificial SequenceSynthetic construct pcDNA3-3XUb-Bla HRV16 (D35A) 69gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgatatc 900gaattcctgc agcccggggg atctaccatg gaaatcttcg tgaagactct gactggtaag 960accatcactc tcgaagtgga gccgagtgac accattgaga atgtcaaggc aaagatccaa 1020gacaaggaag gcatccctcc tgaccagcag aggttgatct ttgctgggaa acagctggaa 1080gatggacgca ccctgtctga ctacaacatc cagaaagagt ccaccctgca cctggtactc 1140cgtctcagag gtgtgcacca cggatctacc atggaaatct tcgtgaagac tctgactggt 1200aagaccatca ctctcgaagt ggagccgagt gacaccattg agaatgtcaa ggcaaagatc 1260caagacaagg aaggcatccc tcctgaccag cagaggttga tctttgctgg gaaacagctg 1320gaagatggac gcaccctgtc tgactacaac atccagaaag agtccaccct gcacctggta 1380ctccgtctca gaggtgtgca ccacggatct accatggaaa tcttcgtgaa gactctgact 1440ggtaagacca tcactctcga agtggagccg agtgacacca ttgagaatgt caaggcaaag 1500atccaagaca aggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaaacag 1560ctggaagatg gacgcaccct gtctgactac aacatccaga aagagtccac cctgcacctg 1620gtactccgtc tcagaggtgt gcaccacgga tccatgggaa ctttgtgttc gcgtattgtg 1680accagtgagc aattacacaa agtcaaagtg gtaacaagga tatatcacaa agccaaacac 1740accaaagctt ggtgcccaag accacccaga gctgttcaat actcacatac acataccacc 1800aactacaaat tgagttcaga agtacacaat gatgtggcta taagacctag aacaaatcta 1860acaactgttg ggcctagtga catgtatgtg catgttggta atctaatata cagaaatcta 1920catttattta actctgacat acatgattcc attttagtgt cttattcatc agctttaatc 1980atataccgaa caagcacaca aggtgatggt tatattccaa catgtaattg cactgaagct 2040acatattact gcaaacacaa aaacaggtac tacccaatta atgtcacacc tcatgactgg 2100tatgagatac aagagagtga atattatcca aaacatatcc agtacaattt actaataggt 2160gaaggaccat gtgaaccagg tgattgtggt gggaaattat tatgcaaaca tggagtgata 2220ggtattatta cagcaggtgg tgagggccat gttgcattca tagatcttag acactttcac 2280tgtgctgaag gatccggggc gtggctgcac ccagaaacgc tggtgaaagt aaaagatgct 2340gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 2400cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 2460tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 2520tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 2580atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 2640ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 2700gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 2760gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 2820gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 2880gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 2940gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 3000cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 3060atcgctgaga taggtgcctc actgattaag cattggtaat ctagagggcc ctattctata 3120gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 3180atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 3240cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 3300ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 3360tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg 3420gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 3480cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 3540tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt 3600ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 3660tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 3720taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 3780tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca 3840aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca 3900ggctccccag gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 3960tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 4020agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 4080ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 4140tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 4200aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc 4260gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 4320gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 4380gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 4440tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 4500agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 4560ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 4620tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 4680acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 4740ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 4800gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 4860ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 4920tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 4980ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 5040ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 5100cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 5160ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 5220ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 5280atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 5340ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 5400tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 5460cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 5520attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 5580tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5640ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5700gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 5760ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5820cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 5880ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 5940accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6000caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6060gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6120tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6180agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6240actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6300gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6360aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6420gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6480aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6540atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6600gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6660atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6720ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6780cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6840agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 6900cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 6960tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7020agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7080gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7140gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 7200ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7260tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 7320tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7380gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7440caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7500atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 7560gtc 7563707053DNAArtificial SequenceSynthetic construct pcDNA3-Ub-Met-Bla HRV16 (C106A) 70gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgg gatgcacgga 1140tccatgggaa ctttgtgttc gcgtattgtg accagtgagc aattacacaa agtcaaagtg 1200gtaacaagga tatatcacaa agccaaacac accaaagctt ggtgcccaag accacccaga 1260gctgttcaat actcacatac acataccacc aactacaaat tgagttcaga agtacacaat 1320gatgtggcta taagacctag aacaaatcta acaactgttg ggcctagtga catgtatgtg 1380catgttggta atctaatata cagaaatcta catttattta actctgacat acatgattcc 1440attttagtgt cttattcatc agatttaatc atataccgaa caagcacaca aggtgatggt 1500tatattccaa catgtaattg cactgaagct acatattact gcaaacacaa aaacaggtac 1560tacccaatta atgtcacacc tcatgactgg tatgagatac aagagagtga atattatcca 1620aaacatatcc agtacaattt actaataggt gaaggaccat gtgaaccagg tgatgctggt 1680gggaaattat tatgcaaaca tggagtgata ggtattatta cagcaggtgg tgagggccat 1740gttgcattca tagatcttag acactttcac tgtgctgaag gatccggggc gtggctgcac 1800ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 1860atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 1920ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 1980gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 2040ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 2100ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 2160gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 2220ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 2280gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 2340ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 2400gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 2460gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 2520caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 2580cattggtaat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc 2640agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 2700cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 2760gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 2820ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga 2880ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt 2940aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 3000gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 3060agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 3120caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 3180tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 3240aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc 3300ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat 3360gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag gcaggcagaa gtatgcaaag 3420catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 3480aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 3540catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 3600ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 3660aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 3720cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 3780cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 3840aatcggctgc tctgatgccg ccgtgttccg

gctgtcagcg caggggcgcc cggttctttt 3900tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 3960gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 4020aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 4080tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 4140ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 4200ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 4260cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 4320tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 4380ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 4440tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 4500tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 4560ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 4620accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 4680atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 4740gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 4800tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 4860ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 4920tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 4980ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 5040tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 5100ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 5160ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 5220gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 5280gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 5340cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 5400ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 5460tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 5520gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 5580tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 5640ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 5700ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 5760ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 5820accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 5880tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 5940cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 6000taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 6060caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 6120gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 6180gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 6240ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 6300attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 6360gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 6420tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 6480agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 6540gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 6600actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 6660tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 6720attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 6780tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 6840tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 6900aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 6960tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 7020cgcacatttc cccgaaaagt gccacctgac gtc 7053717053DNAArtificial SequenceSynthetic construct pcDNA3-Ub-Met-Bla HRV16 (D35A) 71gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgg gatgcacgga 1140tccatgggaa ctttgtgttc gcgtattgtg accagtgagc aattacacaa agtcaaagtg 1200gtaacaagga tatatcacaa agccaaacac accaaagctt ggtgcccaag accacccaga 1260gctgttcaat actcacatac acataccacc aactacaaat tgagttcaga agtacacaat 1320gatgtggcta taagacctag aacaaatcta acaactgttg ggcctagtga catgtatgtg 1380catgttggta atctaatata cagaaatcta catttattta actctgacat acatgattcc 1440attttagtgt cttattcatc agctttaatc atataccgaa caagcacaca aggtgatggt 1500tatattccaa catgtaattg cactgaagct acatattact gcaaacacaa aaacaggtac 1560tacccaatta atgtcacacc tcatgactgg tatgagatac aagagagtga atattatcca 1620aaacatatcc agtacaattt actaataggt gaaggaccat gtgaaccagg tgattgtggt 1680gggaaattat tatgcaaaca tggagtgata ggtattatta cagcaggtgg tgagggccat 1740gttgcattca tagatcttag acactttcac tgtgctgaag gatccggggc gtggctgcac 1800ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 1860atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 1920ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 1980gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 2040ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 2100ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 2160gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 2220ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 2280gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 2340ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 2400gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 2460gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 2520caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 2580cattggtaat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc 2640agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 2700cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 2760gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 2820ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga 2880ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt 2940aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 3000gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 3060agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 3120caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 3180tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 3240aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc 3300ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat 3360gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag gcaggcagaa gtatgcaaag 3420catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 3480aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 3540catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 3600ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 3660aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 3720cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 3780cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 3840aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 3900tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 3960gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 4020aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 4080tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 4140ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 4200ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 4260cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 4320tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 4380ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 4440tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 4500tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 4560ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 4620accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 4680atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 4740gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 4800tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 4860ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 4920tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 4980ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 5040tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 5100ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 5160ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 5220gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 5280gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 5340cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 5400ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 5460tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 5520gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 5580tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 5640ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 5700ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 5760ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 5820accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 5880tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 5940cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 6000taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 6060caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 6120gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 6180gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 6240ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 6300attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 6360gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 6420tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 6480agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 6540gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 6600actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 6660tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 6720attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 6780tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 6840tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 6900aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 6960tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 7020cgcacatttc cccgaaaagt gccacctgac gtc 7053727512DNAArtificial SequenceSynthetic construct pcDNA3-MetUb-Bla HR14 72gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900accatggaga tcttcgtgaa gactctgact ggtaagacca tcactctcga agtggagccg 960agtgacacca ttgagaatgt caaggcaaag atccaagaca aggaaggcat ccctcctgac 1020cagcagaggt tgatctttgc tgggaaacag ctggaagatg gacgcaccct gtctgactac 1080aacatccaga aagagtccac cctgcacctg gtactccgtc tcagaggtgg gatgcacgga 1140tccttgggtc gtgcagcttg tgtgcatgta actgaaatac aaaacaaaga tgctactgga 1200atagataatc acagagaagc aaaattgttc aatgattgga aaatcaacct gtccagcctt 1260gtccaactta gaaagaaact ggaactcttc acttatgtta ggtttgattc tgagtatacc 1320atactggcca ctgcatctca acctgattca gcaaactatt caagcaattt ggtggtccaa 1380gccatgtatg ttccacatgg tgccccgaaa tccaaaagag tgggcgatta cacatggcaa 1440agtgcttcaa accccagtgt attcttcaag gtgggggata catcaaggtt tagtgtgcct 1500tatgtaggat tggcatcagc atataattgt ttttatgatg gttactcaca tgatgatgca 1560gaaactcagt atggcataac tgttctaaac catatgggta gtatggcatt cagaatagta 1620aatgaacatg atgaacacaa aactcttgtc aagatcagag tttatcacag ggcaaagctc 1680gttgaagcat ggattccaag agcacccaga gcactaccct acacatcaat agggcgcaca 1740aattatccta agaatacaga accagtaatt aagaagagga aaggtgacat taaatcctat 1800ggtttaggac ctaggtacgg tgggatttat acatcaaatg ttaaaataat gaattaccac 1860ttgatgacac cagaagacca ccataatctg atagcaccct atccaaatag agatttagca 1920atagtctcaa caggaggaca tggtgcagaa acaataccac actgtaaccg tacatcaggt 1980gtttactatt ccacatatta cagaaagtat taccccataa tttgcgaaaa gcccaccaac 2040atctggattg aaggaagccc ttattaccca agtagatttc aagcaggagt gatgaaaggg 2100gttgggccgg cagagctagg agactgcggt gggattttga gatgcataca tggtcccatt 2160ggattgttaa cagctgaagg tagtggatat gtttgttttg ctgacatacg acagttggag 2220tgtatcgcag aggaacaggg atccggggcg tggctgcacc cagaaacgct ggtgaaagta 2280aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 2340ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 2400gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 2460cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 2520acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 2580gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 2640aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 2700ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 2760ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 2820gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 2880aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 2940aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 3000aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaatc tagagggccc 3060tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag 3120ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 3180tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 3240ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 3300caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg 3360ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 3420tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 3480cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc ggggcatccc 3540tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 3600tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 3660cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 3720ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa aaaatgagct 3780gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga 3840aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca attagtcagc 3900aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 3960caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 4020cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 4080ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 4140cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg 4200atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg 4260ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc 4320cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg 4380tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt 4440tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg 4500cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat 4560catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca

4620ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca 4680ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa 4740ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 4800tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc 4860ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga 4920atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc 4980cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac 5040caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg 5100ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc 5160atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa 5220agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 5280ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc 5340ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 5400cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 5460ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 5520ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 5580gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 5640cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 5700tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 5760cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 5820aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 5880cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 5940gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 6000ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 6060cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 6120aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 6180tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 6240ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 6300tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 6360ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 6420agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 6480atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 6540cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 6600ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 6660ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 6720agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 6780agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 6840gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 6900cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 6960gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 7020tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 7080tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 7140aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 7200cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 7260cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 7320aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 7380ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 7440tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 7500ccacctgacg tc 75127316PRTArtificial SequenceSynthetic construct fusion protein 73His Gly Ser Gly Ala Trp Leu His Pro Glu Thr Leu Val Lys Val Lys1 5 10 157436PRTArtificial SequenceSynthetic construct fusion protein 74Leu Val Leu Arg Leu Arg Gly Val Gly Ser Val Gly Ala Val Gly Ser1 5 10 15Val Gly Asp Glu Val Asp Gly Ser Gly Ala Trp Leu His Pro Glu Thr 20 25 30Leu Val Lys Val 35

Claims

1-37. (canceled)

38. A method of destabilizing a target protein in a cell, comprising; operatively coupling a target protein to a linear multimerized destabilization domain, wherein said linear multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of a destabilization domain.

39. The method of claim 38, wherein said destabilization domain comprises a ubiquitin homolog.

40. The method of claim 39, wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases.

41. The method of claim 39, wherein said ubiquitin homolog comprises a mutation at glycine 76.

42. The method of claim 38, wherein said protein of interest is fused in frame to said multimerized destabilization domain.

43. The method of claim 38, wherein said protein of interest is non-covalently coupled to said multimerized ubiquitin fusion protein.

44. The method of claim 38, wherein said cell is a mammalian cell.

45. The method of claim 38, wherein said cell is a yeast cell.

46. The method of claim 38, wherein said cell is an insect cell.

47. The method of claim 38, wherein said cell is a plant cell.

48. The method of claim 38, wherein said target protein is coupled to said multimerized destabilization domain by a linker.

49. The method of claim 48, wherein said linker is between 1 and 10 amino acid residues.

50. A recombinant DNA molecule, comprising a nucleic acid sequence encoding for; a) a linear multimerized destabilization domain, wherein said linear multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of a destabilization domain, b) a target protein, and c) a linker moiety that operatively couples said multimerized destabilization domain to said reporter moiety, wherein said linker is non-cleavable by a α-NH-ubiquitin protein endoproteases.

51. The method of claim 50, wherein said linker moiety comprises an enzyme modification site for an activity, and modification of said linker moiety by said activity modulates the coupling of said multimerized destabilization domain to said reporter moiety, thereby modulating the stability of said reporter moiety.

52. The method of claim 50, wherein said destabilization domain comprises a ubiquitin homolog.

53. The method of claim 52, wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases.

54. The method of claim 52, wherein said ubiquitin homolog comprises a mutation at glycine 76.

55. A recombinant protein molecule, comprising an amino acid sequence encoding for; a) a linear multimerized destabilization domain, wherein said multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of said destabilization domain, b) a target protein, and c) a linker moiety that operatively couples said multimerized destabilization domain to said reporter moiety, wherein said linker is non-cleavable by a α-NH-ubiquitin protein endoproteases.

56. The method of claim 55, wherein said linker moiety comprises a recognition motif for an activity, and modification of said linker moiety by said activity modulates the coupling of said multimerized destabilization domain to said reporter moiety, thereby modulating the stability of said reporter moiety.

57. The method of claim 55, wherein said destabilization domain comprises a ubiquitin homolog.

58. The method of claim 57, wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases.

59. The method of claim 57, wherein said ubiquitin homolog comprises a mutation at glycine 76.

60. A host cell, comprising a nucleic acid sequence encoding for; a) a linear multimerized destabilization domain, wherein said multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of said destabilization domain, b) a target protein, and c) a linker moiety that operatively couples said multimerized destabilization domain to said reporter moiety, wherein said linker is non-cleavable by a α-NH-ubiquitin protein endoproteases.

61. The method of claim 60, wherein said destabilization domain comprises a ubiquitin homolog.

62. The method of claim 61, wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases.

63. The method of claim 61, wherein said ubiquitin homolog comprises a mutation at glycine 76.

64. A transgenic animal, comprising a nucleic acid sequence encoding for; a linear multimerized destabilization domain, wherein said multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of said destabilization domain, b) a target protein, and c) a linker moiety that operatively couples said multimerized destabilization domain to said reporter moiety, wherein said linker is non-cleavable by a α-NH-ubiquitin protein endoproteases.

65. The method of claim 64, wherein said destabilization domain comprises a ubiquitin homolog.

66. The method of claim 65, wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases.

67. The method of claim 65, wherein said ubiquitin homolog comprises a mutation at glycine 76.

68. A transgenic plant, comprising a nucleic acid sequence encoding for; a) a linear multimerized destabilization domain, wherein said multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of said destabilization domain, b) a target protein, and c) a linker moiety that operatively couples said multimerized destabilization domain to said reporter moiety, wherein said linker is non-cleavable by a α-NH-ubiquitin protein endoproteases.

69. The method of claim 68, wherein said destabilization domain comprises a ubiquitin homolog.

70. The method of claim 69, wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases.

71. The method of claim 69, wherein said ubiquitin homolog comprises a mutation at glycine 76.

72. A method for identifying a modulator of an activity, comprising; a) contacting a cell with a test chemical, wherein said cell comprises, i) at least one destabilization domain, wherein said destabilization domain is non-cleavable by α-NH-ubiquitin protein endoproteases, ii) a reporter moiety, and iii) a linker moiety that operatively couples said destabilization domain to said reporter moiety, wherein said linker moiety comprises a recognition motif for said activity and modification of said linker moiety by said activity modulates the coupling of said destabilization domain to said reporter moiety thereby modulating the stability of said reporter moiety, and wherein said linker moiety is non-cleavable by said α-NH-ubiquitin protein endoproteases, b) detecting said reporter moiety, or a product of said reporter moiety in the presence of said test chemical, and c) comparing said reporter moiety activity from step b) to the reporter moiety activity in a control cell in the absence of said test chemical.

73. The method of claim 72, further comprising the step of contacting said cell with an activator of said activity prior to the addition said test chemical.

74. The method of claim 72, further comprising the step of detecting the viability of said cell.

75. The method of claim 72, wherein said activity is selected from the group consisting of a protease activity, a protein kinase activity and a phosphoprotein phosphatase activity.

76. The method of claim 72, wherein said reporter moiety is selected from the group consisting of a naturally fluorescent protein homolog, a β-lactamase homolog, a β-galactosidase homolog, an alkaline phosphatase homolog, a CAT homolog, and a luciferase homolog.

77-79. (canceled)

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