CAMKK-BETA AS A TARGET FOR TREATING CANCER

Abstract

Provided herein are compounds, compositions, including pharmaceutical compositions, having anti-cancer activity. Also provided are methods for diagnosing, detecting, and treating cancer in a subject, as well as a method for evaluating cancer stage in a subject, wherein the methods include determining the amount of a Ca²+/calmodulin dependent kinase kinase (CaMKK) in a sample. Further provided are methods of screening and identifying a compound that inhibits CaMKK.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to and claims the benefit of priority to U.S. Provisional Patent application Ser. No. 61/374,106, filed Aug. 16, 2010, and U.S. Provisional Patent application Ser. No. 61/379,226, filed Sep. 1, 2010. The content of both applications are incorporated herein by reference.

FIELD

[0003] The disclosure relates to cancer, including diagnostic markers of cancer, methods for the diagnosis of cancer, methods and compounds for the treatment of cancer, methods for identifying cancer stage in a subject, methods for identifying a cancer that is responsive to particular therapies, and methods for evaluating efficacy of cancer therapy.

SEQUENCE LISTING

[0004] An electronic version of the sequence listing ("028193_--9098_SeqList.txt") which is 233,472 bytes in size and created on Aug. 16, 2011, is submitted herewith and is herein incorporated by reference.

BACKGROUND

[0005] Prostate cancer is the most common malignancy in men and is second only to lung cancer in terms of cancer mortalities [Cancer Facts and Figures: American Cancer Society; 2007.]. Early diagnosis of prostate cancer usually allows for successful surgical treatment of localized tumors and thus, good patient outcomes. However, as with many cancers, the treatment of the advanced disease state requires a systemic approach to inhibit the growth and spread of secondary metastases. Prostate cancers express the androgen receptor (AR) and rely on androgens for growth and survival [Isaacs J T, Isaacs W B., Nat Med 2004; 10:26-7]. Consequently, androgen ablation therapies are the standard of care for late-stage disease. While 80% of patients with prostate cancer respond favorably to initial androgen ablation therapy, most patients experience a relapse of the disease within 1-2 years [Isaacs J T, Isaacs W B., Nat Med 2004; 10:26-7.]. Despite the unresponsiveness of the hormone-refractory disease to androgen-deprivation therapy, AR-regulated signaling pathways remain active and are necessary for cancer progression [Chen C. D., et al., Nature Med 2004; 10:33-9.].

[0006] Several approaches are currently used to target the AR signaling axis in prostate cancer. Existing therapies focus on decreasing the levels of circulating androgens and/or competitively blocking the AR transcriptional complex. Specifically, gonadotropin-releasing hormone (GnRH) agonists are used to suppress the testicular production of testosterone whereas antiandrogens, such as bicalutamide, function by competitively inhibiting the interaction of androgens with AR. The initial response to either form of androgen deprivation is very high. Nevertheless, the rapid onset of resistance to these interventions highlights the need for other strategies that target the hormone-independent activities of AR.

[0007] Most of the studies on the role of androgens in prostate cancer have focused on defining the mechanisms underlying the mitotic actions of this class of hormone [Balk S. P., Nucl Recept Signal 2008; 6:e001]. However, there is a growing body of evidence that AR signaling also influences tumor cell migration and invasion. For example, different clinical trials of goserelin (a GnRH analog) in prostate cancer patients demonstrate reduced incidences of distant metastases [Lawton C. A., et al. Int J Radiation Oncology Biol Phys 2001; 49:937-46; Bolla M., et al. The Lancet 2002; 360:103-8.]. Furthermore, it has recently been reported that MDV3100, a second generation AR-antagonist, decreases the number of circulating tumor cells in approximately half of the treated patients having a castration-resistant type cancer [Scher H. I., et al. The Lancet; 375:1437-46].

[0008] Compounds of Formula I are known and have been used as dye molecules. See, for example, U.S. Pat. No. 2,820,037 which describes:

##STR00001##

wherein R₁ is selected from CN, COOH, or COCl. The dye industry has generated a number of compounds that are structurally related to those of Formula I. See, e.g., U.S. Pat. Nos. 2,835,674; 2,965,644; 2,949,467; 3,953,452; 3,960,867; 4,239,868; and 4,336,383.

[0009] Japanese Patent Application No. 2003-012516 (Sumitomo Pharmaceutical Co.) identifies compounds as Ca²+/calmodulin dependent kinase kinase (CaMKK) inhibitors. The compounds are described as Formula II:

##STR00002##

wherein R₁ and R₂ are independently selected from H, halo, alkyl, or haloalkyl; and R₃ is H, alkyl, or substituted alkyl, or three COOR₃ groups can be substituted at any location on the naphthalene ring.

[0010] U.S. Patent Application Publication No. 2010/0105716 discloses methods of treating obesity, insulin resistance, and hyperglycemia by administering a CaMKK inhibitor compound of Formula III:

##STR00003##

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R_7a, R₈, R₉, R₁₀, and R₁₁ are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, heterocyclo, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, alkoxy, halo, mercapto, azido, cyano, formyl, carboxylic acid, hydroxyl, nitro, acyl, aryloxy, alkylthio, amino, alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfoxyl, sulfonyl, sulfonate, sulfonic acid, sulfonamide, urea, alkoxylacylamino, and aminoacyloxy; or a pharmaceutically acceptable salt or prodrug thereof.

[0011] None of these documents disclose or suggest that any of the compounds of Formula I-III would be useful in methods relating to cancer, or that CaMKKβ represents a therapeutic target in the treatment of cancers.

SUMMARY OF THE INVENTION

[0012] In an aspect, the disclosure provides a method of diagnosing prostate cancer in a subject comprising: determining an amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in a sample from the subject; and comparing the amount to a control sample comprising an amount of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in a control sample; wherein the subject is diagnosed as having prostate cancer when the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the sample from the subject is greater than the amount in the control sample.

[0013] In an aspect the disclosure relates to a method for determining disease stage in a subject having prostate cancer, the method comprising: determining an amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in a sample from the subject; and comparing the amount to a control sample comprising an amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit; wherein the disease stage of prostate cancer is determined by the difference in the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the sample from the subject and the amount in the control sample.

[0014] Aspects also relate to a method for predicting the likelihood of success of hormone-based therapeutic treatment of a subject having prostate cancer, the method comprising determining an amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in a sample from the subject; and comparing the amount to a control sample comprising an amount of the CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit. Embodiments provide for a likely successful response to hormone-based therapeutic treatment when the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the sample from the subject is greater than the amount in the control sample.

[0015] In an aspect the disclosure relates to a method for early detection of prostate cancer in a subject comprising obtaining a sample from the subject; determining an amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the sample from the subject; and comparing the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit from the sample from the subject to an amount of the CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, AMPK, and AMPK α1 subunit in a control sample; wherein early detection of prostate cancer is made when the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the sample from the subject is greater than the amount in the control sample.

[0016] In another aspect the disclosure provides a method for identifying a selective inhibitor of CaMKKβ where the method includes contacting CaMKKβ and a substrate therefor, in the presence and in the absence of the test compound, under conditions such that CaMKKβ-dependent phosphorylation of the substrate can be effected, and determining the level of phosphorylation of the substrate resulting from the contacting, and comparing the amount of phosphorylated substrate with a level of phosphorylation of the substrate in the absence of the test compound, wherein an decrease in phosphorylation of the substrate in the presence of the test compound indicates that the test compound is a selective inhibitor of CaMKKβ.

[0017] In an aspect the disclosure provides a method of screening a test compound for anti-cancer activity comprising: contacting CaMKKβ and a substrate therefor in the presence of the test compound, under conditions that allow for CaMKKβ-dependent phosphorylation of the substrate; determining the level of phosphorylation of the substrate resulting from the contacting; and comparing that level with a level of phosphorylation of the substrate obtained in the absence of the test compound, wherein a reduction in the level of phosphorylation of the substrate in the presence of the test compound indicates that the test compound has anti-cancer activity.

[0018] In an aspect the disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of a compound that inhibits activity of a CaMK biological cascade in the subject.

[0019] In an aspect the disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of a compound that inhibits activity of at least one of CaMKK or AMPK.

[0020] In an aspect the disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of a compound that inhibits activity of at least one of CaMKKβ, CaMKKβ splice variant 2, or CaMKKβ splice variant 7.

[0021] In a further aspect the disclosure provides a method of treating prostate cancer in a subject, comprising administering to the subject an effective amount of a compound that inhibits activity of a CaMK biological cascade in the subject.

[0022] In a further aspect the disclosure provides a method of treating prostate cancer in a subject, comprising administering to the subject an effective amount of a compound that inhibits activity of at least one of CaMKK or AMPK.

[0023] In a further aspect the disclosure provides a method of treating prostate cancer in a subject, comprising administering to the subject an effective amount of a compound that inhibits activity of at least one of CaMKKβ, CaMKKβ splice variant 2, or CaMKKβ splice variant 7.

[0024] In yet another aspect, the disclosure relates to a method of treating prostate cancer in a subject, the method comprising administering to the subject an effective amount of an inhibitor of phosphorylated AMPK, phosphorylated AMPKα1 subunit, or phosphorylated AMPKα2 subunit.

[0025] In another aspect, the disclosure provides a method of determining the efficacy of therapy in a patient being treated for prostate cancer, the method comprising: determining an amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in a series of samples from the subject, where the samples are taken from the subject at different time points during the therapy; and comparing the determined amount over the course of the time points; wherein when the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 in the series of samples is about the same or increases over the course of the time points, the therapy is not effective.

[0026] In an aspect the disclosure provides a method of inhibiting androgen-mediated migration of a prostate cancer cell in a subject comprising administering to the subject an effective amount of a compound that inhibits activity of a CaMK biological cascade in the subject.

[0027] In an aspect the disclosure provides a method of inhibiting androgen-mediated migration of a prostate cancer cell in a subject comprising administering to the subject an effective amount of a compound that inhibits activity of at least one of CaMKK or AMPK.

[0028] In another aspect the disclosure provides a method of inhibiting androgen-mediated migration of a prostate cancer cell in a subject comprising administering to the subject an effective amount of an inhibitor of at least one of CaMKKβ, CaMKKβ splice variant 7, or CaMKKβ splice variant 2 or any combination thereof.

[0029] In an aspect the disclosure provides a method of inhibiting androgen-mediated invasion of a prostate cancer cell in a subject comprising administering to the subject an effective amount of a compound that inhibits activity of a CaMK biological cascade in the subject.

[0030] In an aspect the disclosure provides a method of inhibiting androgen-mediated invasion of a prostate cancer cell in a subject comprising administering to the subject an effective amount of a compound that inhibits activity of at least one of CaMKK or AMPK.

[0031] In an aspect the disclosure provides a method of inhibiting androgen-mediated invasion of a prostate cancer cell in a subject comprising administering to the subject an effective amount of an inhibitor of at least one of CaMKKβ, CaMKKβ splice variant 7, or CaMKKβ splice variant 2 or any combination thereof.

[0032] In an aspect the disclosure provides a method of inhibiting metastasis of prostate cancer in a subject comprising administering to the subject an effective amount of a compound that inhibits activity of a CaMK biological cascade in the subject.

[0033] In an aspect the disclosure provides a method of inhibiting metastasis of prostate cancer in a subject comprising administering to the subject an effective amount of a compound that inhibits activity of at least one of CaMKK or AMPK.

[0034] In an aspect the disclosure provides a method of inhibiting metastasis of prostate cancer in a subject comprising administering to the subject an effective amount of an inhibitor of at least one of CaMKKβ, CaMKKβ splice variant 7, or CaMKKβ splice variant 2 or any combination thereof.

[0035] Aspects also relate to a nucleic acid molecule comprising a sequence that binds under stringent conditions to a region that is about 2.3 kb upstream (5') relative to a CaMKKβ transcriptional start site.

[0036] Further aspects relate to an antibody that specifically binds to a C-terminal portion of a CaMKKb.

[0037] Aspects also relate to polynucleotides (e.g., siRNA) that comprise a sequence that is complementary to CaMKKβ, CaMKKα, or AMPK and having kinase-inhibitory activity.

[0038] The disclosure provides for and encompasses additional aspects and embodiments, which will be apparent to those of skill in the art in light of the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1. Androgens increase CaMKKβ levels in an AR-dependent manner. LNCaP or VCaP cells were treated for 24 h with vehicle or increasing concentrations of the synthetic androgen R1881 (A-0.1, 1, and 10 nM; B-0.01, 0.1, 1, and 10 nM). A, after treatment, cells were lysed, and RNA was isolated and reversed transcribed. The expression of CaMKKβ was assessed using qPCR. B, after treatment, cells were subjected to western blot analysis and subsequent densitometry (top). CaMKKβ protein levels were normalized to GAPDH loading control. A and B, results are expressed as fold induction over vehicle-treated cells+SE (n=3). *, significant changes from vehicle-treated cells. C, LNCaP cells were transiently transfected with mock or Stealth siRNAs targeting a negative control (siLacZ) or CaMKKβ (#1-3). Two days later, cells were treated for 24 h+/-10 nM R1881. Whole-cell extracts were subjected to western blot analysis and densitometry (top) as described in B. *, significant changes from mock-transfected cells. D, LNCaP cells were transfected as described in C with mock or Stealth siRNAs targeting LacZ or AR and treated for 24 h. The expression of CaMKKβ was assessed as in A using qPCR.

[0040] FIG. 2. Validation of CaMKKβ protein bands. For CaMKKβ western blot analysis, a different monoclonal CaMKKβ antibody (clone 1A11) was used than in FIG. 1. CaMKKβ protein levels were normalized to GAPDH loading control. Results are expressed as fold induction over vehicle-treated cells+SE (n=2). *, P<0.05 indicates significant changes from mock-transfected cells.

[0041] FIG. 3. CaMKKβ levels are increased in prostate cancer samples. Independent microarrays were analyzed using the Oncomine resource. A, four separate studies determined that CaMKKβlevels were elevated in prostate cancer samples (red) compared to normal prostate controls (blue). B, CaMKKβ levels correlated with disease progression. C, CaMKKβ levels are significantly higher in prostate cancer samples compared to other cancers (a-bladder, b-kidney, c-colon, d-breast, e-esophageal, f-liver, g-lung, h-ovarian, i-pancreatic, j-squamous cell lung). All changes in expression were at the P<0.001 level.

[0042] FIG. 4. The prostate expresses a different functional splice variant of CaMKKβ compared to brain. A, schematic of CaMKKβ splice variants. B, RT-PCR using primers spanning specific exons (indicated in right schematic) was performed on cDNA generated from various tissues and cell lines. C, LNCaP or VCaP cells were treated for 24 h+/-10 nM R1881. Cell lysates were then subjected to western blot analysis and subsequent densitometry (right). Phospho-CaMKI (p-CaMKI) protein levels were normalized to total CaMKI. Results are expressed as fold CaMKI phosphorylation over vehicle-treated cells+SE (n=3). *, significant changes from vehicle-treated cells.

[0043] FIG. 5. The prostate expresses different splice variants of CaMKK compared to brain (expanded FIG. 4A and FIG. 4B). A, schematic of CaMKKβ splice variants. B, RT-PCR using primers spanning specific exon-exon boundaries (indicated in right schematic) was performed on cDNA generated from various tissues and cell lines.

[0044] FIG. 6. CaMKKβ activity in androgen-mediated cell migration in prostate cancer cells. A, LNCaP cells were pretreated for 1 h with vehicle, 10 or 30 mM STO-609 prior to overnight treatment with vehicle, 100 pM or 10 nM R1881. Cell lysates were then subjected to western blot analysis and subsequent densitometry (right). Phospho-CaMKI (p-CaMKI) levels were normalized to total CaMKI. Results are expressed as fold induction/phosphorylation over double vehicle-treated cells+SE (n=2). *, P<0.05 indicates significant changes from vehicle-treated cells. ^#, P<0.05 indicates significant changes from vehicle (no STO-609)-treated cells. B, VCaP cells were plated in 96-well plates and grown for 3 d. Cells were treated +/-1 nM R1881 and +/-30 mM STO-609 on d 3, d 5, and d 7. On d 10, cells were lysed and the relative number of cells was measured with the fluorescent DNA binding dye FluoReporter Blue. Each sample was performed in triplicate, and results from a representative experiment are shown. Results are expressed as relative cell number ±SE (n=2). *, P<0.05 indicates significant changes from vehicle (no R1881)-treated cells. C, VCaP cells were pretreated for 1 h+/-30 mM STO-609 prior to overnight treatment +/-10 nM R1881. Cells were then dissociated and reseeded into the top chamber for a Boyden dual chamber migration assay. Fresh medium with the corresponding treatments was added to the top and bottom chambers while either no chemoattractant or 5% FBS (serum) was added to the bottom chamber. After 16 h, migrated cells were fixed, stained with crystal violet and counted in three different microscopic fields and added together. The results are expressed as mean±SE (n=2). *, P<0.05 indicates significant changes from vehicle (no R1881)-treated cells. D and E, densitometry results for western blots in FIG. 7C and FIG. 7D respectively. *, P<0.05 indicates significant changes from vehicle-treated (D) or GAL4 control (E) cells. ^#, P<0.05 indicates significant changes from control (siLacZ)-transfected cells (D).

[0045] FIG. 7. CaMKKβ activity in the androgen-mediated migration and invasion of prostate cancer cells. A, LNCaP cells were plated in 96-well plates and grown for 3 d. Cells were treated +/-1 nM R1881 and +/-30 mM STO-609 on d 3, d 5, and d 7. On d 10, cells were lysed and the relative number of cells was measured with the fluorescent DNA binding dye FluoReporter Blue. Each sample was performed in triplicate, and results from a representative experiment are shown. Results are expressed as relative cell number ±SE (n=2). *, significant changes from vehicle (no R1881)-treated cells. B, LNCaP cells were pretreated for 1 h+/-30 mM STO-609 prior to overnight treatment+/-10 nM R1881. Cells were then dissociated and reseeded into the top chamber for a Boyden migration or Matrigel extracellular matrix invasion assay. Fresh medium with the corresponding treatments was added to the top and bottom chambers while either no chemoattractant or 5% FBS (serum) was added to the bottom chamber. After 16 h, migrated cells were fixed, stained and counted in three different microscopic fields and added together. The results are expressed as mean±SE (n=3). *, significant changes from vehicle (no R1881)-treated cells. ⁴, significant changes from vehicle (no STO-609)-treated cells. C top, LNCaP cells were transfected with indicated siRNAs. Two days after transfection, cells were treated +/-10 nM R1881 and subjected to a Boyden migration assay as described in B. *, significant changes from vehicle-treated cells. ⁴, significant changes from control (siLacZ)-transfected cells. C bottom, western blot to demonstrate CaMKKβ knockdown. Quantification of these blots is presented in FIG. 6D. D right, LNCaP cells stably expressing either GAL4 (control) or CaMKKβ were subjected to a migration assay as described in B using +/-5% FBS as chemoattractant. The results are expressed as mean±SE (n=3). *, significant changes from LNCaP-GAL4 cells. D left, western blot confirming CaMKK expression. Quantification of these blots is presented in FIG. 6E.

[0046] FIG. 8. Androgen mediated prostate cancer cell migration and functional AR-mediated transcription. A and B, an example of the AR replacement strategy is shown. This method has the advantage of using cells with endogenous androgen signaling as opposed to the common reintroducing of AR into AR-negative cells, which often has artificial biological consequences. Here, cells that express endogenous AR, in this case LNCaPs, were retrovirally infected to create stable cell lines expressing a control (GAL4) or a v5-tagged version of AR (wild type or a DNA-binding domain mutant (C562S)) linked to an IRES-EGFP. Cells were then selected using 2 rounds of flow cytometry. Subsequently, EGFP-positive cells were transfected with chemical siRNAs targeting either a control sequence (siLacZ) or the 3'-UTR of AR (eliminates endogenous receptor). A, a western blot characterization of the resultant cell lines is shown at the right using antibodies for v5 (recognizes only exogenous AR), AR (recognizes both exogenous and endogenous AR) or GAPDH (loading control). B, LNCaP cells used in the AR replacement experiments were also subjected to qPCR analysis using primers targeting the AR 3'UTR (monitors endogenous AR levels). The expression of AR was normalized to 36B4 levels and results are expressed as relative mRNA levels of AR compared to mock-transfected cells+SE (n=2). *, P<0.05 indicates significant changes from mock-transfected cells. C, cells were then subjected to a migration assay as described in FIG. 9. *, P<0.05 indicates significant changes from vehicle-treated cells.

[0047] FIG. 9. Identification of the ARE that regulates CaNIKKβ expression. A, LNCaP cells were pretreated for 1 h with vehicle or 1 mg/mL cycloheximide followed by vehicle or 10 nM R1881 for 24 h. CaMKKβ or CXCR4 mRNA levels were quantified using qPCR. Results are expressed as fold induction over vehicle (no R1881)-treated cells ±SE (n=3). *, significant changes from vehicle-treated cells. B, LNCaP cells were treated with vehicle (V) or 10 nM R1881 for 1 or 4 h. Cross-linked chromatin was immunoprecipitated with indicated antibodies. The precipitated DNA was amplified using primers spanning a region identified using ChIP on Chip data as a potential AR-binding site (indicated in top schematic) or a distal upstream region (negative control). The results are presented as percent input ±SE (n=3). *, significant changes from IgG controls. C, various enhancer luciferase reporter constructs (depicted in top model) were transfected into LNCaP cells and treated overnight +/-10 nM R1881. After treatment, cells were harvested and assayed for luciferase activity. Luciferase values were normalized to J3-galactosidase control. Data are the mean relative light units (RLUs)+SEM for one representative experiment performed in triplicate (n=3). *, significant changes from vehicle-treated cells. D, CaMKKβpromoter constructs (depicted in top model) were transfected into LNCaP cells and then treated overnight with vehicle or 10 nM R1881. After treatment, cells were harvested and assayed for luciferase activity as in C. Emp Vec, empty vector.

[0048] FIG. 10. Identification of the ARE that regulates CaMKKβ expression. A, two CaMKKβ enhancer (fragments D and E from FIG. 9C) luciferase reporter constructs were transfected into LNCaP cells and then pretreated for 30 minutes with vehicle or 10 mM Casodex followed by treatment overnight with vehicle or various concentrations of R1881 (0, 0.1, 1 and 10 nM). After treatment, cells were harvested and assayed for luciferase activity. Luciferase values were normalized to β-galactosidase control. Data are the mean relative light units (RLUs) SEM for one representative experiment performed in triplicate (n=3). *, P<0.05 indicates significant changes from vehicle (no R1881)-treated cells. ^# P<0.05 indicates significant changes from vehicle (no Casodex)-treated cells. B, VCaP cells were transfected, treated and assayed for luciferase activity as in A using the PSA enhancer and CaMKKβ enhancer fragments D and E. C, CaMKKβ enhancer deletion constructs were transfected into LNCaP cells and then treated and assayed for luciferase activity as in A (n=2). Emp Vec, empty vector.

[0049] FIG. 11. Androgen-mediated migration occurs through a CaMKKβ-AMPK-dependent pathway. A, LNCaP cells were pretreated for 1 h+/-30 mM STO-609 prior to overnight treatment +/-10 nM R1881. Cell lysates were then subjected to western blot analysis and subsequent densitometry (right). CaMKKβ levels were normalized to GAPDH. Phospho-CaMKI (p-CaMKI) levels were normalized to total CaMKI. Phospho-AMPK (p-AMPK) levels were normalized to total AMPK. Results are expressed as fold induction/phosphorylation over double vehicle-treated cells+SE (n=3). *, significant changes from vehicle-treated cells. B, LNCaP cells stably expressing either GAL4 or CaMKK were treated overnight +/-10 nM R1881. Cell lysates were then subjected as in A to western blot analysis and densitometry (right). Results are expressed as fold induction/phosphorylation over LNCaP-GAL4 vehicle-treated cells+SE (n=3). *, significant changes from LNCaP-GAL4 vehicle-treated cells. C and D, LNCaP cells were transfected with indicated siRNAs, treated and subjected to a migration assay (top) or western blot analysis (bottom) as in FIG. 7C. *, significant changes from control (siLacZ)-transfected cells. Quantification of the blots is presented in FIG. 12.

[0050] FIG. 12. Androgen-mediated migration occurs through a CaMKKβ-AMPK-dependent pathway. A, VCaP cells were treated for 24 h+/-10 nM R1881. Cell lysates were then subjected to western blot analysis and subsequent densitometry (right). Phospho-CaMKI (p-CaMKI) levels were normalized to total CaMKI. Phospho-AMPK (p-AMPK) levels were normalized to total AMPK. Results are expressed as fold induction/phosphorylation over vehicle-treated cells+SE (n=2). *, P<0.05 indicates significant changes from vehicle-treated cells. B, selection of optimal AMPKα1 and α2 siRNAs. LNCaP cells were transfected as described in FIG. 11 with mock or Stealth siRNAs targeting LacZ (negative control) or AMPKα1 or α2. The expression of AMPK was assessed using qPCR and normalized to 36B4 levels. Results are expressed as fold induction over mock-transfected cells+SE (n=2). *, P<0.05 indicates significant changes from mock-transfected cells. C and D, densitometry results for western blots in FIG. 11C and FIG. 11D, respectively. For AMPKαknockdown (C), siAMPKα1-#1 and siAMPKα2-#1 from B were selected since they produced the greatest knockdowns. *, P<0.05 indicates significant changes from control (siLacZ)-transfected cells.

[0051] FIG. 13. AMPK and androgen-mediated prostate cancer cell migration. A, LNCaP cells were pretreated for 1 h with vehicle or increasing concentrations of compound C (10 or 40 mM) prior to overnight treatment +/-10 nM R1881 or 1 mM AICAR. Cells were then subjected to a migration assay as described in FIG. 7. The results are expressed as mean±SE (n=2). *, P<0.05 indicates significant changes from double vehicle-treated cells. ^#, P<0.05 indicates significant decreases from vehicle (no compound C)-treated cells. B, LNCaP cells were pretreated for 1 h with vehicle, 1, 10 or 40 mM compound C prior to overnight treatment+/-10 nM R1881. Cell lysates were then subjected to western blot analysis and subsequent densitometry (top). ACC is a direct target of AMPK and thus, was used as a readout of AMPK catalytic activity. Phospho-ACC (p-ACC) levels were normalized to total ACC. Results are expressed as fold induction/phosphorylation over double vehicle-treated cells +SE (n=2). *, P<0.05 indicates significant changes from double vehicle-treated cells. C, LNCaP cells were treated overnight +/-1 mM AICAR and then subjected to western blot analysis and densitometry (top) as in B. Phospho-AMPK (p-AMPK) levels were normalized to total AMPK. *, P<0.05 indicates significant changes from vehicle-treated cells.

DETAILED DESCRIPTION

[0052] The inventors have identified Ca²+/calmodulin-dependent protein kinase kinases (CaMKKs), such as CaMKKβ, as viable targets for therapeutic intervention in various cancers such as, for example, prostate cancer, glioblastoma, and myeloid leukemia as well as other cancer types described herein. In a general sense, the disclosure provides an array of compounds and compositions that are active inhibitors of CaMKK and use of such compounds in methods relating to detection, determination of disease stage/progression, prognostic evaluation of hormone therapy, treatment of disease, identification of active agents against various cancers, as well as identification of CaMKK inhibitors, including inhibitors that are selective for a particular CaMKK. For purposes of illustration some particular aspects and embodiments are explicitly described herein, relating to Ca²+/calmodulin-dependent protein kinase kinase β (CaMKKβ) which is shown (a) to be expressed in the prostate, (b) to be regulated by AR, (c) to correspond to prostate cancer progression/disease stage and, accordingly, provides a therapeutic target for prostate cancer.

[0053] As used herein, the term "Ca²+/calmodulin-dependent protein kinase kinase" and/or "CaMKK" are used interchangeably herein and refer to a serine/threonine protein kinase that can phosphorylate and activate members of the Ca²+/calmodulin-dependent protein kinase (CaMK) family of enzymes as well as other protein substrates such as AMPK (e.g., SEQ ID NOs: 21-24). The terms encompass all of the various isoforms, orthologs, and splice variants of CaMKK proteins such as, for example, Ca²+/calmodulin-dependent protein kinase kinase (3 (CaMKKβ, or CaMKK2) Ca²+/calmodulin-dependent protein kinase kinase α (CaMKKα, or CaMKK1), splice variants such as, for example, CaMKKβ splice variants 1-7, CaMKKα splice variants 1-3, and the like (e.g., SEQ ID NOs: 1-20 and 25-46). Some embodiments relate to CaMKKβ that comprises the amino acid sequence of SEQ ID NO:2, or a fragment thereof. The CaMKK amino acid sequences, such as CaMKKβ, can be encoded by any appropriate polynucleotide molecule as determined by the genetic code and codon usage in any particular organism. In embodiments, CaMKKβ is encoded by a polynucleotide comprising SEQ ID NO:1, or a fragment thereof. Some embodiments relate to CaMKKα that comprises the amino acid sequence of SEQ ID NO:16, or a fragment thereof. In some embodiments, CaMKKα like CaMKKβ above, is encoded by any polynucleotide that can be envisioned by one of skill in the art and, in some embodiments, comprises SEQ ID NO:15, or a fragment thereof.

[0054] In some embodiments the disclosure relates to a CaMKK splice variant. Non-limiting examples of CaMKK splice variants include nucleotide sequences of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 17, and 19. Some embodiments relate to "CaMKKβ splice variant 2" and comprise a nucleotide sequence of SEQ ID NO:3, or a fragment thereof. Some embodiments relate to "CaMKKβ splice variant 7" and comprise a nucleotide sequence of SEQ ID NO:13. In these embodiments, the splice variant proteins encoded by SEQ ID NO:3 and SEQ ID NO:13 are identical in sequence. Thus, embodiments of disclosure provide for a polynucleotide that encodes a CaMKKβ splice variant protein comprising SEQ ID NO:4, or a fragment thereof. Similarly, the disclosure relates to polynucleotide sequences that encodes an amino acid sequence of any CaMKK or CaMK protein such as, for example those of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. As noted above, CaMKKβ splice variant 2 and splice variant 7 encode for the same amino acid sequence; thus, in embodiments relating to a CaMKKβ amino acid sequence encoded by splice variant 2 or splice variant 7, reference to an amino acid sequence encoded by either splice variant will also encompass the other (i.e., each term is interchangeable with and inclusive of the other when it relates to the encoded amino acid sequence).

[0055] Any sample can be used in the methods described herein. Embodiments provide for the use of a biological sample (e.g., tissue biopsy, cerebrospinal fluid, blood, sera, sputum, urine and/or tumor biopsies) from a subject with and/or without a cancer (which can be determined using standard clinical tests).

[0056] Embodiments of the disclosure relate to compounds that are inhibitors of a CaMK biochemical cascade. A CaMK biochemical cascade refers to a biochemical activation pathway that typically involves the phosphorylation of a first Ca²+/calmodulin-dependent protein kinase (CaMK) by a second Ca²+/calmodulin-dependent protein kinase (thus, a Ca²+/calmodulin-dependent protein kinase kinase (CaMKK)). The phosphorylated CaMK can subsequently phosphorylate a substrate. CaMK cascades are described in the literature. See, Corcoran, E. E., and Means, A. R., J Biol Chem, (Feb. 2, 2001); 276(5):2975-2978, incorporated herein by reference.

Methods of Treatment

[0057] In an aspect, the disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of a Ca²+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor. In embodiments, the method comprises administering an effective amount of a CaMKK inhibitor that is a selective inhibitor of CaMKKα and/or CaMKKβ. In some embodiments the CaMKK inhibitor is a selective inhibitor of CaMKKα. In some embodiments the CaMKK inhibitor is a selective inhibitor of CaMKKβ. The term "selective inhibitor," including "selective inhibitor of CaMKKβ/α" or "CaMKKβ/αselective inhibitor" relates to a compound (e.g., a small molecule or biological molecule) that has increased inhibitory activity for a target, for example, CaMKKβ or CaMKKα, relative to the inhibitory activity for other CaMKs. For purposes of illustration, when describing embodiments comprising a selective inhibitor of CaMKKβ, examples of "other" CaMKs include natural/physiological substrates of CaMKKβ such as, for example, Ca²+/calmodulin-dependent protein kinases (e.g., CaMKI and CaMKIV), CaMKs that are not substrates of CaMKKβ (e.g., CaMKII and CaMKIII), AMP-activated protein kinase (e.g., AMPKα1 subunit and AMPKα2 subunit) as well as other kinases that can phosphorylate such substrates (CaMKKα). Embodiments also relate to polypeptide fragments comprising a sequence that contains a portion of a CaMKKβ substrate. In such embodiments, the fragment comprises an amino acid that can be phosphorylated. In some embodiments a selective inhibitor comprises a ratio of IC₅₀ concentrations (concentration inhibiting 50% of activity) wherein the ratio of the IC₅₀ concentration for one or more other CaMKs to the IC₅₀ concentration for CaMKKβ is greater than 1. The ratio of IC₅₀ values can be readily determined from data obtained from one or more assay(s) (performed separately, in parallel or series) that is effective to measure activity or abundance of a CaMK or CaMKK (e.g., phosphorylation, mRNA transcription, protein expression, etc.), and can comprise any methods known in the art such as, for example those disclosed in U.S. Pat. No. 7,105,312, which is incorporated herein by reference. The inhibitory activity can be assessed and demonstrated either in vivo and/or in vitro optionally in cell-based or cell-free assay systems.

[0058] In general, the CaMKK inhibitor, including a CaMKK selective inhibitor, can be any type of chemical or biological molecule that exhibits inhibitory activity against one or more CaMKK. Effective CaMKK inhibitors for use in the methods described herein can inhibit the kinase activity of a CaMKK or they can regulate the amount of a CaMKK in a cell. Accordingly, the CaMKK inhibitors can inhibit phosphorylation associated with a CaMK cascade, and/or regulate expression of a CaMKK (e.g., by inhibiting a CaMKK gene promoter, inhibiting CaMKK gene transcription, inhibiting CaMKK mRNA translation, and/or affect CaMKK mRNA stability).

[0059] In some embodiments of this aspect, the method includes at least one selective inhibitor of CaMKKβ that comprises a compound of Formula III:

##STR00004##

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R_7a, R₈, R₉, R₁₀, and R₁₁ are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, heterocyclo, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, alkoxy, halo, mercapto, azido, cyano, formyl, carboxylic acid, hydroxyl, nitro, acyl, aryloxy, alkylthio, amino, alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfoxyl, sulfonyl, sulfonate, sulfonic acid, sulfonamide, urea, alkoxylacylamino, aminoacyloxy, and, for the groups R₇ and R_7a, can optionally be taken together to form oxo; or a pharmaceutically acceptable salt or prodrug thereof. Such compounds are disclosed in U.S. patent application publication US 2010/0105716, incorporated herein by reference.

[0060] In further embodiments R₇ and R_7a together form oxo (C═O).

[0061] In some embodiments, R₇ and R_7a do not form oxo (C═O).

[0062] In some embodiments, R₃ is --COOH, --CH₂COOH, --CH₂CH₂COOH, or an ester thereof.

[0063] In some embodiments, R₁, R₂, R₄, R₅, R₆, R₇, R_7a, R₈, R₉, R₁₀, R₁₁ are all H.

[0064] In some embodiments, at least one, two, or three of R₁, R₂, R₄, R₅, R₆, R₇, R_7a, R₈, R₉, R₁₀, and R₁₁ is not H. Thus, in some embodiments, R₁ is not H; in some embodiments R₂ is not H; in some embodiments R₃ is not H; in some embodiments R₄ is not H; in some embodiments R₅ is not H; in some embodiments R₆ is not H; in some embodiments R₇ is not H; in some embodiments R₉ is not H; in some embodiments R₁₀ is not H; and/or in some embodiments R₁₁ is not H.

[0065] Compounds of Formula III, including further definitions of substituent terms and various formulations thereof are disclosed in U.S. Patent Application Publication No: 2010/0105716 as useful in methods of treating metabolic diseases/disorders including obesity, insulin resistance, hyperglycemia, diabetes, and the like. Synthetic routes and strategies for the compounds of Formula III are known in the art. The disclosure of US 2010/0105716 is incorporated herein by reference.

[0066] Embodiments of this aspect relate to a method comprising a selective inhibitor of CaMKKβ of Formula I:

##STR00005##

wherein R₁ is selected from CN, COOH, or COCl. Compounds of Formula I are described in U.S. Pat. No. 2,820,037 for use as dye molecules, and is incorporated herein by reference.

[0067] Other embodiments of this aspect relate to a method comprising a selective inhibitor of CaMKKβ of Formula II:

##STR00006##

wherein R₁ and R₂ are independently selected from H, halo, alkyl, or haloalkyl; and R₃ is H, alkyl, or substituted alkyl, or three COOR₃ groups can be substituted at any location on the naphthalene ring. Compounds of Formula II are disclosed in Japanese Patent Application No. 2003-012516 as Ca²+/calmodulin dependent kinase kinase (CaMKK) inhibitors; however the reference fails to disclose the use of these compounds as effective in methods relating to cancer. The disclosure of Japanese Patent Application No. 2003-012516 is incorporated herein by reference.

[0068] The compounds of Formulas I-III can be synthesized by any method known in the art such as, for example, the methods described Japanese Patent Application No. 2003-012516; U.S. Pat. No. 2,820,037; U.S. Pat. No. 2,835,674; U.S. Pat. No. 2,965,644; U.S. Pat. No. 2,949,467; U.S. Pat. No. 3,953,452; U.S. Pat. No. 3,960,867; U.S. Pat. No. 4,239,868; and U.S. Pat. No. 4,336,383, each of which is incorporated herein by reference.

[0069] In other embodiments, the CaMKK inhibitor is a biological molecule, such as a polynucleotide having RNAi activity against a CaMKK or a substrate thereof, or an antibody that can specifically bind to a CaMKK or a substrate thereof.

[0070] Nucleic Acids/RNAi

[0071] Embodiments of the disclosure relate to methods that include CaMKK inhibitors, wherein the inhibitors comprise nucleic acid molecules having inhibitory activity against one or more biological molecules involved in a CaMK cascade including CaMK enzymes such as, for example, CaMKI and/or CaMKIV as well as kinases for such molecules (CaMKKα, CaMKKβ, etc.), other biological substrates of CaMKKs (e.g., AMPK), as well as other CaMKs (e.g., CaMKII and CaMKIII). In embodiments, the nucleic acid molecules can include decoy RNAs, dsRNAs, siRNAs, nucleic acid aptamers, antisense nucleic acid molecules, and enzymatic nucleic acid molecules that comprise a sequence that is sufficient allow for binding to a CaMK, AMPK, or CaMKK encoding nucleic acid sequence and inhibit activity thereof (i.e., are complementary to such encoding nucleic acid sequences).

[0072] In embodiments, the inhibitory nucleic acid molecule can bind to a target CaMK, AMPK, or CaMKK nucleic acid sequence under stringent binding conditions. The terms "stringent conditions" "stringent binding conditions" or "stringent hybridization conditions" refers to conditions under which a polynucleotide will hybridize to a target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). An example of stringent conditions include those in which hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C. is performed. Amino acid and polynucleotide identity, homology and/or similarity can be determined using the ClustalW algorithm, MEGALIGN® (Lasergene, Wis.).

[0073] Given a target polynucleotide sequence of a CaMK, CaMKK, or biological substrate thereof, an inhibitory nucleic acid molecule can be designed using motifs and targeted to a region that is anticipated to be effective for inhibitory activity, such as is known in the art.

[0074] Antibodies

[0075] Embodiments of the disclosure relate to methods that include CaMKK inhibitors, wherein the inhibitors comprise antibodies having specific binding activity against one or more biological molecules involved in a CaMK cascade including CaMK enzymes such as, for example, CaMKI and/or CaMKIV as well as kinases for such molecules (CaMKKα, CaMKKβ, etc.), biological substrates of CaMKKs (e.g., AMPK), and CaMKs that are not substrates of CaMKKs (e.g., CaMKII and CaMKIII).

[0076] Preparation of Antibodies

[0077] The antibodies described herein can be produced by any method known in the art, such as by immunization with a full-length CaMK or CaMKK, or fragments thereof. The antibodies can be polyclonal or monoclonal, and/or may be recombinant antibodies. In embodiments, antibodies that are human antibodies can be prepared, for example, by immunization of transgenic animals capable of producing a human antibody (see, for example, International Patent Application, Publication WO 93/12227).

[0078] Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein [Nature (1975); 256:495], and other techniques, e.g., viral or oncogenic transformation of B-lymphocytes.

[0079] Animal systems for preparing hybridomas include mouse. Hybridoma production in the mouse is very well established, and immunization protocols and techniques for isolation of immunized splenocytes for fusion are well known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0080] In embodiments, human monoclonal antibodies directed against a CaMK or CaMKK can be generated using transgenic mice carrying parts of the human immune system rather than the mouse system. These transgenic mice, referred to herein as "HuMab" mice, contain a human immunoglobulin gene minilocus that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci [Lonberg et al., Nature (1994); 368:856-859]. The preparation of HuMab mice is described in detail in Taylor et al., Nucleic Acids Res. (1992); 20:6287-6295; Chen et al., International Immunology (1993); 5:647-656; Tuaillon et al., J. Immunol. (1994); 152:2912-2920; Lonberg et al., Nature (1994); 368:856-859; Lonberg, Handbook of Exp. Pharmacology (1994); 113:49-101; Taylor et al., International Immunology (1994); 6:579-591; Lonberg & Huszar, Intern. Rev. Immunol. (1995); 13:65-93; Harding & Lonberg, Ann N.Y. Acad. Sci. (1995); 764:536-546; Fishwild et al., Nature Biotechnology (1996); 14:845-851, the contents of all of which are hereby incorporated by reference in their entirety. See further U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, as well as U.S. Pat. No. 5,545,807 to Surani et al.; International Patent Application Publication Nos. WO 93/1227, published Jun. 24, 1993; WO 92/22646, published Dec. 23, 1992; and WO 92/03918, published Mar. 19, 1992, the disclosures of all of which are hereby incorporated by reference in their entirety.

[0081] Embodiments provide human monoclonal antibodies that are specific for and neutralize biological activity of human CaMK and/or CaMKK polypeptides. Such antibodies can comprise heavy and light chain amino acid sequences, the light and heavy chain variable regions, and any combination (including all) hypervariable CDR regions, which are specific for and neutralize CaMK and/or CaMKK polypeptides when they bind. Such antibodies can provide an effective immunotherapy for CaMK and CaMKK associated diseases including various cancers, such prostate cancer, glioma, glioblastoma, and myeloid leukemia. Such antibodies also provide a useful reagent for the detection of a CaMK or a CaMKK in a biological sample.

[0082] In an embodiment, the antibodies target an epitope in a region of CaMK and/or CaMKK located in the C-terminal portion. In some embodiments, the antibody recognizes and binds specifically to an epitope in the C-terminal region of CaMKKβ splice variants 2 and/or 7.

[0083] In some embodiments, the antibodies are of the IgG1, IgG2, IgG3, or IgG4 isotype. In other embodiments, the antibodies of the invention are of the IgM, IgA, IgE, or IgD isotype. In certain embodiments, the antibodies are cloned for expression in mammalian cells. In embodiments, the antibodies can be a fragment of an antibody that retains specific binding activity for a CaMK or CaMKK polypeptide and is effective to inhibit biological activity. Such fragments are known in the art and include, for example, single-chain antibodies (scFV), Fab, Fab', Fab₂, and the like.

[0084] Any of the CaMKK inhibitors disclosed herein and which are useful in the methods described herein can be provided as salts such as, for example, basic or acidic addition salts. The selection and formation of such salts are within the ability of one skilled in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 21^st ed., Lippincott Williams & Wilkins, A Wolters Kluwer Company, Philadelphia, Pa. (2005).

[0085] Further, embodiments of the disclosure provide for compositions or formulations comprising any of the CaMKK inhibitors disclosed herein that can are suitable for pharmaceutical use. Further, such formulations can be provided in suitable dosage forms. Such compositions, formulations, and dosage forms are known to those of skill in the art. For example, compounds of Formulas I-III can be provided as a composition or formulation and prepared in a dosage form as described U.S. patent application publication number US 2010/0105716, which is incorporated by reference herein. See, also, Remington: The Science and Practice of Pharmacy, 21^st ed., Lippincott Williams & Wilkins, A Wolters Kluwer Company, Philadelphia, Pa. (2005).

[0086] In an aspect, the disclosure provides a method for screening or identifying a compound having agonist or antagonist activity for CaMKK (including CaMKKβ and/or CaMKKα) that includes contacting CaMKK and a substrate therefor, in the presence and absence of a test compound, under conditions that allow for CaMKK-dependent phosphorylation of the substrate; and determining, directly or indirectly, the level of phosphorylation of the substrate, wherein a reduction in phosphorylation of the substrate in the presence of the test compound is indicative of a CaMKK antagonist (for example, an anticancer agent) and an increase in phosphorylation of the substrate in the presence of the test compound is indicative of a CaMKK agonist. In embodiments, the CaMKK is CaMKKβ.

[0087] In some embodiments of this aspect, the method identifies a compound that is selective for a specific CaMK relative to at least one other CaMK. In further embodiments, the method identifies a compound that is selective for CaMKKβ relative to at least one other CaMK such as, for example, CaMKI, CaMKII, CaMKIII, CaMKIV, or CaMKKα. Yet further embodiments of the method provide identification of a compound that is selective for CaMKKβ splice variant 2 or CaMKKβ splice variant 7, relative to at least one other CaMKKβ isoform, and relative to at least one other CaMK such as, for example, CaMKI, CaMKII, CaMKIII, CaMKIV, or CaMKKα.

[0088] Embodiments of these methods provide compounds having selective antagonist activity for a CaMK wherein the CaMK-dependent phosphorlyation of the substrate is reduced by about 4-fold or more in the presence of the compound compared to phosphorylation in the absence of the compound (e.g., about 4-fold to about 100-fold or more).

[0089] Merely for purposes of illustration of an embodiment of this aspect, an assay system can comprise calmodulin (CaM), calcium, CaMKKβ, and a substrate (such as a synthetic peptide that can be phosphorylated by CaMKKβ such as from either AMPK or CaMKIV). The assay can further comprise evaluation of the test compound(s) that involves AMPK as the enzyme and a peptide from acetyl-CoA-carboxylase (ACC) as the substrate. In particular, assay conditions that allow for phosphorylation are provided (e.g., any appropriate buffer system) and further includes one or the other of CaMKKα and CaMKKβ (i.e., run in parallel), a calcium salt (e.g., CaCl₂), a phosphate source (e.g., ATP, optionally comprising radiolabelled ³²P), calmodulin (CaM, e.g., from bovine), and two substrates (one that can be phosphorylated by both CaMKKα and CaMKKβ, while the other can only be phosphorylated by one or the other of CaMKKα or CaMKKβ). A non-limiting example of a substrate that can be phosphorylated by both CaMKKα and CaMKKβ includes CaMIV, or a peptide fragment thereof (for example, Lys-Lys-Lys-Lys-Glu-His-Gln-Val-Leu-Met-Lys-Thr-Val-Cys-Gly-Thr-Pro-Gly-T- yr). A non-limiting example of a substrate that can be phosphorylated by CaMKKβ and not CaMKKα includes AMPK, or a peptide fragment thereof (for example, Ala-Lys-Pro-Lys-Gly-Asn-Lys-Asp-Tyr-His-Leu-Gln-Thr-Cys-Cys-Gly-Ser-Leu-A- la-Tyr-Arg-Arg-Arg). Any substrate, including fragments thereof can be used in these methods, as long as the substrate can be phosphorylated. Differences between the amount of phosphorylation of the substrates can be used to evaluate the substrate specificity and selectivity of a candidate test compound. Concentrations of the various assay components can vary widely, but are usually in the range of 1 nM to 500 μM (for active reagents including proteins and substrates, phosphate source(s) and test compounds) and in the mM range for other assay components (calcium and magnesium salts/cofactors, reducing agents, buffer systems, etc.). Incubation time and temperature can also be varied depending on the particular activity and sensitivity of the assay components. In embodiments, the temperature can range from about 4° C. to about 30° C., and the incubation time can be on the order of minutes (e.g., 10 minutes) to hours (e.g., 1 hrs, 1.5 hrs, 2 hrs, 2.5 hrs, 3 hrs, 3.5 hrs, etc.). In embodiments, a selective inhibitor will inhibit CaMKKβ activity to a greater extent than it will inhibit CaMKKαactivity. In some embodiments the selective inhibitor will inhibit CaMKKβ activity about anywhere from about 3-100 fold or more, relative to CaMKKαactivity (e.g., about 10-20 fold, about 20-30 fold, about 40-50 fold, about 50-60 fold, about 60-70 fold, about 70-80 fold, about 80-90 fold, about 90-100 fold, or over 100 fold).

[0090] Embodiments of the disclosure provide for detection of CaMKK, such as CaMKKβ and/or CaMKKα in circulating tumor cells (CTCs). CTCs are known in the art and comprise cells that have detached from a primary tumor and circulate in the bloodstream. It is thought that CTCs may indicate potential for metastasis and spread of a primary tumor to different tissues. Thus, circulating tumor cells can be a factor indicating the metastatic spread of cancers, such as carcinomas, and can be used in methods for the detection of, and prognosticate the likelihood of, metastatic disease. See, e.g., Fidler I. J., Nat Rev Cancer (2003); 3:453-8; Sleijfer S., et al., Eur J Cancer (2007); 43:2645-50; Hayes D. F., and Smerage J., Clin Cancer Res (2008); 14:3646-50; Pantel K, et al., Nat Rev Clin Oncol (2009); 6:339-51; Pantel K., and Riethdorf S., Nat Rev Clin Oncol. (2009); 6:190-1; and Panteleakou Z., et al., Mol Med (2009); 15:101-14, all incorporated by reference. Methods for expanding and enriching the number of CTCs in a biological sample are known in the art and allow for measurable amounts of a biochemical marker (a genetic or biochemical signature, such as CaMKKβ) of disease (e.g., an androgen-driven cancer). Accordingly, methods for detecting the presence of a CaMK, such as CaMKKβ/α or phosphorylated AMPK allow for identification of therapies that can be useful in treatment of disease. In embodiments, such methods provide for a method of monitoring the course of a therapeutic treatment, such as administering an inhibitor of CaMKKβ, in a patient undergoing therapy, based on a detectable increase or decrease in the amount of the biochemical marker(s) in a sample comprising CTCs. Such analytic methods include Kaplan Meier Analysis which has been used to correlate overall survival before starting a new line of therapy for patients with metastatic breast, colorectal and prostate cancer. Patients can be divided into those with Favorable and Unfavorable CTC (Unfavorable: >5 CTC/7.5 mL for breast and prostate, >3 CTC/7.5 mL for colon). See, Miller, M. C., et al., J of Oncology. 2010. doi:10.1155/2010/617421. incorporated by reference. Methods known in the art, such as the CellSearch system ("Veridex CellSearch Website". March 2010. http://veridex.com/CellSearch/CellSearchHCP.aspx. Retrieved 2010-03-14, as well as other methods, have been demonstrated as a strong prognostic factor for overall survival in patients with metastatic breast, colorectal or prostate cancer. See, e.g., Paterlini-Brechot P, and Benali N. L., Cancer Lett. (2007); 253:180-204; "Veridex LLC. CellSearch circulating tumor cell kit premarket notification--expanded indications for use--metastatic prostate cancer". March 2010. http://www.fda.gov/cdrh/pdf7/K073338.pdf. Retrieved 2010-03-14; Cristofanilli M., et al., NEJM (2004); 351:781-91; Budd G., et al., Clin Can Res (2006); 12:6404-09; Cohen, S. J., et al., JCO (2008); 26:3213-21; DeBono, J. S., et al., Clin Can Res (2008); 14:6302-9; Allard, W. J., et al., Clin Can Res (2004); 10:6897-6904; and Riethdorf et al., Clin Cancer Res (2007); 13:920-8, all incorporated by reference.

[0091] In an aspect, the disclosure also provides a method for treating conditions or diseases associated with abnormal AMP-activated protein kinase (AMPK) activity, which includes increased phosphorylation of AMPK, by administering an effective amount of at least one compound that inhibits CaMKKβ to a subject having such a condition or disease. Diseases characterized by abnormal AMPK activity include, but are not limited to, various cancers including prostate cancer.

[0092] As used herein, the term "subject" is intended to include human and non-human animals. Exemplary human subjects include a human patient having a disorder, e.g., a disorder described herein, such as cancer, or a normal subject. The term "non-human animals" includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals (such as sheep, dogs, cats, cows, pigs, etc.), and rodents (such as mice, rats, hamsters, guinea pigs, etc.).

[0093] "Treatment" or "treat" refers to both therapeutic treatment and prophylactic or preventative measures. Those subjects in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.

[0094] The terms "treating" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures. Those subjects in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented. When used with reference to a disease or a subject in need of treatment the terms accordingly include, but are not limited to, halting or slowing of disease progression, remission of disease, prophylaxis of symptoms, reduction in disease and/or symptom severity, or reduction in disease length as compared to an untreated subject. In embodiments, the methods of treatment can abate one or more clinical indications of the particular disease being treated. Certain embodiments relating to methods of treating a disease or condition associated with activation of a substrate in a CaMK cascade (CaMKI, CaMKIV, AMPK) and comprise administration of therapeutically effective amounts of a compound that inhibits CaMKKβ, as well as pharmaceutical compositions thereof. In embodiments, the method of treating can relate to any method that prevents further progression of the disease and/or symptoms, slows or reduces the further progression of the disease and/or symptoms, or reverses the disease and/or clinical symptoms associated with expression of CaMKKβ or kinase activity thereof.

[0095] In embodiments, the methods are used to treat cancer in a subject, wherein the subject is a mammal. Yet further embodiments relate to methods wherein the mammal is a human.

[0096] Aspects of the disclosure provide a method of inhibiting CaMKKβ in a cell, including a cell within a subject, comprising contacting the cell with a compound in an amount effective to inhibit CaMKKβ activity. In embodiments, the method provides for inhibiting CaMKKβ activity in a cell in a subject, wherein the method includes administering to the subject a compound, or a pharmaceutically acceptable salt thereof, according to Formula I in an amount effective to inhibit CaMKKβ activity in the cell in the subject. Both the activity of CaMKKβ and AMPK can be monitored by any method familiar to those of skill in the art. In some embodiments CaMKKβ and/or AMPK activity can be monitored by clinical evaluation of the symptoms or stage of a disease associated with abnormal CaMKKβ and/or AMPK activity. In embodiments, the disease is cancer. In further embodiments, the cancer is glioma, glioblastoma, carcinoma, or leukemia. In some embodiments, the cancer is prostate cancer, cancer of the blood or bone marrow, or cancer of the brain/CNS.

[0097] In these embodiments, "inhibiting" or "inhibition" of CaMKKβ means that there is a measurable decrease in the activity of CaMKKβ in the presence of a compound (e.g., through contacting/administration), relative to the activity of CaMKKβ in the absence of the compound. As described above, the decrease in CaMKKβ activity can arise from direct inhibition of kinase activity by, for example, binding of a small molecule inhibitor of Formulas I-III to the active site of CaMKKβ. A decrease in CaMKKβ activity can also arise from inhibition of expression of a CaMKKβ gene via antisense inhibition, gene silencing, disruption or degradation of CaMKKβ mRNA via RNAi (e.g., siRNA). CaMKKβ expression can also be modulated indirectly by manipulating the activity or expression of a regulator of CaMKKβ, such as androgen receptor (AR) or proteins involved in CaMKKβ splicing activity such as Fox2/RTA-1, using any agent having such activity. In embodiments, CaMKKβ can be inhibited by about 10% to about 100% or more (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, etc.) relative to a control. In some embodiments compounds can inhibit CaMKKβ (e.g., IC₅₀) at concentrations from about 0.1 nM to about 500 μM, (e.g., about 0.1 nM to about 250 μM, about 0.5 nM to about 200 μM, about 1.0 nM to about 100 μM, about 10 nM to about 50 μM, or about 100 nM to about 10 μM, and the like).

[0098] In some embodiments the therapeutically effective amount is an amount sufficient to stop or slow the progression of the cancer. In further embodiments, the therapeutically effective amount is an amount sufficient to reduce the number of cancer cells in the subject (i.e., killing of cancer cells). Methods for monitoring the proliferation of cancer cells and progress of cancer in a subject (e.g., tumor size, cell counts, biochemical markers, secondary indications, etc.) are known in the art.

[0099] In various embodiments of the method, the cancer is associated with the activity of CaMKKβ. Non-limiting examples of cancer that are associated with CaMKKβ activity include carcinoma, melanoma, leukemia, myeloid leukemia, glioma, and glioblastoma. In embodiments, the cancer is leukemia, cancer of the prostate or cancer of the brain/central nervous system. In further embodiments, the cancer is prostate cancer.

[0100] In some embodiments, the method of treatment is used as a co-therapy such as, for example, administration in conjunction with radiation, surgery, or other chemotherapeutics. In some embodiments, the method includes administration of a therapeutically effective amount of a compound that inhibits CaMKKβ in combination with an additional anti-cancer agent. A wide variety of anti-cancer (i.e., anti-neoplastic) agents are known in the art and include, for example alkylating agents, antimetabolites, natural antineoplastic agents, hormonal antineoplastic agents, angiogenesis inhibitors, differentiating reagents, RNA inhibitors, antibodies or immunotherapeutic agents, gene therapy agents, small molecule enzymatic inhibitors, biological response modifiers, and anti-metastatic agents.

[0101] In embodiments, the method comprises treating prostate cancer in a subject who is in need of treatment, where the method includes administering to the subject an effective amount of a CaMKK inhibitor in combination with a second treatment. In such embodiments, the second treatment can include such non-limiting examples as surgery, radiation, and chemotherapy. In further embodiments, the method comprises co-administration of an effective amount of a CaMKK inhibitor and a second agent effective against prostate cancer such as, for example, anti-androgens, Selective Androgen Receptor Modulators (SARMs), Selective Androgen Receptor Degraders (SARDs), CYP17 inhibitors, suphatase inhibitors, Src inhibitors, anti-estrogens, estrogens, Selective Estrogen Receptor Modulators (SERMs), Selective Estrogen Receptor Degraders (SERDs), ERb antagonists, aromatase inhibitors, vaccine-based therapeutics such as sipuleucel-T)(Provenge®, and the like. In further embodiments the method comprises administration an effective amount of a CaMKK inhibitor and an active agent selected from MDV3100 (an androgen receptor antagonist from Medivation Inc., San Francisco, Calif.); ARN-509 (an androgen receptor antagonist from Aragon Pharmaceuticals, San Diego, Calif.); bicalutamide (Casodex® a non-steroidal anti-androgen from AstraZeneca); or flutamide (Eulexin® a non-steroidal anti-androgen from Schering-Plough).

[0102] In some embodiments, the method of treatment can be used an adjuvant therapy (i.e., additional treatment) such as, for example, when compounds of any of Formulas I-III, or pharmaceutical compositions thereof, are administered after surgery or other treatments (e.g., radiation, hormone therapy, or chemotherapy). Accordingly, in such embodiments, the method of adjuvant therapy encompasses administering the compounds of Formula I-III to a subject following a primary or initial treatment, and can be administered either alone or in combination with one or more other adjuvant treatments, including, for example surgery, radiation therapy, or systemic therapy (e.g., chemotherapy, immunotherapy, hormone therapy, or biological response modifiers). Those of skill in the art will be able to use statistical evidence to assess the risk of disease relapse before deciding on the specific adjuvant therapy. The aim of adjuvant treatment is to improve disease-specific and overall survival. Because the treatment is essentially for a risk, rather than for provable disease, it is accepted that a proportion of patients who receive adjuvant therapy will already have been effectively treated or cured by their primary surgery. Adjuvant therapy is often given following surgery for many types of cancer including, for example, colon cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, and some gynecological cancers.

[0103] Some embodiments of the method relate to neoadjuvant therapy, which is administered prior to a primary treatment. Effective neoadjuvant therapy is commonly characterized by a reduction in the number of cancer cells (e.g., size of the tumor) so as to facilitate more effective primary treatment such as, for example, surgery.

[0104] The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Some non-limiting examples of cancer include carcinoma, melanoma, lymphoma, blastoma, sarcoma, germ cell tumors, and leukemia or lymphoid malignancies. Non-limiting examples of cancers that fall within these broad categories include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases.

[0105] The term "cancer" also encompasses cell proliferative disorders which are associated with some degree of abnormal cell proliferation, and includes tumors. "Tumor" as used herein, refers to any neoplasm or neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. In embodiments disclosed above, the cancer can be prostate cancer, cancer of the brain/CNS (glioma, glioblastoma, etc.), or leukemia (myeloid leukemia).

[0106] Administration of an effective amount of an inhibitor of CaMKK such as, for example a compound of Formula I-III, such as STO-609 for example, to a subject may be carried out by any means known in the art including, but not limited to intraperitoneal, intravenous, intramuscular, subcutaneous, or transcutaneous injection or oral, nasopharyngeal or transmucosal absorption. Such administration encompasses the administration of a CaMKK inhibitor formulated as a pharmaceutical composition. Delivery (administration route) also includes targeted delivery wherein the CaMKK inhibitor is only active in a targeted region of the body (for example, in the prostate and/or cancerous tissues), as well as sustained release formulations in which the CaMKK inhibitor compound is released over a period of time in a controlled manner. Sustained release formulations and methods for targeted delivery are known in the art and include, for example, use of liposomes, drug loaded biodegradable microspheres, drug-polymer conjugates, drug-specific binding agent conjugates and the like. Pharmaceutically acceptable carriers are well known to those of skill in the art. Determination of particular pharmaceutical formulations and therapeutically effective amounts and dosing regimen for a given treatment is within the ability of one of skill in the art taking into consideration, for example, patient age, weight, sex, ethnicity, organ (e.g., liver and kidney) function, the extent of desired treatment, the stage and severity of the disease and associated symptoms, and the tolerance of the patient for the treatment.

Kits

[0107] In an aspect, the disclosure relates to kits. Such kits can be used in methods of identifying a cancer that can be responsive to a method of treatment comprising administration of a CaMKK inhibitor, methods of identifying a compound as an inhibitor of CaMKK, methods of evaluating efficacy of a therapeutic regimen comprising administration of a CaMKK inhibitor, and the like. Kits can also include appropriate buffer systems and reagents, such as substrates of one or more CaMKKs, phosphate-donating groups (optionally radiolabelled phosphate-donating groups such as ³²P-ATP), a calmodulin and a calcium source, typically a calcium salt, and molecules that can detect the presence of a CaMK or CaMKK (e.g., antibodies). Kits also include instructions for use.

[0108] It will be understood that any numerical value recited herein includes all values from the lower value to the upper value. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

[0109] Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use herein of terms such as "comprising," "including," "having," and variations thereof is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. "Comprising" encompasses the terms "consisting of" and "consisting essentially of." The use of "consisting essentially of" means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.

[0110] All patents publications and references cited herein are hereby fully incorporated by reference.

[0111] While the following examples provide further detailed description of certain embodiments of the invention, they should be considered merely illustrative and not in any way limiting the invention, as defined by the claims.

EXAMPLES

[0112] Materials and Methods.

[0113] Cell Culture and RNA.

[0114] The LNCaP and VCaP human prostate carcinoma cell lines were obtained from ATCC and maintained as recommended. All experiments were performed with cells of passage less than 25. These cells were authenticated by morphological inspection and mycoplasma testing by the ATCC. Furthermore, their response to androgens was authenticated using growth and reporter gene assays. RNA from placenta, skeletal muscle, cerebellum, whole brain and normal prostate was from Clontech (Mountain View, Calif.). RNA from glioblastoma cell lines was a generous gift from Valerie Curtis (Duke University, Durham, N.C.).

[0115] RNA Isolation, cDNA Preparation, and Quantitative and Standard Reverse Transcription (RT)-PCR.

[0116] RNA isolation, cDNA preparation and quantitative RT-PCR (qPCR) were performed as previously described using 36B4 as a control (12). Standard RT-PCR was performed using the Advantage GC 2 Polymerase Mix and PCR Kit (Clontech). All qPCR and RT-PCR primers used in this study are listed in Table 1.

[0117] Western Blot Analysis.

[0118] Western blots were performed as previously described (12) with the exception that a modified radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris (pH 8.0), 200 mM NaCl, 1.5 mM MgCl₂, 1% Triton X-100, 1 mM EGTA, 10% glycerol, 50 mM NaF, 2 mM Na₃VO₄ and protease inhibitors] was used. Results shown are representative blots. For each sample, protein levels were determined by densitometry using the ImageJ software (NIH) and normalizing to indicated controls.

[0119] Small Interfering RNA (siRNA) Transfection of Human Prostate Cells.

[0120] Stealth siRNA (Invitrogen) transfections were performed as previously described (5). The sequences of all siRNAs used in this study are listed in Table 1.

[0121] Chromatin Immunoprecipitation (ChIP).

[0122] ChIP was performed as previously described (4). All primers used for ChIP qPCR analysis are listed in Table 1.

[0123] Transient Transfections and Reporter Gene Assays.

[0124] Transient transfections and reporter gene assays were performed as previously described (4).

[0125] Cell Proliferation Assay.

[0126] Proliferation assays were performed as previously described (12) by measuring the cellular DNA content using the FluoReporter Blue Fluorometric double-stranded DNA Quantitation Kit (Invitrogen) as per the manufacturer's protocol.

[0127] Migration and Invasion Assays.

[0128] Boyden dual chamber migration assays were performed as previously described (4). Invasion assays were performed the same as migration assays except that inserts were layered with 100 ml of Matrigel extracellular matrix (BD Biosciences) prior to reseeding of cells.

[0129] Statistical Analysis.

[0130] Data were analyzed using one-way ANOVA and post hoc Dunnett's test with GraphPad Prism, Version 4 (GraphPad Software, Inc.). Unless otherwise noted, significance was determined at the P<0.05 level.

[0131] Chemicals.

[0132] Methyltrienolone (R1881) was purchased from PerkinElmer (Waltham, Mass.) and dissolved in ethanol. Bicalutamide (Casodex) was provided as a gift from P. Turnbull (GlaxoSmithKline, Research Triangle Park, N.C.) and resuspended in a 1:1 mixture of ethanol and dimethylsulfoxide (DMSO). Cycloheximide was obtained from Sigma (St Louis, Mo.) and dissolved in DMSO. Compound C (in DMSO) was from Calbiochem (San Diego, Calif.). STO-609 was purchased from Tocris (Ellisville, Mo.) and resuspended in 100 mM NaOH. 5-aminoimidazole-4-carboxamide 1-b-D-ribo-furanoside (AICAR) was from Enzo Life Sciences (Plymouth Meeting, Pa.) and dissolved in water.

[0133] Antibodies.

[0134] The CaMKK antibody used, unless otherwise specified, was from BD Biosciences (Palo Alto, Calif.). The CaMKKβ (clone 1A11) antibody was from Abnova (Walnut, Calif.). The v5 antibody was purchased from Invitrogen (Carlsbad, Calif.). The GAPDH and AR antibodies have previously been described (1). Phospho-CaMKI (T177), CaMKI and Lamin A antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Phospho-AMPKα(T172), AMPKα, phospho-acetyl-CoA carboxylase (S79), and acetyl-CoA carboxylase antibodies were from Cell Signaling Technology (Danvers, Mass.).

[0135] Plasmids.

[0136] The CMV-βgal and PSA-Luc plasmids were previously described (2). The pGL4.14 (lacks both basal promoter and enhancers) and pGL4.26 (lacks enhancer, but contains basal promoter) vectors were obtained from Promega (Madison, Wis.). MSCV-GWb-GAL4(DNA-binding domain (DBD))-IRES-EGFP, MSCV-GWb-CaMKKβ--IRES-EGFP, MSCV-GWb-v5-ARwt-IRES-EGFP and MSCV-GWb-v5-AR(C562S)--IRES-EGFP were created using the Invitrogen Gateway recombinase subcloning system according to the manufacturer's instructions. To do this, GAL4(DBD), CaMKKβ, v5-ARwt or v5-AR(C562S) were shuttled from pENTR-GAL4(DBD), pENTR-v5-ARwt, pENTR-v5-AR(C562) or pOTB7-CaMKKβ_prostate splice variant (American Type Culture Collection (ATCC), Manassas, Va.) to MSCV-IRES-EGFP that was converted to a Gateway destination vector. The pGL4.14-CaMKKβ promoter construct was created by PCR amplifying a 2.1 kb genomic sequence that encompassed the CaMKKβ transcriptional start site through the potential AR binding site identified using ChIP on Chip (previously described (3)). This fragment was then cloned into the pGL4.14 vector using NheI and HindIII restriction sites. Subsequent deletion constructs were created by PCR amplifying smaller fragments that were cloned into pGL4.26 using NheI and HindIII restriction sites. Finally, the pGL4.14-CaMKKβ promoter-ARE deletion construct was created from the original pGL4.14-CaMKKβ promoter construct using the ExSite PCR-Based Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.). All primers used for the creation of constructs are listed in Supplementary Table 1. All sequences were confirmed using restriction digests and sequencing.

[0137] Creation of Stable Cell Lines.

[0138] To create LNCaP-GAL4, LNCaP-CAMKKLNCaP-v5-ARwt and LNCaP-v5-AR(C562S) cells, parental cells were infected with retrovirus expressing MSCV-GWb-GAL4(DBD)-IRES-EGFP (negative control), MSCV-GWb-CAMKKβ-IRES-EGFP, MSCV-GWb-v5-ARwt-IRES-EGFP or MSCV-GWb-v5-AR(C562S)-IRES-EGFP. EGFP positive cells were then selected through two rounds of cells sorting using flow cytometry and expression levels were confirmed by western blot and/or qPCR.

TABLE-US-00001 TABLE 1 Primers and siRNA sequences used in these studies Primer/siRNA Sequence (SEQ ID NO) qPCR primers 36B4 Forward: 5'-GGACATGTTGCTGGCCAATAA-3' (SEQ ID NO: 48) Reverse: 5'-GGGCCCGAGACCAGTGTT-3' (SEQ ID NO: 49) CaMKKβ Forward: 5'-TCCAGACCAGCCCGACATAG-3' (SEQ ID NO: 50) Reverse: 5'-CAGGGGTGCAGCTTGATTTC-3' (SEQ ID NO: 51) CXCR4 Forward: 5'-TGGCCTTATCCTGCCTGGTAT-3' (SEQ ID NO: 52) Reverse: 5'-AGGAGTCGATGCTGATCCCAA-3' (SEQ ID NO: 53) AR 3'UTR Forward: 5'-CCATGGCACCTTCAGACTTT-3' (SEQ ID NO: 54) Reverse: 5'-ACTGGGCCATATGAGGATCA-3' (SEQ ID NO: 55) AMPKα1 Forward: 5'-CTCAGTTCCTGGAGAAAGATGG-3' (SEQ ID NO: 56) Reverse: 5'-CCCAGTCAATTCATGTTTGCC-3' (SEQ ID NO: 57) AMPKα2 Forward: 5'-ATGGAATATGTGTCTGGAGGTG-3' (SEQ ID NO: 58) Reverse: 5'-TGGTTTCAGGTCTCGATGAAC-3' (SEQ ID NO: 59) CaMKKβ enhancer ChIP primers distal upstream control Forward: 5'-GCACAGTTTGCACACCTGAA-3' (SEQ ID NO: 60) Reverse: 5'-GCTTTGGATTTAGGCCCTGT-3' (SEQ ID NO: 61) CaMKKβ enhancer Forward: 5'-AACAGGAAAGGACACCCAAA-3' (SEQ ID NO: 62) Reverse: 5'-AAACCATTCTTAGCAGGCCAT-3' (SEQ ID NO: 63) CaMKKβ enhancer and promoter reporter gene primers promoter Fwd: 5'-CGCTAGCAGGGAGGTGGCTGAGCATCAAATA-3' (SEQ ID NO: 64) Rv: 5'-CAAAGCTTTGAGACAGGGTCTCTCTGTGTTGC-3' (SEQ ID NO: 65) fragment A enhancer Fwd: 5'-CGCTAGCGAATTGCAACTGTGAGACCAGGCA-3' (SEQ ID NO: 66) Rv: 5'-CAAAGCTTGTGGCCTTGGGCAAATGACTTGAT-3' (SEQ ID NO: 67) fragment B enhancer Fwd: 5'-CGCTAGCATCAAGTCATTTGCCCAAGGCCAC-3' (SEQ ID NO: 68) Rv: 5'-CAAAGCTTAACACTGTAGCTCACACAGGCAGA-3' (SEQ ID NO: 69) fragment C enhancer Fwd: 5'-CGCTAGCATCAAGTCATTTGCCCAAGGCCAC-3' (SEQ ID NO: 70) Rv: 5'-CAAAGCTTTATTTGATGCTCAGCCACCTCCCT-3' (SEQ ID NO: 71) fragment D/598 bp enhancer Fwd: 5'-CGCTAGCAGGGAGGTGGCTGAGCATCAAATA-3' (SEQ ID NO: 72) Rv: 5'-CAAAGCTTAAATGTGAAAGGCCAGGTGTGGTG-3' (SEQ ID NO: 73) fragment E enhancer Fwd: 5'-CGCTAGCTGCCTGTGTGAGCTACAGTGTTCT-3' (SEQ ID NO: 74) Rv: 5'-CAAAGCTTAAATGTGAAAGGCCAGGTGTGGTG-3' (SEQ ID NO: 75) fragment F enhancer Fwd: 5'-CGCTAGCAGGGAGGTGGCTGAGCATCAAATA-3' (SEQ ID NO: 76) Rv: 5'-CAAAGCTTAACACTGTAGCTCACACAGGCAGA-3' (SEQ ID NO: 77) fragment G enhancer Fwd: 5'-CGCTAGCCACCACACCTGGCCTTTCACATTT-3' (SEQ ID NO: 78) Rv: 5'-CAAAGCTTGCACTTTAAGGCAGGGTCAGCAAA-3' (SEQ ID NO: 79) fragment H enhancer Fwd: 5'-CGCTAGCGTTTCAAGCGATTCTCCTGCCTCA-3' (SEQ ID NO: 80) Rv: 5'-CAAAGCTTTCACGCCTGTAATCCCAGCACTTT-3' (SEQ ID NO: 81) 566 bp enhancer Fwd: 5'-CGCTAGCAGGGAGGTGGCTGAGCATCAAATA-3' (SEQ ID NO: 82) Rv: 5'-CAAAGCTTTACACGGGTGATTACAATCTTAGC-3' (SEQ ID NO: 83) 487 bp enhancer Fwd: 5'-CGCTAGCAGGGAGGTGGCTGAGCATCAAATA-3' (SEQ ID NO: 84) Rv: 5'-CAAAGCTTTGGACAACATGGCAAGACCCATCT-3' (SEQ ID NO: 85) 312 bp enhancer Fwd: 5'-CGCTAGCAGGGAGGTGGCTGAGCATCAAATA-3' (SEQ ID NO: 86) Rv: 5'-CAAAGCTTCTGGATCTCTTTTCCTGGTACTTG-3' (SEQ ID NO: 87) 233 bp enhancer Fwd: 5'-CGCTAGCAGGGAGGTGGCTGAGCATCAAATA-3' (SEQ ID NO: 88) Rv: 5'-CAAAGCTTACACTGTAGCTCACACAGGCAGAA-3' (SEQ ID NO: 89) 152 bp enhancer Fwd: 5'-CGCTAGCAGGGAGGTGGCTGAGCATCAAATA-3' (SEQ ID NO: 90) Rv: 5'-CAAAGCTTTACAAATCCAAACCCTAGCTCAAG-3' (SEQ ID NO: 91) 90 bp enhancer Fwd: 5'-CGCTAGCAGGGAGGTGGCTGAGCATCAAATA-3' (SEQ ID NO: 92) Rv: 5'-CAAAGCTTTGCTGTGAGCCAGGCCCTCCCTGC-3' (SEQ ID NO: 93) 69 bp enhancer Fwd: 5'-CGCTAGCAGGGAGGTGGCTGAGCATCAAATA-3' (SEQ ID NO: 94) Rv: 5'-CAAAGCTTCTGCCCGCTCCTCTCTCTGATGTC-3' (SEQ ID NO: 95) promoter-ARE deletion Forward: 5'-CATACAGAATTGTTTAACAAGTACC-3' (SEQ ID NO: 96) Rv: 5'-TAAATTGCCTGTGTTTTATTAGAACACTG-3' (SEQ ID NO: 97) CaMKKβ RT-PCR primers F(1-6) 5'-ACCTGTAATCCCAGCACTTTCGGA-3' (SEQ ID NO: 98) R(1-6) 5'-CGATCTCGGATCACTGCAACCTCT-3' (SEQ ID NO: 99) F(7) 5'-TGAGCCGAGCCGAGCCGAGCTG-3' (SEQ ID NO: 100) R(1-7) 5'-TCACAGGGCTTCTGGCTTTCGCT-3' (SEQ ID NO: 101) F1 5'-AGCTGAGGACTTGAAGGACCTGAT-3' (SEQ ID NO: 102) R1 5'-AGGTTGTCTTCGCTGCCTTGCTT-3' (SEQ ID NO: 103) R2 5'-ACCTGGGCTGGCTATGTGTATGAA-3' (SEQ ID NO: 104) siRNA sequences CaMKKβ #1 5'-GGACCAUCUGUACAUGGUGUUCGAA-3' (SEQ ID NO: 105) CaMKKβ #2 5'-GCUGACUUUGGUGUGAGCAAUGAAU-3' (SEQ ID NO: 106) CaMKKβ #3 5'-CACCUGGGCAUGGAGUCCUUCAUUG-3' (SEQ ID NO: 107) AR 3'UTR 5'-CAGAUGUCUUCUGCCUGUUAUAACU-3' (SEQ ID NO: 108) AMPKα1 #1 5'-CCCAUCCUGAAAGAGUACCAUUCUU-3' (SEQ ID NO: 109) AMPKα1 #2 5'-CCCUCAAUAUUUAAAUCCUUCUGUG-3' (SEQ ID NO: 110) AMPKα1 #3 5'-ACCAUGAUUGAUGAUGAAGCCUUAA-3' (SEQ ID NO: 111) AMPKα2 #1 5'-GAUGGUGAAUUUCUGAGAACUAGUU-3' (SEQ ID NO: 112) AMPKα2 #2 5'-CCGUAUGACAUUAUGGCUGAAGUUU-3' (SEQ ID NO: 113) CaMKI #1 5'-GGAGATACAGCTCTAGATAAGAATA-3' (SEQ ID NO: 114) CaMKI #2 5'-CCATAGGTGTCATCGCCTACATCTT-3' (SEQ ID NO: 115)

Example 1

Androgens Increase CaMKKβ mRNA and Protein Levels in an AR-Dependent Manner

[0139] In an effort to identify novel prostate cancer therapeutics, we have focused on defining key regulators downstream of AR action that contribute to prostate pathobiology and that may be amenable to pharmacological exploitation. As a first step in this process, we analyzed the expression level of mRNAs encoding targetable signaling molecules using microarray data derived from androgen-treated LNCaP prostate cancer cells (13). These studies suggested that one such candidate, CaMKKβ, was upregulated by androgens. To confirm the significance of this observation, CaMKKβ mRNA levels were analyzed by qPCR following treatment with the synthetic androgen R1881. In both LNCaP and VCaP prostate cancer cell lines, CaMKKβ mRNA levels increased in a dose-dependent manner (FIG. 1A). Further, western immunoblot analysis revealed a corresponding dose-dependent increase in CaMKKβ protein levels in both cell lines (FIG. 1B). The specificity of the antibodies used in this study was verified using three siRNAs targeting CaMKKβ mRNA (FIG. 1C). In addition, analogous immunoblot results were obtained using a second antibody (clone 1A11) directed against CaMKKβ (FIG. 2). Finally, androgen-mediated induction, but not the basal expression, of CaMKKβ mRNA was abrogated in cells in which AR expression was inhibited using a validated siRNA (4) directed against the AR mRNA (FIG. 1D). Taken together, these data demonstrate that androgens, acting through AR, increase both CaMKKβ mRNA and protein levels in multiple cellular models of prostate cancer.

Example 2

Functionally Active Splice Variants of CaMKKβ are Expressed in Response to Androgens in the Prostate

[0140] Given that AR increases CaMKKβ levels in multiple cellular models of prostate cancer, we next determined if its expression correlated with the development of prostate cancer in human samples. Analysis of the clinically annotated prostate cancer data sets accessible through Oncomine revealed that CaMKKβ expression increases with grade (14-17) (FIG. 3A and FIG. 3B). Interestingly, this analysis also revealed that CaMKKβ was consistently overexpressed in prostate tumors, but not other malignancies (FIG. 3C) (18).

[0141] The full-length CaMKKβ protein is encoded by an mRNA composed of 18 exons. Interestingly, the majority of commercially available CaMKKβ antibodies target the C-terminus of the protein that is absent in some functionally active splice variants (19). Thus, given that the expression of CaMKKβ in the prostate has not been reported previously, we hypothesized that the prostate, and prostate cancers, may express a functionally important splice variant(s) of CaMKKβ that was not recognized by the most commonly used antibodies. To test this hypothesis, we performed RT-PCR analysis using primers spanning various exon boundaries to examine the splice variant repertoire in the normal prostate and in prostate cancer cells. In this manner, it was demonstrated that unlike in brain, which expresses a longer variant, both normal prostate and prostate cancer cells predominantly express shorter variants of CaMKKβ (FIG. 4A, FIG. 4B, and FIG. 5). The variants found are equivalent to the previously described CaMKKβ splice variants 2 and 7 that lack exon 16 (of note, splice variants 2 and 7 make identical protein products) (19). Interestingly, these shorter variants were also found in brain tumors (FIG. 4B). A complete analysis of the additional variants expressed in the prostate/prostate cancer is described in FIG. 5. Phosphorylation of the classical CaMKKβ target CaMKI was observed in both androgen-treated LNCaP and VCaP cells (FIG. 4C), indicating that the CaMKKβ variant expressed in prostate cancer cells is functionally active.

Example 3

CaMKKβ is Necessary and Sufficient for AR-Mediated Prostate Cancer Cell Migration and Invasion

[0142] Given that the expression of CaMKKβ is upregulated by androgens and is elevated in prostate cancer, we next wanted to assess its potential role(s) in processes of pathological importance in this disease. As a first step, we evaluated the ability of the CaMKK antagonist STO-609 to inhibit the androgen-mediated cellular growth of prostate cancer cells. However, at a concentration that suppressed CaMKKβ activity (FIG. 6A), this drug had no significant effect on LNCaP and VCaP cell number over the seven-day period of this assay (FIG. 7A and FIG. 6B).

[0143] In addition to proliferation, androgens increase the migration of prostate cancer cells (4, 20). Since CaMKKβ has recently been implicated in cell migration during neuronal development (21, 22), we next asked whether CaMKKβ is involved with AR-meditated prostate cancer cell migration and/or invasion. Using Boyden dual chamber migration assays, treatment with the CaMKK antagonist STO-609 blocked the androgen-mediated migration of both LNCaP (FIG. 7B, top) and VCaP prostate cancer cells (FIG. 6C). STO-609 also inhibited androgen-mediated invasion of LNCaP cells through a Matrigel extracellular matrix (FIG. 7B, bottom). Furthermore, knockdown of CaMKKβ suppressed, while its overexpression increased, both basal and androgen-stimulated cell migration (FIG. 7C, FIG. 7D, FIG. 6D, and FIG. 6E). These findings highlight a heretofore-unrecognized role for CaMKKβ in prostate cancer cell migration and invasion.

Example 4

Definition of the Molecular Mechanism for AR-Mediated CaMKKβ mRNA Expression

[0144] Using a knockdown/replacement strategy, it was demonstrated that expression of wild-type AR, but not a transcriptionally inactive DNA binding mutant (C562S), was able to complement the knockdown of endogenously expressed AR in an LNCaP cell migration assay (FIG. 8). Further, at a concentration that inhibits the expression of secondary androgen target genes (ex. CXCR4 (4)), cycloheximide treatment did not block the R1881-mediated increase in CaMKKβ mRNA levels (FIG. 9A). Together, these data indicate that CaMKKβ is a primary AR target gene.

[0145] By mining our previously published ChIP on Chip data (23), we identified a putative AR binding region located ˜2.3 kb upstream of the CaMKKβ transcriptional start site (FIG. 9B, top). No other AR binding was detected within the CaMKKβ gene or within 100 kb in either direction of the gene. The validity of this AR-binding site was confirmed using ChIP assays, which showed that AR was recruited to this region of the promoter within one hour following R1881 treatment (FIG. 9B, bottom). Given these data, we focused on characterizing the functionality of the putative ARE identified. To this end, we cloned overlapping regions of CaMKKβ's 5' upstream region and tested their ability to confer androgen responsiveness to an enhancerless luciferase reporter gene. In this manner, we determined that a construct incorporating a fragment, -2231 to -1632 (D), and an overlapping fragment, -2019 to -1632 (E), contained an AR-dependent enhancer (FIG. 9C). Both fragments D and E demonstrated androgen responsiveness in a dose-dependent manner that was suppressed by the antiandrogen Casodex (FIG. 10A). Similar results were obtained in VCaP cells (FIG. 10B). Deletion analysis further narrowed down the androgen-responsive region to a 79 by stretch of DNA that included a sequence, GTAACAtgaTGTAAA, that resembled the consensus androgen response element (ARE) AGAACAnnnTGTTCT (FIG. 10C). Deletion of the 15 by ARE in the full-length CaMKKβ promoter construct (-2231 to +83) completely abolished the androgen responsiveness (FIG. 9D). Thus, in the context of prostate cancer cells, CaMKKβ is a direct target of AR.

Example 5

Androgens Promote Prostate Cancer Cell Migration Through an AR-CaMKKβ-AMPK Signaling Axis

[0146] CaMKI, CaMKIV and, more recently, AMPK have been shown to be downstream targets of CaMKKβ (24). Since CaMKIV is not expressed in the prostate (data not shown), we tested whether AR-CaMKKβ signaling led to increased CaMKI and/or AMPK signaling. Western blot analysis revealed that androgens increased the phosphorylation of both CaMKI and AMPK at their CaMKKβ activation loop target sites (T177 and T172 respectively) in both LNCaP and VCaP cells, an effect that was reversed by pretreatment with STO-609 (FIG. 11A and FIG. 12A). Interestingly, we found that overexpression of CaMKKβ alone was sufficient to increase the phosphorylation/activity of AMPK, but not CaMKI (FIG. 11B). These findings indicated that AMPK, rather than CaMKI, could be regulating cell migration because CaMKKβ overexpression alone was also sufficient to increase migration (FIG. 7D). To verify this, we used our most efficacious siRNAs (FIG. 12B) to knockdown both isoforms of the catalytic subunit of AMPK (FIG. 11C, bottom and FIG. 12C) or CaMKI (FIG. 11D, bottom and FIG. 12D). In this manner, it was demonstrated that loss of AMPK, but not CaMKI, resulted in decreased prostate cancer cell migration (FIG. 11C and FIG. 11D). In support of these findings, cotreatment of cells with the AMPK antagonist compound C, at a concentration that inhibited its kinase activity, completely abolished androgen-mediated cell migration (FIG. 13A and FIG. 13B). Conversely, treatment of LNCaP cells with the AMP mimetic AICAR alone was sufficient to increase cell migration (FIG. 13A and FIG. 13C). These data highlight a central role for AMPK in prostate cancer cell migration. Definition of the mechanism(s) by which AMPK interfaces with the cellular processes responsible for migration and invasion is currently under investigation.

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Sequence CWU 1

1

11515620DNAArtificial SequenceSynthetic 1gagcctgggg aggtcgaggg tgcagcgagc cgtgatcgtg ctactgcact ccagcctggg 60caacacagag agaccctgtc tcaaaacaaa caaacaaaca aacaaacaaa caaacaaaaa 120aaacaaagaa aaaaaaatgg gagtgggccg ggcgcggtga ctcacacctg taatcccagc 180actttcggag gccaaggcgg gtggatcacg aggtcaggaa ttcaagatta gcctggacaa 240catggtgaaa ccccatctct acgaaaaata caaaaattag ccaagtatgg tggccggcgc 300ctgtaatccc agctactcgg gagactgagg cagagaactg cttgaacctg ggaggcagag 360gttgcagtga tccgagatcg cgtcactgca ctccagcgtg ggcgacagag cgagactccg 420tttcagaaaa gaaaaaaaaa aaaaaaaaaa agggagtcgg ggtggagctc tcattggctc 480gttgcatgtg agtgtcccta cggcctagaa atacaagaga agcacatcgg aacgggctgg 540aaatccaccc agttaactag agggctttga accttttatt aacttggagg ttgactctcc 600tgtcaactcg attccctttt ggctgtttgg cagggtcagt gagacatccc ctgggtcgct 660cgaccccgta ggacggttca gggagccctc caggtcttcg tttctcctct tccccgcaca 720gtgctgttat ccagctgggg gatccaacgc acacttaagg ctccagcaaa gtggctccgc 780tgccggatgg gagtgcccca gtgtgctgga tgaagctggc gcatgcacca tgtcatcatg 840tgtctctagc cagcccagca gcaaccgggc cgccccccag gatgagctgg ggggcagggg 900cagcagcagc agcgaaagcc agaagccctg tgaggccctg cggggcctct catccttgag 960catccacctg ggcatggagt ccttcattgt ggtcaccgag tgtgagccgg gctgtgctgt 1020ggacctcggc ttggcgcggg accggcccct ggaggccgat ggccaagagg tcccccttga 1080cacctccggg tcccaggccc ggccccacct ctccggtcgc aagctgtctc tgcaagagcg 1140gtcccagggt gggctggcag ccggtggcag cctggacatg aacggacgct gcatctgccc 1200gtccctgccc tactcacccg tcagctcccc gcagtcctcg cctcggctgc cccggcggcc 1260gacagtggag tctcaccacg tctccatcac gggtatgcag gactgtgtgc agctgaatca 1320gtataccctg aaggatgaaa ttggaaaggg ctcctatggt gtcgtcaagt tggcctacaa 1380tgaaaatgac aatacctact atgcaatgaa ggtgctgtcc aaaaagaagc tgatccggca 1440ggccggcttt ccacgtcgcc ctccaccccg aggcacccgg ccagctcctg gaggctgcat 1500ccagcccagg ggccccattg agcaggtgta ccaggaaatt gccatcctca agaagctgga 1560ccaccccaat gtggtgaagc tggtggaggt cctggatgac cccaatgagg accatctgta 1620catggtgttc gaactggtca accaagggcc cgtgatggaa gtgcccaccc tcaaaccact 1680ctctgaagac caggcccgtt tctacttcca ggatctgatc aaaggcatcg agtacttaca 1740ctaccagaag atcatccacc gtgacatcaa accttccaac ctcctggtcg gagaagatgg 1800gcacatcaag atcgctgact ttggtgtgag caatgaattc aagggcagtg acgcgctcct 1860ctccaacacc gtgggcacgc ccgccttcat ggcacccgag tcgctctctg agacccgcaa 1920gatcttctct gggaaggcct tggatgtttg ggccatgggt gtgacactat actgctttgt 1980ctttggccag tgcccattca tggacgagcg gatcatgtgt ttacacagta agatcaagag 2040tcaggccctg gaatttccag accagcccga catagctgag gacttgaagg acctgatcac 2100ccgtatgctg gacaagaacc ccgagtcgag gatcgtggtg ccggaaatca agctgcaccc 2160ctgggtcacg aggcatgggg cggagccgtt gccgtcggag gatgagaact gcacgctggt 2220cgaagtgact gaagaggagg tcgagaactc agtcaaacac attcccagct tggcaaccgt 2280gatcctggtg aagaccatga tacgtaaacg ctcctttggg aacccattcg agggcagccg 2340gcgggaggaa cgctcactgt cagcgcctgg aaacttgctc accaaaaaac caaccaggga 2400atgtgagtcc ctgtctgagc tcaaggaagc aaggcagcga agacaacctc cagggcaccg 2460acccgccccc cgtgggggag gaggaagtgc tcttgtgaga ggcagtccct gcgtggaaag 2520ttgctgggcc cccgcccccg gctcccccgc acgcatgcat ccactgcggc cggaggaggc 2580catggagccc gagtagctgc ctggatcgct cgacctcgca tgcgcgccgc gtcgcctctg 2640gggggctgct gcaccgcgtt tccatagcag catgtcctac ggaaacccag cacgtgtgta 2700gagcctcgat cgtcatctct ggttatttgt tttttccttt gttgttttaa aggggacaaa 2760aaaaaaaaaa ggacttgact ccatgacgtc gaccgtggcc gctggctggc tggacaggcg 2820ggtgtgagga gttgcagacc caaacccacg tgcattttgg gacaattgct ttttaaaacg 2880tttttatgcc aaaaatcctt cattgtgatt ttcagaacca cgtcagatat accaagtgac 2940tgtgtgtggg gtttgacaac tgtggaaagg cgagcagaaa actccggcgg tctgaggcca 3000tggaggtggt tgctgcattt gagagggagt agggggctag atgtggctcc tagtgcaaac 3060cggaaaccat ggcaccttcc agagccgtgg tctcaaggag tcagagcagg gctggccctc 3120agtagctgca gggagctttg atgcaactta tttgtaagaa ggatttttaa attttttatg 3180ggtagaattg tagtcaggaa aacagaaagg gcttgaaatt taataagtgc tgctggaagg 3240ggattttcca agcctggaag ggtattcagc agctgtggtg gggaaacatt tctcctgaaa 3300gactgaacgt gtttcttcat gacagctgct caaagcaggt ttctgagata gctgaccgag 3360ctctggtaaa tctctttgtc aaattacgaa aacttcaggg tgaaatccta tgcttccatg 3420tacattacat ggcttaagat taaacaaaaa catttttcaa gtctctaact agagtgaact 3480ctagagcaca gtagttcaga aactatttag agcttccagg atatatttca cagcttcagg 3540catgtgatca gttagagccg atgaaaccta tgcccgcctg tatatatatt agcagcttag 3600ctagttcata acctgtatat tctaaagact gctaaggttt tgttttcatt ttaaatccta 3660gctgattgtt gtggtcaatg aaatacccag tttctggagg gccaggtggg aaatgctttc 3720actggaccaa cacacaaatg atcatcctga ggatctgagc ttccctagac tccacacaat 3780aaccttgggg caccctttta gagaagactg ttgaaaccca cagcactcgt tggggtatga 3840ggaaaccagg gcttggcaca ggaagttccc ctttgtagct aaaagtccag aaagaaaggg 3900ttcatctttt tgacttccaa ctgatattgg gaagtttggt tgaggttcaa gtgtgactcc 3960ttccagagcc acaggtaggg gagtgtgaag ttgaggggga ggaaagctgg aaggactctg 4020ccttgggaga ttcccagctc tgctttccag cgcttggtgg aatctgggct ggggaaagac 4080ggcaccggga aactctgctt ccccattgtt tccatctgat cagctgtggt gtgaggactt 4140ctcagacaaa ggcaaggcct cgtgcccctg cccagcccat tcatggagcc ctgggccttc 4200ttggcttcca tagatcctaa gctcttgact gtagtttagc cagacttgtt ttgctatctt 4260ataagcagtt cagaattagg gaatgctggt tttgaagagc aaaggacagg tagtctagag 4320agggtcgtct ggcctgcttg ctgggtcttt gtaacccagc acttcctctt gccctcctgg 4380ctttatgttt atggggagag gactcaatag ctccacccct tctggcacca gatggggctt 4440ggttagtttg caataagcac cttgcagagg ttaaagccag cgggtcccta gtcttaggcc 4500cagcctgctt gtgtgggctc tggcctggcc tggtggctgg cccagggggc agcagtgctt 4560agagcttctg cagggcttct cttgtttaca cagctgcatc agacaatgcc atttctcccc 4620accacggaac cttccatcta agatttcttc cagggaatgc cagcaatcag gcagcaccca 4680gctgtggggg cagtggggtg ggggagaccc acattgatga cttttttttt ttcttttaat 4740gaagaaacac caaagaaagc tgtggaaagg acctgcccca catgaaaagg ataagccaag 4800atggctgtaa acacagagca tttgagctgc cactcttgga gcacattgat ttttcaaaag 4860ccagctctgt caggaaagga ggtgctgtta tgagcagctc ttccagtggg caaagaggac 4920gcccataatt tcttccattg ctagctcatc tgtgggacca atttggtgta agcaacctgt 4980ggcctgcact tgtggcctcg aaggaagcac aaaccctcca tccacttccc atttcctctg 5040cccttttcca cctccccctt ccatcccacc agctgccagt ggctcccaga aagccttatt 5100gagccccttg ttgacacttg gggctgcgga ggcctctccc tactggtctg gcctttcctg 5160agaggcaggt cttccgtcct cagagccttt ctggaacaag gagaatgcct gtgcaggtgg 5220acacacaggc ctggcctgtc gctctcactt gtcttccagc ggggagcttc acgttgccga 5280gtggaagaac catgacctcc acttgcttcc aaggtgctag ggaagtttca gggtacgctg 5340gttcccctct ccagctggag gccgagtttc tggggactgc agatttttct actctgtgat 5400cgattcaatg cccgatgctt ctgtttcatt cccgaccctt tctactatgc attttccttt 5460tatcaggtgt ataaagttaa atactgtgta tttatcacta aaaagtacat gaacttaaga 5520gacaactaag cctttcgtgt ttttccacag gtgtttaagc ttctctgtac agttgaaata 5580aacagacagc aaaatggtgc caaaaaaaaa aaaaaaaaaa 56202588PRTArtificial SequenceSynthetic 2Met Ser Ser Cys Val Ser Ser Gln Pro Ser Ser Asn Arg Ala Ala Pro 1 5 10 15 Gln Asp Glu Leu Gly Gly Arg Gly Ser Ser Ser Ser Glu Ser Gln Lys 20 25 30 Pro Cys Glu Ala Leu Arg Gly Leu Ser Ser Leu Ser Ile His Leu Gly 35 40 45 Met Glu Ser Phe Ile Val Val Thr Glu Cys Glu Pro Gly Cys Ala Val 50 55 60 Asp Leu Gly Leu Ala Arg Asp Arg Pro Leu Glu Ala Asp Gly Gln Glu 65 70 75 80 Val Pro Leu Asp Thr Ser Gly Ser Gln Ala Arg Pro His Leu Ser Gly 85 90 95 Arg Lys Leu Ser Leu Gln Glu Arg Ser Gln Gly Gly Leu Ala Ala Gly 100 105 110 Gly Ser Leu Asp Met Asn Gly Arg Cys Ile Cys Pro Ser Leu Pro Tyr 115 120 125 Ser Pro Val Ser Ser Pro Gln Ser Ser Pro Arg Leu Pro Arg Arg Pro 130 135 140 Thr Val Glu Ser His His Val Ser Ile Thr Gly Met Gln Asp Cys Val 145 150 155 160 Gln Leu Asn Gln Tyr Thr Leu Lys Asp Glu Ile Gly Lys Gly Ser Tyr 165 170 175 Gly Val Val Lys Leu Ala Tyr Asn Glu Asn Asp Asn Thr Tyr Tyr Ala 180 185 190 Met Lys Val Leu Ser Lys Lys Lys Leu Ile Arg Gln Ala Gly Phe Pro 195 200 205 Arg Arg Pro Pro Pro Arg Gly Thr Arg Pro Ala Pro Gly Gly Cys Ile 210 215 220 Gln Pro Arg Gly Pro Ile Glu Gln Val Tyr Gln Glu Ile Ala Ile Leu 225 230 235 240 Lys Lys Leu Asp His Pro Asn Val Val Lys Leu Val Glu Val Leu Asp 245 250 255 Asp Pro Asn Glu Asp His Leu Tyr Met Val Phe Glu Leu Val Asn Gln 260 265 270 Gly Pro Val Met Glu Val Pro Thr Leu Lys Pro Leu Ser Glu Asp Gln 275 280 285 Ala Arg Phe Tyr Phe Gln Asp Leu Ile Lys Gly Ile Glu Tyr Leu His 290 295 300 Tyr Gln Lys Ile Ile His Arg Asp Ile Lys Pro Ser Asn Leu Leu Val 305 310 315 320 Gly Glu Asp Gly His Ile Lys Ile Ala Asp Phe Gly Val Ser Asn Glu 325 330 335 Phe Lys Gly Ser Asp Ala Leu Leu Ser Asn Thr Val Gly Thr Pro Ala 340 345 350 Phe Met Ala Pro Glu Ser Leu Ser Glu Thr Arg Lys Ile Phe Ser Gly 355 360 365 Lys Ala Leu Asp Val Trp Ala Met Gly Val Thr Leu Tyr Cys Phe Val 370 375 380 Phe Gly Gln Cys Pro Phe Met Asp Glu Arg Ile Met Cys Leu His Ser 385 390 395 400 Lys Ile Lys Ser Gln Ala Leu Glu Phe Pro Asp Gln Pro Asp Ile Ala 405 410 415 Glu Asp Leu Lys Asp Leu Ile Thr Arg Met Leu Asp Lys Asn Pro Glu 420 425 430 Ser Arg Ile Val Val Pro Glu Ile Lys Leu His Pro Trp Val Thr Arg 435 440 445 His Gly Ala Glu Pro Leu Pro Ser Glu Asp Glu Asn Cys Thr Leu Val 450 455 460 Glu Val Thr Glu Glu Glu Val Glu Asn Ser Val Lys His Ile Pro Ser 465 470 475 480 Leu Ala Thr Val Ile Leu Val Lys Thr Met Ile Arg Lys Arg Ser Phe 485 490 495 Gly Asn Pro Phe Glu Gly Ser Arg Arg Glu Glu Arg Ser Leu Ser Ala 500 505 510 Pro Gly Asn Leu Leu Thr Lys Lys Pro Thr Arg Glu Cys Glu Ser Leu 515 520 525 Ser Glu Leu Lys Glu Ala Arg Gln Arg Arg Gln Pro Pro Gly His Arg 530 535 540 Pro Ala Pro Arg Gly Gly Gly Gly Ser Ala Leu Val Arg Gly Ser Pro 545 550 555 560 Cys Val Glu Ser Cys Trp Ala Pro Ala Pro Gly Ser Pro Ala Arg Met 565 570 575 His Pro Leu Arg Pro Glu Glu Ala Met Glu Pro Glu 580 585 35577DNAArtificial SequenceSynthetic 3gagcctgggg aggtcgaggg tgcagcgagc cgtgatcgtg ctactgcact ccagcctggg 60caacacagag agaccctgtc tcaaaacaaa caaacaaaca aacaaacaaa caaacaaaaa 120aaacaaagaa aaaaaaatgg gagtgggccg ggcgcggtga ctcacacctg taatcccagc 180actttcggag gccaaggcgg gtggatcacg aggtcaggaa ttcaagatta gcctggacaa 240catggtgaaa ccccatctct acgaaaaata caaaaattag ccaagtatgg tggccggcgc 300ctgtaatccc agctactcgg gagactgagg cagagaactg cttgaacctg ggaggcagag 360gttgcagtga tccgagatcg cgtcactgca ctccagcgtg ggcgacagag cgagactccg 420tttcagaaaa gaaaaaaaaa aaaaaaaaaa agggagtcgg ggtggagctc tcattggctc 480gttgcatgtg agtgtcccta cggcctagaa atacaagaga agcacatcgg aacgggctgg 540aaatccaccc agttaactag agggctttga accttttatt aacttggagg ttgactctcc 600tgtcaactcg attccctttt ggctgtttgg cagggtcagt gagacatccc ctgggtcgct 660cgaccccgta ggacggttca gggagccctc caggtcttcg tttctcctct tccccgcaca 720gtgctgttat ccagctgggg gatccaacgc acacttaagg ctccagcaaa gtggctccgc 780tgccggatgg gagtgcccca gtgtgctgga tgaagctggc gcatgcacca tgtcatcatg 840tgtctctagc cagcccagca gcaaccgggc cgccccccag gatgagctgg ggggcagggg 900cagcagcagc agcgaaagcc agaagccctg tgaggccctg cggggcctct catccttgag 960catccacctg ggcatggagt ccttcattgt ggtcaccgag tgtgagccgg gctgtgctgt 1020ggacctcggc ttggcgcggg accggcccct ggaggccgat ggccaagagg tcccccttga 1080cacctccggg tcccaggccc ggccccacct ctccggtcgc aagctgtctc tgcaagagcg 1140gtcccagggt gggctggcag ccggtggcag cctggacatg aacggacgct gcatctgccc 1200gtccctgccc tactcacccg tcagctcccc gcagtcctcg cctcggctgc cccggcggcc 1260gacagtggag tctcaccacg tctccatcac gggtatgcag gactgtgtgc agctgaatca 1320gtataccctg aaggatgaaa ttggaaaggg ctcctatggt gtcgtcaagt tggcctacaa 1380tgaaaatgac aatacctact atgcaatgaa ggtgctgtcc aaaaagaagc tgatccggca 1440ggccggcttt ccacgtcgcc ctccaccccg aggcacccgg ccagctcctg gaggctgcat 1500ccagcccagg ggccccattg agcaggtgta ccaggaaatt gccatcctca agaagctgga 1560ccaccccaat gtggtgaagc tggtggaggt cctggatgac cccaatgagg accatctgta 1620catggtgttc gaactggtca accaagggcc cgtgatggaa gtgcccaccc tcaaaccact 1680ctctgaagac caggcccgtt tctacttcca ggatctgatc aaaggcatcg agtacttaca 1740ctaccagaag atcatccacc gtgacatcaa accttccaac ctcctggtcg gagaagatgg 1800gcacatcaag atcgctgact ttggtgtgag caatgaattc aagggcagtg acgcgctcct 1860ctccaacacc gtgggcacgc ccgccttcat ggcacccgag tcgctctctg agacccgcaa 1920gatcttctct gggaaggcct tggatgtttg ggccatgggt gtgacactat actgctttgt 1980ctttggccag tgcccattca tggacgagcg gatcatgtgt ttacacagta agatcaagag 2040tcaggccctg gaatttccag accagcccga catagctgag gacttgaagg acctgatcac 2100ccgtatgctg gacaagaacc ccgagtcgag gatcgtggtg ccggaaatca agctgcaccc 2160ctgggtcacg aggcatgggg cggagccgtt gccgtcggag gatgagaact gcacgctggt 2220cgaagtgact gaagaggagg tcgagaactc agtcaaacac attcccagct tggcaaccgt 2280gatcctggtg aagaccatga tacgtaaacg ctcctttggg aacccattcg agggcagccg 2340gcgggaggaa cgctcactgt cagcgcctgg aaacttgctc acgaagcaag gcagcgaaga 2400caacctccag ggcaccgacc cgccccccgt gggggaggag gaagtgctct tgtgagaggc 2460agtccctgcg tggaaagttg ctgggccccc gcccccggct cccccgcacg catgcatcca 2520ctgcggccgg aggaggccat ggagcccgag tagctgcctg gatcgctcga cctcgcatgc 2580gcgccgcgtc gcctctgggg ggctgctgca ccgcgtttcc atagcagcat gtcctacgga 2640aacccagcac gtgtgtagag cctcgatcgt catctctggt tatttgtttt ttcctttgtt 2700gttttaaagg ggacaaaaaa aaaaaaagga cttgactcca tgacgtcgac cgtggccgct 2760ggctggctgg acaggcgggt gtgaggagtt gcagacccaa acccacgtgc attttgggac 2820aattgctttt taaaacgttt ttatgccaaa aatccttcat tgtgattttc agaaccacgt 2880cagatatacc aagtgactgt gtgtggggtt tgacaactgt ggaaaggcga gcagaaaact 2940ccggcggtct gaggccatgg aggtggttgc tgcatttgag agggagtagg gggctagatg 3000tggctcctag tgcaaaccgg aaaccatggc accttccaga gccgtggtct caaggagtca 3060gagcagggct ggccctcagt agctgcaggg agctttgatg caacttattt gtaagaagga 3120tttttaaatt ttttatgggt agaattgtag tcaggaaaac agaaagggct tgaaatttaa 3180taagtgctgc tggaagggga ttttccaagc ctggaagggt attcagcagc tgtggtgggg 3240aaacatttct cctgaaagac tgaacgtgtt tcttcatgac agctgctcaa agcaggtttc 3300tgagatagct gaccgagctc tggtaaatct ctttgtcaaa ttacgaaaac ttcagggtga 3360aatcctatgc ttccatgtac attacatggc ttaagattaa acaaaaacat ttttcaagtc 3420tctaactaga gtgaactcta gagcacagta gttcagaaac tatttagagc ttccaggata 3480tatttcacag cttcaggcat gtgatcagtt agagccgatg aaacctatgc ccgcctgtat 3540atatattagc agcttagcta gttcataacc tgtatattct aaagactgct aaggttttgt 3600tttcatttta aatcctagct gattgttgtg gtcaatgaaa tacccagttt ctggagggcc 3660aggtgggaaa tgctttcact ggaccaacac acaaatgatc atcctgagga tctgagcttc 3720cctagactcc acacaataac cttggggcac ccttttagag aagactgttg aaacccacag 3780cactcgttgg ggtatgagga aaccagggct tggcacagga agttcccctt tgtagctaaa 3840agtccagaaa gaaagggttc atctttttga cttccaactg atattgggaa gtttggttga 3900ggttcaagtg tgactccttc cagagccaca ggtaggggag tgtgaagttg agggggagga 3960aagctggaag gactctgcct tgggagattc ccagctctgc tttccagcgc ttggtggaat 4020ctgggctggg gaaagacggc accgggaaac tctgcttccc cattgtttcc atctgatcag 4080ctgtggtgtg aggacttctc agacaaaggc aaggcctcgt gcccctgccc agcccattca 4140tggagccctg ggccttcttg gcttccatag atcctaagct cttgactgta gtttagccag 4200acttgttttg ctatcttata agcagttcag aattagggaa tgctggtttt gaagagcaaa 4260ggacaggtag tctagagagg gtcgtctggc ctgcttgctg ggtctttgta acccagcact 4320tcctcttgcc ctcctggctt tatgtttatg gggagaggac tcaatagctc caccccttct 4380ggcaccagat ggggcttggt tagtttgcaa taagcacctt gcagaggtta aagccagcgg 4440gtccctagtc ttaggcccag cctgcttgtg tgggctctgg cctggcctgg tggctggccc 4500agggggcagc agtgcttaga gcttctgcag ggcttctctt gtttacacag ctgcatcaga 4560caatgccatt tctccccacc acggaacctt ccatctaaga tttcttccag ggaatgccag 4620caatcaggca gcacccagct gtgggggcag tggggtgggg gagacccaca ttgatgactt 4680tttttttttc ttttaatgaa gaaacaccaa agaaagctgt ggaaaggacc tgccccacat 4740gaaaaggata agccaagatg gctgtaaaca cagagcattt gagctgccac tcttggagca 4800cattgatttt tcaaaagcca gctctgtcag gaaaggaggt gctgttatga gcagctcttc 4860cagtgggcaa agaggacgcc cataatttct tccattgcta gctcatctgt gggaccaatt 4920tggtgtaagc aacctgtggc ctgcacttgt ggcctcgaag gaagcacaaa ccctccatcc 4980acttcccatt tcctctgccc ttttccacct cccccttcca tcccaccagc tgccagtggc 5040tcccagaaag ccttattgag ccccttgttg acacttgggg ctgcggaggc ctctccctac 5100tggtctggcc tttcctgaga ggcaggtctt ccgtcctcag agcctttctg gaacaaggag 5160aatgcctgtg caggtggaca cacaggcctg gcctgtcgct ctcacttgtc ttccagcggg 5220gagcttcacg ttgccgagtg gaagaaccat gacctccact tgcttccaag gtgctaggga 5280agtttcaggg tacgctggtt cccctctcca gctggaggcc gagtttctgg ggactgcaga 5340tttttctact ctgtgatcga ttcaatgccc gatgcttctg tttcattccc gaccctttct 5400actatgcatt ttccttttat caggtgtata aagttaaata ctgtgtattt atcactaaaa 5460agtacatgaa

cttaagagac aactaagcct ttcgtgtttt tccacaggtg tttaagcttc 5520tctgtacagt tgaaataaac agacagcaaa atggtgccaa aaaaaaaaaa aaaaaaa 55774541PRTArtificial SequenceSynthetic 4Met Ser Ser Cys Val Ser Ser Gln Pro Ser Ser Asn Arg Ala Ala Pro 1 5 10 15 Gln Asp Glu Leu Gly Gly Arg Gly Ser Ser Ser Ser Glu Ser Gln Lys 20 25 30 Pro Cys Glu Ala Leu Arg Gly Leu Ser Ser Leu Ser Ile His Leu Gly 35 40 45 Met Glu Ser Phe Ile Val Val Thr Glu Cys Glu Pro Gly Cys Ala Val 50 55 60 Asp Leu Gly Leu Ala Arg Asp Arg Pro Leu Glu Ala Asp Gly Gln Glu 65 70 75 80 Val Pro Leu Asp Thr Ser Gly Ser Gln Ala Arg Pro His Leu Ser Gly 85 90 95 Arg Lys Leu Ser Leu Gln Glu Arg Ser Gln Gly Gly Leu Ala Ala Gly 100 105 110 Gly Ser Leu Asp Met Asn Gly Arg Cys Ile Cys Pro Ser Leu Pro Tyr 115 120 125 Ser Pro Val Ser Ser Pro Gln Ser Ser Pro Arg Leu Pro Arg Arg Pro 130 135 140 Thr Val Glu Ser His His Val Ser Ile Thr Gly Met Gln Asp Cys Val 145 150 155 160 Gln Leu Asn Gln Tyr Thr Leu Lys Asp Glu Ile Gly Lys Gly Ser Tyr 165 170 175 Gly Val Val Lys Leu Ala Tyr Asn Glu Asn Asp Asn Thr Tyr Tyr Ala 180 185 190 Met Lys Val Leu Ser Lys Lys Lys Leu Ile Arg Gln Ala Gly Phe Pro 195 200 205 Arg Arg Pro Pro Pro Arg Gly Thr Arg Pro Ala Pro Gly Gly Cys Ile 210 215 220 Gln Pro Arg Gly Pro Ile Glu Gln Val Tyr Gln Glu Ile Ala Ile Leu 225 230 235 240 Lys Lys Leu Asp His Pro Asn Val Val Lys Leu Val Glu Val Leu Asp 245 250 255 Asp Pro Asn Glu Asp His Leu Tyr Met Val Phe Glu Leu Val Asn Gln 260 265 270 Gly Pro Val Met Glu Val Pro Thr Leu Lys Pro Leu Ser Glu Asp Gln 275 280 285 Ala Arg Phe Tyr Phe Gln Asp Leu Ile Lys Gly Ile Glu Tyr Leu His 290 295 300 Tyr Gln Lys Ile Ile His Arg Asp Ile Lys Pro Ser Asn Leu Leu Val 305 310 315 320 Gly Glu Asp Gly His Ile Lys Ile Ala Asp Phe Gly Val Ser Asn Glu 325 330 335 Phe Lys Gly Ser Asp Ala Leu Leu Ser Asn Thr Val Gly Thr Pro Ala 340 345 350 Phe Met Ala Pro Glu Ser Leu Ser Glu Thr Arg Lys Ile Phe Ser Gly 355 360 365 Lys Ala Leu Asp Val Trp Ala Met Gly Val Thr Leu Tyr Cys Phe Val 370 375 380 Phe Gly Gln Cys Pro Phe Met Asp Glu Arg Ile Met Cys Leu His Ser 385 390 395 400 Lys Ile Lys Ser Gln Ala Leu Glu Phe Pro Asp Gln Pro Asp Ile Ala 405 410 415 Glu Asp Leu Lys Asp Leu Ile Thr Arg Met Leu Asp Lys Asn Pro Glu 420 425 430 Ser Arg Ile Val Val Pro Glu Ile Lys Leu His Pro Trp Val Thr Arg 435 440 445 His Gly Ala Glu Pro Leu Pro Ser Glu Asp Glu Asn Cys Thr Leu Val 450 455 460 Glu Val Thr Glu Glu Glu Val Glu Asn Ser Val Lys His Ile Pro Ser 465 470 475 480 Leu Ala Thr Val Ile Leu Val Lys Thr Met Ile Arg Lys Arg Ser Phe 485 490 495 Gly Asn Pro Phe Glu Gly Ser Arg Arg Glu Glu Arg Ser Leu Ser Ala 500 505 510 Pro Gly Asn Leu Leu Thr Lys Gln Gly Ser Glu Asp Asn Leu Gln Gly 515 520 525 Thr Asp Pro Pro Pro Val Gly Glu Glu Glu Val Leu Leu 530 535 540 55491DNAArtificial SequenceSynthetic 5gagcctgggg aggtcgaggg tgcagcgagc cgtgatcgtg ctactgcact ccagcctggg 60caacacagag agaccctgtc tcaaaacaaa caaacaaaca aacaaacaaa caaacaaaaa 120aaacaaagaa aaaaaaatgg gagtgggccg ggcgcggtga ctcacacctg taatcccagc 180actttcggag gccaaggcgg gtggatcacg aggtcaggaa ttcaagatta gcctggacaa 240catggtgaaa ccccatctct acgaaaaata caaaaattag ccaagtatgg tggccggcgc 300ctgtaatccc agctactcgg gagactgagg cagagaactg cttgaacctg ggaggcagag 360gttgcagtga tccgagatcg cgtcactgca ctccagcgtg ggcgacagag cgagactccg 420tttcagaaaa gaaaaaaaaa aaaaaaaaaa agggagtcgg ggtggagctc tcattggctc 480gttgcatgtg agtgtcccta cggcctagaa atacaagaga agcacatcgg aacgggctgg 540aaatccaccc agttaactag agggctttga accttttatt aacttggagg ttgactctcc 600tgtcaactcg attccctttt ggctgtttgg cagggtcagt gagacatccc ctgggtcgct 660cgaccccgta ggacggttca gggagccctc caggtcttcg tttctcctct tccccgcaca 720gtgctgttat ccagctgggg gatccaacgc acacttaagg ctccagcaaa gtggctccgc 780tgccggatgg gagtgcccca gtgtgctgga tgaagctggc gcatgcacca tgtcatcatg 840tgtctctagc cagcccagca gcaaccgggc cgccccccag gatgagctgg ggggcagggg 900cagcagcagc agcgaaagcc agaagccctg tgaggccctg cggggcctct catccttgag 960catccacctg ggcatggagt ccttcattgt ggtcaccgag tgtgagccgg gctgtgctgt 1020ggacctcggc ttggcgcggg accggcccct ggaggccgat ggccaagagg tcccccttga 1080cacctccggg tcccaggccc ggccccacct ctccggtcgc aagctgtctc tgcaagagcg 1140gtcccagggt gggctggcag ccggtggcag cctggacatg aacggacgct gcatctgccc 1200gtccctgccc tactcacccg tcagctcccc gcagtcctcg cctcggctgc cccggcggcc 1260gacagtggag tctcaccacg tctccatcac gggtatgcag gactgtgtgc agctgaatca 1320gtataccctg aaggatgaaa ttggaaaggg ctcctatggt gtcgtcaagt tggcctacaa 1380tgaaaatgac aatacctact atgcaatgaa ggtgctgtcc aaaaagaagc tgatccggca 1440ggccggcttt ccacgtcgcc ctccaccccg aggcacccgg ccagctcctg gaggctgcat 1500ccagcccagg ggccccattg agcaggtgta ccaggaaatt gccatcctca agaagctgga 1560ccaccccaat gtggtgaagc tggtggaggt cctggatgac cccaatgagg accatctgta 1620catggtgttc gaactggtca accaagggcc cgtgatggaa gtgcccaccc tcaaaccact 1680ctctgaagac caggcccgtt tctacttcca ggatctgatc aaaggcatcg agtacttaca 1740ctaccagaag atcatccacc gtgacatcaa accttccaac ctcctggtcg gagaagatgg 1800gcacatcaag atcgctgact ttggtgtgag caatgaattc aagggcagtg acgcgctcct 1860ctccaacacc gtgggcacgc ccgccttcat ggcacccgag tcgctctctg agacccgcaa 1920gatcttctct gggaaggcct tggatgtttg ggccatgggt gtgacactat actgctttgt 1980ctttggccag tgcccattca tggacgagcg gatcatgtgt ttacacagta agatcaagag 2040tcaggccctg gaatttccag accagcccga catagctgag gacttgaagg acctgatcac 2100ccgtatgctg gacaagaacc ccgagtcgag gatcgtggtg ccggaaatca agatcctggt 2160gaagaccatg atacgtaaac gctcctttgg gaacccattc gagggcagcc ggcgggagga 2220acgctcactg tcagcgcctg gaaacttgct caccaaaaaa ccaaccaggg aatgtgagtc 2280cctgtctgag ctcaaggaag caaggcagcg aagacaacct ccagggcacc gacccgcccc 2340ccgtggggga ggaggaagtg ctcttgtgag aggcagtccc tgcgtggaaa gttgctgggc 2400ccccgccccc ggctcccccg cacgcatgca tccactgcgg ccggaggagg ccatggagcc 2460cgagtagctg cctggatcgc tcgacctcgc atgcgcgccg cgtcgcctct ggggggctgc 2520tgcaccgcgt ttccatagca gcatgtccta cggaaaccca gcacgtgtgt agagcctcga 2580tcgtcatctc tggttatttg ttttttcctt tgttgtttta aaggggacaa aaaaaaaaaa 2640aggacttgac tccatgacgt cgaccgtggc cgctggctgg ctggacaggc gggtgtgagg 2700agttgcagac ccaaacccac gtgcattttg ggacaattgc tttttaaaac gtttttatgc 2760caaaaatcct tcattgtgat tttcagaacc acgtcagata taccaagtga ctgtgtgtgg 2820ggtttgacaa ctgtggaaag gcgagcagaa aactccggcg gtctgaggcc atggaggtgg 2880ttgctgcatt tgagagggag tagggggcta gatgtggctc ctagtgcaaa ccggaaacca 2940tggcaccttc cagagccgtg gtctcaagga gtcagagcag ggctggccct cagtagctgc 3000agggagcttt gatgcaactt atttgtaaga aggattttta aattttttat gggtagaatt 3060gtagtcagga aaacagaaag ggcttgaaat ttaataagtg ctgctggaag gggattttcc 3120aagcctggaa gggtattcag cagctgtggt ggggaaacat ttctcctgaa agactgaacg 3180tgtttcttca tgacagctgc tcaaagcagg tttctgagat agctgaccga gctctggtaa 3240atctctttgt caaattacga aaacttcagg gtgaaatcct atgcttccat gtacattaca 3300tggcttaaga ttaaacaaaa acatttttca agtctctaac tagagtgaac tctagagcac 3360agtagttcag aaactattta gagcttccag gatatatttc acagcttcag gcatgtgatc 3420agttagagcc gatgaaacct atgcccgcct gtatatatat tagcagctta gctagttcat 3480aacctgtata ttctaaagac tgctaaggtt ttgttttcat tttaaatcct agctgattgt 3540tgtggtcaat gaaataccca gtttctggag ggccaggtgg gaaatgcttt cactggacca 3600acacacaaat gatcatcctg aggatctgag cttccctaga ctccacacaa taaccttggg 3660gcaccctttt agagaagact gttgaaaccc acagcactcg ttggggtatg aggaaaccag 3720ggcttggcac aggaagttcc cctttgtagc taaaagtcca gaaagaaagg gttcatcttt 3780ttgacttcca actgatattg ggaagtttgg ttgaggttca agtgtgactc cttccagagc 3840cacaggtagg ggagtgtgaa gttgaggggg aggaaagctg gaaggactct gccttgggag 3900attcccagct ctgctttcca gcgcttggtg gaatctgggc tggggaaaga cggcaccggg 3960aaactctgct tccccattgt ttccatctga tcagctgtgg tgtgaggact tctcagacaa 4020aggcaaggcc tcgtgcccct gcccagccca ttcatggagc cctgggcctt cttggcttcc 4080atagatccta agctcttgac tgtagtttag ccagacttgt tttgctatct tataagcagt 4140tcagaattag ggaatgctgg ttttgaagag caaaggacag gtagtctaga gagggtcgtc 4200tggcctgctt gctgggtctt tgtaacccag cacttcctct tgccctcctg gctttatgtt 4260tatggggaga ggactcaata gctccacccc ttctggcacc agatggggct tggttagttt 4320gcaataagca ccttgcagag gttaaagcca gcgggtccct agtcttaggc ccagcctgct 4380tgtgtgggct ctggcctggc ctggtggctg gcccaggggg cagcagtgct tagagcttct 4440gcagggcttc tcttgtttac acagctgcat cagacaatgc catttctccc caccacggaa 4500ccttccatct aagatttctt ccagggaatg ccagcaatca ggcagcaccc agctgtgggg 4560gcagtggggt gggggagacc cacattgatg actttttttt tttcttttaa tgaagaaaca 4620ccaaagaaag ctgtggaaag gacctgcccc acatgaaaag gataagccaa gatggctgta 4680aacacagagc atttgagctg ccactcttgg agcacattga tttttcaaaa gccagctctg 4740tcaggaaagg aggtgctgtt atgagcagct cttccagtgg gcaaagagga cgcccataat 4800ttcttccatt gctagctcat ctgtgggacc aatttggtgt aagcaacctg tggcctgcac 4860ttgtggcctc gaaggaagca caaaccctcc atccacttcc catttcctct gcccttttcc 4920acctccccct tccatcccac cagctgccag tggctcccag aaagccttat tgagcccctt 4980gttgacactt ggggctgcgg aggcctctcc ctactggtct ggcctttcct gagaggcagg 5040tcttccgtcc tcagagcctt tctggaacaa ggagaatgcc tgtgcaggtg gacacacagg 5100cctggcctgt cgctctcact tgtcttccag cggggagctt cacgttgccg agtggaagaa 5160ccatgacctc cacttgcttc caaggtgcta gggaagtttc agggtacgct ggttcccctc 5220tccagctgga ggccgagttt ctggggactg cagatttttc tactctgtga tcgattcaat 5280gcccgatgct tctgtttcat tcccgaccct ttctactatg cattttcctt ttatcaggtg 5340tataaagtta aatactgtgt atttatcact aaaaagtaca tgaacttaag agacaactaa 5400gcctttcgtg tttttccaca ggtgtttaag cttctctgta cagttgaaat aaacagacag 5460caaaatggtg ccaaaaaaaa aaaaaaaaaa a 54916545PRTArtificial SequenceSynthetic 6Met Ser Ser Cys Val Ser Ser Gln Pro Ser Ser Asn Arg Ala Ala Pro 1 5 10 15 Gln Asp Glu Leu Gly Gly Arg Gly Ser Ser Ser Ser Glu Ser Gln Lys 20 25 30 Pro Cys Glu Ala Leu Arg Gly Leu Ser Ser Leu Ser Ile His Leu Gly 35 40 45 Met Glu Ser Phe Ile Val Val Thr Glu Cys Glu Pro Gly Cys Ala Val 50 55 60 Asp Leu Gly Leu Ala Arg Asp Arg Pro Leu Glu Ala Asp Gly Gln Glu 65 70 75 80 Val Pro Leu Asp Thr Ser Gly Ser Gln Ala Arg Pro His Leu Ser Gly 85 90 95 Arg Lys Leu Ser Leu Gln Glu Arg Ser Gln Gly Gly Leu Ala Ala Gly 100 105 110 Gly Ser Leu Asp Met Asn Gly Arg Cys Ile Cys Pro Ser Leu Pro Tyr 115 120 125 Ser Pro Val Ser Ser Pro Gln Ser Ser Pro Arg Leu Pro Arg Arg Pro 130 135 140 Thr Val Glu Ser His His Val Ser Ile Thr Gly Met Gln Asp Cys Val 145 150 155 160 Gln Leu Asn Gln Tyr Thr Leu Lys Asp Glu Ile Gly Lys Gly Ser Tyr 165 170 175 Gly Val Val Lys Leu Ala Tyr Asn Glu Asn Asp Asn Thr Tyr Tyr Ala 180 185 190 Met Lys Val Leu Ser Lys Lys Lys Leu Ile Arg Gln Ala Gly Phe Pro 195 200 205 Arg Arg Pro Pro Pro Arg Gly Thr Arg Pro Ala Pro Gly Gly Cys Ile 210 215 220 Gln Pro Arg Gly Pro Ile Glu Gln Val Tyr Gln Glu Ile Ala Ile Leu 225 230 235 240 Lys Lys Leu Asp His Pro Asn Val Val Lys Leu Val Glu Val Leu Asp 245 250 255 Asp Pro Asn Glu Asp His Leu Tyr Met Val Phe Glu Leu Val Asn Gln 260 265 270 Gly Pro Val Met Glu Val Pro Thr Leu Lys Pro Leu Ser Glu Asp Gln 275 280 285 Ala Arg Phe Tyr Phe Gln Asp Leu Ile Lys Gly Ile Glu Tyr Leu His 290 295 300 Tyr Gln Lys Ile Ile His Arg Asp Ile Lys Pro Ser Asn Leu Leu Val 305 310 315 320 Gly Glu Asp Gly His Ile Lys Ile Ala Asp Phe Gly Val Ser Asn Glu 325 330 335 Phe Lys Gly Ser Asp Ala Leu Leu Ser Asn Thr Val Gly Thr Pro Ala 340 345 350 Phe Met Ala Pro Glu Ser Leu Ser Glu Thr Arg Lys Ile Phe Ser Gly 355 360 365 Lys Ala Leu Asp Val Trp Ala Met Gly Val Thr Leu Tyr Cys Phe Val 370 375 380 Phe Gly Gln Cys Pro Phe Met Asp Glu Arg Ile Met Cys Leu His Ser 385 390 395 400 Lys Ile Lys Ser Gln Ala Leu Glu Phe Pro Asp Gln Pro Asp Ile Ala 405 410 415 Glu Asp Leu Lys Asp Leu Ile Thr Arg Met Leu Asp Lys Asn Pro Glu 420 425 430 Ser Arg Ile Val Val Pro Glu Ile Lys Ile Leu Val Lys Thr Met Ile 435 440 445 Arg Lys Arg Ser Phe Gly Asn Pro Phe Glu Gly Ser Arg Arg Glu Glu 450 455 460 Arg Ser Leu Ser Ala Pro Gly Asn Leu Leu Thr Lys Lys Pro Thr Arg 465 470 475 480 Glu Cys Glu Ser Leu Ser Glu Leu Lys Glu Ala Arg Gln Arg Arg Gln 485 490 495 Pro Pro Gly His Arg Pro Ala Pro Arg Gly Gly Gly Gly Ser Ala Leu 500 505 510 Val Arg Gly Ser Pro Cys Val Glu Ser Cys Trp Ala Pro Ala Pro Gly 515 520 525 Ser Pro Ala Arg Met His Pro Leu Arg Pro Glu Glu Ala Met Glu Pro 530 535 540 Glu 545 75448DNAArtificial SequenceSynthetic 7gagcctgggg aggtcgaggg tgcagcgagc cgtgatcgtg ctactgcact ccagcctggg 60caacacagag agaccctgtc tcaaaacaaa caaacaaaca aacaaacaaa caaacaaaaa 120aaacaaagaa aaaaaaatgg gagtgggccg ggcgcggtga ctcacacctg taatcccagc 180actttcggag gccaaggcgg gtggatcacg aggtcaggaa ttcaagatta gcctggacaa 240catggtgaaa ccccatctct acgaaaaata caaaaattag ccaagtatgg tggccggcgc 300ctgtaatccc agctactcgg gagactgagg cagagaactg cttgaacctg ggaggcagag 360gttgcagtga tccgagatcg cgtcactgca ctccagcgtg ggcgacagag cgagactccg 420tttcagaaaa gaaaaaaaaa aaaaaaaaaa agggagtcgg ggtggagctc tcattggctc 480gttgcatgtg agtgtcccta cggcctagaa atacaagaga agcacatcgg aacgggctgg 540aaatccaccc agttaactag agggctttga accttttatt aacttggagg ttgactctcc 600tgtcaactcg attccctttt ggctgtttgg cagggtcagt gagacatccc ctgggtcgct 660cgaccccgta ggacggttca gggagccctc caggtcttcg tttctcctct tccccgcaca 720gtgctgttat ccagctgggg gatccaacgc acacttaagg ctccagcaaa gtggctccgc 780tgccggatgg gagtgcccca gtgtgctgga tgaagctggc gcatgcacca tgtcatcatg 840tgtctctagc cagcccagca gcaaccgggc cgccccccag gatgagctgg ggggcagggg 900cagcagcagc agcgaaagcc agaagccctg tgaggccctg cggggcctct catccttgag 960catccacctg ggcatggagt ccttcattgt ggtcaccgag tgtgagccgg gctgtgctgt 1020ggacctcggc ttggcgcggg accggcccct ggaggccgat ggccaagagg tcccccttga 1080cacctccggg tcccaggccc ggccccacct ctccggtcgc aagctgtctc tgcaagagcg 1140gtcccagggt gggctggcag ccggtggcag cctggacatg aacggacgct gcatctgccc 1200gtccctgccc tactcacccg tcagctcccc gcagtcctcg cctcggctgc cccggcggcc 1260gacagtggag tctcaccacg tctccatcac gggtatgcag gactgtgtgc agctgaatca 1320gtataccctg aaggatgaaa ttggaaaggg ctcctatggt gtcgtcaagt tggcctacaa 1380tgaaaatgac aatacctact atgcaatgaa ggtgctgtcc aaaaagaagc tgatccggca 1440ggccggcttt ccacgtcgcc ctccaccccg aggcacccgg ccagctcctg gaggctgcat 1500ccagcccagg ggccccattg agcaggtgta ccaggaaatt gccatcctca agaagctgga 1560ccaccccaat gtggtgaagc tggtggaggt cctggatgac cccaatgagg accatctgta 1620catggtgttc gaactggtca accaagggcc cgtgatggaa gtgcccaccc tcaaaccact 1680ctctgaagac caggcccgtt tctacttcca ggatctgatc aaaggcatcg agtacttaca 1740ctaccagaag atcatccacc gtgacatcaa accttccaac ctcctggtcg gagaagatgg 1800gcacatcaag atcgctgact ttggtgtgag caatgaattc aagggcagtg acgcgctcct 1860ctccaacacc gtgggcacgc ccgccttcat ggcacccgag tcgctctctg agacccgcaa 1920gatcttctct gggaaggcct tggatgtttg ggccatgggt gtgacactat actgctttgt 1980ctttggccag tgcccattca tggacgagcg gatcatgtgt ttacacagta agatcaagag 2040tcaggccctg gaatttccag accagcccga catagctgag gacttgaagg acctgatcac 2100ccgtatgctg gacaagaacc ccgagtcgag gatcgtggtg ccggaaatca agatcctggt 2160gaagaccatg atacgtaaac gctcctttgg

gaacccattc gagggcagcc ggcgggagga 2220acgctcactg tcagcgcctg gaaacttgct cacgaagcaa ggcagcgaag acaacctcca 2280gggcaccgac ccgccccccg tgggggagga ggaagtgctc ttgtgagagg cagtccctgc 2340gtggaaagtt gctgggcccc cgcccccggc tcccccgcac gcatgcatcc actgcggccg 2400gaggaggcca tggagcccga gtagctgcct ggatcgctcg acctcgcatg cgcgccgcgt 2460cgcctctggg gggctgctgc accgcgtttc catagcagca tgtcctacgg aaacccagca 2520cgtgtgtaga gcctcgatcg tcatctctgg ttatttgttt tttcctttgt tgttttaaag 2580gggacaaaaa aaaaaaaagg acttgactcc atgacgtcga ccgtggccgc tggctggctg 2640gacaggcggg tgtgaggagt tgcagaccca aacccacgtg cattttggga caattgcttt 2700ttaaaacgtt tttatgccaa aaatccttca ttgtgatttt cagaaccacg tcagatatac 2760caagtgactg tgtgtggggt ttgacaactg tggaaaggcg agcagaaaac tccggcggtc 2820tgaggccatg gaggtggttg ctgcatttga gagggagtag ggggctagat gtggctccta 2880gtgcaaaccg gaaaccatgg caccttccag agccgtggtc tcaaggagtc agagcagggc 2940tggccctcag tagctgcagg gagctttgat gcaacttatt tgtaagaagg atttttaaat 3000tttttatggg tagaattgta gtcaggaaaa cagaaagggc ttgaaattta ataagtgctg 3060ctggaagggg attttccaag cctggaaggg tattcagcag ctgtggtggg gaaacatttc 3120tcctgaaaga ctgaacgtgt ttcttcatga cagctgctca aagcaggttt ctgagatagc 3180tgaccgagct ctggtaaatc tctttgtcaa attacgaaaa cttcagggtg aaatcctatg 3240cttccatgta cattacatgg cttaagatta aacaaaaaca tttttcaagt ctctaactag 3300agtgaactct agagcacagt agttcagaaa ctatttagag cttccaggat atatttcaca 3360gcttcaggca tgtgatcagt tagagccgat gaaacctatg cccgcctgta tatatattag 3420cagcttagct agttcataac ctgtatattc taaagactgc taaggttttg ttttcatttt 3480aaatcctagc tgattgttgt ggtcaatgaa atacccagtt tctggagggc caggtgggaa 3540atgctttcac tggaccaaca cacaaatgat catcctgagg atctgagctt ccctagactc 3600cacacaataa ccttggggca cccttttaga gaagactgtt gaaacccaca gcactcgttg 3660gggtatgagg aaaccagggc ttggcacagg aagttcccct ttgtagctaa aagtccagaa 3720agaaagggtt catctttttg acttccaact gatattggga agtttggttg aggttcaagt 3780gtgactcctt ccagagccac aggtagggga gtgtgaagtt gagggggagg aaagctggaa 3840ggactctgcc ttgggagatt cccagctctg ctttccagcg cttggtggaa tctgggctgg 3900ggaaagacgg caccgggaaa ctctgcttcc ccattgtttc catctgatca gctgtggtgt 3960gaggacttct cagacaaagg caaggcctcg tgcccctgcc cagcccattc atggagccct 4020gggccttctt ggcttccata gatcctaagc tcttgactgt agtttagcca gacttgtttt 4080gctatcttat aagcagttca gaattaggga atgctggttt tgaagagcaa aggacaggta 4140gtctagagag ggtcgtctgg cctgcttgct gggtctttgt aacccagcac ttcctcttgc 4200cctcctggct ttatgtttat ggggagagga ctcaatagct ccaccccttc tggcaccaga 4260tggggcttgg ttagtttgca ataagcacct tgcagaggtt aaagccagcg ggtccctagt 4320cttaggccca gcctgcttgt gtgggctctg gcctggcctg gtggctggcc cagggggcag 4380cagtgcttag agcttctgca gggcttctct tgtttacaca gctgcatcag acaatgccat 4440ttctccccac cacggaacct tccatctaag atttcttcca gggaatgcca gcaatcaggc 4500agcacccagc tgtgggggca gtggggtggg ggagacccac attgatgact tttttttttt 4560cttttaatga agaaacacca aagaaagctg tggaaaggac ctgccccaca tgaaaaggat 4620aagccaagat ggctgtaaac acagagcatt tgagctgcca ctcttggagc acattgattt 4680ttcaaaagcc agctctgtca ggaaaggagg tgctgttatg agcagctctt ccagtgggca 4740aagaggacgc ccataatttc ttccattgct agctcatctg tgggaccaat ttggtgtaag 4800caacctgtgg cctgcacttg tggcctcgaa ggaagcacaa accctccatc cacttcccat 4860ttcctctgcc cttttccacc tcccccttcc atcccaccag ctgccagtgg ctcccagaaa 4920gccttattga gccccttgtt gacacttggg gctgcggagg cctctcccta ctggtctggc 4980ctttcctgag aggcaggtct tccgtcctca gagcctttct ggaacaagga gaatgcctgt 5040gcaggtggac acacaggcct ggcctgtcgc tctcacttgt cttccagcgg ggagcttcac 5100gttgccgagt ggaagaacca tgacctccac ttgcttccaa ggtgctaggg aagtttcagg 5160gtacgctggt tcccctctcc agctggaggc cgagtttctg gggactgcag atttttctac 5220tctgtgatcg attcaatgcc cgatgcttct gtttcattcc cgaccctttc tactatgcat 5280tttcctttta tcaggtgtat aaagttaaat actgtgtatt tatcactaaa aagtacatga 5340acttaagaga caactaagcc tttcgtgttt ttccacaggt gtttaagctt ctctgtacag 5400ttgaaataaa cagacagcaa aatggtgcca aaaaaaaaaa aaaaaaaa 54488498PRTArtificial SequenceSynthetic 8Met Ser Ser Cys Val Ser Ser Gln Pro Ser Ser Asn Arg Ala Ala Pro 1 5 10 15 Gln Asp Glu Leu Gly Gly Arg Gly Ser Ser Ser Ser Glu Ser Gln Lys 20 25 30 Pro Cys Glu Ala Leu Arg Gly Leu Ser Ser Leu Ser Ile His Leu Gly 35 40 45 Met Glu Ser Phe Ile Val Val Thr Glu Cys Glu Pro Gly Cys Ala Val 50 55 60 Asp Leu Gly Leu Ala Arg Asp Arg Pro Leu Glu Ala Asp Gly Gln Glu 65 70 75 80 Val Pro Leu Asp Thr Ser Gly Ser Gln Ala Arg Pro His Leu Ser Gly 85 90 95 Arg Lys Leu Ser Leu Gln Glu Arg Ser Gln Gly Gly Leu Ala Ala Gly 100 105 110 Gly Ser Leu Asp Met Asn Gly Arg Cys Ile Cys Pro Ser Leu Pro Tyr 115 120 125 Ser Pro Val Ser Ser Pro Gln Ser Ser Pro Arg Leu Pro Arg Arg Pro 130 135 140 Thr Val Glu Ser His His Val Ser Ile Thr Gly Met Gln Asp Cys Val 145 150 155 160 Gln Leu Asn Gln Tyr Thr Leu Lys Asp Glu Ile Gly Lys Gly Ser Tyr 165 170 175 Gly Val Val Lys Leu Ala Tyr Asn Glu Asn Asp Asn Thr Tyr Tyr Ala 180 185 190 Met Lys Val Leu Ser Lys Lys Lys Leu Ile Arg Gln Ala Gly Phe Pro 195 200 205 Arg Arg Pro Pro Pro Arg Gly Thr Arg Pro Ala Pro Gly Gly Cys Ile 210 215 220 Gln Pro Arg Gly Pro Ile Glu Gln Val Tyr Gln Glu Ile Ala Ile Leu 225 230 235 240 Lys Lys Leu Asp His Pro Asn Val Val Lys Leu Val Glu Val Leu Asp 245 250 255 Asp Pro Asn Glu Asp His Leu Tyr Met Val Phe Glu Leu Val Asn Gln 260 265 270 Gly Pro Val Met Glu Val Pro Thr Leu Lys Pro Leu Ser Glu Asp Gln 275 280 285 Ala Arg Phe Tyr Phe Gln Asp Leu Ile Lys Gly Ile Glu Tyr Leu His 290 295 300 Tyr Gln Lys Ile Ile His Arg Asp Ile Lys Pro Ser Asn Leu Leu Val 305 310 315 320 Gly Glu Asp Gly His Ile Lys Ile Ala Asp Phe Gly Val Ser Asn Glu 325 330 335 Phe Lys Gly Ser Asp Ala Leu Leu Ser Asn Thr Val Gly Thr Pro Ala 340 345 350 Phe Met Ala Pro Glu Ser Leu Ser Glu Thr Arg Lys Ile Phe Ser Gly 355 360 365 Lys Ala Leu Asp Val Trp Ala Met Gly Val Thr Leu Tyr Cys Phe Val 370 375 380 Phe Gly Gln Cys Pro Phe Met Asp Glu Arg Ile Met Cys Leu His Ser 385 390 395 400 Lys Ile Lys Ser Gln Ala Leu Glu Phe Pro Asp Gln Pro Asp Ile Ala 405 410 415 Glu Asp Leu Lys Asp Leu Ile Thr Arg Met Leu Asp Lys Asn Pro Glu 420 425 430 Ser Arg Ile Val Val Pro Glu Ile Lys Ile Leu Val Lys Thr Met Ile 435 440 445 Arg Lys Arg Ser Phe Gly Asn Pro Phe Glu Gly Ser Arg Arg Glu Glu 450 455 460 Arg Ser Leu Ser Ala Pro Gly Asn Leu Leu Thr Lys Gln Gly Ser Glu 465 470 475 480 Asp Asn Leu Gln Gly Thr Asp Pro Pro Pro Val Gly Glu Glu Glu Val 485 490 495 Leu Leu 92981DNAArtificial SequenceSynthetic 9gagcctgggg aggtcgaggg tgcagcgagc cgtgatcgtg ctactgcact ccagcctggg 60caacacagag agaccctgtc tcaaaacaaa caaacaaaca aacaaacaaa caaacaaaaa 120aaacaaagaa aaaaaaatgg gagtgggccg ggcgcggtga ctcacacctg taatcccagc 180actttcggag gccaaggcgg gtggatcacg aggtcaggaa ttcaagatta gcctggacaa 240catggtgaaa ccccatctct acgaaaaata caaaaattag ccaagtatgg tggccggcgc 300ctgtaatccc agctactcgg gagactgagg cagagaactg cttgaacctg ggaggcagag 360gttgcagtga tccgagatcg cgtcactgca ctccagcgtg ggcgacagag cgagactccg 420tttcagaaaa gaaaaaaaaa aaaaaaaaaa agggagtcgg ggtggagctc tcattggctc 480gttgcatgtg agtgtcccta cggcctagaa atacaagaga agcacatcgg aacgggctgg 540aaatccaccc agttaactag agggctttga accttttatt aacttggagg ttgactctcc 600tgtcaactcg attccctttt ggctgtttgg cagggtcagt gagacatccc ctgggtcgct 660cgaccccgta ggacggttca gggagccctc caggtcttcg tttctcctct tccccgcaca 720gtgctgttat ccagctgggg gatccaacgc acacttaagg ctccagcaaa gtggctccgc 780tgccggatgg gagtgcccca gtgtgctgga tgaagctggc gcatgcacca tgtcatcatg 840tgtctctagc cagcccagca gcaaccgggc cgccccccag gatgagctgg ggggcagggg 900cagcagcagc agcgaaagcc agaagccctg tgaggccctg cggggcctct catccttgag 960catccacctg ggcatggagt ccttcattgt ggtcaccgag tgtgagccgg gctgtgctgt 1020ggacctcggc ttggcgcggg accggcccct ggaggccgat ggccaagagg tcccccttga 1080cacctccggg tcccaggccc ggccccacct ctccggtcgc aagctgtctc tgcaagagcg 1140gtcccagggt gggctggcag ccggtggcag cctggacatg aacggacgct gcatctgccc 1200gtccctgccc tactcacccg tcagctcccc gcagtcctcg cctcggctgc cccggcggcc 1260gacagtggag tctcaccacg tctccatcac gggtatgcag gactgtgtgc agctgaatca 1320gtataccctg aaggatgaaa ttggaaaggg ctcctatggt gtcgtcaagt tggcctacaa 1380tgaaaatgac aatacctact atgcaatgaa ggtgctgtcc aaaaagaagc tgatccggca 1440ggccggcttt ccacgtcgcc ctccaccccg aggcacccgg ccagctcctg gaggctgcat 1500ccagcccagg ggccccattg agcaggtgta ccaggaaatt gccatcctca agaagctgga 1560ccaccccaat gtggtgaagc tggtggaggt cctggatgac cccaatgagg accatctgta 1620catggtgttc gaactggtca accaagggcc cgtgatggaa gtgcccaccc tcaaaccact 1680ctctgaagac caggcccgtt tctacttcca ggatctgatc aaaggcatcg agtacttaca 1740ctaccagaag atcatccacc gtgacatcaa accttccaac ctcctggtcg gagaagatgg 1800gcacatcaag atcgctgact ttggtgtgag caatgaattc aagggcagtg acgcgctcct 1860ctccaacacc gtgggcacgc ccgccttcat ggcacccgag tcgctctctg agacccgcaa 1920gatcttctct gggaaggcct tggatgtttg ggccatgggt gtgacactat actgctttgt 1980ctttggccag tgcccattca tggacgagcg gatcatgtgt ttacacagta agatcaagag 2040tcaggccctg gaatttccag accagcccga catagctgag gacttgaagg acctgatcac 2100ccgtatgctg gacaagaacc ccgagtcgag gatcgtggtg ccggaaatca agctgcaccc 2160ctgggtcacg aggcatgggg cggagccgtt gccgtcggag gatgagaact gcacgctggt 2220cgaagtgact gaagaggagg tcgagaactc agtcaaacac attcccagct tggcaaccgt 2280gatcctggtg aagaccatga tacgtaaacg ctcctttggg aacccattcg agggcagccg 2340gcgggaggaa cgctcactgt cagcgcctgg aaacttgctc accaaaaaac caaccaggga 2400atgtgagtcc ctgtctgagc tcaagaccta gaaaataagt ccccttcctg cctgttgcaa 2460agtaacgtaa gagttccctc acccgagtgg atgcagacct tcttgctgtc agccaccctt 2520ccttcataca catagccagc ccaggtgacc agaacctccc aggacagatg aggctttgtg 2580tccttatgag actgggagaa cctgctgggc acccctgctg caggtgctgt ggtgggtggg 2640gaccccactg cccttcccac tgagcacatc atggctacct gacttggtgg gagctccagg 2700cagtcacttc tgtttcttaa acatagcttt actgaggtac aattcacata ccatgtaatt 2760cacccacggg aagtgtatga ttcagtggtt tctaatacag acttctgcag ccattaccac 2820cgtcaacttt acgacatttt catcagccca agaagacacc ctacactcct tagctgtccc 2880catccaactc ccccacccca gtaaccactc agaataggta tggatttgcc tattctggac 2940gtttcgtata aatggcgtca tacactaaaa aaaaaaaaaa a 298110533PRTArtificial SequenceSynthetic 10Met Ser Ser Cys Val Ser Ser Gln Pro Ser Ser Asn Arg Ala Ala Pro 1 5 10 15 Gln Asp Glu Leu Gly Gly Arg Gly Ser Ser Ser Ser Glu Ser Gln Lys 20 25 30 Pro Cys Glu Ala Leu Arg Gly Leu Ser Ser Leu Ser Ile His Leu Gly 35 40 45 Met Glu Ser Phe Ile Val Val Thr Glu Cys Glu Pro Gly Cys Ala Val 50 55 60 Asp Leu Gly Leu Ala Arg Asp Arg Pro Leu Glu Ala Asp Gly Gln Glu 65 70 75 80 Val Pro Leu Asp Thr Ser Gly Ser Gln Ala Arg Pro His Leu Ser Gly 85 90 95 Arg Lys Leu Ser Leu Gln Glu Arg Ser Gln Gly Gly Leu Ala Ala Gly 100 105 110 Gly Ser Leu Asp Met Asn Gly Arg Cys Ile Cys Pro Ser Leu Pro Tyr 115 120 125 Ser Pro Val Ser Ser Pro Gln Ser Ser Pro Arg Leu Pro Arg Arg Pro 130 135 140 Thr Val Glu Ser His His Val Ser Ile Thr Gly Met Gln Asp Cys Val 145 150 155 160 Gln Leu Asn Gln Tyr Thr Leu Lys Asp Glu Ile Gly Lys Gly Ser Tyr 165 170 175 Gly Val Val Lys Leu Ala Tyr Asn Glu Asn Asp Asn Thr Tyr Tyr Ala 180 185 190 Met Lys Val Leu Ser Lys Lys Lys Leu Ile Arg Gln Ala Gly Phe Pro 195 200 205 Arg Arg Pro Pro Pro Arg Gly Thr Arg Pro Ala Pro Gly Gly Cys Ile 210 215 220 Gln Pro Arg Gly Pro Ile Glu Gln Val Tyr Gln Glu Ile Ala Ile Leu 225 230 235 240 Lys Lys Leu Asp His Pro Asn Val Val Lys Leu Val Glu Val Leu Asp 245 250 255 Asp Pro Asn Glu Asp His Leu Tyr Met Val Phe Glu Leu Val Asn Gln 260 265 270 Gly Pro Val Met Glu Val Pro Thr Leu Lys Pro Leu Ser Glu Asp Gln 275 280 285 Ala Arg Phe Tyr Phe Gln Asp Leu Ile Lys Gly Ile Glu Tyr Leu His 290 295 300 Tyr Gln Lys Ile Ile His Arg Asp Ile Lys Pro Ser Asn Leu Leu Val 305 310 315 320 Gly Glu Asp Gly His Ile Lys Ile Ala Asp Phe Gly Val Ser Asn Glu 325 330 335 Phe Lys Gly Ser Asp Ala Leu Leu Ser Asn Thr Val Gly Thr Pro Ala 340 345 350 Phe Met Ala Pro Glu Ser Leu Ser Glu Thr Arg Lys Ile Phe Ser Gly 355 360 365 Lys Ala Leu Asp Val Trp Ala Met Gly Val Thr Leu Tyr Cys Phe Val 370 375 380 Phe Gly Gln Cys Pro Phe Met Asp Glu Arg Ile Met Cys Leu His Ser 385 390 395 400 Lys Ile Lys Ser Gln Ala Leu Glu Phe Pro Asp Gln Pro Asp Ile Ala 405 410 415 Glu Asp Leu Lys Asp Leu Ile Thr Arg Met Leu Asp Lys Asn Pro Glu 420 425 430 Ser Arg Ile Val Val Pro Glu Ile Lys Leu His Pro Trp Val Thr Arg 435 440 445 His Gly Ala Glu Pro Leu Pro Ser Glu Asp Glu Asn Cys Thr Leu Val 450 455 460 Glu Val Thr Glu Glu Glu Val Glu Asn Ser Val Lys His Ile Pro Ser 465 470 475 480 Leu Ala Thr Val Ile Leu Val Lys Thr Met Ile Arg Lys Arg Ser Phe 485 490 495 Gly Asn Pro Phe Glu Gly Ser Arg Arg Glu Glu Arg Ser Leu Ser Ala 500 505 510 Pro Gly Asn Leu Leu Thr Lys Lys Pro Thr Arg Glu Cys Glu Ser Leu 515 520 525 Ser Glu Leu Lys Thr 530 112852DNAArtificial SequenceSynthetic 11gagcctgggg aggtcgaggg tgcagcgagc cgtgatcgtg ctactgcact ccagcctggg 60caacacagag agaccctgtc tcaaaacaaa caaacaaaca aacaaacaaa caaacaaaaa 120aaacaaagaa aaaaaaatgg gagtgggccg ggcgcggtga ctcacacctg taatcccagc 180actttcggag gccaaggcgg gtggatcacg aggtcaggaa ttcaagatta gcctggacaa 240catggtgaaa ccccatctct acgaaaaata caaaaattag ccaagtatgg tggccggcgc 300ctgtaatccc agctactcgg gagactgagg cagagaactg cttgaacctg ggaggcagag 360gttgcagtga tccgagatcg cgtcactgca ctccagcgtg ggcgacagag cgagactccg 420tttcagaaaa gaaaaaaaaa aaaaaaaaaa agggagtcgg ggtggagctc tcattggctc 480gttgcatgtg agtgtcccta cggcctagaa atacaagaga agcacatcgg aacgggctgg 540aaatccaccc agttaactag agggctttga accttttatt aacttggagg ttgactctcc 600tgtcaactcg attccctttt ggctgtttgg cagggtcagt gagacatccc ctgggtcgct 660cgaccccgta ggacggttca gggagccctc caggtcttcg tttctcctct tccccgcaca 720gtgctgttat ccagctgggg gatccaacgc acacttaagg ctccagcaaa gtggctccgc 780tgccggatgg gagtgcccca gtgtgctgga tgaagctggc gcatgcacca tgtcatcatg 840tgtctctagc cagcccagca gcaaccgggc cgccccccag gatgagctgg ggggcagggg 900cagcagcagc agcgaaagcc agaagccctg tgaggccctg cggggcctct catccttgag 960catccacctg ggcatggagt ccttcattgt ggtcaccgag tgtgagccgg gctgtgctgt 1020ggacctcggc ttggcgcggg accggcccct ggaggccgat ggccaagagg tcccccttga 1080cacctccggg tcccaggccc ggccccacct ctccggtcgc aagctgtctc tgcaagagcg 1140gtcccagggt gggctggcag ccggtggcag cctggacatg aacggacgct gcatctgccc 1200gtccctgccc tactcacccg tcagctcccc gcagtcctcg cctcggctgc cccggcggcc 1260gacagtggag tctcaccacg tctccatcac gggtatgcag gactgtgtgc agctgaatca 1320gtataccctg aaggatgaaa ttggaaaggg ctcctatggt gtcgtcaagt tggcctacaa 1380tgaaaatgac aatacctact atgcaatgaa ggtgctgtcc aaaaagaagc tgatccggca 1440ggccggcttt ccacgtcgcc ctccaccccg aggcacccgg ccagctcctg gaggctgcat 1500ccagcccagg ggccccattg agcaggtgta ccaggaaatt gccatcctca agaagctgga 1560ccaccccaat gtggtgaagc tggtggaggt cctggatgac cccaatgagg accatctgta 1620catggtgttc gaactggtca accaagggcc cgtgatggaa gtgcccaccc tcaaaccact 1680ctctgaagac caggcccgtt tctacttcca ggatctgatc aaaggcatcg agtacttaca 1740ctaccagaag atcatccacc gtgacatcaa accttccaac ctcctggtcg gagaagatgg 1800gcacatcaag atcgctgact ttggtgtgag caatgaattc aagggcagtg acgcgctcct 1860ctccaacacc gtgggcacgc ccgccttcat ggcacccgag

tcgctctctg agacccgcaa 1920gatcttctct gggaaggcct tggatgtttg ggccatgggt gtgacactat actgctttgt 1980ctttggccag tgcccattca tggacgagcg gatcatgtgt ttacacagta agatcaagag 2040tcaggccctg gaatttccag accagcccga catagctgag gacttgaagg acctgatcac 2100ccgtatgctg gacaagaacc ccgagtcgag gatcgtggtg ccggaaatca agatcctggt 2160gaagaccatg atacgtaaac gctcctttgg gaacccattc gagggcagcc ggcgggagga 2220acgctcactg tcagcgcctg gaaacttgct caccaaaaaa ccaaccaggg aatgtgagtc 2280cctgtctgag ctcaagacct agaaaataag tccccttcct gcctgttgca aagtaacgta 2340agagttccct cacccgagtg gatgcagacc ttcttgctgt cagccaccct tccttcatac 2400acatagccag cccaggtgac cagaacctcc caggacagat gaggctttgt gtccttatga 2460gactgggaga acctgctggg cacccctgct gcaggtgctg tggtgggtgg ggaccccact 2520gcccttccca ctgagcacat catggctacc tgacttggtg ggagctccag gcagtcactt 2580ctgtttctta aacatagctt tactgaggta caattcacat accatgtaat tcacccacgg 2640gaagtgtatg attcagtggt ttctaataca gacttctgca gccattacca ccgtcaactt 2700tacgacattt tcatcagccc aagaagacac cctacactcc ttagctgtcc ccatccaact 2760cccccacccc agtaaccact cagaataggt atggatttgc ctattctgga cgtttcgtat 2820aaatggcgtc atacactaaa aaaaaaaaaa aa 285212490PRTArtificial SequenceSynthetic 12Met Ser Ser Cys Val Ser Ser Gln Pro Ser Ser Asn Arg Ala Ala Pro 1 5 10 15 Gln Asp Glu Leu Gly Gly Arg Gly Ser Ser Ser Ser Glu Ser Gln Lys 20 25 30 Pro Cys Glu Ala Leu Arg Gly Leu Ser Ser Leu Ser Ile His Leu Gly 35 40 45 Met Glu Ser Phe Ile Val Val Thr Glu Cys Glu Pro Gly Cys Ala Val 50 55 60 Asp Leu Gly Leu Ala Arg Asp Arg Pro Leu Glu Ala Asp Gly Gln Glu 65 70 75 80 Val Pro Leu Asp Thr Ser Gly Ser Gln Ala Arg Pro His Leu Ser Gly 85 90 95 Arg Lys Leu Ser Leu Gln Glu Arg Ser Gln Gly Gly Leu Ala Ala Gly 100 105 110 Gly Ser Leu Asp Met Asn Gly Arg Cys Ile Cys Pro Ser Leu Pro Tyr 115 120 125 Ser Pro Val Ser Ser Pro Gln Ser Ser Pro Arg Leu Pro Arg Arg Pro 130 135 140 Thr Val Glu Ser His His Val Ser Ile Thr Gly Met Gln Asp Cys Val 145 150 155 160 Gln Leu Asn Gln Tyr Thr Leu Lys Asp Glu Ile Gly Lys Gly Ser Tyr 165 170 175 Gly Val Val Lys Leu Ala Tyr Asn Glu Asn Asp Asn Thr Tyr Tyr Ala 180 185 190 Met Lys Val Leu Ser Lys Lys Lys Leu Ile Arg Gln Ala Gly Phe Pro 195 200 205 Arg Arg Pro Pro Pro Arg Gly Thr Arg Pro Ala Pro Gly Gly Cys Ile 210 215 220 Gln Pro Arg Gly Pro Ile Glu Gln Val Tyr Gln Glu Ile Ala Ile Leu 225 230 235 240 Lys Lys Leu Asp His Pro Asn Val Val Lys Leu Val Glu Val Leu Asp 245 250 255 Asp Pro Asn Glu Asp His Leu Tyr Met Val Phe Glu Leu Val Asn Gln 260 265 270 Gly Pro Val Met Glu Val Pro Thr Leu Lys Pro Leu Ser Glu Asp Gln 275 280 285 Ala Arg Phe Tyr Phe Gln Asp Leu Ile Lys Gly Ile Glu Tyr Leu His 290 295 300 Tyr Gln Lys Ile Ile His Arg Asp Ile Lys Pro Ser Asn Leu Leu Val 305 310 315 320 Gly Glu Asp Gly His Ile Lys Ile Ala Asp Phe Gly Val Ser Asn Glu 325 330 335 Phe Lys Gly Ser Asp Ala Leu Leu Ser Asn Thr Val Gly Thr Pro Ala 340 345 350 Phe Met Ala Pro Glu Ser Leu Ser Glu Thr Arg Lys Ile Phe Ser Gly 355 360 365 Lys Ala Leu Asp Val Trp Ala Met Gly Val Thr Leu Tyr Cys Phe Val 370 375 380 Phe Gly Gln Cys Pro Phe Met Asp Glu Arg Ile Met Cys Leu His Ser 385 390 395 400 Lys Ile Lys Ser Gln Ala Leu Glu Phe Pro Asp Gln Pro Asp Ile Ala 405 410 415 Glu Asp Leu Lys Asp Leu Ile Thr Arg Met Leu Asp Lys Asn Pro Glu 420 425 430 Ser Arg Ile Val Val Pro Glu Ile Lys Ile Leu Val Lys Thr Met Ile 435 440 445 Arg Lys Arg Ser Phe Gly Asn Pro Phe Glu Gly Ser Arg Arg Glu Glu 450 455 460 Arg Ser Leu Ser Ala Pro Gly Asn Leu Leu Thr Lys Lys Pro Thr Arg 465 470 475 480 Glu Cys Glu Ser Leu Ser Glu Leu Lys Thr 485 490 134923DNAArtificial SequenceSynthetic 13gcgcgcccgc cgcccgggcg gaggagagga gcgcgcggcc gcgcagagca agctgagccg 60agccgagccg agctgggggc gcagagcgcg ggaggcggcg gcggcgcgga gcccaggtgg 120ctccgctgcc ggatgggagt gccccagtgt gctggatgaa gctggcgcat gcaccatgtc 180atcatgtgtc tctagccagc ccagcagcaa ccgggccgcc ccccaggatg agctgggggg 240caggggcagc agcagcagcg aaagccagaa gccctgtgag gccctgcggg gcctctcatc 300cttgagcatc cacctgggca tggagtcctt cattgtggtc accgagtgtg agccgggctg 360tgctgtggac ctcggcttgg cgcgggaccg gcccctggag gccgatggcc aagaggtccc 420ccttgacacc tccgggtccc aggcccggcc ccacctctcc ggtcgcaagc tgtctctgca 480agagcggtcc cagggtgggc tggcagccgg tggcagcctg gacatgaacg gacgctgcat 540ctgcccgtcc ctgccctact cacccgtcag ctccccgcag tcctcgcctc ggctgccccg 600gcggccgaca gtggagtctc accacgtctc catcacgggt atgcaggact gtgtgcagct 660gaatcagtat accctgaagg atgaaattgg aaagggctcc tatggtgtcg tcaagttggc 720ctacaatgaa aatgacaata cctactatgc aatgaaggtg ctgtccaaaa agaagctgat 780ccggcaggcc ggctttccac gtcgccctcc accccgaggc acccggccag ctcctggagg 840ctgcatccag cccaggggcc ccattgagca ggtgtaccag gaaattgcca tcctcaagaa 900gctggaccac cccaatgtgg tgaagctggt ggaggtcctg gatgacccca atgaggacca 960tctgtacatg gtgttcgaac tggtcaacca agggcccgtg atggaagtgc ccaccctcaa 1020accactctct gaagaccagg cccgtttcta cttccaggat ctgatcaaag gcatcgagta 1080cttacactac cagaagatca tccaccgtga catcaaacct tccaacctcc tggtcggaga 1140agatgggcac atcaagatcg ctgactttgg tgtgagcaat gaattcaagg gcagtgacgc 1200gctcctctcc aacaccgtgg gcacgcccgc cttcatggca cccgagtcgc tctctgagac 1260ccgcaagatc ttctctggga aggccttgga tgtttgggcc atgggtgtga cactatactg 1320ctttgtcttt ggccagtgcc cattcatgga cgagcggatc atgtgtttac acagtaagat 1380caagagtcag gccctggaat ttccagacca gcccgacata gctgaggact tgaaggacct 1440gatcacccgt atgctggaca agaaccccga gtcgaggatc gtggtgccgg aaatcaagct 1500gcacccctgg gtcacgaggc atggggcgga gccgttgccg tcggaggatg agaactgcac 1560gctggtcgaa gtgactgaag aggaggtcga gaactcagtc aaacacattc ccagcttggc 1620aaccgtgatc ctggtgaaga ccatgatacg taaacgctcc tttgggaacc cattcgaggg 1680cagccggcgg gaggaacgct cactgtcagc gcctggaaac ttgctcacga agcaaggcag 1740cgaagacaac ctccagggca ccgacccgcc ccccgtgggg gaggaggaag tgctcttgtg 1800agaggcagtc cctgcgtgga aagttgctgg gcccccgccc ccggctcccc cgcacgcatg 1860catccactgc ggccggagga ggccatggag cccgagtagc tgcctggatc gctcgacctc 1920gcatgcgcgc cgcgtcgcct ctggggggct gctgcaccgc gtttccatag cagcatgtcc 1980tacggaaacc cagcacgtgt gtagagcctc gatcgtcatc tctggttatt tgttttttcc 2040tttgttgttt taaaggggac aaaaaaaaaa aaaggacttg actccatgac gtcgaccgtg 2100gccgctggct ggctggacag gcgggtgtga ggagttgcag acccaaaccc acgtgcattt 2160tgggacaatt gctttttaaa acgtttttat gccaaaaatc cttcattgtg attttcagaa 2220ccacgtcaga tataccaagt gactgtgtgt ggggtttgac aactgtggaa aggcgagcag 2280aaaactccgg cggtctgagg ccatggaggt ggttgctgca tttgagaggg agtagggggc 2340tagatgtggc tcctagtgca aaccggaaac catggcacct tccagagccg tggtctcaag 2400gagtcagagc agggctggcc ctcagtagct gcagggagct ttgatgcaac ttatttgtaa 2460gaaggatttt taaatttttt atgggtagaa ttgtagtcag gaaaacagaa agggcttgaa 2520atttaataag tgctgctgga aggggatttt ccaagcctgg aagggtattc agcagctgtg 2580gtggggaaac atttctcctg aaagactgaa cgtgtttctt catgacagct gctcaaagca 2640ggtttctgag atagctgacc gagctctggt aaatctcttt gtcaaattac gaaaacttca 2700gggtgaaatc ctatgcttcc atgtacatta catggcttaa gattaaacaa aaacattttt 2760caagtctcta actagagtga actctagagc acagtagttc agaaactatt tagagcttcc 2820aggatatatt tcacagcttc aggcatgtga tcagttagag ccgatgaaac ctatgcccgc 2880ctgtatatat attagcagct tagctagttc ataacctgta tattctaaag actgctaagg 2940ttttgttttc attttaaatc ctagctgatt gttgtggtca atgaaatacc cagtttctgg 3000agggccaggt gggaaatgct ttcactggac caacacacaa atgatcatcc tgaggatctg 3060agcttcccta gactccacac aataaccttg gggcaccctt ttagagaaga ctgttgaaac 3120ccacagcact cgttggggta tgaggaaacc agggcttggc acaggaagtt cccctttgta 3180gctaaaagtc cagaaagaaa gggttcatct ttttgacttc caactgatat tgggaagttt 3240ggttgaggtt caagtgtgac tccttccaga gccacaggta ggggagtgtg aagttgaggg 3300ggaggaaagc tggaaggact ctgccttggg agattcccag ctctgctttc cagcgcttgg 3360tggaatctgg gctggggaaa gacggcaccg ggaaactctg cttccccatt gtttccatct 3420gatcagctgt ggtgtgagga cttctcagac aaaggcaagg cctcgtgccc ctgcccagcc 3480cattcatgga gccctgggcc ttcttggctt ccatagatcc taagctcttg actgtagttt 3540agccagactt gttttgctat cttataagca gttcagaatt agggaatgct ggttttgaag 3600agcaaaggac aggtagtcta gagagggtcg tctggcctgc ttgctgggtc tttgtaaccc 3660agcacttcct cttgccctcc tggctttatg tttatgggga gaggactcaa tagctccacc 3720ccttctggca ccagatgggg cttggttagt ttgcaataag caccttgcag aggttaaagc 3780cagcgggtcc ctagtcttag gcccagcctg cttgtgtggg ctctggcctg gcctggtggc 3840tggcccaggg ggcagcagtg cttagagctt ctgcagggct tctcttgttt acacagctgc 3900atcagacaat gccatttctc cccaccacgg aaccttccat ctaagatttc ttccagggaa 3960tgccagcaat caggcagcac ccagctgtgg gggcagtggg gtgggggaga cccacattga 4020tgactttttt tttttctttt aatgaagaaa caccaaagaa agctgtggaa aggacctgcc 4080ccacatgaaa aggataagcc aagatggctg taaacacaga gcatttgagc tgccactctt 4140ggagcacatt gatttttcaa aagccagctc tgtcaggaaa ggaggtgctg ttatgagcag 4200ctcttccagt gggcaaagag gacgcccata atttcttcca ttgctagctc atctgtggga 4260ccaatttggt gtaagcaacc tgtggcctgc acttgtggcc tcgaaggaag cacaaaccct 4320ccatccactt cccatttcct ctgccctttt ccacctcccc cttccatccc accagctgcc 4380agtggctccc agaaagcctt attgagcccc ttgttgacac ttggggctgc ggaggcctct 4440ccctactggt ctggcctttc ctgagaggca ggtcttccgt cctcagagcc tttctggaac 4500aaggagaatg cctgtgcagg tggacacaca ggcctggcct gtcgctctca cttgtcttcc 4560agcggggagc ttcacgttgc cgagtggaag aaccatgacc tccacttgct tccaaggtgc 4620tagggaagtt tcagggtacg ctggttcccc tctccagctg gaggccgagt ttctggggac 4680tgcagatttt tctactctgt gatcgattca atgcccgatg cttctgtttc attcccgacc 4740ctttctacta tgcattttcc ttttatcagg tgtataaagt taaatactgt gtatttatca 4800ctaaaaagta catgaactta agagacaact aagcctttcg tgtttttcca caggtgttta 4860agcttctctg tacagttgaa ataaacagac agcaaaatgg tgccaaaaaa aaaaaaaaaa 4920aaa 492314541PRTArtificial SequenceSynthetic 14Met Ser Ser Cys Val Ser Ser Gln Pro Ser Ser Asn Arg Ala Ala Pro 1 5 10 15 Gln Asp Glu Leu Gly Gly Arg Gly Ser Ser Ser Ser Glu Ser Gln Lys 20 25 30 Pro Cys Glu Ala Leu Arg Gly Leu Ser Ser Leu Ser Ile His Leu Gly 35 40 45 Met Glu Ser Phe Ile Val Val Thr Glu Cys Glu Pro Gly Cys Ala Val 50 55 60 Asp Leu Gly Leu Ala Arg Asp Arg Pro Leu Glu Ala Asp Gly Gln Glu 65 70 75 80 Val Pro Leu Asp Thr Ser Gly Ser Gln Ala Arg Pro His Leu Ser Gly 85 90 95 Arg Lys Leu Ser Leu Gln Glu Arg Ser Gln Gly Gly Leu Ala Ala Gly 100 105 110 Gly Ser Leu Asp Met Asn Gly Arg Cys Ile Cys Pro Ser Leu Pro Tyr 115 120 125 Ser Pro Val Ser Ser Pro Gln Ser Ser Pro Arg Leu Pro Arg Arg Pro 130 135 140 Thr Val Glu Ser His His Val Ser Ile Thr Gly Met Gln Asp Cys Val 145 150 155 160 Gln Leu Asn Gln Tyr Thr Leu Lys Asp Glu Ile Gly Lys Gly Ser Tyr 165 170 175 Gly Val Val Lys Leu Ala Tyr Asn Glu Asn Asp Asn Thr Tyr Tyr Ala 180 185 190 Met Lys Val Leu Ser Lys Lys Lys Leu Ile Arg Gln Ala Gly Phe Pro 195 200 205 Arg Arg Pro Pro Pro Arg Gly Thr Arg Pro Ala Pro Gly Gly Cys Ile 210 215 220 Gln Pro Arg Gly Pro Ile Glu Gln Val Tyr Gln Glu Ile Ala Ile Leu 225 230 235 240 Lys Lys Leu Asp His Pro Asn Val Val Lys Leu Val Glu Val Leu Asp 245 250 255 Asp Pro Asn Glu Asp His Leu Tyr Met Val Phe Glu Leu Val Asn Gln 260 265 270 Gly Pro Val Met Glu Val Pro Thr Leu Lys Pro Leu Ser Glu Asp Gln 275 280 285 Ala Arg Phe Tyr Phe Gln Asp Leu Ile Lys Gly Ile Glu Tyr Leu His 290 295 300 Tyr Gln Lys Ile Ile His Arg Asp Ile Lys Pro Ser Asn Leu Leu Val 305 310 315 320 Gly Glu Asp Gly His Ile Lys Ile Ala Asp Phe Gly Val Ser Asn Glu 325 330 335 Phe Lys Gly Ser Asp Ala Leu Leu Ser Asn Thr Val Gly Thr Pro Ala 340 345 350 Phe Met Ala Pro Glu Ser Leu Ser Glu Thr Arg Lys Ile Phe Ser Gly 355 360 365 Lys Ala Leu Asp Val Trp Ala Met Gly Val Thr Leu Tyr Cys Phe Val 370 375 380 Phe Gly Gln Cys Pro Phe Met Asp Glu Arg Ile Met Cys Leu His Ser 385 390 395 400 Lys Ile Lys Ser Gln Ala Leu Glu Phe Pro Asp Gln Pro Asp Ile Ala 405 410 415 Glu Asp Leu Lys Asp Leu Ile Thr Arg Met Leu Asp Lys Asn Pro Glu 420 425 430 Ser Arg Ile Val Val Pro Glu Ile Lys Leu His Pro Trp Val Thr Arg 435 440 445 His Gly Ala Glu Pro Leu Pro Ser Glu Asp Glu Asn Cys Thr Leu Val 450 455 460 Glu Val Thr Glu Glu Glu Val Glu Asn Ser Val Lys His Ile Pro Ser 465 470 475 480 Leu Ala Thr Val Ile Leu Val Lys Thr Met Ile Arg Lys Arg Ser Phe 485 490 495 Gly Asn Pro Phe Glu Gly Ser Arg Arg Glu Glu Arg Ser Leu Ser Ala 500 505 510 Pro Gly Asn Leu Leu Thr Lys Gln Gly Ser Glu Asp Asn Leu Gln Gly 515 520 525 Thr Asp Pro Pro Pro Val Gly Glu Glu Glu Val Leu Leu 530 535 540 153583DNAArtificial SequenceSynthetic 15ctgggcccca gcgaggcggt ggggcggggc ggggcggggc ggggcgcgca gcaggagcga 60gtggggccgc ccgccgggcc gcggacactg tcgcccggcg cccaggttcc caacaaggct 120acgcagaaga acccccttga ctgaagcaat ggaggggggt ccagctgtct gctgccagga 180tcctcgggca gagctggtag aacgggtggc agccatcgat gtgactcact tggaggaggc 240agatggtggc ccagagccta ctagaaacgg tgtggacccc ccaccacggg ccagagctgc 300ctctgtgatc cctggcagta cttcaagact gctcccagcc cggcctagcc tctcagccag 360gaagctttcc ctacaggagc ggccagcagg aagctatctg gaggcgcagg ctgggcctta 420tgccacgggg cctgccagcc acatctcccc ccgggcctgg cggaggccca ccatcgagtc 480ccaccacgtg gccatctcag atgcagagga ctgcgtgcag ctgaaccagt acaagctgca 540gagtgagatt ggcaagggtg cctacggtgt ggtgaggctg gcctacaacg aaagtgaaga 600cagacactat gcaatgaaag tcctttccaa aaagaagtta ctgaagcagt atggctttcc 660acgtcgccct cccccgagag ggtcccaggc tgcccaggga ggaccagcca agcagctgct 720gcccctggag cgggtgtacc aggagattgc catcctgaag aagctggacc acgtgaatgt 780ggtcaaactg atcgaggtcc tggatgaccc agctgaggac aacctctatt tggtgtttga 840cctcctgaga aaggggcccg tcatggaagt gccctgtgac aagcccttct cggaggagca 900agctcgcctc tacctgcggg acgtcatcct gggcctcgag tacttgcact gccagaagat 960cgtccacagg gacatcaagc catccaacct gctcctgggg gatgatgggc acgtgaagat 1020cgccgacttt ggcgtcagca accagtttga ggggaacgac gctcagctgt ccagcacggc 1080gggaacccca gcattcatgg cccccgaggc catttctgat tccggccaga gcttcagtgg 1140gaaggccttg gatgtatggg ccactggcgt cacgttgtac tgctttgtct atgggaagtg 1200cccattcatc gacgatttca tcctggccct ccacaggaag atcaagaatg agcccgtggt 1260gtttcctgag gagccagaaa tcagcgagga gctcaaggac ctgatcctga agatgttaga 1320caagaatccc gagacgagaa ttggggtgcc agacatcaag ttgcaccctt gggtgaccaa 1380gaacggggag gagccccttc cttcggagga ggagcactgc agcgtggtgg aggtgacaga 1440ggaggaggtt aagaactcag tcaggctcat ccccagctgg accacggtga tcctggtgaa 1500gtccatgctg aggaagcgtt cctttgggaa cccgtttgag ccccaagcac ggagggaaga 1560gcgatccatg tctgctccag gaaacctact ggtgaaagaa gggtttggtg aagggggcaa 1620gagcccagag ctccccggcg tccaggaaga cgaggctgca tcctgagccc ctgcatgcac 1680ccagggccac ccggcagcac actcatcccg cgcctccaga ggcccacccc tcatgcaaca 1740gccgcccccg caggcagggg gctggggact gcagccccac tcccgcccct cccccatcgt 1800gctgcatgac ctccacgcac gcacgtccag ggacagactg gaatgtatgt catttggggt 1860cttgggggca gggctcccac gaggccatcc tcctcttctt ggacctcctt ggcctgaccc 1920attctgtggg gaaaccgggt gcccatggag cctcagaaat gccacccggc tggttggcat 1980ggcctggggc aggaggcaga ggcaggagac caagatggca ggtggaggcc aggcttacca 2040caacggaaga gacctcccgc tggggccggg caggcctggc tcagctgcca caggcatatg 2100gtggagaggg gggtaccctg cccaccttgg ggtggtggca ccagagctct tgtctattca 2160gacgctggta tgggggctcg gacccctcac tggggacagg gccagtgttg gagaattctg

2220attccttttt tgttgtcttt tacttttgtt tttaacctgg gggttcgggg agaggccctg 2280cttgggaaca tctcacgagc tttcctacat cttccgtggt tcccagcaca gcccaagatt 2340atttggcagc caagtggatg gaactaactt tcctggactg tgtttcgcat tcggcgttat 2400ctggaaagtg gactgaacgg aatcaagctc tgagcagagg cctgaagcgg aagcaccaca 2460tcgtccctgc ccatctcact ctctcccttg atgatgcccc tagagctgag gctggagaag 2520acaccagggc tgactttgac cgagggccat ggacgcgaca ggcctgtggc cctgcgcatg 2580ctgaaataac tggaacccag cctctcctcc tacaccggcc tacccatctg ggcccaagag 2640ctgcactcac actcctacaa cgaaggacaa actgtccagg tcggagggat cacgagacac 2700agaacctgga ggggtgtgca cgctggcagg tggcctctgc ggcaattgcc tcaccctgag 2760gacatcagca gtcagcctgc tcagagcggg ggtgctggag cgcgtgcaga cacagctctt 2820ccggagcagc cttcaccttc tctctgggat cagtgtccgg ctggccgacg tggcatttgc 2880tgaccgaatg ctcatagagg ttgaccccca cagggtcacg caggactcgg acactgccct 2940ggaaacatgg atggacaagg gcttttggcc acaggtgtgg gtgtcctgtt ggaggagggc 3000ttgtttggag aagggaggct ggctggggga gaaacccgga tcccgctgca tctccgcgcc 3060tgtgggtgca tgtcgcgtgc tcatctgttg cacacagctc actcgtatgt cctgcactgg 3120tacatgcatc tgtaatacag tttctacgtc tatttaaggc taggagccga atgtgcccca 3180ttgtcagtgg gtccacgttt ctccccggct cctctgggct aaggcagtgt ggcccgaagc 3240ttaaaaagtt actcggtact gtttttaaga acacttttat agagttagtg gaaggcaagt 3300taagagccaa tcactgatcc ccaagtgttt cttgagcatc tggtctgggg ggaccacttt 3360gatcggaccc acccttggaa agctcagggg taggcccagg tgggatgctc accctgtcac 3420tgagggtttt ggttggcatc gttgtttttg aatgtagcac aagcgatgag caaactctat 3480aagagtgttt taaaaattaa cttcccagga agtgagttaa aaacaataaa agccctttct 3540tgagttaaaa agaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 358316505PRTArtificial SequenceSynthetic 16Met Glu Gly Gly Pro Ala Val Cys Cys Gln Asp Pro Arg Ala Glu Leu 1 5 10 15 Val Glu Arg Val Ala Ala Ile Asp Val Thr His Leu Glu Glu Ala Asp 20 25 30 Gly Gly Pro Glu Pro Thr Arg Asn Gly Val Asp Pro Pro Pro Arg Ala 35 40 45 Arg Ala Ala Ser Val Ile Pro Gly Ser Thr Ser Arg Leu Leu Pro Ala 50 55 60 Arg Pro Ser Leu Ser Ala Arg Lys Leu Ser Leu Gln Glu Arg Pro Ala 65 70 75 80 Gly Ser Tyr Leu Glu Ala Gln Ala Gly Pro Tyr Ala Thr Gly Pro Ala 85 90 95 Ser His Ile Ser Pro Arg Ala Trp Arg Arg Pro Thr Ile Glu Ser His 100 105 110 His Val Ala Ile Ser Asp Ala Glu Asp Cys Val Gln Leu Asn Gln Tyr 115 120 125 Lys Leu Gln Ser Glu Ile Gly Lys Gly Ala Tyr Gly Val Val Arg Leu 130 135 140 Ala Tyr Asn Glu Ser Glu Asp Arg His Tyr Ala Met Lys Val Leu Ser 145 150 155 160 Lys Lys Lys Leu Leu Lys Gln Tyr Gly Phe Pro Arg Arg Pro Pro Pro 165 170 175 Arg Gly Ser Gln Ala Ala Gln Gly Gly Pro Ala Lys Gln Leu Leu Pro 180 185 190 Leu Glu Arg Val Tyr Gln Glu Ile Ala Ile Leu Lys Lys Leu Asp His 195 200 205 Val Asn Val Val Lys Leu Ile Glu Val Leu Asp Asp Pro Ala Glu Asp 210 215 220 Asn Leu Tyr Leu Val Phe Asp Leu Leu Arg Lys Gly Pro Val Met Glu 225 230 235 240 Val Pro Cys Asp Lys Pro Phe Ser Glu Glu Gln Ala Arg Leu Tyr Leu 245 250 255 Arg Asp Val Ile Leu Gly Leu Glu Tyr Leu His Cys Gln Lys Ile Val 260 265 270 His Arg Asp Ile Lys Pro Ser Asn Leu Leu Leu Gly Asp Asp Gly His 275 280 285 Val Lys Ile Ala Asp Phe Gly Val Ser Asn Gln Phe Glu Gly Asn Asp 290 295 300 Ala Gln Leu Ser Ser Thr Ala Gly Thr Pro Ala Phe Met Ala Pro Glu 305 310 315 320 Ala Ile Ser Asp Ser Gly Gln Ser Phe Ser Gly Lys Ala Leu Asp Val 325 330 335 Trp Ala Thr Gly Val Thr Leu Tyr Cys Phe Val Tyr Gly Lys Cys Pro 340 345 350 Phe Ile Asp Asp Phe Ile Leu Ala Leu His Arg Lys Ile Lys Asn Glu 355 360 365 Pro Val Val Phe Pro Glu Glu Pro Glu Ile Ser Glu Glu Leu Lys Asp 370 375 380 Leu Ile Leu Lys Met Leu Asp Lys Asn Pro Glu Thr Arg Ile Gly Val 385 390 395 400 Pro Asp Ile Lys Leu His Pro Trp Val Thr Lys Asn Gly Glu Glu Pro 405 410 415 Leu Pro Ser Glu Glu Glu His Cys Ser Val Val Glu Val Thr Glu Glu 420 425 430 Glu Val Lys Asn Ser Val Arg Leu Ile Pro Ser Trp Thr Thr Val Ile 435 440 445 Leu Val Lys Ser Met Leu Arg Lys Arg Ser Phe Gly Asn Pro Phe Glu 450 455 460 Pro Gln Ala Arg Arg Glu Glu Arg Ser Met Ser Ala Pro Gly Asn Leu 465 470 475 480 Leu Val Lys Glu Gly Phe Gly Glu Gly Gly Lys Ser Pro Glu Leu Pro 485 490 495 Gly Val Gln Glu Asp Glu Ala Ala Ser 500 505 173529DNAArtificial SequenceSynthetic 17agcagaacag agtatgcaat ttgggaagct gtggtgtggc tgcagtggag agttcccaac 60aaggctacgc agaagaaccc ccttgactga agcaatggag gggggtccag ctgtctgctg 120ccaggatcct cgggcagagc tggtagaacg ggtggcagcc atcgatgtga ctcacttgga 180ggaggcagat ggtggcccag agcctactag aaacggtgtg gaccccccac cacgggccag 240agctgcctct gtgatccctg gcagtacttc aagactgctc ccagcccggc ctagcctctc 300agccaggaag ctttccctac aggagcggcc agcaggaagc tatctggagg cgcaggctgg 360gccttatgcc acggggcctg ccagccacat ctccccccgg gcctggcgga ggcccaccat 420cgagtcccac cacgtggcca tctcagatgc agaggactgc gtgcagctga accagtacaa 480gctgcagagt gagattggca agggtgccta cggtgtggtg aggctggcct acaacgaaag 540tgaagacaga cactatgcaa tgaaagtcct ttccaaaaag aagttactga agcagtatgg 600ctttccacgt cgccctcccc cgagagggtc ccaggctgcc cagggaggac cagccaagca 660gctgctgccc ctggagcggg tgtaccagga gattgccatc ctgaagaagc tggaccacgt 720gaatgtggtc aaactgatcg aggtcctgga tgacccagct gaggacaacc tctatttggt 780gtttgacctc ctgagaaagg ggcccgtcat ggaagtgccc tgtgacaagc ccttctcgga 840ggagcaagct cgcctctacc tgcgggacgt catcctgggc ctcgagtact tgcactgcca 900gaagatcgtc cacagggaca tcaagccatc caacctgctc ctgggggatg atgggcacgt 960gaagatcgcc gactttggcg tcagcaacca gtttgagggg aacgacgctc agctgtccag 1020cacggcggga accccagcat tcatggcccc cgaggccatt tctgattccg gccagagctt 1080cagtgggaag gccttggatg tatgggccac tggcgtcacg ttgtactgct ttgtctatgg 1140gaagtgccca ttcatcgacg atttcatcct ggccctccac aggaagatca agaatgagcc 1200cgtggtgttt cctgaggagc cagaaatcag cgaggagctc aaggacctga tcctgaagat 1260gttagacaag aatcccgaga cgagaattgg ggtgccagac atcaagttgc acccttgggt 1320gaccaagaac ggggaggagc cccttccttc ggaggaggag cactgcagcg tggtggaggt 1380gacagaggag gaggttaaga actcagtcag gctcatcccc agctggacca cggtgatcct 1440ggtgaagtcc atgctgagga agcgttcctt tgggaacccg tttgagcccc aagcacggag 1500ggaagagcga tccatgtctg ctccaggaaa cctactggtg aaagaagggt ttggtgaagg 1560gggcaagagc ccagagctcc ccggcgtcca ggaagacgag gctgcatcct gagcccctgc 1620atgcacccag ggccacccgg cagcacactc atcccgcgcc tccagaggcc cacccctcat 1680gcaacagccg cccccgcagg cagggggctg gggactgcag ccccactccc gcccctcccc 1740catcgtgctg catgacctcc acgcacgcac gtccagggac agactggaat gtatgtcatt 1800tggggtcttg ggggcagggc tcccacgagg ccatcctcct cttcttggac ctccttggcc 1860tgacccattc tgtggggaaa ccgggtgccc atggagcctc agaaatgcca cccggctggt 1920tggcatggcc tggggcagga ggcagaggca ggagaccaag atggcaggtg gaggccaggc 1980ttaccacaac ggaagagacc tcccgctggg gccgggcagg cctggctcag ctgccacagg 2040catatggtgg agaggggggt accctgccca ccttggggtg gtggcaccag agctcttgtc 2100tattcagacg ctggtatggg ggctcggacc cctcactggg gacagggcca gtgttggaga 2160attctgattc cttttttgtt gtcttttact tttgttttta acctgggggt tcggggagag 2220gccctgcttg ggaacatctc acgagctttc ctacatcttc cgtggttccc agcacagccc 2280aagattattt ggcagccaag tggatggaac taactttcct ggactgtgtt tcgcattcgg 2340cgttatctgg aaagtggact gaacggaatc aagctctgag cagaggcctg aagcggaagc 2400accacatcgt ccctgcccat ctcactctct cccttgatga tgcccctaga gctgaggctg 2460gagaagacac cagggctgac tttgaccgag ggccatggac gcgacaggcc tgtggccctg 2520cgcatgctga aataactgga acccagcctc tcctcctaca ccggcctacc catctgggcc 2580caagagctgc actcacactc ctacaacgaa ggacaaactg tccaggtcgg agggatcacg 2640agacacagaa cctggagggg tgtgcacgct ggcaggtggc ctctgcggca attgcctcac 2700cctgaggaca tcagcagtca gcctgctcag agcgggggtg ctggagcgcg tgcagacaca 2760gctcttccgg agcagccttc accttctctc tgggatcagt gtccggctgg ccgacgtggc 2820atttgctgac cgaatgctca tagaggttga cccccacagg gtcacgcagg actcggacac 2880tgccctggaa acatggatgg acaagggctt ttggccacag gtgtgggtgt cctgttggag 2940gagggcttgt ttggagaagg gaggctggct gggggagaaa cccggatccc gctgcatctc 3000cgcgcctgtg ggtgcatgtc gcgtgctcat ctgttgcaca cagctcactc gtatgtcctg 3060cactggtaca tgcatctgta atacagtttc tacgtctatt taaggctagg agccgaatgt 3120gccccattgt cagtgggtcc acgtttctcc ccggctcctc tgggctaagg cagtgtggcc 3180cgaagcttaa aaagttactc ggtactgttt ttaagaacac ttttatagag ttagtggaag 3240gcaagttaag agccaatcac tgatccccaa gtgtttcttg agcatctggt ctggggggac 3300cactttgatc ggacccaccc ttggaaagct caggggtagg cccaggtggg atgctcaccc 3360tgtcactgag ggttttggtt ggcatcgttg tttttgaatg tagcacaagc gatgagcaaa 3420ctctataaga gtgttttaaa aattaacttc ccaggaagtg agttaaaaac aataaaagcc 3480ctttcttgag ttaaaaagaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 352918505PRTArtificial SequenceSynthetic 18Met Glu Gly Gly Pro Ala Val Cys Cys Gln Asp Pro Arg Ala Glu Leu 1 5 10 15 Val Glu Arg Val Ala Ala Ile Asp Val Thr His Leu Glu Glu Ala Asp 20 25 30 Gly Gly Pro Glu Pro Thr Arg Asn Gly Val Asp Pro Pro Pro Arg Ala 35 40 45 Arg Ala Ala Ser Val Ile Pro Gly Ser Thr Ser Arg Leu Leu Pro Ala 50 55 60 Arg Pro Ser Leu Ser Ala Arg Lys Leu Ser Leu Gln Glu Arg Pro Ala 65 70 75 80 Gly Ser Tyr Leu Glu Ala Gln Ala Gly Pro Tyr Ala Thr Gly Pro Ala 85 90 95 Ser His Ile Ser Pro Arg Ala Trp Arg Arg Pro Thr Ile Glu Ser His 100 105 110 His Val Ala Ile Ser Asp Ala Glu Asp Cys Val Gln Leu Asn Gln Tyr 115 120 125 Lys Leu Gln Ser Glu Ile Gly Lys Gly Ala Tyr Gly Val Val Arg Leu 130 135 140 Ala Tyr Asn Glu Ser Glu Asp Arg His Tyr Ala Met Lys Val Leu Ser 145 150 155 160 Lys Lys Lys Leu Leu Lys Gln Tyr Gly Phe Pro Arg Arg Pro Pro Pro 165 170 175 Arg Gly Ser Gln Ala Ala Gln Gly Gly Pro Ala Lys Gln Leu Leu Pro 180 185 190 Leu Glu Arg Val Tyr Gln Glu Ile Ala Ile Leu Lys Lys Leu Asp His 195 200 205 Val Asn Val Val Lys Leu Ile Glu Val Leu Asp Asp Pro Ala Glu Asp 210 215 220 Asn Leu Tyr Leu Val Phe Asp Leu Leu Arg Lys Gly Pro Val Met Glu 225 230 235 240 Val Pro Cys Asp Lys Pro Phe Ser Glu Glu Gln Ala Arg Leu Tyr Leu 245 250 255 Arg Asp Val Ile Leu Gly Leu Glu Tyr Leu His Cys Gln Lys Ile Val 260 265 270 His Arg Asp Ile Lys Pro Ser Asn Leu Leu Leu Gly Asp Asp Gly His 275 280 285 Val Lys Ile Ala Asp Phe Gly Val Ser Asn Gln Phe Glu Gly Asn Asp 290 295 300 Ala Gln Leu Ser Ser Thr Ala Gly Thr Pro Ala Phe Met Ala Pro Glu 305 310 315 320 Ala Ile Ser Asp Ser Gly Gln Ser Phe Ser Gly Lys Ala Leu Asp Val 325 330 335 Trp Ala Thr Gly Val Thr Leu Tyr Cys Phe Val Tyr Gly Lys Cys Pro 340 345 350 Phe Ile Asp Asp Phe Ile Leu Ala Leu His Arg Lys Ile Lys Asn Glu 355 360 365 Pro Val Val Phe Pro Glu Glu Pro Glu Ile Ser Glu Glu Leu Lys Asp 370 375 380 Leu Ile Leu Lys Met Leu Asp Lys Asn Pro Glu Thr Arg Ile Gly Val 385 390 395 400 Pro Asp Ile Lys Leu His Pro Trp Val Thr Lys Asn Gly Glu Glu Pro 405 410 415 Leu Pro Ser Glu Glu Glu His Cys Ser Val Val Glu Val Thr Glu Glu 420 425 430 Glu Val Lys Asn Ser Val Arg Leu Ile Pro Ser Trp Thr Thr Val Ile 435 440 445 Leu Val Lys Ser Met Leu Arg Lys Arg Ser Phe Gly Asn Pro Phe Glu 450 455 460 Pro Gln Ala Arg Arg Glu Glu Arg Ser Met Ser Ala Pro Gly Asn Leu 465 470 475 480 Leu Val Lys Glu Gly Phe Gly Glu Gly Gly Lys Ser Pro Glu Leu Pro 485 490 495 Gly Val Gln Glu Asp Glu Ala Ala Ser 500 505 192535DNAArtificial SequenceSynthetic 19ctgggcccca gcgaggcggt ggggcggggc ggggcggggc ggggcgcgca gcaggagcga 60gtggggccgc ccgccgggcc gcggacactg tcgcccggcg cccaggttcc caacaaggct 120acgcagaaga acccccttga ctgaagcaat ggaggggggt ccagctgtct gctgccagga 180tcctcgggca gagctggtag aacgggtggc agccatcgat gtgactcact tggaggaggc 240agatggtggc ccagagccta ctagaaacgg tgtggacccc ccaccacggg ccagagctgc 300ctctgtgatc cctggcagta cttcaagact gctcccagcc cggcctagcc tctcagccag 360gaagctttcc ctacaggagc ggccagcagg aagctatctg gaggcgcagg ctgggcctta 420tgccacgggg cctgccagcc acatctcccc ccgggcctgg cggaggccca ccatcgagtc 480ccaccacgtg gccatctcag atgcagagga ctgcgtgcag ctgaaccagt acaagctgca 540gagtgagatt ggcaagggtg cctacggtgt ggtgaggctg gcctacaacg aaagtgaaga 600cagacactat gcaatgaaag tcctttccaa aaagaagtta ctgaagcagt atggctttcc 660acgtcgccct cccccgagag ggtcccaggc tgcccaggga ggaccagcca agcagctgct 720gcccctggag cgggtgtacc aggagattgc catcctgaag aagctggacc acgtgaatgt 780ggtcaaactg atcgaggtcc tggatgaccc agctgaggac aacctctatt tggccctgca 840gaaccaggcc cagaatatcc agttagattc aacaaatatc gccaagcccc actccctgct 900tccctctgag cagcaagaca gtggatccac gtgggctgcg cgctcagtgt ttgacctcct 960gagaaagggg cccgtcatgg aagtgccctg tgacaagccc ttctcggagg agcaagctcg 1020cctctacctg cgggacgtca tcctgggcct cgagtacttg cactgccaga agatcgtcca 1080cagggacatc aagccatcca acctgctcct gggggatgat gggcacgtga agatcgccga 1140ctttggcgtc agcaaccagt ttgaggggaa cgacgctcag ctgtccagca cggcgggaac 1200cccagcattc atggcccccg aggccatttc tgattccggc cagagcttca gtgggaaggc 1260cttggatgta tgggccactg gcgtcacgtt gtactgcttt gtctatggga agtgcccatt 1320catcgacgat ttcatcctgg ccctccacag gaagatcaag aatgagcccg tggtgtttcc 1380tgaggagcca gaaatcagcg aggagctcaa ggacctgatc ctgaagatgt tagacaagaa 1440tcccgagacg agaattgggg tgccagacat caagttgcac ccttgggtga ccaagaacgg 1500ggaggagccc cttccttcgg aggaggagca ctgcagcgtg gtggaggtga cagaggagga 1560ggttaagaac tcagtcaggc tcatccccag ctggaccacg gtgatcctgg tgaagtccat 1620gctgaggaag cgttcctttg ggaacccgtt tgagccccaa gcacggaggg aagagcgatc 1680catgtctgct ccaggaaacc tactggtgta agtactggtg ggccagggac tgccgggcac 1740tccctggagt tgggtgggga ggtctgaggc ccatcctccc actctcactg tcgttgggcc 1800aaggccagag cctggggact tggccaggtc tcggtgttgg ccccatttgc atctctgtcc 1860ccaaggttag tcggggctag aagggacctt ttgggcccag ctcttgcttc attcctgggg 1920ccagcatccc tcacacacac acttccaggg atgaggagct cacgcagccc ctccatggga 1980caggaagacc cttcttccat gcagcttgat gtcactctct cactgggtcc agcccctctg 2040gggcttcaaa tctgtggccc cctcagccct tggcagcctg gcagaggttt gcagacaggc 2100tgatgttggc ttcctgtagg aggctggcgg gctgtagagg aggggtgctg gcccctctgc 2160ctggccctgg ggactgttgg ctgctctccc aagtggccca ggctgcctgc agccattgct 2220ggggctctgt gcccagtcag cactttgtga gtgcttgttc agtgagtaag cagggacagg 2280ctggccggtg gaccacggga gaggaacccg cattggccga gggctcccta tggtgagcca 2340cgcctgtggg ttcaccacct cctaggaggg tccagaaaag cagctcccca agcctgtgcg 2400cctcgtcctc agcagatcca ccttcttcac tataataaaa gccagtctgg gatgctaaaa 2460aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520aaaaaaaaaa aaaaa 253520520PRTArtificial SequenceSynthetic 20Met Glu Gly Gly Pro Ala Val Cys Cys Gln Asp Pro Arg Ala Glu Leu 1 5 10 15 Val Glu Arg Val Ala Ala Ile Asp Val Thr His Leu Glu Glu Ala Asp 20 25 30 Gly Gly Pro Glu Pro Thr Arg Asn Gly Val Asp Pro Pro Pro Arg Ala 35 40 45 Arg Ala Ala Ser Val Ile Pro Gly Ser Thr Ser Arg Leu Leu Pro Ala 50 55 60 Arg Pro Ser Leu Ser Ala Arg Lys Leu Ser Leu Gln Glu Arg Pro Ala 65 70 75 80 Gly Ser Tyr Leu Glu Ala Gln Ala Gly Pro Tyr Ala Thr Gly Pro Ala 85 90 95 Ser His Ile Ser Pro Arg Ala Trp Arg Arg Pro Thr Ile Glu Ser His 100 105 110 His Val Ala Ile Ser Asp Ala Glu Asp Cys Val Gln Leu Asn Gln

Tyr 115 120 125 Lys Leu Gln Ser Glu Ile Gly Lys Gly Ala Tyr Gly Val Val Arg Leu 130 135 140 Ala Tyr Asn Glu Ser Glu Asp Arg His Tyr Ala Met Lys Val Leu Ser 145 150 155 160 Lys Lys Lys Leu Leu Lys Gln Tyr Gly Phe Pro Arg Arg Pro Pro Pro 165 170 175 Arg Gly Ser Gln Ala Ala Gln Gly Gly Pro Ala Lys Gln Leu Leu Pro 180 185 190 Leu Glu Arg Val Tyr Gln Glu Ile Ala Ile Leu Lys Lys Leu Asp His 195 200 205 Val Asn Val Val Lys Leu Ile Glu Val Leu Asp Asp Pro Ala Glu Asp 210 215 220 Asn Leu Tyr Leu Ala Leu Gln Asn Gln Ala Gln Asn Ile Gln Leu Asp 225 230 235 240 Ser Thr Asn Ile Ala Lys Pro His Ser Leu Leu Pro Ser Glu Gln Gln 245 250 255 Asp Ser Gly Ser Thr Trp Ala Ala Arg Ser Val Phe Asp Leu Leu Arg 260 265 270 Lys Gly Pro Val Met Glu Val Pro Cys Asp Lys Pro Phe Ser Glu Glu 275 280 285 Gln Ala Arg Leu Tyr Leu Arg Asp Val Ile Leu Gly Leu Glu Tyr Leu 290 295 300 His Cys Gln Lys Ile Val His Arg Asp Ile Lys Pro Ser Asn Leu Leu 305 310 315 320 Leu Gly Asp Asp Gly His Val Lys Ile Ala Asp Phe Gly Val Ser Asn 325 330 335 Gln Phe Glu Gly Asn Asp Ala Gln Leu Ser Ser Thr Ala Gly Thr Pro 340 345 350 Ala Phe Met Ala Pro Glu Ala Ile Ser Asp Ser Gly Gln Ser Phe Ser 355 360 365 Gly Lys Ala Leu Asp Val Trp Ala Thr Gly Val Thr Leu Tyr Cys Phe 370 375 380 Val Tyr Gly Lys Cys Pro Phe Ile Asp Asp Phe Ile Leu Ala Leu His 385 390 395 400 Arg Lys Ile Lys Asn Glu Pro Val Val Phe Pro Glu Glu Pro Glu Ile 405 410 415 Ser Glu Glu Leu Lys Asp Leu Ile Leu Lys Met Leu Asp Lys Asn Pro 420 425 430 Glu Thr Arg Ile Gly Val Pro Asp Ile Lys Leu His Pro Trp Val Thr 435 440 445 Lys Asn Gly Glu Glu Pro Leu Pro Ser Glu Glu Glu His Cys Ser Val 450 455 460 Val Glu Val Thr Glu Glu Glu Val Lys Asn Ser Val Arg Leu Ile Pro 465 470 475 480 Ser Trp Thr Thr Val Ile Leu Val Lys Ser Met Leu Arg Lys Arg Ser 485 490 495 Phe Gly Asn Pro Phe Glu Pro Gln Ala Arg Arg Glu Glu Arg Ser Met 500 505 510 Ser Ala Pro Gly Asn Leu Leu Val 515 520 215085DNAArtificial SequenceAMPK alpha 1 variant 1 21agcgccatgc gcagactcag ttcctggaga aagatggcga cagccgagaa gcagaaacac 60gacgggcggg tgaagatcgg ccactacatt ctgggtgaca cgctgggggt cggcaccttc 120ggcaaagtga aggttggcaa acatgaattg actgggcata aagtagctgt gaagatactc 180aatcgacaga agattcggag ccttgatgtg gtaggaaaaa tccgcagaga aattcagaac 240ctcaagcttt tcaggcatcc tcatataatt aaactgtacc aggtcatcag tacaccatct 300gatattttca tggtgatgga atatgtctca ggaggagagc tatttgatta tatctgtaag 360aatggaaggc tggatgaaaa agaaagtcgg cgtctgttcc aacagatcct ttctggtgtg 420gattattgtc acaggcatat ggtggtccat agagatttga aacctgaaaa tgtcctgctt 480gatgcacaca tgaatgcaaa gatagctgat tttggtcttt caaacatgat gtcagatggt 540gaatttttaa gaacaagttg tggctcaccc aactatgctg caccagaagt aatttcagga 600agattgtatg caggcccaga ggtagatata tggagcagtg gggttattct ctatgcttta 660ttatgtggaa cccttccatt tgatgatgac catgtgccaa ctctttttaa gaagatatgt 720gatgggatct tctatacccc tcaatattta aatccttctg tgattagcct tttgaaacat 780atgctgcagg tggatcccat gaagagggcc acaatcaaag atatcaggga acatgaatgg 840tttaaacagg accttccaaa atatctcttt cctgaggatc catcatatag ttcaaccatg 900attgatgatg aagccttaaa agaagtatgt gaaaagtttg agtgctcaga agaggaagtt 960ctcagctgtc tttacaacag aaatcaccag gatcctttgg cagttgccta ccatctcata 1020atagataaca ggagaataat gaatgaagcc aaagatttct atttggcgac aagcccacct 1080gattcttttc ttgatgatca tcacctgact cggccccatc ctgaaagagt accattcttg 1140gttgctgaaa caccaagggc acgccatacc cttgatgaat taaatccaca gaaatccaaa 1200caccaaggtg taaggaaagc aaaatggcat ttaggaatta gaagtcaaag tcgaccaaat 1260gatattatgg cagaagtatg tagagcaatc aaacaattgg attatgaatg gaaggttgta 1320aacccatatt atttgcgtgt acgaaggaag aatcctgtga caagcactta ctccaaaatg 1380agtctacagt tataccaagt ggatagtaga acttatctac tggatttccg tagtattgat 1440gatgaaatta cagaagccaa atcagggact gctactccac agagatcggg atcagttagc 1500aactatcgat cttgccaaag gagtgattca gatgctgagg ctcaaggaaa atcctcagaa 1560gtttctctta cctcatctgt gacctcactt gactcttctc ctgttgacct aactccaaga 1620cctggaagtc acacaataga attttttgag atgtgtgcaa atctaattaa aattcttgca 1680caataaacag aaaactttgc ttatttcttt tgcagcaata agcatgcata ataagtcaca 1740gccaaatgct tccatttgta atcaagttat acataattat aaccgagggc tggcgttttg 1800gaatgcaatt tgcacaggga ttggaacatg atttatagtt aaaagcctaa tatgcagaaa 1860tgaattaaga tcattttgtt gttcattgtg cagtatgtat atagcataat atacacagtg 1920aattataggt ctcaggctta cttgattttt ggctatttta tatttagtgt acacagggct 1980ttgaaatatt aatttacata aaggccttca tatattatta cgtgttatat attacgtgtt 2040ataaatttat tcaataaata tttgcctaga attcccaaga cctttatagg tgattttgtt 2100ttctgggctc cttaacttca taaatagcta gtatcttcca gcagtagtaa cagtctggat 2160aacttcttcc atatccctcc ctctttgttt ttttgagaca gtgtcacttt gtcacccagg 2220ctggagtgca atggtgtggt ctcggctcac tgcaacctcc acctcccggg ttcaagtgat 2280tctcccgcct cagcttcctg agtagctgga actacaggcg tgtgccacca cacccggcta 2340atttttcgta tttttagtgt agacggggtt tcactatgtt gcccaggctg gtctcgaact 2400cctgaccgcg tgatccacca cctcagcttc ccaaagtggt gggattacag gcgtgagcca 2460ccgcacccgg cctccatatc ccccttttaa aattctgtag tgtatggtaa gtcatatcag 2520atatcagacc taatttaaat ttcattttag ctttacaagt ccaaaaacac agaatttata 2580tattcagata ctctagcact aattttagtc ttaaaatatt cccacgatat tctgtacaca 2640aaatgttctt tttgttacaa gagctgagtt gcatatactg tagataaatc atattatttt 2700tgccaatttc acaaattcct ctggcccatc atgtcagtca ttattgagta tatgcacaca 2760ttgctactta tttgattatg tatcttttaa attgattcag tgcatagaaa actatctctt 2820acaaacttta agtgctctga tatgacttcc cccccaaatt ttattatgaa catttttaaa 2880aacagaaaaa ttgaaaaact gtttggtaag cacatgtata tctaccattt agattcagca 2940gttgttaatg ttttgtcatt tgttttctct atacctatat atgtatagat acagctagtt 3000atgcatatat atgcatatat gtgtttgttt gtgtatgtat atatgctttt ttccccctga 3060accatttgga tgttacagac atacttatca ccgtgaaaat acttcaagta tctcctacag 3120ataatgacat tctcctaaaa atccgtaata ccattgtaaa agtaataatt ccccaatatc 3180atctaatcaa gccatattta aatttctgaa gttaactcca aatttcttta tagctgatta 3240tttcaaacta ggatccaatt aaagtttaca tatgacactt ggttataact ctttagttgg 3300atataacatt attattattt tgataaaata tggaacaaat caattctatt aataagtggt 3360cacatttgtt ttgggcttaa attacttttt aaagatactg gattttccta agatttctga 3420tttacactga tatttttttt tgtcattctt aattgcatca cacaatagat gtaaatgaag 3480atgtagtcac ctcagataaa attggtatcg tgtatgataa tattgtatca tttatatttg 3540ccttatgtta actttaagaa attgattttt ttgtattaat cattttccca ttgcaacaga 3600gctatatttt ttctatttta agaatcatat tttaggatta tttttggcaa atacagtgag 3660cacttatgta accagatgat aatgaactca aatgtcatga tagcttgcat aaatggtgac 3720tctagtagat ttgactcaag cacttctaga atcatgcact gaattcaaaa gaaaaatctt 3780gctgcttttt gtccagggct tgttctattc aacttctaat ttgaaagctg tacaaagtaa 3840tagaagttcc atttaaatat gagttcaaaa ctgtatttac tttttatgtg gccctctctt 3900taggggattc taattttact tagggtctct aagtgcagca taatgttcct gatgttaaca 3960gaagactgta tttttaaagt tacaaatttg tatatggaat taagtaatgg cgctatatac 4020gctgttgtgg ggagggggga agaaaaggag gaaccaatta aataggacct tttaaaaatt 4080gttaattttg taaactttgc ttctcttata agttattgtg attcatttta gttactgtgt 4140tttattttga aaatatttaa atattgcact tctataaata gtatgataaa tgcacagaca 4200attgcagtaa attctttttt aagctaggat atttgaaatg acaacctttg gttaagtgtg 4260tcaaggttgc aacagaattt tcacaatttt tttgttgttt gcaaattgtt actaatattg 4320aagaggtaag ggaggcaatg caaatgattt ttaatctttt tttattatct tttcagcagt 4380ttatattttt tgtgacttta tgcaaccata tttttacttt gtcttgacaa ctgaaagatg 4440tataaggttt tttgccagaa atgtactgta tacatagttt taagtataac agattttact 4500gatatgtaaa aattttgcca ttaaaataaa tgatttctca ctgagaggaa cttttctacc 4560aggttggggc atatgggagc ttaatatatc atatctaatt taaaataatt tcactgaaat 4620aaactccatt gcttttacct aatttttttc ttgagatgct tttgtagttt ttcagagttt 4680tagatgattt tatacaaaat cctctgccta gcactgctct ttttgatgtt gtagtgacac 4740catttacatt gaattaatgc ttggtagcct ggggctagat gtggaactcc atggatctgt 4800gttctgactg gcacctttgg aatgaaagaa aagtgtgtgc tgtccaaatt ttttcccctt 4860aattctttcc ctcatcttct cacccataat agaaatttta tttccattgt gagttctgac 4920aagaatgaaa ttccacatac aacataactg taaattgttg gtaggtagaa gttaatattt 4980gtggttcatg tatattttga ccagagtata tttaagtata taatttcagc ttccttgatt 5040tagaaatatg atataataaa gaaaaactcc atttatcatc tgtta 508522559PRTArtificial SequenceAMPK alpha 1 isoform 1 22Met Arg Arg Leu Ser Ser Trp Arg Lys Met Ala Thr Ala Glu Lys Gln 1 5 10 15 Lys His Asp Gly Arg Val Lys Ile Gly His Tyr Ile Leu Gly Asp Thr 20 25 30 Leu Gly Val Gly Thr Phe Gly Lys Val Lys Val Gly Lys His Glu Leu 35 40 45 Thr Gly His Lys Val Ala Val Lys Ile Leu Asn Arg Gln Lys Ile Arg 50 55 60 Ser Leu Asp Val Val Gly Lys Ile Arg Arg Glu Ile Gln Asn Leu Lys 65 70 75 80 Leu Phe Arg His Pro His Ile Ile Lys Leu Tyr Gln Val Ile Ser Thr 85 90 95 Pro Ser Asp Ile Phe Met Val Met Glu Tyr Val Ser Gly Gly Glu Leu 100 105 110 Phe Asp Tyr Ile Cys Lys Asn Gly Arg Leu Asp Glu Lys Glu Ser Arg 115 120 125 Arg Leu Phe Gln Gln Ile Leu Ser Gly Val Asp Tyr Cys His Arg His 130 135 140 Met Val Val His Arg Asp Leu Lys Pro Glu Asn Val Leu Leu Asp Ala 145 150 155 160 His Met Asn Ala Lys Ile Ala Asp Phe Gly Leu Ser Asn Met Met Ser 165 170 175 Asp Gly Glu Phe Leu Arg Thr Ser Cys Gly Ser Pro Asn Tyr Ala Ala 180 185 190 Pro Glu Val Ile Ser Gly Arg Leu Tyr Ala Gly Pro Glu Val Asp Ile 195 200 205 Trp Ser Ser Gly Val Ile Leu Tyr Ala Leu Leu Cys Gly Thr Leu Pro 210 215 220 Phe Asp Asp Asp His Val Pro Thr Leu Phe Lys Lys Ile Cys Asp Gly 225 230 235 240 Ile Phe Tyr Thr Pro Gln Tyr Leu Asn Pro Ser Val Ile Ser Leu Leu 245 250 255 Lys His Met Leu Gln Val Asp Pro Met Lys Arg Ala Thr Ile Lys Asp 260 265 270 Ile Arg Glu His Glu Trp Phe Lys Gln Asp Leu Pro Lys Tyr Leu Phe 275 280 285 Pro Glu Asp Pro Ser Tyr Ser Ser Thr Met Ile Asp Asp Glu Ala Leu 290 295 300 Lys Glu Val Cys Glu Lys Phe Glu Cys Ser Glu Glu Glu Val Leu Ser 305 310 315 320 Cys Leu Tyr Asn Arg Asn His Gln Asp Pro Leu Ala Val Ala Tyr His 325 330 335 Leu Ile Ile Asp Asn Arg Arg Ile Met Asn Glu Ala Lys Asp Phe Tyr 340 345 350 Leu Ala Thr Ser Pro Pro Asp Ser Phe Leu Asp Asp His His Leu Thr 355 360 365 Arg Pro His Pro Glu Arg Val Pro Phe Leu Val Ala Glu Thr Pro Arg 370 375 380 Ala Arg His Thr Leu Asp Glu Leu Asn Pro Gln Lys Ser Lys His Gln 385 390 395 400 Gly Val Arg Lys Ala Lys Trp His Leu Gly Ile Arg Ser Gln Ser Arg 405 410 415 Pro Asn Asp Ile Met Ala Glu Val Cys Arg Ala Ile Lys Gln Leu Asp 420 425 430 Tyr Glu Trp Lys Val Val Asn Pro Tyr Tyr Leu Arg Val Arg Arg Lys 435 440 445 Asn Pro Val Thr Ser Thr Tyr Ser Lys Met Ser Leu Gln Leu Tyr Gln 450 455 460 Val Asp Ser Arg Thr Tyr Leu Leu Asp Phe Arg Ser Ile Asp Asp Glu 465 470 475 480 Ile Thr Glu Ala Lys Ser Gly Thr Ala Thr Pro Gln Arg Ser Gly Ser 485 490 495 Val Ser Asn Tyr Arg Ser Cys Gln Arg Ser Asp Ser Asp Ala Glu Ala 500 505 510 Gln Gly Lys Ser Ser Glu Val Ser Leu Thr Ser Ser Val Thr Ser Leu 515 520 525 Asp Ser Ser Pro Val Asp Leu Thr Pro Arg Pro Gly Ser His Thr Ile 530 535 540 Glu Phe Phe Glu Met Cys Ala Asn Leu Ile Lys Ile Leu Ala Gln 545 550 555 235130DNAArtificial SequenceAMPK alpha 1 variant 2 23agcgccatgc gcagactcag ttcctggaga aagatggcga cagccgagaa gcagaaacac 60gacgggcggg tgaagatcgg ccactacatt ctgggtgaca cgctgggggt cggcaccttc 120ggcaaagtga aggttggcaa acatgaattg actgggcata aagtagctgt gaagatactc 180aatcgacaga agattcggag ccttgatgtg gtaggaaaaa tccgcagaga aattcagaac 240ctcaagcttt tcaggcatcc tcatataatt aaactgtacc aggtcatcag tacaccatct 300gatattttca tggtgatgga atatgtctca ggaggagagc tatttgatta tatctgtaag 360aatggaagga aatctgatgt acctggagta gtaaaaacag gctccacgaa ggagctggat 420gaaaaagaaa gtcggcgtct gttccaacag atcctttctg gtgtggatta ttgtcacagg 480catatggtgg tccatagaga tttgaaacct gaaaatgtcc tgcttgatgc acacatgaat 540gcaaagatag ctgattttgg tctttcaaac atgatgtcag atggtgaatt tttaagaaca 600agttgtggct cacccaacta tgctgcacca gaagtaattt caggaagatt gtatgcaggc 660ccagaggtag atatatggag cagtggggtt attctctatg ctttattatg tggaaccctt 720ccatttgatg atgaccatgt gccaactctt tttaagaaga tatgtgatgg gatcttctat 780acccctcaat atttaaatcc ttctgtgatt agccttttga aacatatgct gcaggtggat 840cccatgaaga gggccacaat caaagatatc agggaacatg aatggtttaa acaggacctt 900ccaaaatatc tctttcctga ggatccatca tatagttcaa ccatgattga tgatgaagcc 960ttaaaagaag tatgtgaaaa gtttgagtgc tcagaagagg aagttctcag ctgtctttac 1020aacagaaatc accaggatcc tttggcagtt gcctaccatc tcataataga taacaggaga 1080ataatgaatg aagccaaaga tttctatttg gcgacaagcc cacctgattc ttttcttgat 1140gatcatcacc tgactcggcc ccatcctgaa agagtaccat tcttggttgc tgaaacacca 1200agggcacgcc atacccttga tgaattaaat ccacagaaat ccaaacacca aggtgtaagg 1260aaagcaaaat ggcatttagg aattagaagt caaagtcgac caaatgatat tatggcagaa 1320gtatgtagag caatcaaaca attggattat gaatggaagg ttgtaaaccc atattatttg 1380cgtgtacgaa ggaagaatcc tgtgacaagc acttactcca aaatgagtct acagttatac 1440caagtggata gtagaactta tctactggat ttccgtagta ttgatgatga aattacagaa 1500gccaaatcag ggactgctac tccacagaga tcgggatcag ttagcaacta tcgatcttgc 1560caaaggagtg attcagatgc tgaggctcaa ggaaaatcct cagaagtttc tcttacctca 1620tctgtgacct cacttgactc ttctcctgtt gacctaactc caagacctgg aagtcacaca 1680atagaatttt ttgagatgtg tgcaaatcta attaaaattc ttgcacaata aacagaaaac 1740tttgcttatt tcttttgcag caataagcat gcataataag tcacagccaa atgcttccat 1800ttgtaatcaa gttatacata attataaccg agggctggcg ttttggaatg caatttgcac 1860agggattgga acatgattta tagttaaaag cctaatatgc agaaatgaat taagatcatt 1920ttgttgttca ttgtgcagta tgtatatagc ataatataca cagtgaatta taggtctcag 1980gcttacttga tttttggcta ttttatattt agtgtacaca gggctttgaa atattaattt 2040acataaaggc cttcatatat tattacgtgt tatatattac gtgttataaa tttattcaat 2100aaatatttgc ctagaattcc caagaccttt ataggtgatt ttgttttctg ggctccttaa 2160cttcataaat agctagtatc ttccagcagt agtaacagtc tggataactt cttccatatc 2220cctccctctt tgtttttttg agacagtgtc actttgtcac ccaggctgga gtgcaatggt 2280gtggtctcgg ctcactgcaa cctccacctc ccgggttcaa gtgattctcc cgcctcagct 2340tcctgagtag ctggaactac aggcgtgtgc caccacaccc ggctaatttt tcgtattttt 2400agtgtagacg gggtttcact atgttgccca ggctggtctc gaactcctga ccgcgtgatc 2460caccacctca gcttcccaaa gtggtgggat tacaggcgtg agccaccgca cccggcctcc 2520atatccccct tttaaaattc tgtagtgtat ggtaagtcat atcagatatc agacctaatt 2580taaatttcat tttagcttta caagtccaaa aacacagaat ttatatattc agatactcta 2640gcactaattt tagtcttaaa atattcccac gatattctgt acacaaaatg ttctttttgt 2700tacaagagct gagttgcata tactgtagat aaatcatatt atttttgcca atttcacaaa 2760ttcctctggc ccatcatgtc agtcattatt gagtatatgc acacattgct acttatttga 2820ttatgtatct tttaaattga ttcagtgcat agaaaactat ctcttacaaa ctttaagtgc 2880tctgatatga cttccccccc aaattttatt atgaacattt ttaaaaacag aaaaattgaa 2940aaactgtttg gtaagcacat gtatatctac catttagatt cagcagttgt taatgttttg 3000tcatttgttt tctctatacc tatatatgta tagatacagc tagttatgca tatatatgca 3060tatatgtgtt tgtttgtgta tgtatatatg cttttttccc cctgaaccat ttggatgtta 3120cagacatact tatcaccgtg aaaatacttc aagtatctcc tacagataat gacattctcc 3180taaaaatccg taataccatt gtaaaagtaa taattcccca atatcatcta atcaagccat 3240atttaaattt ctgaagttaa ctccaaattt ctttatagct gattatttca aactaggatc 3300caattaaagt ttacatatga cacttggtta taactcttta gttggatata acattattat 3360tattttgata aaatatggaa caaatcaatt ctattaataa gtggtcacat ttgttttggg 3420cttaaattac tttttaaaga tactggattt tcctaagatt tctgatttac actgatattt 3480ttttttgtca ttcttaattg catcacacaa tagatgtaaa tgaagatgta gtcacctcag 3540ataaaattgg

tatcgtgtat gataatattg tatcatttat atttgcctta tgttaacttt 3600aagaaattga tttttttgta ttaatcattt tcccattgca acagagctat attttttcta 3660ttttaagaat catattttag gattattttt ggcaaataca gtgagcactt atgtaaccag 3720atgataatga actcaaatgt catgatagct tgcataaatg gtgactctag tagatttgac 3780tcaagcactt ctagaatcat gcactgaatt caaaagaaaa atcttgctgc tttttgtcca 3840gggcttgttc tattcaactt ctaatttgaa agctgtacaa agtaatagaa gttccattta 3900aatatgagtt caaaactgta tttacttttt atgtggccct ctctttaggg gattctaatt 3960ttacttaggg tctctaagtg cagcataatg ttcctgatgt taacagaaga ctgtattttt 4020aaagttacaa atttgtatat ggaattaagt aatggcgcta tatacgctgt tgtggggagg 4080ggggaagaaa aggaggaacc aattaaatag gaccttttaa aaattgttaa ttttgtaaac 4140tttgcttctc ttataagtta ttgtgattca ttttagttac tgtgttttat tttgaaaata 4200tttaaatatt gcacttctat aaatagtatg ataaatgcac agacaattgc agtaaattct 4260tttttaagct aggatatttg aaatgacaac ctttggttaa gtgtgtcaag gttgcaacag 4320aattttcaca atttttttgt tgtttgcaaa ttgttactaa tattgaagag gtaagggagg 4380caatgcaaat gatttttaat ctttttttat tatcttttca gcagtttata ttttttgtga 4440ctttatgcaa ccatattttt actttgtctt gacaactgaa agatgtataa ggttttttgc 4500cagaaatgta ctgtatacat agttttaagt ataacagatt ttactgatat gtaaaaattt 4560tgccattaaa ataaatgatt tctcactgag aggaactttt ctaccaggtt ggggcatatg 4620ggagcttaat atatcatatc taatttaaaa taatttcact gaaataaact ccattgcttt 4680tacctaattt ttttcttgag atgcttttgt agtttttcag agttttagat gattttatac 4740aaaatcctct gcctagcact gctctttttg atgttgtagt gacaccattt acattgaatt 4800aatgcttggt agcctggggc tagatgtgga actccatgga tctgtgttct gactggcacc 4860tttggaatga aagaaaagtg tgtgctgtcc aaattttttc cccttaattc tttccctcat 4920cttctcaccc ataatagaaa ttttatttcc attgtgagtt ctgacaagaa tgaaattcca 4980catacaacat aactgtaaat tgttggtagg tagaagttaa tatttgtggt tcatgtatat 5040tttgaccaga gtatatttaa gtatataatt tcagcttcct tgatttagaa atatgatata 5100ataaagaaaa actccattta tcatctgtta 513024574PRTArtificial SequenceAMPK alpha 1 isoform 2 24Met Arg Arg Leu Ser Ser Trp Arg Lys Met Ala Thr Ala Glu Lys Gln 1 5 10 15 Lys His Asp Gly Arg Val Lys Ile Gly His Tyr Ile Leu Gly Asp Thr 20 25 30 Leu Gly Val Gly Thr Phe Gly Lys Val Lys Val Gly Lys His Glu Leu 35 40 45 Thr Gly His Lys Val Ala Val Lys Ile Leu Asn Arg Gln Lys Ile Arg 50 55 60 Ser Leu Asp Val Val Gly Lys Ile Arg Arg Glu Ile Gln Asn Leu Lys 65 70 75 80 Leu Phe Arg His Pro His Ile Ile Lys Leu Tyr Gln Val Ile Ser Thr 85 90 95 Pro Ser Asp Ile Phe Met Val Met Glu Tyr Val Ser Gly Gly Glu Leu 100 105 110 Phe Asp Tyr Ile Cys Lys Asn Gly Arg Lys Ser Asp Val Pro Gly Val 115 120 125 Val Lys Thr Gly Ser Thr Lys Glu Leu Asp Glu Lys Glu Ser Arg Arg 130 135 140 Leu Phe Gln Gln Ile Leu Ser Gly Val Asp Tyr Cys His Arg His Met 145 150 155 160 Val Val His Arg Asp Leu Lys Pro Glu Asn Val Leu Leu Asp Ala His 165 170 175 Met Asn Ala Lys Ile Ala Asp Phe Gly Leu Ser Asn Met Met Ser Asp 180 185 190 Gly Glu Phe Leu Arg Thr Ser Cys Gly Ser Pro Asn Tyr Ala Ala Pro 195 200 205 Glu Val Ile Ser Gly Arg Leu Tyr Ala Gly Pro Glu Val Asp Ile Trp 210 215 220 Ser Ser Gly Val Ile Leu Tyr Ala Leu Leu Cys Gly Thr Leu Pro Phe 225 230 235 240 Asp Asp Asp His Val Pro Thr Leu Phe Lys Lys Ile Cys Asp Gly Ile 245 250 255 Phe Tyr Thr Pro Gln Tyr Leu Asn Pro Ser Val Ile Ser Leu Leu Lys 260 265 270 His Met Leu Gln Val Asp Pro Met Lys Arg Ala Thr Ile Lys Asp Ile 275 280 285 Arg Glu His Glu Trp Phe Lys Gln Asp Leu Pro Lys Tyr Leu Phe Pro 290 295 300 Glu Asp Pro Ser Tyr Ser Ser Thr Met Ile Asp Asp Glu Ala Leu Lys 305 310 315 320 Glu Val Cys Glu Lys Phe Glu Cys Ser Glu Glu Glu Val Leu Ser Cys 325 330 335 Leu Tyr Asn Arg Asn His Gln Asp Pro Leu Ala Val Ala Tyr His Leu 340 345 350 Ile Ile Asp Asn Arg Arg Ile Met Asn Glu Ala Lys Asp Phe Tyr Leu 355 360 365 Ala Thr Ser Pro Pro Asp Ser Phe Leu Asp Asp His His Leu Thr Arg 370 375 380 Pro His Pro Glu Arg Val Pro Phe Leu Val Ala Glu Thr Pro Arg Ala 385 390 395 400 Arg His Thr Leu Asp Glu Leu Asn Pro Gln Lys Ser Lys His Gln Gly 405 410 415 Val Arg Lys Ala Lys Trp His Leu Gly Ile Arg Ser Gln Ser Arg Pro 420 425 430 Asn Asp Ile Met Ala Glu Val Cys Arg Ala Ile Lys Gln Leu Asp Tyr 435 440 445 Glu Trp Lys Val Val Asn Pro Tyr Tyr Leu Arg Val Arg Arg Lys Asn 450 455 460 Pro Val Thr Ser Thr Tyr Ser Lys Met Ser Leu Gln Leu Tyr Gln Val 465 470 475 480 Asp Ser Arg Thr Tyr Leu Leu Asp Phe Arg Ser Ile Asp Asp Glu Ile 485 490 495 Thr Glu Ala Lys Ser Gly Thr Ala Thr Pro Gln Arg Ser Gly Ser Val 500 505 510 Ser Asn Tyr Arg Ser Cys Gln Arg Ser Asp Ser Asp Ala Glu Ala Gln 515 520 525 Gly Lys Ser Ser Glu Val Ser Leu Thr Ser Ser Val Thr Ser Leu Asp 530 535 540 Ser Ser Pro Val Asp Leu Thr Pro Arg Pro Gly Ser His Thr Ile Glu 545 550 555 560 Phe Phe Glu Met Cys Ala Asn Leu Ile Lys Ile Leu Ala Gln 565 570 251501DNAArtificial SequenceCaMKI alpha 25ggagagagcc gccgagccga gccgagcccc agctccagca agagcgcggg cgggtggccc 60aggcacgcag cggtgaggac cgcggccaca gctcggcgcc aaccaccgcg ggcctcccag 120ccagccccgc ggcggggcag ccgcaggagc cctggctgtg gtcggggggc agtgggccat 180gctgggggca gtggaaggcc ccaggtggaa gcaggcggag gacattagag acatctacga 240cttccgagat gttctgggca cgggggcctt ctcggaggtg atcctggcag aagataagag 300gacgcagaag ctggtggcca tcaaatgcat tgccaaggag gccctggagg gcaaggaagg 360cagcatggag aatgagattg ctgtcctgca caagatcaag caccccaaca ttgtagccct 420ggatgacatc tatgagagtg ggggccacct ctacctcatc atgcagctgg tgtcgggtgg 480ggagctcttt gaccgtattg tggaaaaagg cttctacacg gagcgggacg ccagccgcct 540catcttccag gtgctggatg ctgtgaaata cctgcatgac ctgggcattg tacaccggga 600tctcaagcca gagaatctgc tgtactacag cctggatgaa gactccaaaa tcatgatctc 660cgactttggc ctctccaaga tggaggaccc gggcagtgtg ctctccaccg cctgtggaac 720tccgggatac gtggcccctg aagtcctggc ccagaagccc tacagcaagg ctgtggattg 780ctggtccata ggtgtcatcg cctacatctt gctctgcggt taccctccct tctatgacga 840gaatgatgcc aaactctttg aacagatttt gaaggccgag tacgagtttg actctcctta 900ctgggacgac atctctgact ctgccaaaga tttcatccgg cacttgatgg agaaggaccc 960agagaaaaga ttcacctgtg agcaggcctt gcagcaccca tggattgcag gagatacagc 1020tctagataag aatatccacc agtcggtgag tgagcagatc aagaagaact ttgccaagag 1080caagtggaag caagccttca atgccacggc tgtggtgcgg cacatgagga aactgcagct 1140gggcaccagc caggaggggc aggggcagac ggcgagccat ggggagctgc tgacaccagt 1200ggctgggggg ccggcagctg gctgttgctg tcgagactgc tgcgtggagc cgggcacaga 1260actgtccccc acactgcccc accagctcta gggccctgga cctcgggtca tgatcctctg 1320cgtgggaggg cttgggggca gcctgctccc cttccctccc tgaaccggga gtttctctgc 1380cctgtcccct cctcacctgc ttccctacca ctcctcactg cattttccat acaaatgttt 1440ctattttatt gttccttctt gtaataaagg gaagataaaa ccaaaaaaaa aaaaaaaaaa 1500a 150126370PRTArtificial SequenceCaMKI alpha 26Met Leu Gly Ala Val Glu Gly Pro Arg Trp Lys Gln Ala Glu Asp Ile 1 5 10 15 Arg Asp Ile Tyr Asp Phe Arg Asp Val Leu Gly Thr Gly Ala Phe Ser 20 25 30 Glu Val Ile Leu Ala Glu Asp Lys Arg Thr Gln Lys Leu Val Ala Ile 35 40 45 Lys Cys Ile Ala Lys Glu Ala Leu Glu Gly Lys Glu Gly Ser Met Glu 50 55 60 Asn Glu Ile Ala Val Leu His Lys Ile Lys His Pro Asn Ile Val Ala 65 70 75 80 Leu Asp Asp Ile Tyr Glu Ser Gly Gly His Leu Tyr Leu Ile Met Gln 85 90 95 Leu Val Ser Gly Gly Glu Leu Phe Asp Arg Ile Val Glu Lys Gly Phe 100 105 110 Tyr Thr Glu Arg Asp Ala Ser Arg Leu Ile Phe Gln Val Leu Asp Ala 115 120 125 Val Lys Tyr Leu His Asp Leu Gly Ile Val His Arg Asp Leu Lys Pro 130 135 140 Glu Asn Leu Leu Tyr Tyr Ser Leu Asp Glu Asp Ser Lys Ile Met Ile 145 150 155 160 Ser Asp Phe Gly Leu Ser Lys Met Glu Asp Pro Gly Ser Val Leu Ser 165 170 175 Thr Ala Cys Gly Thr Pro Gly Tyr Val Ala Pro Glu Val Leu Ala Gln 180 185 190 Lys Pro Tyr Ser Lys Ala Val Asp Cys Trp Ser Ile Gly Val Ile Ala 195 200 205 Tyr Ile Leu Leu Cys Gly Tyr Pro Pro Phe Tyr Asp Glu Asn Asp Ala 210 215 220 Lys Leu Phe Glu Gln Ile Leu Lys Ala Glu Tyr Glu Phe Asp Ser Pro 225 230 235 240 Tyr Trp Asp Asp Ile Ser Asp Ser Ala Lys Asp Phe Ile Arg His Leu 245 250 255 Met Glu Lys Asp Pro Glu Lys Arg Phe Thr Cys Glu Gln Ala Leu Gln 260 265 270 His Pro Trp Ile Ala Gly Asp Thr Ala Leu Asp Lys Asn Ile His Gln 275 280 285 Ser Val Ser Glu Gln Ile Lys Lys Asn Phe Ala Lys Ser Lys Trp Lys 290 295 300 Gln Ala Phe Asn Ala Thr Ala Val Val Arg His Met Arg Lys Leu Gln 305 310 315 320 Leu Gly Thr Ser Gln Glu Gly Gln Gly Gln Thr Ala Ser His Gly Glu 325 330 335 Leu Leu Thr Pro Val Ala Gly Gly Pro Ala Ala Gly Cys Cys Cys Arg 340 345 350 Asp Cys Cys Val Glu Pro Gly Thr Glu Leu Ser Pro Thr Leu Pro His 355 360 365 Gln Leu 370 271888DNAArtificial SequencePNCK/CaMKI beta variant 1 27gacttggatt gacatagaga gctgcaggag ggtgacatga tctcaataaa aggaatgctc 60tggctgctgg gtagagaaga aagcgagggt gagggattaa tggtggagcc agagggagcc 120cagtgagtgc cgggagccca gggtggcaga gctgtggtgc agggttgcag ggtcctggag 180ctaggcatgg aggctgcttt cgggcaagtg gccgggtcag cctgtcctcg gagaggcggt 240gaaggcagag actggaaggc agagagcctt gccgacctgt ggccgaagag ctctccggga 300gacagccacc ggtggtgcaa aggccctggg gccggcccag ccgggccgca gctccgggag 360gcggcgcgag cgagcagtgg gctgggcggt ggcggccggc acccgagccg gatcccggcg 420attgccttac aagacatgct gctgctgaag aaacacacgg aggacatcag cagcgtctac 480gagatccgcg agaggctcgg ctcgggtgcc ttctccgagg tggtgctggc ccaggagcgg 540ggctccgcac acctcgtggc cctcaagtgc atccccaaga aggccctccg gggcaaggag 600gccctggtgg agaacgagat cgcagtgctc cgtaggatca gtcaccccaa catcgtcgct 660ctggaggatg tccacgagag cccttcccac ctctacctgg ccatggaact ggtgacgggt 720ggcgagctgt ttgaccgcat catggagcgc ggctcctaca cagagaagga tgccagccat 780ctggtgggtc aggtccttgg cgccgtctcc tacctgcaca gcctggggat cgtgcaccgg 840gacctcaagc ccgaaaacct cctgtatgcc acgccctttg aggactcgaa gatcatggtc 900tctgactttg gactctccaa aatccaggct gggaacatgc taggcaccgc ctgtgggacc 960cctggatatg tggccccaga gctcttggag cagaaaccct acgggaaggc cgtagatgtg 1020tgggccctgg gcgtcatctc ctacatcctg ctgtgtgggt accccccctt ctacgacgag 1080agcgaccctg agctcttcag ccagatcctg agggccagct atgagtttga ctctcctttc 1140tgggatgaca tctcagaatc agccaaagac ttcatccggc accttctgga gcgagacccc 1200cagaagaggt tcacctgcca acaggccttg cggcaccttt ggatctctgg ggacacagcc 1260ttcgacaggg acatcttagg ctctgtcagt gagcagatcc ggaagaactt tgctcggaca 1320cactggaagc gagccttcaa tgccacctcg ttcctgcgcc acatccggaa gctggggcag 1380atcccagagg gcgagggggc ctctgagcag ggcatggccc gccacagcca ctcaggcctc 1440cgtgctggcc agccccccaa gtggtgatgc ccaggcagat gccgaggcca agtggactga 1500cccccagatt tccttccctt ggatgctttc ggtcccctcc cccaacccct ccccctgggg 1560ctggcctctg ctggattttg agatttgagg gtgtggcgca tggcgctggg gttggaatgg 1620ggcaccccca agtctgtccc caggctctgc cctgcctggg ggcagtggct cccctcccct 1680gttgcctctc ccgcccctgc cccccccgcc ccgccaaaag ccgagggggt gctggcaggc 1740gggcctcagg ggctgtcttt cctgcacggc tgttgtgtgc ttcgctgagt gtgggtggtc 1800ctgcttgtgt catggtcatg gccttccagc cccctccagt tttccccaaa ccaataaaga 1860aagatacagc aaaaaaaaaa aaaaaaaa 188828426PRTArtificial SequencePNCK/CaMKI beta variant 1 28Met Glu Ala Ala Phe Gly Gln Val Ala Gly Ser Ala Cys Pro Arg Arg 1 5 10 15 Gly Gly Glu Gly Arg Asp Trp Lys Ala Glu Ser Leu Ala Asp Leu Trp 20 25 30 Pro Lys Ser Ser Pro Gly Asp Ser His Arg Trp Cys Lys Gly Pro Gly 35 40 45 Ala Gly Pro Ala Gly Pro Gln Leu Arg Glu Ala Ala Arg Ala Ser Ser 50 55 60 Gly Leu Gly Gly Gly Gly Arg His Pro Ser Arg Ile Pro Ala Ile Ala 65 70 75 80 Leu Gln Asp Met Leu Leu Leu Lys Lys His Thr Glu Asp Ile Ser Ser 85 90 95 Val Tyr Glu Ile Arg Glu Arg Leu Gly Ser Gly Ala Phe Ser Glu Val 100 105 110 Val Leu Ala Gln Glu Arg Gly Ser Ala His Leu Val Ala Leu Lys Cys 115 120 125 Ile Pro Lys Lys Ala Leu Arg Gly Lys Glu Ala Leu Val Glu Asn Glu 130 135 140 Ile Ala Val Leu Arg Arg Ile Ser His Pro Asn Ile Val Ala Leu Glu 145 150 155 160 Asp Val His Glu Ser Pro Ser His Leu Tyr Leu Ala Met Glu Leu Val 165 170 175 Thr Gly Gly Glu Leu Phe Asp Arg Ile Met Glu Arg Gly Ser Tyr Thr 180 185 190 Glu Lys Asp Ala Ser His Leu Val Gly Gln Val Leu Gly Ala Val Ser 195 200 205 Tyr Leu His Ser Leu Gly Ile Val His Arg Asp Leu Lys Pro Glu Asn 210 215 220 Leu Leu Tyr Ala Thr Pro Phe Glu Asp Ser Lys Ile Met Val Ser Asp 225 230 235 240 Phe Gly Leu Ser Lys Ile Gln Ala Gly Asn Met Leu Gly Thr Ala Cys 245 250 255 Gly Thr Pro Gly Tyr Val Ala Pro Glu Leu Leu Glu Gln Lys Pro Tyr 260 265 270 Gly Lys Ala Val Asp Val Trp Ala Leu Gly Val Ile Ser Tyr Ile Leu 275 280 285 Leu Cys Gly Tyr Pro Pro Phe Tyr Asp Glu Ser Asp Pro Glu Leu Phe 290 295 300 Ser Gln Ile Leu Arg Ala Ser Tyr Glu Phe Asp Ser Pro Phe Trp Asp 305 310 315 320 Asp Ile Ser Glu Ser Ala Lys Asp Phe Ile Arg His Leu Leu Glu Arg 325 330 335 Asp Pro Gln Lys Arg Phe Thr Cys Gln Gln Ala Leu Arg His Leu Trp 340 345 350 Ile Ser Gly Asp Thr Ala Phe Asp Arg Asp Ile Leu Gly Ser Val Ser 355 360 365 Glu Gln Ile Arg Lys Asn Phe Ala Arg Thr His Trp Lys Arg Ala Phe 370 375 380 Asn Ala Thr Ser Phe Leu Arg His Ile Arg Lys Leu Gly Gln Ile Pro 385 390 395 400 Glu Gly Glu Gly Ala Ser Glu Gln Gly Met Ala Arg His Ser His Ser 405 410 415 Gly Leu Arg Ala Gly Gln Pro Pro Lys Trp 420 425 291727DNAArtificial SequenceCaMKI delta/CKLiK variant 1 29agccggcgcg cggcggcggc aggaagtctg tgcccgagaa cagcagaaat aagagccagg 60gagggaccgc ggccgcggcg gcggcggcga gagcgaaaga ggaaactgca gaggaggaag 120ctgcgccgca gcccgagccg cccggcatcc ccgccgcctc tgcgcccgcg ccgcgccccc 180ggcgccccct ccccagcgcg cccccggccg ctcctccgcg ccgcgctcgt cggccatggc 240ccgggagaac ggcgagagca gctcctcctg gaaaaagcaa gctgaagaca tcaagaagat 300cttcgagttc aaagagaccc tcggaaccgg ggccttttcc gaagtggttt tagctgaaga 360gaaggcaact ggcaagctct ttgctgtgaa gtgtatccct aagaaggcgc tgaagggcaa 420ggaaagcagc atagagaatg agatagccgt cctgagaaag attaagcatg aaaatattgt 480tgccctggaa gacatttatg aaagcccaaa tcacctgtac ttggtcatgc agctggtgtc 540cggtggagag ctgtttgacc ggatagtgga gaaggggttt tatacagaga aggatgccag 600cactctgatc cgccaagtct tggacgccgt gtactatctc cacagaatgg gcatcgtcca 660cagagacctc aagcccgaaa atctcttgta ctacagtcaa gatgaggagt ccaaaataat

720gatcagtgac tttggattgt caaaaatgga gggcaaagga gatgtgatgt ccactgcctg 780tggaactcca ggctatgtcg ctcctgaagt cctcgcccag aaaccttaca gcaaagccgt 840tgactgctgg tccatcggag tgattgccta catcttgctc tgcggctacc ctccttttta 900tgatgaaaat gactccaagc tctttgagca gatcctcaag gcggaatatg agtttgactc 960tccctactgg gatgacatct ccgactctgc aaaagacttc attcggaacc tgatggagaa 1020ggacccgaat aaaagataca cgtgtgagca ggcagctcgg cacccatgga tcgctggtga 1080cacagccctc aacaaaaaca tccacgagtc cgtcagcgcc cagatccgga aaaactttgc 1140caagagcaaa tggagacaag catttaatgc cacggccgtc gtcagacata tgagaaaact 1200acacctcggc agcagcctgg acagttcaaa tgcaagtgtt tcgagcagcc tcagtttggc 1260cagccaaaaa gactgtgcgt atgtagcaaa accagaatcc ctcagctgac actgaagacg 1320agcctggggt ggagaggagg gagccggcat ctgccgagca cctcctgttt gccaggcgct 1380ttctatactt aatcccatgt catgcgaccc taggactttt tttaacatgt aatcactggg 1440ctgggtgcag tggctcacgc ctgtaatccc aacactttgg gaggctgagg caggaggact 1500gtttgagttc aggagtttta agaccagcct gaccaacatg gtgaaacccc atctctacta 1560aaatataaaa attagccggg tgtggtggcg agcacctgta atgtcagcta cttgggaggc 1620tgaggcagga gaatcacttg aacccaggaa gcggaggttg caatgagctg agatcacacc 1680actgcactcc agcctgggtg acagattgag actccctctc aaaaaaa 172730357PRTArtificial SequenceCaMKI delta/CKLiK variant 1 30Met Ala Arg Glu Asn Gly Glu Ser Ser Ser Ser Trp Lys Lys Gln Ala 1 5 10 15 Glu Asp Ile Lys Lys Ile Phe Glu Phe Lys Glu Thr Leu Gly Thr Gly 20 25 30 Ala Phe Ser Glu Val Val Leu Ala Glu Glu Lys Ala Thr Gly Lys Leu 35 40 45 Phe Ala Val Lys Cys Ile Pro Lys Lys Ala Leu Lys Gly Lys Glu Ser 50 55 60 Ser Ile Glu Asn Glu Ile Ala Val Leu Arg Lys Ile Lys His Glu Asn 65 70 75 80 Ile Val Ala Leu Glu Asp Ile Tyr Glu Ser Pro Asn His Leu Tyr Leu 85 90 95 Val Met Gln Leu Val Ser Gly Gly Glu Leu Phe Asp Arg Ile Val Glu 100 105 110 Lys Gly Phe Tyr Thr Glu Lys Asp Ala Ser Thr Leu Ile Arg Gln Val 115 120 125 Leu Asp Ala Val Tyr Tyr Leu His Arg Met Gly Ile Val His Arg Asp 130 135 140 Leu Lys Pro Glu Asn Leu Leu Tyr Tyr Ser Gln Asp Glu Glu Ser Lys 145 150 155 160 Ile Met Ile Ser Asp Phe Gly Leu Ser Lys Met Glu Gly Lys Gly Asp 165 170 175 Val Met Ser Thr Ala Cys Gly Thr Pro Gly Tyr Val Ala Pro Glu Val 180 185 190 Leu Ala Gln Lys Pro Tyr Ser Lys Ala Val Asp Cys Trp Ser Ile Gly 195 200 205 Val Ile Ala Tyr Ile Leu Leu Cys Gly Tyr Pro Pro Phe Tyr Asp Glu 210 215 220 Asn Asp Ser Lys Leu Phe Glu Gln Ile Leu Lys Ala Glu Tyr Glu Phe 225 230 235 240 Asp Ser Pro Tyr Trp Asp Asp Ile Ser Asp Ser Ala Lys Asp Phe Ile 245 250 255 Arg Asn Leu Met Glu Lys Asp Pro Asn Lys Arg Tyr Thr Cys Glu Gln 260 265 270 Ala Ala Arg His Pro Trp Ile Ala Gly Asp Thr Ala Leu Asn Lys Asn 275 280 285 Ile His Glu Ser Val Ser Ala Gln Ile Arg Lys Asn Phe Ala Lys Ser 290 295 300 Lys Trp Arg Gln Ala Phe Asn Ala Thr Ala Val Val Arg His Met Arg 305 310 315 320 Lys Leu His Leu Gly Ser Ser Leu Asp Ser Ser Asn Ala Ser Val Ser 325 330 335 Ser Ser Leu Ser Leu Ala Ser Gln Lys Asp Cys Ala Tyr Val Ala Lys 340 345 350 Pro Glu Ser Leu Ser 355 312507DNAArtificial SequenceCaMKI gamma 31tggagcagct aatcctcaca gacctgtagg agctggagtg ggagctcaag caggattctt 60cccgagtccc tggcatcctc agaagcttca actctggagg caatgggtcg aaaggaagaa 120gatgactgca gttcctggaa gaaacagacc accaacatcc ggaaaacctt catttttatg 180gaagtgctgg gatcaggagc tttctcagaa gttttcctgg tgaagcaaag actgactggg 240aagctctttg ctctgaagtg catcaagaag tcacctgcct tccgggacag cagcctggag 300aatgagattg ctgtgttgaa aaagatcaag catgaaaaca ttgtgaccct ggaggacatc 360tatgagagca ccacccacta ctacctggtc atgcagcttg tttctggtgg ggagctcttt 420gaccggatcc tggagcgggg tgtctacaca gagaaggatg ccagtctggt gatccagcag 480gtcttgtcgg cagtgaaata cctacatgag aatggcatcg tccacagaga cttaaagccc 540gaaaacctgc tttaccttac ccctgaagag aactctaaga tcatgatcac tgactttggt 600ctgtccaaga tggaacagaa tggcatcatg tccactgcct gtgggacccc aggctacgtg 660gctccagaag tgctggccca gaaaccctac agcaaggctg tggattgctg gtccatcggc 720gtcatcacct acatattgct ctgtggatac cccccattct atgaagaaac ggagtctaag 780cttttcgaga agatcaagga gggctactat gagtttgagt ctccattctg ggatgacatt 840tctgagtcag ccaaggactt tatttgccac ttgcttgaga aggatccgaa cgagcggtac 900acctgtgaga aggccttgag tcatccctgg attgacggaa acacagccct ccaccgggac 960atctacccat cagtcagcct ccagatccag aagaactttg ctaagagcaa gtggaggcaa 1020gccttcaacg cagcagctgt ggtgcaccac atgaggaagc tacacatgaa cctgcacagc 1080ccgggcgtcc gcccagaggt ggagaacagg ccgcctgaaa ctcaagcctc agaaacctct 1140agacccagct cccctgagat caccatcacc gaggcacctg tcctggacca cagtgtagca 1200ctccctgccc tgacccaatt accctgccag catggccgcc ggcccactgc ccctggtggc 1260aggtccctca actgcctggt caatggctcc ctccacatca gcagcagcct ggtgcccatg 1320catcaggggt ccctggccgc cgggccctgt ggctgctgct ccagctgcct gaacattggg 1380agcaaaggaa agtcctccta ctgctctgag cccacactcc tcaaaaaggc caacaaaaaa 1440cagaacttca agtcggaggt catggtacca gttaaagcca gtggcagctc ccactgccgg 1500gcagggcaga ctggagtctg tctcattatg tgattcctgg agcctgtgcc tatgtcactg 1560caattttcag gagacatatt caactcctct gctcttccaa acctggtgtc tatccggcag 1620agggaggaag gcagagcaag tggagcaggg cttagcagga gcagtttctg gccagaagca 1680ccagcctgct gccagcgggg cagcccctca taggaggccc aggagggagc cccaaggcgt 1740agaagccttg ttgaagctgt gagcaggaga agcggtgccc accagcttcc aggtctccct 1800gacctgcctg ctctatgccc cacaccctac gtgccgtggc tctgtgcagt gtacgtagat 1860agctctcgcc tgggtctgtg ctgtttgtcg tgaaaagctt aatgggctgg ccaggctgtg 1920tcaccttctc caagcaaagc catatggagc atctacccag actcccactc tgcacacact 1980cactcccacc tctcaagcct ccaacctctt ggccagattg ggctcattaa tgtcgttgcc 2040tgcccatctg catgaatgac aggcagctcc ccatggtggt ctgcctgtga gctcttcaag 2100ttctaatcct taactccagg attagctccc aagtgcgctg agacccagcc agcacacttc 2160tggcccttct ccctgcctca atctaaaagc agtgccacac cctccaaagt ggaatagaaa 2220gaagttcatg agtaagggct gcaaggaatt cttatcctgg ccacatgtcc tccgtgcaca 2280cacccaatgg agttaacctt ggaagttgac tattttaatg tctgccagga gttctaatcc 2340tgcctctgtt cccttttctc tccttgaaag tccagcacac cattcttgtc cttccccagt 2400ttcctcgccc tccacccctc cagcttcatg ctcagtgttg tgcttaataa aatggacata 2460tttttctcta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 250732476PRTArtificial SequenceCaMKI gamma 32Met Gly Arg Lys Glu Glu Asp Asp Cys Ser Ser Trp Lys Lys Gln Thr 1 5 10 15 Thr Asn Ile Arg Lys Thr Phe Ile Phe Met Glu Val Leu Gly Ser Gly 20 25 30 Ala Phe Ser Glu Val Phe Leu Val Lys Gln Arg Leu Thr Gly Lys Leu 35 40 45 Phe Ala Leu Lys Cys Ile Lys Lys Ser Pro Ala Phe Arg Asp Ser Ser 50 55 60 Leu Glu Asn Glu Ile Ala Val Leu Lys Lys Ile Lys His Glu Asn Ile 65 70 75 80 Val Thr Leu Glu Asp Ile Tyr Glu Ser Thr Thr His Tyr Tyr Leu Val 85 90 95 Met Gln Leu Val Ser Gly Gly Glu Leu Phe Asp Arg Ile Leu Glu Arg 100 105 110 Gly Val Tyr Thr Glu Lys Asp Ala Ser Leu Val Ile Gln Gln Val Leu 115 120 125 Ser Ala Val Lys Tyr Leu His Glu Asn Gly Ile Val His Arg Asp Leu 130 135 140 Lys Pro Glu Asn Leu Leu Tyr Leu Thr Pro Glu Glu Asn Ser Lys Ile 145 150 155 160 Met Ile Thr Asp Phe Gly Leu Ser Lys Met Glu Gln Asn Gly Ile Met 165 170 175 Ser Thr Ala Cys Gly Thr Pro Gly Tyr Val Ala Pro Glu Val Leu Ala 180 185 190 Gln Lys Pro Tyr Ser Lys Ala Val Asp Cys Trp Ser Ile Gly Val Ile 195 200 205 Thr Tyr Ile Leu Leu Cys Gly Tyr Pro Pro Phe Tyr Glu Glu Thr Glu 210 215 220 Ser Lys Leu Phe Glu Lys Ile Lys Glu Gly Tyr Tyr Glu Phe Glu Ser 225 230 235 240 Pro Phe Trp Asp Asp Ile Ser Glu Ser Ala Lys Asp Phe Ile Cys His 245 250 255 Leu Leu Glu Lys Asp Pro Asn Glu Arg Tyr Thr Cys Glu Lys Ala Leu 260 265 270 Ser His Pro Trp Ile Asp Gly Asn Thr Ala Leu His Arg Asp Ile Tyr 275 280 285 Pro Ser Val Ser Leu Gln Ile Gln Lys Asn Phe Ala Lys Ser Lys Trp 290 295 300 Arg Gln Ala Phe Asn Ala Ala Ala Val Val His His Met Arg Lys Leu 305 310 315 320 His Met Asn Leu His Ser Pro Gly Val Arg Pro Glu Val Glu Asn Arg 325 330 335 Pro Pro Glu Thr Gln Ala Ser Glu Thr Ser Arg Pro Ser Ser Pro Glu 340 345 350 Ile Thr Ile Thr Glu Ala Pro Val Leu Asp His Ser Val Ala Leu Pro 355 360 365 Ala Leu Thr Gln Leu Pro Cys Gln His Gly Arg Arg Pro Thr Ala Pro 370 375 380 Gly Gly Arg Ser Leu Asn Cys Leu Val Asn Gly Ser Leu His Ile Ser 385 390 395 400 Ser Ser Leu Val Pro Met His Gln Gly Ser Leu Ala Ala Gly Pro Cys 405 410 415 Gly Cys Cys Ser Ser Cys Leu Asn Ile Gly Ser Lys Gly Lys Ser Ser 420 425 430 Tyr Cys Ser Glu Pro Thr Leu Leu Lys Lys Ala Asn Lys Lys Gln Asn 435 440 445 Phe Lys Ser Glu Val Met Val Pro Val Lys Ala Ser Gly Ser Ser His 450 455 460 Cys Arg Ala Gly Gln Thr Gly Val Cys Leu Ile Met 465 470 475 334918DNAArtificial SequenceCaMKII alpha isoform 1 33ggttgccatg gggacctgga tgctgacgaa ggctcgcgag gctgtgagca gccacagtgc 60cctgctcaga agccccgggc tcgtcagtca aaccggttct ctgtttgcac tcggcagcac 120gggcaggcaa gtggtcccta ggttcgggag cagagcagca gcgcctcagt cctggtcccc 180cagtcccaag cctcacctgc ctgcccagcg ccaggatggc caccatcacc tgcacccgct 240tcacggaaga gtaccagctc ttcgaggaat tgggcaaggg agccttctcg gtggtgcgaa 300ggtgtgtgaa ggtgctggct ggccaggagt atgctgccaa gatcatcaac acaaagaagc 360tgtcagccag agaccatcag aagctggagc gtgaagcccg catctgccgc ctgctgaagc 420accccaacat cgtccgacta catgacagca tctcagagga gggacaccac tacctgatct 480tcgacctggt cactggtggg gaactgtttg aagatatcgt ggcccgggag tattacagtg 540aggcggatgc cagtcactgt atccagcaga tcctggaggc tgtgctgcac tgccaccaga 600tgggggtggt gcaccgggac ctgaagcctg agaatctgtt gctggcctcc aagctcaagg 660gtgccgcagt gaagctggca gactttggcc tggccataga ggtggagggg gagcagcagg 720catggtttgg gtttgcaggg actcctggat atctctcccc agaagtgctg cggaaggacc 780cgtacgggaa gcctgtggac ctgtgggctt gtggggtcat cctgtacatc ctgctggttg 840ggtacccccc gttctgggat gaggaccagc accgcctgta ccagcagatc aaagccggcg 900cctatgattt cccatcgccg gaatgggaca ctgtcacccc ggaagccaag gatctgatca 960ataagatgct gaccattaac ccatccaaac gcatcacagc tgccgaagcc cttaagcacc 1020cctggatctc gcaccgctcc accgtggcat cctgcatgca cagacaggag accgtggact 1080gcctgaagaa gttcaatgcc aggaggaaac tgaagggagc cattctcacc acgatgctgg 1140ccaccaggaa cttctccgga gggaagagtg ggggaaacaa gaagagcgat ggtgtgaaga 1200aaagaaagtc cagttccagc gttcagttaa tggaatcctc agagagcacc aacaccacca 1260tcgaggatga agacaccaaa gtgcggaaac aggaaattat aaaagtgaca gagcagctga 1320ttgaagccat aagcaatgga gattttgagt cctacacgaa gatgtgcgac cctggcatga 1380cagccttcga acctgaggcc ctggggaacc tggttgaggg cctggacttc catcgattct 1440attttgaaaa cctgtggtcc cggaacagca agcccgtgca caccaccatc ctgaatcccc 1500acatccacct gatgggcgac gagtcagcct gcatcgccta catccgcatc acgcagtacc 1560tggacgctgg cggcatccca cgcaccgccc agtcggagga gacccgtgtc tggcaccgcc 1620gggatggcaa atggcagatc gtccacttcc acagatctgg ggcgccctcc gtcctgcccc 1680actgagggac caggctgggg tcgctgcgtt gctgtgccgc agagatccac tctgtccgtg 1740gagtggagct gctggttctc ccaggtggat tttgctggaa ttctcccatg tcatcacccc 1800accaccgtca cttctgtacc tgcatcaaga aaacctgctt gttcacaaaa gtcatcgcaa 1860cttcagagcg aacggccaca tctccccacc tctcaccccc accctctccc ctgccaggct 1920ggggcttcct caggcatggg tgtccacagc actggccccc tctccccagc ctcagctgct 1980gtccgcctga tctgtcttgg gctgtaggct agaatgcccg ggctggtgcc caccaggggc 2040tggggagaag gaggggtggc atgatgagga aggcagcatc cgtccgtccc tctcccagac 2100ctctcctctt ccagtgtccc cggggaaggg cagatgacac tcccttcccc ctaagccaac 2160cgcactgaag gagtggggag aagagcatac gccaggagcc tcctgcctca aagtgctccc 2220ctaagtcttc ttcctcctgt gctgacctca gggtggtctg acccttccct cggtgtgggg 2280gatgtggccc tctcaggtgc ccctacttgc tttctgcttc cttctggtga agtccacctc 2340caacattaac ctgcccaccc cacccccgtc atccctggag aattccagct ttgtcgtatc 2400tcagagaggg aatctaattg tttttggggg gcaaaagaaa gcaacgttta ggtatcactt 2460ctacttggac cgcatgcctt tttatagcca aatttctgtg tatttcgtaa atggatttcg 2520cgttaatgga tatttatgta ataactagac ttctcagatt attgtgagaa gggtcaggtt 2580ggaaggggtg taggaagagg ggtgaggggt agtttttttc tgttctagtt tttttttttt 2640tttttgtcat ctctgaggtg gaccttgtca cctgtggtta ttggggccaa ggtggactca 2700gctccgggga gaagggcctc tctgccattt cggtcccaag gtgagctgac acaggcgttc 2760cttttgggac tgtggaagca tcagatgcca gcactgactc aggaacagca agtcagggca 2820gagaggagga gggaggctgt caggatggaa atacctggac ttttctttgc ttccctcgca 2880aactggggtc ttctctaccg aacttcccag gatttcatct caccatatct gtgtgccgcc 2940cccagcaccc cccacccacc tctggggggc ccgtgagcgt gtgtcttcat tgcctctctc 3000cccttggcgt ctgatgacca cagcaaagca ctgggaattt ctactcttca tgcctcatcc 3060tgcagcctcg ggttcgcatt ctctctttct tttcctcttt ccctctttcc ctgggattga 3120ctctgagtgg aataccttgg cacatccact aggatctact gtctgcactg ttttctttgc 3180atgactttat acgcagtaag tatgttgaaa acaaacaaaa agaagaaaac actcaacaaa 3240accaatctac atgttttgga ctaaaaaaaa aaatagaggt tgtattctca gtgtccgact 3300cggaattatg ttgctgcctc tctgtgcttt tggcctctgt gtggccgtgt tttgccagca 3360tgagatactg tcccctctgg aggattttag gggaggaaga gccacgtccc cagggattgg 3420aggaggctcc ggtaccctcg accctcctgg gtgttggttg gagcagaact ggtgaggatg 3480tttgatccga gattttctga gctctcccca atcaccagct gtctgctggg ttcttttctc 3540aagtcctgct gcccaggccc aggtgagaca ggcaacgcca ggtctgcagg ccaggagaga 3600tgctgcccag gcctcctggt ttccaagctg gtccatcact ggcctctgtc cttggcagag 3660accttgctgc ccaggcccag gggcaggctc ttggcctgcc ccaggcccag agggcttccc 3720agtaaggccc agtgatccca ttatcccagg ggcaaaacca cctgtcccct tttgagctgc 3780cagttcccta cagccatccc cagtcaaggg tgagggtgtg gccttcacca ggggctgctg 3840taattaccga gcaaggtctg agctcttctt cagcctcagt tccctcattg gttaaaaggg 3900ttctttgttc ccatccagcc gatgaaggag caaacgtctg gctatgtgaa gcctaattta 3960cctgcaggaa ctggcaggga tagtcactgg ctggactcct gtttacttct agacctggtc 4020aggctccatc ccctccccca cctgcccctg attcccctcg tcggtgcctg tcaactgctt 4080ttcagcagtg gactgcaggg gaaagagcag tgatttgggg tgagtaggct tcaattccca 4140gctctgacca gacttgctgt gtgaccttgg gcaagttcct ttccctcttt ggagcttggt 4200ttccctgcca gaggaaactg agctggagga gcctgaggtc ctgcctttca ttggctgaca 4260cacctcctgt ccactgtgtc actctccaag tgccagagaa gtggaggcag atcgctaccc 4320caggctgaga tggcccccac tgtgaaggcc acgcctgtgg gtgggcagcc acctggtgcc 4380accacagggc accagggatg atcctgatgt ggcaggcagg ggagactcac agaaaaatct 4440gcccagagcc taccctcacc agacaaactc tgtgctcctc caaaacatcc tttagatgca 4500aaataataat aataataata ataaataaat aaataaaaat ccaaacccaa gtcaaaacct 4560tggctccagc atgaaaacac gtttacagga aagtgttctc ctgggtttgt gcccaccatg 4620gtgcgaatcc tgacccaagg cctcctgtct cccttcaaag ggagaccctt ttgggggatg 4680agtttgccag actccccgtg ctggtttctt tgttactatt tgtttggggt tttgttttag 4740ttcttttttt ttttcttttc ttttttaaaa atatgtggct gtgaacttga atgaacactg 4800ctcaaacttt ctgctattgg ggggggcggg tgggatggga agaaggggcg tttgttttat 4860tcttggtgtt ttcagtgcaa taaatagcta caaacttctg tgcaaaaaaa aaaaaaaa 491834489PRTArtificial SequenceCaMKII alpha isoform 1 34Met Ala Thr Ile Thr Cys Thr Arg Phe Thr Glu Glu Tyr Gln Leu Phe 1 5 10 15 Glu Glu Leu Gly Lys Gly Ala Phe Ser Val Val Arg Arg Cys Val Lys 20 25 30 Val Leu Ala Gly Gln Glu Tyr Ala Ala Lys Ile Ile Asn Thr Lys Lys 35 40 45 Leu Ser Ala Arg Asp His Gln Lys Leu Glu Arg Glu Ala Arg Ile Cys 50 55 60 Arg Leu Leu Lys His Pro Asn Ile Val Arg Leu His Asp Ser Ile Ser 65 70 75 80 Glu Glu Gly His His Tyr Leu Ile Phe Asp Leu Val Thr Gly Gly Glu 85 90 95 Leu Phe Glu Asp Ile Val Ala Arg Glu Tyr Tyr Ser Glu Ala Asp Ala 100 105 110 Ser His Cys Ile Gln Gln Ile Leu Glu Ala Val Leu His Cys His Gln 115 120 125 Met Gly Val Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Ala 130 135 140

Ser Lys Leu Lys Gly Ala Ala Val Lys Leu Ala Asp Phe Gly Leu Ala 145 150 155 160 Ile Glu Val Glu Gly Glu Gln Gln Ala Trp Phe Gly Phe Ala Gly Thr 165 170 175 Pro Gly Tyr Leu Ser Pro Glu Val Leu Arg Lys Asp Pro Tyr Gly Lys 180 185 190 Pro Val Asp Leu Trp Ala Cys Gly Val Ile Leu Tyr Ile Leu Leu Val 195 200 205 Gly Tyr Pro Pro Phe Trp Asp Glu Asp Gln His Arg Leu Tyr Gln Gln 210 215 220 Ile Lys Ala Gly Ala Tyr Asp Phe Pro Ser Pro Glu Trp Asp Thr Val 225 230 235 240 Thr Pro Glu Ala Lys Asp Leu Ile Asn Lys Met Leu Thr Ile Asn Pro 245 250 255 Ser Lys Arg Ile Thr Ala Ala Glu Ala Leu Lys His Pro Trp Ile Ser 260 265 270 His Arg Ser Thr Val Ala Ser Cys Met His Arg Gln Glu Thr Val Asp 275 280 285 Cys Leu Lys Lys Phe Asn Ala Arg Arg Lys Leu Lys Gly Ala Ile Leu 290 295 300 Thr Thr Met Leu Ala Thr Arg Asn Phe Ser Gly Gly Lys Ser Gly Gly 305 310 315 320 Asn Lys Lys Ser Asp Gly Val Lys Lys Arg Lys Ser Ser Ser Ser Val 325 330 335 Gln Leu Met Glu Ser Ser Glu Ser Thr Asn Thr Thr Ile Glu Asp Glu 340 345 350 Asp Thr Lys Val Arg Lys Gln Glu Ile Ile Lys Val Thr Glu Gln Leu 355 360 365 Ile Glu Ala Ile Ser Asn Gly Asp Phe Glu Ser Tyr Thr Lys Met Cys 370 375 380 Asp Pro Gly Met Thr Ala Phe Glu Pro Glu Ala Leu Gly Asn Leu Val 385 390 395 400 Glu Gly Leu Asp Phe His Arg Phe Tyr Phe Glu Asn Leu Trp Ser Arg 405 410 415 Asn Ser Lys Pro Val His Thr Thr Ile Leu Asn Pro His Ile His Leu 420 425 430 Met Gly Asp Glu Ser Ala Cys Ile Ala Tyr Ile Arg Ile Thr Gln Tyr 435 440 445 Leu Asp Ala Gly Gly Ile Pro Arg Thr Ala Gln Ser Glu Glu Thr Arg 450 455 460 Val Trp His Arg Arg Asp Gly Lys Trp Gln Ile Val His Phe His Arg 465 470 475 480 Ser Gly Ala Pro Ser Val Leu Pro His 485 354586DNAArtificial SequenceCaMKII beta isoform 1 35aggtgtgcgg cgcgctcctg gcgaggacgg agcgagcaga tctcgcgtgc gctcgccgcc 60cggcgcagcc cagcccggcc cccgcctggc gccgcgagcc gaggtgtctc ccgcgcccgc 120gcccgtgtcg ccgccgtgcc cgcgagcggg agccggagtc gccgccgccc gagcgcagcc 180gagcgcacgc cgagcccgtc cgccgccgcc atggccacca cggtgacctg cacccgcttc 240accgacgagt accagctcta cgaggatatt ggcaaggggg ctttctctgt ggtccgacgc 300tgtgtcaagc tctgcaccgg ccatgagtat gcagccaaga tcatcaacac caagaagctg 360tcagccagag atcaccagaa gctggagaga gaggctcgga tctgccgcct tctgaagcat 420tccaacatcg tgcgtctcca cgacagcatc tccgaggagg gcttccacta cctggtcttc 480gatctggtca ctggtgggga gctctttgaa gacattgtgg cgagagagta ctacagcgag 540gctgatgcca gtcactgtat ccagcagatc ctggaggccg ttctccattg tcaccaaatg 600ggggtcgtcc acagagacct caagccggag aacctgcttc tggccagcaa gtgcaaaggg 660gctgcagtga agctggcaga cttcggccta gctatcgagg tgcaggggga ccagcaggca 720tggtttggtt tcgctggcac accaggctac ctgtcccctg aggtccttcg caaagaggcg 780tatggcaagc ctgtggacat ctgggcatgt ggggtgatcc tgtacatcct gctcgtgggc 840tacccaccct tctgggacga ggaccagcac aagctgtacc agcagatcaa ggctggtgcc 900tatgacttcc cgtcccctga gtgggacacc gtcactcctg aagccaaaaa cctcatcaac 960cagatgctga ccatcaaccc tgccaagcgc atcacagccc atgaggccct gaagcacccg 1020tgggtctgcc aacgctccac ggtagcatcc atgatgcaca gacaggagac tgtggagtgt 1080ctgaaaaagt tcaatgccag gagaaagctc aagggagcca tcctcaccac catgctggcc 1140acacggaatt tctcagtggg cagacagacc accgctccgg ccacaatgtc caccgcggcc 1200tccggcacca ccatggggct ggtggaacaa gccaagagtt tactcaacaa gaaagcagat 1260ggagtcaagc cccagacgaa tagcaccaaa aacagtgcag ccgccaccag ccccaaaggg 1320acgcttcctc ctgccgccct ggagcctcaa accaccgtca tccataaccc agtggacggg 1380attaaggagt cttctgacag tgccaatacc accatagagg atgaagacgc taaagccccc 1440agggtccccg acatcctgag ctcagtgagg aggggctcgg gagccccaga agccgagggg 1500cccctgccct gcccatctcc ggctcccttt agccccctgc cagccccatc ccccaggatc 1560tctgacatcc tgaactctgt gagaaggggt tcaggaaccc cagaagccga gggccccctc 1620tcagcggggc ccccgccctg cctgtctccg gctctcctag gccccctgtc ctccccgtcc 1680cccaggatct ctgacatcct gaactctgtg aggaggggct cagggacccc agaagccgag 1740ggcccctcgc cagtggggcc cccgccctgc ccatctccga ctatccctgg ccccctgccc 1800accccatccc ggaagcagga gatcattaag accacggagc agctcatcga ggccgtcaac 1860aacggtgact ttgaggccta cgcgaaaatc tgtgacccag ggctgacctc gtttgagcct 1920gaagcactgg gcaacctggt tgaagggatg gacttccaca gattctactt cgagaacctg 1980ctggccaaga acagcaagcc gatccacacg accatcctga acccacacgt gcacgtcatt 2040ggagaggatg ccgcctgcat cgcttacatc cggctcacgc agtacattga cgggcagggc 2100cggccccgca ccagccagtc tgaggagacc cgcgtgtggc accgccgcga cggcaagtgg 2160cagaacgtgc acttccactg ctcgggcgcg cctgtggccc cgctgcagtg aagagctgcg 2220ccctggtttc gccggacaga gttggtgttt ggagcccgac tgccctcggg cacacggcct 2280gcctgtcgca tgtttgtgtc tgcctcgttc cctcccctgg tgcctgtgtc tgcagaaaaa 2340caagaccaga tgtgatttgt taaaaaaaaa caaaaaaaaa aaaaaaaaaa acaagatgac 2400gacgacaacc acaaaaaaaa ttgacatcag atgaaatgaa aaaaaaaaaa aacaaaaaaa 2460actaaaggaa ggaaaaagct gtaaaaatca ctggcattcg tggggccact ccccacccaa 2520gctccacgtg tgtccgtctg tgctcctggc ctctggggga ccagctggga catgaacttg 2580tctgccaggc ccccgtcgcg tgctgaacgg tgttagtttg taggtaacgc acacacccca 2640cacctaaggt gtctgcatcc tcctgccaac gcatgggctc cacgtggtgt gctcgctggc 2700tgtcgtgact gtcagctgtc tcttgggagg ggctgtgggg gcccgctggg ctgcctcctt 2760tcccgctagt tgtgcctgag agttgctgtt gttcctgctt tcccttccct tcctttcatc 2820ccctgaaggg ctaggtgtgg gttttccgtg cccggtatcc ccacacaccc agcacggaca 2880acccttcggc agagcccagg ccggcccctc accccctgga gtattgaaac tggagtcccg 2940tccccaaggc cttcagagat gcccctacac acccagggct ccagctctgg tccttctggg 3000ggagtaaagt gcaaagaggg gcacagctta gttttgggcc tctcgccgag caagagacag 3060cactgctggc tacagctcca acacagccag ctgtggcaag aggactctgc ctgggctggc 3120ccccctcctg tgtgaggtgt ctgtcccttc tctgctggcc agcagcagat gcactggcag 3180ctcccaaccc tgtttccgcc cctcggccct cccccagcct gttcggcttc tctgcagccc 3240gcaaggggga gcagactttt gacaaaggac tgcgggcctc gctcaagtcc ctgagccccc 3300agctgaagct gggaggggag gccaggcttt gtgtctgggc atattcgtct gctgatgggg 3360tttggggaag cctggggctt ggggtttggt cgggtggtgc agctagtggc agagcgggat 3420cagaggtggt ggctgcccag cttctgggct gagacaaggg tctgtgcagg ggtttactga 3480agtgggagtg cctttggaat ctgggccggg agcagaaggg agcaaaagct acagtgggag 3540ccagcctagg gcacatggga ggcgtgaggg cagtgctgcc cgtgcagtgt caggtgtgcc 3600agtgccttgg cgggctgcag tgcgtgtgag ggcaccttct aggtgggcca gggatgcagc 3660tatggagata aggcgggctg gggacagaaa caggtgggca cagggcccag gacaccagcg 3720gatggagggc agggtctagc cctgtgctcc tgagcgtcgg ctgcctgggt tcgaggcggt 3780gggtccccgg ccccttgtga tggtgtgtac catgggggag ctcggggaca gggcaagccc 3840gagcatggtg gggctgcagg gtgggtctga agccaggttg ggtgggggtg gtcacaagcc 3900ctgactgcag agggtcaggg gctcctgccc cagtgcctgc ccactttcaa ttcacattgt 3960tttcaacaag gattttcttt atcttcccct acaaatcaag ccaagggagg ggcacagaat 4020ggggaacagg acacaggatc ctaaactcca aggggactgt ccaccgatga acactcagag 4080tggacaccat cttccgtcca cgctgtgccc aggacagctg tccccatcca tgaacacagg 4140gtaaacatct gccgggctcc gcaccagtgg ctccctgggc catgggacag cggcagggct 4200caccacggac agcacgtggc ccagcagccg gccaccctgg cgtcctgggg cctcctcccc 4260tcctctccct ctcaccttgt cacctccacg gagctgcctg tctgggataa tttggggatt 4320ttttttctgg gggataattc ttttgcatga cccctaaaga gcaagccaca ccggtctgct 4380agctaggtgt ccgcggtgtg gtggtggcgg ccgctggcca gcgctgcaag gggtcggctg 4440cccacggtgc tggctggcct cccctcctct ctctttttgc tgagtttcat tgtcttttct 4500ttctgagcct tgtaagtgta caaaaattat tcttattttg ttctgtctcg ggaaactgca 4560aataaaagaa aaacaggaca aactgc 458636666PRTArtificial SequenceCaMKII beta isoform 1 36Met Ala Thr Thr Val Thr Cys Thr Arg Phe Thr Asp Glu Tyr Gln Leu 1 5 10 15 Tyr Glu Asp Ile Gly Lys Gly Ala Phe Ser Val Val Arg Arg Cys Val 20 25 30 Lys Leu Cys Thr Gly His Glu Tyr Ala Ala Lys Ile Ile Asn Thr Lys 35 40 45 Lys Leu Ser Ala Arg Asp His Gln Lys Leu Glu Arg Glu Ala Arg Ile 50 55 60 Cys Arg Leu Leu Lys His Ser Asn Ile Val Arg Leu His Asp Ser Ile 65 70 75 80 Ser Glu Glu Gly Phe His Tyr Leu Val Phe Asp Leu Val Thr Gly Gly 85 90 95 Glu Leu Phe Glu Asp Ile Val Ala Arg Glu Tyr Tyr Ser Glu Ala Asp 100 105 110 Ala Ser His Cys Ile Gln Gln Ile Leu Glu Ala Val Leu His Cys His 115 120 125 Gln Met Gly Val Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu 130 135 140 Ala Ser Lys Cys Lys Gly Ala Ala Val Lys Leu Ala Asp Phe Gly Leu 145 150 155 160 Ala Ile Glu Val Gln Gly Asp Gln Gln Ala Trp Phe Gly Phe Ala Gly 165 170 175 Thr Pro Gly Tyr Leu Ser Pro Glu Val Leu Arg Lys Glu Ala Tyr Gly 180 185 190 Lys Pro Val Asp Ile Trp Ala Cys Gly Val Ile Leu Tyr Ile Leu Leu 195 200 205 Val Gly Tyr Pro Pro Phe Trp Asp Glu Asp Gln His Lys Leu Tyr Gln 210 215 220 Gln Ile Lys Ala Gly Ala Tyr Asp Phe Pro Ser Pro Glu Trp Asp Thr 225 230 235 240 Val Thr Pro Glu Ala Lys Asn Leu Ile Asn Gln Met Leu Thr Ile Asn 245 250 255 Pro Ala Lys Arg Ile Thr Ala His Glu Ala Leu Lys His Pro Trp Val 260 265 270 Cys Gln Arg Ser Thr Val Ala Ser Met Met His Arg Gln Glu Thr Val 275 280 285 Glu Cys Leu Lys Lys Phe Asn Ala Arg Arg Lys Leu Lys Gly Ala Ile 290 295 300 Leu Thr Thr Met Leu Ala Thr Arg Asn Phe Ser Val Gly Arg Gln Thr 305 310 315 320 Thr Ala Pro Ala Thr Met Ser Thr Ala Ala Ser Gly Thr Thr Met Gly 325 330 335 Leu Val Glu Gln Ala Lys Ser Leu Leu Asn Lys Lys Ala Asp Gly Val 340 345 350 Lys Pro Gln Thr Asn Ser Thr Lys Asn Ser Ala Ala Ala Thr Ser Pro 355 360 365 Lys Gly Thr Leu Pro Pro Ala Ala Leu Glu Pro Gln Thr Thr Val Ile 370 375 380 His Asn Pro Val Asp Gly Ile Lys Glu Ser Ser Asp Ser Ala Asn Thr 385 390 395 400 Thr Ile Glu Asp Glu Asp Ala Lys Ala Pro Arg Val Pro Asp Ile Leu 405 410 415 Ser Ser Val Arg Arg Gly Ser Gly Ala Pro Glu Ala Glu Gly Pro Leu 420 425 430 Pro Cys Pro Ser Pro Ala Pro Phe Ser Pro Leu Pro Ala Pro Ser Pro 435 440 445 Arg Ile Ser Asp Ile Leu Asn Ser Val Arg Arg Gly Ser Gly Thr Pro 450 455 460 Glu Ala Glu Gly Pro Leu Ser Ala Gly Pro Pro Pro Cys Leu Ser Pro 465 470 475 480 Ala Leu Leu Gly Pro Leu Ser Ser Pro Ser Pro Arg Ile Ser Asp Ile 485 490 495 Leu Asn Ser Val Arg Arg Gly Ser Gly Thr Pro Glu Ala Glu Gly Pro 500 505 510 Ser Pro Val Gly Pro Pro Pro Cys Pro Ser Pro Thr Ile Pro Gly Pro 515 520 525 Leu Pro Thr Pro Ser Arg Lys Gln Glu Ile Ile Lys Thr Thr Glu Gln 530 535 540 Leu Ile Glu Ala Val Asn Asn Gly Asp Phe Glu Ala Tyr Ala Lys Ile 545 550 555 560 Cys Asp Pro Gly Leu Thr Ser Phe Glu Pro Glu Ala Leu Gly Asn Leu 565 570 575 Val Glu Gly Met Asp Phe His Arg Phe Tyr Phe Glu Asn Leu Leu Ala 580 585 590 Lys Asn Ser Lys Pro Ile His Thr Thr Ile Leu Asn Pro His Val His 595 600 605 Val Ile Gly Glu Asp Ala Ala Cys Ile Ala Tyr Ile Arg Leu Thr Gln 610 615 620 Tyr Ile Asp Gly Gln Gly Arg Pro Arg Thr Ser Gln Ser Glu Glu Thr 625 630 635 640 Arg Val Trp His Arg Arg Asp Gly Lys Trp Gln Asn Val His Phe His 645 650 655 Cys Ser Gly Ala Pro Val Ala Pro Leu Gln 660 665 375820DNAArtificial SequenceCaMKII delta variant 1 37aaaggaggga gtgcgagaga tccacgaagg gacaggcttg gagtcgctag agggaggtgt 60gggaccagcg aggagggggc ttcgccaggg agggggtgct ggcaggcgga gggagcggcg 120ggaggaggcg ccggaggagg agacggaggc ctggggacgg cagaagaggc ttcgcctgag 180ccgagcgctc tttctctcgc cgcgccgtct tgaagccgcg cgggctcgtg agcagcgcga 240ggccgccaag gtgcctcgct tcgccggagc cgctgccgcc cgccggaggg aagccggcct 300cgggcgcgca cgctcgtcgg agccccggcg cgccccgcgc ctgagcctgc tgacagcggc 360cgctgggctc aggctgtccg ctctgggctc cgcggcctcg gccccgctgc actccacctc 420cgccccctcg gactccctcc cctctgcttc tactcctcct gctccagtgc ggatcgtttc 480gcaactgctt gccactcgtc ccgtgcctgg ctgtttttcc atttcccggc cccctcttct 540tgagtacttt accccctgca tttggggaca gggactggaa aaggggcggg tggagcgtcc 600agtggagaag aaggaagcga ggcccgcagg aggaggagga tcggcggact gtggggagga 660gaccccacgc caccctttct ggtcatctcc cctcccgccc cgcccctgcg cacactccct 720cgcgggcgag ctactttcgg accaggaaag taagagcggc cctgggtgac agcgccgcgg 780ggccagtccc ggggttagcc gcgcgtctgc tcgcttctgg tccgtcgcgc tcccagccag 840ggcacagccc ggaccgagga tggcttcgac cacaacctgc accaggttca cggacgagta 900tcagcttttc gaggagcttg gaaagggggc attctcagtg gtgagaagat gtatgaaaat 960tcctactgga caagaatatg ctgccaaaat tatcaacacc aaaaagcttt ctgctaggga 1020tcatcagaaa ctagaaagag aagctagaat ctgccgtctt ttgaagcacc ctaatattgt 1080gcgacttcat gatagcatat cagaagaggg ctttcactac ttggtgtttg atttagttac 1140tggaggtgaa ctgtttgaag acatagtggc aagagaatac tacagtgaag ctgatgccag 1200tcattgtata cagcagattc tagaaagtgt taatcattgt cacctaaatg gcatagttca 1260cagggacctg aagcctgaga atttgctttt agctagcaaa tccaagggag cagctgtgaa 1320attggcagac tttggcttag ccatagaagt tcaaggggac cagcaggcgt ggtttggttt 1380tgctggcaca cctggatatc tttctccaga agttttacgt aaagatcctt atggaaagcc 1440agtggatatg tgggcatgtg gtgtcattct ctatattcta cttgtggggt atccaccctt 1500ctgggatgaa gaccaacaca gactctatca gcagatcaag gctggagctt atgattttcc 1560atcaccagaa tgggacacgg tgactcctga agccaaagac ctcatcaata aaatgcttac 1620tatcaaccct gccaaacgca tcacagcctc agaggcactg aagcacccat ggatctgtca 1680acgttctact gttgcttcca tgatgcacag acaggagact gtagactgct tgaagaaatt 1740taatgctaga agaaaactaa agggtgccat cttgacaact atgctggcta caaggaattt 1800ctcagcagcc aagagtttgt tgaagaaacc agatggagta aaggagtcaa ctgagagttc 1860aaatacaaca attgaggatg aagatgtgaa agcacgaaag caagagatta tcaaagtcac 1920tgaacaactg atcgaagcta tcaacaatgg ggactttgaa gcctacacaa aaatctgtga 1980cccaggcctt actgcttttg aacctgaagc tttgggtaat ttagtggaag ggatggattt 2040tcaccgattc tactttgaaa atgctttgtc caaaagcaat aaaccaatcc acactattat 2100tctaaaccct catgtacatc tggtagggga tgatgccgcc tgcatagcat atattaggct 2160cacacagtac atggatggca gtggaatgcc aaagacaatg cagtcagaag agactcgtgt 2220gtggcaccgc cgggatggaa agtggcagaa tgttcatttt catcgctcgg ggtcaccaac 2280agtacccatc aagtaaatat ttccaggctg tcagcttctt tgttaataca cccatgctaa 2340atttcaacag tgccacttct gcattctctg ttctcaaggc acctggatgg tgaccctggg 2400ccgtcctctc ctcctcttca tgcatgtttc tgagtgcatg aagttgtgaa ggtcctacat 2460gtaatgcata tgtgatgcat catcttatca tatattcctt cctatacatt gtttacactt 2520caactacggg gatgttccac acaaacttaa attactgttg gcaaaacaat agggggagat 2580tagacaaaaa aaaaaatcca caatattcca agtacaactc ttcatcaagt ttctctgtta 2640atgccaagat ttaacagact taagaactat tgttctctga atgacagttg taagagaaat 2700gtaaattttt tagaactctt tgctgttaat ctgttttggt ttgtttggtt tttttttttt 2760tttttaaggt aaaaaaaaaa tacaccttca gtttcctggt gtgatcctgg ttaaaatgga 2820tgatttttca ttgaaagttt tgctgattaa caattaaagt gggatgatat gtgggcaaaa 2880tcacttatga aagtagaagc aagaatcagt tggtttgcta ccacataaag ccatgctgtt 2940tttggtcaaa ctgtgtaaac tggaaaaatt cacatcattt ctgagtttaa tcactttagg 3000atatattcac attgttttgg tgaatttgct gaattgaatt gtttttcttt ctcaaatctg 3060tgatctcttt tctttatcct gtttctttgt tcctttcgtt tgctttctta tttttctttt 3120gttccattct tttcttactt ttttcccttt tccttttttg gggaggctgg ctagtagtgt 3180gtgagaaaag aatagaagtg aaatttgcat aatgaatgta aaagggaaat aaaagtcttt 3240tgaaggtagc tatactagca cttttgatca tcttcagggc ccacaaaaat gttgtcaaga 3300ttttaaaggt ttataattct gcttaagctc tagtttggac ttaggtatcc taactatgtt 3360ggaggtattt gcattgttta aagttaggat aaaagcaagt tcctcctgtg actgcaacgt 3420cttactgatt gggacagttg ccaggaggat accaacttga tagcagaggg ggttttatgc 3480aaacgcactc acctccgcct tggggaatga aagggtcact tctgcatcat cactagctag 3540ttttctagtg ttagagaggc ttacaaatgt ttgccattct cataagtgtt ttgaacttga 3600tctttgtgac ttgtgctttt ttagcttctc tcttgaatca gagtatcatt gtcttcctcc 3660aaggagttag aatttcccag tttaaaacaa aaagggaaat gtcctaggtt ttctttgtgc

3720ttctcatttt tcctttgttg attcaattcc tgtgattttt gttctcttcc ctgaagtgct 3780ttacagtgca tggaatctcc atcattgtta ttttaacgat agtaattcac agtcctcaga 3840agcctatttt taaagcagaa gcaaaaaaga aaaacaaaat aacaaaaaca acccttcctc 3900ttttctctca tctcacctct ctgtgttgat tactaatcat cttagatatt attgctagtg 3960gatgtatggt agatgggttg aagcttttct gataattatt acacaattta aaacaacata 4020tatatttaaa ataaatatat acagtaaata tattgagcca tgttaacctg ccaatgagat 4080ctgtgaaaaa ataatggcct catttttctc tttttaattt cttttaccct tttgtgaagc 4140agctatacgt ggcatacatg tatttaaaga aaaaaaaata gatgtagagt gtttttttta 4200cacttttaac ttagcatgtg gtgttgaagt attactgtag atcaagtttg tcttccgcac 4260taagatgtga ggaaattgtg atttgttctc tccaccacaa atgaattaca catttattat 4320cttctatcat tttgaaacac tgcagtttac catgggacac tgtatatatt tcttgccata 4380atggtaaagg actgattgat atatttaaga gttaataaat ttgtgatttc tgctgacagt 4440gcgtccatct ttatttcttc agaagaggta ctgtatgtat gcctgcatag tgctggccag 4500tgtcaagggc agtgtgtcct actctggtct catttagtac ataacaattt gcacttggtg 4560agaatggcaa gttaattgtt ctctgtgagc aaaacaatgg tctcttctgg gaaaatgttg 4620ctgagaacaa tatagttaac aactaagact cctaaaagct tctctaaact gtaccctcca 4680atccagcctt cacatggctg cttttttttt ttttttttaa tacgaacctg tccttgtaac 4740actttgatgt tatcatttct gggatacagg caagcacccc agctcctgct actccccagc 4800ttgaacttga gcatacatgg atgctcagct tcttttgatt tgctaaaaac atcacacttg 4860ctcacatgcc tgtttatgct gttcatgttg tttatgtttc ttacctagaa taaatagtct 4920cttcccctac ttcttttccc gacttcttac tttttcctaa gattcagtgt acagcatcat 4980gctccacagc aaaccttcct aggccctatt ctgggcttgc cttccctctc aaaacctaca 5040taatagattg tatttacctc tcctgtcaac cacattgttt tgaaaatata tttctatttg 5100tgtctcctct actgcagtat aatgtctcca tgggcaagaa ctgtgtattc atcattgcat 5160tcctaaaccc aaaccaaggc caggaatgga gatatcattg ataaatagtt gttgaattga 5220ggccaagccc ttttgataac agaagcctca aggggtaccc agatagtcct tgttttaatg 5280atgggttctc tcaccactgt cttgatgctc tgagcaagtt acctcttccc tctgaccctc 5340agtttccata tttgtaaaat gagaataaac ataccaactt aataaagata ttgtgaggat 5400taatgggtac agagtgacta gaatgatatt tgatagaaat taaatggtag cagtataact 5460attctgatca ctgacattaa tattcctatt gttattattc tttgctcacg agggtataca 5520actcttgttt tgctgttggg ctgccctctt tatgtaggtt tactgttaat gctgaggata 5580tactcggact caaatgtctc agcagaaggc tgagagacac caaatgaagt ggtcatctag 5640ctgaatgtag gaaaaatgaa atgtagtagc aaatcagtat attctaagga aattttcaag 5700gaatattaat cttcacccaa attttgaatt tttatgtaaa aaattataat ttaagggtaa 5760acatagatga cacagctttc gagtgatttc attgaataaa attctactga cttctatgaa 582038478PRTArtificial SequenceCaMKII delta isoform 1 38Met Ala Ser Thr Thr Thr Cys Thr Arg Phe Thr Asp Glu Tyr Gln Leu 1 5 10 15 Phe Glu Glu Leu Gly Lys Gly Ala Phe Ser Val Val Arg Arg Cys Met 20 25 30 Lys Ile Pro Thr Gly Gln Glu Tyr Ala Ala Lys Ile Ile Asn Thr Lys 35 40 45 Lys Leu Ser Ala Arg Asp His Gln Lys Leu Glu Arg Glu Ala Arg Ile 50 55 60 Cys Arg Leu Leu Lys His Pro Asn Ile Val Arg Leu His Asp Ser Ile 65 70 75 80 Ser Glu Glu Gly Phe His Tyr Leu Val Phe Asp Leu Val Thr Gly Gly 85 90 95 Glu Leu Phe Glu Asp Ile Val Ala Arg Glu Tyr Tyr Ser Glu Ala Asp 100 105 110 Ala Ser His Cys Ile Gln Gln Ile Leu Glu Ser Val Asn His Cys His 115 120 125 Leu Asn Gly Ile Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu 130 135 140 Ala Ser Lys Ser Lys Gly Ala Ala Val Lys Leu Ala Asp Phe Gly Leu 145 150 155 160 Ala Ile Glu Val Gln Gly Asp Gln Gln Ala Trp Phe Gly Phe Ala Gly 165 170 175 Thr Pro Gly Tyr Leu Ser Pro Glu Val Leu Arg Lys Asp Pro Tyr Gly 180 185 190 Lys Pro Val Asp Met Trp Ala Cys Gly Val Ile Leu Tyr Ile Leu Leu 195 200 205 Val Gly Tyr Pro Pro Phe Trp Asp Glu Asp Gln His Arg Leu Tyr Gln 210 215 220 Gln Ile Lys Ala Gly Ala Tyr Asp Phe Pro Ser Pro Glu Trp Asp Thr 225 230 235 240 Val Thr Pro Glu Ala Lys Asp Leu Ile Asn Lys Met Leu Thr Ile Asn 245 250 255 Pro Ala Lys Arg Ile Thr Ala Ser Glu Ala Leu Lys His Pro Trp Ile 260 265 270 Cys Gln Arg Ser Thr Val Ala Ser Met Met His Arg Gln Glu Thr Val 275 280 285 Asp Cys Leu Lys Lys Phe Asn Ala Arg Arg Lys Leu Lys Gly Ala Ile 290 295 300 Leu Thr Thr Met Leu Ala Thr Arg Asn Phe Ser Ala Ala Lys Ser Leu 305 310 315 320 Leu Lys Lys Pro Asp Gly Val Lys Glu Ser Thr Glu Ser Ser Asn Thr 325 330 335 Thr Ile Glu Asp Glu Asp Val Lys Ala Arg Lys Gln Glu Ile Ile Lys 340 345 350 Val Thr Glu Gln Leu Ile Glu Ala Ile Asn Asn Gly Asp Phe Glu Ala 355 360 365 Tyr Thr Lys Ile Cys Asp Pro Gly Leu Thr Ala Phe Glu Pro Glu Ala 370 375 380 Leu Gly Asn Leu Val Glu Gly Met Asp Phe His Arg Phe Tyr Phe Glu 385 390 395 400 Asn Ala Leu Ser Lys Ser Asn Lys Pro Ile His Thr Ile Ile Leu Asn 405 410 415 Pro His Val His Leu Val Gly Asp Asp Ala Ala Cys Ile Ala Tyr Ile 420 425 430 Arg Leu Thr Gln Tyr Met Asp Gly Ser Gly Met Pro Lys Thr Met Gln 435 440 445 Ser Glu Glu Thr Arg Val Trp His Arg Arg Asp Gly Lys Trp Gln Asn 450 455 460 Val His Phe His Arg Ser Gly Ser Pro Thr Val Pro Ile Lys 465 470 475 393821DNAArtificial SequenceCaMKII gamma variant 1 39agtctcgcgg tgctgccggg ctcagccccg tctcctcctc ttgctccctc ggccgggcgg 60cggtgactgt gcaccgacgt cggcgcgggc tgcaccgccg cgtccgcccg cccgccagca 120tggccaccac cgccacctgc acccgtttca ccgacgacta ccagctcttc gaggagcttg 180gcaagggtgc tttctctgtg gtccgcaggt gtgtgaagaa aacctccacg caggagtacg 240cagcaaaaat catcaatacc aagaagttgt ctgcccggga tcaccagaaa ctagaacgtg 300aggctcggat atgtcgactt ctgaaacatc caaacatcgt gcgcctccat gacagtattt 360ctgaagaagg gtttcactac ctcgtgtttg accttgttac cggcggggag ctgtttgaag 420acattgtggc cagagagtac tacagtgaag cagatgccag ccactgtata catcagattc 480tggagagtgt taaccacatc caccagcatg acatcgtcca cagggacctg aagcctgaga 540acctgctgct ggcgagtaaa tgcaagggtg ccgccgtcaa gctggctgat tttggcctag 600ccatcgaagt acagggagag cagcaggctt ggtttggttt tgctggcacc ccaggttact 660tgtcccctga ggtcttgagg aaagatccct atggaaaacc tgtggatatc tgggcctgcg 720gggtcatcct gtatatcctc ctggtgggct atcctccctt ctgggatgag gatcagcaca 780agctgtatca gcagatcaag gctggagcct atgatttccc atcaccagaa tgggacacgg 840taactcctga agccaagaac ttgatcaacc agatgctgac cataaaccca gcaaagcgca 900tcacggctga ccaggctctc aagcacccgt gggtctgtca acgatccacg gtggcatcca 960tgatgcatcg tcaggagact gtggagtgtt tgcgcaagtt caatgcccgg agaaaactga 1020agggtgccat cctcacgacc atgcttgtct ccaggaactt ctcagctgcc aaaagcctat 1080tgaacaagaa gtcggatggc ggtgtcaagc cacagagcaa caacaaaaac agtctcgtaa 1140gcccagccca agagcccgcg cccttgcaga cggccatgga gccacaaacc actgtggtac 1200acaacgctac agatgggatc aagggctcca cagagagctg caacaccacc acagaagatg 1260aggacctcaa agctgccccg ctccgcactg ggaatggcag ctcggtgcct gaaggacgga 1320gctcccggga cagaacagcc ccctctgcag gcatgcagcc ccagccttct ctctgctcct 1380cagccatgcg aaaacaggag atcattaaga ttacagaaca gctgattgaa gccatcaaca 1440atggggactt tgaggcctac acgaagattt gtgatccagg cctcacttcc tttgagcctg 1500aggcccttgg taacctcgtg gaggggatgg atttccataa gttttacttt gagaatctcc 1560tgtccaagaa cagcaagcct atccatacca ccatcctaaa cccacacgtc cacgtgattg 1620gggaggacgc agcgtgcatc gcctacatcc gcctcaccca gtacatcgac gggcagggtc 1680ggcctcgcac cagccagtca gaagagaccc gggtctggca ccgtcgggat ggcaagtggc 1740tcaatgtcca ctatcactgc tcaggggccc ctgccgcacc gctgcagtga gctcagccac 1800aggggcttta ggagattcca gccggaggtc caaccttcgc agccagtggc tctggagggc 1860ctgagtgaca gcggcagtcc tgtttgtttg aggtttaaaa caattcaatt acaaaagcgg 1920cagcagccaa tgcacgcccc tgcatgcagc cctcccgccc gcccttcgtg tctgtctctg 1980ctgtaccgag gtgtttttta catttaagaa aaaaaaaaaa gaaaaaaaga ttgtttaaaa 2040aaaaaaggaa tccataccat gatgcgtttt aaaaccaccg acagcccttg ggttggcaag 2100aaggcaggag tatgtatgag gtccatcctg gcatgagcag tggctcaccc accggccttg 2160aagaggtgag cttggcctct ctggtcccca tggacttagg gggaccaggc aagaactctg 2220acagagcttt gggggccgtg atgtgattgc agctcctgag gtggcctgct taccccaggt 2280ctaggaatga acttctttgg aacttgcata ggcgcctaga atggggctga tgagaacatc 2340gtgaccatca gacctacttg ggagagaacg cagagctccc agcctgctgt ggaggcagct 2400gagaagtggt ggcctcagga ctgagagccc ggacgttgct gtactgtctt gtttagtgta 2460gaagggaaga gaattggtgc tgcagaagtg tacccgccat gaagccgatg agaaacctcg 2520tgttagtctg acatgcactc actcatccat ttctatagga tgcacaatgc atgtgggccc 2580taatattgag gccttatccc tgcagctagg agggggaggg gttgttgctg ctttgcttcg 2640tgttttcttc taacctggca aggagagagc caggccctgg tcagggctcc cgtgccgcct 2700ttggcggttc tgtttctgtg ctgatctgga ccatctttgt cttgcctttt cacggtagtg 2760gtccccatgc tgaccctcat ctgggcctgg gccctctgcc aagtgcccct gtgggatggg 2820aggagtgagg cagtgggaga agaggtggtg gtcgtttcta tgcattcagg ctgcctttgg 2880ggctgcctcc cttcttattc ttccttgctg cacgtccatc tcttttcctg tctttgagat 2940tgacctgact gctctggcaa gaagaagagg tgtccttaca gaggcctctt tactgaccaa 3000ctgaagtata gacttactgc tggacaatct gcatgggcat cacccctccc cgcatgtaac 3060ccaaaagagg tgtccagagc caaggcttct accttcattg tccctctctg tgctcaagga 3120gttccattcc aggaggaaga gatctatacc ctaagcagat agcaaagaag ataatggagg 3180agcaattggt catggccttg gtttccctca aaacaacgct gcagatttat ctgcacaaac 3240atctccactt ttgggggaaa ggtgggtaga ttccagttcc ctggactacc ttcaggaggc 3300acgagagctg ggagaagagg caaagctaca ggtttacttg ggagccagct gagaagagag 3360cagactcaca ggtgctggtg cttggattta gccaggctcc tccgagcacc tcatgcatgt 3420cccagcccct gggccctagc cctttcctgc cctgcagtct gcagtgccag cacgcaaatc 3480ccttcaccac agggtttcgt tttgctggct tgaagacaaa tggtcttaga attcattgag 3540acccatagct tcatatggct gctccagccc cacttcttag cattcttact cctcttctgg 3600ggctaatgtc agcatctata gacaatagac tattaaaaaa tcacctttta aacaagaaac 3660ggaaggcatt tgatgcagaa tttttgcatg acaacataga aataatttaa aaatagtgtt 3720tgttctgaat gttggtagac ccttcatagc tttgttacaa tgaaaccttg aactgaaaat 3780atttaataaa ataaccttta aacagtcaaa aaaaaaaaaa a 382140556PRTArtificial SequenceCaMKII gamma isoform 1 40Met Ala Thr Thr Ala Thr Cys Thr Arg Phe Thr Asp Asp Tyr Gln Leu 1 5 10 15 Phe Glu Glu Leu Gly Lys Gly Ala Phe Ser Val Val Arg Arg Cys Val 20 25 30 Lys Lys Thr Ser Thr Gln Glu Tyr Ala Ala Lys Ile Ile Asn Thr Lys 35 40 45 Lys Leu Ser Ala Arg Asp His Gln Lys Leu Glu Arg Glu Ala Arg Ile 50 55 60 Cys Arg Leu Leu Lys His Pro Asn Ile Val Arg Leu His Asp Ser Ile 65 70 75 80 Ser Glu Glu Gly Phe His Tyr Leu Val Phe Asp Leu Val Thr Gly Gly 85 90 95 Glu Leu Phe Glu Asp Ile Val Ala Arg Glu Tyr Tyr Ser Glu Ala Asp 100 105 110 Ala Ser His Cys Ile His Gln Ile Leu Glu Ser Val Asn His Ile His 115 120 125 Gln His Asp Ile Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu 130 135 140 Ala Ser Lys Cys Lys Gly Ala Ala Val Lys Leu Ala Asp Phe Gly Leu 145 150 155 160 Ala Ile Glu Val Gln Gly Glu Gln Gln Ala Trp Phe Gly Phe Ala Gly 165 170 175 Thr Pro Gly Tyr Leu Ser Pro Glu Val Leu Arg Lys Asp Pro Tyr Gly 180 185 190 Lys Pro Val Asp Ile Trp Ala Cys Gly Val Ile Leu Tyr Ile Leu Leu 195 200 205 Val Gly Tyr Pro Pro Phe Trp Asp Glu Asp Gln His Lys Leu Tyr Gln 210 215 220 Gln Ile Lys Ala Gly Ala Tyr Asp Phe Pro Ser Pro Glu Trp Asp Thr 225 230 235 240 Val Thr Pro Glu Ala Lys Asn Leu Ile Asn Gln Met Leu Thr Ile Asn 245 250 255 Pro Ala Lys Arg Ile Thr Ala Asp Gln Ala Leu Lys His Pro Trp Val 260 265 270 Cys Gln Arg Ser Thr Val Ala Ser Met Met His Arg Gln Glu Thr Val 275 280 285 Glu Cys Leu Arg Lys Phe Asn Ala Arg Arg Lys Leu Lys Gly Ala Ile 290 295 300 Leu Thr Thr Met Leu Val Ser Arg Asn Phe Ser Ala Ala Lys Ser Leu 305 310 315 320 Leu Asn Lys Lys Ser Asp Gly Gly Val Lys Pro Gln Ser Asn Asn Lys 325 330 335 Asn Ser Leu Val Ser Pro Ala Gln Glu Pro Ala Pro Leu Gln Thr Ala 340 345 350 Met Glu Pro Gln Thr Thr Val Val His Asn Ala Thr Asp Gly Ile Lys 355 360 365 Gly Ser Thr Glu Ser Cys Asn Thr Thr Thr Glu Asp Glu Asp Leu Lys 370 375 380 Ala Ala Pro Leu Arg Thr Gly Asn Gly Ser Ser Val Pro Glu Gly Arg 385 390 395 400 Ser Ser Arg Asp Arg Thr Ala Pro Ser Ala Gly Met Gln Pro Gln Pro 405 410 415 Ser Leu Cys Ser Ser Ala Met Arg Lys Gln Glu Ile Ile Lys Ile Thr 420 425 430 Glu Gln Leu Ile Glu Ala Ile Asn Asn Gly Asp Phe Glu Ala Tyr Thr 435 440 445 Lys Ile Cys Asp Pro Gly Leu Thr Ser Phe Glu Pro Glu Ala Leu Gly 450 455 460 Asn Leu Val Glu Gly Met Asp Phe His Lys Phe Tyr Phe Glu Asn Leu 465 470 475 480 Leu Ser Lys Asn Ser Lys Pro Ile His Thr Thr Ile Leu Asn Pro His 485 490 495 Val His Val Ile Gly Glu Asp Ala Ala Cys Ile Ala Tyr Ile Arg Leu 500 505 510 Thr Gln Tyr Ile Asp Gly Gln Gly Arg Pro Arg Thr Ser Gln Ser Glu 515 520 525 Glu Thr Arg Val Trp His Arg Arg Asp Gly Lys Trp Leu Asn Val His 530 535 540 Tyr His Cys Ser Gly Ala Pro Ala Ala Pro Leu Gln 545 550 555 412168DNAArtificial SequenceCaMKIV 41ctctctcgct cctgcgttcg caggcggcgg ctggcggccg gcttctcgct cgggcagcgg 60cggcggcggc ggcggcggct tccggagtcc cgctgcgaag atgctcaaag tcacggtgcc 120ctcctgctcc gcctcgtcct gctcttcggt caccgccagt gcggccccgg ggaccgcgag 180cctcgtcccg gattactgga tcgacggctc caacagggat gcgctgagcg atttcttcga 240ggtggagtcg gagctgggac ggggtgctac atccattgtg tacagatgca aacagaaggg 300gacccagaag ccttatgctc tcaaagtgtt aaagaaaaca gtggacaaaa aaatcgtaag 360aactgagata ggagttcttc ttcgcctctc acatccaaac attataaaac ttaaagagat 420atttgaaacc cctacagaaa tcagtctggt cctagaactc gtcacaggag gagaactgtt 480tgataggatt gtggaaaagg gatattacag tgagcgagat gctgcagatg ccgttaaaca 540aatcctggag gcagttgctt atctacatga aaatgggatt gtccatcgtg atctcaaacc 600agagaatctt ctttatgcaa ctccagcccc agatgcacca ctcaaaatcg ctgattttgg 660actctctaaa attgtggaac atcaagtgct catgaagaca gtatgtggaa ccccagggta 720ctgcgcacct gaaattctta gaggttgtgc ctatggacct gaggtggaca tgtggtctgt 780aggaataatc acctacatct tactttgtgg atttgaacca ttctatgatg aaagaggcga 840tcagttcatg ttcaggagaa ttctgaattg tgaatattac tttatctccc cctggtggga 900tgaagtatct ctaaatgcca aggacttggt cagaaaatta attgttttgg atccaaagaa 960acggctgact acatttcaag ctctccagca tccgtgggtc acaggtaaag cagccaattt 1020tgtacacatg gataccgctc aaaagaagct ccaagaattc aatgcccggc gtaagcttaa 1080ggcagcggtg aaggctgtgg tggcctcttc gcgcctggga agtgccagca gcagccatgg 1140cagcatccag gagagccaca aggctagccg agacccttct ccaatccaag atggcaacga 1200ggacatgaaa gctattccag aaggagagaa aattcaaggc gatggggccc aagccgcagt 1260taagggggca caggctgagc tgatgaaggt gcaagcctta gagaaagtta aaggtgcaga 1320tataaatgct gaagaggccc ccaaaatggt gcccaaggca gtggaggatg ggataaaggt 1380ggctgacctg gaactagagg agggcctagc agaggagaag ctgaagactg tggaggaggc 1440agcagctccc agagaagggc aaggaagctc tgctgtgggt tttgaagttc cacagcaaga 1500tgtgatcctg ccagagtact aaacagcttc cttcagatct ggaagccaaa caccggcatt 1560ttatgtactt tgtccttcag caagaaaggt gtggaagcat gatatgtact atagtgattc 1620tgtttttgag gtgcaaaaaa catacatata taccagttgg taattctaac ttcaatgcat 1680gtgactgctt tatgaaaata atagtgtctt ctatggcatg taatggatac ctaataccga 1740tgagttaaat cttgcaagtt aacacaacgt aacacttaaa agcatacatt ttcagcaacc 1800agtggcacat atttgaagtg aatagtagca aattgttttt gctttgaaaa tctagccatc 1860ctacatcctt tggatttctt cacaaggcag taattccttt gaactactgc ttagctaata 1920ctaggtagtg ctaaaagaca tgttcccata acttttacaa cattttactt tttatcattg 1980atgtgttcaa actgtttaca aggagatgct tatagatgat agttgtacat atgtgcaaaa 2040aaaaatccac ttgcaatggt aagaaattga agtatcctta aaggccatga agccatatgt 2100ccctaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160aaaaaaaa

216842473PRTArtificial SequenceCaMKIV 42Met Leu Lys Val Thr Val Pro Ser Cys Ser Ala Ser Ser Cys Ser Ser 1 5 10 15 Val Thr Ala Ser Ala Ala Pro Gly Thr Ala Ser Leu Val Pro Asp Tyr 20 25 30 Trp Ile Asp Gly Ser Asn Arg Asp Ala Leu Ser Asp Phe Phe Glu Val 35 40 45 Glu Ser Glu Leu Gly Arg Gly Ala Thr Ser Ile Val Tyr Arg Cys Lys 50 55 60 Gln Lys Gly Thr Gln Lys Pro Tyr Ala Leu Lys Val Leu Lys Lys Thr 65 70 75 80 Val Asp Lys Lys Ile Val Arg Thr Glu Ile Gly Val Leu Leu Arg Leu 85 90 95 Ser His Pro Asn Ile Ile Lys Leu Lys Glu Ile Phe Glu Thr Pro Thr 100 105 110 Glu Ile Ser Leu Val Leu Glu Leu Val Thr Gly Gly Glu Leu Phe Asp 115 120 125 Arg Ile Val Glu Lys Gly Tyr Tyr Ser Glu Arg Asp Ala Ala Asp Ala 130 135 140 Val Lys Gln Ile Leu Glu Ala Val Ala Tyr Leu His Glu Asn Gly Ile 145 150 155 160 Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Tyr Ala Thr Pro Ala 165 170 175 Pro Asp Ala Pro Leu Lys Ile Ala Asp Phe Gly Leu Ser Lys Ile Val 180 185 190 Glu His Gln Val Leu Met Lys Thr Val Cys Gly Thr Pro Gly Tyr Cys 195 200 205 Ala Pro Glu Ile Leu Arg Gly Cys Ala Tyr Gly Pro Glu Val Asp Met 210 215 220 Trp Ser Val Gly Ile Ile Thr Tyr Ile Leu Leu Cys Gly Phe Glu Pro 225 230 235 240 Phe Tyr Asp Glu Arg Gly Asp Gln Phe Met Phe Arg Arg Ile Leu Asn 245 250 255 Cys Glu Tyr Tyr Phe Ile Ser Pro Trp Trp Asp Glu Val Ser Leu Asn 260 265 270 Ala Lys Asp Leu Val Arg Lys Leu Ile Val Leu Asp Pro Lys Lys Arg 275 280 285 Leu Thr Thr Phe Gln Ala Leu Gln His Pro Trp Val Thr Gly Lys Ala 290 295 300 Ala Asn Phe Val His Met Asp Thr Ala Gln Lys Lys Leu Gln Glu Phe 305 310 315 320 Asn Ala Arg Arg Lys Leu Lys Ala Ala Val Lys Ala Val Val Ala Ser 325 330 335 Ser Arg Leu Gly Ser Ala Ser Ser Ser His Gly Ser Ile Gln Glu Ser 340 345 350 His Lys Ala Ser Arg Asp Pro Ser Pro Ile Gln Asp Gly Asn Glu Asp 355 360 365 Met Lys Ala Ile Pro Glu Gly Glu Lys Ile Gln Gly Asp Gly Ala Gln 370 375 380 Ala Ala Val Lys Gly Ala Gln Ala Glu Leu Met Lys Val Gln Ala Leu 385 390 395 400 Glu Lys Val Lys Gly Ala Asp Ile Asn Ala Glu Glu Ala Pro Lys Met 405 410 415 Val Pro Lys Ala Val Glu Asp Gly Ile Lys Val Ala Asp Leu Glu Leu 420 425 430 Glu Glu Gly Leu Ala Glu Glu Lys Leu Lys Thr Val Glu Glu Ala Ala 435 440 445 Ala Pro Arg Glu Gly Gln Gly Ser Ser Ala Val Gly Phe Glu Val Pro 450 455 460 Gln Gln Asp Val Ile Leu Pro Glu Tyr 465 470 431522DNAArtificial SequencePNCK/CaMKI beta variant 2 43agcagctgta ggcctgggat gtggaggcgt gtatgcggtg gcctcgctgg gaggcgtccg 60tctggggaca tgctgctgct gaagaaacac acggaggaca tcagcagcgt ctacgagatc 120cgcgagaggc tcggctcggg tgccttctcc gaggtggtgc tggcccagga gcggggctcc 180gcacacctcg tggccctcaa gtgcatcccc aagaaggccc tccggggcaa ggaggccctg 240gtggagaacg agatcgcagt gctccgtagg atcagtcacc ccaacatcgt cgctctggag 300gatgtccacg agagcccttc ccacctctac ctggccatgg aactggtgac gggtggcgag 360ctgtttgacc gcatcatgga gcgcggctcc tacacagaga aggatgccag ccatctggtg 420ggtcaggtcc ttggcgccgt ctcctacctg cacagcctgg ggatcgtgca ccgggacctc 480aagcccgaaa acctcctgta tgccacgccc tttgaggact cgaagatcat ggtctctgac 540tttggactct ccaaaatcca ggctgggaac atgctaggca ccgcctgtgg gacccctgga 600tatgtggccc cagagctctt ggagcagaaa ccctacggga aggccgtaga tgtgtgggcc 660ctgggcgtca tctcctacat cctgctgtgt gggtaccccc ccttctacga cgagagcgac 720cctgagctct tcagccagat cctgagggcc agctatgagt ttgactctcc tttctgggat 780gacatctcag aatcagccaa agacttcatc cggcaccttc tggagcgaga cccccagaag 840aggttcacct gccaacaggc cttgcggcac ctttggatct ctggggacac agccttcgac 900agggacatct taggctctgt cagtgagcag atccggaaga actttgctcg gacacactgg 960aagcgagcct tcaatgccac ctcgttcctg cgccacatcc ggaagctggg gcagatccca 1020gagggcgagg gggcctctga gcagggcatg gcccgccaca gccactcagg cctccgtgct 1080ggccagcccc ccaagtggtg atgcccaggc agatgccgag gccaagtgga ctgaccccca 1140gatttccttc ccttggatgc tttcggtccc ctcccccaac ccctccccct ggggctggcc 1200tctgctggat tttgagattt gagggtgtgg cgcatggcgc tggggttgga atggggcacc 1260cccaagtctg tccccaggct ctgccctgcc tgggggcagt ggctcccctc ccctgttgcc 1320tctcccgccc ctgccccccc cgccccgcca aaagccgagg gggtgctggc aggcgggcct 1380caggggctgt ctttcctgca cggctgttgt gtgcttcgct gagtgtgggt ggtcctgctt 1440gtgtcatggt catggccttc cagccccctc cagttttccc caaaccaata aagaaagata 1500cagcaaaaaa aaaaaaaaaa aa 152244360PRTArtificial SequencePNCK/CaMKI beta isoform b 44Met Trp Arg Arg Val Cys Gly Gly Leu Ala Gly Arg Arg Pro Ser Gly 1 5 10 15 Asp Met Leu Leu Leu Lys Lys His Thr Glu Asp Ile Ser Ser Val Tyr 20 25 30 Glu Ile Arg Glu Arg Leu Gly Ser Gly Ala Phe Ser Glu Val Val Leu 35 40 45 Ala Gln Glu Arg Gly Ser Ala His Leu Val Ala Leu Lys Cys Ile Pro 50 55 60 Lys Lys Ala Leu Arg Gly Lys Glu Ala Leu Val Glu Asn Glu Ile Ala 65 70 75 80 Val Leu Arg Arg Ile Ser His Pro Asn Ile Val Ala Leu Glu Asp Val 85 90 95 His Glu Ser Pro Ser His Leu Tyr Leu Ala Met Glu Leu Val Thr Gly 100 105 110 Gly Glu Leu Phe Asp Arg Ile Met Glu Arg Gly Ser Tyr Thr Glu Lys 115 120 125 Asp Ala Ser His Leu Val Gly Gln Val Leu Gly Ala Val Ser Tyr Leu 130 135 140 His Ser Leu Gly Ile Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu 145 150 155 160 Tyr Ala Thr Pro Phe Glu Asp Ser Lys Ile Met Val Ser Asp Phe Gly 165 170 175 Leu Ser Lys Ile Gln Ala Gly Asn Met Leu Gly Thr Ala Cys Gly Thr 180 185 190 Pro Gly Tyr Val Ala Pro Glu Leu Leu Glu Gln Lys Pro Tyr Gly Lys 195 200 205 Ala Val Asp Val Trp Ala Leu Gly Val Ile Ser Tyr Ile Leu Leu Cys 210 215 220 Gly Tyr Pro Pro Phe Tyr Asp Glu Ser Asp Pro Glu Leu Phe Ser Gln 225 230 235 240 Ile Leu Arg Ala Ser Tyr Glu Phe Asp Ser Pro Phe Trp Asp Asp Ile 245 250 255 Ser Glu Ser Ala Lys Asp Phe Ile Arg His Leu Leu Glu Arg Asp Pro 260 265 270 Gln Lys Arg Phe Thr Cys Gln Gln Ala Leu Arg His Leu Trp Ile Ser 275 280 285 Gly Asp Thr Ala Phe Asp Arg Asp Ile Leu Gly Ser Val Ser Glu Gln 290 295 300 Ile Arg Lys Asn Phe Ala Arg Thr His Trp Lys Arg Ala Phe Asn Ala 305 310 315 320 Thr Ser Phe Leu Arg His Ile Arg Lys Leu Gly Gln Ile Pro Glu Gly 325 330 335 Glu Gly Ala Ser Glu Gln Gly Met Ala Arg His Ser His Ser Gly Leu 340 345 350 Arg Ala Gly Gln Pro Pro Lys Trp 355 360 452242DNAArtificial SequenceCaMKI delta/CKLiK variant 2 45agccggcgcg cggcggcggc aggaagtctg tgcccgagaa cagcagaaat aagagccagg 60gagggaccgc ggccgcggcg gcggcggcga gagcgaaaga ggaaactgca gaggaggaag 120ctgcgccgca gcccgagccg cccggcatcc ccgccgcctc tgcgcccgcg ccgcgccccc 180ggcgccccct ccccagcgcg cccccggccg ctcctccgcg ccgcgctcgt cggccatggc 240ccgggagaac ggcgagagca gctcctcctg gaaaaagcaa gctgaagaca tcaagaagat 300cttcgagttc aaagagaccc tcggaaccgg ggccttttcc gaagtggttt tagctgaaga 360gaaggcaact ggcaagctct ttgctgtgaa gtgtatccct aagaaggcgc tgaagggcaa 420ggaaagcagc atagagaatg agatagccgt cctgagaaag attaagcatg aaaatattgt 480tgccctggaa gacatttatg aaagcccaaa tcacctgtac ttggtcatgc agctggtgtc 540cggtggagag ctgtttgacc ggatagtgga gaaggggttt tatacagaga aggatgccag 600cactctgatc cgccaagtct tggacgccgt gtactatctc cacagaatgg gcatcgtcca 660cagagacctc aagcccgaaa atctcttgta ctacagtcaa gatgaggagt ccaaaataat 720gatcagtgac tttggattgt caaaaatgga gggcaaagga gatgtgatgt ccactgcctg 780tggaactcca ggctatgtcg ctcctgaagt cctcgcccag aaaccttaca gcaaagccgt 840tgactgctgg tccatcggag tgattgccta catcttgctc tgcggctacc ctccttttta 900tgatgaaaat gactccaagc tctttgagca gatcctcaag gcggaatatg agtttgactc 960tccctactgg gatgacatct ccgactctgc aaaagacttc attcggaacc tgatggagaa 1020ggacccgaat aaaagataca cgtgtgagca ggcagctcgg cacccatgga tcgctggtga 1080cacagccctc aacaaaaaca tccacgagtc cgtcagcgcc cagatccgga aaaactttgc 1140caagagcaaa tggagacaag catttaatgc cacggccgtc gtcagacata tgagaaaact 1200acacctcggc agcagcctgg acagttcaaa tgcaagtgtt tcgagcagcc tcagtttggc 1260cagccaaaaa gactgtctgg caccttccac gctctgtagt ttcatttctt cttcgtcggg 1320ggtctcagga gttggagccg agcggagacc caggcccacc actgtgacgg cagtgcactc 1380tggaagcaag tgactggccc tggaggtggg gcccggggtc ggggctgggg aaggggagcc 1440ccagggtcgc cagagccgcg agccactcca gcgagacccc accttgcatg gtgccccttc 1500ctgcatagga ctggaagacc gaagtttttt tatggccata ttttctactg caattctgaa 1560gtgttcattt ctcacaaact gtactgactc gaggggcgct gatttcatag gatctggtgc 1620tgtatatacg aatcttgcaa agctctaact gaacggacct tcttattcct ctcccctaac 1680accatcgttt ccactcttct cagtgtaggt aaccgtctat ggtgtgtttt ttcattaatg 1740acaaaaaaaa aaaggtttca actggattat ttaaatattg gtaaatattg tgcattaggg 1800tttgtttttc cttttaagaa gtatgtcctt tgtatctcta agttacatga cctatatctt 1860ttcctcttta atagtagttt tatgttaacc tttaagagat ttgtttttcc tcaaaggaga 1920atttaaaggt attttttaaa attctaataa gaggatcagc cgggtgcaat gactcatgcc 1980tgtaatccca gcacgttggg aggccaagtc gggcggatca caaggtcagg agatcaaggc 2040catcctggcc aacatggtga aaccccacgt ctactaaaaa tacaaaaaat tagccgggcg 2100tggtggcaca cacctgtagt cccggctact cgggaggctg aggcaggaga attgcttgaa 2160cccgggagac ggaggttgca gtgagctgag atcgtgccac tgcactccag cctgggtgac 2220agagcaagac tctgtctcaa aa 224246385PRTArtificial SequenceCaMKI delta/CKLiK isoform 2 46Met Ala Arg Glu Asn Gly Glu Ser Ser Ser Ser Trp Lys Lys Gln Ala 1 5 10 15 Glu Asp Ile Lys Lys Ile Phe Glu Phe Lys Glu Thr Leu Gly Thr Gly 20 25 30 Ala Phe Ser Glu Val Val Leu Ala Glu Glu Lys Ala Thr Gly Lys Leu 35 40 45 Phe Ala Val Lys Cys Ile Pro Lys Lys Ala Leu Lys Gly Lys Glu Ser 50 55 60 Ser Ile Glu Asn Glu Ile Ala Val Leu Arg Lys Ile Lys His Glu Asn 65 70 75 80 Ile Val Ala Leu Glu Asp Ile Tyr Glu Ser Pro Asn His Leu Tyr Leu 85 90 95 Val Met Gln Leu Val Ser Gly Gly Glu Leu Phe Asp Arg Ile Val Glu 100 105 110 Lys Gly Phe Tyr Thr Glu Lys Asp Ala Ser Thr Leu Ile Arg Gln Val 115 120 125 Leu Asp Ala Val Tyr Tyr Leu His Arg Met Gly Ile Val His Arg Asp 130 135 140 Leu Lys Pro Glu Asn Leu Leu Tyr Tyr Ser Gln Asp Glu Glu Ser Lys 145 150 155 160 Ile Met Ile Ser Asp Phe Gly Leu Ser Lys Met Glu Gly Lys Gly Asp 165 170 175 Val Met Ser Thr Ala Cys Gly Thr Pro Gly Tyr Val Ala Pro Glu Val 180 185 190 Leu Ala Gln Lys Pro Tyr Ser Lys Ala Val Asp Cys Trp Ser Ile Gly 195 200 205 Val Ile Ala Tyr Ile Leu Leu Cys Gly Tyr Pro Pro Phe Tyr Asp Glu 210 215 220 Asn Asp Ser Lys Leu Phe Glu Gln Ile Leu Lys Ala Glu Tyr Glu Phe 225 230 235 240 Asp Ser Pro Tyr Trp Asp Asp Ile Ser Asp Ser Ala Lys Asp Phe Ile 245 250 255 Arg Asn Leu Met Glu Lys Asp Pro Asn Lys Arg Tyr Thr Cys Glu Gln 260 265 270 Ala Ala Arg His Pro Trp Ile Ala Gly Asp Thr Ala Leu Asn Lys Asn 275 280 285 Ile His Glu Ser Val Ser Ala Gln Ile Arg Lys Asn Phe Ala Lys Ser 290 295 300 Lys Trp Arg Gln Ala Phe Asn Ala Thr Ala Val Val Arg His Met Arg 305 310 315 320 Lys Leu His Leu Gly Ser Ser Leu Asp Ser Ser Asn Ala Ser Val Ser 325 330 335 Ser Ser Leu Ser Leu Ala Ser Gln Lys Asp Cys Leu Ala Pro Ser Thr 340 345 350 Leu Cys Ser Phe Ile Ser Ser Ser Ser Gly Val Ser Gly Val Gly Ala 355 360 365 Glu Arg Arg Pro Arg Pro Thr Thr Val Thr Ala Val His Ser Gly Ser 370 375 380 Lys 385 472349DNAArtificial SequenceCaMKK2 promoter region 47agggaggtgg ctgagcatca aataaatggt acagtccttg ctttaagaca tcagagagag 60gagcgggcag ggagggcctg gctcacagca tttcaggaaa ctatcaggaa aagcaactct 120tgagctaggg tttggatttg taggggcact tcccaacctt tcttacttaa ttggctttta 180agtttacaca gctgtgtgta tatctaagtt tctgcctgtg tgagctacag tgttctaata 240aaacacaggc aatttagtaa catgatgtaa acatacagaa ttgtttaaca agtaccagga 300aaagacatcc agtaacagga aaggacaccc aaatctattt tggttacctt agtaaagcag 360ggttgccaaa ctatttattg cccataagct aaatctgtca tgccacctgt tttttatggc 420ctgctaagaa tggtttttac gtttttcttt tttctttttg tcaagatggg tcttgccatg 480ttgtccaggc tggtctcaaa cttctaggct caagtgatcc tgccccctcg gcctcccaga 540gtgctaagat tgtaatcacg cgtgtaggtg taagccacca cacctggcct ttcacatttt 600tcaatggtta aataaaaacc aaaacagtaa tattttgtga cttatgaaaa tcatatgaaa 660ttcagttttc agtgtctgta aataaagttt tattggaaca ctgccgcatg cattcattta 720ctatttcctg tggctgctag gatggcaggg ttgagtagct gcattacaga gcctatggct 780tgcaaagcct aaaatattta ctatctggcc ctttacaaag tttgctgacc ctgccttaaa 840gtgccccaga taatctccac caggcctgat acatattttt tttggggggg gggggggaaa 900tatgagaata tagaaaaata cgggactatg catcccccat tcagaattga ctattgctaa 960cagtttgcca tttgcctcac agccatttat aagtactaaa aggaataaaa ctttacaaca 1020ttccttatgc tccctgtccc gttctgtgct tttttccttc tttgtggcac actgtcatga 1080atttaatgta tattcttctc tttgacttta aaaatcttgt atgtaagcat gtgtccatga 1140acagtatcta ctgcatggga ttttaaggta cacacaaatt gtacatcatg ttgtattctg 1200caacttgctg ttttactcaa cattatgttt ttgggatttc cttatgtcag tacagatcca 1260gtgcacccct tttatctatt agatggtatt ccatcaaaca actataatga gtttcattta 1320tccattcctt tgatgaacat ataggaagtt gttcccaggt ggtttttttt ttgtgttttt 1380gttttgtttt gttttgtttt tgagatggag tcttgctctg tcacccaggc tggagtgcag 1440tggcatgatc tcggctcact gcaacctctg cctcccgggt ttcaagcgat tctcctgcct 1500cagcctcctg agtagttggg attacaggcg cgcgccacca tgcctggcta atttttgcat 1560ttttttttta agtagagacg gggtttcacc atgctggtca ggctggactt gaactcctga 1620cttcgtgatc tgcccacctt ggcctcccaa agtgctggga ttacaggcgt gagccactgt 1680gcccagactt tttttttttt ttttgaaaca gtcttggtct gtcgtccagg ctggagggta 1740gtggtgcgat ctctctgcct cccggttcaa gtggttctcg tgcttcagcc tcctgagtag 1800ctgggattac gggcaccgcc accacaccgg gctaattttt gtatttttga tagagatggg 1860gtttcaccat gctggccagg ctagtctcga acttctggcc tcaagtgatc tctccgcccg 1920ggcctcccaa agtgctggga tcataggcgt gagccaccat gcccggcctc aggtttttgc 1980catttccagc aaatgcatgc acgttgagta acatcagaag aggtggatgg ccgagcatgt 2040tggctcatgc ctataatccc agcactttgg gaggcagagg caggaggatc acttgagccc 2100aggagtttga gaccagcctg ggcaacatag ggagaactct gtctctacaa aaaatagaaa 2160aaattagcca ggtgtggtgg tgtgtgcctg tagtcccagc tactcaggag gctgaggctg 2220gaggatcacc tgagcctggg gaggtcgagg gtgcagcgag ccgtgatcgt gctactgcac 2280tccagcctgg gcaacacaga gagaccctgt ctcaaaacaa acaaacaaac aaacaaacaa 2340acaaacaaa 23494821DNAArtificial SequenceSynthetic 48ggacatgttg ctggccaata a 214918DNAArtificial SequenceSynthetic 49gggcccgaga ccagtgtt 185020DNAArtificial SequenceSynthetic 50tccagaccag cccgacatag 205120DNAArtificial SequenceSynthetic 51caggggtgca gcttgatttc 205221DNAArtificial SequenceSynthetic 52tggccttatc ctgcctggta t

215321DNAArtificial SequenceSynthetic 53aggagtcgat gctgatccca a 215420DNAArtificial SequenceSynthetic 54ccatggcacc ttcagacttt 205520DNAArtificial SequenceSynthetic 55actgggccat atgaggatca 205622DNAArtificial SequenceSynthetic 56ctcagttcct ggagaaagat gg 225721DNAArtificial SequenceSynthetic 57cccagtcaat tcatgtttgc c 215822DNAArtificial SequenceSynthetic 58atggaatatg tgtctggagg tg 225921DNAArtificial SequenceSynthetic 59tggtttcagg tctcgatgaa c 216020DNAArtificial SequenceSynthetic 60gcacagtttg cacacctgaa 206120DNAArtificial SequenceSynthetic 61gctttggatt taggccctgt 206220DNAArtificial SequenceSynthetic 62aacaggaaag gacacccaaa 206321DNAArtificial SequenceSynthetic 63aaaccattct tagcaggcca t 216431DNAArtificial SequenceSynthetic 64cgctagcagg gaggtggctg agcatcaaat a 316532DNAArtificial SequenceSynthetic 65caaagctttg agacagggtc tctctgtgtt gc 326631DNAArtificial SequenceSynthetic 66cgctagcgaa ttgcaactgt gagaccaggc a 316732DNAArtificial SequenceSynthetic 67caaagcttgt ggccttgggc aaatgacttg at 326831DNAArtificial SequenceSynthetic 68cgctagcatc aagtcatttg cccaaggcca c 316932DNAArtificial SequenceSynthetic 69caaagcttaa cactgtagct cacacaggca ga 327031DNAArtificial SequenceSynthetic 70cgctagcatc aagtcatttg cccaaggcca c 317132DNAArtificial SequenceSynthetic 71caaagcttta tttgatgctc agccacctcc ct 327231DNAArtificial SequenceSynthetic 72cgctagcagg gaggtggctg agcatcaaat a 317332DNAArtificial SequenceSynthetic 73caaagcttaa atgtgaaagg ccaggtgtgg tg 327431DNAArtificial SequenceSynthetic 74cgctagctgc ctgtgtgagc tacagtgttc t 317532DNAArtificial SequenceSynthetic 75caaagcttaa atgtgaaagg ccaggtgtgg tg 327631DNAArtificial SequenceSynthetic 76cgctagcagg gaggtggctg agcatcaaat a 317732DNAArtificial SequenceSynthetic 77caaagcttaa cactgtagct cacacaggca ga 327831DNAArtificial SequenceSynthetic 78cgctagccac cacacctggc ctttcacatt t 317932DNAArtificial SequenceSynthetic 79caaagcttgc actttaaggc agggtcagca aa 328031DNAArtificial SequenceSynthetic 80cgctagcgtt tcaagcgatt ctcctgcctc a 318132DNAArtificial SequenceSynthetic 81caaagctttc acgcctgtaa tcccagcact tt 328231DNAArtificial SequenceSynthetic 82cgctagcagg gaggtggctg agcatcaaat a 318332DNAArtificial SequenceSynthetic 83caaagcttta cacgggtgat tacaatctta gc 328431DNAArtificial SequenceSynthetic 84cgctagcagg gaggtggctg agcatcaaat a 318532DNAArtificial SequenceSynthetic 85caaagctttg gacaacatgg caagacccat ct 328631DNAArtificial SequenceSynthetic 86cgctagcagg gaggtggctg agcatcaaat a 318732DNAArtificial SequenceSynthetic 87caaagcttct ggatctcttt tcctggtact tg 328831DNAArtificial SequenceSynthetic 88cgctagcagg gaggtggctg agcatcaaat a 318932DNAArtificial SequenceSynthetic 89caaagcttac actgtagctc acacaggcag aa 329031DNAArtificial SequenceSynthetic 90cgctagcagg gaggtggctg agcatcaaat a 319132DNAArtificial SequenceSynthetic 91caaagcttta caaatccaaa ccctagctca ag 329231DNAArtificial SequenceSynthetic 92cgctagcagg gaggtggctg agcatcaaat a 319332DNAArtificial SequenceSynthetic 93caaagctttg ctgtgagcca ggccctccct gc 329431DNAArtificial SequenceSynthetic 94cgctagcagg gaggtggctg agcatcaaat a 319532DNAArtificial SequenceSynthetic 95caaagcttct gcccgctcct ctctctgatg tc 329625DNAArtificial SequenceSynthetic 96catacagaat tgtttaacaa gtacc 259729DNAArtificial SequenceSynthetic 97taaattgcct gtgttttatt agaacactg 299824DNAArtificial SequenceSynthetic 98acctgtaatc ccagcacttt cgga 249924DNAArtificial SequenceSynthetic 99cgatctcgga tcactgcaac ctct 2410022DNAArtificial SequenceSynthetic 100tgagccgagc cgagccgagc tg 2210123DNAArtificial SequenceSynthetic 101tcacagggct tctggctttc gct 2310224DNAArtificial SequenceSynthetic 102agctgaggac ttgaaggacc tgat 2410323DNAArtificial SequenceSynthetic 103aggttgtctt cgctgccttg ctt 2310424DNAArtificial SequenceSynthetic 104acctgggctg gctatgtgta tgaa 2410525RNAArtificial SequenceSynthetic 105ggaccaucug uacauggugu ucgaa 2510625RNAArtificial SequenceSynthetic 106gcugacuuug gugugagcaa ugaau 2510725RNAArtificial SequenceSynthetic 107caccugggca uggaguccuu cauug 2510825RNAArtificial SequenceSynthetic 108cagaugucuu cugccuguua uaacu 2510925RNAArtificial SequenceSynthetic 109cccauccuga aagaguacca uucuu 2511025RNAArtificial SequenceSynthetic 110cccucaauau uuaaauccuu cugug 2511125RNAArtificial SequenceSynthetic 111accaugauug augaugaagc cuuaa 2511225RNAArtificial SequenceSynthetic 112gauggugaau uucugagaac uaguu 2511325RNAArtificial SequenceSynthetic 113ccguaugaca uuauggcuga aguuu 2511425DNAArtificial SequenceSynthetic 114ggagatacag ctctagataa gaata 2511525DNAArtificial SequenceSynthetic 115ccataggtgt catcgcctac atctt 25

Claims

1. A method of diagnosing prostate cancer in a subject comprising: a) obtaining a sample from the subject; b) determining an amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the sample from the subject; and c) comparing the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit from the sample from the subject to an amount of the CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in a control sample; wherein the subject is diagnosed as having prostate cancer when the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the sample from the subject is greater than the amount in the control sample.

2. The method of claim 1, wherein the determining comprises detecting the amount of mRNA expression.

3. The method of claim 1, wherein the determining comprises detecting the amount of at least one protein of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the samples.

4. The method of claim 1, wherein the sample from the subject comprises prostate tissue.

5. The method of claim 3, wherein the detecting comprises an antibody that specifically binds to a C-terminal portion of the amino acid sequence encoded by CaMKKβ, CaMKKβ splice variant 2, or CaMKKβ splice variant 7.

6-16. (canceled)

17. A method of screening a test compound for its anti-cancer activity comprising: i) contacting CaMKKβ and a substrate therefor in the presence of the test compound, under conditions that allow for CaMKKβ-dependent phosphorylation of the substrate, and ii) determining the level of phosphorylation of the substrate resulting from step (i) and comparing the level with a level of phosphorylation of the substrate obtained in the absence of the test compound, wherein a reduction in the level of phosphorylation of the substrate in the presence of the test compound indicates that the test compound has said anti-cancer activity.

18. The method according to claim 17 wherein said substrate is a peptide substrate.

19. The method according to claim 17 wherein the CaMKKβ and the substrate are present in a cell free system.

20. The method according to claim 17 wherein the CaMKKβ and the substrate are present in a cell.

21. The method according to claim 17 wherein, in step (i), the CaMKKβ and the substrate are contacted with the test compound in the presence of calmodulin and calcium under conditions such that CaMKKβ-dependent phosphorylation of the substrate can be effected.

22-26. (canceled)

27. A method of treating prostate cancer in a subject, comprising administering to the subject an effective amount of a compound that inhibits activity of at least one of CaMKKβ, CaMKKβ splice variant 2, or CaMKKβ splice variant 7.

28. The method of claim 27, wherein the compound is selected from an inhibitory RNA or an antibody that specifically binds to a C-terminal portion of CaMKKβ, CaMKKβ splice variant 2, or CaMKKβ splice variant 7.

29. The method of claim 27, wherein the compound has Formula III: ##STR00007## wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R_7a, R₈, R₉, R₁₀, and R₁₁ are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, heterocyclo, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, alkoxy, halo, mercapto, azido, cyano, formyl, carboxylic acid, hydroxyl, nitro, acyl, aryloxy, alkylthio, amino, alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfoxyl, sulfonyl, sulfonate, sulfonic acid, sulfonamide, urea, alkoxylacylamino, and aminoacyloxy; or a pharmaceutically acceptable salt or prodrug thereof.

30. The method of claim 29, wherein the compound is STO-609.

31. The method of claim 1, wherein the method further comprises administering an effective amount of a second agent selected from anti-androgens, Selective Androgen Receptor Modulators (SARMs), Selective Androgen Receptor Degraders (SARDs), CYP17 inhibitors, suphatase inhibitors, Src inhibitors, anti-estrogens, estrogens, Selective Estrogen Receptor Modulators (SERMs), Selective Estrogen Receptor Degraders (SERDs), ERb antagonists, aromatase inhibitors, and a vaccine.

32. The method of claim 31, wherein the second agent is selected from MDV3100, ARN-509, bicalutamide, and flutamide.

33-41. (canceled)

42. A method of inhibiting proliferation of a prostate cancer cell in a subject, the method comprising administering to the subject an effective amount of an inhibitor of AMPK, AMPKα1 subunit, or AMPKα2 subunit, or any combination thereof.

43. The method of claim 42 wherein the inhibitor is an inhibitory RNA.

44. A method of determining the efficacy of therapy in a patient being treated for prostate cancer, the method comprising: a) obtaining a series of samples from the subject; a) determining an amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the series of samples from the subject, where the samples are taken from the subject at different time points during the therapy; and b) comparing the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 from the series of samples from the subject; wherein when the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 in the series of samples is about the same or increases the therapy is determined to be not effective.

45-47. (canceled)

48. A nucleic acid molecule comprising a sequence that binds under stringent conditions to a region that is about 2.3 kb upstream (5') relative to a CaMKKβtranscriptional start site.

49. The nucleic acid molecule of claim 48 wherein the region is from about -2231 to about -1632 upstream (5') relative to a CaMKKβ transcriptional start site.

50. The nucleic acid molecule of claim 48 wherein the region is from about -2019 to about -1632 upstream (5') relative to a CaMKKβ transcriptional start site.

51. The nucleic acid molecule of claim 50 comprising the sequence 5'-GTA ACA TGA TGT AAA-3'.

52. The nucleic acid molecule of claim 48, wherein the nucleic acid molecule is selected from decoy RNA, dsRNA, a nucleic acid aptamer, an antisense nucleic acid molecule, and an enzymatic nucleic acid molecule.

53. A nucleic acid molecule comprising a double stranded siRNA that down-regulates expression of a CaMKKβ gene via RNA interference (RNAi), wherein: a) each strand of the siRNA molecule is independently about 18 to about 28 nucleotides in length; and b) one strand of the siRNA molecule comprises a sequence that binds under stringent conditions to a CaMKKβ RNA of the CaMKKβ gene and directs cleavage of the CaMKKβ RNA via RNA interference.

54. The nucleic acid molecule of claim 53 comprising the siRNA sequences of SEQ ID NOs. 105-113.

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