Synthesis of cannflavins and other prenylflavonoids

Abstract

This disclosure relates to engineered prenyltransferases useful in the biocatalytic synthesis of prenylflavonoids, such as Cannflavins A, B, and C. This disclosure also relates to the compositions, cell-free systems, and cell-free processes comprising the engineered prenyltransferases that can be used in the preparation of prenylflavonoids. The engineered prenyltransferases of the present disclosure comprise amino acid substitutions that provide for the prenylation of a range of prenylflavonoid precursor, such as the substrate chrysoeriol, using a range of prenyl donor compounds, such as GPP and DMAPP. The engineered prenyltransferases also comprise amino acid substitutions that allow for prenylation at high temperatures, thereby facilitating higher yield and efficiency prenylflavonoid production in cell- free processes.

Description

SYNTHESIS OF CANNFLAVINS AND OTHER PRENYLFLAVONOIDSTECHNICAL FIELD

[0001] This disclosure relates to engineered prenyltransferases useful in the biocatalytic synthesis of prenylflavonoids, such as Cannflavins A, B, and C. This disclosure also relates to the compositions, cell-free systems, and cell-free processes comprising the engineered prenyltransferases that can be used in the preparation of prenylflavonoids.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The inventions disclosed herein were made with government support under grant award number 1R43GM153108 from the National Institute of General Medical Sciences of the U.S. National Institute of Health. The government has certain rights in the inventions.REFERENCE TO SEQUENCE LISTING

[0003] The official copy of the Sequence Listing is submitted concurrently with the specification as an ST26 formatted .xml file via EFS-Web, with a file name of “15041-006PV1.xml”, a creation date of February 6, 2025, and a size of 378,787 bytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.BACKGROUND

[0004] Cannflavins are a group of prenylated flavonoids derived from Cannabis sativa that have garnered significant interest due to their potent anti-inflammatory and antioxidant properties. The Cannabis plant predominantly makes three different cannflavins, Cannflavin A, Cannflavin B, and Cannflavin C, which differ in prenyl group identity or regiospecificity as shown in the structures depicted below.Cannflavin ACannflavin B

[0005] Cannflavins A, B, and C (shown above) are the primary compounds within this group, with Cannflavin A (CF-A) and Cannflavin B (CF-B) being the most extensively studied. These three Cannflavin compounds exhibit distinctive pharmacological activities that differentiate them from cannabinoids like THC, CBD, and other phytocannabinoids (e.g. CBG, THCV, CBDV, CBN). When comparing the anti-inflammatory properties of cannflavins to those of cannabinoids like THC and CBD, cannflavins appear to act by a different mechanism. While common cannabinoids (THC, CBD, etc.) primarily interact with the endocannabinoid system or voltage gated ion channels to exert their effects, cannflavins inhibit pro-inflammatory pathways directly by targeting the activity of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1. This results in a more potent suppression of inflammatory mediators such as prostaglandin E2 (PGE2) and leukotrienes (Werzet al., 2014).Consequently, cannflavins have demonstrated stronger anti-inflammatory effects in vitro compared to THC, CBD, and CBG, highlighting their potential as more effective antiinflammatory agents.

[0006] Yet, the biological effects of cannflavins extend beyond their anti-inflammatory properties. Studies have shown that CF-A and CF-B possess antioxidative, neuroprotective, and anticancer activities. CF-A, in particular, has demonstrated the ability to protect neuronal cells from oxidative stress-induced damage, making it a promising candidate for treating neurodegenerative diseases such as Alzheimer's and Parkinson's (Baban et al., 2021).Additionally, cannflavins have been investigated for their potential to enhance the efficacy ofconventional cancer therapies by modulating cell signaling pathways involved in tumor growth and metastasis (Yasmin-Karim et al., 2018; Faiz et al., 2024).

[0007] The biosynthesis of cannflavins A and B in Cannabis sativa follows the general flavonoid biosynthetic pathway depicted in FIG. 1 , beginning with the conversion of phenylalanine to flavonoids via the phenylpropanoid pathway. An example of such biosynthesis was disclosed by Rea et al., “Biosynthesis of cannflavins A and B from Cannabis sativa L,” Phytochemistry, Volume 164, 2019, Pages 162-171. First, phenylalanine undergoes several enzymatic transformations to p-coumaric acid, which is then activated to its CoA thioester and converted into naringenin chaicone by chaicone synthase (CHS). Naringenin chaicone is subsequently converted to naringenin by chaicone isomerase (CHI), the core structure for various flavonoids (Jez et al., 2000).

[0008] To eventually produce cannflavins, the core structure naringenin is modified via hydroxylation and oxidation to luteolin, followed by methylation to chrysoeriol, and finally sitespecific prenylation. In the case of CF-A and CF-B, an O-methyltransferase catalyzes the regio-specific methylation of a hydroxyl group of luteolin at the 3’-hydroxyl (Choi et al., 2004) to yield chrysoeriol. Numerous methyltransferases from a variety of plants besides Cannabis have been demonstrated to methylate luteolin to produce chrysoeriol. The methylation of small molecules and flavonoids like luteolin modulates the pharmacological properties of the core flavonoid structure by affecting solubility, stability, and bioavailability. Additionally, in the Cannabis plant, a membrane-bound prenyltransferase plays a crucial role by adding a prenyl group regio-specifically to the flavonoid core, enhancing and modulating biological activity further. For instance, in the synthesis of CF-A, an integral membrane prenyltransferase catalyzes the regio-specific transfer of geranyl pyrophosphate (GPP) to the flavonoid core, specifically at the C-6 position. Similarly, an integral membrane prenyltransferase can also catalyze the regio-specific transfer of dimethylallyl pyrophosphate (DMAPP) to yield CF-B. This prenylation step is critical for the biological efficacy of CF-A and CF-B, as the prenyl group significantly enhances the lipophilicity of the flavonoid and subsequently its ability to interact with cell membranes and proteins (Flores-Sanchez & Verpoorte, 2008). Both the methylation and the prenylation reactions are critical for producing bioactive cannflavins with the desired therapeutic effects and are the major handles to tailor their drug properties, especially when those reactions are performed regio-specifically. Regio-specific methylation or prenylation is difficult to achieve via organic chemical synthesis, but such regio-specificity can often be achieved efficiently in high regio-specific excess via enzyme catalysis.

[0009] The total chemical synthesis of cannflavins is challenging. The primary challenges in the chemical synthesis of cannflavins are achieving the correct prenylation regioselectivity to ensure the prenyl group is added at the desired position and ensuring regiospecific methylation of the correct hydroxyl group. Recent synthetic approaches have focused onoptimizing flavonoid scaffold modification (methylation and prenylation) to achieve higher yields and purity, addressing challenges in regioselectivity and stereoselectivity that are critical for producing bioactive compounds (Choi et al., 2004 & Minassi et al., 2008). To our knowledge, however, no commercially viable chemical route to any cannflavin exists.Accordingly, there is a need for alternative enzymatic routes that can produce cannflavins more efficiently and with greater purity.SUMMARY

[0010] This summary is intended to introduce the subject matter of the present disclosure, but does not cover each and every embodiment, combination, or variation that is contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0011] This disclosure relates to engineered prenyltransferases useful in the biocatalytic synthesis of prenylflavonoids, such as the Cannflavins A, B, and C. The engineered prenyltransferases of the present disclosure comprise amino acid substitutions, as compared to a naturally occurring enzyme comprising the amino acid sequence of SEQ ID NO: 2, and referred to herein as “scPT”. These amino acid substitutions confer the ability of the engineered scPT to prenylate a range of prenylflavonoid precursor substrates, including chrysoeriol, using a range of prenyl donor compounds, including GPP and DMAPP. The present disclosure also provides an engineered scPT with further amino acid substitutions that confer high temperature stability, thereby facilitating higher yield and higher efficiency when utilized in cell-free biocatalytic systems and processes for the production of prenylflavonoids, such as Cannflavins A, B, and C. Accordingly, the present disclosure also provides compositions, cell-free systems, and cell-free processes that include an engineered scPT.

[0012] In at least one embodiment, the present disclosure provides an engineered scPT comprising an amino acid sequence of at least 80% identity to SEQ ID NO: 2 and at least one amino acid mutation as compared to SEQ ID NO: 2 at a position corresponding to position of SEQ ID NO: 2 selected from V47, S51, M129, Q161, S177, F213, S214, C230, I234, G286, V294, Q295, and L298; optionally, wherein the amino acid mutation is selected from V47I, S511, S51T, M129A, M129C, M129Q, Q161A, Q161E, Q161L, Q161Y, S177G, S177T, F213A, F213M, S214A, S214L, S214W, C230A, C230T, I234M, G286A, V294A, V294E, V294I, V294T, Q295D, L298M, and L298Y. In at least one embodiment, the amino acid sequence comprises a set of amino acid mutations as compared to SEQ ID NO: 2 at a set of positions corresponding to positions of SEQ ID NO: 2 selected from: (a) Q161, S214, C230, G286, and V294; (b) Q161, S177, S214, C230, G286, and V294; (c) Q161, S177, F213, S214, C230, G286, V294, and L298; (d) S51, M129, Q161, S177, S214, C230, V294; (e) V47,S177, C230, V294, and Q295; and (f) S51, M129, Q161, S177, S214, C230, V294. In at least one embodiment, the set of amino acid mutations as compared to SEQ ID NO: 2 are selected from: (a) Q161A, S214A, C230T, G286A, V294T; (b) Q161A, S177T, S214A, C230T, G286A, V294T; (c) Q161A, S177T, F213A, S214W, C230A, G286A, V294A, L298Y; (d) S51T, M129A, Q161L, S177G, S214A, C230T, V294E; (e) V47I, S177T, C230T, V294I, Q295D; and (f) S51T, M129C, Q161Y, S177T, S214A, C230T, V294E.

[0013] In at least one embodiment, the present disclosure provides an engineered scPT comprising an amino acid sequence of at least 90% identity to SEQ ID NO: 2 and at least one amino acid mutation as compared to SEQ ID NO: 2 at a position corresponding to position of SEQ ID NO: 2 selected from: V47, S64, M106, K119, Y121, F123, M162, S164, K169, N173, Y175, Y216, K284, Y288, and Q295; optionally, wherein the mutation is selected from: V47I, S64L, S64V, M106A, K119A, Y121F, F123Y, M162A, S164A, K169N, K169R, N173L, Y175F, Y216A, K284L, Y288F, and Q295D. In at least one embodiment, the amino acid sequence comprises a set amino acid mutations as compared to SEQ ID NO: 2 selected from: (a) V47L, V49T, S51E, S64L, S66Q, M106S, K119A, Y121F, F123Y, M162G, S164A, K169N, Y216A, K284L, Y288M, Q295M; (b) V47L, V49T, D110G, K119A, Y121L, K169N, K284L, Y288M, Q295M; (c) S51A, D62G, S64Y, S66Q, M106S, A108G, D110A, K118V, K119A, Y121T, F123L, S164A, K169N, N173L, Y175F, Y216T, K284L, Y288F; (d) S51E, S64L, M106A, K119A, Y121F, F123Y, M162G, S164A, K169N, N173L, Y175F, Y216A, K284L; (e) D62G, S64Y, S66Q, M106S, A108G, D110A, K118V, K119A, F123L, S164A, K169N, N173L, Y175F, K284L, Y288F; (f) S64V, K119A, Y121F, S164A, K169R, Y216A, K284L; and (g) S64L, K119A, K169N, K284L;S51A, D62G, S64C, S66Q, M106S, K118V, K119Q, Y121F, F123L, S164A, K169R, N173L, Y175F, Y216T, K284L, Y288F.

[0014] In at least one embodiment, the present disclosure provides an engineered scPT comprising an amino acid sequence of at least 90% identity to SEQ ID NO: 2 and at least one amino acid mutation as compared to SEQ ID NO: 2 at a position corresponding to position of SEQ ID NO: 2 selected from: S64, M106, Y121, F123, M129, Q161, M162, S164, K169, F213, S214, V271, V294, Q295, and L298, optionally, wherein the mutation is selected from: S64V, M106A, Y121F, F123M, M129C, Q161G, Q161I, Q161K, Q161L, Q161S, M162A, S164A, K169N, K169R, F213C, F213H, F213S, S214M, S214Y, V271A, V294K, Q295A, and L298N. In at least one embodiment, the amino acid sequence comprises a set of amino acid mutations as compared to SEQ ID NO: 2 selected from: (a) V49I, S51A, Q161A, S177T, F213A, S214A, C230T, G286A, V294T, L298N; (b) V49I, S51A, M129C, Q161A, S177T, F213A, S214A, C230T, G286A, V294T, L298N; (c) V49I, M129C, Q161A, S177T, F213A, S214A, C230T, G286A, V294T, L298N; (d) S51A, Q161A, S177T, F213A, S214A, C230T, G286A, V294T S51A, M129A, Q161A, S177T, M129A, Q161A, S177T, S214A, C230T,G286A, V294T; (e) Q161A, S177T, S214A, C230T, G286A, V294T; and (f) F213C, S214A, C230T, V271A, G286A, V294T, L298N.

[0015] In at least one embodiment, the present disclosure provides an engineered scPT wherein the amino acid sequence further comprises an amino acid mutation as compared to SEQ ID NO: 2 at a position corresponding to a position of SEQ ID NO: 2 selected from: A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301K, F302Y, D303A. In at least one embodiment, the amino acid sequence comprises a set of amino acid mutations as compared to SEQ ID NO: 2 selected from: (a) A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301K, F302Y, D303A; (b) A5E, C25V, T69V, Q99A, A181E, K251R, G297E, A301K, F302Y, D303A; (c) A4Q, E80A, A133E, A137K, E150P, A153K, R154K, A181E, Q182E, E185A, A186P, E246P; (d) A4Q, D28E, A133E, A137K, E150P, A153K, R154K, A181D, Q182E, E185S, A186D, E246P; (e) M14I, A24P, Y31W, T69P, T77I, V911, T98I, S136A, S214A, E222D, G224S, C230T, N236T, G286A, V294T, G297K; (f) C25V, T69V, Q99A, A181E, K251R, H253T, G297E, A301K, F302Y; and (g) C25V, T69V, Q99A, A181P, K251R, H253T, G297E, A301K, F302Y, D303N.

[0016] In at least one embodiment of an engineered scPT of the present disclosure, the engineered scPT comprises an amino acid sequence having at least 50% sequence identity, at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 87.5% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to an amino acid sequence having a SEQ ID NO: selected from even-numbered SEQ ID NOs: 4-194.

[0017] In at least one embodiment of an engineered scPT of the present disclosure, the engineered scPT has activity capable of converting chrysoeriol and a prenyl donor compound to a prenylflavonoid. In at least one embodiment, the engineered scPT is capable of converting chrysoeriol and a prenyl donor compound to a prenylflavonoid with a conversion rate of at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87.5%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In at least one embodiment, the prenylflavonoid is a cannflavin; optionally, wherein the cannflavin is selected from Cannflavin A, Cannflavin B, Cannflavin C, and a combination thereof.

[0018] In at least one embodiment of an engineered scPT of the present disclosure, the engineered scPT has activity capable of converting chrysoeriol and a prenyl donor compound to a cannflavin in a purity of at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the total product compounds.

[0019] In at least one embodiment of an engineered scPT of the present disclosure, the engineered scPT is active for at least 1 hour at a temperature 40 °C or greater, 45 °C or greater, 50 °C or greater, 55 °C or greater, 60 °C or greater, 65 °C or greater, 70 °C or greater, or 75 °C or greater.

[0020] In at least one embodiment of an engineered scPT of the present disclosure, the engineered scPT is active for at least 1 hour at a temperature of between about 50 °C and about 100 °C, between about 50 °C and about 90 °C, between about 50 °C and about 80 °C, between about 60 °C and about 80 °C, or between about 55 °C and about 75 °C.

[0021] In at least one embodiment, the present disclosure provides a composition for the production of a flavonoid comprising an engineered scPT of the present disclosure. In at least one embodiment of the compositions of the present disclosure, the flavonoid is a prenylflavonoid. In at least one embodiment of the compositions of the present disclosure, the flavonoid is a cannflavin; optionally, wherein the cannflavin is Cannflavin A, Cannflavin B, Cannflavin C, or a combination thereof.

[0022] In at least one embodiment of the compositions of the present disclosure, the composition further comprises a flavonoid precursor compound; optionally, wherein the flavonoid precursor compound is chrysoeriol.

[0023] In at least one embodiment of the compositions of the present disclosure, the composition further comprises a prenyl donor compound; optionally, wherein the prenyl donor comprises a prenyl alcohol. In at least one embodiment, wherein the prenyl alcohol is selected from isoprenol, prenol, an a / p unsaturated alcohol derivative, a p / y unsaturated alcohol derivative, or a mixture thereof. In at least one embodiment, the prenyl donor is selected from dimethylallyl pyrophosphate (DMAPP), and geranyl pyrophosphate (GPP).

[0024] In at least one embodiment of the compositions of the present disclosure, the composition further comprises luteolin.

[0025] In at least one embodiment of the compositions of the present disclosure, the composition further comprises a methyltransferase; optionally, wherein the methyltransferase is roMT-9.

[0026] In at least one embodiment of the compositions of the present disclosure, the composition further comprises an enzyme selected from tkMAT, gsPPase, gsMtn, bsLuxS, and a combination thereof.

[0027] In at least one embodiment of the compositions of the present disclosure, the composition further comprises methionine and methionine adenosyltransferase.

[0028] In at least one embodiment of the compositions of the present disclosure, the composition further comprises S-adenosyl methionine (SAM) and a SAM-dependent methyltransferase (MT).

[0029] In at least one embodiment of the compositions of the present disclosure, the composition further comprises adenosine triphosphate (ATP).

[0030] In at least one embodiment of the compositions of the present disclosure, the composition is substantially free of cells.

[0031] In at least one embodiment, the present disclosure provided a method for the biosynthesis of a prenylflavonoid, wherein the method comprises: contacting chrysoeriol under suitable reaction conditions with an engineered scPT and a prenyl donor compound, whereby the chrysoeriol is converted to a prenylflavonoid.

[0032] In at least one embodiment of the method, the method further comprises: contacting an alcohol under suitable reaction conditions with a path of enzymes comprising an ThiM (e.g., ecThiM), an IPK (e.g., mtIPK) and / or an IDI (e.g., eclDI) and an FPPS_S82F (e.g., gsFPPS), whereby the alcohol is converted to a prenyl donor compound. In at least one embodiment, the alcohol is selected from isoprenol, prenol, an a / p unsaturated alcohol derivative, a p / y unsaturated alcohol derivative, or a combination thereof. In at least one embodiment, the alcohol is a combination of alcohols, wherein the combination is selected from: isoprenol and prenol; isoprenol and an a / p unsaturated alcohol derivative; prenol and a p / y unsaturated alcohol derivative; prenol and an a / p unsaturated alcohol derivative; and an a / p unsaturated alcohol derivative and a p / y unsaturated alcohol derivative.

[0033] In at least one embodiment of the method, the prenyl donor compound is dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), or a combination thereof.

[0034] In at least one embodiment of the method, the method further comprises: contacting luteolin under suitable reaction conditions with a SAM-dependent methyltransferase (“MT”) and S-adenosyl-methionine (“SAM”), whereby the chrysoeriol is produced.

[0035] In at least one embodiment of the method, the method further comprises contacting from methionine, adenosine triphosphate (ATP), and a methionine adenosyltransferase, whereby the SAM is produced.

[0036] In at least one embodiment of the method, the method further comprises: contacting naringenin under suitable reaction conditions with oxygen and the enzymes “F3’H” and “FNS”, whereby the luteolin is produced.

[0037] In at least one embodiment of the method, the method further comprises: contacting phenylalanine under suitable reaction conditions with ATP, malonyl-CoA, oxygen, and the enzymes “PAL,” “C4H,” “4CL,” “CHS,” and “CHI,” whereby the naringenin is produced.

[0038] In at least one embodiment of the method, the prenyl donor compound is GPP, and the chryseriol is converted to the prenylflavonoid, Cannflavin A.

[0039] In at least one embodiment of the method, the prenyl donor compound is DMAPP, and the chryseriol is converted to the prenylflavonoid, Cannflavin B.

[0040] In at least one embodiment of the method, the prenyl donor is GPP, and the chryseriol is converted to the prenylflavonoid, Cannflavin C.

[0041] In at least one embodiment of the method, the engineered prenyltransferase (“scPT”) comprises an engineered scPT of the present disclosure or a composition of the present disclosure.

[0042] In at least one embodiment of the method, the suitable reaction conditions comprise a cell free solution.

[0043] In at least one embodiment of the method, the suitable reaction conditions comprise a temperature of between about 50 °C and about 100 °C, between about 50 °C and about 90 °C, between about 50 °C and about 80 °C, between about 60 °C and about 80 °C, or between about 55 °C and about 75 °C.

[0044] Any combination of inventive features disclosed in the preceding paragraphs of this section or the paragraphs of the following sections is within the scope of this disclosure.

[0045] These, as well as other components, steps, features, objects, benefits, and advantages, will become apparent after reviewing the detailed description of illustrative embodiments, the accompanying drawings, and the claims.BRIEF DESCRIPTION OF DRAWINGS

[0046] The drawings of this disclosure are illustrative examples. They do not illustrate all examples. Other examples may be used in addition or instead. Details that may be apparent or unnecessary may be omitted to save space or for more effective illustration. Some examples may be practiced with additional components or steps and / or without all the illustrated components or steps. When the same numeral appears in different drawings, it refers to the same or similar components or steps. The drawings are not necessarily to scale.

[0047] FIG. 1 depicts an exemplary biosynthetic pathway capable of producing Cannflavin A or Cannflavin B starting from phenylalanine. The enzymes active in the various steps of the pathway include PAL, C4H, 4CL, CHS, CHI, FNS, F3’H, OMT, CAS1, and CAS2 , and are described further in the description below.

[0048] FIG. 2 depicts an exemplary cell-free biocatalytic cascade for the synthesis of Cannflavin A and other prenylflavonoids. Luteolin may first be methylated by the activity of a methyl transferase (MT), such as flavonoid 3'-O-methyltransferase, to chrysoeriol. Chrysoeriol may then be prenylated with geranyl pyrophosphate (GPP) by the activity of an engineered prenyl transferase (e.g., scPT). The methylation co-factor S-adenosyl methionine (SAM) may be produced in situ from ATP and methionine. The prenyl donor GPP may be produced in situ by using an allylic alcohol (e.g. an a / p unsaturated alcohol like prenol, or its derivatives) and a p / y unsaturated alcohol (e.g. isoprenol, or its derivatives).

[0049] FIG. 3A depicts an exemplary reaction scheme for the methylation of luteolin using ATP, Met, and a five-enzyme cascade (tkMAT, roMT-9, gsMtn, bsLuxS, gsPPase) to form chrysoeriol. FIG. 3B depicts results of an exemplary HPLC analysis (using a C-18 RP column, H2O to acetonitrile gradient), carried out as described in Example 1, showing the HPLC trace for the substrate standard, luteolin, the HPLC trace for the product standard for chrysoeriol, and the HPLC trace for the biosynthesis of FIG. 3A.

[0050] FIG. 4A, 4B, and 4C depict HPLC traces (obtained using a C-18 RP column, H2O to acetonitrile gradient) showing production of the prenylated product CF-A. FIG. 4A: HPLC trace for the standard for the substrate, chrysoeriol; FIG. 4B: HPLC trace for the standard for Cannflavin A; and FIG. 4C: the product of the biosynthetic reaction of substrates, chrysoeriol and GPP, with an engineered scPT polypeptide as described in Example 2.

[0051] For a further understanding of the present disclosure's nature, objects, and advantages, reference should be made to the following detailed description, read in conjunction with this disclosure's drawings.DETAILED DESCRIPTION

[0052] Reference will now be made in detail to certain embodiments of the disclosure, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the disclosure to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the disclosure as defined by the claims.

[0053] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The disclosure is in no way limited to the methods and materials described. Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and / or use are to be understood as modified by the word "about" in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary, percent, "parts of," and ratio values are by weight; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammaticalvariations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.

[0054] It must also be noted that, as used in the specification and the appended claims, the singular form "a," "an," and "the" comprise plural referents unless the context indicates otherwise. That is, reference to a component in the singular is intended to include a plurality of components. That is, in this disclosure, the indefinite article “a” and the phrases “one or more” and “at least one” are synonymous and mean “at least one.” Similarly, an element preceded by an “a” or an “an” does not, without further constraints, preclude the existence of additional elements of the identical type. Thus, for example, a reference to “a polynucleotide” includes a plurality of such polynucleotides, and a reference to “the enzyme” includes a reference to one or more enzymes, and so forth.

[0055] As used herein, the term “about” means that the amount or value in question may be the specific value designated or some other value in its neighborhood. For example, the term “about” denoting a particular value represents a range within ± 5% of the value. For example, the phrase “about 100” indicates a range of 100 ± 5, i.e. , the value is in the range of 95 to 105. Generally, when the term “about” is used, it can be expected that similar results or effects according to the invention can be obtained, for example, within a range of ± 5% of the indicated value.

[0056] As used herein, the term “and / or” means that all or only one of the elements of said group may be present. For example, “A and / or B” shall mean “only A, or only B, or both A and B”. In the case of “only A,” the term also covers the possibility that B is absent, i.e., “only A, but not B.”

[0057] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications outlined in this specification, including in the following claims, are approximate, not exact. They are intended to have a reasonable range consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0058] As will be understood by one skilled in the art, for any purposes, such as providing a written description, all ranges disclosed herein also encompass any possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. As will also be understood by one skilled in the art of all languages, such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed herein. Finally, as will be understood by one skilled in the art, a range includes each member. Thus, for example, a group with 1-3articles refers to groups with 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0059] While various aspects and embodiments have been disclosed herein, others will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustration purposes and are not intended to be limiting.

[0060] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference for the subject matter referenced and, in their entirety, are made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and take precedence over any such contradictory material.

[0061] Concerning the use of substantially any plural and / or singular terms herein, those with skill in the art can translate from the plural to the singular and / or from the singular to the plural as appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for clarity.

[0062] The phrase “means for” when used in a claim is intended to and should be interpreted to embrace the corresponding structures and materials that have been described and their equivalents. Similarly, when used in a claim, the phrase “step for” is intended to and should be interpreted to embrace the corresponding acts described and their equivalents. The absence of these phrases from a claim means that the claim is not intended to and should not be interpreted as limited to these corresponding structures, materials, or acts, or to their equivalents.

[0063] In at least some of the embodiments disclosed herein, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions, and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the disclosed subject matter.

[0064] The scope of protection is limited solely by the claims. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language used in the claims when interpreted in light of this specification and the prosecution history that follows, except where specific meanings have been set forth, and to encompass all structural and functional equivalents.

[0065] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generallyintended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Those within the art will further understand that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and, in the absence of such recitation, no such intent is present. For example, as an aid to understanding, the following appended claims may use the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0066] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0067] None of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended coverage of such subject matter is hereby disclaimed. Except asjust stated in this paragraph, nothing that has been said or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0068] The abstract is provided to help the reader quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, various features in the detailed description are grouped in various embodiments to streamline the disclosure. This method of disclosure should not be interpreted as requiring claimed embodiments to require more features than are expressly recited in each claim. Instead, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed embodiment. Thus, the following claims are hereby incorporated into the detailed description, with each claim standing on its own as separately claimed subject matter.

[0069] The scope of this disclosure includes any combination of inventive features disclosed in the preceding paragraphs of this section or the paragraphs of the following sections.

[0070] The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.

[0071] The phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element outlined in that clause; other elements are not excluded from the claim as a whole.

[0072] The phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the essential and novel characteristic(s) of the claimed subject matter.

[0073] The phrase “composed of” means “including” or “consisting of.” Typically, this phrase denotes that an object is formed from a material.

[0074] Concerning the terms “comprising,” “consisting of,” and “consisting essentially of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms. Similarly, the terms “comprises,” “comprising,” and any other variation in connection with a list of elements in the specification or claims indicate that the list is not exclusive and that other elements may be included. For example, “comprising A” includes “consisting of A” or “consisting essentially of A.”

[0075] The term “one or more” means “at least one,” and the term “at least one” means “one or more.” The terms “one or more” and “at least one” include “plurality” as a subset.

[0076] Relational terms such as “first,” “second,” and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual relationship or order between them.

[0077] The terms “substantially,” “generally,” or “about” may be used herein to describe disclosed or claimed embodiments. The term “substantially” may modify a value or relative characteristic disclosed or claimed in the present disclosure. In such instances, “substantially” may signify that the value or relative characteristic it modifies is within ± 0%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% or 10% of the value or relative characteristic.

[0078] It should also be appreciated that integer ranges explicitly include all intervening integers. For example, the integer range 1-10 explicitly contains 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range 1 to 100 contains 1, 2, 3, 4 ...10...20... 50 ... 76...83 . . . 97, 98, 99, 100. Similarly, when any range is called for, intervening numbers that are increments of the difference between the upper and lower limits divided by 10 can be taken as alternative upper or lower limits. For example, if the range is 1.1. to 2.1 the following numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 can be selected as lower or upper limits.

[0079] In this disclosure, the following acronyms and abbreviations are used.

[0080] Ade: adenine

[0081] AdoMet: S-adenosyl methionine

[0082] ATP: adenosine triphosphate

[0083] CBD: cannabidiol

[0084] THC: tetrahydrocannabinol

[0085] CF-A: cannflavin A

[0086] CF-B: cannflavin B

[0087] CF-C: cannflavin C

[0088] DMAPP: dimethylallyl pyrophosphate

[0089] DPD: 4,5-dihydroxy-2,3-pentanedione

[0090] GPP: geranyl pyrophosphate

[0091] HCY: homocysteine

[0092] PPi: pyrophosphate

[0093] RHC: ribosyl-homocysteine

[0094] SAH: S-adenosyl homocysteine

[0095] SAM: S-adenosyl methionine

[0096] Met: methionine

[0097] Pj: inorganic monophosphate

[0098] MT: methyltransferase

[0099] PT: prenyltransferase

[0100] HPLC: high-pressure liquid chromatography

[0101] pM: micromolar

[0102] mM: millimolar

[0103] PDB: protein data bank

[0104] SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0105] ProteinMPNN: Protein Message- Passing Neural Network; a deep learning method to redesign protein sequences based on structure with a local or online platform.

[0106] This disclosure relates to the synthesis of cannflavins and other prenylflavonoids. This disclosure relates to an engineered enzyme that can catalyze synthesis reactions for the production of cannflavins and other prenylflavonoids. This disclosure relates to an engineered prenyltransferase. This disclosure also relates to an engineered prenyltransferase that can catalyze synthesis reactions for the production of cannflavins and other prenylflavonoids. This disclosure further relates to a cell-free system and / or a cell-free composition suitable for the synthesis of cannflavins and other prenylflavonoids.

[0107] “Precursor,” as used herein, may refer to a (chemical) compound, a molecule, or a solution that is involved in a (chemical) reaction to yield a product. For example, the precursor(s) may include a feedstock, a substrate, a catalyst (e.g., an enzyme), a buffer solution, or the like, or a combination thereof. Examples of precursors useful in the embodiments of the present disclosure can include luteolin, chrysoeriol, SAM, ATP, a methyltransferase, a prenyltransferase (e.g., the engineered scPTs of this disclosure), methionine, DMAPP, GPP, and a composition thereof.

[0108] “Product,” as used herein, may refer to a (chemical) compound, a molecule, or a composition produced after a precursor(s) undergoes a chemical reaction(s). Examples of products may be a prenylflavonoid(s) (e.g., a cannflavin(s)) or a composition thereof. A product of a chemical reaction, for example, chrysoeriol, may be a precursor of another chemical reaction.

[0109] In this disclosure, cannflavins and other prenylflavonoids may be prepared by reacting a precursor(s) in a cell-free system using a cell-free composition. The “system,” as used herein, may refer to a solid enclosure, for example, a steel, plastic, or glass vessel. In this exemplary embodiment, a precursor may be synthesized using a cell (e.g., a bacteria) or a plant or through traditional organic chemistry. Such precursor, in a partially or substantially isolated form, may be used in the production of the cannflavins, cannflavin derivatives, and other prenylflavonoids of this disclosure. This synthesis approach hereafter is referred to as a “cell-free” synthesis approach.

[0110] In this disclosure, cannflavins and other prenylflavonoids may also be prepared by reacting a precursor(s) using a composition that includes cells. Such cells may host a precursor(s) during chemical reactions, which provide products of this disclosure. This synthesis approach hereafter is referred to as a “cell-based” synthesis approach.

[0111] “Conversion,” as used herein, refers to converting a precursor to a product. For example, conversion may refer to an enzymatic conversion of a substrate(s) to a corresponding product(s). “Percent conversion” refers to a percent amount of a precursor converted to a product. For example, a percent conversion may refer to a percent amount of a substrate converted to a product within a period of time under specified conditions. The “enzymatic activity” or “activity” of an enzymatic conversion may be expressed as the “percent conversion” of a substrate to a product.

[0112] “Substrate,” as used herein in the context of an enzyme-mediated process, may refer to a precursor acted on by an enzyme. In the context of an engineered prenyltransferase (polypeptide) of the present disclosure, a substrate acted on by such engineered prenyltransferase may include a range of precursor(s). A “cannflavin precursor” or a “cannflavin precursor substrate,” as used herein, may refer to a precursor acted on by an engineered enzyme in a biosynthetic step for producing cannflavins and other prenylflavonoids of this disclosure.

[0113] An “enzyme” means any substance, typically composed substantially or mainly of amino acids making up a protein or polypeptide that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions.

[0114] “Nucleic acid” or “polynucleotide,” as used herein, interchangeably refers to two or more nucleosides covalently linked together. The nucleic acid may substantially comprise ribonucleosides (e.g., RNA), substantially comprise 2'-deoxyribonucleotides (e.g., DNA), ora mixture thereof. The nucleoside units of the nucleic acid may be linked together via phosphodiester linkages (e.g., as in naturally occurring nucleic acids), or the nucleic acid may include one or more non-natural linkages (e.g., phosphorothioester linkage). Nucleic acid or polynucleotide may include single-stranded or double-stranded molecules or molecules with both single-stranded and double-stranded regions. Nucleic acid or polynucleotide may include molecules composed of naturally occurring nucleobases (i.e. , adenine, guanine, uracil, thymine, and cytosine) or may include molecules such as one or more modified and / or synthetic nucleobases, such as inosine, xanthine, hypoxanthine, etc.

[0115] “Protein,” “polypeptide,” and “peptide” are used herein interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). As used herein, “protein,” or “polypeptide,” or “peptide”polymer may include D-amino acids, L-amino acids, or a mixture thereof. A protein or polypeptide can function as an enzyme.

[0116] “Naturally occurring” or “wild-type,” as used herein, for example with reference to nucleic acid and protein molecules, refers to a form of the molecule that exists in nature without human interaction. For example, a naturally occurring nucleic acid or a naturally occurring amino acid is the molecule present in an organism that may be isolated from a source in nature and that has not been intentionally modified by human manipulation.

[0117] “Engineered,” or “synthesized,” as used herein, refers to the form that exists due to human activity. As such, engineered / synthesized precursor / product may refer to a precursor / product prepared by human activity. For example, an engineered / synthesized nucleic acid sequence or an engineered / synthesized amino acid sequence is a sequence prepared by human manipulation.

[0118] “Rosetta Design” refers to a computational protein design software that may be used to identify mutations and sequences compatible with engineering a given protein backbone. A description of the Rosetta Design algorithm and software implementation is found in e.g., Leaver-Fay, et al. (2011). ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. In Methods in enzymology (Vol. 487, pp. 545-574. Academic Press; and online at: rosettacommons.org / software / licensing-faq / )

[0119] “Percentage of sequence identity” or “percent sequence identity” may be used interchangeably herein to refer to values that quantify comparisons of the sequences of polynucleotides, or polypeptides, and may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (or gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage values may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences, or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0120] Those skilled in the art may appreciate that many established algorithms are available to align two sequences. Optimal alignment of sequences for comparison may be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482,by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that may be suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively.

[0121] Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits may then be extended in both directions along each sequence to increase the cumulative alignment score.Cumulative scores may be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0).

[0122] For amino acid sequences, a scoring matrix may used to calculate the cumulative score. Extension of the word hits in each direction may be halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. As defaults, the BLASTN program (for nucleotide sequences) may use a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. As defaults for amino acid sequence alignment calculations, the BLASTP program may use a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary determination of sequence alignment and % sequence identity may employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison, Wis.) using the default parameters provided.

[0123] “Reference sequence” may refer to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length nucleic acid sequence or a full-length polypeptidesequence. A reference sequence may typically be at least 20 nucleotide positions or amino acid residue units in length but may also be the entire length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides (1) may each comprise a sequence (i.e., a portion of the complete sequence) that may be similar between the two sequences and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide may typically be performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity.

[0124] “Comparison window” may refer to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (or gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

[0125] “Substantial identity” or “substantially identical” may refes to a polynucleotide sequence or a polypeptide sequence that may have at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity, as compared to a reference sequence over a comparison window of at least 20 nucleoside or amino acid residue positions, frequently over a window of at least 30-50 positions, wherein the percentage of sequence identity may be calculated by comparing the reference sequence to a sequence that includes deletions or additions that total 20 percent or less of the reference sequence over the window of comparison.

[0126] “Corresponding to,” “reference to,” or “relative to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence, may refer to the numbering of the residues of a specified reference sequence when the given amino acid sequence or polynucleotide sequence may be compared to the reference sequence. In other words, the residue number or residue position of a given polymer may be designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid sequence or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered prenyltransferase, may be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps may be present, the numbering of the residue in the given amino acid or polynucleotide sequence may be made with respect to the reference sequence to which it has been aligned.

[0127] “Isolated,” as used herein in reference to a precursor / product, may mean that the precursor / product (e.g., a prenylflavonoid) is substantially separated from otherprecursors / products that naturally / synthetically accompany it, e.g., protein, lipids, and polynucleotides. This term may embrace nucleic acids or amino acids that may have been removed or purified from their naturally / synthetically occurring environment or expression system (e.g., host cell or in vitro synthesis).

[0128] “Substantially pure” may refer to a composition in which a desired precursor / product is the predominant chemical compound present (i.e., on a molar or weight basis, it may be more abundant than any other individual chemical compound present in the composition), and is generally a substantially purified composition when the object precursor / product may comprise at least about 50 mole percent or at least 50 weight percent of the total chemical compounds present in the composition.

[0129] Prenylation refers to a chemical reaction where a prenyl group is transferred from a prenyl donor compound (e.g., GPP or DMAPP) to a prenyl acceptor compound (e.g., chrysoeriol) to form a prenylated compound. In the context of the present disclosure, prenylation refers to the regiospecific transfer of a prenyl group donor compound to an acceptor substrate that is catalyzed by a prenyltransferase enzyme.

[0130] The disclosure provides a method of producing a prenylated compound comprising contacting a precursor with a prenyl-group having the general structure:

[0131] Prenylated natural products include a large class of bioactive molecules with demonstrated medicinal properties. Examples include prenyl indoles, prenylflavonoids, prenyl-stilbenoids, prenylated terpenoids, and cannabinoids. Plant-derived prenyl compounds are difficult to isolate due to the structural similarity of contaminating molecules and the variable composition between crops. These challenges are further exacerbated when attempting to isolate low-abundance compounds. Many chemical syntheses have previously been developed to address the challenges of making prenylated natural products. Still, they may be commercially not viable for drug manufacturing due to manufacturing complexity and low yields.

[0132] Synthetic biochemistry, or the development and use of a cell-free mixture of enzymes (or “exozyme biosolution”), in which complex biochemical conversions may be performed cell-free using a mixture of enzymes, may provide potential advantages over traditional metabolic engineering, including a higher level of flexibility in pathway design, greater control over component optimization, more rapid design-build-test cycles and freedom from cell toxicity ofintermediates or products. The disclosure provides a cell-free system for producing cannflavins and other prenylflavonoids.

[0133] A “mutation” means any process or mechanism resulting in a mutant protein, enzyme, polynucleotide, gene, or cell. This may include any mutation in which a protein, enzyme, polynucleotide, or gene sequence is altered and any detectable change in a cell arising from such a mutation.

[0134] This disclosure relates to engineered prenyltransferase enzymes having at least one amino acid substitution as compared to a naturally occurring prenyltransferase. Such amino acid substitutions may be referred to as “mutations” or “amino acid differences” in this disclosure. The engineered prenyltransferase enzymes (“engineered scPTs”), of this disclosure have at least one amino acid mutation or amino acid difference (or substitution) in their amino acid sequence relative to the amino acid sequence of the naturally occurring (or wild-type) prenyltransferase from which they are derived.

[0135] The term “substrate” or “suitable substrate” refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme. The term includes a single compound and combinations of compounds, such as solutions, mixtures, and other materials that contain at least one substrate or derivatives thereof.Further, the term “substrate” encompasses not only compounds that provide a starting material but also intermediate and end-products, as described herein.

[0136] The terms “regioselective” and “regioselectivity,” as used in a “regioselective reaction,” refers to a bond-making or bond-breaking reaction that occurs preferentially at a particular specific location on a molecule. A reaction between a first and second substrate may yield two or more reaction products (e.g., a first product, a second product, etc.) Regioselectivity of a reaction can be determined based on the relative molar amount of different products formed in the reaction, wherein the difference between the product is due to bond-making or bond-breaking at different locations of the reactant molecule. For example, in an enzymatic reaction wherein a first substrate and a second substrate react to form a product mixture of a first product and a second product and wherein the molar ratio of the first product A to the second product B is greater than 1:1, the reaction is regioselective to product A. Wherein the molar ratio of the first product to the second product is at least 9:1 or greater, the reaction has 90% or greater regioselectivity for the first product.

[0137] For the purpose of amino acid position numbering, SEQ ID NO: 2 is used as the reference sequence. For example, the mention of amino acid position 49 is in reference to SEQ ID NO: 2, but in the context of a different prenyltransferase sequence (a target sequence or other template sequence), the corresponding amino acid position for variant creation may have the same or different position number, (e.g. 48, 49 or 50). In some cases, the original amino acid and its position on the SEQ ID NO: 2 reference template may precisely correlatewith the original amino acid and position on the target prenyltransferase. In other cases, the original amino acid and its position on the SEQ ID NO: 2 template may correlate with the original amino acid, but its position on the target may not be in the corresponding template position. However, the corresponding amino acid on the target may be a predetermined distance in sequence space from the position on the template, such as within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid positions from the template position but would be assumed to occupy the same or very similar position in three-dimensional space. It is understood that additional alignments may be generated with prenyltransferase sequences not specifically disclosed herein, and such alignments may be used to understand and generate new prenyltransferase variants in view of the current disclosure.

[0138] In some modes of practice, the alignments may allow one to understand common or similar amino acids in the vicinity of the target amino acid, and those amino acids may be viewed as “sequence motifs” having a certain amount of identity or similarity between the template and target sequences. Those sequence motifs may be used to describe portions of prenyltransferase sequences where variant amino acids are located, and the type of variation(s) that may be present in the motif.

[0139] In some embodiments, a prenyltransferase template into which one or more variations (also referred to herein as mutation or substitution) are introduced to create a variant is a prenyltransferase sequence having at least 50% identity, or at least 60% identity, or at least 65% identity, or at least 70% identity, or at least 75% identity, or at least 80% identity, or at least 85% identity, or at least 87.5% identity, or at least 90% identity, or at least 92.5% identity, or at least 95% identity to SEQ ID NO: 2.

[0140] One, or more than one, amino acid variation may be described relative to the location of a particular amino acid in a wild-type prenyltransferase template sequence. Identification of locations in the template that, when substituted with variant amino acids, provide desired activity and regioselectivity may be determined by testing methods as described herein.

[0141] Described herein are engineered prenyltransferases that may serve as alternate production catalysts for the in vitro and / or in vivo prenylation of natural products of various biological origins (e.g. plant, microbial, fungal, etc), such as polyphenols, such as flavonoids, stilbenoids, and bibenzyls or synthetically derived substrates that are not found in nature. In addition, the approaches described herein may provide the basis for exploring novel prenylation chemistry and bioactivity of natural and novel synthetic prenylated aromatic compounds by means of structure-based enzyme engineering.

[0142] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning. A LaboratoryManual, Cold Springs Harbor Press (Cold Springs Harbor, N Y 1989), each of which is incorporated herein by reference. For the purposes of the present invention, the following terms are defined below.

[0143] “Variants” of the sequences described herein are biologically active sequences that have a peptide sequence that differs from the sequence of a native or wild-type sequence, by virtue of an insertion, deletion, modification and / or substitution of one or more amino acids within the native sequence. Such variants generally have less than 100% sequence identity with a native sequence. Ordinarily, however, a biologically active variant may have an amino acid sequence with at least about 50% sequence identity with the sequence of a corresponding naturally occurring sequence, or at least about 60% sequence identity, or at least about 65% sequence identity, or at least about 70% sequence identity, or at least about 75% sequence identity, or at least about 80% sequence identity, or at least about 85% sequence identity, or at least about 87.5% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 96% sequence identity, or at least about 97% sequence identity, or at least about 98% sequence identity, or at least about 99% sequence identity. The variants nucleotide fragments of any length retain the biological activity of the corresponding native sequence. Variants also include sequences wherein one or more amino acids are added at either end of or within a native sequence. Variants also include sequences where a number of amino acids are deleted and optionally substituted by one or more different amino acids.

[0144] Prenyltransferases Engineered for Prenylflavonoid Synthesis Activity

[0145] The present disclosure provides recombinant polypeptides having prenyltransferase activity and exhibiting activity in regio-specifically prenylating the flavonoid precursor substrates, such as chrysoeriol, using a range of prenyl donor compounds, such as GPP and DMAPP, to form prenylflavonoid compounds such as Cannflavin A, Cannflavin B, and Cannflavin C.

[0146] The recombinant polypeptides prenyltransferase activity of the present disclosure are derived from the naturally occurring soluble prenyltransferase, “NphB.” NphB is isolated from Streptomyces sp. CL190 (UniProt: A0A2Z4JFA9) and is a polypeptide with the amino acid sequence of SEQ ID NO: 2. See e.g., US7361483B2, which is hereby incorporated by reference herein in its entirety. Previously, the soluble prenyltransferase, NphB has been engineered with activity capable of regio-specifically prenylating aromatic polyketides, such as OA with the prenyl donor, GPP to form the cannabinoid compound, CBGA. See e.g., WO2019173770A1; WO2019183152A1; W02020028722A1, WO2021134024A1, each of which is hereby incorporated by reference herein in its entirety. Engineered NphB has been used in cell-free biosynthesis systems and methods for the preparation of cannabinoid compounds. See e.g., W02020028722A1 and WO2021134024A1.

[0147] In one exemplary embodiment, the recombinant polypeptides with prenyltransferase activity of the present disclosure are capable of converting the methylated luteolin substrate, chrysoeriol (compound 2), to Cannflavin A (compound 1) as shown in Scheme 1.Scheme 1CH3H3C0H 0OH 0H3C GPP PP, HHO0O0_ HO 00Hengineered PT00(2)CH3 (1) CH3

[0148] A range of exemplary prenyltransferases with activity capable of catalyzing the conversion depicted in Scheme 1 have been designed, synthesized and screened as described in the Example 2 below. The amino acid substitutions and surprising technical effect of activity in the production of Cannflavin A (CF-A) from chrysoeriol and GPP (as in Scheme 1) of these engineered prenyltranferases are summarized in Table 1A below and Table 3 of Example 2 below.

[0149] TABLE 1A: scPT polypeptides with prenyltransferase activityAA NT AASubstitutions SEQ SEQ (relative to ID ID Name SEQ ID NO: 2) AA Sequence NO: NO:P1 n / a MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 1 2TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM1 Q161A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 3 4 S177T, S214A, TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSC230T, HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVSG286A, V294T MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVAMTSMDYKKRQVNLY FTELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFAVYPTLNWETGKIDRLTFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLAAYYHITDTQRGLLKAFDSLEDM2 Q161A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 5 6S177T, F213A, TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSS214W, HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS C230A, MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPSG286A, MPPAVAENAELFARYGLDKVAMTSMDYKKRQVNLYV294A, L298Y FTELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSAWVYPTLNWETGKIDRLAFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLAAYYHITDAQRGYLKAFDSLEDM3 S51T, M129A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 7 8 Q161L, TFQDTLVEGGSVVVFTMASGRHSTELDFSISVPTSS177G, HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVSS214A, MFAIDGEVTGGFKKTYAFFPTDNAPGVAELSAIPSC230T, V294E MPPAVAENAELFARYGLDKVLMTSMDYKKRQVNLY FGELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFAVYPTLNWETGKIDRLTFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDEQRGLLKAFDSLEDM4 V47I, S177T, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 9 10 C230T, V294I, TFQDTLVEGGSIVVFSMASGRHSTELDFSISVPTSQ295D HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FTELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLTFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDIDRGLLKAFDSLEDM5 S51T, M129C, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 11 12 Q161Y, TFQDTLVEGGSVVVFTMASGRHSTELDFSISVPTSS177T, S214A, HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVSC230T, V294E MFAIDGEVTGGFKKTYAFFPTDNCPGVAELSAIPS MPPAVAENAELFARYGLDKVYMTSMDYKKRQVNLY FTELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFAVYPTLNWETGKIDRLTFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDEQRGLLKAFDSLEDM6 C230T MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 13 14TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLTFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM7 L298M MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 15 16TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGMLKAFDSLEDM8 S51I MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 17 18TFQDTLVEGGSVVVFIMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM9 G286A MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 19 20TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLAAYYHITDVQRGLLKAFDSLEDM10 C230A MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 21 22TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLAFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM11 M129Q MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 23 24TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNQPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM12 Q161E MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 25 26TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVEMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM13 V294E MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 27 28TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDEQRGLLKAFDSLEDM14 S177T MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 29 30TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FTELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM15 S214L MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 31 32TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFLVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM16 I234M MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 33 34TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVMSNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM17 V294A MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 35 36TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDAQRGLLKAFDSLEDM18 F213M MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 37 38TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSMSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM19 V294I MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 39 40TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDIQRGLLKAFDSLEDM20 S214A MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 41 42TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFAVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM21 M129A MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 43 44TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNAPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM22 V47I MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 45 46TFQDTLVEGGSIVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM23 Q161Y MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 47 48TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVYMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM24 F213A MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 49 50TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSASVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVQRGLLKAFDSLEDM25 Q295D MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 51 52TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTS HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDVDRGLLKAFDSLEDM1-ST Q161A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 53 54S214A, TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSC230T, HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS G286A, V294T MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVAMTSMDYKKRQVNLY FSELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFAVYPTLNWETGKIDRLTFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLAAYYHITDTQRGLLKAFDSLEDM1-SA Q161A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 55 56S177T, C230T, TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSG286A, V294T HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVAMTSMDYKKRQVNLY FTELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFSVYPTLNWETGKIDRLTFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLAAYYHITDTQRGLLKAFDSLEDM1-CT Q161A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 57 58S177T, S214A, TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSG286A, V294T HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVAMTSMDYKKRQVNLY FTELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFAVYPTLNWETGKIDRLCFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLAAYYHITDTQRGLLKAFDSLED MIQ161A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 59 60 GA S177T, S214A, TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSC230T, V294T HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVAMTSMDYKKRQVNLY FTELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFAVYPTLNWETGKIDRLTFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLGAYYHITDTQRGLLKAFDSLEDM1-VT Q161A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 61 62S177T, S214A, TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSC230T, G286A HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVAMTSMDYKKRQVNLY FTELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFAVYPTLNWETGKIDRLTFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLAAYYHITDVQRGLLKAFDSLEDM1- S177T, S214A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLS 63 64 QA C230T, TFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSG286A, V294T HGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVS MFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPS MPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLY FTELSAQTLEAESVLALVRELGLHVPNELGLKFCK RSFAVYPTLNWETGKIDRLTFAVISNDPTLVPSSD EGD I E KFHNYATKAP YAY VGE KRTL VYGLTL S PRE EYYKLAAYYHITDTQRGLLKAFDSLED

[0150] In another exemplary embodiment, the recombinant polypeptides with prenyltransferase activity of the present disclosure are engineered with activity capable of converting the methylated luteolin substrate, chrysoeriol (compound 2), to Cannflavin B (compound 3) as shown in Scheme 2.Scheme 2OH O CH3OH OH3COHoDMAPP P P . OHuHO OO engineered PTO CH3CH3(2) (3)

[0151] A range of exemplary prenyltransferases engineered to accept chrysoeriol and DMAPP as substrates and have activity capable of catalyzing the conversion depicted in Scheme 2 have been designed as described in Example 4 below. The amino acid substitutions, and sequences associated with the production of Cannflavin B (CF-B) from chrysoeriol and DMAPP (as in Scheme 2) of these engineered prenyltranferases are summarized in Table 1B below.

[0152] TABLE 1B: Recombinant polypeptides engineered for CF-B synthesis activityAA NT AASubstitutions SEQ SEQ (relative to SEQ ID ID Name ID NO: 2) AA Sequence NO: NO:B1 S64V, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 65 66 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFVISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB2 K119A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 67 68 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKATYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB3 K284L, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 69 70 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLEAESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYLLGAYYHITDVQR GLLKAFDSLEDB4 K284L, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 71 72 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYLLGAYYHITDVQR GLLKAFDSLEDB5 K119A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 73 74 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKATYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB6 K169R, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 75 76 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKRRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB7 S64L, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 77 78 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFLISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB8 K169R, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 79 80 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKRRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB9 K169N, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 81 82 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKNRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQRGLLKAFDSLEDB10 Y121F, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 83 84 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTFAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB11 M106A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 85 86 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSAFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB12 F123Y, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 87 88 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAYFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB13 M162A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 89 90 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAE L F AR Y GL D KVQ AT SMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB14 S164A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 91 92 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTAMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB15 Y216A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 93 94 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVAPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB16 K169N, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 95 96 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGC230T, G286A, EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENV294T AELFARYGLDKVQMTSMDYKNRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB17 Y121F, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 97 98 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTFAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB18 N173L, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 99 100 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVLLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB19 Y175F, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 101 102 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLFFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB20 S164A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 103 104 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTAMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB21 Y216A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 105 106 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVAPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB22 V47I, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 107 108 S177T, S214A, QDTLVEGGSIVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLEAESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB23 N173L, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 109 110 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVLLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB24 Y175F, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 111 112 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLFFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQR GLLKAFDSLEDB25 Y288F, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 113 114 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAFYHITDVQR GLLKAFDSLEDB26 Q295D, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 115 116 S177T, S214A, QDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPC230T, G286A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGV294T EVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAEN AELFARYGLDKVQMTSMDYKKRQVNLYFSELSAQTLE AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWE TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKA PYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVDR GLLKAFDSLEDB27 S51A, D62G, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 117 118 S64Y, S66Q, QDTLVEGGSVVVFAMASGRHSTELGFYIQVPTSHGDPM106S, A108G, YATVVEKGLFPATGHPVDDLLADTQKHLPVSSFGIAGD110A, K118V, EVTGGFVATTALFPTDNMPGVAELSAIPSMPPAVAENK119A, Y121T, AELFARYGLDKVQMTAMDYKNRQVLLFFSELSAQTLEF123L, S164A, AESVLALVRELGLHVPNELGLKFCKRSFSVTPTLNWEK169N, N173L, TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAY175F, Y216T, PYAYVGEKRTLVYGLTLSPKEEYYLLGAFYHITDVQRK284L, Y288F, GLLKAFDSLEDQ161A, S177T,S214A, C230T,G286A, V294TB28 D62G, S64Y, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 119 120 S66Q, M106S, QDTLVEGGSVVVFSMASGRHSTELGFYIQVPTSHGDPA108G, D110A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSSFGIAGK118V, K119A, EVTGGFVATYALFPTDNMPGVAELSAIPSMPPAVAENF123L, S164A, AELFARYGLDKVQMTAMDYKNRQVLLFFSELSAQTLEK169N, N173L, AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWEY175F, K284L, TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAY288F, Q161A, PYAYVGEKRTLVYGLTLSPKEEYYLLGAFYHITDVQRS177T, S214A, GLLKAFDSLEDC230T, G286A,V294TB29 S51A, D62G, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 121 122 S64C, S66Q, QDTLVEGGSVVVFAMASGRHSTELGFCIQVPTSHGDPM106S, K118V, YATVVEKGLFPATGHPVDDLLADTQKHLPVSSFAIDGK119Q, Y121F, EVTGGFVQTFALFPTDNMPGVAELSAIPSMPPAVAENF123L, S164A, AELFARYGLDKVQMTAMDYKRRQVLLFFSELSAQTLEK169R, N173L, AESVLALVRELGLHVPNELGLKFCKRSFSVTPTLNWEY175F, Y216T, TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAK284L, Y288F, PYAYVGEKRTLVYGLTLSPKEEYYLLGAFYHITDVQRQ161A, S177T, GLLKAFDSLEDS214A, C230T,G286A, V294TB30 V47L, V49T, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 123 124 S51E, S64L, QDTLVEGGSLVTFEMASGRHSTELDFLIQVPTSHGDPS66Q, M106S, YATVVEKGLFPATGHPVDDLLADTQKHLPVSSFAIDGK119A, Y121F, EVTGGFKATFAYFPTDNMPGVAELSAIPSMPPAVAENF123Y, M162G, AELFARYGLDKVQGTAMDYKNRQVNLYFSELSAQTLES164A, K169N, AESVLALVRELGLHVPNELGLKFCKRSFSVAPTLNWEY216A, K284L, TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAY288M, PYAYVGEKRTLVYGLTLSPKEEYYLLGAMYHITDVMRQ295M, GLLKAFDSLEDQ161A, S177T,S214A, C230T,G286A, V294TB31 S51E, S64L, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 125 126 M106A, K119A, QDTLVEGGSVVVFEMASGRHSTELDFLISVPTSHGDPY121F, F123Y, YATVVEKGLFPATGHPVDDLLADTQKHLPVSAFAIDGM162G, S164A, EVTGGFKATFAYFPTDNMPGVAELSAIPSMPPAVAENK169N, N173L, AELFARYGLDKVQGTAMDYKNRQVLLFFSELSAQTLEY175F, Y216A, AESVLALVRELGLHVPNELGLKFCKRSFSVAPTLNWEK284L, Q161A, TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAS177T, S214A, PYAYVGEKRTLVYGLTLSPKEEYYLLGAYYHITDVQRC230T, G286A, GLLKAFDSLEDV294TB32 V47L, V49T, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 127 128 D110G, K119A, QDTLVEGGSLVTFSMASGRHSTELDFSISVPTSHGDPY121L, K169N, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIGGK284L, Y288M, EVTGGFKATLAFFPTDNMPGVAELSAIPSMPPAVAENQ295M, AELFARYGLDKVQMTSMDYKNRQVNLYFSELSAQTLEQ161A, S177T, AESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWES214A, C230T, TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAG286A, V294T PYAYVGEKRTLVYGLTLSPKEEYYLLGAMYHITDVMR GLLKAFDSLEDB33 S64L, K119A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 129 130 K169N, K284L, QDTLVEGGSVVVFSMASGRHSTELDFLISVPTSHGDPQ161A, S177T, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGS214A, C230T, EVTGGFKATYAFFPTDNMPGVAELSAIPSMPPAVAENG286A, V294T AELFARYGLDKVQMTSMDYKNRQVNLYFSELSAQTLEAESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVYGLTLSPKEEYYLLGAYYHITDVQR GLLKAFDSLEDB34 S64V, K119A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL ST F 131 132 Y121F, S164A, QDTLVEGGSVVVFSMASGRHSTELDFVISVPTSHGDPK169R, Y216A, YATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGK284L, Q161A, EVTGGFKATFAFFPTDNMPGVAELSAIPSMPPAVAENS177T, S214A, AELFARYGLDKVQMTAMDYKRRQVNLYFSELSAQTLEC230T, G286A, AESVLALVRELGLHVPNELGLKFCKRSFSVAPTLNWEV294T TGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVYGLTLSPKEEYYLLGAYYHITDVQRGLLKAFDSLED

[0153] In another exemplary embodiment, the recombinant polypeptides with prenyltransferase activity of the present disclosure are engineered with activity capable of converting the methylated luteolin substrate, chrysoeriol (compound 2), to Cannflavin C (compound 4) as shown in Scheme 3.Scheme 3°H° OH OHO 0 °H GPPHO 0engineered PTCH3 CH CH3(2) (4)H3C CH3

[0154] A range of exemplary prenyltransferases engineered to accept chrysoeriol and GPP as substrates and having activity capable of catalyzing the conversion depicted in Scheme 3 have been designed as described in Example 4 below. The amino acid substitutions, and sequences associated with the production of Cannflavin C (CF-C) from chrysoeriol and GPP (as in Scheme 3) of these engineered prenyltranferases are summarized in Table 1C below.

[0155] TABLE 1C: Recombinant polypeptides engineered for CF-C synthesis activityAA NT AASubstitutions SEQ SEQ (relative to ID ID Name SEQ ID NO: 2) AA Sequence NO: NO:C1 V49I, S51A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 133 134 M129C, DTLVEGGSVVIFAMASGRHSTELDFSISVPTSHGDPYAQ161Y, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTS177T, F213A, GGFKKTYAFFPTDNCPGVAELSAIPSMPPAVAENAELFS214Y, ARYGLDKVYMTSMDYKKRQVNLYFTELSAQTLEAESVLC230A, ALVRELGLHVPNELGLKFCKRSAYVYPTLNWETGKIDRV294E, L298N, LAFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGEG286AKRTLVYGLTLSPKEEYYKLAAYYHITDEQRGNLKAFDSLEDS51A, M129A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 135 136 Q161I, S177T, DTLVEGGSVVVFAMASGRHSTELDFSISVPTSHGDPYAF213C, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTS214M, GGFKKTYAFFPTDNAPGVAELSAIPSMPPAVAENAELFC230A, ARYGLDKVIMTSMDYKKRQVNLYFTELSAQTLEAESVLV271A, ALVRELGLHVPNELGLKFCKRSCMVYPTLNWETGKIDRG286A, LAFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGEV294E, L298N KRTLAYGLTLSPKEEYYKLAAYYHITDEQRGNLKAFDSLEDQ161E, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 137 138 S177T, S214A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAC230T, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTV294K, G286A GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELF ARYGLDKVEMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDKQRGLLKAFDS LEDV49I, M129C, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 139 140 Q161Y, DTLVEGGSVVIFSMASGRHSTELDFSISVPTSHGDPYAS177T, F213A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTS214Y, GGFKKTYAFFPTDNCPGVAELSAIPSMPPAVAENAELFC230A, ARYGLDKVYMTSMDYKKRQVNLYFTELSAQTLEAESVLV294E, L298N, ALVRELGLHVPNELGLKFCKRSAYVYPTLNWETGKIDRG286A LAFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDEQRGNLKAFDS LEDM129A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 141 142 Q161E, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNAPGVAELSAIPSMPPAVAENAELFV294K, G286A ARYGLDKVEMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDKQRGLLKAFDS LEDS51A, Q161A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 143 144 S177T, F213A, DTLVEGGSVVVFAMASGRHSTELDFSISVPTSHGDPYAS214Y, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230A, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFV294E, G286A ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSAYVYPTLNWETGKIDR LAFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDEQRGLLKAFDS LEDV49I, S51A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 145 146 Q161A, DTLVEGGSVVIFAMASGRHSTELDFSISVPTSHGDPYAS177T, F213A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTS214Y, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFC230A, ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVLV294E, L298N, ALVRELGLHVPNELGLKFCKRSAYVYPTLNWETGKIDRG286A LAFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVYGLTLSPKEEYYKLAAYYHITDEQRGNLKAFDSLED08 S51A, Q161G, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 147 148 S177T, F213A, DTLVEGGSVVVFAMASGRHSTELDFSISVPTSHGDPYAS214Y, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230A, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFV294E, G286A ARY GL DKVGMT SMDY KKRQ VNL Y FT EL S AQT LE AE S VL ALVRELGLHVPNELGLKFCKRSAYVYPTLNWETGKIDR LAFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDEQRGLLKAFDS LED09 S214Y, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 149 150 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, C230T, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTG286A, V294T GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELF ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFYVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC10 L298N, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 151 152 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGNLKAFDS LEDC11 S214M, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 153 154 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, C230T, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTG286A, V294T GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELF ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFMVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC12 F213C, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 155 156 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSCAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC13 M162A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 157 158 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAATSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC14 Q161S, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 159 160 S177T, S214A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELF G286A, V294T ARYGLDKVSMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC15 F213H, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 161 162 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSHAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC16 Q161K, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 163 164 S177T, S214A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAC230T, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTG286A, V294T GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELF ARYGLDKVKMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC17 V271A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 165 166 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLAYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC18 M129C, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 167 168 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNCPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC19 V294K, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 169 170 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, G286A GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELF ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDKQRGLLKAFDS LEDC20 F213S, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 171 172 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVLALVRELGLHVPNELGLKFCKRSSAVYPTLNWETGKIDRLTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC21 F123M, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 173 174 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAMFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC22 Q161I, S177T, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 175 176 S214A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAC230T, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTG286A, V294T GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELF ARYGLDKVIMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC23 Q161L, S177T, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 177 178 S214A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAC230T, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTG286A, V294T GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELF ARYGLDKVLMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC24 Q161G, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 179 180 S177T, S214A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAC230T, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTG286A, V294T GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFARY GL DKVGMT SMDY KKRQ VNL Y FT EL S AQT LE AE S VL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC25 Q295A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 181 182 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTARGLLKAFDS LEDC26 S64V, Q161A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 183 184 S177T, S214A, DTLVEGGSVVVFSMASGRHSTELDFVISVPTSHGDPYAC230T, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTG286A, V294T GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELF ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLEDC27 K169R, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 185 186 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKRRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC28 S164A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 187 188 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTAMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC29 Y121F, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 189 190 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTFAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC30 K169N, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 191 192 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKNRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDS LEDC31 M106A, MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQ 193 194 Q161A, DTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYAS177T, S214A, TVVEKGLFPATGHPVDDLLADTQKHLPVSAFAIDGEVTC230T, GGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFG286A, V294T ARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVL ALVRELGLHVPNELGLKFCKRSFAVYPTLNWETGKIDR LTFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGE KRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLED

[0100] A range of exemplary engineered scPT with activity capable of catalyzing the conversion depicted in Scheme 1 with increased thermostability have been designed, synthesized and screened as described in the Examples below. The engineered scPT include additional amino acid substitutions (e.g., A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301 K, F302Y, D303A) that result in the surprising technical effect of activity in the production of Cannflavin A (CF-A) from chrysoeriol and GPP (as in Scheme 1) when using higher biocatalytic reaction mixture temperatures (e.g., 50 C - 70 C). The mutationsand sequences of these thermostable engineered scPT enzymes are summarized in Table 1D below.

[0156] TABLE 1D: Thermostable prenyltransferase polypeptides with CF-A synthesis activity AA NT AASubstitutions SEQ SEQ (relative to SEQ ID ID Name ID NO: 2) AA Sequence NO: NO:S1 Q161A, S214A, MSEAADVERVYAAIEEAAGLLGVPCARDKIWPLL 195 196 C230T, G286A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPV294T, S136A, PSHGDPYAIVVEKGLFPATGHPIDDLLADIQKHLM14I, E222D, PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELAG224S, N236T, AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKRA24P, G297K, QVNLYFSELSAQTLEAESVLALVRELGLHVPNELY31W, T69P, GLKFCKRS FAVY PTLNWDTSKIDRLT FAVI STDPT77I, V911, T98I TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRKLLKAFDSLE DS2 Q161A, S214A, MS EAEDVE RVYAAMEE AAGLLGVAVARDKI Y PLL 197 198 C230T, G286A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPV294T, A5E, VSHGDPYATVVEKGLFPATGHPVDDLLADTAKHLC25V, T69V, PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSQ99A, A181E, AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKRK251R, G297E, QVNLYFSELSEQTLEAESVLALVRELGLHVPNELA301 K, F302Y, GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDPD303A TLVPSSDEGDIERFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRELLKKYASLE DS3 Q161A, S214A, MS EAEDVE RVYAAMEE AAGLLGVAVARDKI Y PLL 199 200 C230T, G286A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPV294T, A5E, VSHGDPYATVVEKGLFPATGHPVDDLLADTAKHLC25V, T69V, PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSQ99A, A181E, AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKRK251R, H253T, QVNLYFSELSEQTLEAESVLALVRELGLHVPNELG297N, A301K, GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDPF302Y, D303A TLVPSSDEGDIERFTNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRNLLKKYASLE DS4 Q161A, S214A, MS EAADVE RVYAAMEE AAGLLGVAVARDKI Y PLL 201 202 C230T, G286A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPV294T, C25V, VSHGDPYATVVEKGLFPATGHPVDDLLADTAKHLT69V, Q99A, PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSA181P, K251R, AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKRH253T, G297E, QVNLYFSELSPQTLEAESVLALVRELGLHVPNELA301 K, F302Y, GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDPD303N TLVPSSDEGDIERFTNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRELLKKYNSLE DS5 Q161A, S214A, MS EQADVE RVYAAMEE AAGLLGVACARDKI Y PLL 203 204 C230T, G286A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPV294T, A4Q, TSHGDPYATVVAKGLFPATGHPVDDLLADTQKHLE80A, A133E, PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVEELSA137K, E150P, KIPSMPPAVAENAPLFKKYGLDKVAMTSMDYKKRA153K, R154K, QVNLYFSELSEETLAPESVLALVRELGLHVPNELA181E, Q182E, GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDPE185A, A186P, TLVPSSDPGDIEKFHNYATKAPYAYVGEKRTLVYE246P GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLEDS6 Q161A, S214A, MS EQADVE RVYAAMEE AAGLLGVACARE KI Y PLL 205 206 C230T, G286A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPV294T, A4Q, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLD28E, A133E, PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVEELSA137K, E150P, KIPSMPPAVAENAPLFKKYGLDKVAMTSMDYKKRA153K, R154K, QVNLYFSELSDETLSDESVLALVRELGLHVPNELA181D, Q182E, GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDPE185S, A186D, TLVPSSDPGDIEKFHNYATKAPYAYVGEKRTLVYE246P GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLEDS7 Q161A, S214A, MS EAADVE RVYAAMEE AAGLLGVAVARDKI Y PLL 207 208 C230T, G286A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPV294T, C25V, VSHGDPYATVVEKGLFPATGHPVDDLLADTAKHLT69V, Q99A, PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSA181E, K251R, AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKRH253T, G297E, QVNLYFSELSEQTLEAESVLALVRELGLHVPNELA301 K, F302Y GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIERFTNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRELLKKYDSLE DS8 C25V, Q161A, MS EAADVE RVYAAMEE AAGLLGVAVARDKI Y PLL 209 210 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS9 F302Y, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 211 212 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAYDSLE DS10 Q99A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 213 214 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTAKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDPTLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVYGLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLEDS11 T69V, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 215 216 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, VSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS12 K251R, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 217 218 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIERFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS13 A301K, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 219 220 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKKFDSLE DS14 H253T, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 221 222 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFTNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS15 A181E, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 223 224 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSEQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS16 R154K, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 225 226S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAl P SMP PAVAENAE L FAKYGLDKVAMT SMD Y KKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS17 Q182E, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 227 228 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAETLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS18 E246P, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 229 230 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDPGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS19 E185S, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 231 232 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLSAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS20 A153K, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 233 234 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFKRYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS21 A137K, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 235 236 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSKIPSMPPAVAENAELFARYGLDKVAMTSMDYKKRQVNLYFTELSAQTLEAESVLALVRELGLHVPNELGLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS22 E150P, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 237 238 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAl PSMP PAVAENAPL FARYGLDKVAMT SMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS23 D303A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 239 240 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFASLE DS24 A181D, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 241 242 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSDQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS25 D28E, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARE KI Y PLL 243 244 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS26 E80A, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 245 246 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVAKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLEDS27 A186D, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 247 248 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEDESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS28 A181P, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 249 250 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSPQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS29 A133E, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 251 252 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVEELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRGLLKAFDSLE DS30 G297N, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 253 254 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRNLLKAFDSLE DS31 G297E, Q161A, MS EAADVE RVYAAMEE AAGLLGVACARDKI Y PLL 255 256 S177T, S214A, STFQDTLVEGGSVVVFSMASGRHSTELDFSISVPC230T, G286A, TSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLV294T PVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELS AIPSMPPAVAENAELFARYGLDKVAMTSMDYKKR QVNLYFTELSAQTLEAESVLALVRELGLHVPNEL GLKFCKRS FAVY PTLNWETGKI DRLT FAVI SNDP TLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVY GLTLSPKEEYYKLAAYYHITDTQRELLKAFDSLED

[0157] As illustrated by the exemplary polypeptides of Tables 1 A-1 D, the engineered scPT polypeptides of the present disclosure have one or more amino acid residue differences intheir amino acid sequence as compared to the naturally-occurring scPT polypeptide of SEQ ID NO: 2 that confer improved acceptance of precursor substrates (e.g., chrysoeriol, GPP, DMAPP) and improved activity in the rate of conversion of these precursor substrates to desired prenylflavonoid product compounds (e.g., cannflavins A, B, or C).

[0158] For example, the exemplary engineered scPT polypeptides of Table 1 A have one or more residue differences in residue positions that result in increased activity and specificity in the conversion of the precursor substrate, chrysoeriol and the prenyl donor, GPP to the prenylflavonoid product, cannflavin A, wherein the residue positions selected from V47, S51, M129, Q161, S177, F213, S214, C230, I234, G286, V294, Q295, and L298. In some embodiments, the specific amino acid residue difference at the selected position can be selected from V47I, S511, S51T, M129A, M129C, M129Q, Q161A, Q161E, Q161L, Q161Y, S177G, S177T, F213A, F213M, S214A, S214L, S214W, C230A, C230T, I234M, G286A, V294A, V294E, V294I, V294T, Q295D, L298M, and L298Y. Additionally, it is contemplated that the engineered scPT polypeptide can comprise a set of amino acid residue differences relative to SEQ ID NO: 2 selected from a combination of amino acid residue differences found in any one of the exemplary engineered scPT polypeptides of Table 1A. Accordingly, in at least one embodiment the specific set of amino acid residue differences can be selected from: (a) Q161A, S214A, C230T, G286A, V294T; (b) Q161A, S177T, S214A, C230T, G286A, V294T; (c) Q161A, S177T, F213A, S214W, C230A, G286A, V294A, L298Y; (d) S51T, M129A, Q161L, S177G, S214A, C230T, V294E; (e) V47I, S177T, C230T, V294I, Q295D; and (f) S51T, M129C, Q161Y, S177T, S214A, C230T, V294E.

[0159] In another embodiment, the exemplary engineered scPT polypeptides of Table 1B have one or more residue differences in residue positions that result in increased activity in the conversion of the precursor substrate, chrysoeriol and the prenyl donor, DMAPP to the prenylflavonoid product, cannflavin B, wherein the residue positions selected from V47, S64, M106, K119, Y121, F123, M162, S164, K169, N173, Y175, Y216, K284, Y288, and Q295. In some embodiments, the specific amino acid residue difference at the selected position can be selected from V47I, S64L, S64V, M106A, K119A, Y121F, F123Y, M162A, S164A, K169N, K169R, N173L, Y175F, Y216A, K284L, Y288F, and Q295D. Additionally, it is contemplated that the engineered scPT polypeptide can comprise a set of amino acid residue differences relative to SEQ ID NO: 2 selected from a combination of amino acid residue differences found in any one of the exemplary engineered scPT polypeptides of Table 1B. Accordingly, in at least one embodiment the specific set of amino acid residue differences can be selected from: (a) V47L, V49T, S51E, S64L, S66Q, M106S, K119A, Y121F, F123Y, M162G, S164A, K169N, Y216A, K284L, Y288M, Q295M; (b) V47L, V49T, D110G, K119A, Y121L, K169N, K284L, Y288M, Q295M; (c) S51A, D62G, S64Y, S66Q, M106S, A108G, D110A, K118V, K119A, Y121T, F123L, S164A, K169N, N173L, Y175F, Y216T, K284L, Y288F; (d) S51E, S64L, M106A, K119A, Y121F, F123Y, M162G, S164A, K169N, N173L, Y175F, Y216A, K284L; (e)D62G, S64Y, S66Q, M106S, A108G, D110A, K118V, K119A, F123L, S164A, K169N, N173L, Y175F, K284L, Y288F; (f) S64V, K119A, Y121F, S164A, K169R, Y216A, K284L; and (g) S64L, K119A, K169N, K284L;S51A, D62G, S64C, S66Q, M106S, K118V, K119Q, Y121F, F123L, S164A, K169R, N173L, Y175F, Y216T, K284L, Y288F.

[0160] In another embodiment, the exemplary engineered scPT polypeptides of Table 10 have one or more residue differences in residue positions that result in increased activity in the conversion of the precursor substrate, chrysoeriol and the prenyl donor, GPP to the prenylflavonoid product, cannflavin C, wherein the residue positions selected from S64, M106, Y121, F123, M129, Q161, M162, S164, K169, F213, S214, V271, V294, Q295, and L298. In some embodiments, the specific amino acid residue difference at the selected position can be selected from S64V, M106A, Y121F, F123M, M129C, Q161G, Q161I, Q161K, Q161L, Q161S, M162A, S164A, K169N, K169R, F213C, F213H, F213S, S214M, S214Y, V271A, V294K, Q295A, and L298N. Additionally, it is contemplated that the engineered scPT polypeptide can comprise a set of amino acid residue differences relative to SEQ ID NO: 2 selected from a combination of amino acid residue differences found in any one of the exemplary engineered scPT polypeptides of Table 1C. Accordingly, in at least one embodiment the specific set of amino acid residue differences can be selected from: (a) V49I, S51A, Q161A, S177T, F213A, S214A, C230T, G286A, V294T, L298N; (b) V49I, S51A, M129C, Q161A, S177T, F213A, S214A, C230T, G286A, V294T, L298N; (c) V49I, M129C, Q161A, S177T, F213A, S214A, C230T, G286A, V294T, L298N; (d) S51A, Q161A, S177T, F213A, S214A, C230T, G286A, V294T S51A, M129A, Q161A, S177T, M129A, Q161A, S177T, S214A, C230T, G286A, V294T; (e) Q161A, S177T, S214A, C230T, G286A, V294T; and (f) F213C, S214A, C230T, V271A, G286A, V294T, L298N.

[0161] As further described below, it is also contemplated that any of the engineered scPT polypeptides of the present disclosure (e.g., the exemplary polypeptides of Tables 1A-1D) can further have in combination with the amino acid residue differences at the foregoing residue positions, one or more thermostability conferring amino residue differences as compared to SEQ ID NO: 2 selected from A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301K, F302Y, D303A. Accordingly, it is contemplated that in at least one embodiment, the exemplary engineered scPT polypeptides of Table 1A can have the one or more residue differences in residue positions that result in increased activity in the conversion of the precursor substrate, chrysoeriol and the prenyl donor, GPP to the prenylflavonoid product, cannflavin A (e.g., differences at positions V47, S51, M129, Q161, S177, F213, S214, C230, I234, G286, V294, Q295, and L298), and also have one or more thermostability conferring amino residue differences as compared to SEQ ID NO: 2 (e.g., A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301K, F302Y, D303A). It is to be understood that the amino acid residue differences relative to SEQ ID NO: 2 at residue positions associated with increased thermostability can be used in various combinations to form recombinantprenyltransferase polypeptides having desirable enzymatic characteristics, for example combination of increased thermostability, and increased conversion rate, product yield, and / or utilization of prenyl group donor substrate.

[0162] Examples of such engineered scPT polypeptides with mutations conferring increased thermostability are provided in Table 1D and illustrated in the Examples. In at least one embodiment such an engineered scPT with increased thermostability and activity in the conversion of the precursor substrate, chrysoeriol and the prenyl donor, GPP to the prenylflavonoid product, cannflavin A, the amino acid sequence comprises a set of amino acid mutations as compared to SEQ ID NO: 2 selected from: (a) A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301K, F302Y, D303A; (b) A5E, C25V, T69V, Q99A, A181E, K251R, G297E, A301K, F302Y, D303A; (c) A4Q, E80A, A133E, A137K, E150P, A153K, R154K, A181E, Q182E, E185A, A186P, E246P; (d) A4Q, D28E, A133E, A137K, E150P, A153K, R154K, A181D, Q182E, E185S, A186D, E246P; (e) M14I, A24P, Y31W, T69P, T77I, V911, T98I, S136A, S214A, E222D, G224S, C230T, N236T, G286A, V294T, G297K; (f) C25V, T69V, Q99A, A181E, K251R, H253T, G297E, A301K, F302Y; and (g) C25V, T69V, Q99A, A181P, K251R, H253T, G297E, A301K, F302Y, D303N. It is further contemplated that an engineered scPT polypeptide with increased thermostability can comprise a set of amino acid residue differences relative to SEQ ID NO: 2 selected from A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301K, F302Y, D303A, or selected from any combination of these found in an exemplary engineered scPT polypeptide of Table 1 D, and a combination of amino acid residue differences relative to SEQ ID NO: 2 found in any one of the exemplary engineered scPT polypeptides of Tables 1A-1D.

[0163] In at least one embodiment, the present disclosure provides an engineered scPT polypeptide having activity in the conversion of the precursor substrate, chrysoeriol and a prenyl donor (e.g., GPP, DMAPP) to a prenylflavonoid product, such as cannflavin A, B, or C, and / or has increased thermostability, wherein the polypeptide comprises an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, or greater, sequence identity to SEQ ID NO: 2, and one or more of the amino acid residue differences as compared to SEQ ID NO: 2 selected from the amino acid differences listed in Tables 1A-1D (e.g., V47I, S511, S51T, M129A, M129C, M129Q, Q161A, Q161E, Q161L, Q161Y, S177G, S177T, F213A, F213M, S214A, S214L, S214W, C230A, C230T, I234M, G286A, V294A, V294E, V294I, V294T, Q295D, L298M, and L298Y). In some embodiments, the engineered scPT polypeptide comprises an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, or greater, sequence identity to SEQ ID NO: 2, and a combination of amino acid differences found in any one of the engineered scPT polypeptide sequences of Table 1A-1C, and further comprises one or more thermostability conferring amino acid differences selected from: A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301K, F302Y, and D303A.

[0164] In at least one embodiment, the engineered scPT polypeptide comprises one or more amino acid residue differences as compared to SEQ ID NO: 2 selected from Tables 1A-1 D, and comprises an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, or greater, sequence identity to an amino acid sequence having a SEQ ID NO: selected from even-numbered SEQ ID NOs: 4-256.

[0165] Based on the correlation of recombinant polypeptide functional information provided herein with the sequence information provided in Tables 1A-1D and the accompanying Sequence Listing, one of ordinary skill can recognize that the present disclosure provides a range of engineered scPT polypeptides having prenyltransferase activity, and optionally, increased thermostability, wherein the polypeptide comprises an amino acid sequence comprising one or more of the amino acid differences or sets of amino acid differences relative to SEQ ID NO: 2 disclosed in any one of even-numbered SEQ ID NO: 4-256, and otherwise have at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of even-numbered SEQ ID NO: 4-256.

[0166] Thus, in at least one embodiment, an engineered scPT polypeptide of the present disclosure having prenyltransferase activity, and optionally, increased thermostability, can have an amino acid sequence comprising one or more of the amino acid differences or sets of amino acid differences relative to SEQ ID NO: 2 disclosed in any one of even-numbered SEQ ID NO: 4-256, and additionally have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1- 14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at other residue positions. In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences at the other residue positions.

[0167] In addition to the residue positions specified above, any of the engineered scPT polypeptides disclosed herein can further comprise other residue differences relative to SEQ ID NO:2 at other residue positions that may be known in the art. Residue differences at these other residue positions can provide for additional variations in the amino acid sequence without adversely affecting the ability of the recombinant polypeptide to carry out the desired biocatalytic conversion (e.g., conversion of compound (2) to compound (1)). In some embodiments, the recombinant polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residue differences at other amino acid residue positions as compared to SEQ ID NO: 2. In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, and 40 residue differences at other residue positions. The residue difference at these other positions can include conservative changes or nonconservative changes. In some embodiments, the residue differences can compriseconservative substitutions and non-conservative substitutions as compared to the naturally-occurring scPT polypeptide of SEQ ID NO: 2.

[0168] Amino acid residue differences at other positions relative to the naturally-occurring scPT polypeptide of SEQ ID NO: 2 and the effect of these differences on enzyme function are provide by other recombinant prenyltransferase polypeptides disclosed in international patent applications with publication nos. WO2019173770A1, WO2019183152A1, WQ2020028722A1 , WO2021178976A2, WQ2021063396A1, and WQ2021134024A1, each of which is hereby incorporated by reference herein in its entirety. Accordingly, in some embodiments, one or more of the amino acid differences provided in the recombinant polypeptides of WQ2019173770A1, WO2019183152A1, WQ2020028722A1,WO2021178976A2, WQ2021063396A1 , and WQ2021134024A1 could also be introduced into a recombinant prenyltransferase polypeptide of the present disclosure.

[0169] In some embodiments, the present disclosure provides an engineered scPT polypeptide capable of converting compound (2) to compound (1) with increased thermostability relative to the activity of the polypeptide of SEQ ID NO: 2, which comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, with the proviso that the amino acid sequence excludes any one or more of the engineered prenyltransferase polypeptides disclosed in any one or more of international patent applications, WQ2019173770A1, WO2019183152A1 , WQ2020028722A1 , WQ2021178976A2, WQ2021063396A1 , or WQ2021134024A1.

[0170] In some embodiments, the engineered scPT polypeptides of the disclosure can be in the form of fusion polypeptides in which the engineered polypeptides are fused to other polypeptides, such as, by way of example and not limitation, antibody tags (e.g., myc epitope), purification sequences (e.g., His tags for binding to metals), and cell localization signals (e.g., secretion signals). Thus, the recombinant polypeptides described herein can be used with or without fusions to other polypeptides. It is also contemplated that the recombinant polypeptides described herein are not restricted to the genetically encoded amino acids. In addition to the genetically encoded amino acids, the polypeptides described herein may be comprised, either in whole or in part, of naturally-occurring and / or synthetic non-encoded amino acids.

[0171] In another aspect, the present disclosure provides polynucleotides encoding the engineered scPT polypeptides having prenyltransferase activity, and optionally, increased thermostability, as described herein. In at least one embodiment, the polynucleotide comprises a sequence encoding an exemplary engineered scPT polypeptide having prenyltransferase activity as disclosed in any one of Tables 1A-1D and the accompanying Sequence Listing. Exemplary polynucleotide sequences encoding the engineered scPTvariants of the Tables 1A-1D are provided in Table 1E as odd-numbered SEQ ID NO: 3-255 and the accompanying Sequence Listing.

[0172] TABLE 1E: Exemplary polynucleotide sequences encoding the engineered scPT polypeptides of the present disclosureSEQ IDName NT sequence NO:P1 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 1 (scPT) GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM1 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 3 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM2 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 5 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCAGCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTACACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGGCGTGGGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAGCGTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACGCCCAACGCGGATACTTGAAAGCGTTTGACTCATTGGAG GATTAAM3 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 7 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTACGATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATGCGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCTGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCGGTGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGAGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM4 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 9 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCATCGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACATCGACCGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM5 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 11 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTACGATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATTGCCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTTACATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATCTCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTTCATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGAGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM6 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 13 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM7 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 15 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGAATGTTGAAAGCGTTTGACTCATTGGAG GATTAAM8 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 17 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTATCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTTGTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATATTACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM9 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 19 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM10 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 21 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAGCGTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM11 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 23 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATCAGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAGGATTAAM12 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 25 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGAGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM13 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 27 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM14 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 29 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTTTAGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM15 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 31 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACGTTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCCGGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATG TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM16 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 33 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGATGAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM17 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 35 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT T ACC AT AT C ACC GAC AT C C AAC GC GG AC T T T T GAAAGC GT T T GACT C AT T GG AG GATTAAM18 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 37 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCTGTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCTATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM19 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 39 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATGCGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM20 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 41 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCATCGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM21 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 43 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCAGTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTTACATGACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM22 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 45 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGGCGAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM23 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 47 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTGACCGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM24 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 49 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTACACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTACCCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM25 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 51 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM1-ST ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 53GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM1-SA ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 55GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTAGTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTTCATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTTGTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM1-CT ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 57GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTATGCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAA MIATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 59 GA GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGGAGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAM1-VT ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 61GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACGTTCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAGGATTAAM1- ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 63 QA GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCAAATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB1 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 65 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCGTAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB2 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 67 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGGCCACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB3 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 69 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACGTTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCCGGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATCTGCTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB4 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 71 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATCTGCTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB5 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 73 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGGCCACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB6 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 75 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCTGTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCTATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGCGTCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB7 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 77 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTTAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB8 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 79 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGCGTCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB9 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 81 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCAGTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATGACAAGTATGGATTACAAGAATCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB10 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 83 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATTTGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB11 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 85 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCGCGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB12 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 87 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTATTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTACACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTACCCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB13 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 89 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGGCG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB14 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 91 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAGCTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB15 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 93 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTGCG CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTTCATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTTGTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB16 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 95 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAATCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB17 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 97 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATTTGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB18 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 99 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTACTGCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAGGATTAAB19 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 101 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTTCTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB20 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 103 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAGCTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB21 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 105 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTGCG CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB22 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 107 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACGTTTCAGGACACACTGGTAGAAGGAGGCTCCATCGTTGTGTTTAGCATGGCTTCCGGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB23 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 109 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTACTGCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB24 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 111 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTTCTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB25 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 113 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCTGTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCTATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATTT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB26 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 115 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGGACCGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB27 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 117 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTGCCATGGCTTCC GGTCGTCACAGTACAGAGCTTGGCTTCTACATTCAAGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCAGCTTTGGG ATCGCGGGCGAAGTCACCGGCGGCTTCGTTGCCACAACCGCTCTGTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAGCTATGGATTACAAGAATCGTCAGGTACTGCTGTTCTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTACC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATCTGCTGGCTGCATTT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB28 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 119 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGGCTTCTACATTCAAGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCAGCTTTGGG ATCGCGGGCGAAGTCACCGGCGGCTTCGTTGCCACATATGCTCTGTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCAGTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATGACAGCTATGGATTACAAGAATCGTCAGGTACTGCTGTTCTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATCTGCTGGCTGCATTT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB29 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 121 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTGCCATGGCTTCC GGTCGTCACAGTACAGAGCTTGGCTTCTGTATTCAAGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCAGCTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCGTTCAAACATTTGCTCTGTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAGCTATGGATTACAAGCGTCGTCAGGTACTGCTGTTCTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTACC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATCTGCTGGCTGCATTT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB30 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 123 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCTTAGTTACCTTTGAGATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTTAATTCAAGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCAGCTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGGCCACATTTGCTTATTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGGGC ACAGCTATGGATTACAAGAATCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTGCG CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATCTGCTGGCTGCAATG TACCATATCACCGACACGATGCGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB31 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 125 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTGAGATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTTAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCGCGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGGCCACATTTGCTTATTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGGGC ACAGCTATGGATTACAAGAATCGTCAGGTACTGCTGTTCTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTACACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTGCGCCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATCTGCTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB32 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 127 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCTTAGTTACCTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGGCGGCGAAGTCACCGGCGGCTTCAAGGCCACACTGGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAATCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATCTGCTGGCTGCAATG TACCATATCACCGACACGATGCGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB33 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 129 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTTAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGGCCACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAATCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATCTGCTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAB34 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 131 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCGTAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGGCCACATTTGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAGCTATGGATTACAAGCGTCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTGCG CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTTCATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTTGTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATCTGCTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAA ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 133 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTATCTTTGCCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATTGCCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTTACATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGGCGTACGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAGCGTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACGAGCAACGCGGAAATTTGAAAGCGTTTGACTCATTGGAG GATTAA ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 135 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTGCCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATGCGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTATCATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTGCATGGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAGCGTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GCTTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACGAGCAACGCGGAAATTTGAAAGCGTTTGACTCATTGGAG GATTAA ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 137 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGAGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACAAGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAGGATTAAATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 139 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTATCTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATTGCCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTTACATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGGCGTACGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAGCGTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACGAGCAACGCGGAAATTTGAAAGCGTTTGACTCATTGGAG GATTAA ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 141 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATGCGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGAGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACAAGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAA ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 143 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTGCCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGGCGTACGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAGCGTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACGAGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAA ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 145 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACGTTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTATCTTTGCCATGGCTTCCGGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGGCGTACGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAGCGTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACGAGCAACGCGGAAATTTGAAAGCGTTTGACTCATTGGAG GATTAAC8 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 147 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTGCCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGGTATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGGCGTACGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAGCGTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACGAGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC9 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 149 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTTACGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC10 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 151 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCTGTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCTATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGAAATTTGAAAGCGTTTGACTCATTGGAG GATTAAC11 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 153 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTATGGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC12 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 155 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTGCGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC13 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 157 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCAGTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGGCGACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC14 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 159 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTAGCATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC15 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 161 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGCATGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC16 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 163 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTAAGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTACACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTACCCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC17 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 165 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GCTTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC18 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 167 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATTGCCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC19 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 169 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTTCATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTTGTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACAAGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC20 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 171 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGAGCGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC21 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 173 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTATGTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC22 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 175 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTATCATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAGGATTAAC23 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 177 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTCTGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC24 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 179 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGGTATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC25 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 181 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGGCGCGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC26 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 183 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACGTTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCCGGTCGTCACAGTACAGAGCTTGATTTCGTAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC27 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 185 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGCGTCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC28 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 187 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAGCTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC29 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 189 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCTGTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCTATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATTTGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC30 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 191 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAATCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAC31 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 193 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCGCGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS1 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATTGAGGAAGCCGCA 195 GGGCTGCTGGGGGTGCCATGTGCTCGCGACAAAATCTGGCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCCCATCGCATGGG GACCCCTATGCCATCGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT ATCGATGACCTGCTTGCTGATATCCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGGCTGCTATCCCGAGTATGCCGCCCGCAGTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATGACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGACACTAGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCACCGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCAAGCTTTTGAAAGCGTTTGACTCATTGGAG GATTAA ATGTCCGAGGCCGAGGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 197 GGGCTGCTGGGGGTGGCGGTGGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCGTTTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTGCGAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GAGCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAACGTTTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGAGCTTTTGAAAAAGTATGCGTCATTGGAG GATTAA ATGTCCGAGGCCGAGGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 199 GGGCTGCTGGGGGTGGCGGTGGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCGTTTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTGCGAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GAGCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAACGTTTT ACCAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCAATCTTTTGAAAAAGTATGCGTCATTGGAG GATTAA ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 201 GGGCTGCTGGGGGTGGCGGTGGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCGTTTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTGCGAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA CCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTACACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTACCCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAACGTTTT ACCAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGAGCTTTTGAAAAAGTATAATTCATTGGAG GATTAA ATGTCCGAGCAAGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 203 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGCGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGAGGAGTTGTCGAAGATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTCCACTGTTCAAGAAGTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GAGGAGACCCTGGCGCCAGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATCCGGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAA ATGTCCGAGCAAGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 205 GGGCTGCTGGGGGTGGCGTGTGCTCGCGAGAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGAGGAGTTGTCGAAGATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTCCACTGTTCAAGAAGTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GACGAGACCCTGAGCGACGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATCCGGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAA ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 207 GGGCTGCTGGGGGTGGCGGTGGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCGTTTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTGCGAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCAGCGAGCTTTCA GAGCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAACGTTTTACCAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTTGTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGAGCTTTTGAAAAAGTATGACTCATTGGAG GATTAAS8 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 209 GGGCTGCTGGGGGTGGCGGTGGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS9 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 211 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTATGACTCATTGGAG GATTAAS10 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 213 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTGCGAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAGGATTAAS11 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 215 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCGTTTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS12 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 217 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAACGTTTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS13 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 219 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAAAGTTTGACTCATTGGAG GATTAAS14 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 221 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACGTTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCCGGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT ACCAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS15 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 223 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GAGCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS16 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 225 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTAAGTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS17 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 227 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCTGTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCTATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTGAGACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS18 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 229 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATCCGGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS19 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 231 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGAGCGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS20 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 233 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCAGTGGCCGAGAACGCTGAGCTGTTCAAGCGTTATGGCCTTGATAAGGTTGCGATGACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS21 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 235 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGAAGATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS22 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 237 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTCCACTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS23 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 239 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTACACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTACCCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGCGTCATTGGAG GATTAAS24 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 241 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GACCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS25 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 243 GGGCTGCTGGGGGTGGCGTGTGCTCGCGAGAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS26 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 245 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGCGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTTCATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTTGTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS27 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 247 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGACGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS28 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 249 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA CCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS29 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 251 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGAGGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGGACTTTTGAAAGCGTTTGACTCATTGGAGGATTAAS30 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 253 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCAATCTTTTGAAAGCGTTTGACTCATTGGAG GATTAAS31 ATGTCCGAGGCCGCAGATGTAGAACGCGTTTACGCAGCTATGGAGGAAGCCGCA 255 GGGCTGCTGGGGGTGGCGTGTGCTCGCGACAAAATCTATCCCCTTCTTTCGACG TTTCAGGACACACTGGTAGAAGGAGGCTCCGTTGTTGTGTTTAGCATGGCTTCC GGTCGTCACAGTACAGAGCTTGATTTCTCAATTTCCGTACCCACGTCGCATGGG GACCCCTATGCCACTGTTGTGGAGAAGGGGTTGTTTCCAGCGACAGGGCACCCT GTCGATGACCTGCTTGCTGATACTCAAAAACATTTACCCGTCAGCATGTTTGCT ATCGATGGCGAAGTCACCGGCGGCTTCAAGAAGACATATGCTTTTTTCCCAACA GATAATATGCCTGGCGTAGCAGAGTTGTCGGCTATCCCGAGTATGCCGCCCGCA GTGGCCGAGAACGCTGAGCTGTTCGCTCGTTATGGCCTTGATAAGGTTGCGATG ACAAGTATGGATTACAAGAAGCGTCAGGTAAACCTGTACTTCACGGAGCTTTCA GCTCAAACCCTGGAAGCCGAGTCCGTATTAGCCCTTGTACGTGAGTTGGGCTTA CACGTACCAAACGAGCTTGGACTGAAATTCTGTAAACGCTCGTTTGCTGTTTAC CCTACATTAAATTGGGAGACTGGCAAGATTGATCGTTTAACCTTCGCAGTGATC TCCAACGATCCGACTCTGGTGCCGAGCTCTGATGAAGGAGATATTGAAAAATTT CATAATTATGCTACAAAGGCCCCGTATGCATATGTTGGCGAAAAGCGCACTCTT GTCTATGGCCTGACTTTAAGTCCCAAGGAGGAGTACTATAAACTGGCTGCATAT TACCATATCACCGACACGCAACGCGAGCTTTTGAAAGCGTTTGACTCATTGGAGGATTAA

[0173] In at least one embodiment, the polynucleotide comprises a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of odd-numbered SEQ ID NO: 1-255. In at least one embodiment, the polynucleotide comprises a codon degenerate sequence of a sequence selected from the group consisting of odd-numbered SEQ ID NO: 1-255.

[0174] The polynucleotides encoding the engineered scPT polypeptides of the present disclosure may be operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression constructs containing a heterologous polynucleotide encoding the recombinant polypeptide can be introduced into appropriate host cells to express the corresponding polypeptide. Because of the knowledge of the codons corresponding to thevarious amino acids, availability of a protein sequence provides a description of all the polynucleotides capable of encoding the subject. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the improved prenyltransferase enzymes disclosed herein. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences of even-numbered SEQ ID NO: 4-256 presented in Tables 1A-1D.

[0175] The codons can be selected to fit the host cell in which the polypeptide is being produced. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. It is contemplated that all codons need not be replaced to optimize the codon usage of the recombinant polypeptide since the natural sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues. Consequently, codon optimized polynucleotides encoding the engineered scPT polypeptide may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full length coding region.

[0176] The present disclosure provides an expression vector comprising a polynucleotide encoding a engineered scPT polypeptide having prenyltransferase activity, and optionally, increased thermostability, and one or more expression regulating regions such as a promoter, a terminator, a replication origin, or the like, depending on the type of hosts into which they are to be introduced. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the recombinant polypeptide at such sites. Alternatively, a polynucleotide sequence of the present disclosure may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. The recombinant expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide sequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0177] The expression vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. In at least one embodiment, the expression vector further comprises one or more selectable markers, which permit easy selection of transformed cells.

[0178] The present disclosure also provides a host cell comprising a polynucleotide or expression vector encoding a recombinant engineered scPT polypeptide of the present disclosure, wherein the polynucleotide is operatively linked to one or more control sequences for expression of the polypeptide having prenyltransferase activity in the host cell. Host cells for use in expressing the polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, bacterial cells, such as E. coli, Bacillus subtilis, or fungal cells, such as Saccharomyces cerevisiae or Pichia pastoris, insect cells, such as Drosophila S2 and Spodoptera Sf9, animal cells, such as CHO, COS, BHK, 293, and plant cells. Appropriate culture mediums and growth conditions for the abovedescribed host cells are well known in the art.

[0179] In at least one embodiment, the present disclosure provides a method for producing a prenylflavonoid comprising: (a) culturing in a suitable medium a recombinant host cell of the present disclosure; and (b) recovering the produced prenylflavonoid. As disclosed elsewhere herein, increased thermostability of the engineered scPT polypeptides of the present disclosure provides at least the following benefits for the use of these enzymes: simplified heat purification (allowing more efficient enzyme preparation), increased biosynthetic reaction lifetime (allowing less enzyme to be used in biosynthesis and more complete reactions), higher temperature biosynthetic reaction (allowing increased reaction rate to completion).

[0180] In at least one embodiment, the engineered scPT polypeptides with prenyltransferase activity, and optionally, increased thermostability, of the present disclosure can be used in cell-free, in vitro biosynthesis of prenylflavonoid compounds. The prenyltransferase catalyzed transfer of a prenyl group from a prenyl donor substrate, such as GPP or DMAPP, to a flavonoid precursor compound, such as chrysoeriol, is a critical enzymatic step in the biosynthesis of many flavonoid compounds of interest, including the prenylflavonoids, Cannflavin A, B, and C. Accordingly, it is contemplated that the recombinant scPTpolypeptides of the present disclosure (e.g., scPTs of Tables 1A-1D), which have been engineered to prenylate flavonoid substrates such, as chrysoeriol, with prenyl donors, such as GPP and DMAPP, optionally, with increased thermostability, can be used in a range of in vitro, cell-free systems for the biosynthesis of prenylflavonoid compounds requiring a prenyltransferase step, such as cannflavins.

[0181] FIG. 1 depicts a schematic overview of the molecular inputs / outputs and enzymes involved in an exemplary system for the biosynthesis of prenylflavonoid compounds, Cannflavin A or Cannflavin B. This biosynthetic scheme begins with phenylalanine as the starting input substrate, and carries out a chain of enzymatic conversions to naringenin, then to luteolin, then to chrysoeriol, and finally, the prenylation of chrysoeriol with either GPP or DMAPP as prenyl donor, to either Cannflavin A or Cannflavin B. In at least one embodiment, it is contemplated that the engineered scPT polypeptides of the present disclosure can be used in a cell-free process for the synthesis of Cannflavin A or B starting from phenylalanine or any of the other precursors to chrysoeriol as depicted in FIG. 1.

[0182] The final two steps of the biosynthetic scheme of FIG. 1 are the methylation of luteolin to form chrysoeriol and the prenylation of chrysoeriol to form either Cannflavin A or Cannflavin B. FIG. 2 depicts how these final two steps can be incorporated in a cell-free system or process for the biosynthesis of Cannflavin A that starts from the precursor substrate, luteolin. As shown in FIG. 2, luteolin is methylated to chrysoeriol by a methyltransferase using S-adenosyl methionine as co-substrate, and then the chrysoeriol is prenylated with GPP by the activity of an engineered scPT polypeptide of the present disclosure. The GPP prenyl donor can be formed is situ from a precursor isoprenol via an enzyme cascade as described elsewhere herein.

[0183] A cell-free biocatalysis cascade process for the biosynthesis of Cannflavin A can be based on the scheme depicted in FIG. 2. In such a process, low-cost luteolin (3',4',5,7-tetrahydroxyflavone), which can be obtained from plant extraction, is methylated regioselectively at the 3'-OH position using a reaction mixture of SAM (or AdoMet) and a SAM (or AdoMet)-dependent methyltransferase (MT) to yield the chrysoeriol (4',5,7-Trihydroxy-3-methoxyflavone). In one alternative depicted in the scheme of FIG. 2, the SAM used in this biocatalytic reaction step may be produced in situ from methionine and ATP using a methionine adenosyltransferase (MAT). This in situ production of the co-substrate SAM provides the unexpected advantage of eliminating enzyme precipitation that can occur when commercially obtained SAM is used in the biocatalytic reaction at the desired concentrations. Production of the SAM in situ from ATP and methionine, avoids this problem and thereby making it possible to reach commercially desired SAM concentrations.

[0184] The next biocatalytic step depicted in the scheme of FIG. 2 uses the chrysoeriol product of the first step and a prenyl donor compound GPP and the activity of an engineeredscPT to catalyze the regioselective prenylation of the chrsyoeriol thereby forming the prenylflavonoid product, Cannflavin A. As noted in the scheme of FIG. 2, the prenyl donor compound, GPP may be synthesized biocatalytically in situ from the precursor isoprenol by employing a cell-free four-enzyme cascade, as disclosed in e.g., US 2024 / 0076699 A1, which is hereby incorporated by reference herein for all purposes.

[0185] Additionally, the enzyme cascades used for in situ production of substrates as described in FIG. 1 and FIG. 2 and elsewhere in the present disclosure contain multiple thermodynamically favored and substantially irreversible steps. When carried out under cell-free in vitro conditions, these enzyme cascades can lead to more substantial and complete conversion of the substrate at the solubility limit of each respective compound. Accordingly, the biosynthetic processes of this disclosure generally require only slightly over-stoichiometric input of such precursor chemicals that may run reactions to completion, yielding a better molecule economy.

[0186] In at least one embodiment of the process based on the scheme of FIG. 2, the SAM forming and the prenyl-donor compound forming reactions may be combined in a single cell-free reaction along with one (or multiple) soluble engineered scPT polypeptide (e.g., as disclosed in Table 1A) to catalyze the regioselective C-prenylation forming the product Cannflavin A (or derivative). As noted elsewhere herein, engineered scPT polypeptides may be prepared to catalyze the transfer prenyl groups from donor compounds other than GPP onto substrates other than chrysoeriol, to form alternative prenylflavonoids, e.g., Cannflavin B, D, or other prenylflavonoid molecule derivatives. Exemplary prenylation of flavonoids, other than chrysoeriol, include prenylation of naringenin, genistein, and apigenin, which have been disclosed in e.g., Valliere et al., “A cell-free platform for the prenylation of natural products and application to cannabinoid production,” Nat. Commun. 10, 565 (2019); and in U.S. Patent No.11,479,760.

[0187] The present disclosure also provides novel approaches for cell-free, biocatalytic synthesis of prenylflavonoids, such as the Cannflavins A, B, and C. Such approaches may have the potential to significantly advance the production and application of these bioactive compounds. Such approaches may also potentially be broadly applied to other prenyl flavonoid compounds, which also have bioactivities relevant to healthcare. By addressing current limitations in synthetic biology and chemical synthetic pathways, such approaches disclosed here may facilitate the large-scale production of cannflavins, enabling further exploration of their therapeutic benefits and the development of new medical treatments. For example, the novel approaches of this disclosure may be used for the (enzymatic) synthesis of cannflavins and other prenylflavonoids at commercially relevant titers and scales.

[0188] Table 2 provides a list of exemplary enzymes that can be used in a cell-free system or process based on biosynthetic schemes of FIG. 1 and FIG. 2 that incorporates the engineered scPT polypeptides of the present disclosure.

[0189] TABLE 2: Enzymes useful in biocatalytic processes of the disclosureAbbrev. SourceName Name Organism NCBI # or E.C. bsLuxS S-ribosylhomocysteine lyase Bacillus subtilis 034667 gsMtn MTA / SAH nucleosidase Geobacillus A0A0K9HF05 stearothermophilusgsPPase pyrophosphatase Geobacillus 005724stearothermophilustkMAT methionine Thermococcus Q5JF22adenosyltransferase kodakarensisroMT-9 flavonoid 3'-O- Oryza sativa Q6ZD89methyltransferase 9scPT aromatic prenyltransferase Streptomyces sp. CL190 Q4R2T2PAL phenylalanine ammoniaCannabis sativa 4.3.1.24lyaseC4H cinnamic acid 4-hydroxylase Cannabis sativa 1.14.14.91 4CL 4-coumaric acid:CoA ligase Cannabis sativa 6.2.1.12CHS chaicone synthase Cannabis sativa 2.3.1.74 CHI chaicone isomerase Cannabis sativa 5.5.1.6FNS flavone synthase Cannabis sativa 1.14.11.22 F3’H flavonoid 3' hydroxylase Cannabis sativa 1.14.14.82 OMT-21 flavonoid 3'-O- Cannabis sativa A0A7J6DZY9 methyltransferase 21PT-3 prenyltransferase 3 Cannabis sativa DAC76713.1 ecThiM hydroxyethylthiazole kinase Escherichia coli K12 P76423 mtIPK isopentyl phosphate kinase Methanothermobacter 026153thermautotrophicuseclDI isopentenyl-diphosphate Escherichia coli K12 NP_417365Delta-isomerasegsFPPS farnesyl pyrophosphate Geobacillus KOR95521synthase (S82F) stearothermophilusNCBI# (in bold) are listed for those enzymes characterized in the literature related to Cannflavin synthesis,.Enzyme classification numbers (E.C.) are listed for all other enzymes that are generallyknown to be part of the flavonoid biosynthesis pathways

[0190] Cell free biosynthesis methods utilizing the soluble prenyltransferase, NphB, from which the engineered polypeptides of the present disclosure are derived, are described in Valliere et al. “A bio-inspired cell-free system for cannabinoid production from inexpensive inputs,” Nature Chemical Biology Vol. 16, Dec. 2020, 1427-1433; and W02020 / 028722A1 , which is hereby incorporated by reference herein in its entirety. Indeed, the increased thermostability of the engineered scPT polypeptides of the present disclosure, including the exemplary polypeptides of Table 1D, allows them to be incorporated directly into cell-freebiosynthesis methods that require prenylation of a flavonoid precursor, such as chrysoeriol, with prenyl donors, such as GPP or DMAPP. Moreover, using the engineered scPT polypeptides of the present disclosure, the cell-free biosynthesis methods can be carried at higher temperatures resulting higher rates of conversion. Such uses of the engineered NphB polypeptides of the present disclosure for cell-free biosynthesis of prenylflavonoids, such as Cannflavins A, B, and C, are described elsewhere herein and exemplified in the Examples.

[0191] As described elsewhere herein, the engineered scPT polypeptides with prenyltransferase activity on the substrate chrysoeriol, and optionally, increased thermostability, of the present disclosure can be incorporated in any biosynthesis method requiring a prenyltransferase catalyzed biocatalytic step. In at least one embodiment, the engineered scPT polypeptides (e.g., exemplary polypeptides of Tables 1A or 1D) can be used in a method for preparing a prenylflavonoid of compound (1) (Cannflavin A).CH3H3COH OH3COH HO OO(1) CH3

[0192] This biosynthetic method comprises contacting an engineered polypeptide of the present disclosure (e.g., polypeptide of any one of even-numbered SEQ ID NO: 4-64 or 196-256) under suitable reactions conditions, with the prenyl donor compound GPP and a flavonoid precursor compound (2) (chrysoeriol)OH OOH HO OO(2)CH3.

[0193] In at least one embodiment, the engineered scPT polypeptides (e.g., exemplary polypeptides of Tables 1B) can be used in a method for preparing a prenylflavonoid of compound (3) (Cannflavin B).CH3OH OH3COH HO O O CH3(3)

[0194] This biosynthetic method comprises contacting an engineered polypeptide of the present disclosure (e.g., polypeptide of any one of even-numbered SEQ ID NO: 66-132) under suitable reactions conditions, with the prenyl donor compound, DMAPP compound and a flavonoid precursor compound (2) (chrysoeriol)OH OOH HO OO(2)CH3.

[0195] In at least one other embodiment, the engineered scPT polypeptides (e.g., exemplary polypeptides of Tables 1C) can be used in a method for preparing a prenylflavonoid of compound (4) (Cannflavin C).OH OOH HO O O CH3CH3(4)H3C CH3

[0196] This biosynthetic method comprises contacting an engineered polypeptide of the present disclosure (e.g., polypeptide of any one of even-numbered SEQ ID NO: 134-194) under suitable reactions conditions, with the prenyl donor compound, GPP compound and a flavonoid precursor compound (2) (chrysoeriol).OH OOH HO OO(2)CH3

[0197] The present disclosure contemplates ranges of suitable reaction conditions that can be used in the methods, including but not limited to ranges of pH, temperature, buffer, solvent system, substrate loading, polypeptide loading, co-substrate or co-factor loading, atmosphere, and reaction time. The present disclosure also contemplates that the methods comprising the biocatalytic conversion of a substrate compound (2) to a product compound (1), compound (3), or compound (4) using an engineered scPT polypeptide of the disclosure can further comprise additional chemical or biocatalytic steps carried out on the product compound, product compound work-up, extraction, isolation, purification, and / or crystallization, each of which can be carried out under a range of conditions.

[0198] Further suitable reaction conditions for carrying out the biocatalytic conversion of a substrate compound (2) to a product compound (1), compound (3), or compound (4) using an engineered scPT polypeptide described herein can be readily optimized by routine experimentation that includes, but is not limited to, contacting the engineered polypeptide and substrate under experimental reaction conditions of concentration, pH, temperature, solvent conditions, and detecting the production of the desired compound, for example, using the methods described in the Examples provided herein.

[0199] The increased thermostability of the engineered scPT polypeptides of the present disclosure can also provide increased biosynthetic reaction lifetimes, which allows for the use of less enzyme, and / or allows for more complete enzymatic reactions resulting in higher product purity. Thus, it is contemplated that the use of the engineered prenyltransferase enzymes in a method for the conversion of a substrate compound (2) to a product compound (1), compound (3), or compound (4) using an engineered scPT polypeptides can result in the preparation of the product compound of in very high purity. Accordingly, in at least one embodiment, the engineered scPT polypeptides (e.g., exemplary polypeptides of Tables 1A or 1 D) can be used in a biosynthetic process for preparing the compound (1) with a purity of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or even higher.

[0200] Generally, a biosynthetic reaction involving the prenyltransferase catalyzed conversion of a flavonoid precursor compound (2) to a Cannaflavin A product of compound (1) can be carried out in accordance with reaction conditions for cell-free biosynthesis cannabinoids using prenylation by a similar engineered prenyltransferase known in the art (see e.g., Valliereet al. 2020; or W02020028722A1) or as described herein. However, in view of their increased thermostability of the engineered polypeptides of the present disclosure, it is contemplated that the suitable reaction conditions can include temperature of the reaction solution up to about 45 °C. Thus, in some embodiments of the method, the suitable reaction conditions can include a temperature range of about 20 °C to about 45 °C. In one embodiment, the suitable reaction conditions comprise a temperature of about 37 °C.

[0201] It is also contemplated that the increased thermostability of the engineered scPT polypeptides of the present disclosure can allow a range of substrate loading in the reaction. Thus, in some embodiments of the method of preparing a prenylflavonoid compound of any of Cannflavins A, B, or C, the suitable reaction conditions can comprise a cannflavin precursor substrate (e.g. a flavonoid) loading of at least about 0.6 g / L, at least about 1.2 g / L, at least about 2 g / L, at least about 6 g / L, at least about 12 g / L, at least about 18 g / L, at least about 24 g / L, at least about 30 g / L or even greater. Specifically, where the flavonoid precursor substrate is chrysoeriol, the substrate loading can be at least about 0.6 g / L, at least about 1.2 g / L, at least about 2 g / L, at least about 6 g / L, at least about 12 g / L, at least about 18 g / L, at least about 24 g / L, at least about 30 g / L, or even greater.

[0202] The increased thermostability of the engineered scPT polypeptides of the present disclosure can allow reactions to be carried out at higher temperatures, resulting in higher rates of biocatalytic conversion. Additionally, increased thermostability of the engineered scPT polypeptides facilitates their heat-based purification from cell lysates. Thus, it is contemplated that in some embodiments the prenyltransferase catalyzed conversion of a flavonoid precursor compound (2) to a prenylflavonoid product of compounds (1), (2), or (3), can be carried out with lower concentrations of the engineered scPT polypeptide.Accordingly, in at least one embodiment of the method, the suitable reaction conditions comprise an engineered scPT polypeptide concentration of about 0.1 g / L to about 5 g / L, or an even lower concentration.

[0203] As noted elsewhere herein, suitable pH and buffer conditions for the use of soluble prenyltransferases in the cell-free biosynthesis of cannabinoids are known in the art, and these conditions can also be used with the engineered scPT polypeptides in the processes for biosynthesis of cannflavins of the present disclosure. Accordingly, in at least one embodiment a method of producing a prenylflavonoid, such as Cannflavin A, B, or C, using the engineered polypeptides of the present disclosure, the suitable reaction conditions can comprise: (a) a pH of about 5.0 to about 11.0, or about 4.0 to 10.0; and / or a buffer solution of about 0.05 M Tris-Cl pH 8.0 to about 0.5 M Tris-CI pH 8.0. In at least one embodiment, the suitable reaction conditions for preparing the cannflavin compound, Cannflavin A, comprise: chrysoeriol, GPP, 0.1 M buffer (e.g., Tris), pH 8.0, and the engineered scPT polypeptide at 28 °C for at least 1 hour. It is contemplated that identical or very similar conditions for the biosynthetic production of cannflavins B or C except with DMAPP replacing GPP for preparing cannflavin B. Suitablereaction conditions for the various engineered scPT polypeptides of the present disclosure can be easily determined using routine techniques for optimizing biocatalytic reaction conditions well-known to one of ordinary skill.EXAMPLES

[0204] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.EXAMPLE 1. A cell-free five-enzyme cascade system for synthesis of chrysoeriol.

[0205] This example illustrates the use of a cell-free five-enzyme cascade system for the bioconversion of luteolin to chrysoeriol using the biosynthetic scheme as depicted in FIG. 3A.The five-enzyme cascade system drives the specific 3’-O-methylation of luteolin to chrysoeriol with a high yield. Additional details of this five-enzyme system and its use can be found in e.g., Kim, et al. (2006). Flavonoid 3'-O-methyltransferase from rice: cDNA cloning, characterization and functional expression. Phytochemistry, 67(4), 387-394.

[0206] The five enzymes used and their concentrations were as follows: tkMAT (10 pM), gsPPase (about 0.25 pM), roMT-9 (about 8.75 pM), gsMtn (about 0.625 pM), and bsLuxS (about 0.25 pM). The starting bioconversion reaction mixture contained about 7 mM luteolin (TCI America) as the substrate, used cofactor feedstocks of ATP (about 7.5 mM;FisherScientific) and methionine (about 7.5 mM; FisherScientific), and used as buffers Tris-HCI at pH 8 (about 200 mM), MgCh (about 20 mM), KCI (about 50 mM), and SAM (about 0.05 mM; Millipore-Sigma). The bioconversion reaction was carried out at about 28 °C for about 24 hours.

[0207] The product compound, chrysoeriol was crystallized as follows: to remove the product from the reaction mixture (1 VE; volume equivalent) enzymes were precipitated adding 0.25 VE ethanol and filtered, subsequently 0.75 VE methanol was added and chrysoeriol was eventually crystalized at 4C by adding ice-cold ddH2O. The identity of the crystals was confirmed as the product by HPLC analysis (ThermoFisher Ultimate HPLC: C-18 RP column, H2O to acetonitrile gradient, UV= 260nm) and comparison with the standards of luteolin and chrysoeriol

[0208] As shown in FIG. 3B, HPLC traces of the luteolin and chrysoeriol standard (purchased from TCI America and Toronto Research Chemicals, respectively) with the HPLC trace of thebioconversion reaction indicated that approximately 99% conversion to the desired chrysoeriol product.EXAMPLE 2. Engineering promiscuous aromatic prenyltransferases for manufacturing of Cannflavin A.

[0209] This example illustrates the preparation of engineered prenyltransferase enzymes capable of prenylating chrysoeriol to form Cannflavin A.

[0210] The naturally-occurring promiscuous aromatic prenyltransferase from Streptomyces sp. (strain CL190) (“scPT”) having the amino acid sequence of SEQ ID NO: 2 and the known 3D structure (PDB# 1ZB6) was engineered with site-directed mutations to accept chrysoeriol as a prenyl acceptor of geranyl pyrophosphate (GPP). The Rosetta Design algorithm (see e.g., Leaver-Fay, et al. (2011). ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. In Methods in enzymology (Vol. 487, pp. 545-574). Academic Press.), was used to design a mutant library consisting of specific site mutations of the scPT sequence that were likely to increase acceptance of the chryseriol substrate were identified. The library of mutations were introduced into DNA sequences encoding the naturally occurring scPT of SEQ ID NO: 2. The corresponding library of mutated DNA sequences encoding the mutated scPT polypeptides were introduced in a plasmid vector (pET28a). The mutant plasmid library was expressed recombinantly in E. coli (BL21 DE3 Gold). To screen the mutant library, small scale expression and purifications were performed. Briefly, 50 mL of LB media was inoculated with 25 pL of a saturated culture of BL21 DE3 Gold harboring the scPT mutant expression plasmid (pET28a). The cultures were incubated at 37° C. until the QD600 reached 0.4-0.6. The expression of the scPT constructs was induced with the addition of 1 mM IPTG, followed by incubation for 18 hours at 18° C. Cells were harvested by centrifugation at 2500xg. The pellets were re-suspended in 500 pL of lysis buffer: 50 mM [Tris pH 8.0], 150 mM NaCI, and 5 mM imidazole and lysed by sonication. The cell lysate was clarified by centrifugation at 20,000xg for 10 minutes at 4° C., and the supernatant was incubated at 4° C. with 50 PL of NiNTA resin. The supernatant / resin was applied to the columns and washed with 500 pL of lysis buffer. The enzyme was eluted using 200 pL of elution buffer (50 mM Tris [pH 8.0], 150 mM NaCI, 250 mM imidazole, and 10% (v / v) glycerol).

[0211] The eluted enzymes were isolated and screened for Cannflavin A production in an assay employing chrysoeriol and GPP as the prenyl-acceptor and prenyl-donor pair, respectively. Briefly, the desired bioconversion activity was assayed under the following conditions: ~5 mM geranyl pyrophosphate, ~3.5 mM chrysoeriol, 5 mM MgCh, 50 mM Tris pH 8.0, ~6 pM scPT mutant in a final volume of 100 pL. All enzymes were diluted to the same stock concentration using elution buffer so the final concentration of imidazole was the same in each reaction. The reactions were incubated for 48 hours at 28° C, then quenched byadding 900 pL methanol and subjected to HPLC analysis. A typical HPLC trace of the enzyme assay product (FIG. 4C) was compared to standard for the substrate chrysoeriol (FIG. 4A) and the desired product, Cannflavin A (FIG. 4B).

[0212] Amino acid sequences of these 25 scPT mutants are disclosed in Table 1A, and in the accompanying Sequence Listing as even-numbered SEQ ID NO: 4-52. Table 3 summarizes the activity of the 25 variants in the prenylation of chrysoeriol to produce Cannflavin A.

[0213] TABLE 3SEQ Production Production Amino Acid DifferencesID of target of side NO: (as compared to SEQ ID NO: 2) cannflavin1products14 Q161A, S177T, S214A, C230T, G286A, V294T +++ - 6 Q161A, S177T, F213A, S214W, C230A, G286A, ++ - V294A, L298Y8 S51T, M129A, Q161L, S177G, S214A, C230T, ++ + V294E10 V47I, S177T, C230T, V294I, Q295D - - 12 S51T, M129C, Q161Y, S177T, S214A, C230T, - + V294E14 C230T - + 16 L298M - - 18 S51I - - 20 G286A ++ - 22 C230A + + 24 M129Q ++ + 26 Q161E28 S177T30 S214L32 I234M34 F213M36 V294I + + 38 S214A - + 40 M129A + - 42 V47I ++ + 44 Q161Y + + 46 F213A - - 48 Q295D - - 50 V294T - - 52 Q161A ++ - “+” indicates low abundance product.“++” indicates medium abundance product.“+++” indicates high abundance product.indicates product not detected / not stable.

[0214] As shown by the results in Table 3, the scPT variant of SEQ ID NO: 4, which has a set of six mutations (Q161A, S177T, S214A, C230T, G286A, V294T) relative to the naturallyoccurring scPT of SEQ ID NO: 2, exhibited -90% conversion of chrysoeriol to Cannflavin A, as confirmed by HPLC comparison with an authentic Cannflavin A standard.

[0215] To identify a minimal set of mutations capable of achieving the high bioconversion rate of SEQ ID NO: 4, a set of six variants were prepared in which each of the six individual mutations of the engineered were reverted to the wild-type sequence, and the effects of each reversion on enzyme activity were assessed. Table 4 summarizes the activity of these 6 variants having even-numbered SEQ ID NO: 54-64 in the prenylation of chrysoeriol to produce Cannflavin A.

[0216] TABLE 4Amino Acid DifferencesSEQ ID Production of NO: (as compared to SEQ ID NO: 4) Cannflavin154 Q161A, S214A, C230T, G286A, V294T +++56 Q161A, S177T, C230T, G286A, V294T ++58 Q161A, S177T, S214A, G286A, V294T ++60 Q161A, S177T, S214A, C230T, V294T ++62 Q161A, S177T, S214A, C230T, G286A ++64 S177T, S214A, C230T, G286A, V294T -1Cannflavin Production“+” indicates low abundance product.“++” indicates medium abundance product.“+++” indicates high abundance product.indicates product not detected / not stable.

[0217] As shown by the assay results of Table 4, the mutation S177T was not essential for activity. The scPT variant M1-ST of SEQ ID NO: 54, which has only five mutations (Q161A, S214A, C230T, G286A, V294T) exhibits high activity in the bioconversion of the chrysoeriol substrate to Cannflavin A.EXAMPLE 3. Increasing thermal stability of promiscuous aromatic prenyltransferases.

[0218] This example illustrates engineering variants of the promiscuous aromatic prenyltransferase scPT that exhibit increased thermal stability, and allow for the bioconversion of chrysoeriol to Cannflavin A at higher temperatures.

[0219] The thermostable variants were engineered from the scPT variant M1-ST of SEQ ID NO: 54 described in Example 2. To further enhance the thermostability of this optimized enzyme, we utilized the ProteinMPNN method to design thermostable variants of SEQ ID NO: 54 having amino acid sequences of even-numbered SEQ ID NO: 196-256. The variants were computationally generated using ProteinMPNN, with one variant designed based on mutations described in in U.S. 11,518,983 B1 (which is hereby incorporated by referenceherein for all purposes). The variant enzymes were expressed, prepared, and assayed as outlined in Example 2. Assays for increased thermostability were carried out according to methods for assaying thermostable prenyltransferases described in U.S. 11,518,983 B1. Table 5 summarizes the mutations and assay results for these variants in the prenylation of chrysoeriol to produce Cannflavin A and under thermostability challenging conditions.

[0220] TABLE 5SEQ ProductionAmino Acid DifferencesID of target ThermoNO: (as compared to SEQ ID NO: 2) cannflavin1stability22 n / a + not stable at 55C for 1 h 54 Q161A, S214A, C230T, G286A, V294T +++ + 196 Q161A, S214A, C230T, G286A, V294T, S136A, + + + M14I, E222D, G224S, N236T, A24P, G297K,Y31W, T69P, T77I, V911, T98I198 Q161A, S214A, C230T, G286A, V294T, A5E, + + + + C25V, T69V, Q99A, A181E, K251R, G297E,A301 K, F302Y, D303A200 Q161A, S214A, C230T, G286A, V294T, A5E, + + +++ C25V, T69V, Q99A, A181E, K251R, H253T,G297N, A301K, F302Y, D303A202 Q161A, S214A, C230T, G286A, V294T, C25V, + + + + T69V, Q99A, A181P, K251R, H253T, G297E,A301 K, F302Y, D303N204 Q161A, S214A, C230T, G286A, V294T, A4Q, + + + E80A, A133E, A137K, E150P, A153K, R154K,A181E, Q182E, E185A, A186P, E246P206 Q161A, S214A, C230T, G286A, V294T, A4Q, + + + D28E, A133E, A137K, E150P, A153K, R154K,A181D, Q182E, E185S, A186D, E246P208 Q161A, S214A, C230T, G286A, V294T, C25V, + + + + T69V, Q99A, A181E, K251R, H253T, G297E,A301 K, F302Y210 C25V, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T212 F302Y, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T214 Q99A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T216 T69V, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T218 K251R, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T220 A301K, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T222 H253T, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T224 A181E, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T226 R154K, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T228 Q182E, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T230 E246P, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T232 E185S, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T234 A153K, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T236 A137K, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T238 E150P, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T240 D303A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T242 A181D, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T244 D28E, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T246 E80A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T248 A186D, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T250 A181P, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T252 A133E, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T254 G297N, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T256 G297E, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T1Cannflavin Production“+” indicates low abundance product.“++” indicates medium abundance product.“+++” indicates high abundance product.indicates product not detected / not stable.“n.d.” is “not determined.”2Thermostability“+” indicates < 20% activity retained after heat treatment.“++” indicates < 50% activity retained after heat treatment.“+++” indicates < 80% activity retained after heat treatment.“n.d.” is “not determined.”

[0221] Of the seven variants prepared, the variant “S3” having SEQ ID NO: 200, demonstrated superior thermostability as confirmed by SDS-PAGE analysis at about 55 °C and about 75°C (data not shown). The variant also exhibited medium abundance production of Cannflavin A as confirmed by HPLC-based activity assays following heat treatment at about 55°C. The variant of SEQ ID NO: 200 includes 11 thermostabilizing mutations (A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301K, F302Y, and D303A) and five substrate specific mutations (Q161A, S214A, C230T, G286A, V294T). The thermostable variant of SEQID NO: 200 can allow the production of Cannflavin A under elevated temperatures, thereby providing significant advantages in biosynthetic efficiency for industrial applications requiring robust biocatalysts.EXAMPLE 4. Engineering promiscuous aromatic prenyltransferases for manufacturing of Cannflavin B and Cannflavin C.

[0222] This example illustrates the design of engineered scPT mutants with engineered structural features suitable for use in the biosynthetic manufacturing of Cannflavin B or Cannflavin C. These mutants were designed by using the Rosetta Design algorithm as described in Example 2. The amino acid sequences of these mutants are listed in Table 2B and 2C.

[0223] A total of 34 scPT variants having amino acid sequence of even-numbered SEQ ID NO: 66-132 were designed to convert chrysoeriol to Cannflavin B and use in a process for the biosynthesis of Cannflavin B. These scPT variants can be screened to determine activity and stability in synthesizing Cannflavin B using the approaches disclosed in Example 2. Table 6 summarizes the features of these 34 scPT variant designs.

[0224] TABLE 6SEQ Production Production ID Amino Acid Differences of target of side NO: (as compared to SEQ ID NO: 2) cannflavin1products266 S64V, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T68 K119A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T70 K284L, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T72 K284L, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T74 K119A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T76 K169R, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T78 S64L, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T80 K169R, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T82 K169N, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T84 Y121F, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T86 M106A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294T88 F123Y, Q161A, S177T, S214A, C230T, G286A, n.d. n.d.V294TM162A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TS164A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TY216A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TK169N, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TY121F, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TN173L, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TY175F, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TS164A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TY216A, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TV47I, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TN173L, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TY175F, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TY288F, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TQ295D, Q161A, S177T, S214A, C230T, G286A, n.d. n.d. V294TS51A, D62G, S64Y, S66Q, M106S, A108G, D110A, n.d. n.d. K118V, K119A, Y121T, F123L, S164A, K169N,N173L, Y175F, Y216T, K284L, Y288F, Q161A,S177T, S214A, C230T, G286A, V294TD62G, S64Y, S66Q, M106S, A108G, D110A, n.d. n.d. K118V, K119A, F123L, S164A, K169N, N173L,Y175F, K284L, Y288F, Q161A, S177T, S214A,C230T, G286A, V294TS51A, D62G, S64C, S66Q, M106S, K118V, K119Q, n.d. n.d. Y121F, F123L, S164A, K169R, N173L, Y175F,Y216T, K284L, Y288F, Q161A, S177T, S214A,C230T, G286A, V294TV47L, V49T, S51E, S64L, S66Q, M106S, K119A, n.d. n.d. Y121F, F123Y, M162G, S164A, K169N, Y216A,K284L, Y288M, Q295M, Q161A, S177T, S214A,C230T, G286A, V294TS51E, S64L, M106A, K119A, Y121F, F123Y, n.d. n.d. M162G, S164A, K169N, N173L, Y175F, Y216A,K284L, Q161A, S177T, S214A, C230T, G286A,V294TV47L, V49T, D110G, K119A, Y121L, K169N, n.d. n.d. K284L, Y288M, Q295M, Q161A, S177T, S214A,C230T, G286A, V294TS64L, K119A, K169N, K284L, Q161A, S177T, n.d. n.d.S214A, C230T, G286A, V294T132 S64V, K119A, Y121F, S164A, K169R, Y216A, n.d. n.d. K284L, Q161A, S177T, S214A, C230T, G286A,V294T“+” indicates low abundance product.“++” indicates medium abundance product.“+++” indicates high abundance product.indicates product not detected / not stable.“n.d.” is “not determined.”

[0225] ...

Claims

CLAIMSWhat is claimed is:

1. An engineered prenyltransferase enzyme (“scPT”) comprising an amino acid sequence of at least 80% identity to SEQ ID NO: 2 and at least one amino acid mutation as compared to SEQ ID NO: 2 at a position corresponding to position of SEQ ID NO: 2 selected from V47, S51, M129, Q161, S177, F213, S214, C230, I234, G286, V294, Q295, and L298; optionally, wherein the amino acid mutation is selected from V47I, S51I, S51T, M129A, M129C, M129Q, Q161A, Q161E, Q161L, Q161Y, S177G, S177T, F213A, F213M, S214A, S214L, S214W, C230A, C230T, I234M, G286A, V294A, V294E, V294I, V294T, Q295D, L298M, and L298Y.

2. The engineered scPT of claim 1 , wherein the amino acid sequence comprises a set of amino acid mutations as compared to SEQ ID NO: 2 at a set of positions corresponding to positions of SEQ ID NO: 2 selected from:Q161, S214, C230, G286, and V294;Q161, S177, S214, C230, G286, and V294;Q161, S177, F213, S214, C230, G286, V294, and L298;S51, M129, Q161, S177, S214, C230, V294;V47, S177, C230, V294, and Q295; andS51, M129, Q161, S177, S214, C230, V294.

3. The engineered scPT of claim 3, wherein the set of amino acid mutations as compared to SEQ ID NO: 2 are selected from:Q161A, S214A, C230T, G286A, V294T;Q161A, S177T, S214A, C230T, G286A, V294T;Q161A, S177T, F213A, S214W, C230A, G286A, V294A, L298Y;S51T, M129A, Q161L, S177G, S214A, C230T, V294E;V47I, S177T, C230T, V294I, Q295D; andS51T, M129C, Q161Y, S177T, S214A, C230T, V294E.

4. An engineered scPT, comprising an amino acid sequence of at least 90% identity to SEQ ID NO[[s]]: P1 and at least one amino acid mutation as compared to SEQ ID NO: 2 at a position corresponding to position of SEQ ID NO: 2 selected from: V47, S64, M106, K119, Y121, F123, M162, S164, K169, N173, Y175, Y216, K284, Y288, and Q295; optionally, wherein the mutation is selected from: V47I, S64L, S64V, M106A, K119A, Y121F, F123Y, M162A, S164A, K169N, K169R, N173L, Y175F, Y216A, K284L, Y288F, and Q295D.

5. The engineered scPT of claim 4, wherein the amino acid sequence comprises a set amino acid mutations as compared to SEQ ID NO: 2 selected from:V47L, V49T, S51E, S64L, S66Q, M106S, K119A, Y121F, F123Y, M162G, S164A, K169N, Y216A, K284L, Y288M, Q295M;V47L, V49T, D110G, K119A, Y121L, K169N, K284L, Y288M, Q295M;S51A, D62G, S64Y, S66Q, M106S, A108G, D110A, K118V, K119A, Y121T, F123L, S164A, K169N, N173L, Y175F, Y216T, K284L, Y288F;S51E, S64L, M106A, K119A, Y121F, F123Y, M162G, S164A, K169N, N173L, Y175F, Y216A, K284L;D62G, S64Y, S66Q, M106S, A108G, D110A, K118V, K119A, F123L, S164A, K169N, N173L, Y175F, K284L, Y288F;S64V, K119A, Y121F, S164A, K169R, Y216A, K284L; andS64L, K119A, K169N, K284L;S51A, D62G, S64C, S66Q, M106S, K118V, K119Q, Y121F, F123L, S164A, K169R, N173L, Y175F, Y216T, K284L, Y288F.

6. An engineered scPT comprising an amino acid sequence of at least 90% identity to SEQ ID NO[[s]]: P1 and at least one amino acid mutation as compared to SEQ ID NO: 2 at a position corresponding to position of SEQ ID NO: 2 selected from: S64, M106, Y121, F123, M129, Q161, M162, S164, K169, F213, S214, V271, V294, Q295, and L298, optionally, wherein the mutation is selected from: S64V, M106A, Y121F, F123M, M129C, Q161G, Q161I, Q161K, Q161L, Q161S, M162A, S164A, K169N, K169R, F213C, F213H, F213S, S214M, S214Y, V271A, V294K, Q295A, and L298N.

7. The engineered scPT of claim 6, wherein the amino acid sequence comprises a set of amino acid mutations as compared to SEQ ID NO: 2 selected from:V49I, S51A, Q161A, S177T, F213A, S214A, C230T, G286A, V294T, L298N;V49I, S51A, M129C, Q161A, S177T, F213A, S214A, C230T, G286A, V294T, L298N; V49I, M129C, Q161A, S177T, F213A, S214A, C230T, G286A, V294T, L298N;S51A, Q161A, S177T, F213A, S214A, C230T, G286A, V294T S51A, M129A, Q161A, S177T, M129A, Q161A, S177T, S214A, C230T, G286A, V294T;Q161A, S177T, S214A, C230T, G286A, V294T; andF213C, S214A, C230T, V271A, G286A, V294T, L298N.

8. The engineered scPT of any one of claims 1-7, wherein the amino acid sequence further comprises an amino acid mutation as compared to SEQ ID NO: 2 at a position corresponding to a position of SEQ ID NO: 2 selected from: A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301K, F302Y, D303A.

9. The engineered scPT of any one of claims 1-8, wherein the amino acid sequence comprises a set of amino acid mutations as compared to SEQ ID NO: 2 selected from:A5E, C25V, T69V, Q99A, A181E, K251R, H253T, G297N, A301K, F302Y, D303A;A5E, C25V, T69V, Q99A, A181E, K251R, G297E, A301K, F302Y, D303A;A4Q, E80A, A133E, A137K, E150P, A153K, R154K, A181E, Q182E, E185A, A186P, E246P;A4Q, D28E, A133E, A137K, E150P, A153K, R154K, A181D, Q182E, E185S, A186D, E246P;M14I, A24P, Y31W, T69P, T77I, V911, T98I, S136A, S214A, E222D, G224S, C230T, N236T, G286A, V294T, G297K;C25V, T69V, Q99A, A181E, K251R, H253T, G297E, A301K, F302Y; andC25V, T69V, Q99A, A181P, K251R, H253T, G297E, A301K, F302Y, D303N.

10. The engineered scPT of any one of claims 1-9, wherein the engineered scPT comprises an amino acid sequence having at least 50% sequence identity, at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 87.5% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence selected from even-numbered SEQ ID NO: 4-194.

11. The engineered scPT of any one of claims 1-10, wherein the engineered scPT has activity capable of converting chrysoeriol and a prenyl donor compound to a prenylflavonoid.

12. The engineered scPT of the claim 11 , wherein the scPT is capable of converting chrysoeriol and a prenyl donor compound to a prenylflavonoid with a conversion rate of at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87.5%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

13. The engineered scPT of any one of claims 11-12, wherein the prenylflavonoid is a cannflavin.

14. The engineered scPT of claim 13, wherein the flavonoid is selected from Cannflavin A, Cannflavin B, Cannflavin C, and a combination thereof.

15. The engineered scPT of any one of claims 1-14, wherein engineered scPT of has activity capable of converting chrysoeriol and a prenyl donor compound to a cannflavin in a purity of at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the total product compounds.

16. The engineered scPT of any one of claims 1-15, wherein the scPT is active for at least 1 hour at a temperature 40 °C or greater, 45 °C or greater, 50 °C or greater, 55 °C or greater, 60 °C or greater, 65 °C or greater, 70 °C or greater, or 75 °C or greater.

17. The engineered scPT of any one of claims 1-16, wherein the scPT is active for at least 1 hour at a temperature of between about 50 °C and about 100 °C, between about 50 °C and about 90 °C, between about 50 °C and about 80 °C, between about 60 °C and about 80 °C, or between about 55 °C and about 75 °C.

18. A composition for the production of a flavonoid, comprising an engineered scPT of any one of claims 1-17.

19. A composition for the production of a flavonoid of claim 18, wherein the flavonoid is a prenylflavonoid.

20. A composition for the production of a flavonoid of any one of claims 18-19, wherein the flavonoid is a cannflavin.

21. A composition for the production of a flavonoid of any one of claims 18-20, wherein the flavonoid is Cannflavin A, Cannflavin B, Cannflavin C, ora combination thereof.

22. A composition for the production of a flavonoid of any one of claims 18-21 , wherein the composition further comprises a flavonoid precursor compound; optionally, wherein the flavonoid precursor compound is chrysoeriol.

23. A composition for the production of a flavonoid of any one of claims 18-22, wherein the composition further comprises a prenyl donor compound.

24. A composition for the production of a flavonoid of claim 23, wherein the prenyl donor comprises a prenyl alcohol.

25. A composition for the production of a flavonoid of claim 24, wherein the prenyl alcohol is selected from isoprenol, prenol, an a / p unsaturated alcohol derivative, a p / y unsaturated alcohol derivative, an alcohol with a functionalized allylic group, or a mixture thereof.

26. A composition for the production of a flavonoid of any one of claims 23-25, wherein the prenyl donor is selected from dimethylallyl pyrophosphate (DMAPP), and geranyl pyrophosphate (GPP).

27. A composition for the production of a flavonoid of any one of claims 23-26, wherein the prenyl donor comprises geranyl pyrophosphate (GPP).

28. A composition for the production of a flavonoid of any one of claims 18-27, wherein the composition further comprises luteolin.

29. A composition for the production of a flavonoid of any one of claims 18-28, wherein the composition further comprises a methyltransferase; optionally, wherein the methyltransferase is roMT-9.

30. A composition for the production of a flavonoid of any one of claims 18-29, wherein the composition further comprises an enzyme selected from tkMAT, gsPPase, gsMtn, bsLuxS, and a combination thereof.

31. A composition for the production of a flavonoid of any one of claims 18-30, wherein the composition further comprises methionine and methionine adenosyltransferase.

32. A composition for the production of a flavonoid of any one of claims 18-31 , wherein the composition further comprises S-adenosyl methionine (SAM) and a SAM-dependent methyltransferase (MT).

33. A composition for the production of a flavonoid of any one of claims 18-32, wherein the composition further comprises adenosine triphosphate (ATP).

34. A composition for the production of a flavonoid of any one of claims 18-33, wherein the composition is substantially free of cells.

35. A method for the biosynthesis of a prenylflavonoid, wherein the method comprises:contacting chrysoeriol under suitable reaction conditions with an engineered scPT and a prenyl donor compound, whereby the chrysoeriol is converted to a prenylflavonoid.

36. The method of claim 35, wherein the method further comprises:contacting an alcohol under suitable reaction conditions with a path of enzymes comprising ThiM, IPK and / or IDI and FPPS_S82F, whereby the alcohol is converted to a prenyl donor compound.

37. The method of claim 36, wherein the alcohol is selected from isoprenol, prenol, an a / p unsaturated alcohol derivative, a p / y unsaturated alcohol derivative, or a combination thereof.

38. The method of any one of claims 35-37, wherein the prenyl donor compound is dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), ora combination thereof.

39. The method of any one of claims 35-38, wherein the method further comprises: contacting luteolin under suitable reaction conditions with a SAM-dependent methyltransferase (“MT”) and S-adenosyl-methionine (“SAM”), whereby the chrysoeriol is produced.

40. The method of claim 39, wherein the method further comprisescontacting from methionine, adenosine triphosphate (ATP), and a methionine adenosyltransferase, whereby the SAM is produced.

41. The method of any one of claims 39-40, wherein the method further comprises: contacting naringenin under suitable reaction conditions with oxygen and the enzymes “F3’H” and “FNS”, whereby the luteolin is produced.

42. The method of claim 41 , wherein the method further comprises:contacting phenylalanine under suitable reaction conditions with ATP, malonyl-CoA, oxygen, and the enzymes “PAL,” “C4H,” “4CL,” “CHS,” and “CHI,” whereby the naringenin is produced.

43. The method of any one of claims 35-42, wherein the prenyl donor compound is GPP, and the chryseriol is converted to the prenylflavonoid, Cannflavin A.

44. The method of any one of claims 35-42, wherein the prenyl donor compound is DMAPP, and the chryseriol is converted to the prenylflavonoid, Cannflavin B.

45. The method of any one of claims 35-42, wherein the prenyl donor compound GPP and the chryseriol is converted to the prenylflavonoid, Cannflavin C.

46. The method of any one of claims 35-45, wherein the engineered prenyltransferase (“scPT”) comprises an engineered scPT of any one of claims 1-17 or a composition of any one of claims 18-34.

47. The method of any one of claims 35-46, wherein the suitable reaction conditions comprise a cell free solution.

48. The method of any one of claims 35-47, wherein the suitable reaction conditions comprise a temperature of between about 50 °C and about 100 °C, between about 50 °C and about 90 °C, between about 50 °C and about 80 °C, between about 60 °C and about 80 °C, or between about 55 °C and about 75 °C.

49. A polynucleotide encoding an engineered scPT polypeptide of any one of claims 1-17.

50. The polynucleotide of claim 49, wherein the polynucleotide comprises a sequence of at least 80% identity to a sequence selected odd-numbered SEQ ID NO: 3-255.

51. An expression vector comprising the polynucleotide of any one of claims 49-50.

52. A host cell comprising the polynucleotide of any one of claims 49-51.

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