PROTEIN AND PEPTIDE DELIVERY SYSTEMS AND METHODS FOR MAKING AND USING THEM

RELATED APPLICATIONS

This Patent Convention Treaty (PCT) International Application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial No. (USSN) 63/347,873, filed June 1, 2022. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Department of Defense, Office of Naval Research (ONR) grant nos. N00014-17-1-2677; and N00014-20- 1-2120; and, NSF grant no. 1942251. The government has certain rights in the invention.

TECHNICAL FIELD

This invention generally relates to microbiology and bioengineering. In alternative embodiments, provided are chimeric products of manufacture and methods for delivering a proteinaceous cargo, a polypeptide or peptide, or a compound to or into a cell, for example, a eukaryotic cell such as a mammalian or a human cell, or to a plant cell, or to an individual in need thereof. In alternative embodiments, products of manufacture as provided herein comprise: (a) a recombinant bacterial Contractile Injection System (CIS) or a Metamorphosis Associated Contractile structure (MAC) formed or configured to comprise a tube having an inner core, (b) a Metamorphosis- Inducing Factor 1 (Mifl) protein positioned in the inner core of the tube of the CIS or MAC, (c) a chaperone 605 protein non-covalently associated with the Mifl protein positioned in the inner core of the tube of the CIS or MAC, and (d) a proteinaceous cargo, or a heterologous protein or peptide, or compound, non-covalently associated or covalently associated or linked to the Mifl .

BACKGROUND

Many bacteria interact with target organisms using syringe-like structures called Contractile Injection Systems (CIS). CIS structurally resemble headless bacteriophages and share evolutionarily related proteins such as the tail tube, sheath, and baseplate complex. Recent evidence shows that CIS are specialized to puncture membranes and often deliver effectors to target-cells. In many cases, CIS mediate trans-kingdom interactions between bacteria and eukaryotes, however the effectors delivered to target cells and their mode of action are often unknown.

A CIS mediating the beneficial relationship between the gram-negative bacterium Pseudoalteramonas luteoviolacea and marine tubeworm Hydroides elegans was recently characterized (Shikuma et al., 2014, 2016); and this CIS was named “Metamorphosis Associated Contractile structure” (MACs), because they stimulate the metamorphosis of Hydroides (Shikuma et al., 2014). While MACs provide an example of CIS-eukaryote interactions, the range of hosts targeted by CIS like MACs as well as the identity and mode of action of effectors that mediate these interactions remain poorly understood.

SUMMARY

In alternative embodiments, provided are chimeric products of manufacture for delivering a proteinaceous cargo, or a heterologous protein or peptide, or a compound, into a cell, comprising:

(a) a recombinant bacterial Contractile Injection System (CIS) or a Metamorphosis Associated Contractile structure (MAC) formed or configured to comprise a tube having an inner core,

(b) a Metamorphosis-Inducing Factor 1 (Mifl) protein positioned in the inner core of the tube of the CIS or MAC,

(c) a chaperone 605 protein non-covalently associated with or covalently associated with or linked to the Mifl protein positioned in the inner core of the tube of the CIS or MAC, and

(d) a proteinaceous cargo, or a heterologous protein or peptide, or compound, non-covalently associated or covalently associated or linked to the Mifl, wherein optionally the proteinaceous cargo, the heterologous protein or peptide, or drug is chemically linked or electrostatically linked to the Mifl, and optionally the compound is or comprises a small molecule, a lipid, a saccharide, a nucleic acid, a drug or a marker, optionally a detectable marker or a detectable moiety, and optionally the proteinaceous cargo, the heterologous protein or peptide has enzymatic activity, optionally a lipase activity, and optionally the proteinaceous cargo, the heterologous protein or peptide has binding activity, optionally heterologous protein or peptide comprises an antibody or antigen binding fragment, and optionally the Mifl protein is encoded by a nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 1, or between about 80% to 100% sequence identity to SEQ ID NO: 1, and optionally the Mifl protein comprises a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:2, or between about 80% to 100% sequence identity to SEQ ID NO:2, and optionally CIS or MAC proteins are encoded by a nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:5, or between about 80% to 100% sequence identity to SEQ ID NO:5, and optionally the chaperone 605 protein is encoded by a nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:3, or between about 80% to 100% sequence identity to SEQ ID NO:3, and optionally the chaperone 605 protein comprises a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 4, or between about 80% to 100% sequence identity to SEQ ID NO:4.

In alternative embodiments, provided are liposomes or lipid-comprising nanoparticle comprising, or incorporating or expressing on its outer surface, a chimeric product of manufacture as provided herein.

In alternative embodiments, provided are protoplasts or a spheroplasts comprising, or incorporating or expressing on its outer surface, a chimeric product of manufacture as provided herein. In alternative embodiments, provided are cells comprising, or expressing on its extracellular surface, a chimeric product of manufacture as provided herein, wherein optionally the cell is a microbial cell or a eukaryotic cell, and optionally the microbial cell is a bacterial cell or a yeast cell, or a human cell.

In alternative embodiments, provided are methods for delivering a proteinaceous cargo, or a protein or a peptide, or a compound, to a cell, optionally to a eukaryotic, mammalian or human cell, or to a plant cell, or to an individual in need thereof, comprising contacting the cell with: a chimeric product of manufacture as provided herein, a liposome or lipid-comprising nanoparticle as provided herein, a protoplast or a spheroplast as provided herein, or a cell as provided herein, under conditions wherein the proteinaceous cargo, or the protein or peptide, or the compound, is delivered into the cell.

In alternative embodiments of methods as provided herein:

- the proteinaceous cargo, or the protein or peptide, comprises or is an antibody or an enzyme or an active biological agent;

- the contacting of the formulation or composition with the cell eukaryotic cell is in vitro, ex vivo, or in vivo,' and/or

- the eukaryotic cell is a mammalian, human or an animal cell.

In alternative embodiments, provided are pharmaceutical compositions or formulations comprising: a chimeric product of manufacture as provided herein, a liposome or lipid-comprising nanoparticle as provided herein, a protoplast or a spheroplast as provided herein, or a cell as provided herein.

In alternative embodiments, provided are kits comprising: a chimeric product of manufacture as provided herein, a liposome or lipid-comprising nanoparticle as provided herein, a protoplast or a spheroplast as provided herein, or a cell as provided herein, wherein optionally the kit further comprises instructions for practicing a method as provided herein.

In alternative embodiments, provided are uses of: a chimeric product of manufacture as provided herein, a liposome or lipid-comprising nanoparticle as provided herein, a protoplast or a spheroplast as provided herein, or a cell as provided herein, for delivery of a proteinaceous cargo, a protein or peptide, or a compound, into a cell, wherein optionally the delivery of the proteinaceous cargo, or the protein or peptide, or compound, into the cell is in vitro, ex vivo, or in vivo.

In alternative embodiments, provided are products of manufacture for use in delivering a proteinaceous cargo, a protein or peptide, or a compound, into a cell, wherein the product of manufacture is or comprises: a chimeric product of manufacture as provided herein, a liposome or lipid-comprising nanoparticle as provided herein, a protoplast or a spheroplast as provided herein, or a cell as provided herein.

The details of one or more embodiments as provided herein are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments as provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

FIG. 1 A-C illustrates Mifl alpha fold prediction;

FIG. 1 A schematically illustrates ALPHAF0LD2™ prediction of the effector protein Mifl; FIG. IB graphically illustrates the predicted IDDT local superposition-free score for each residue 1-943;

FIG. 1C graphically illustrates predicted alignment error of predicted residues vs scored residues;

FIG. ID graphically illustrates sequence coverage of predicted residues; and

FIG. 1E-F graphically illustrate images of negative staining transmission electron microscopy of purified Mif 1.

FIG. 2A-B illustrate domains of Mifl required for Hydroides metamorphosis:

FIG. 2A graphically illustrates data where two hundred amino acid residues were systematically removed from Mifl in order to determine their role in Mifl effector loading; and

FIG. 2B graphically illustrates data from metamorphosis assays of extracted MACs complexes with the various mutants (including Mifl knockouts, as indicated) were tested and assessed for their ability to induce metamorphosis.

FIG. 3 A-C illustrate that Mifl amino acid residues are required for binding with the MACs loading protein 1 (Mlpl):

FIG. 3 A schematically illustrates the design of Mifl protein fragments to be expressed in E. coli with the full Mlpl protein for recombinant protein analysis., and identifies the Mifl amino acid residues are required for binding with the MACs loading protein 1;

FIG. 3B illustrates a Western blot showing the presence of Mlpl tagged with a S-tag. Ni ²⁺ agarose pull-down using Mifl or Mifl fragments was washed of unbound protein and the resultant preparation was blotted for the presence of Mlpl 1, total lysate was used for comparison of pull-down protein versus total expressed protein; and

FIG. 3C illustrates a Western blot showing data from where a reciprocal S-tag was also used as bait and the Mifl or Mifl fragments were blotted by 6XHis tag antibody.

FIG. 4A-B illustrate images of the N- and C-termini of Mifl are toxic when overexpressed in E. coli:

FIG. 4 A illustrates E. coli expressing recombinant mifl, mifl fragments A-E, JF50 0605 or gfp genes from an IPTG inducible promoter in a pET15b vector in the presence or absence of 0.1 mM IPTG. Bacteria were grown overnight and then spotted by 1/5 serial dilutions starting at OD 1.0; and

FIG. 4B illustrates E. coli expressing recombinant mifl fragments Al-3, Cl-3 from an IPTG inducible promoter in a pET15b vector in the presence or absence of 0.1 mM IPTG; bacteria were grown overnight and then spotted by 1/5 serial dilutions starting at OD 1.0.

FIG. 5 A-D illustrate that Mifl binds membrane lipids and possesses lipase activity:

FIG. 5 A illustrates images of lipid spotted membrane with various membrane lipids;

FIG. 5B illustrates images of Far western using purified Mifl protein and Mifl specific antibody shows binding to both PI3P and PA; and

FIG. 5C illustrates images of a lipid cleavage assay with purified Mifl protein or chaperone (12605) protein, incubated for 1 hour with decanoic acid-PNPP substrate, cleavage and PnPP (4 -nitrophenyl phosphate) release occurs if acyl-ester linkage is hydrolyzed.

FIG. 6A-E illustrate that Mifl possesses lipase activity:

FIG. 6A graphically illustrates data from a lipid cleavage assay with purified Mifl protein or chaperone (12605) protein, or a GFP control protein incubated with Tween-20 in the presence of Ca ²⁺ ;

FIG. 6B graphically illustrates data from a assay where purified proteins were incubated for 1 hour with decanoic acid-PNPP substrate, and cleavage and PnPP (4- nitrophenyl phosphate) release occurs if acyl-ester linkage is hydrolyzed;

FIG. 6C graphically illustrates data from a PLD specific lipid cleavage assay with phosphatidylcholine substrate to assess enzymatic cleavage site of lipases by presence of choline release;

FIG. 6D graphically illustrates data from a Phospholipase A2 specific cleavage assay with Mifl, Buffer, or a control protein GH1; and

FIG. 6E graphically illustrates data from a Phospholipase C specific cleavage assay with Buffer, Mifl or 605 control protein.

FIG. 7A-B illustrates Mifl fragment analysis for lipase activity: FIG. 7A graphically illustrates a Pnpp-decanoic acid lipase assay with purified proteins from BL21 plysE E. coli the average of 4 technical replicates is shown; and

FIG. 7B graphically illustrates a tween-20 esterase assay of individually purified Mifl fragments; the average of 3 technical replicates is shown.

FIG. 8 schematically illustrates a table of psiBLAST hits from full length Mifl which identified full length Mifl domain from psiBLAST hits (DUF4157).

FIG. 9A-B illustrates related strains of bacteria with similar Mifl homologues stimulate Hydroides metamorphosis:

FIG. 9A schematically illustrates a maximum likelihood tree showing the relatedness of Mifl homologs in marine bacteria; and

FIG. 9B graphically illustrates data from a metamorphosis assay of marine bacteria possessing a Mifl homolog related to P. Luteo.

FIG. 10 schematically illustrates the alignment of Mifl and A. coli hemolysin E pore forming toxin via PHYRE2™ (Protein Homology/ Analog Y Recognition Engine) (Creative Commons Attribution-2.0); SEQ ID NO:6 illustrates the query sequence, and SEQ ID NO: 7 illustrates the template sequence.

FIG. 11 graphically illustrates data showing metamorphosis of larvae is not affected by lipids produced during lipase assay; metamorphosis assay of Hydroides with lipids isolated after incubation with purified recombinant protein.

Like reference symbols in the various drawings indicate like elements.

Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.

DETAILED DESCRIPTION

In alternative embodiments, provided are chimeric products of manufacture and methods for delivering a proteinaceous cargo, a protein or a peptide, or compound such as a drug or a marker, to a cell such as a eukaryotic cell such as a human cell, or to an individual in need thereof. In alternative embodiments, methods as provided herein comprise use of chimeric products of manufacture as provided herein to deliver a proteinaceous cargo, a protein or a peptide, or compound such as a drug or a marker, to a cell such as a eukaryotic cell such as a human cell, or to an individual in need thereof.

Generating and Manipulating Nucleic Acids

In alternative embodiments, nucleic acids used to generate protein components of products of manufacture as provided herein, including (a) a recombinant bacterial Contractile Injection System (CIS) or a Metamorphosis Associated Contractile structure (MAC) formed or configured to comprise a tube having an inner core, (b) a Metamorphosis-Inducing Factor 1 (Mifl) protein positioned in the inner core of the tube of the CIS or MAC, (c) a chaperone 605 protein non-covalently associated with the Mifl protein positioned in the inner core of the tube of the CIS or MAC, and (d) a proteinaceous cargo, or a heterologous protein or peptide, or compound, non- covalently associated or covalently associated or linked to the Mifl . In alternative embodiments, nucleic acids used to practice methods as provided herein.

In alternative embodiments, nucleic acids used to practice embodiments as provided herein, for example, encoding components of products of manufacture as provided herein, for example, comprising nucleic acids encoding MACs or CIS, Mifl, chaperone 605 protein and/or payload, are isolated and/or manipulated by, or inserted into bacteria and expressed, for example, by cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. The nucleic acids and genes used to practice this invention, including DNA, RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system or gene therapy delivery vehicle can be used, including for example, viral (for example, AAV constructs or hybrids) bacterial, fungal, mammalian, yeast, insect or plant cell expression systems or expression vehicles.

Alternatively, nucleic acids used to practice methods as provided herein, or to make products of manufacture, compositions or recombinant bacteria as provided herein, can be synthesized in vitro by well-known chemical synthesis techniques, as described in, for example, Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Patent No. 4,458,066.

Techniques for the manipulation of nucleic acids as provided herein, or to make compositions or recombinant bacteria as provided herein, such as, for example, subcloning, labeling probes (for example, random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, for example, Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED ), Vols. 1- 3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used to practice methods as provided herein, or to make compositions or recombinant bacteria as provided herein, is to clone from operons or genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, for example, genomic clones or cDNA clones. Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, for example, mammalian artificial chromosomes (MACs), see, for example, U.S. Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, for example, Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); Pl artificial chromosomes, see, for example, Woon (1998) Genomics 50:306- 316; Pl-derived vectors (PACs), see, for example, Kern (1997) Biotechniques 23: 120-124; cosmids, recombinant viruses, phages or plasmids.

In alternative embodiments, a heterologous peptide or polypeptide joined or fused to a protein made by a method or a recombinant bacteria as provided herein can be an N-terminal identification peptide which imparts a desired characteristic, such as fluorescent detection, increased stability and/or simplified purification. Peptides and polypeptides made by a method or a recombinant bacteria as provided herein can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, for example, producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like. Detection and purification facilitating domains include, for example, metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affmity purification system (Immunex Corp, Seattle WA). The inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego CA) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification. For example, an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see for example, Williams (1995) Biochemistry 34: 1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-414). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see for example, Kroll (1993) DNA Cell. Biol., 12:441-53.

Nucleic acids or nucleic acid sequences used to practice embodiments as provided herein can be an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin. Compounds use to practice this invention include “nucleic acids” or “nucleic acid sequences” including oligonucleotide, nucleotide, polynucleotide, or any fragment of any of these; and include DNA or RNA (for example, mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which may be single-stranded or doublestranded; and can be a sense or antisense strand, or a peptide nucleic acid (PNA), or any DNA-like or RNA-like material, natural or synthetic in origin, including, for example, iRNA, ribonucleoproteins (for example, for example, double stranded iRNAs, for example, iRNPs). Nucleic acids or nucleic acid sequences used to practice embodiments as provided herein include nucleic acids or oligonucleotides containing known analogues of natural nucleotides. Nucleic acids or nucleic acid sequences used to practice embodiments as provided herein include nucleic-acid-like structures with synthetic backbones, see for example, Mata (1997) Toxicol. Appl. Pharmacol. 144: 189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156. Nucleic acids or nucleic acid sequences used to practice embodiments as provided herein include “oligonucleotides” including a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that may be chemically synthesized. Compounds use to practice this invention include synthetic oligonucleotides having no 5' phosphate, and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated.

In alternative aspects, methods and recombinant bacteria as provided herein comprise use of "expression cassettes" comprising a nucleotide sequences capable of affecting expression of the nucleic acid, for example, a structural gene or a transcript (for example, encoding a Contractile Injection System (CIS)) in a host compatible with such sequences. Expression cassettes can include at least a promoter operably linked with the polypeptide coding sequence or inhibitory sequence; and, in one aspect, with other sequences, for example, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, for example, enhancers.

In alternative aspects, expression cassettes used to practice embodiments as provided herein also include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like. In alternative aspects, a "vector" used to practice embodiments as provided herein can comprise a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. In alternative aspects, a vector used to practice embodiments as provided herein can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. In alternative aspects, vectors used to practice embodiments as provided herein can comprise viral or bacterial nucleic acids and/or proteins, and/or membranes (for example, a cell membrane, a viral lipid envelope, etc.). In alternative aspects, vectors used to practice embodiments as provided herein can include, but are not limited to replicons (for example, RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (for example, plasmids, viruses, and the like, see, for example, U.S. Patent No. 5,217,879), and can include both the expression and non-expression plasmids. In alternative aspects, the vector used to practice embodiments as provided herein can be stably replicated by the cells during mitosis as an autonomous structure, or can be incorporated within the host's genome.

In alternative aspects, “promoters” used to practice this invention include all sequences capable of driving transcription of a coding sequence (for example, for a Contractile Injection System (CIS)) in a cell, for example, a bacterial cell. Thus, promoters used in the constructs of the invention include cv.s-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter used to practice this invention can be a cv.s-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3’ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.

Bacterial Contractile Injection System (CIS) or Metamorphosis Associated Contractile structures (MAC)

In alternative embodiments, products of manufacture as provided herein comprise a Bacterial Contractile Injection System (CIS) or a Metamorphosis Associated Contractile structure (MAC), which are a toxin-delivery particle that evolved from a bacteriophage tail, as described for example in Geller, A.M., Pollin, I., Zlotkin, D. et al. The extracellular contractile injection system is enriched in environmental microbes and associates with numerous toxins. Nat Commun 12, 3743 (2021). In alternative embodiments, the CIS or MAC is homologous to a bacteria from which the CIS or MAC is isolated for use in a product of manufacture as provided herein, or the CIS or MAC is heterologous to a bacteria, and coding sequence

Bacterial CISs as provided herein can be extracellular CISs (eCISs) or type VI secretion systems (T6SSs), as described for example in Xu et al, Nature Microbiology volume 7, pgs 397-410 (2022). eCISs resemble headless phage particles that, are assembled in the bacterial cytoplasm and then released into the medium upon cell lysis, and upon binding to a target cell via tail fibres, and eCISs contract and puncture the target’s cell envelope. T6SSs remain intracellular and are anchored to the inner membrane, injecting pay loads by a cell-cell contact-dependent mechanism.

In alternative embodiments, the CIS or MAC structure comprises a contractile sheath enveloping a rigid tube that is sharpened by a spike-shaped protein complex at its tip. The spike complex forms the centerpiece of a baseplate complex that, terminates the sheath and the tube. The baseplate anchors the tail to the target cell membrane with the help of fibrous proteins emanating from it and triggers contraction of the sheath. The contracting sheath drives the tube with its spiky tip through the target cell membrane, thus resulting in injection of a payload through the tube.

In alternative embodiments, the protein subunits that comprise a CIS or MAC complex can encoded by an operon having a nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:5, or between about 80% to 100% sequence identity to SEQ ID NO: 5.

Metamorphosis-Inducing Factor 1 (Mifl ) proteins

In alternative embodiments, products of manufacture as provided herein comprise a Metamorphosis-Inducing Factor 1 (Mifl) protein positioned in the inner core of the tube of the CIS or MAC; and a proteinaceous cargo, or a heterologous protein or peptide, or compound, is non-covalently associated or covalently associated or linked to the Mifl .

In alternative embodiments, the Mifl protein is encoded by a nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO: 1, or between about 80% to 100% sequence identity to SEQ ID NO: 1, or optionally the Mifl protein comprises a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:2, or between about 80% to 100% sequence identity to SEQ ID NO:2.

Chaperone 605 proteins

In alternative embodiments, products of manufacture as provided herein comprise chaperone 605 proteins, which are associated with the Mifl protein component of the product of manufacture as provided herein. A chaperone 605 protein can be non-covalently associated with or covalently associated with or linked to the Mifl protein positioned in the inner core of the tube of the CIS or MAC.

In alternative embodiments, the chaperone 605 protein is encoded by a nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:3, or between about 80% to 100% sequence identity to SEQ ID NO:3, and/or the chaperone 605 protein comprises a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity to SEQ ID NO:4, or between about 80% to 100% sequence identity to SEQ ID NO:4.

Sequence Identity Determinations

In alternative embodiments, a sequence identity is calculated using a sequence comparison algorithm consisting of a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall-p blastp-d "nr pataa"-F F, and all other options are set to default. In alternative embodiments, protein and/or nucleic acid sequence homologies are calculated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).

In alternative embodiments, the sequence identity (homology) is calculated using BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. 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 are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are 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. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3 and expectations (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison of both strands.

In alternative embodiments, protein and nucleic acid sequence homologies (sequence identity) are evaluated using the Basic Local Alignment Search Tool ("BLAST") In particular, five specific BLAST programs are used to perform the following task: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six- frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

SEQ ID NO: 1 (Mifl protein) is:

ATGCAACAACAAGAACAGGAGCAAGCTCCTACCTTTCAAAGTTATCCCAC

GCGCACTGCGTTATCAGTGAGCAATGCATTAGACTCTGATTCTGTGAGTGT

CGATCTACTCTCATCGGGCGTTGATGTTGCAAAGCGGTATGCCTCACAAGT

GGTTTGGGATCATTTTAATGGCGATGCAACAGCTAAGCTGATTATCTCAAG

TGTATTTAATTATGCGGTGACATCATTAAAGTCCTTGAACCCATGGGTGAC

GGCAATCTCACAAGCACTTTTACTTTTGGCAAAAGTTCCGCCTGGTGTTGT

TTCCGCCGTTTTATGGGCCATTGGAAAAATCTGGCTTTGGGCTGCAAATAA

ATTTTATAACGGTGGTTGGATAGCCGCTGCGTGGGGAGATATTGATGAGC

CATATATTTATCAATGGTTAAAAAAAGGCAGTGATGCACATGGGGCATTA

CGGGCACTCGTGGATGATTTAAAAGCTTGGGTTAAGTATATTCAAGATAA

GCTTGCCAGTAGCGTTGCACGTTTAATTGGTGTCTCGGATTCAAGTAGTGA

AGATGAGCAAAGCGATGAGCAACAAACGGATCAAGATGCACAAACATCA

CCTAATATAGTTGATAATAAGTTCATTTCGTTGGGGATAAACCAACCTGAA

TTATTGGACTGGGAAGAGGGGGCAAGTAAGCCCAAGCGCGCTGGACTAC

ATGGTACGGGTGTTGCCAAGGTCAATCTGCTTGGTCAGCAATTTGGTGGTG

ATATAGATGTAAAATTACCATTTGGCAGTGGCTGGGAAGTGGCTGTATCT

CCGGTTTTTGCAAGCCAACAAGGGGTGGGTTTTAGTGGTTATATTGAAGC

GCATCAAGTATTGGGCGATGAGCTGACCATTAATGAAGAAGGCCTTGCTC

GTTTTAAAGCAAGCATTGTTGGGCTAAACATTGCGAACAAGCGGGTTTAC

TCCGATTTAATGTCACTTGATTACAATAAGCAGCAAAAAGAAGTTCATTTT

GCAGGTGATGCACATGTGCCGCTTTGGGATAAAAAGAAACTGGATGGTGA

GTTTGATTTAAAATTGGATACTGCAGGTAAATTCAAATCAGGCAGGACCA

AAATCTCTTCTAAAGATACATTCGAAGTAATCCCCAAGTTTTTGTCTATCA

GCAACCCGAGTGGTGAAGTAGAAGTTAAAGAGGATGCTTCACCAGAGTTT

GAGGTGAGTACTGACGCTGCGCTTTACGGCTTACCCGCTGGTGTGAAAGC

ATCAATTACGCAAGCAAAAGTGATGTATCACGATGAGCAATTAGGCGGTG

AGGTTGAGCAGGCTGATGTGGAGATCCCAATTAGCAAACACACTACAATG

ACGCTGGCCATGACGAAGGCTAAGTTTACAAAAGATGCAATTGAAGCTGA

AAATGCTTTTATGGATCTCGATCATGATAATGCCAAAGTGCTTAATGAAAC

GGCCATTGTCGAGTCAGACTTTTTCTCTGGAAACTTCGATTTTGGAAAAGT

GTTTGAGCTTAAGCAGTTGAAAGTCCACCAAGGGTTAAACGCGCTTAAAT

TTGCTGGTGGTAAATTCTCCTACGAGAAGGATAGCAACAATGGTTTACAG

TTACTTAGAGCACGTATTTTAGGCCTTGAGGCTTACTATAATAGAAAAGAT

CGTGAAGGTGGGATCACTGGAGAATGGCACAAAGGGATTGATTTTCCGGC

TTTCTCTTTGGACTTTCCCGTTGCGGCTGGTGTTGCAGGAGTTGGAGTTGA

TATTACCGGGGGGTTTGAGTTTGGTGCGAGCATCGGCGCTAAGCTTAAAA

ATGAAGAGCAGCATAACACAGAGACACAAATGCTTTTCTCTGTGAATGGT

GCAGCTTCGGCCAGTGCCAAAGCTAAAGCGAGAATTGAGGCTGGTGCTTT

TGTGGGGGTCCCTTATCTTGCAAAGGTTCAAGGTGGTGTATTTGGTGAGAT

CCGAGGTGGTGTTGAAGGTAAGGTTGAAACAGAAGGGCGTTTGAAATACA

CGCGTGGCAAAGGCGGTGATAGCTTTGGTCAATTTGCCATTGATAGTGATT

ATCCAATGGAGGCGAGCTTTAGCCTAACCGGCTCATTAGAAGCGGAAGTA

GGTGCATCGATAAAAGCCAAAGTACTGACATTTGAAAAAGAAATTGCGTC

GGTATCTATTGGTGATTGGACACTGGGTGAGTATACGCTTAATGGTAAAA

TCAAAAAAGATCCTAATGGGAAAGGGTATATCGTTGAGCGCTCTAAAGGG

GAGTTTACCGAAGGCAAACCAAAACCTGCCGAGGTAACTAAGGAGGCAC

TCCCAGTAGATAAGTGGGTTAATCAGTTAGAAAAAGATCATACTCATATT

GAATACACCGAAGATGAAGCCAAAAGAAAGTTACCTGAGGTTGCACGTA

AAACGGTATATAGTCACCTGAGTCCAATGCAAAAAGAGGATGTAAGTGAG CGATATACTCATTTGCTCAAGTTGATATCGCAGCAAAATAAGTTCACTGAA ATTTATCGAGAAACCAAAGCTGATAGGGATAAGTTACCGACCAGTGGCAC TTATATTTGGACCTCTAAAGTTTGGAATGAAGCGGTACAGCGCAAGAGTT TCTGGATTTTTGAAATGAGTAAGAGTAAAGCAAAAGTGGCGGATAAGCTG GATAAATATCACAAAACGCACTCTATTTTAGAGCGGAGACTGTTACTTGC AAAGCTAGAAGAGATAGCCTCGGACTATCGCAAAAACCGCTCGAAAAAC GCAGAAAATAAAGAAGCTGCCGGGGAGTTTTTAGAGAGTATTGAAAAAG AAAGGCTCATTTTAATGTAA

SEQ ID N0:2 (Mifl protein) is:

MQQQEQEQAPTFQSYPTRTALSVSNALDSDSVSVDLLSSGVDVAKRYASQVV WDHFNGDATAKLIISSVFNYAVTSLKSLNPWVTAISQALLLLAKVPPGVVSAV LWAIGKIWLWAANKFYNGGWIAAAWGDIDEPYIYQWLKKGSDAHGALRAL VDDLKAWVKYIQDKLASSVARLIGVSDSSSEDEQSDEQQTDQDAQTSPNIVD NKFISLGINQPELLDWEEGASKPKRAGLHGTGVAKVNLLGQQFGGDIDVKLP FGSGWEVAVSPVFASQQGVGFSGYIEAHQVLGDELTINEEGLARFKASIVGLN IANKRVYSDLMSLDYNKQQKEVHFAGDAHVPLWDKKKLDGEFDLKLDTAG KFKSGRTKISSKDTFEVIPKFLSISNPSGEVEVKEDASPEFEVSTDAALYGLPAG VKASITQAKVMYHDEQLGGEVEQADVEIPISKHTTMTLAMTKAKFTKDAIEA ENAFMDLDHDNAKVLNETAIVESDFFSGNFDFGKVFELKQLKVHQGLNALKF AGGKFSYEKDSNNGLQLLRARILGLEAYYNRKDREGGITGEWHKGIDFPAFS LDFPVAAGVAGVGVDITGGFEFGASIGAKLKNEEQHNTETQMLFSVNGAASA SAI<AI<ARIEAGAFVGVPYLAI<VQGGVFGEIRGGVEGI<VETEGRL I<YTRGI<G GDSFGQFAIDSDYPMEASFSLTGSLEAEVGASIKAKVLTFEKEIASVSIGDWTL GEYTLNGKIKKDPNGKGYIVERSKGEFTEGKPKPAEVTKEALPVDKWVNQLE KDHTHIEYTEDEAKRKLPEVARKTVYSHLSPMQKEDVSERYTHLLKLISQQN KFTEIYRETKADRDKLPTSGTYIWTSKVWNEAVQRKSFWIFEMSKSKAKVAD KLDKYHKTHSILERRLLLAKLEEIASDYRKNRSKNAENKEAAGEFLESIEKER LILM

SEQ ID N0:3 (chaperone 605 protein) is:

ATGATGTCAGATATGGACGCCTCATACTTGCTTGATAGCGAAGAGCAGAA GCAAAGAAACAAAGCTCGTCGTCAAAAAAGAAATCGAACGGAAGAAGCG CCTTCTCGTTTTCGAGAACGCAGCCCAATTCGGCAGCCAAAGCATAACCT

ATCTCGGCAAAACTCAGATATGAGTATGCAAGAAGAAGATATTGAGACAA TTGTGCTGGATGATTTAGATTACAGTGAGTCGATTTCCGAAGATGAGTATG ACAACTTTATTCCTATATTAGACCAGTACTCGACTGATTTGACGTCGACTG AGAGAGTTGAACAAGCAGTTGAAGTCATAGGTAGCAGCTATTTTGATGTG TTGATGGATGAAGAGCAAGTTTATGATGTCGCACAACGGTACTATCAAGT GGTTGGCGCGAGGCTTAGCCACATTTACAAACTGCTTCTGCCAGAGAGCA AGGATGTTGACTTTGCGTTCAAAGCCATCAATCAAGTGGCTCAAAAAGAC ATGCCTGGAGTTAACTTAATTCAAAACTTTGCTTATCAATACAACCCGTAT TTAATAGGGACTTCATTCTCAATTAACCCAGCATGGAATATTGCCATACCT GCTGGTGGCAGCGAAGAAAGGGTACGTGAGACTGCTGGTTCTGATTCGAT AAATGATACAAAAATTAAGTTTTTTGCAGCCAAAGATTTTAAGTATAAAA ATGCATCCGGTGCTGATGTAACGATTAGTAATATCGTTGGTGATAAAGTTG AGGCTCGATTATCTCGTCTAGCCCCCAGAGGCGAGTCTGCTGAAGTCAAT GCCGATCAAAGCGATTTGATGAAGAATAAGCTTGTTTACAATAACAACGG GAATAATGCCAATGAAAAAGGCTGGATCCGTGGCCACCTTCTCAACGACA ATTTAGGCGGCTCAGCATTGAAATTTAATTTGTATCCTATTACAGGATCTG CGAATAAAGAGCATCACGCTCGAGTTGAATCTCATGTAAAAAACCTCGTT GAAGCCGGTTATGTTGTTGAATATAAAGTAGAAGTAGTACCAACAGTCCC CGCACCTCATCAAACAGAAACCGCCCCTGGGTACAAGAGCGGAGCTGCGC CTAAGGCTAATTTAGTATGCGAGGTAAAAGTCTTAAGTGATATCAGTACG GATGCTGTGAGTAGTTACCACCCTGAAGGGTCGTTTTCAGTGACCATCACG TCTGAATACAAAACCAAACATGCCTCTACAGGAGATGTGTCGACTTTAAA GGCATTTACAAACAAAGCGGTTAAAAGCAAAGACAACACGGTTGACAGC AAGGCATATATGCGACCTTGGACAACGAGAAAAGGGCTTACTAAGCCGCT GGGTCTTGCACATAAAGATGATAAACACATTCGAGATAAAGACTATAAAG

ATTGGACAGATAAGCAAAAAAATAGCAAGTTTTGGACATGGGACCAGAC ACGAATTGATGCGCTTAAAGCGGCACTAAAAGGCTAA

SEQ ID N0:4 (chaperone 605 protein) is:

MMSDMDASYLLDSEEQKQRNKARRQKRNRTEEAPSRFRERSPIRQPKHNLSR QNSDMSMQEEDIETIVLDDLDYSESISEDEYDNFIPILDQYSTDLTSTERVEQA VEVIGSSYFDVLMDEEQVYDVAQRYYQVVGARLSHIYKLLLPESKDVDFAFK AINQVAQKDMPGVNLIQNFAYQYNPYLIGTSFSINPAWNIAIPAGGSEERVRE TAGSDSINDTKIKFFAAKDFKYKNASGADVTISNIVGDKVEARLSRLAPRGES

AEVNADQSDLMI<NI<LVYNNNGNNANEI<GWIRGHLLNDNLGGSALI <FNLYP ITGSANI<EHHARVESHVI<NLVEAGYVVEYI<VEVVPTVPAPHQTETAPG YI<S GAAPKANLVCEVKVLSDISTDAVSSYHPEGSFSVTITSEYKTKHASTGDVSTL KAFTNKAVKSKDNTVDSKAYMRPWTTRKGLTKPLGLAHKDDKHIRDKDYK DWTDKQKNSKFWTWDQTRIDALKAALKG

SEQ ID NO:5 (CIS or MAC operon) is:

1 TCACGCCTTT TCGATGCTGT AAAGTGCATC AGCAACCTGT TTCAGATCAA CCTCACCTAC

61 AAAATCATTC GATAAAGTTA AAATAGCATT CAAATTATTT TTGAGTTTTT CATGACACAT

121 TGGTTCAGGT TTAAGCGCTG TTTGCAGCAA ATGCAAAGAC ATATCGGCTA TCAATAATCT

181 TTGCTTCTCA GACCAATCAT CATCTCCTTC AACGGCAGTG TGATGGACGA TTGCTTGCTC

241 AACTTCATCG TGGATCCCCC AACTCAAGGT TAAGATCTCC TGTCTTTTTA CTTTATCCAT

301 AGCTCCCTCC TATTTCTTAA AAGGTTGCTA CATACTCAGC CAAATTTCAA TTGACAATTC

361 GTCATATACC AGTTCAATTT TTACTATTTA TTTAAAGGCT CTTAACACTC GCATTCCCAC

421 TCTACGACTT TGGATTTTTT GGAAAGTAGT TATCCGGTTG CTTAGGACAC AATTCAAATT

481 CTCGGGTATT TTTGTAAGGC AGCTTCATAA ACCCTGCTAC ACCATAGTCT TGAATACTCA

541 CTGTGTATTG AATAATCTCC TTTAAAAGCG CTGCAAAGGC GACCTCTTGC TCAGCATCAA

601 GCAATCGATA ATAGTAGTGA ATTTTCAATC GGTCACTCAG TGACTCAAAA ATAATACAGC

661 CTGTGTGGTG AATTTTATTT AGCTCAAATA CACACTGGAT AATTTGCTTA GGCAATTGCA

721 GTTGCTCATC AGGGTCTTTA CCATCTTCAA AATAAGAATG AGGTCGCGTC ACCTCTGACG

781 CAGCCACATG GCTCACTGTA TACTGCAACT CAGGAAGCTG CTCAAAGATA TAAGCACCTG

841 TTTCGTCTCG TTCTAATTGA ATTGTTTGCC TCGCATCAAA TAAACAACAC TTAAAAAGAC

901 CTTTTTGCTC AATCCCCTGC GCCAGCATAA CCGTGGCGTT GACTGCATCT GTAAACGCAC

961 TTTGTAAACT ATCATCATGC AGCATCAAAC TGGACTCAAC AAATAAAGAC GCTTGCTGCC

1021 AATGAAAGCT CAGTCCCCCT ACAACCGGAT ATTTAGCTAA TCGACTAATA GAAGCTAAAA

1081 ATGCTTTTTC AGTGTCCCCC GTATCAAATA GGAACTCTTT ATAATACCTA TCTAGCTGTG

1141 CATCATTTAC ATCCAGTAAT TGTTGATTAT GATCGGCACG ACGTAAGAAC TGTTTTCTTG

1201 AACTATAAAC GACAGTATCT GGCAAATCAG TTTGTGAAGA AGTCCAAAAT GGTATATCCG

1261 CTTTGAGTTG AGGTGAGTCA ATATCAGGCA AGTATACTTT GGCCATCGCA GGCATATTAC

1321 GCATAAAATA ATCACCTGCT GCATCGCGGA CCACTTTATC TTTACTCAAC ATACCATCAA

1381 TAACTCTTGA AAACTCCACG GGCAATCCCA AGCTGGTTGC TGGAATTACT TGCGCACCAA

1441 AGCGACATGA TTGTGCGCTT GCCAGCGCAT AGATAGTCGA AGCAACGCCT TGCTCATCAA

1501 AGCGCGGAGA AGACCGTGAA CCCGACATTT GTTCATCACC AATAAAATAC ACATCCCCCA

1561 TTCGAGCATT CGTACTGGCG ATATCAGAGG ACATTAAATC CATGATATTA CTGGCGACAG

1621 GCTCACCATG TACGTCTATT TGAGCATAAA CAGAGCTACC CCAATCAACC AAAGAGAACG

1681 CATCAGATTG CTGATCCCAA ACAATATTTG AAGGTTTTAT GTCCCCATGA ACCACAGGCT

1741 GTAAGGACAT ACCATTTTTT CGTTCTCTCA AGTCTAACAA AACATTGCGT AACTTCAGGG

1801 CTAAATGAAC CAAGACATGT GGCTTTAGCC GCCCTTGTTT TAACGAAATT TGCTCAAGGT

1861 CTTCACCTTG GGCTCGAGCC ATCATTAATA TTCCCTGTTT TTTCACACGT TCAAATGCAA

1921 AAAACTCTGG CACCATAGGA TTATTAATTT GAGATAACAT ATAAGCTTCA TCTTCAAGCC

1981 GATCTCTTAC GCTCTGCGCC AGCGTGATAC GAGAAAATTT GAATACCCAC TGCGCGCCAC 2041 TGTCTTCAAC ACCTGCAAAT ACAAACCCAA AGGCACCAGA GCCTATAAGT TCAACATCTT

2101 GATACCCAAG CAACGACAAT TGCTTTTTAC AAATGTTTAG CCATTGACGG TGTTTTTTTG

2161 CGTCATGGTG AGATAGCAAA TATATCGATT GCTGTTCATT AATGTAGAAA TTGTGAATTG

2221 ACTGTTTGCT CACGGCGCGA CTTAATAAAA CTAATTTGAT GAATATATTG AAAAAAAGGC

2281 CGACAAATGT CGACCTTTTT TCATAAATAA TAGCGTTAAA TTAAACTGCT TTACGCTTTT

2341 TCAATTTTTG CCCATGAGTC ACGAAGACCC ACCGTTTGGT TAAACGTCAA AGCTTCAGAC

2401 TTATTGTCTC GGCTGTAATA GCCTAGGCGT TCAAACTGAA AACCTTGTTC AGCATTCGCT

2461 TTGGCAAGTG AGGGCTCAAG TTTAGCATTT GCAATCACAA CCAATGAATC TGGGTTTAGC

2521 GTAGTTGCAA AATCTTCTGC GGCAGCTGGG TTTGGTACAT TGAATAGACG GTCATACTGA

2581 CGTACTTCCG CTTCGATGCA ATGTGACGCT GAAACCCAAT GAATTACGCC TTTAACTTTA

2641 CGGCCATCTG CTGGGTTTTT ACCAAGCGTG TCGGCATCAT ATGTACAATA AATAGTTGTA

2701 ATATTGCCTG CATCATCTTC TTCAATGCGC TCCGCTTTGA TCACATAAGC ATTACGTAAA

2761 CGCACTTCTT TACCCAACAC TAAACGCTTA AACTTTTTGT TCGCTTCAAC GCGGAAGTCT

2821 TCACGTTCAA TGAAAATTTC ACGAGTAAAT GGCAATTCAC GCTCACCCAT TTCTAATGTT

2881 GGGTGATTTG GAGCAGATAA CATCTCCACT TGGTCTGCAT CATAATTTTC GATAACAATT

2941 TTAACTGGAT CTAAAACAGC CATCGCACGT GGTGCATTTT CATTTAGGTC ATCACGGATA

3001 CATGCTTCAA GCATCCCCAT TTCAACCATG TTATCTTGCT TTGTAATACC AATACGCTTA

3061 CAGAATTCAC GAATAGATGC CGGTGTGTAA CCACGACGAC GTAGGCCCGC AATGGTAGGC

3121 ATACGCGGAT CATCCCAGCC TTCTACCTGA CCGTTCACGA CTAAGTCATT AAGCTTACGC

3181 TTGGACATCA CGGTATATTC TAGGTTTAAA CGAGAAAACT CAATCTGCTG CGGTTGACAC

3241 TCAATGCTGA TGTTCTCAAG TACCCAATCG TATAAACGAC GGTTGTCTTG GAATTCAAGC

3301 GTACACAATG AATGCGTGAT CCCTTCTAGC GCATCTGAAA TACAGTGCGT GAAGTCATAC

3361 ATTGGATAAA TGCACCACTT GTCACCAGTC TGATGGTGAT GAACAAAGCG TATACGGTAA

3421 ATAATCGGAT CACGAAGTAC CATAAACGAG CTTGCCATGT CGATTTTTGC TCGCAACACA

3481 CACTCGCCTT CTTTAAACTC GCCGTTTTTC ATTTTTTCGA AAAGTGCTAA GTTCTCTTCA

3541 GGTGAGGTAT CTCTGTGTGG GCTATTTTTA CCAGGCTCAG TCAGTGTACC ACGATATTCA

3601 CGTGCTTGTT CGGGAGACAA GAAGCAGACA TATGCGAGGC CCTTTTCGAT CAGCTCTACA

3661 GCATAGCTGT ACAATTTGTC GAAATAGTTT GATGAATAGC AAATTTCACC ATCCCATTCA

3721 AAGCCTAGCC ATTTAACATC TTCTTGAATT GAGTTTACGT AATCGATGTC TTCTTTTTCT

3781 GGGTTAGTAT CGTCGAAACG TAAATTACAA AGACCTTTAT AGTCCTGAGC AATGCCAAAA

3841 TTTAGGCAGA TAGACTTTGC ATGGCCAATG TGTAAAAAAC CATTCGGCTC TGGCGGGAAA

3901 CGCGTATGTG TCGATGCGTG TTTGCCACTT GCCAGATCTC CGTCAATGAT ATTTCTGATG

3961 AAGTTTGTTG GGCGATTCTC TGTATCCGCC ATAGGAGGTC TTTCCTCTGA ATTTGTAGCT

4021 TGTTAACTGA CGCATTATTA CAAATTAATC AGGTAACTTA CAGCGCTTAT CGACCCTTAA

4081 AGTCGATTTA TTTAATGTTT TTTGTATGCT GCACAATTTA CATACAACTT CATTAGTTGT

4141 TCCTTCCAAG CGGTTTTTCA AGTGGGCATC ATGCGATAAC AAATATCAAA ATAATTTTAA

4201 AAGAATGACA CCGCGCTAAC TCACTTTGCA ACTCACGCCA TTAACGTCTT CAAAAACCAA

4261 AATGATAACT CCCAATTTAC ACATACCACA GTGTATCAAG CTTATTCACT AATGAGGTTC

4321 ACCCATCACA GACGGTTTGT TTCATTTTTT CCTCATTGGC ACAAGCAAAC ACCTCTAGAC

4381 AACAAGCTCC GCTTATTAAA ACTAGAAAAA GCGCAAAATC GCCTTATGCC ACAATTAAAA

4441 ATAAAGCATA CAATTTTTTG TTCACTTTTC ATTCATAATG TGTTTTATTT ATAGGGTCTA

4501 ATCCAACTTA AATGGAAATA GGTTGGGCAG TTAAGTCAAT TAGAGGTAAT CAAGGAGATA

4561 TGATGATGAA AAGTATGTTG GCACTCTCAC TAGGTTTGAG TGTGTCTTTT AACGCCATTG

4621 CGCACAGCTC AGAGAAAAAA GTTCAGAACG CTAGCTATAA GGTTTGCCAT TACAAACTCG

4681 CGGCGGCTAA GACTAAAGTA TCTTCATACC CGTTATGGGA GAGAAAAAAA GGCACACGTG

4741 GCAGAGTTGT CCACTCAGTT GGACGCTTTC TGAACCCGAG AATAAAGCAC CAATTGGCGT

4801 GTACATTTAT CTTTATACGA CATACCACTA AACTTATTTT ATTGCGAAAT CCGCCACACC

4861 CTTGATGCGT TAAATGAAAT AATACTGCCA TAACATCATT TTATAGAGCT CAATAACGAT

4921 GAGCTCCCCG ATAAAAATCC ATACAAATTC ATTAACTTCT AAATGTATAG AAAATAAAAT

4981 AAGATTCAAA AGTATTACTT TTTGCTATTT TAGTACATCT TATTTTTCTT GAATATGCTT

5041 ACACTTTTTT GGACATATAC AGCATGTAAA GGAAAACTAA ATGCAAAATA CAACGAAAAT

5101 ATTGAGTATT TTAGGATTAA CGGCCACCAT AAGCTCTTCA TTCGTCTATG CTCAAGGTCA

5161 TGAAAATTCG GCCATTCGAG TTATGATAGA CCAACAAGGG ACCCCCTATC TTCACAAAGA

5221 CGATAATGTT GGAACATGGC AAGCAATTCC ACTGCCCTAT TATCAAAAGG CTGTAGCCGC

5281 TTCCGGTGGT TATTTTCACT ATCGCCATGA AAGTGGCAAC AGTCCAGGTT ATGCCTTCGA

5341 TGAAGAGGCC ACATTCGCAG TAGGCGAATA TGGCATGATA TTTAGATATC AAAACGATGC 5401 ATGGCAACAC ATTTTTGGAT GTTGGGATGC GAGGGATGTA TCATCTCACA GCCCTAAATA

5461 CGCTTATTGT GTTAATGCCA GTGGTGATTT GAAACGCTTC AATCTTAACA ATGGTAATTT

5521 TGGTGGGAGT TATGGCATTG ACGGCAAAAA AATCACCAAA GTGGATGTCA ATCGACAAGG

5581 AGATGTTTGG GCACTCACAC AAGACAATGG AGTTTATGTG CGCAGAGGCG ATCACTGGTC

5641 ACAAGTAGAG GTTGTCTGCC CTAGAGCGTG TTCCTTTAAA GATATCGCCG TCGGCGCTGG

5701 CCAAATATAC CTCACGGCTC ATATCGTTAA AAATACCTTA GGCGAGCAAC AAGTCTACCA

5761 ATTAAATGGC TCGAAATTAG AAAAATTCGG AGATTTTTAT AATATCGAGG TTGATAGAGA

5821 TAACACCATT TGGGCGATTT CAAATGAATC TAGGACACTG CACTACAAAC GCCCCGGCAT

5881 GATTAACTTT ATTGAAGATC ACCGTATAAA TAGTGTGTCA GCCAACGACA TAGGTGGTTA

5941 GTTTTACTGC GAACTTACAT AGCTTGAGCG CTTAACGCTA AAAAGTGCCA AGCTATTTGT

6001 GTGTAAAACC GAGCGCATTG AACTTATTTG CCACTCGGTC TCCACCAAGC CATTAGAAAA

6061 AAGATGACTA AGCCAAAAGG TATTAAAGCC GCAATACTAC TAACATACCC TTGTACATGC

6121 ACTAGCTTTT CAAATGGTGC AATGACGCGT CTATTTTGAG TGATATTTAC TATTTTATTA

6181 TTATTTGAAG CGTCATACCA GACTCGTACT CTGCCATTGG GTAACCCTTG CTTAACCTGT

6241 CTATAAAATG GAAAATATTC ATTGACAGTA AAAGGGACAT TCTTAATCGT AAATTGAATT

6301 CTTTCAGAGC AATTTCGTCG CTTGTTGCAC GGTATTTCTT ATACAATGAC CGGCGCAGCT

6361 TGAGACACAA TTGAGAAATC CGCCAGAATC GGTCGATTAA GCTCTGTTAA ATATTGTACT

6421 AAAAAAGTAG CTATTAACCC TGCTACAAAG ATAACAATAG AAATGGTCAC TTTTTGCATA

6481 CTTTCACTCG CTTTAAAGCG CGACATTCAA AATCGATCCT AATTGTAAAA AGCTGCCGAA

6541 GCAGCTTTTT AAGTTGTAGT CAAATAGGCC AAACGCTGCT TAAATCGTTC ACTTGGCACT

6601 GTATTCCACA GCTCTCTATC AGACATCTCT TTACTAAACG CCTCAAGGTC ATTTTGTACT

6661 ACATAGCGCT GAATTCGTCT GCGCGTTGCT GGGGTGTAAT CCCCTCGGTA GCGTAAATCA

6721 ATCAAAATTT CCTTGATCTT GGGGTGAAGG TTTTCCCAGT CCACGGGCCC ATAGACTTCA

6781 ACACAATCAG CTTTATCACA TATTCTCTTG ACGTCTTTTG ACATGACTTC GTAGGTTTGC

6841 TCGAATAAAA TCACTTGCTC CTTTGGGCTC ACTTCAAAGC CTTCAAGACC ATGAGTCTTT

6901 ATAAACTGCT GTGCCTGTGA CCCATATAAG CCTGAAGCTT TTGCCAAAAC TTCTGCATCC

6961 TCAAAGGCAA CGCCAGCTTC GCTGAGCGAT TTGAGTATTT GTTTTGAGCT GCGATGTTTG

7021 AGGTCAAATC CTCGGCCAAT TGTCAGGCCA GAATACTTTG AAGGCACATG TAACACGCGA

7081 CTATGGTAAC GCCCACCTTC TTGCCCTTCT TGTTCAAAGG TAAATTGGCC AACTTTCGGC

7141 TGTTGTATTG TCATAGTGCT TTCCTTAATT GTTCTAGTTC ATTGCTTAAA CTAGTAGTAT

7201 AAATGACTAC CTATATTGGG TAATAAACCC ATAATTCAAG TTTTAGCAGC AAGAATCATG

7261 TTTTCTAATC GCCTATTTTC AATGAGCGAG AAATAATCAA CTCCTGCGAT GAGCCACTCG

7321 ATGAGTTTGT TTTCAGGTAA TGTGTTTATC TGATTTAAAG TACACTCACA CATTTGGCTA

7381 CTTAAATTAA TATTAAAGTA GTTTTGCATT TCACTGAGCA TAACTGAGAG GTAGCCTTGA

7441 GCATTAATTA ATAACAGTAA CCCTGCGGCT TGTTGATTCT GGATTTCCTC ACCTTTGAGC

7501 TCTGACCAAA GTTCAAATGC ATAAATATGT TCAAATATTC GTTCAACCTC CTTTTCACTA

7561 AACCCATGGT TATACAGACT TTGCCAGTAA ATCAATGGGT CTGAATTACA ATATAAACTT

7621 TGACGCGTTT CGCTCGTGTT AGAAAACGCA TATTGAAATG CCTCTTGATA ATTTGCCACT

7681 GTGACTCTCC TTAGCATATT TAAACACCGA CGCAAACAGA TGAGTTTTGT GACAAAAAAC

7741 CAAGACGTGT ATTTGACCTT TTATTAGCTT AACTTCCCTC TGATCTGTAA AAAAACCAGG

7801 CTTAGAAAAT CCGATGCCAT GGACTTAAAT TCACTGTGGT CCTCAAAATT GGGCGGAAAG

7861 GGTAGATGTG GAAACTGCAT ATCCGCAGTC GGTATATTTT GATAGTAATA ATCATCAGCA

7921 AAAAATAGAG GCTGAGTCTG GCTGGTAACA AGATCTGTAA TATATTTTGC ATATATAAAC

7981 GCATGGTTCG GCCTTGTAAA AATCAACAAT CTATTTTGCG TTAAACCAAT ATCTTGATCG

8041 TCTTGATTAC CTGAGAATAA TAAAGATACT TGCGTGATCA CTTCAATCGC TGAACTTTCT

8101 TCATATAACG TCTGTTTTTC TATCTCGATA TCTGCGGTAT ACCCTTGTTC AATGAGTAAC

8161 GATTTTACTA ACTCTAGATG TGGTAATACC CTGCTTGTAA ATAAGTTATT CAAATGTTGG

8221 AGCAAATACC GACGATTATA CTCCAAATAA GCTTGTACTT TATTTTCCGC AACTTGATAC

8281 TGCTGTTGGC GCACCTGATG TTCCTCGATT AAGGCGCGTA GGTTTGAATG ATTATTTACA

8341 GGCATACACT TCTCCATATT GAATTGGGAC TGGCTCAGAG TTTGCGAGTC CATTTGATAA

8401 TTGGTTTTGG GCGTTGAAGC GCACCACGTC AAAAGATGCG CTTTCATTTT AAGTTTTATT

8461 GTGAATTTAA GGTCATGGCT TGTGAACGCT ATGCTAAAAG CCGTAATCCC ACTCGATGTT

8521 AATAAGATCG GTGTCTAACC AAGGTAATTG CACTATCCCT AGGCCCCAAG GTAGTTTCAT

8581 AAGTAGTACA TCTTGAGGTT TGTTCTCGAC AACAATACTC ACACCATTGG CAGTGAGCTT

8641 TACTTCTCCC CTCCTCTGCA AGAACATGTG TCTAAATGCA TCGATGGGCA TATCTTTGAG

8701 CGCTTCCCAG CGGTTTATCG CAGCTTTAAT GAGGGTGTGT AATTCATCTT GTGCTTCGCT 8761 ATCTATAACA AGCGTCTCTT CATCAAACTC ATGGTCAAGT TCTAGGCCTA GCAAGGCATT 8821 AATCACATAC GTTTCTGCGC TATTTGCCTC AATCCCAGCC AGTTGACACA ATAAAGCATG 8881 CGCTTTTTGT TGAGCAGCTA AATCCGCAAA CGTGATCTGA CCGCTTTCAG CTGTAATAAG 8941 TAACTCTAAC TTAGTGAATA AAATTTCTAA AAATGGCCAC AGCAGCACCA CACCGGCGTC 9001 GTCACTAATA AGACTCTCTG TTGATTGCTG ATATTGATGT CGTAGCTCAT TTAACATGTG 9061 TTCACTGTGC GCTTTTAAAG GCTCAATACC AGAATGTTTC AGGTTATTTT GATACCTACG 9121 TACCTGTTTT GACAGGCATG CAGAAATACG TTCGACCCGA CGCTGTTCTT GTAGCCGCTG 9181 CGACATCTCT TTTACGACTG AGTGTGATAT TCCCTTTTCA ACCACTTTAA GGTCTGGTTT 9241 ATTGCTCATA TCTGTGCGTA TTGCAGACAC CGAACTATTG GTGCTTTGAC CCAGCTCTGC 9301 GCAGTTCAAT GCAACCACAG CATCATCATC CGAAGTCACC TCCCCAAGCA AGGCGCCCTT 9361 TTGTTTTTAG TTGCTTGAAG CCGTTCCTCA TAATGAGTTA ATAATCGAGT CACATCGGTA 9421 AAAGTTCGAG CTTCAACATA TTCACTGATG TCATGTATAG TAATAAAGTT TTGTTCAGAA 9481 CTTGTTTGGT GAACTTGCTG ATGACTTCCT GCGTTCAAAT ACACATCTGA CAGGCCATCA 9541 TGGTGTACAT TTACTAAACT TCCTGAGTCA TCTTTTGCAA AGTGACTATC AGGTACCAGT 9601 GATACATCTA ACAACTCTAG CCACCACATT TGCCCTTTCT GTGCTTGCGT TTTCAAATAA 9661 GGGTTTAGCT GATAGCTCTC CTCCCGCACA GTATTCAGTT TGACAATTGC ACTATTCAAA 9721 ATCTCATTTA ATGACGGTGT ACTTAGTGAT AGACAATGGA TCGAACACAC TTTTTGCCAA 9781 CTGCGCAGGT ATATCACCAT ATACACAAGC AGTTCATGAT TATCGTGTAG CTGGATACTC 9841 GCTTGATTGA CTTTGATAGT GTGCTTAAGT GCATCACAGT ATCGTAATAA TTGAGAGGCG 9901 ATGCTCAACA TTCTGGCCAA TTCAAGACTT GAAGATTTAG ACAGCGACTG ACGAGCTACT 9961 TCTGGTACCG TTCCACTGTT ACTTGATTTA TTTTTACTAC TTAAACTGTG CTGCGACTCA 10021 CTCAGTGCTT GCTGCTGCTC TTGAGCTTGT GACTGCATTT TATCAGCTAA CTCACTTTGT 10081 AATTTTTGAA CGTCACGGAT CTGATAAATC GCCTGTTCAA GGTCTTGACT AAGCTGTTCA 10141 CAAACAGAGA GCAAACTTGC AATTGAAGAC AAGCTTTTAG CAGTGTGTGT ATAAACACCG 10201 TCGTAACACT GATTGATATT TTGCGTTGCC AAAACCGTCG TTGAACTCAA ATCTGAAGCA 10261 TATTTTCTGA GCAGCGACTT TAACTTTTCT AATCTTGCAC CTTTCAATTC GGTGTTCAAT 10321 ATCAATCTTT TACTGTCATT TAATCGTGGA GCTACCGCCA ACAACGTTAA TCTGAGCTGT 10381 GCAGCCTCTA CCTGCTCCAA ATATTTTTCA GATGTGCTTC CACACACAAT TCGCAGTGAA 10441 GAGTCAAATT CACTTACGCT AGTGAGAGAC AACGGTGTTG TAACTTCAGA CCCGTCTTCA 10501 AGCTTATGAG TGTTTTTTTC ACGCCGCTCT ATTAAGTTTG AAGTATCGTC TTTTAGGAGC 10561 AAATGATTAG CCACTGAACC TTCGCTGATA CGGATGGCAC TGTGCTTACC CAGTATGTAT 10621 TCATTATCTA ACCAGCGTTC AACCTGTTGA AGCAGTTTTT CATAGCTCGG TAACGCCACA 10681 GCAAATGTAA AGTCACTGAT TGCATGAATT GAAGGTCCGA ACATACTCTT AAATTGGTCG 10741 ATATATTGCC AAGGTATATT TTTGATTTGG CACAACACCG CATATAAACG ACGAGCATAT 10801 ACCGGCCTAG ATTCAATTAC TTTTTACGTT TTATCCATCT GTATTCACGA GCGATCAATG 10861 CCAGTGCATA ATTAACAGCT CGGCGCTTAG ACAACAAGAC TTGCTCCGTA CCGAGCTGTG 10921 ATTTCAACAG GTGTTTCAAA GGGGTCAGCA CAGATGCTGG TAAGCGCTGC TCAATATCGT 10981 GCTGAGCAAC AAACTCACGC CACAGCACTT GACTATAAAA AGGCAATTCA CCGGCAGTAA 11041 CATGTGTATC AATAAGCTTA AGTAACCCTT CTAAGGCTCG GATAGTTTTT CTGCAACCAC 11101 TACCAAGCTG CCCCTGCAGC TGAGCAAGCA AATATGAAGC CTCTTTTTGA CGCGTTTGGT 11161 TAACTGTCGT TTGTTGCTCT CCAACAGGAC TATAATCATC CAACAGTTTA GCTAATCCGC 11221 TAGGCTGATA AGGGGTTATC AGTGCACGAA CACTTGCCAC AGCCCTTTCC TTAACCTTTG 11281 CTTTATCAAT CAGCTGCACT AATGCCTGAC AGTCAGAACG GGTCAATTCA GTCGAAGCTT 11341 TAACCGTGAA CCAAGTTGAT AAATAGCGAA GAGATATATT GTTGTTTTTT ATTCCACTGA 11401 CCACACACCT TCTTATTAGC TCTGAAAACT CTGGCTGCGC TACAGACATT TGAAAGCTTT 11461 TTAACACGAG CGATAAACTG TGCTGGCGAG TATTCAAATC TGATGCATGT ATGATCCGAA 11521 GTGTTTCGAT ATCCTGCCAT AGCTCAATGC ACTCAGGTAG TGACAACTTG GACAAAAATG 11581 CCTCAGAGCT TCTAGAAATG AATCCATCGC TAATAACAGA AAATGACGCG GCAAGGTAAT 11641 CAGGATCTTG ATATATCGCC TCGACAAACC GCCTTCGCAT ACTTGGCCAT TCTGCACTGT 11701 TGAGCACCAT CAAAAATAAC GCTGGCTGGT GCTGTAAACT ATGCCAACAA GCCGCCAATA 11761 CTCTTTGCCA AATATGCGCA CTCTGCTCGC TTTTTGCACG TTTTAACACA TCGTTCAACA 11821 TCAGCGGCGC CAAAAGGTAA TTCTGTTTTT GAGCGCCACT CAAGTACTTA ATTAATTGCT 11881 GCTCAACCGC AGTATAAAAG GCAGGACTAA ACACCCTATT AAAATAATTA CTTGGGTCAC 11941 GTGCTAACGC ATTTAGGTCA ATATCTGGTA GCTCTATTTC AATATCTTCC AAAGTCACAG 12001 GCGCGATACC TGACTCCGCG ATTGGCCAGT CTAATTCAAG TTTATTTATT TCCTTATACA 12061 ACCAGTTTTT AAACAGTGAC TCTAGGTCAG CAATACTGTG CTGACCTAAA CTCACTAAGT 12121 GTGCAGGGGC TTCTAATTCA GCCCCTATTT GACCTATTGC TAGCTTCATG GTTAAACGTG 12181 GTCGTTACGC CAAAAGAAGC GAGAGTGATT CATAGGCTTG TCTAAATACA GCGCATGCCA 12241 CAAGAAATCA CCATGGTGTT CTTCTTGCTC TTCGAGGTGA TAGCAGCCCA TGCTAAAGAT 12301 ATGGTCTCCG TCACCTTGCT GTGCCGTATT ACGTCTCCAG AACCCTTTAC AGATCAAGTT 12361 TTGACATTTA TAAGCGCCGC CACCATGTAG CTGTGCAGTA TTATTGATTG CAACTACTTC 12421 ACTGTCATCA CAATGTGCAA CAGCACCTCC GCGTTCGGCT TGGCAATTGT GTGCTTGAAT 12481 ATTGGCATAT CTCAAGCCAT AAGCCGCCCC ACCTTGACGG CCTGCTCGAC AATTGCGGAT 12541 ATTGTTCGCG GTGATGTTAG TCACATCTTC GCCATAAATC GCTCCACCAT CACCACTGAC 12601 TGCATGGTTC TCAATCACAC AATTAAGTTG TATTTGATCG GCGTAGCGTA AGTTAAATGC 12661 ACCGCCGTTT CCTTCATAAG CACTGAGCTC TCTGGACACT TCGTGGATTG CACCATCAAA 12721 TGTGAAGCCG TGCATTTCAA CATGTTTAAC CATCGCGTCT CTGTCACCAC GGATCAAGAA 12781 GCGACATCCT GCATGTGCTT TGACAATGCG TGTTTCACGC TCATTAAAAC CTACAATGCT 12841 CAAGTTTGAA CCGACTTCAA CGGCATTTTT CAGTTTATAG GCTCTAGAAA CCTGGCGATG 12901 ACCATTGTGT GGGAATAATA AGATCGTCAT ATTATTCAAG CAATCTCTCG CAAATATCTC 12961 GTCAAACTCT TCTTGATTAT GGATCGCAAC AACCTTCTTA CCGCGTCCAA AGCGCCATGC 13021 ATGTTCATCC ACATAGGCTT TAACAGCAGC TTGAGTGGCA AGTACATGAT CTGACGCCAT 13081 CTCACCGCCC AGTTCACAGT CTGACGATAT TGCGTCAATC TGCGGCCCTT GTGGTAATTG 13141 AAGTTGCTGT GGTGCAATCT GGCCTTGAAC AGCACAATCA CCCACAATTT CAGCCCCCTG 13201 TTGTAGGTTA AGCTTACTCT CGAGGACCAA CTCACCAGTC ACAGTCAACG AGTCCGATAC 13261 GCGTGCATCG CCACGGATAT CTAACTTGTG CTCAGGTGCT TTACAGCCAA TACCAACATT 13321 CGCATTGCCA TCAACAATCA GCTCTGAATT ACCGCGCACA GCCACATCTA GCGCAGCTTC 13381 CCCTTCACTG GTATTAACTG ACACCATCGC GCTGTCATTA TGTGAAATGC CCACTTTAAC 13441 ACTGTGTTCT ACCGCTAAGC ATGCAGCATT TACGGCACCA CTGATATACG AATCACCTTT 13501 AACATGCGTG TCGTTACCAA AGTTTGTTTC ACCTTGCACG GCTAAATGAC TATGAATATC 13561 TACATGCGGT TTAAACACGA CCTGATTTGC CGCATCTAAG CCTTCAACGA CTAAGCTGCC 13621 ACGTACCGTT GTATCTTTAT CAAAGTGAAC CGCTTCTTTA AAGAATGCAT TACCCCTTAC 13681 TGTCAGCTCC TCACCTAGGT GCAAGCTTTT AGCAAAGTCG GCGTCAGCCA GAAGATTTAG 13741 TTTGCGCTCT ATGGTTGTTA GGCCCTCAAT TGTGACAGTA TCTTTAAATA AGGCATCATC 13801 GTATACGGTC AAGTCAGAGC GCAGCTCCAC ATCACCTAAA ATCTTCGCGC CATCTAAACA 13861 CTCAAGTTGG CCGGTACCAC GCAATACCCC ATTGAGCTCA ATATCACCTT CGACCTTAAT 13921 ATCACCTAAA ATATCCAGCT CAGCTTCCGG TTGTGCATTG CGGATCCCCA CTTTGCCATC 13981 TTGAAATACA ATTGGGTTAA CCCCTTTGTG ATAAACGGCT AAGCCATACT CAAAGTGGTT 14041 TTGCTCGACA CTAAACTGGG CGTTACGGCG TATTGTAGAT GCGTCTTCAT CTTGCGCGTG 14101 CATAGCCAGG CCAATTCTGG TTTCACCCTG AAATTCTGAA GTGCCATAAA TGTTCACATT 14161 ACTTCTGAAG CTGGCATACT CATTGACGTG TAGACTATCA TCTATTTCGA CGCGACCATT 14221 AACAGTCAAA TCCCTTCTAA AACAGGCATC TTCCCCTACA TCTAAAATGT AATCTGGGCG 14281 CTCTACACCC AATCCTAAAT TGCCATGGCT AAACACCAAC TGTGCCTTAC CACTTGGGTC 14341 ATCTATACGA AGTGCTTGTT GATCCGTTTC ACAGATATGC ACTCGCGCAG TCGCTTGATA 14401 TGGTGCTAAG GACAAATTTC CCGAGATAGT CGCATCGGCT CTAAAACTAG CATCATTATT 14461 TACGGTCAAC GCATCGCGAC CTTCAGTGCC CTCAATTTCG ACACTACCAC CAGCATAATG 14521 CTCGCCAGAT ACTGTTAAGT TCTCCCCAAC TTGAGCATTG CGTCTCACTG CCAAGTTAGA 14581 CTTGATTTCG CTATTACCAT CTACCGTGTG ATTAGCATCA AACTGCACGT CACCCGTTGT 14641 ATGTAGTTCA CCCTCTAGTA TTGATTCTCC AGATACTGTC AGAGCAGCGC GCTGTACATC 14701 TCCTGAGTTG TTAGTCACAC TCACATGCAG TCCGTTTTTC AACTCTGCAT GACCCATCAA 14761 CTTAGATTTT CCGTCTACTG TTAACGCAAT CCCATTATCT CTAATAGTGA GTTGGCCCGT 14821 AATATCAGCC AAACCAGTAA CCGCAAAATT AACGCCACCT TCTGGGATCA TTTGATTAAT 14881 AGCAAGCTGG CCTGCAGCTG CCAATATTTC CGCATCTTGT GCTTGATTAG TGATTCTAAA 14941 TACGGGGTTT TCTGATAATT GAGTGAGCTC AAATTGGCTA TTCGCAAAAT TGACTGCGAT 15001 GTGCTCGTTT TCAACAATGG TAAACTCATC AAATAAATGT GTATTACCAT ACACGTGCAA 15061 ATCTGCGGAC TTCTGCTCAG TACCCACTTG AGTTTGTATC AAGGTCGCAC TGAAACCACC 15121 AAATTCACTG TTCTCACTGG CAACGTAAAC GCCTTTAAGG GCCGTGATAT CTTTATCAAA 15181 CAATGCAGAT TCACCGACAT GTAAGCTCTG CTGCGGGTTA TTTTGGAACA AACCAATTTG 15241 CTTGCCCGTT GCATTGATGA CTGACGTTGT GTTTAAGCCG TCAAAAAAGC TGACATCCAC 15301 ACTTTGTGGT TGGCCGTTGT GGGCTAGCAC AGCCAATTTT GCGTTGGTTG TCCAATCACT 15361 TTGAACCGTG TTAGATGCAA TCACCACATG TTCACTAAAG TCCGCCTGGT AAATAGTCGC 15421 CTCATGAGAA ACGTCTAGAT CAGGCGTTGT GATATCCCCT TTAACCGTCA AAGATTGAGC 15481 TTGGCCTGTA CTACCCATCA CTGCTTGGTC ATTATCAATA CGCAGTAATG GATTACCTTC 15541 TTTATCAGCA ACTAGCAAAG ACGGGTGATT TGTGGCTGTA CTTTTCACCG TCAACGTCGC 15601 TTCATGGTTG CCTACCTGCT CACCCACAGT GAGATTATGC CTCACTACAC CATCGGTTGC 15661 TTTTACTAAG GTATCGGTCG AAATCTCCTC ACTCGCATGC AATGATTTAA ACGTAGCATG 15721 TGCAGTGACA TCGAGGGTGG TATTAATAAG TGCGCCTGCA TCTGTTTGTA CCGGTGTGCT 15781 AGGCGTTTCG CCGACTTGAA GCTGGCGCGT ACGTGTTTGA CCGCTAACTT GGGCATCTTG 15841 ACTAACCGAC AGCGCATCAA GCACCGTACT CCCCGCTGTT AAGGTATCTG TAACCTGCAC 15901 AGTCTCCGCA TTGACTTGTC CAGCAGCAGT CAAATCATCT GAAATGTATG TATGTGCAAA 15961 CATATTGATC GACTGTTCAC CCGCTAGTGG CATATTCGTG ACTTTCACCA AATTACGACT 16021 ACCATCACCA ATATTCAGTA AGTCACCGTT GCCAGCCTGA ATATCCACTT TTGCATTCGG 16081 TGTTTGCGCT CCCACCGACA GCTTGCCGTT GACATACGCA TCACTTTCTA CGTTCAAATC 16141 GCCCGTAATG TCTGCTGCTT GTTCTATGCT CACAGTCCCA GAAAAGTGCG CGCGCCCGTC 16201 TTCATTTAGC GAGATGTGAG GGTGGTCTTT ATCTTGTCCA ACTAATACCG AATCCGCAAC 16261 ATGGAAGCGC GCTTTAGGCA CACTTGTACC CACCCCCACT TTACCTTCTG GGTTGATAAT 16321 AAAAGGCGTG TTATCTTGGT TTAAATCATC CACTCGGAGT AGATCGCTCG TGTCTGATGA 16381 ACGTTTGCCA ATATGCAATT TCGCCAAGGT GCTATTTTCA TCTAGGCCAA CACCTAACAT 16441 CGGCGCTTCT ACATGCTCTG TAAAGATGGC ATTTTGCGCA CTGAGCTCAC CGAGTAACAT 16501 GGTACGCTCT GCGACCGATA GCATCTGAGT TGATGTATTA CCGCTCACAC TGGCATCATT 16561 CACAATAGAG ATTGAGTCAG CTTGGATATG ACCTTCAACG GCAACATCTG CGTTCACTTC 16621 AACATCACCA TAAGCGGTAA TTTTTGGATT ATCATCATCA TCTAAGCCCG CTTCCAATGT 16681 CTGGATAGTG CCTTTACTGA CAGATAACGC GCCCGCTCTT AAGTGCTGAT TAATATCCGC 16741 CGTTTGTGAC TTTAAGCCCT CCGTTAACTT TGCGCTGCCA GAGACGCGTA AAACCTCTTC 16801 TGCGTCAGGT AATACTTTGA CTTTGTCATA CACAGAAACG CTTTGCGCAT CGGCACTGAG 16861 CACCTGCATT TCGCTATCAC CATCACAAAC AACCAGTTCG TGATCAATTT TGACGGCATC 16921 AGTATCCACG CGTAAACGCG GTGTCTCACC ATCTGGATTG ACTGTGAGAG GCGATGTAAC 16981 CGCTATTTCT TGAGTTTCTC CGACTCTCTC TACTGCGATC GGGTCGTCTA TTTGATTAAA 17041 GCCTGAGCGA ATAAGAGATT CAAAATCATC CCCTGTCGGC GTTTTGAGAT CATCAAACTT 17101 TTCAATTAAT TCTGCGCGGC TGCGCTTAGT TACTATAGAT GTTTGGTCAC ATTCGACTAA 17161 CACTTCTTGG TCACTGACTT TGTTCATTCT TCGTTTCCTT CAGGATCTAT GCGATTTGGC 17221 ACGCTGGTCG CCACGATAAA TTTCGAAACC ACCGCGGTAT TTGAGTTAAT TTGTGAAAAA 17281 GACACAGGTA ATATTGGTTT AAGAAAAGGG AGTGGAGAGT ATCCAACCGT AAACCGAGTA 17341 TTGCTTGATT GTGCAAATGC ATTAGGTGTA CAAATGTGGT CGATACACAA CTGTGTGGCT 17401 AAATACAATA CTTGATCTTC GCTAAACCCA TCAGGGTAAA GCGCACTGTG TTCATTGATA 17461 ATTTGCGCAA CCGCGCTGCG TACTTGCGCA TAACTTTGCT CATCAATTTC AATTTCATTA 17521 GGCAAAGGTT CATATGCATA CTCAGACAAT ATCTGCACAA GATACGCTTC GAGTACTTCC 17581 ATACCTTGAG GCTTAGCCAG CACGTAATTA ATGATGTTTT CAATCATGGT ACCTGGCTCA 17641 GATACCAGCA CAGTTTCCAA CAAACCGCCG ATATCTGCTG CGACACTGGC AATATGAGCT 17701 TGGCCTTCTG CAAACCTTTC ACGTTGTGCC TGACCGTAAA AATTCAACCA ACCAAAATAT 17761 AAGCGTTCAA ATAAACTCAT CTGCTCTCGC GTCAACCAGA CACATTGAGC AGACAGGTGC 17821 AAAGGCTTAA ACTTGCCTAT TTGACTTTCC ACTAACGCTC TAAACGCGTT ATTTTGTAAA 17881 CGAGTCGTCC AGTTAGGTAA GACACAATAT AAATTGCCCG ACGAGTCATT CCCCAGTAGA 17941 TCGCTTTCCA ATAAATAAAA GCTCTCTCTG TTATGGGTCG CTAGCGGCAT CTGGCCATGG 18001 TGCGAAAAAC CAAGGCTGCG CATCACTAAG CGACTCAGGC TCGCAGTATG CGCTGTTTTT 18061 GGTTCTTTCG ATAAATAATC AAAACCGCGA CAACGATTTT CTCCCAAAGC GCCAATCTCA 18121 TTGAGTAATC GACACTTATC AATGTACTGG CGCAGTAATA CTAGACGTAA TAATAGATCC 18181 TCATGTTCGC TCGGATCATT CTGGCAGGCA CCAATCACAT TGACATAAAA ACCAAATACT 18241 TCGCGATATT TCAGCAGGTT TGCATTCGGA AGTTTAATGG CAAAACGTGC CAGCAAATGC 18301 GCGATACTGT CGTTCAAGCG CGTTAACTGA GCTACTGAAA AGTCACTTTC TAATTGCTGC 18361 TGTAAACCTT GTTGTTGATA CGCGTCTAAT GCCAATTTTA CTAGGTGATT AACGCCACTA 18421 ATGCCTGTGA CCGCCTGACT CAATTGCGTG TGCGGTAAAG CTTTGACTCT GTGCCAAAAC 18481 TCATCAAGTT GCCCTTGCGA AAGCGCTTCG CTGGACAACA TTCGATCCAT AATCTGCCCT 18541 AACCGCTCGA AATGCGTATA TTGAGGCAAA GCCAAAATAT GAGGTAAAAC ATCCAGTTGC 18601 TTGTGCTGAT CAGCCAATAT TTGATCAAAC AAGTGTAAAT ACCCTTTAAA TTGTAATAAT 18661 TGTGCTTTTT GATCAGAGTC GATATCTCGG TTTAAAGACT GCTCTGTCAG CGCATAAACT 18721 TGTGGCAATT CATGCTGCAG TGAACGGTAT TGACTCAAAG GTAAGAATCG ATGTGACTCT 18781 TGATTTACTT GCTCGGCGAG CCCTGATGTG TGTTCAACGA TGCGCTCGCC GATTGCATAC 18841 CTAATGGACG CAATTTCATC TTCATCAAGT ACATAACTTT GACCTTCAAT GACTAAAGAA 18901 ATAGCATCAA AGAATTGTGT ACTCAAAAAT CCTAACTCAG GTGCCAAGTT TACTGAACTA 18961 GGGTCTAAGT CTTGAACTTC AACTTGCCAG TATTCATAAC CTGGCTGAGC ACCTTGATCT 19021 ACGATAAATC TAAACTCTTC AATCTGTTCA ATATTCGGGT GAACTCGCAG TGCATCTAAA 19081 ATATCGGAGC TATAGAGGTG CTTCTTAACC GGTGTGCTCT CTAAATCTTC GTCTAAAATG 19141 AAGCCATTAC TTAAAGATGG ACCTACGTAG ATATCCTCTA CATACATCCC TTTATCCAAT 19201 AATTCCTGAC TAGTGTAACG GGTTAAATTA GGCGATATTT CAAGTGCGAC TTTACTCAAA 19261 ATATCGGCCA TCACGTGTAC AATGTCTTGC GCATCTTTGA GTACCAAATG CATACCCAAT 19321 TGCACAGGTA CAGACTGATA AAATTCAATC TTACCCAGTT GATGCGTTAT ACAACGAGCT 19381 GCGGAAAACG TGTGATGGAT TTTTTCTCGC AAGCTATCTT GTGATGCCTG TGCCGATGGT 19441 GAAGTCAAAA CAATCACATC AAAAATTGTC GCGCCAGTCG CGCGTTCATC GCGAACAGAA 19501 ATCGTAATGT TCTTTATCTC TGTGATATCA AGCAACAAAC GCCTATAGTC AGCAAGCGTA 19561 ACGCAATGGC TAAACATCAC TTGCTCCAAG TTTAAAAACT GCGTCGGTGC GAGAGACTCA 19621 CCAGGCTCAA CCGCGATAAG ATCCTCAACG GTATAATTGA GTTTATAGCT TAAATCCGAT 19681 AGCACATAAG CCAATACTTC AAGCAAGGTA ATGCCTGGAT CAAATACATT GTGGTCGCTC 19741 CATGTATCGG GTGCGAGCTT TTGTAGCTGT GCTATCCCCT GTGCTCGCAA ACCATCTAGA 19801 TCTTGCCCAG GGTTTAGCAC AGCATCTGAA GCAATTTTCA GTGCGTTGGT TTCCATGATT 19861 TTCCTTTGGA AAGTAAGTCG ATATGGGAGA TGACGCCAAA CCTTCAAGAT TGTGAAACGG 19921 TTTATTAATT TGGCGTCGCT TTAAATGTAA GTAGCTTATT GCACAACAAA ATTATGCTGA 19981 ATTTTCCATT TTCCAATCCC TTCAAATACG TCACCATCTA CATTCGTTAA AGAGATTTTA 20041 TGATTGCGTG TCGGAACTAA AATTTCGCGG CTTTCATTAG GCGAAATTTG AGAGTATTGC 20101 CTGATCAGAT CATCCATACG TTTACCTCGG ATCACCTGTA CCATGTTGAC CGCTTGATGG 20161 GCTTCTAACG CCTGTGCTAC ATCCGCCAAA TAGATGGTTT GCGATAACTC AGCATCCGGT 20221 TTGTTCCATG GGGTTAAGGT ATCCACAATT AAGTCGTTTA GTTCAACTAC GGTGGTCTGA 20281 ATATCAAATC TTGGGTCAAT CTGAATGATG ATTTCTAGCT GCACTTCAAT ATATGTTGGA 20341 TCTTCAACTT TAACATTGAG ATATGGCGCA CAGCGGGCTG TCACTTCATC CTGTATTTTA 20401 CGCATCAAGT AACGAGGTAC TTTGGGTTGT AAAATATTCA CATCATGGTT AAGCGGAATG 20461 ACAGTGATGT TTACACCATC AGCATTTTGA TAGGCATTCA CAGAGTGTAA TTCAGGGAAA 20521 GTCTGCAACG TAAAATGCTC ATAATCCCAC CCCGTTTGAA GCCTATCTCT GTGCCTTAAG 20581 CGCTCACTGA CACGCCGATA TAAGCGACTA TCATCTTCTT TGACCTTGGC ACCAAAGCTA 20641 TCAAATGGCT GTTCAATCGA CGCGATTAAA GGGTCCGTCA CAGTTAATTT ACTAATGCTC 20701 CCAGCTGCCA ACGGTTTTAA ATAATGAGAT TGAGCATGTT CAGCTGAACT AAGCGTAATT 20761 TGGACGGCCT GAGCATAGAC GCCTTTTAGT TTTGAATAGA TAGGGTTGAC ATAGCCATCT 20821 TGCAGTACCT CTGAATCAAT GACGGCTCTT ATCCAGATCT TACCGTCTTG ATTAAACTGA 20881 TCCGCTAAAT CAAATTCAGG TAAAGAGAAA ATAATGATCC CCGAATCTAG CAAGTCATAG 20941 GTATTGTCTT GTAATATTCG CCCTTCTTCA CCGCTGCCTT GAGAGTCTTC TCGACTAAAG 21001 AGGTGCCATT GCCCTTGATT GTAGTATTCC CACTTAAACT GCGGAGGCTC GGAAAAATTG 21061 TAGCCATCAA CCGCTTCTAG TTGAAACAGT ACGCTGCATT GGCCAGGTGT TGGAAGATCT 21121 CCAATAGCTA GATACAAGTA ACCCAAATCG TCTATTTTCG GCATTAAATG AAGCGTATTT 21181 TGCTGCTCCA ATATTTGCAG CTGTCTGCCA ATAGGTGAAA TATGTTCTAT CCATATAGAG 21241 TTACCTATGG CAGCCCTTTG CGCCCGAGAA ATACTTTGCG TTCGATAATG TAATTTGACT 21301 GAGTCTAATA ATGGGGTATA CGGCTCTGGT ACCTGCGTTG GCATAAAATC ACCATCCGCA 21361 GGATTCCAAT TTGCTAACTT AATACTATTT TGAAACGCAT AATACTCAGA TACCTTAGTA 21421 TATTGCGGGT GACCAAAATC TTGATTGGAT AAAGCCAAAG TGTACCATTT GGGCCATAAT 21481 TTGGCTTGGG TCAAATTGAA TGGCAAAGAC AGATAATCCA CATCACCATT GTCGTCATCG 21541 TAAGTAAAAA CCAAACGGTT AGTCGCGATA TTATTCACAA CATCAGGTAA TTGACCGGTG 21601 TTGTCTTGCA CTATCAACGT GCGCTCATCG GGTGTCAATT CATTATCAGA CGCTTGCCAT 21661 ACCAAGTCGT TATAAAATAA GTCCGCAACC AATTTGCTCT GCTCACTGCT ATAAGTCACG 21721 TCCGATTGCG ATATCAATAC TTGGTTTCTT GGCCAAGCCT CACTCGCAAG TTGAACAGAC 21781 TCAGTTGGTG GTGTCATTGC ACTCTGATAG TCCATATAAG TTTGGTAATG CGCGTCAAAA 21841 TCTGCCGGTC TACCCACCCA ATTAAATTCA ATACTTGCCC TTGTCACTCT TTTACTTAAC 21901 AGCTCAGGGT GCGTGAATTC GAACTTCTCT GCCAGCTGAG GGGTAAAACC AAATGGTTCA 21961 AAGGGCTTAG TGGTATCTAA AAAGCCAGTA CCATTATTGG CAACCAAGCC ACTTGCACCA 22021 AGTACGGTCA CCGCCATTCG TACTTCTTGA ATTTCGCTGA CAGCTAAGTT TGCCAGTGCT 22081 TCCACTCTTT CATTAAGATC CAATGCATAC AGCGCGTCGT ATTTTGACTG CTTTAACACA 22141 AAGGCGATAT AAGGGAGCAC CATGTCTTTG CCAATCATTA AATCAGCGAC AGGGGCAATG 22201 GGCGCAAATA GCTCATCAAC ATGAATCACC AGTTCATTCT CACCATTGAG GCTAAGCTGT

22261 TCTGAGCTTA ACAGCACCGC TTCTTCTTCG GTACTTAACC AAATATCAAA GCTCTCAATC

22321 CAAAGTGATA AATCAATGGG CGTTTCGCCA AATTCATCGG TAGAAAAACG CAGTGAGATA

22381 ATGCGTTTAC CACTACTCAA AAAGAGATCC TGAGAAGCAA CTTTAAAGCC AATCTCTACC

22441 GGTAATTGAG TTTCTGACAT TTGTTTATCG CCAAATGTCA TTGCACTACT TTCAAGCTCA

22501 ATCCCCAATT CAGTATCCAG TAAGACATTG CGACAGATCT TCACGCCACT TGCAAAGTGC

22561 GCTTTGTTCA CCGTTGTCAC CGTCTCTACC ACTGCACGAT TTATCGCACT GGGTTCAGCC

22621 AATTGGTAAA TTCGCTCTTC GCCGTTGTCA TCTTTGCCGC CATCAAACTG AGTGCCAGGT

22681 TGCAGCGTCA GCGACTCGAC CTCGTTTAGT TCGATTAAAA CATGGGCTGA ATCAGGCGTT

22741 GCACTTTGCA ACTCAAAGCC CAATACATCG CGATAATAAT GGTCAAGGTG GCGTTGAACA

22801 AAATCGTTAA ACTGCTGTTG GGTATGCTGT AACAGGTCAA CAAATGTAAG TAGTAAAGAC

22861 AGGTCTGGTC GGCTCGCCAA TCGGGTCGTA AGTTCATCGC CGCTTCCTAC CAGAATTAAT

22921 TTGGCCAGTG CGGCAAACTT ACCTGGCTCA GGTAACACTT TGTACCAAGT TTGTACTGGC

22981 TGCCACGTTG CTTGACCAAT AAATTGGGTG TGTTGCGCGA CAGAAGACAA GTAAGACAGC

23041 CATTGCTCAA TCGTACGCGG TTCTATACTG AAAAAATCAC TGGAAAGCTC GGGGATCTGT

23101 CGTGCGGTTT GACTTAACCC TTTACCGTTG CCAGAGATAA GTGATTGACT ACGAGACTTA

23161 AAAGTCATGA GATTACCTTA TTAATGTTAT TAATGAGCAT ACCTATGCAC TACTAGGTCA

23221 ATTTAACCTT GGTAAAAGGA TAAGCGGTGC GGGTCTGCAT ATTGCTTGGC GAGCAAGGTC

23281 GAAATGGGTG ATTCTGTGGG TGATACCAAA TGTTTGAAAC GCGGGTTTAA ACAGACTGCT

23341 CGGCGAGATA AAACGGAAAC ACCATATTGC TGCGGCTATT GGTTTTGCGG ATCAAATAAG

23401 TGAGCTCTAT TAATAAGGCA CCTTCGTACA CATCTGACAT ATCAAACGAA ATATCCTCAA

23461 GGATAATTCT CGGCTCATAG TTTAAAAGCA CCGCAGAAAC TTCTTGCTTA AGCGTGACCA

23521 GTGCGGACTC ACTGACATCC TCAAAAACAA AATTAGATAA ACCGGAACCC AGCTCGCTCC

23581 AATAAGGCCG CTCGCCTTTG AGTGTATTGA GCGCAATAGA AATGGCCTGT TTAACCAATG

23641 TCTCTCCGTC TTCCATATCA GGCCCTGAAT CCGGGCTGGT AAACTGTGGT GGGAATCCCC

23701 ACCCTCTACC CAGAAATGAA TTGTTCATAG CTACCTCCTC GTTATCCTAT TTCCATCTTG

23761 TCTGTGGCAA CCACCACTTT TGCACTTTCA ATGGTAATTT TACTGTCCAT TTTTATCTGA

23821 CTATCATCAG GTGTTTTAAT CACCACATCC GTTTTAGCAG TCATACCCGC CTTGTCTTTT

23881 TGAGATAAAG CATAACCTGA GCTTTCGCCT AACGAGATTG AGTGGTCTTT ACTTCCTTCC

23941 ATGATGATGT GTGGTTTTTT AAAGGTGAAC TTCAGCGCGG TGGCTTGTTC TTTAAACAGC

24001 AACCCTCGAA CATCATATTC CTGTTTGTAT CCACCCAAAG GAGGAATGGC CACTGGGTTG

24061 TGACATGCCC CAACAATCAC AGGATGTCTG GCATCCCCCC CGATAAAGTC CACCAGCACC

24121 TCAGTGTCTG GGTTAGGGGG TAAAAATAAC CCTTCTTCCT TGCCAACATA AGTAGTCAAA

24181 AGCCTTGCCC AAATAGGCTC CGTGGTTAAT GTATTTAAAG TGATGGGTAT GCGATGTAAT

24241 TTTTCTTTAT CGTCTTTAAA CGGCGCAACG GTAGCAACCA GCATTGGCGT AGCTGGCAAT

24301 TCAGTCCATT TAGACCAGCC ACTTTTCGTT AAAGATAAAC CCAGAGTAAA TTCACAGAAC

24361 CAGCCTTGGT TACTTAAATG ATGGTGAATT TCTGTCACCA TGTAGTCCGC TTTGTTGCTT

24421 TCCCCAGCTC CCGATACTTT GATTGCATCG AGTAGTTTCA GTGTATGCAA AGCGGTGGCT

24481 TCAGACATAT CAACTTGAAT ACGGCCTTGG CTCGTATCTA ATAAACGATA TACTTGCTCG

24541 GCTTTGGCCT TGGCAGCCAA CTCGACTTTG TCAATGGGCG CTTGTGATTT GATGACGATA

24601 TCTGCGGTCG CGCCCTCTTT TTTCTCACCC GAGTCATAGT TTTCAAGTAA CATGGCTTGG

24661 GTTTTAATAT CCCATGCACT GTGGTCAACT TTTTGCGCAT ATGAGCTATT GTCCATACAA

24721 AGCTCAAATT CAGTACACCC ATCCATCGCC ACATTAAACT CTGTTGCCGC CGGTTTGTGC

24781 GTTGTCAAAT CAACGATGTT GATCCCTTCT TCTTCATAAA GCGCAAACCC ATTGGTCAGG

24841 ATGCGATTCA TGATCACTTC CCACGGTTTT TTCTCAACCA TGAGATATTG ATAGTGTTTT

24901 ATTTTACTGT CCGCGACGGT TTTTTTACCG CCACAGGGTT TGAGCAAATC ATTGATAATG

24961 TCGGTGTCAG TAGAGTCTGG CTTGTAGAGC TTACTTATTT GGGACTGACA GAGCTTATTG

25021 GCTTCACCTT TAGCGAATAC GGTAATATAG GGCTCACTAC AATAACCAAC TTTGCCTCCT

25081 GTAATGACAC CAACAAATAA TGTCATTTGC GCATCGTCAC CAAAACCGGC TTTGACTTCG

25141 ATGGACTTAC CGGGCTGAAA ATCTTTATTC AAATCCAGTT TAAAGCTTTC CTCAGCCATA

25201 TCGCCATCTT CGAATACAAT TGACAACTCA GAAACCTGGT TTAGCCCACG ATGGGTGACG

25261 ACTTCACGTA CACCGACTTC CAGTTTCTTC TCGCTACCAT CAACTAATAT CGTATAACGA

25321 ATATCCATAT CATCGCTCCA ATGGTGGGTA GGTAATATAG TCACCCACTT CAGCGCCTCG

25381 GAGCGAGGGC AATCTATTTA CACTCGCTAC TTGATGAACA TAGTTTGCTT TCCCGTATAT

25441 GGAAGTAACT TTCGAAACCA AACTGTCCCC ATCTAAAAAT TGTAATTCGT GCGTGAGATC

25501 TGGCGAGCTT TTACCTGTTC TGAGTTTAAT TTCTTTGCTC GATAGACACT CAGAGATATT 25561 ACAAACAACC TGAGCTTTGA CTCGATTACC TTTGGAGTTA ACCAACTCAG TAATGACATT

25621 AATATCGCTG AACATGCCAT GAAAACCGCC ATCGGCACCA TGATTAAGGA CCATATTCAA

25681 GGGCATAATA GTGACGAACG CAGGCAAGTG CGTTTCCCCT TGTACCGCAA GGCCATATTT

25741 TAGCATTTCC TGAATACTGT CTTCCGTTGA CGTTTCACCG CTCATGGTCA TTGCATATTG

25801 AGAGGGGGTT TCAATAATCG TGTTATCAAG TAAAAACGTA ATCGTTAAAA CGGACGGGGG

25861 GTGATTTTTG ATAACGTGCA GAGCCGCTTT CCGAGCCAAT GGCCTTTGAG TCTTTTAAAC

25921 AATTTTGATG AGTGACATTG AGTGATTTAG GATCATATGG AAGCGTTAAC TCACCTAATT

25981 TATTGCCATC GCCACGCTCA ATTTTTTTAT AAAAAGTCAC TTTACATAGT GGTAAGCCAC

26041 TTGCGTTAAT GCCGTCTGCT TTATTCATAA TTAGGGCCTC TTTTCCTTAC CTTGACGTGC

26101 AAATCTAAAC TCAATATGTC GTTTTAAATC TGACTCTAAT TGGTGTAATA AGTCTTGCAA

26161 TTCGGACCAG CTTACTGTGT TGCTATAATA CTCAGACTGT GGTGATTGCT GCGCCTCGTT

26221 GCCGGACACA CTGGCAGAGT TTTCCACCGT TGATTGGGCA GGTTCGTCAG CATGGATCCG

26281 TGCTTTAACT CGAACATTGT TACACTTAAC TTCCATCTAT TGAGCTCCTT ACACTGGCAC

26341 ACCTGGAATA GGCACAGCCA CCAAATCGCG ATAACTAAAA CTAAGAGACT CAATAAGCAC

26401 ATCATTTCTA CTCGCATCAA TCCCCTCCCA TTCCCAGCTT TCTAAAAATG CATCTTGCAC

26461 AAGCCAAGCT TGCGATGGTA AGTAGTTATC ATCTAAGGTA GTGATCAAAA TCTGATTTTT

26521 AACCAAGTAG GTATTCCAAG CATCCCCAAA TACGAGATTT TGCAACATCA AAGGACTGCC

26581 ACCTTGAAAA ACGCCACGCT TTAAAGTGAG TGTTTTCGCT TGTTTTTGAT TCGCAATGCT

26641 GACGCGGTTA TTGGTATAGT CGATACTGCG GTTCATTTTT AGACCACTGA CTTCTTTAAA

26701 CAGAATATCA ATGGGATTAG GAATGCCTGC GGCCACAATA CCAACTAAAA ATCGATACCC

26761 GACCATGGGA GTTCTAGGGT CTTGAAACAT ATTAGCCACC TAAGCGGGCA CTTTCGTGCC

26821 CTAAAAGAGA GTTAAAAATT AATGGAACTC AATCTTGATG TCATCTGCCA TCATCTCTAA

26881 AGACTCAACA CTGGCCTCGT TAGAGCCACC ATTGATACTC GGAGCGGTCA GTTTTTTAGG

26941 AAATGCATTA ATCACTTTCC ATGTTACCAA TGGCTGAGAC CTGTCTTCAT TGGTCAATGA

27001 AATTGTGATG TCTTTTTTAT CAACTAAGTT CAAGCTGATA GATGAGATCC AATCGTAGAA

27061 TTGGCTCTTC TGTTTAACCA GACCACGCTT CAAAGTAATG TTGACGTCTG TTGGTTGCCC

27121 AGGCATGTGT TTTTTGCCAT AGCCATCTTT ATACGTAATT GTTTCAACGC CGATGTCTAG

27181 ACCAGATACT TCTGAAAATG GAATGCTCTC TTCGCCGAAA CTGACAACAA ATCGATAGAC

27241 GGGAATTGGA TATTCTGCTG CGATATCTGC TTTAGTAGTA GCCATGTAAT AACTCCAAAA

27301 ATTCGGTATT TACAGCTGTG CTGTAGGAAA AATAAATATC TATTTGACGC CATGGTTCAG

27361 CGCCAAATAG CAATGTTTAA TTAGCTAATT GCCTTTAGGC GCAAGAATTA ACCTTCAAGG

27421 CTCTTATGAG AGAATGTTAG AACGATGAAT TCAGCTGGGC GCACTGCCGC AAGGCCAATT

27481 TCAATGTTCA TTAAGCCATT GTTGATGTCA TCTTCAGTCA TTGTTTGACC AAGGCCAACG

27541 TTGACGAAAA ATGCTTGCTC TGGTGTTTCA CCAAAGAAAG CACCTGAGCG CCAAAGCCCT

27601 TCTAGATAAC TTTCAATCAT GGTTTTAAGT TTTAGCCATG TGAATGGCGT GTTAGGCTCA

27661 AATACTGCGA AGTGCGTTGC TTTTTGTACA GACTCTTCAA CCATGTTAAA TAGGCGACGT

27721 ACAGACACAT AACGCCACTC GTTATCGTTA CCCGCTAGCG TACGAGCACC CCAAACAAGC

27781 GTTCCTTTAC CGACGAATGT ACGGATAGCA TTGATAGACT TACCAGAAGT CGCATCTACG

27841 TTGAGGTTTT CTTGATCTGC ATTATCAATC GCCAGTTTAG GCATTAATAC TTGTGCAAGT

27901 GCAGCATTCG CTGGCGCTTT CCAAACACCA CGATCTTTAT CTGTTTTAGC CATCACACCC

27961 GCGATTGCTG GTGAAGGTGG TAAATCAAGG TAGTTTTTAC CAAGTTCTGC TTTTACTTGG

28021 TTATACACAT CTGCTGATGA GAATGGGTAA GCCGGGTCAC CGATTGCAGC AAGCTGCAGC

28081 TGAGGAAGCA TGATGTCAGT TAATTCGAAA TCATCTGAGA TAGTAATTGC AGAGCCGCCT

28141 GGTGTCGCTG TTACTTCACC GTCTGCTAGA TCAACTTGCT CACCATCAGT AAGGCGATAA

28201 TATTTACTAC CGTCTGTTGC AAATACCGTT TCTGGCACTA AGTAACGATT ACCTGATGGG

28261 TCATATTCAG CATCCGTTGG GTCGTTCGCC CCTGCAGGGT TATAAAGGTA ATCTTGGCCA

28321 ATTGCAGCCC AAGTGCCGTC GTCACCCACA CAAACCAGTT CATGTGTGTT CGCTTTTGGC

28381 CACCAAGCTT GCGCTGTTAG TGGAGTATTA ACAACTACTT TCTTCGCGTC ATATGCACGT

28441 GCCATTGTTG TTGTTAAATA TGGGTAGTAA GCTGCACCAT ATTTAAGGCC CAATGTAGCG

28501 CCCGCTCTCA ATGCTTTTGA ATCATCCGCG ATTGGGTCAA TGCTGTCTGC ATCCGCTTGC

28561 ATTTGAACGT CAACCAGTGC AAAGCGATCC ATTCTTTTTT CAGCATGAAC AAGCGCCTTA

28621 TTTTGCACTT CATAATGCTT AACAGTGCTC AGGCCAATGG CTTCTGGACA AGAGATAAGT

28681 GTGACTTCAT CTATTTTGTT TAGTAATGTG ATAGCCGCTG TGAACTTTGC TGCTTGCTCA

28741 GCTAGCAGGT TTATTTCACC AGTCACTGGA TCTGCTGAAA GAAGTCCTGC GTCTGGCGCA

28801 CCGATACTTA CCACATAACA CGCACCGCCA CCATTGGCAA AGAAGTGACT CACTGCTTGG

28861 TGTAAAAAGA AATATGCATC CGCAAGCATT GTGCCATCTG ACGTGACATT GAAGCCACCA 28921 GTTGACTTAG GCGCAACTTT AAAGCTCTCT TGATATGCGC CACCAAATAC AGCTTCAAAT 28981 TCAACCATGC TCGTAATACG AGTTGGTGAA TTAAGGTAAG CAACATTACC AGCATCATCG 29041 AGCTTTGTTG TATAACCGAT GAAAGCAGGG ATAGCAGTAG CCACTTCTGC AACTGACGGA 29101 GGCAGAGTTG ACTTCTCTTG GACGTAGACG TCTGGGGTTT TATATTGAGG CATAGTTTTT 29161 CCTTACGTTG ATAATTACAT TCTAAAGAGC CGATTAGGCA AGAATTTTGT GGGAAAAGAG 29221 GCACAAAGCT GTTCGGTCAT AACAGTTCAG GTGGCAAATC AATGGGAACA AACCCACCCT 29281 TTCCTTTGTG CCTCTGAAAT CGAAGAGCCT TTGCACGGCT CCGCCACATC AGTCCCAACT 29341 GATGTGTATG TATTAATTAA TTTGTACTGC TATGACGCCA TGGCCTAGAG GGTGTGAAAA 29401 TTAAAATGAA TATTTTTTAA TTTATTGATT TATAGTATTT TTGACTGAAT ATTTCTCTGT

29461 GAATTAACCC GCCCCAAGAG CGCTTTACAG ACGAGATAGG GTAAAGTCGG TGTAATCATG 29521 ATATGAAATG CGAAAACGAC TAAATTGAAT AGAAACGATT TGAAAAACCA GTTAAATATA 29581 CGCTCATAAG TAGTGCGTAG GCACTATGCA GTGACTCGCA TAAGTCAATT TGAGTGATTG 29641 ACAAATTCAC TTGATGACAA TTCAGCTGTG CTTGACTAGC AAGTAGATCA AGATATTCTC 29701 TGTCAAAGCG GTTATTAATG GCTTGCGCCT GTAACCAACT GTCTCTGTTG GTCGCGATAG 29761 ACACATAATT AAGTTCATAA CTGTCGATGA GTGGTTCACT GAAATTTGCA AAAAGCGCAT 29821 TTAAAACAAA TCGGTTGCCT CCATAGGCCG TTATGTTTCT GTAGTCTGTG TATTCTACAT 29881 ATTCACCTTC CTCATTAAGC GCTTCCCAAT GTTCCAACGC CTTTTGCTCG AAGAACCTAT 29941 GAGGTGACTG ATTTAAGGTT GCTTGCTGCT TTTCATCAAG CATCCTTGCA TGATACTGCA 30001 GTTCGTAAAA GCAAAGCCGA CATAAAGTTG CACTTCTGGC CAATAGGTAG CCTTGGGCAT 30061 CAACGTATAA CTCGGCCCCT CTGGGTACCG CAATATTAAT TCCGTAAAAA GCATCACTAT 30121 AAATATCGAA ACTCAACGAA GATATATCGA GGATAAAATT GCCATAAACT GATTTAAATA 30181 ATTGGTCAAC CTGATTAAAT TCATTGGGTG TGGCACTACT TAAAGTCAGT TCAGCGAGCG 30241 TGCGCGATTT CACTTTAACA AATTCAAAGC CCTGATATTT TACCGAGTAT AATGGTGGCA 30301 ACAACTCATT GTCCGTGTCT AATTCAGAAC CAGCATGCGC TTTAGTTACA AACAAATTTG 30361 ATAAACGGGG TAATTGCACC ATGTTTCGCT GTCTGTGCTT CGGCTGTCTT TGAGCTTTTG 30421 TCACTTTATC TAGCGTGAAC AGCTCAGAAG TGCTTTTTAA TCCTTCGGGT AATACCCTCA 30481 ACTTAGAATT AACCAGCTTA GCTAAATTGG TTGCTGGGAT CACCCAACTG ACATTAGCGG 30541 CGCCATTTTC TAATCCGCCG TTCCCTATAC CTACCAACCT GCCCTGGCTA TCAAATACTG 30601 GGGCACCTGA AAAACCAGGT AAAAGACTAC CGTCCAAGTA GTAAATAGGA AACTCCACAT 30661 CGGGTATATT GGTTTTTGCT AGCGTATCGA CGGCATAAGG TGGTAAAAAC TGCTTTAATA 30721 CTTCTGGCTT TGCGTACCCT TTCAATAATT CACGTGTACT CATTGCCAGC GCGCCATGGT 30781 GAAAGCCCAA TGCAGTCACA TGCTCTCGAT ACTCTGGCTT CGCTTGCTTA ATTTGATTTA 30841 AAGGGCGCCA CCCTTTTGGT GGGTTAACGA CCTCAAGTAA CACTAAATCT GCTTCAGGGA 30901 AAACACGAGA CACCTTCGCA AGGCGTCTTT TCTTCCCAAA ATCTATGATA ATTTTACTGC 30961 GTGGGTGCGG GTCCATCACG TGCAAAGACG TCACGACCCA ATGGTTTTTT TGCCACAAAA 31021 ACCCAGAAGC GACTCCCATT GCATTGTTCG GTTTTTTGAC AACGATTCTG ACTGTACTCT 31081 TGCTCAGAAC ATCAGGCTCT AAACTGGCAA AGGCATATTT AACGGAAAAA AACGTCAACA 31141 ACAGTGACAG CAAAAACAGT GACTTTGTCA TACTATCAAC CCTGCTTGAC TGGACAGACC 31201 ATTTCATGAA CGAACTTCAG CGCATGGAGC TTATTCACCT CTGATAGTCT TTGTTCATTT 31261 AACTGTTGCA AGGTTTGATG CCAAGCTTGC GACAATATAT GTTGGGCTTC ATCAAACTCA 31321 GTTATGTTTG CAAGCTCCCT ATCATCTAAG CAAGGTTTTT CATCGCCGAG TTCACCACAA 31381 ACCGTATCCC GTACCGCCAA TAAAATAGAC TTTTGGTCGC CCGGCTCCAC TTGTCGCACT 31441 ATCGACTCAC TCAGAGTCGC CCACTCATCG CCAGTGAGGG TAAAATTATT AATCACCTCT 31501 TCCAGCTCAA GGCTACGATC TGTATATCTT AACTCTTCGC AACCCGTCAC AGATACACTG 31561 GCACTGAATA GCACGCTATT ATGCTGGCTA GATAACAAGT TTGCGTGCTC TGAAAAGTGA 31621 AAGGATCCGC TGGATGATGC GCCCTCAGCG CTACTGACAT GCTCTAGTGT CATGCCAAAC 31681 TCTTTTAAGG TCAAAAGTTG TAGAGCTTGC TGATGTGTGT AACCTGCTGC TAAGTATTGT 31741 GCTTTAAGTT CCAACAACGA AGGCGTAAAT ACATTGTGGG CACGTACATA AATCCCCATG 31801 TACGCCCCAA CAACACAGGC AACCCCAAAA GCACCAATAC GCAGCGCCTT AGCATCATTA 31861 AAGTGACGAT CATTCAGTCC CAAAATAGCA GCCAGTACTG CCGTCAATGC ACCGAGTAAC 31921 ATTGTCACTG TCGGCGTCAC ACTTGTGCCC ATAATTACAC CGAACAAAAG GCCAATACCT 31981 GCCCCGCCAA AACAGGCGAT TTTAAGGTTT ACTTTCCTTT CCAATATGAT TTCCTTTGAC 32041 GCGTTAATTA CCTTATTAAC AGATTAGGGG TTTTATACTT CCGCTTACGA CGCTTGGTAT 32101 TTTTTCCACC AAACTTATAT TCATATTGTG CCGAGAGTGT ATGTGTGTGT CCGCTATTAC 32161 TCAGCCATAA CTTTTCGAAT TTGACTGTGA TCAATGTACT TGCATCAAGA CGGTATTCAG 32221 CCTCTGACAC TAACGCCTGT TGTGATTGAC TTCCCCAGTA GCTCCGCTCA ATATAGTTTG 32281 GAAAGACAGA GTGCCGGCCA CCCACATACA AGGTTAGAAA ATGCGAAAAT TGATACCCGT

32341 ACTCAATAAG TAGAGAATGA TAAACCTGAC TTGTGTCGTC TAACGACTCA CTCTCAAAAT

32401 TCAACTCTCG AAAGCTTAGT ACATAATCTA GATCCACTTT TGCATTTTCA AAACGCTGTT

32461 GATGACTCAA TAAAATAGCA CCACCTAACT CTTTATCCTT AAAGCGACTT TGTTGATGCT

32521 GGTATTGTTC AGCACCTGCA TACACATCAA CCCCGAATGC GTACCCATGG GATAAGTCAT

32581 ATTCGTAAAA TAGTTCGAGC CTAGATTTAT GTCGCCGATA ATTCACTGAG TCAATAAATT

32641 GCGCTTCGCC AACACGGCTT ATTTGGCCTT TAAGCTTGGT ATGATCTAAC GTTAGCTCCG

32701 CATAAATAGA TCCACTCGAA AACAACTTGC CACCCGCACT GAAGCGTCCT TCAAGCGTAT

32761 TGGAATCCCC AGTGAAAACA TCACGACTTA TTGCTTGGTT AACAGGCTCT CTGCGCAAAG

32821 AAATCGATTG ATTGTATGCC GAGATATTAC CAAAGTAGTT GACCTTTTTA TGCCAAGGCA

32881 TACTGGTTGA GCGCTTTTCT TTAAGCAAAA CAAGAGGCTG AGCACTCAAG CCCATACTCA

32941 TCAGCAACAT GCTAAGCAAG GCGAAGACAG TCGATGGTGT TTTTACATCA CGTGGTGGCT

33001 TTTTATTACC CATTTCCACC GGTCACCCCC GTTACATTCA CAGCCTGAAT GTCTGCTTGT

33061 AGCTTTATAT TTTCAGCTTT TAATAAATCA AAATTGATCG GTTGGGTTTG TGCCTCATGT

33121 AACCTGCGAT TGATGTCGTC TGGGATATTT TTCACTAATC CACTTTGCGC TAACTTAACG

33181 GAGCTTGCCA TCAGTGCCGT TTTTAACGCT GATAACTTAT CTCGGTTCGC TGCATCTTCT

33241 GGCTGGGTGC TAAATTGCTT AATTGCTGTG ATAAGCGGAG CTAATTGTGC GGTATTTTCG

33301 GCTACCGCCG CATTGACTAA ACTCAATAGC AACAGAGTCT GTTCTTGCTC AGTACTGAGT

33361 TGGTTAACAG GCGTTAGCTC AGTAGCCAGT ATATTACGCT GCTGATCAGG CAAATTAAGT

33421 GCCGTCAAAA CCACTTGACT GGCTTGCTGA GCAATCGAGT CAAACTCGCC TAGGCTTGTG

33481 CCTTGGTTTA ATTTATTATT GATTTGGCCA TACACCAAAC TCGTTAAAGC ATTTACTTGC

33541 ACAGAGCCAA TTAAATTATT GGACGAAGCC ATATAATTAA GCTGTAAATG AGCAAGCTCA

33601 GGGGCTGTGA CGACTTCTTG ATAGTTAGCA ATTAAGCCAC CATCGCTATC AAAGCACTCA

33661 ACTGCATCAC AAACCATTGT TGTGTCTTCT ATTGCTTTGA CCTCAATGTA AAAAACGCTG

33721 TTTTCGCTCA CACTGAACTG ACTGTCAAAT GACCCAAATT CATCACTTTG GCCTTGCCAA

33781 ATGGTTGCAT GTTTTGCATC AGAAATAATA ATAGAGGCAT AAGCCATTGT GCCTTTGAGC

33841 AAACTGCCAT TTATTAAGAG TTCATTACTG CCTCTTATCA CCCCCTCTTT GTAACACCCC

33901 ACCGGCATAC ATATCAGCAA AGTTATGCTG AGAAAACGAG ACAACTTAAC ATTATTCATG

33961 AGATTTCCTT TCATCCCCTC TATAAATGGG CATTTAAGTA TGTGTATAAA ATGAAAAAAG

34021 TGCAGTAAAT TGACTTTTTA TTTTGAAATA ACAATCGGCT GGGTCGGTTC ATATTTGACC

34081 GTAAGTTCAG TCGGAGGGCC AGGATCCGGT ACTCCCTGCG GATTAAGTGC GGGGATCACG

34141 CTCAATGCGC ACACAATATC ACCCTTAAGC ACAATTGGCG CGCCGCCTTT AAATGTCACT

34201 TCGGATAAAA CAATATCGTT AATTGCACTG AATGTGACCG CTCCACCGGC CGCATAGGCA

34261 CCATTAATAT AAGGCGACAT ATTAACTTTC TTGGTCAAGA ATTTTTCTAT GTCATCTTGA

34321 ACGGCTATCT CCCCTTGGCA AATTAGTCCT TTGGAAGCCA CTTCTAAAGG GCCTGCGGTT

34381 GGAACAACCA AGACGCCTTT ATCTAGCAAC AAGTTAAAAA CGGTACTTTC ATTGAGAATT

34441 TTTATCATTA TTGATACCTA GCGCAATGCG ATAAACGGGT GTGCTTTAGA GTTTTTATGC

34501 TTTTACTCCT CACAAAGTCA CTACTCAAAA GCTGATGGAG ATGAACGCCT TTGGGCGACT

34561 CTAGATACAA CGGCGCTGAG CCTAAGTTGT GAATTTTATT TTCGCAATGG ACTTATCCGT

34621 TGCGAAGTAA GACTAGCGAC CATACTTTTT GATAATCGAC CAAAGTCCTA ATTACAGTTT

34681 GAATTCCATC CCATAACTTA GAAGTGTTCG ATTTTCAGCC AAAAGGACCT TTATGTTGAC

34741 GTCAAATACG TTAAAAACCA TTCTGTTCAG TACAAGCTTA TTGCTCACTT CGCACGTACA

34801 TGCTGCTCCA AAATCTGAAG AGCCACCACT GCTGTTAATT GGTGCTTCAT TCGCGAATGC

34861 AAAAATGCCT TATTTCGATA ACCTTGAAGC GCCATTAAAT GGTATAGCAA TCAATTCAGG

34921 GAAGTACTTA TCACTGGGTA ATGCCTTGAT CAGAGAGCCT CGACTATCTG GGCATCTGAT

34981 AAACGAAGGC CAAGCTGGTG CGACAACATT TGACAGGCTC ACTTGCTTTC CGGGACCAGA

35041 ATGTGTGGGC CCAGGCTGGG AAGGATATGA AAAGCTATTT ACCAAGGCTT TAAGCAGAGT

35101 AACTTCATTT TCCGGTGAAG TCTCTGCAGA GTATATCGTT ATTATTCGCG GTAATGATTG

35161 TAACCACCCT GATGCATTCG GTATTCCGAT GGCAGACACA TCGGAATGTA CCATTGAGCA

35221 AATGAACTCG TACATTGATA CATTTGTCAG TGTCGCTAAT CGTGCATTAG ATGCTGGGAT

35281 CACTCCCATA TTTTCAAAAG CACCGGCCTA TGACGCCATA GATTGGGAAA CATTACGAAG

35341 CCGATTCAAC TGGCCTTGGA TAATCAGTAA AGAAAATTAC GAGACATTTT CTGAACTCAG

35401 GCTAAACCGT CTCCGAGCTG AAGTGCCCAA TGCCATTTTT TTGGATATCT GGAAAGGGTT

35461 TGAGCCCATG GATGACGGAT TGCACCCGAA TAGGAAAACC ATGCAACGCG CGGCTAAGCG

35521 TATTGCAAAA GCCATCAAAA AACACCGTAA GCACAGCGCA GAATAAATAT TAAACTGTCT

35581 ATAATATAGG GCCGCCTTCC CCTTCCCTGC GCGTAAAGCG GCCCTCTTCG CCATGATTAT 35641 TGCTATCTAA TATTGCTTGC GTGCCAATCG GCGTAAATAT TTTTGTTGCA TGGCATCCAC

35701 TTTAATCGTG TTTTTTACCC AAGTCTCGGC AACACTATTT AACTGATTGA ATGTATTTAT

35761 ACTTTTCTGA TAGCGGTAAT TCTGTAAAAC CGAATGTCCA ATACCACAAG CTTGCGCTCG

35821 AGCGACCAGA CTTCTGACCT TATTCTCCCC TTCACCGACA TTGACTAACA CATCAATCAA

35881 GCCGAGCTCA TACAGCTCTT TGGCACTAAA TCGCTTATCT GAATACATGA CTTCCATCAA

35941 GGCACTACGC GATGTTTTGT GCTGCAACTT CGCAATTAAA AGGTGCTCTA GGCCATGATT

36001 AAACAGCGTA GAAGGATACA TAAAGACTGC ACCTTCCTCC GCTACAACAC AATCCGTACA

36061 TAAGGCAACT TCCATACCTG TACCGTAAGC ACGACCTTGT ATCAATGCGA TACTGGTCAC

36121 ATTGTGTTTC GCGCCGTGTA AAATTCGCTG CAAAAGCGTC ATATAAGTAA ACGCATAGTC

36181 TTTGAGCCAA ACGCCATCTT GCTCAGACAC ACAATTTGCT AAGCTACCTA AGTCTCCCCC

36241 CAAACTGAAT ACACCTGCTT TGTCACTGCT GAGGACCTTA ATTACACGCT TTTGTGTATT

36301 AGATTCTGGT AAAAAGAAAT GGCTCAGCTG TCTTAATAAC GATTTTGTGA AATAAGGTGC

36361 CTTATTTAGC TGCATTGTCG CCCAATGCAA GGAGCCAGTG CGCATGATTG CCAAAGGCTC

36421 ACAGTTATAA ACGGAAGTTG CTCTCACCTG CACGAATTTT TTACCCTTTC AAATTAGATC

36481 GATAACCCGA CGTACTAGAC TGAGCTTTGT GAACAATTCG TTAAAGTAAT GGCTAGATAA

36541 AGTCGACACC CTGCCACTGA GCTTCTACTT TTAAAGAAAA CACACGGCCA CATGCAATGA

36601 AAATTCAACT TTTAAGTTTT ATACTTTTTT CAAGCGCCAG CTTAGCTGAT GCGCAAGTCG

36661 CTAAGGTCAT GCTTGCTAAA GAGCAAGTCT TGGCCACATC GGGTTCGGTT GAGCGCAGTT

36721 TGAGCCGTAA GTCCCCTATC TATCGCGCAG ACATACTAAA AACAGGAAAA AATGCCCGAG

36781 CACAATTTAG GTTTTCTGAT GGTACGATTT TGTCACTGGG TGAACATACT CAATTTATTG

36841 TCGATCAATT TGAACATGAA ACAGTCTCTG AAGCGCACTT TGAATTTATA CAAGGCGCAT

36901 TCAGAGTCGT GACAGGTCAA ATCACTCAGG TTACCAATCC CGATTTTAAA ATTAAAACAC

36961 CGATGGGCTC TATTGGGATC AGAGGCACTG ACTTTTGGGG AGGCAATTTA TATAGCGAAG

37021 ACACAATCGA TGTTATTTTG CTAGACAGTG AACACCCGCT TGTTGTTGAA AATGAATACG

37081 GCCGCGTCAC CATATCACCC CCTGGGTTAG GTACAACGCT GACTTTTGGA AAGCCACCAA

37141 GCAAACCAGA GAAATGGTCA GATAAAAAGT TACAAGATGC GGTAAAAACC ATTCAATAAG

37201 TTTCTCTCTT GGATGAGCTT TAAGTCACTT CATAAAAATA TAGAAAATAT TTATTCCTTT

37261 GTCTGGCCAG ACTAATTATT GCACTTTAGA AAAGCACAAT AATTTACATT AAATTAAAAT

37321 GGTTGATTAT TTTTAAAAAA TGAAGTCATC AGTAAAGCTA ATTGAAGCTT ATGAATTACA

37381 TATTCAATCA AATTAACTAA TTAATTAAAC CACTTGAAAA AACCATCAAA ACATTGATTT

37441 ATATACATAT AAATTCTTAA CCCCTACAAT TTAACTTTTA CATATCACCA ATTTGCTGAT

37501 GTTCATTTTG TTTGATACTT TGCATCTCGT TAACACTTTG GGCCCAAGTG ATAACTCACT

37561 AAAACTAACA AGTAATAAAC AACTGAATTA AAGAACGACC AAAGGAAAAA AATGAAAATG

37621 TTTAAATTAG GTACACTTGC AGCTGCTTTA TTAACCACAG TAAGTGCCGT TGCTGCGCCT

37681 ATCAACGTTG CTGATACAAA TGGCTATGAC CGCCACACTG TTTACTCACA CGGCCCAGTA

37741 AGCCGCGTAG TGATCACCAG TGAGTTTGCG CACAACTTCT CAGCTGACAT TGAACTTCGC

37801 GACGGCGCTC GTTGTGATAA TGGTAACCTA TATTCAAACG TTGACAGCAG CCATGTTGTA

37861 AATGTTGGTG TTCAAGCAAA TGCGTCTAAC GCAAATGCAG CAACAATCGG TGTGACTAAC

37921 GCAATTGCAG AAGACAAATT AACACACAAC AAAACACTTC ACTTAAGCTG CTATGATGCA

37981 GACAATACTT GGTATAACGT TCTGGTAAAC GTACCTGGTG CGCCTATTGT AAATTGGGAC

38041 ATTACAGTTG AGCCTGCAGG TGAGTTCGTA AACCAGCCAT ATTCATTTGG TTACCATTCA

38101 GCATTCCGTG TTAAGAGCAC GCTTAATGTG AACAACCAGA ATAAGAGCCA GTCATACTGC

38161 TACACAGTTG CTAACCGTGG TCTATCTCTA GGCCTATTCC ACGGTAGTGA TACTTCGAAT

38221 ACTTTCCATT CAGATGTATT CACGCAAGAC AAAGTGTACA GCAATGACAC TGCACAACCT

38281 GTTCTTTACC AAATTGTTCA GTGTGAAAAT GCTGCTGGTA AAACGATGGC TGTTAAAGTA

38341 TTCAACTTAA CAGATCCAAA CGGTATCTAC ACTTATGAAG ACCAACTTAT CGTTAAGTAA

38401 GTAGTGACGA GAAACGATTT GAAAATGGCA GCTTAAATTA GCTGCCATTT GTTTTGATAT

38461 CTTAAAGTTG AGAATGGTTA AACGTGGATA CGGAAATAAA TACACGGTGG CCAAGCGCTT

38521 TTTTATCAGG GCGCTGATAA ACATAACGTC GATATTCAGA AAGGCTTGCT GGTGCCCCAT

38581 CAAAGCCAGA CAACATTACA CGAGAATGGT CGGGTCTATT CTGCGCAGGC AAAAAGGTCT

38641 CATCACCAAA ACTAAAACTC GGTAGTTCAG AAACCAAAGC ACCAGAGACG TGCCATTTTA

38701 TTGGGTACTT GCTCGTATCA AAACTAAATT CCCATGCTGC GCTCTGAATA CGTGTATCTA

38761 ACCCAGTAAA GTCATAACTT ACTGCATCCC CCTGCTCTAG AGAAGAAATA GCCACAGTTA

38821 ATTGCTGTGC CAGCTCAGTA GATGCAATTG GAAACGTCAA TTCGCCAATT TGGTCTACTT

38881 TATCAAAGCG CAGTGTCACG CCATTTGATA TAGCGACAGG GAGCCCCTCA TAATGGAACA

38941 ATATACTCTG GTAGTTACTG AGTTCATGTT CAGCAAACCC TGTTGGGTAC ATTTTCAGAT 39001 TGACACCACC TTCAACTGAA AAATCAAAAT CTTGAAAAAA GTGATTGCCT ATCTTATCAA 39061 CAGCACTCAA ATAGACTTTC GATTTTTGCT CAAATTGTGA CGTAGATAAA TCTGCACTGT 39121 GGTGCACAAA GTCACTGCCA TTTACTGTGA TTGTATTTAC ACCTTCGGGC AAATCAATTA 39181 CCGCTAGTAG GCTAGAACCA TAGCCAGGTT GGTCGATAGT ATAATATACC TTTTCGTCAT 39241 TTCCACAGAC CTTAACAACA TCTTGATAGT CTTGTATATC ACCACCGTTA GATCTTGAAA 39301 CACGAAACCT GCTATGTGTT ACTTTAAGTG CACTCAGATC AACGTCTATT TCTTTACATT 39361 TACAATAAGC AGACAGGTCC TGAAATTCGA TTGTCGTAAA ATCAGCGCCT GAAGAAATAT 39421 CTAACTTGGT ATATATTTTT CGAAAATTTT CAAAGCCACC GTTATTCTCC ATGCCAATAA 39481 TAGAGGCATG TTTAGCGCCT GCGGGAATTT GCTGACTAAA ATACCCGCTA CTATCACTCG 39541 AATGTGACGC AACAACTTGA CCGTTGTCGT CGTGAAATAC AATACTCGCA TTAGGGTAAG 39601 CAACCAAACC GCACTCTGTG CGCTTTTTAA CATTTAATGT CAGAGTTGGC TGTGCATCTG 39661 GCTTTGTTAC AACAGGATTA TTGTCTTGTG GATCACTTGA AGACCCACCG CATCCTGCCA 39721 GCGCAAATAT CGAAGCCAAA ATAGGGGCAA CTGTCAATTG GTTTACTTTC ATGAAGTTCA 39781 TAACTAAAAA AATTTTTCGA ATTGTACACG AAATGCAACT CGTTTGTATT TTATTTAGAG 39841 ACAAAAATAG AATGCAAGCT CATGTTTGTC AGGATCTTAA GTCGTTTACA TACCTACTGC 39901 CACATTAATG AGAACATAAT AAGATGTAGT AAGTACAAAT TTAACCGAGC TGCTGCCGTG 39961 ATTACATATT AAATCACTCG ATTTAAGCAA GCGGAATTAA TTGAACCAAA CAAGGTATAA 40021 TTGGGTTGGG AAAATACGCT CCACAACTTC TCCATTTTCG CCTTTGCTTA AATTTAAGAA 40081 AAGATAAAGT AAAAATTAAC TTTCTTGTAT ATAATTAATT AGTTAAAAAT TAAACCCACA 40141 TTTAATTAAA GAACAACCAG ACAGCCTAAA ACCTCTTAAC CAAATTCAGT TAAAATAAGA 40201 ATTAAATATG AATAAACTTG CAAGAATAAC TATTCTAACT TTCTGCCTAA TCGCACTACA 40261 AGGCTGCTAT TTTTTTAGTG AAAATGTAAT TTTAGAAAAT TGCTTTTCTC CATTTTCAAT 40321 TTCTAAGCAA GCACTTTCAA AAGACGTCGA GACCCGTATC TTACCTCCGA GTAGCAACTT 40381 AATTGATGAT GGCTATCGCT TTACTCTAAT TGATAAGTCT TACGATTATG GCAAATATAG

40441 ACAATCTACA GGGAATATTA ACTTTACAGG GAGCTTAATA TTAAGTGTAC CAAATTGGGC 40501 TCACAAATAT ATTGGTGGAA ACCCAAAAGG TTTATACTTT GAAATAAAAA ACGGTGGGCA 40561 AAAAGGTATA ACTAATTTTT TAACAATGGA CTACTATATG CCCAACCACC TAGTTAGTAT 40621 TAAACCGCAC TGTAAGGATG AGCTGATAAA AACGTGTCCA AACCAAAAAT GCAGCTATAA 40681 AAATATATTA AATTAAAAGT AAACCTCTTT TTTGGGGTTA TAAAAAAAGC AGCCATCAGG 40741 CTGCTTTTAT TGAATCAAAG AGCGAAGGGC CATCTCTGAC CGCGTGTGTG TATCGCCAAT 40801 TAGGCTTTGG CATGGATCAA TGTTCCTTTT ACATGTTGTA TCGATACGAG CAGTTATGAA 40861 CTCACAGCCA CTCAAATCTA ACTAGCAACA TAAAGAACTC ACTAGCCAAT ATCTATTACG 40921 CCTTATTACA CTCATTGTCA TTTTTTCAAT TATCAGAACA AGTTAGTTTC TGACTAACAG 40981 ATCATTACAT TAAAATGAGC CTTTCTTTTT CAATACTCTC TAAAAACTCC CCGGCAGCTT 41041 CTTTATTTTC TGCGTTTTTC GAGCGGTTTT TGCGATAGTC CGAGGCTATC TCTTCTAGCT 41101 TTGCAAGTAA CAGTCTCCGC TCTAAAATAG AGTGCGTTTT GTGATATTTA TCCAGCTTAT 41161 CCGCCACTTT TGCTTTACTC TTACTCATTT CAAAAATCCA GAAACTCTTG CGCTGTACCG 41221 CTTCATTCCA AACTTTAGAG GTCCAAATAT AAGTGCCACT GGTCGGTAAC TTATCCCTAT 41281 CAGCTTTGGT TTCTCGATAA ATTTCAGTGA ACTTATTTTG CTGCGATATC AACTTGAGCA 41341 AATGAGTATA TCGCTCACTT ACATCCTCTT TTTGCATTGG ACTCAGGTGA CTATATACCG 41401 TTTTACGTGC AACCTCAGGT AACTTTCTTT TGGCTTCATC TTCGGTGTAT TCAATATGAG 41461 TATGATCTTT TTCTAACTGA TTAACCCACT TATCTACTGG GAGTGCCTCC TTAGTTACCT 41521 CGGCAGGTTT TGGTTTGCCT TCGGTAAACT CCCCTTTAGA GCGCTCAACG ATATACCCTT 41581 TCCCATTAGG ATCTTTTTTG ATTTTACCAT TAAGCGTATA CTCACCCAGT GTCCAATCAC 41641 CAATAGATAC CGACGCAATT TCTTTTTCAA ATGTCAGTAC TTTGGCTTTT ATCGATGCAC

41701 CTACTTCCGC TTCTAATGAG CCGGTTAGGC TAAAGCTCGC CTCCATTGGA TAATCACTAT 41761 CAATGGCAAA TTGACCAAAG CTATCACCGC CTTTGCCACG CGTGTATTTC AAACGCCCTT 41821 CTGTTTCAAC CTTACCTTCA ACACCACCTC GGATCTCACC AAATACACCA CCTTGAACCT 41881 TTGCAAGATA AGGGACCCCC ACAAAAGCAC CAGCCTCAAT TCTCGCTTTA GCTTTGGCAC 41941 TGGCCGAAGC TGCACCATTC ACAGAGAAAA GCATTTGTGT CTCTGTGTTA TGCTGCTCTT 42001 CATTTTTAAG CTTAGCGCCG ATGCTCGCAC CAAACTCAAA CCCCCCGGTA ATATCAACTC 42061 CAACTCCTGC AACACCAGCC GCAACGGGAA AGTCCAAAGA GAAAGCCGGA AAATCAATCC 42121 CTTTGTGCCA TTCTCCAGTG ATCCCACCTT CACGATCTTT TCTATTATAG TAAGCCTCAA 42181 GGCCTAAAAT ACGTGCTCTA AGTAACTGTA AACCATTGTT GCTATCCTTC TCGTAGGAGA 42241 ATTTACCACC AGCAAATTTA AGCGCGTTTA ACCCTTGGTG GACTTTCAAC TGCTTAAGCT 42301 CAAACACTTT TCCAAAATCG AAGTTTCCAG AGAAAAAGTC TGACTCGACA ATGGCCGTTT 42361 CATTAAGCAC TTTGGCATTA TCATGATCGA GATCCATAAA AGCATTTTCA GCTTCAATTG

42421 CATCTTTTGT AAACTTAGCC TTCGTCATGG CCAGCGTCAT TGTAGTGTGT TTGCTAATTG

42481 GGATCTCCAC ATCAGCCTGC TCAACCTCAC CGCCTAATTG CTCATCGTGA TACATCACTT

42541 TTGCTTGCGT AATTGATGCT TTCACACCAG CGGGTAAGCC GTAAAGCGCA GCGTCAGTAC

42601 TCACCTCAAA CTCTGGTGAA GCATCCTCTT TAACTTCTAC TTCACCACTC GGGTTGCTGA

42661 TAGACAAAAA CTTGGGGATT ACTTCGAATG TATCTTTAGA AGAGATTTTG GTCCTGCCTG

42721 ATTTGAATTT ACCTGCAGTA TCCAATTTTA AATCAAACTC ACCATCCAGT TTCTTTTTAT

42781 CCCAAAGCGG CACATGTGCA TCACCTGCAA AATGAACTTC TTTTTGCTGC TTATTGTAAT

42841 CAAGTGACAT TAAATCGGAG TAAACCCGCT TGTTCGCAAT GTTTAGCCCA ACAATGCTTG

42901 CTTTAAAACG AGCAAGGCCT TCTTCATTAA TGGTCAGCTC ATCGCCCAAT ACTTGATGCG

42961 CTTCAATATA ACCACTAAAA CCCACCCCTT GTTGGCTTGC AAAAACCGGA GATACAGCCA

43021 CTTCCCAGCC ACTGCCAAAT GGTAATTTTA CATCTATATC ACCACCAAAT TGCTGACCAA

43081 GCAGATTGAC CTTGGCAACA CCCGTACCAT GTAGTCCAGC GCGCTTGGGC TTACTTGCCC

43141 CCTCTTCCCA GTCCAATAAT TCAGGTTGGT TTATCCCCAA CGAAATGAAC TTATTATCAA

43201 CTATATTAGG TGATGTTTGT GCATCTTGAT CCGTTTGTTG CTCATCGCTT TGCTCATCTT

43261 CACTACTTGA ATCCGAGACA CCAATTAAAC GTGCAACGCT ACTGGCAAGC TTATCTTGAA

43321 TATACTTAAC CCAAGCTTTT AAATCATCCA CGAGTGCCCG TAATGCCCCA TGTGCATCAC

43381 TGCCTTTTTT TAACCATTGA TAAATATATG GCTCATCAAT ATCTCCCCAC GCAGCGGCTA

43441 TCCAACCACC GTTATAAAAT TTATTTGCAG CCCAAAGCCA GATTTTTCCA ATGGCCCATA

43501 AAACGGCGGA AACAACACCA GGCGGAACTT TTGCCAAAAG TAAAAGTGCT TGTGAGATTG

43561 CCGTCACCCA TGGGTTCAAG GACTTTAATG ATGTCACCGC ATAATTAAAT ACACTTGAGA

43621 TAATCAGCTT AGCTGTTGCA TCGCCATTAA AATGATCCCA AACCACTTGT GAGGCATACC

43681 GCTTTGCAAC ATCAACGCCC GATGAGAGTA GATCGACACT CACAGAATCA GAGTCTAATG

43741 CATTGCTCAC TGATAACGCA GTGCGCGTGG GATAACTTTG AAAGGTAGGA GCTTGCTCCT

43801 GTTCTTGTTG TTGCATTTCA ATAATTTCCT ATTGTTGCTT ATGTAGAGAA TAGAAACTTA

43861 GAAGAGGTGT TGAGTAGAAG CGCAAATGCA TGTGTGAAGC GTGTATTTCT AATATCAGTG

43921 AGAGTTAACT TAGCCTTTTA GTGCCGCTTT AAGCGCATCA ATTCGTGTCT GGTCCCATGT

43981 CCAAAACTTG CTATTTTTTT GCTTATCTGT CCAATCTTTA TAGTCTTTAT CTCGAATGTG

44041 TTTATCATCT TTATGTGCAA GACCCAGCGG CTTAGTAAGC CCTTTTCTCG TTGTCCAAGG

44101 TCGCATATAT GCCTTGCTGT CAACCGTGTT GTCTTTGCTT TTAACCGCTT TGTTTGTAAA

44161 TGCCTTTAAA GTCGACACAT CTCCTGTAGA GGCATGTTTG GTTTTGTATT CAGACGTGAT

44221 GGTCACTGAA AACGACCCTT CAGGGTGGTA ACTACTCACA GCATCCGTAC TGATATCACT

44281 TAAGACTTTT ACCTCGCATA CTAAATTAGC CTTAGGCGCA GCTCCGCTCT TGTACCCAGG

44341 GGCGGTTTCT GTTTGATGAG GTGCGGGGAC TGTTGGTACT ACTTCTACTT TATATTCAAC

44401 AACATAACCG GCTTCAACGA GGTTTTTTAC ATGAGATTCA ACTCGAGCGT GATGCTCTTT

44461 ATTCGCAGAT CCTGTAATAG GATACAAATT AAATTTCAAT GCTGAGCCGC CTAAATTGTC

44521 GTTGAGAAGG TGGCCACGGA TCCAGCCTTT TTCATTGGCA TTATTCCCGT TGTTATTGTA

44581 AACAAGCTTA TTCTTCATCA AATCGCTTTG ATCGGCATTG ACTTCAGCAG ACTCGCCTCT

44641 GGGGGCTAGA CGAGATAATC GAGCCTCAAC TTTATCACCA ACGATATTAC TAATCGTTAC

44701 ATCAGCACCG GATGCATTTT TATACTTAAA ATCTTTGGCT GCAAAAAACT TAATTTTTGT

44761 ATCATTTATC GAATCAGAAC CAGCAGTCTC ACGTACCCTT TCTTCGCTGC CACCAGCAGG

44821 TATGGCAATA TTCCATGCTG GGTTAATTGA GAATGAAGTC CCTATTAAAT ACGGGTTGTA

44881 TTGATAAGCA AAGTTTTGAA TTAAGTTAAC TCCAGGCATG TCTTTTTGAG CCACTTGATT

44941 GATGGCTTTG AACGCAAAGT CAACATCCTT GCTCTCTGGC AGAAGCAGTT TGTAAATGTG

45001 GCTAAGCCTC GCGCCAACCA CTTGATAGTA CCGTTGTGCG ACATCATAAA CTTGCTCTTC

45061 ATCCATCAAC ACATCAAAAT AGCTGCTACC TATGACTTCA ACTGCTTGTT CAACTCTCTC

45121 AGTCGACGTC AAATCAGTCG AGTACTGGTC TAATATAGGA ATAAAGTTGT CATACTCATC

45181 TTCGGAAATC GACTCACTGT AATCTAAATC ATCCAGCACA ATTGTCTCAA TATCTTCTTC

45241 TTGCATACTC ATATCTGAGT TTTGCCGAGA TAGGTTATGC TTTGGCTGCC GAATTGGGCT

45301 GCGTTCTCGA AAACGAGAAG GCGCTTCTTC CGTTCGATTT CTTTTTTGAC GACGAGCTTT

45361 GTTTCTTTGC TTCTGCTCTT CGCTATCAAG CAAGTATGAG GCGTCCATAT CTGACATCAT

45421 AGTGCGCGGT CCTTTAGTAG TTTACGAATA TTTGAGCTCT GCTGAATAAG GCTTTTCTGA

45481 ATAAATAAAA CGGAGGGTAT TGTGCTCATT GACTTTAAGC ATGGGTGCTT ACCAATGAAA

45541 ATGTATTGTC TAGATCGACT CGAAGCAGCT TAAAATGAGT GCTTGCTTCA CTTTCTAATT

45601 CACAAGTGCT AATTGATAAA ATACTTCTTG CTCAGCCCCT TCGCCTATGG CCTCTTCCAA

45661 CGCGTTAAAT CCAACTTGCG TTAAAATGTG CCGAGAGGCC ATGTTATGTA CCCAAGCAGA 45721 GGTCTTAATA TGGGAAATAT TCAGTTTGAG CGAGCGCTGT TTTAATGTGG CTACTGCCAA

45781 GCTAACCCCT ATTTTTCCAA AACCACGATG CTGGTAATCG CACCCCACCC AAAATGATAA

45841 GTGTGCATGA CGTTGCGAAT TTTGATTGTC AATCGCATCT GGCAGCGGAT AAAAATCCAC

45901 AACAATTGCC CCCACAAAAC CAAAAGACTC ATGAACGAGT GCAAAATGCG CCTTTTCACC

45961 ACGCAGCCCC TCTTCAAGCC AAGTCGGCCA AACTGCTTGT AATTGATTAA ACTGTTCAAT

46021 TTTCAAACCA CGCAGGCGTT CGGCAATATC ATCTTGTCGA TACTGAATAT AAAACTCACC

46081 TAACTGATGC GCCCCAAGTG GTAAAATTCT GAGCCCTGAC ACGTCGCATC GAGTGCACCG

46141 AAGTCCTTTA TCAAACCAAG ATAGTTGGTT AGTTTGCTGC TGATAAGTAG CAATATCGAT

46201 ATTAAGCTGC TTTACTTGCG CATCTTCAGG GCTGAAATCA AGTGCCAATT CTGCGCTTTG

46261 ACTCGCCACT TCTTGCTCTC CAGTAGCCCA AGCCACCAGT GCCAAATTAT GTAAATATGC

46321 TGTACTAGGC CCATTAACTT CCATACAAGC CAGCATACAG TTTTTCGCTA ATCCCCAATG

46381 GCTAAGATCA ATGGCCAACA ATCCTAGTGC AAAAGCGAAA GACTCTTGTC TACAGTCAAC

46441 AATGTGATTT TGCCAAACTT GAGAGAGTAC ACCGCACCAC TGTATACGAT TTTCAACAGA

46501 CACCCCCTGC TTTAGCAATT GTGGGAGGAA AATCTGCAAC ATATTCGGGT CATATTGACA

46561 GAGTCTAACA TACGCTAACA TTTGCGCCTC TGTTAAGCTC TGAGTGCAGC TTTTTAAGCT

46621 TTCATAGATA TTTACCTGGC AACTGGGTAA ATGCTCAGAG CAGGCTTTCT CAAATATATG

46681 CTGCGTGTAA ATAAATTCCT CTGGGTTTTG ATGGCCTAAC CTCATACACG TCGCCATCTC

46741 TGTGCCTTCG TGTTTTTTTA TTTGGGTATG GGTTACAGCC GAACTCACAT AACTTAATGC

46801 CTTCAAATCT AAAGGTAACT CCACCCCAGT TTGTGTAATC ACTGTGGGTA GATTAAGCCC

46861 GAGCTCTGGC GTCAAATGAT CGCTAATTAA ATGACATACC CCTTTATCAA AAGTCTGCTC

46921 TAAACGCATA CATACTTGTA CCGCATTGAT TGGAAGCGCG ATAGGTTTAC AGCCCCCGCT

46981 TGCCAACTCT TTTCTCAATA TGTCGTCAAA TGGCAAAGCT AAGTCGCCGT GCCCGACTTC

47041 AAGATCAATA CTCGTCTTGC ACCACTGATA TGCAAGTCGA ATTTCATCCT CTGCTTTATC

47101 CGAGGGTGAG TGCGCTTTCA TACGCCAAAG CAGCGGCAGC TCTTGGTTAT TAGCCGCTTC

47161 AGTGCTTGCA GCACTGTTGC TGGGACCGAT ATTTGCTGTA GTTGCTATCT CAGCTCGGTA

47221 AAGGTCTTGG TAGTGTGTGT GATATATCGC CTGTGGCAAC TTAGAAAGCA CACCATGAGC

47281 TATCATAAAT GCTGGATTTT CGCCGAATGC AAGCATCGTT TCGTCAATAT CTTGATGCGG

47341 TAACGGTTGT TCTGAAAAGA TACTCCAATC TAGTATGTGT GCTTTTCCAA TCTGATGATA

47401 ATGGCTGAAA TCGGGGTGGC ACAAGAGCTG TGCCTGTGCC GTTTTATCTG TTTCCATCAA

47461 CAACAAACAC ACATTGAATT CTTCCAAGCC TTGCTGTGGT CCCTCTTCCA AAAGTTTATT

47521 CATTAACGCC AAACCAAAAG CGTAACGCGC CTCTCCAAGG CACAATAAAT ATATTGGCTC

47581 AGACTGGTTT AACAGATTAT TGCCTTTAAT GTCGTGTAGC CAAGCTACAA GCAGATCAAA

47641 ATAAAGGGCG ATCCAAGCTT GATTATGCCA GCAATCGTTT TGTGGTGACG GGTCTTGTGC

47701 TGACCAGATA TCAGCCTCTG GCGCTTTGTT AAATACACAC TTTTGTTTTT GTTGTTCAGT

47761 CAAAAAAGTC GCTTTGGGTA CTGGCTTTAA TTGCGCATAA CCGACGCCAT TAAACACCTC

47821 ATTAAGCTTT TTTTCGCGTG CTTCTTCTGT CATGTATATG CCCTGTTTTT TGGTTTGTTA

47881 ACGTCACGGC AGTTTAATTA TATAGTGAAG TTGGATAGCT CCACCTGGCG GGGCATTTAG

47941 CAGTGGTCTT CCATCTTTAC CATCGCATAA CCCGTAACCT AGTGGTAGTG AATGACCGTG

48001 GTATAGAAAA ACTGCCCCAC TTGGCACCCC ATTTGTCTGG GTTGCTTTTG TTTTTAGCGA

48061 TGTCGTCTGT GTGTGTTCAG CAAGCCAAAG TGTGCCCTCC ACAATTAAAC TGCCACTGAT

48121 CTTCAAGTTT TGAGTGATTT CTAGGCCCGT GCTGCGCTTT TCAATACCAT CTTCACCTTG

48181 CAATAAACAT GCATCTAGCC ATTGGGCAAA CTGTGACTCT GTCGGCTTTG CGCCGCGCTT

48241 AAAAAAAGAT TTAAGCGTTT TTCGAAGCTG CATTGCCATT TAAACTCCTT GCATGAGGTT

48301 AAGAAAGTTT AAGATCGCTT GCAGTGCTGA TCTCATTTTT AGGAAGGTGA AGTGCTTGCC

48361 AAACCTGGCC GGTATGAGAT ATTTCAGTAA CTTTAAAGTA ACTTTCAGCG CTCTCATTAT

48421 GTGCTGTATT TACTGATACT ATGTTACCTA GCTCCAGCTC AATAGCTGCA CGTTGCCATA

48481 GACCAGATTC ACTTATCACA TATATGCCAT TCTCTGTTTT ATCTCTTTGT GCTGTGAGTA

48541 ATACATACAT TTGCCAATAA CTTGGTGGTA GAGGTAAATG TATATTCTGA TCAGTATTGA

48601 AAACCACATC TAAGACGCCA ATGAACCCAA CAAATCGTTC TATATGTCGT TGGTGCCTCA

48661 TAAATCTATC TAGTAATTGA TTAACATTGA CGTACCCTTC AGCCATATTC ATAATCCCTC

48721 GTAAATAACT GGCCAAGCAC ATCGCTTGAC CAGTTCATAA TTAAACTGCG ATCGCGCCGT

48781 CACTTAAGGT GACTTCACTC CACTGATCGC CTTCCATCAA TTTATAAAAC ACAGTAGATG

48841 AAGTCGGCGG TGTACCAAGT AACTCAACTA AGTTACCTAG CTCAATGTTT GGATACGTCA

48901 CTTTGTTAAG TGTGTCAGCA ATTCGTTGAT ACACACCATT TTGAGAAGGA GTAGATTGCT

48961 TGGTAAGCAA AATCTTGTCG CCATCGGCAA GATTCAACGT GTTGGTTAAA TAGTTGTCAT

49021 TGTTTGCTGT CGACAAATCG ACATTCGCAT CTTTTTTAGC GCTATTCACA CGGTCAATAA 49081 TACCGGTATA ACGATAAAGT GATTTCGTTG CTGTCATATT CACTGCAATT TGTTCAGCAT 49141 CAATCGCTGC GAGAATACCA GAGAGCGTTT TTTGAGCCAT AAGGCCTCCT TGATAAATGT 49201 TGCGTGTACA TACACACGCA ACGGGTTAGA AAATCGAATT AGCTATCGGG AACAGTCCTA 49261 ACTGGACGGC GTAATGGTGA AGTACCTGTG ATGAGTTAAA AATGAAACTT AAGGGATATA 49321 TTCAAAAAGA CGATAATCTG CCACTTGATA GTGCACAAAC GCACTGGTGC CATTGTCATT 49381 ACCTATGCTT GTGATAATGC CAACGACTCC ACCTTCGAAT ACCACAGTAC CTCCAACAAA 49441 ATCGGTAATC GCTAAATCAA TTTGATTATC AGTAAACCAG TTACGACTAC CAATGTTATT 49501 AAAACCCGGG TTGCTATTGT GGTGGGTATG GGCAGAGATA GATGAAACAC TCGGCTTATC 49561 TTTAAGGTAA TTTTTTTCCT CTTTGCCCTT TGTATATAGT GCTAACATGC GTCGACCGGC 49621 AGGTGCACCA TGTTGTGATA ACGTTTGCTC GGCAACTATA TATTGCCTTT CATCTAATAT 49681 TTTGGTTGTA TCCCACAAAC TGTTTTCTAC TAAGTTACCT AAATAAGTAA CTACACTTTG 49741 AGATATTAAG AAGCGTTCTT TACTTTGCGG GTTATATACT TTTCGAACGT AGCCAAGGTG 49801 ACTTTGAATA CTGCCCGACA CGCTTTGCCA AGCACTGCCT ATATATTCCC AAACATCGTT 49861 ATTTTGTGCG TCATATAGTT TTGATACATC TAAGCCGCCC CGTAAATCGA TGTCAAGATA 49921 AGTTGAATTA GGCTGAGAGT AAGTGATGTA GCCATAGGAT TGCTTTCCCT TGAAGCGGTG 49981 CAAATCAGTA TCATTATTTA AATCAACCAC AATTTCACTA GCCGACTGAA AGCTGCCAGT 50041 TTGATTCCAA GGGGTCACAT AGAATTTAAA AATATCGCCT TGACGCTGAA CCTTTACACG 50101 AACCCGTTTA TTGCTCCAAC TACCACCGGT TCCAATATTA TACTGCCCTA AAAGCTTATC 50161 AGACCCAGAG CCCACCCCAG CATAACCAAA ATATACACCA CACCCTGTCG CGTTGGGAGG 50221 AGTACCACCT GTATTCAACA CAATAGTGAG TACATAATTT ATGTTTCCTT CTCTGACAAA 50281 TGCGGCAACC ACACCAATGG TATCATTGTC AGTACTATCA GAGGATAACG TCGCTTCTAG 50341 TGTATAGTTA TCGACTTTTT CTGCGGATAC AAACCCGTTT GAAGGTGAAA CATTAAGTGG 50401 CATCACGACA CTATCTGTAG AAGCTTGGTA AAACCAGGCT TTTGCGTTAG CACTATTGCG 50461 AGCTTCAGCG TCTTGTTTAT TTAAGTAGTA CTCATTGCCG TTAAATCTCG CCCAATTGTT 50521 GAATATATCT TGTACCGTCG GAGGTCTGTA ATCTTGTCGA GCTTGATCCG CTTGTTGTTG 50581 AGTACGGTAT ATATATGATT CAAGCTCAGG CAATGGCGTG ATATTGATGT AGACGACACC 50641 CGTTTGCTCA ACACCATGGC CGTTACGCAC TTTATAGTTA AATTGCGCAG GTTGTTCTGC 50701 AAGACCCGTA GACAGAAAGG TAATTGTATC GCCACTCAGA GATACAGTTC CGCCCTGCGC 50761 ACTCGATACA CCGATTAATG ACAAGCCAGT ACCAGAACCA TCTTCATCAT TGGCAATGAG 50821 TTGCTGCTTA CTGATAAATA CAGACTCACC CTGCTGCAAA CTGAACGTAT CAGGGTTACA 50881 CACGATAGGT GGAACAGCAA CGACTGACAT TGTGACGAAG TGTGTTTTTC GGATCCCAAT 50941 ACTATTTTCT ACCACATAAT TAAAACCTGC CGCGCTCCCG ATCCCACTAT TTGAAGTAAA 51001 CTCAATGTTA GCTCCCTCTA TACGCACTGT GCCAAACAGA GGGGTATGTA CGGCTATCAA 51061 TCGCACTGGA TCTTGCCCGT TGCTATATTC ATCAAAGCTC CCTTCGAGGA TATTAGCAAT 51121 CGGAATGATT GCCGTGCGCT GCGTATAAAC TTCAAAGGTT TTCACCTCTA GGCGTATCGT 51181 TTGCGCAGAT TTTCCATAAA ACTCGCTAAT TGCAATCGTG TCTCCCGGTA CTTTTGTGAC 51241 GTGCTCACTA TAAAAACCAA GCTCATAAGG CTGAGCATCA CGGTATTCAT CACCGATATC 51301 TTTAAGTGAT ATTGGTCCAC TTGTTTGTAA AGTCATTATG CTTCACTCCC CTTAGTTAGA 51361 CTCAAATTTC ACTATTTTTT GCTCAAGGCC CACCACTTTC TCATTGAGCT CTTTTATGCT 51421 TTCAACTAAC AACCCCACTA AATTCCCATA AGCCACAGAC ATATATTCAT CTTGCTCATA 51481 GACAGCTTCG GGTATGACTT GCTCAACATT TTGTGCGATC AGTCCGGTAT AACGTCTACC 51541 AGTATTAACA TCTGCTCGTT CAAATGTAAC ACCTTCCAGT TTGTGAATCG CAGCCAACGC 51601 ATTTTCAATC GGTTTGATAT TGGATTTCAG GCGAGCATCA GAGAATGCAG TAACGTCTCC 51661 TGTTGCTGTT ATCGCTCCTG TGACAGCCAT ATCGCCCGAG AATGTCGCTT GGGTAATGTC 51721 GACAGAACCT ACTAACTGAG TATTGACCGA TTTCAAAAAG CGTTGATCTG ACTCTGTTTT 51781 AGTATAACGA TTTGTTGAGT CCGCAGTTCG GTCGGTAACT TCTTGCTGCA AAGCATTCTG 51841 TAACGTAGCA ACTTGTTGGC TTCTCGTATT AGACTCATTC GCTAACTCTG TTTCTATCTG 51901 AGCGCTTCGG GCATCATTTG TTAAAACGTA ATTGGCAAAA GCTTGATCCG ACTCTGTGTC 51961 GACAGAGTTG ATGAGATCAA CAATTTCTTT AAACGAGTCA GCATCGGCAT CACTGGCTTC 52021 TAAAATACTG TCAATACGCT CCTTTTGAAC CGTAATTTTA GTGTTGTAAT CTGCTTTTAA 52081 AGCATCCGAG CCGGAAGATT GCTGCGTTTT AAATTGCGCA GCATCCAATA ATTTATCAGC 52141 CAAACCCGCC TCGATATCAG CTGGGGTCGC AACATCGACA GAAATTTGGT TGTTGCGTGC 52201 TTTTAAACCA TCGCCAACAA GTGCAACCTG CAACGAGTCT TGCCAGTGAT TTTCACTTTT 52261 ACCAGAGTCA TTGGTAAATT GAGACGTCAA TACAAATACT TTATGTTGAT GGGAAGTACC 52321 TGCTGAGATA CATATAGCAA GACCCTGATA TAATGAAATG GCCGCAGCAG GCAACGCCAA 52381 AGATTTATCT AATTGAACTT GCCAAATTTG ATTTTCTGAT GGATCTTCTT GTGCGGTTAA 52441 TAAAATCAGA TCGCCTGCCT GAAGTGCAAT ATCATCCAGA GTATTAAGTG GTTGCGAGAC

52501 ATCAATATTA ACCGACGTTT CAGCGCAAGT GATCGTCACT TCATTGAATG TATACACACC

52561 CAGCGACTCT ACTTTTTGAT TTAACCACTG ACCGTGATCT GCACTGAGTA TTTTATCCGT

52621 GCCGCCAGAT TTTAAATCAT TTGTATCGGC GAAATTTAAT CTTTGAGGCG GCACTTGCCC

52681 AACGGTCAAT TGCCCTGCAT CTAAATCGGT CAACGAGCCG CCATGTCCGG TTATACTTTT

52741 TGCAGTGATA CTTGAATCAC TTGCCACTTG TGTTAAATCG ATATGAGCTG GCAGGTGTGC

52801 ATTATTCAGA TTTGTGATGC CTTCACCATC CCCAACCAAA TAGTCTGCAG TCAACTTACT

52861 ATTTGTCGTG TCTCCTTGCT GAGTTAAATC AACTTCAACA GGAAAGCGGG CGTTTTGAAT

52921 ATGTTCTGAG TAATACTCAG GTACGTCAAA GTTTTCAACT TTGTACCAAT TCAAGTTTTG

52981 ACTGGTTTCG TTCAGCATGA CATAGTGAAA GTAGCTGGCA CTGTTATCCA ATGACCTATT

53041 TGCCTTAATT AACATGCCTT CATATTCATT AATGGTTTGC TTGGTCAACG TCGCGGATCT

53101 TTCTACCGAA GATGCATCGT AAATGTAGAC ACCATTTTGA GTCTCGTCTT GCTGAAGATA

53161 TATGATCACT CGCTCACCAG GTACTAGTGA GTATGGTGAT AAAATTTCAG TCAAATCTGA

53221 TTGCAAGTCA AGGTTGGACT CACTGATTGC GCAATCTACT GTAGTACCGA ATAAATGGGC

53281 CAGTAAGCTA AATGTCGTCG TATTGTCCGT TGCCGCATCT ATGAGTTTAC GAAATTCCTC

53341 TTGTGTAGGA ATGTCTCCAT TTTCAAAATA GCCGCGTAAC TCCGTATTCA GTTCTGAATG

53401 TTTCATAGTT AACCTTTGAT AATTAGTCGT GTATTAAGGG GACTTGCTAC TGGAAGGGAG

53461 GAAGTTCAGT AGCAAGAAAA TATTGCGGGT AAACCAAGCT TGGATTAAAC CGATTTGTTA

53521 TCCATTTTCG TTTCAATATC TTGAACAATC GGTAATATAT CTGGCGTGAA TACTGGCACA

53581 GGTACTGACA AAGACAGCAA CAGTGTAGGC TTGGGTGTGG ATCCTAATGC CTGCCAGACA

53641 TGCCCACTTA CTTTTTCGCT ATGTTCATTA CCGAACAACT GCGTTCGAAT GCCATAAGGC

53701 TTATCTTCCA CGCCATACAT CGTAAGAATG TCTTCTGGCA AAAAGTCATA AGCCCCTAAA

53761 CCACACAGTA AGTAACTCAA CACCATATGC TCTAACTCAG CACTTCCCAT TTCCGCTTTA

53821 CTCCAGACCG TCAGCATATA AGTTAATGAT ACAAATCGAG GCTCTTTATA ATGCACTCTA

53881 TGAGTCTGAG CTGTATTTAG ATAGCTTCTC GGTGGCTCAC TTTGTCTGCG TGTCGTGTCC

53941 TCTGCTACAC CAATTAGATA GCAGTTTACT GTCGGCTCAT CACTTACCTG TTTGTCATAA

54001 AAAGTCTTGT CGGGCGCGAC AAAACTCAAT GCAATATCCC CTTCTAACTC AGTATCTCCG

54061 CCTTTGCTCA TAGACAGAAT CCGCTCAGTC AAAAACGTCT TCAGTGCTTG TTGAGTGTTA

54121 TAAACTATTT TTGGGTCCAT CTTAATTTCC TTGCAACCAA GCTTTAATTT CATGCTGTGC

54181 CATAAACCCT TCATGCTGTT TCGCAAGCTC TCTTTTGAGC GCTCTGGCTA ATGATTCCGG

54241 GTCAATACAG CTATGCTGTT CTATCAAACG TTGAAGTAAC GCTGACTCTG TAATATTGAT

54301 AATTTGTGCT GCACTTAGCT CAAACCTGTG CGCCAAACTG CCAATAGATT TGGTCAACGC

54361 ATCGTTTGAA ATACCGCTCA ACATACGCTG CCATATCTCT TCCCGCTGCT GAGCTGAAGG

54421 CATAGTAAAC TCAATCACAT TATGAAAACG CCTCAAAAAC GCGTCATCTA AGTTTGATTT

54481 TAAGTTGGTG CTTAACAGTA ATAACCCACT GTAGTGCTCC ATTTTCTGTA AGAGGTAACT

54541 GACCCCCATG TTGGCATTTT TGTCCTGACT GGATTCAACA GCACTACGTT TTGCAAACAC

54601 AGCATCCGCC TCATCAAACA TCAACACTGC ATTGTGTTTT TGAGCTTGGT CGAATAGCTT

54661 CGCTAGGTGC TTTTCAGTTT CACCAATCCA TTTACTGGCA ATATTAGCTA AATTAACGAC

54721 ATATAAAGGA AGCTGTAGTT CACCTGCAAT AGCTTCAGCA GCCATTGACT TACCTGTACC

54781 AGGGCGGCCC CAAAAAATGG CCTTGCAACC TGGTGTAAAG CGCTGAAGTA AGGATTGTAA

54841 TTCGGCTTGT CTATCAATTC GGCCGACTAA TTCATAGAGT TGACTGTGTA CTGCTGGAGA

54901 CAACACCATG TCAGACAACT TGAACCTTGG TTCAGAAAGT TTCGCTAGTT CTTCAGGTCC

54961 TTTATTTAGC TCTTCCAAAC ACTGCTGCTG TAATTCACGC CAAAAATCAG CGTTATTCTG

55021 GGGGGTAACT TTAGCGGTTT GCGCTAACCC TGACATACGA TAAATTGGTA CCGGGTACCT

55081 CGTGGCGATA CACCGTGCTT TTGCTTCATC AGGCTCTAAA GACAATTTCA GCCAGGCGCT

55141 TACTAACGAC TTATGTGAAG GCGGTTGACA CTCAATCACA TGAAACAAGC TTGTATCTCG

55201 ATGCGTGTTT GGGGCCTTTA AAGTAAAAAA TACAACCGGG TTTGAGCACC CTGTCCACAC

55261 TCCAGCATTT TTCGCAGTGC ACAAGCATAT GTAGCATGAA GTTTTTCAAT AAATATAAAC

55321 ACGCATTTGG AGTTCGCATT TAATATTAAC CCAACCAACG ACAGTACGAT TTCTGACAAG

55381 GTCAGATCTT GCGCTTGCTC ATCTAAAAAA TAACCAAAAT CCGCCCCTGA TAACAAAGCA

55441 AGTTGCTCTG TGTACCACTG AGCCATCCGA GGGTCAGGTG TATCAAGTTC AAATAACTGC

55501 GAATCGCTTA AGTCAATTTT AGGATTAAAA CACGATGTAA AAGCCTCATC CTGAACCGTA

55561 GACGAGCCCA GCTTTACAAG GTGTTCATTA CTCAAAGTGA CTTGACCGGT ATGCAAAAAC

55621 TGCCTGAGCT CTGTATGTAA ACTGGCACTT TCAGTCAGCA ACTTTTTTTC AGAGCATTGT

55681 AATAAATGCC AATCGAATAC TTGGCCACAC AATACATCTT GCGAAATTAA CTCTCGTTTA

55741 GACCCCCGCT GGCAAAGAAA AAGCAACTTA TCGAGACTTA ACATTGGCCC TTGCTCATAC 55801 CAGCTAAGAC CGATGTAAGG CATCAAAATA TCCGGCTCAA GAGTTTGAAT ATAAACAAGA

55861 GCAATCAACC TAGTTTCATG TGATGTGAGA GAAAAACGCC CTTCAACATA CTTAAATCTA

55921 GGGGTGTTAA CCAGCGTATT TAATGCGTCG TCAAGTTGAC TTAATCGTTC CTGTATATTG

55981 TCATCATCAT TTGCTAAAGC GATACCAACC TGGAAAAAGC GCAATGATAA CTGCTTTTCG

56041 GCTTTAAATT GTTTTGCTGA GTCGTCACGC ATTACCATTT TATCACCCAT GTTTCATTTG

56101 AATGGCGAGC TCCATAGCTC TATCACAACA CGGATCTACC CAGCCGCAGT GCCAAACTTC

56161 ATGCGCAGCT AATGAAAAGT CCTCAACACA CAAGGCCGCT AGTAGCTTTT TTAACCCTTT

56221 TAAGCCATCG ATACCAATGC TAAACGCCAT ATTAAGCAGG ACCAGTTTTC TAGACTCACT

56281 CAATCGATTA AAAATTGGTA CCTCTACTGA CAGAGTTCGA GCTAAATAGG CAATATCGTT

56341 TTCAAGCATA TATTCTGCCT CTTGCTCGCT AATACCTACC TGCTCTATTT GCCTGCCGAT

56401 GCCAACAACA AGTGCTCCGT TGCTGTTTCT CACCGGTTTA ACAGTAAGTC CTACATGGCA

56461 AATCAATTGT ACCTTCAATG CTTCAAGGCT TTGACGAGAC ACACAGTGAG TCATGACACC

56521 TCCTTAGAAA GCAAAAAATC TGAAGGCACA GATATCATCA AGCTTTGCTC CAGTCCTTCA

56581 ATCTCGATAA CAAATACGGC GTCATTTTTG CGCTCAACAA GTTTTCCTTG GTAGCCTTTA

56641 AGGGCACCCA CAGTGATTTC TACAGCACTC CCAACGGCAA ATCGAGTGGG TACTGGTTCG

56701 CACTGGTAAC CTGACGACAT CACCGTTTTA ATTTTTACAA TTTCTGCATT ACTCACCATG

56761 CTGGGCTTAC CGTTAAATTT AATAAAGTCA ACGAAGCCAC TCAATCGCTT TACATGGTGA

56821 TATTCAAAAT CATCGACATA AACAAATACA TAAGATTTAA ATAACGGTTT CGCTATTGAT

56881 TTAATTCGGT CGCTCCACTG TTTTTTCTCG ACTACTAATG GTAAAAAAAC CTCACAGCCA

56941 CTCTTTATAA GTGAAACCTG CTCCGCGAAT TTTTTTTCCG TATTCGGTTT GGTGTAAACT

57001 ACATACCAGT TTCTTATTGT TGTCATCACA AATCCCAGAT ACGCTCCGCC ACATCACCCC

57061 CAGTATGAAG TAACTTAAAC GGCAGTTAGT TTTTAGCTAT AGCAATAAAA ACTTGTCAAT

57121 TTCCTATTGC TGGTTTAGTA CCAAGCAGAC GGAAATAGTT ATTTTTTGTG AAAATTAAAT

57181 TTTTAATAGT TTGAAAAAAG TGACCTTACA TAATAAGGCC ACTTTACAGG TTGGGCTTAT

57241 GTTCAAAAAC CTATCAAGGT ATAAACGTAA GACTGAGATA TTTGTGAATT TATTTATTGA

57301 GCTTTTCGGT TAAATATTTA AATTAAAAAA TTTATACGCC ACCAGACTAT CAAAAGTCCG

57361 TTAGCTATAT CTCTTATATT CTTCTTTTAG ACTTGTCAGC TGCCTTCTCG CGAGCTGCAC

57421 ACGTTTTCTA ACATTTTCTA GTGATAATTG AAGGCGCCTG GCGATTTCAG GGTAATCCAT

57481 TTCATATATA AATTTATATT GCATCACAAG ACGTAAATCC CTTGGCAAAA TAGAGATCTC

57541 ATTTACTAAC CGTGTATACA AGCTAGAATT CCAATGATCG CTTTCTAGTG AAGTAGATTG

57601 ATTATCTGCG AAGAAAAAAT GGTCCGGTAA TTCAGATACA TGATTAACAA TATCTTGGTG

57661 TTTTGCTTTA GCGCGATGTT CGTCCATGCA GAGATTGTGA GCGATGCGAC ATAACCATGC

57721 GAATTCATTC TCGATAGTAT AATCAGCACG ATTGTATGCT CTAAATGCCT TTTCACAGGT

57781 TTGTGCCAGT ACATCTTCTA CCTTGTCGCT GTCATTTCTT AACCACCGAT GACAACAACT

57841 TAATAATTTA TCTTTGTTTT CAAGCCAAAC TTTCCAAAAT TCACTATGTC CTTTTAGTTG

57901 CCGTTGTTTT AAGGCAACCA ACTCCCCTGT ATGTAACATC ATTTCTCCAA TAAAAAAATT

57961 TATATCGGAG GAAAGACGCG CTATTACCAT TTATGTGAAA ACAAAATTAA TACAATATAT

58021 AAACCATTGT TTAATTGATG TATAACCCTA TTCTGTTTAA CACTCAGTAT TTCAAATGAG

58081 AAGCAAAGCG CGTTGTAATG ATAAAACTTT ACAATAAAAT CGGCATTTAG ATTGAGGTTG

58141 GATAGAATAC CTAATCCAAT TGTGGGAGCG CTTACTCATA TCCAAAGTAA CACTATATGT

58201 GAAGCGCTTT TGACATTTAA GTGTTCAGCG CCGCTATCTC ACGTTCAATA GCTGGTGTGA

58261 TCAACAACTC TACCTCACCC CTATGAGCAT ATGAGATACG CAGTCGCCCT TGGTATGTAC

58321 TTGCAACTAA CGCCAGACCA GCAGGCGGCA ATGGTACACT AAAGTTATAC ACATCAATAA

58381 TCTTAAAAGA GCCAATATAG CTTAAGTTAA ATTCCAATTT ACCTAGGTTG GATAAGATCA

58441 CATTACCATT GAGAAACAAT CTTTTATAAC TCTCAACAAG TAATTTAAGC GCACATTTTG

58501 GTACTAGATT TATTGAGTAA GACTGAATCC TCTGCTCGGC TTGATGCACC TTATACCCTT

58561 CAAGTTTCAA CTCTTGCAAA CGCGCCTGGT ACGCCTTAAT CCAACAACTT TCGCCTCCGA

58621 TCTCTGAACC GAATGGCATA ATTAAATTAT TATATGAGCC AGCAAGATCC TTTTTAGCTA

58681 TTCCCCTAAG CGAAATAGTT TCAAGTACAG TGTATTTTTG ACCATTATTT AGTGCTTTAT

58741 TTAAGACATA ATTAAGCGCG ACATTCACGG AAACCTGATG CCGTTTAGAC CAAATTTTCA

58801 GTGCTTTAGT CGCGTCCAAA CCGAAGTCAT GATAATCAAC ACGCCAACAC GCTGAGTCTG

58861 CAATCTCAAT CTTTGTTTTC GATTTAAATA AATCAATGGC CAATCTAATA CCAGCATGAA

58921 ACAATGCTTT TGTACCAACC TTATCAAAAA GCATATGCTC TTCACAAAAG CTATTATCGA

58981 TACTCGAAAT AGGCTGGTTA AGTAACAGTG AATGTAACTG TTCAAATAAA ATGTAGCCGC

59041 TACGTGCATC TGAGGCCGTA TGTGAGGAAA GAAATATCAA TGCGCTGTAC TGTTTAAACC

59101 GGATACAAAC AAATTGAACT GGCGATCCAA CGTATGGATC AACTGGGTTG TCAATCGCGT 59161 ACTGTCGCAA TGCGACATGC CATTGCTTGA CGTCTAGAAA GCGATCGGTC CAATCCAGTT

59221 GTGACTTTTC AGGGTAAAGC GTCTCATCAT ACAGCCAAAA ATACTTGAGT CCTTTTCTGA

59281 CAATACGACT TTGCATTATT GGGTGTAAGC TTGCAATTTT ATCCACACAG TTAGACAACT

59341 CGTCCATGCC ATAATGGCCT TCTAAAATGA CAAGGTCGCA AGAATTAGCA TTTTTATTAT

59401 TTGCTTCGGC TAAAGCTAAA AAGTTGAGTG CTCGATCACT CAAAGGTAAT AGGTTCATTT

59461 ATTAAGTTCC TGTTTAACAC ACAAAAACTT TAAACGAGAA TCATTTACTT GTCTAATCTG

59521 TTATCCTAAT TAGCAGCACA AACATGATTA ATCGCCTACA AATTATTACC AGAACTAACT

59581 CAAGTTTTGT GACAACTTTT GTTCCAGCGC AATAAAATCG TCACGTAATT TCAATAATAT

59641 GAAGTCAAAT TAGGTGTATC GTTAAGGATT AAAAATGGCA TCATTGAATT TACATCGCGT

59701 CTATATTCCA ACTAATGCTC GCAATAACCA CTACATTCTG GCTGAGTTTA AACCTGATGA

59761 CTCGTTTTAC AGCCACTTTG ATGACTTAGA AAGTGCATAT CAAAGGCTTG CGAGAAAGTT

59821 ATTTGCACTG TGTGATGAAT ATGAACTCTA TAACGTTCAG CTTATCGTGA ACGACAAGTT

59881 GCCTGTTGTC CGATATCATG AAGAAGCATA TAGCTTACAA ACAGATAAAC AAATACTGTT

59941 TTTTACAACC CCAAATACCA TGAAGCCCAT AAAATTTAAT CAAGACGAGG GCCACAAAGC

60001 GAGAAAGATC CGTTTATTAT TTTTAGTCAA CGGGTGATGA ATTAAGAGCA AATGCAGCAG

60061 CATTTCACAG CAAAGTAAAG CGCACATTAG ATGCCTTACA AACACAATAC GAAAAAGAGA

60121 ACATGCGCTT TAAAGTAAGG GACCATCAAC ACCTTACATA CGATATATTC TCGAAAATAA

60181 AAGGACACCG AGAAACATAT GGCTATAAGT TAAGAAGCTT ATATCCCAGA TACCAGGCAA

60241 GAAACTGCTC ACTGCCAGAG GCACACAGTG AAATCACTTA TGTTACTTTT TCAGTACCTA

60301 TCACCAGAGC GATAAAAACT GAATATCAAC ACTTATTGAG ACCAGGGGAT TATTCTGGGT

60361 TTTACCGACA TATTGAAGAC AAACTATTGA CGACCTGTAC TCAGCTACAG CTTTCTCATG

60421 TTGGGTTTGT CGCTGATGGT AGAATGCCAA TCATTAGAAA CAGTCAAATT GATAAGTCGG

60481 CACACAATAG AGAGCTACAA AAGCTAAGCT TTGATACATC TTTAGCAGAT GGTCAAACCC

60541 ATACAATTTG GGACGCACAA CATTTATGTG ATGTCATGCA TTTTGTCATC GTGGCCAGTG

60601 ATGCGGATAA CAAAGATGCT GGCTATGGTA AATTTATGAA TAATGTAGAA ACTATGGTTC

60661 GACGATTTAT TACCCAGCTA CCTATAAACC CTGAGAAACA GGATGTGACA ATGCGGTTCT

60721 TCCAGCATAT TAGTTATACT TACTAATCTC ATTAGGCAGT CTAAAGCGGG TGGTCAAAAT

60781 GCAACCACCT GCCCTTTTGC AGTACGTTTA ACATGGATTA AACGACTCTC TAATTCAGCA

60841 AAGATATTAC CAAAGTCCCC TTCCTCAGAG CATATCAGCG CCCCTGACAT TTGCAGTGTT

60901 GTCACATTTT CCTGTGTGGC AAATACATGC TGTAATACCG CTAATCGTTT TTGTAAAACT

60961 GGCTTTTTCA TCCCTTCACA CAAAATGACA AATTCTTGCC CACTCACTCG ACATACAAGA

61021 TCGCCATCAT CCCTAAACTG CATTGATAGC TGCTGCGCTG CAAATTTAAT TGACTTAGCA

61081 TCCATATCAG AGACAGTCAT GGCCTCACTT TGTTCAGGAC TGATCTCAAT AAGCGCCATA

61141 GCATAGTCGA GATTCTTCTT TGAGATCCCC TCAAACTGCT CAATAAAATA CCGCCTGTTA

61201 TATAGCCCTG TCACAGTATC TTTATAGTTC AAAGACTGCG CTAAATGGTG TTTGGAACGC

61261 CTAAAGTAAA CAATCACAAC GAACAAAATA ACAATAAGCA GCGCTATGAT AATGCGAGCA

61321 TGCATTAGCG CTTTGACAAC TTCGTCTTCA GCGGCCGTGT TAATTTGGCG CGCTGATCTG

61381 ATTGGTTGCT CGAATACAAC TGCTTTGTGT TCAGGTTTGC TTTGAATTTT TAGTTCACTT

61441 TCTTCAAGCA AGCGAATGTA GGTGCGTAGC GTGGAAATTG TTTGCGGCAA ATCTTGTTTA

61501 GATTGATAAA CATCGGCTTC AATTTTTAAA GTTTGTCGAG TATAGCGATT ATTAAGCGTA

61561 CTATTATACG ACTTTAACAC CTGCTTAGAT AACGCGATTT GCTCTAACGC TTGATCGAAT

61621 AACTTCATCT GAGCAAACGC AAAGGCTAAA TTATTATGTA ACACAATGAT GTGAGCAGGA

61681 TTAATAATGG CGTTATGTTG TGTTTGTCGA GCAGCTTGCA GATATTTAAT CGCATCCGTA

61741 AAATTTTTAT TTGAGAGCGC TATTTTACCG AGCCCAGATA AGGCCCAAAA CTCGTATCTA

61801 GGCAAGTTAT GTAACTCAGA GACGTTATAC ATTTTCTGGT AACACTGCTG GGCTTTTTCA

61861 ATGAGTTGAG CACGTAACAA CGTTAAGCAC AAATTATACT TAATAGGGAG ACCGTCCAAC

61921 AAATTGTGAG ACTGCTGAGC CAAATCCTGT GCCTGATGTG CATAACCCAA CGCTTTAACA

61981 TAAAACTTTA GCGTCGAATA GTAGGCTGTG GAGCTATCAT ACACCATCAT TCTCGTAATG

62041 GGATCGGACT TATTTTCGCT CCGCGTGAGC AGCGCTTGAG CTGATTGAAG TGATTTGAGC

62101 GCAAAACTGT ATTCCTCACG CCAAACAGAA ATAATCGCCT CCATTGCATA GGTTCGGATC

62161 AGAAAATCGG TTGCTTTATT TTGTGTAAAG CACAACCTAG CTTGTTTGAT ATATTGCTTT

62221 GCCTTATCTA ACTCTCCCTG CTCTATGGAA AAAAAAGCAC GATACAAGAA CCAATATCCA

62281 GACGATAGCT TTAAAGGAGA TGCTTGAACT TGCACTTTTG CCGAATCATA AACAGCCTCA

62341 GCCGTTTCAA TATCCCCTCT AAAAAAAGCG GCGCGTGCTT TCAATGTGAG CCATAAGACT

62401 TCATCTGTCG GCTCTGGTGG TACTTGAGTC GAAAGCAGCG TTGATGGCGA TGTTGAAGCT

62461 GCAGCTTCGT ACTCCAAAAG CTGCTTTTTT GAAACCCCGA ATGCAGAACA CGTGAATAGT 62521 AAAGAAACTG CGAAAAAAAT GAATAGTTGC AAATATAAAA GCCCGTATTT TATCTCTCGC

62581 TTTAAATGTA ATCCTTTATT ACGAAATGTA CAATAATTAC GGGCTTTATA TCCATAAGAA

62641 AGCTCATTGG CTGTTATAGA TACTAAGGAT CTATTAAAGC CGGTTTAGAT TTGCTAGCAG

62701 ATGTTTTTAT CGTGTCTTGA GTCGATTTTA ACGTCTCCGT ACTCCTCTGC TGAGGAGGTT

62761 GAGGCGATGG TGCTTGACTG TATAACGAAG GTTGGCTCTC TTGTGTAATT GTAGACATGA

62821 GCTGCTGATT TAATCGTTGG CTTTGTGAGA TAGTGTCTTT GATATCTTTA TTTTGCTGCT

62881 GTAATTGCTC AGCGAGATCA GCTCGAGTTA GCCCACTTTT TACTTTATCT TTGGTTTTCT

62941 GATCTGTGCT GCCCATCAGC TGGTTAACTA CTTCAACTAA TCGACTCAAC ACGCTGTATA

63001 TCACATCTGC AGAAAAACCG CCGAGTAATG CAGCAAGTGG CTTATGAAAG TCACCCAAAG

63061 AAGAGTGATT TTGAGTTATC TCTTGGGTTG GGATCAGCTC CGCAATCATC AATCCCGCCA

63121 TAAAGCCCAT AATCACCATA GACCAATAAG TAGAATCAAA CTTGGGATCG TAATTGCATA

63181 ATCGAATAAA ATTTCGAGAT TTATGCAAAC AAGCAAATGT TGCTCCTAGC CCCGCGCAGC

63241 AGAGTAAAAA TAACTGATTC AGTAAGAGCA CCCTGCCTTC TGAGTTAAAT AAACCTTCAT

63301 TAATTGTTTT CTGATTGACT TCAGGTGATA TAGACAGAGA AATAATAGAA ATTAAAAAGA

63361 AAAGCGCCAA CAATGACATA TGTCGAACTA ACTTAACAGG CCCTAAAAAC CTTAACCAAC

63421 TCACCGAAGT GGACTCCTTA TCCATAATAG CAAGCGTCAA TGGCTTAGCT GGATAAACCA

63481 GTAAAGATAA CTCTTTATGG CACTTCGTCA GGGCAGTTAA CTCGTGCTCT AACATTAAGC

63541 TATAGCTATC TCCCTCTCTC ACCTCTTGCT TATTTTGTAA TTGATAAACT GTGTTCGGGA

63601 TATAGCTTGG CATGGTCTTT CCTTCGCCTG AAGCGTAGCG GATCATGACA TCGCATTCTG

63661 TCAGTAAATG ACTAACAAAG TCCTTCTTAG ACATAGTGTT ACCTATATTA AATGGCATCG

63721 AGTTGACGTT TATAACAGGG AATATAGTGT CACTTCAAGC ACATTTAATT ACAACTAACT

63781 GTAAAAGATA AATTAGGTCT TAGTTTAAAA TAAAACTAGT AATAAGAGAT GAAAGACACC

63841 AAGTAACGGC CGCGCAGATA AGCTTAAGCT TTTGAATAAT CCATTAAAGG TTATTAGCGT

63901 AATTAAGTGA TAGTGCGCTC CTCGTTTTGT GTAACGAGAT CCACCTCTAG ATATTAGGTG

63961 GGATCAATGT CAATTGGGTT TAATCGCAAC TTATGACGAG TCGTAGATTG GCTCGCCTTT

64021 TAACGGTTCT TCTAAGACTC ATTTAATTGC TAAATAATCA TCGTGCAGAG TTTGCTTAGC

64081 TAGTGATTAT ATTACGCTCT CCAAATTCAG TTTGAAAAGT ATGAACGCTA TTTAGGACTG

64141 AACAATAAAA ACAAAAAAGG CTCCCTAAGG AGCCTTTTTA CCTGAATGGT AAATGATTAC

64201 CAACCAGCTT TTTCTTTAAG TGCAGAACCG ATCTCAGCTA GAGAACGAAC AGTCTTAACG

64261 CCTGCTTCTT CTAGAGCTGC GAACTTCTCA TCAGCAGTAC CTTTACCGCC TGAGATGATT

64321 GCACCAGCGT GACCCATACG CTTGCCCGGA GGAGCAGTAA CACCAGCAAT GTAAGAAACC

64381 ACTGGCTTAG TCACGTTGTG TTTGATGTAC TCTGCAGCTT CTTCTTCTGC TGTACCACCG

64441 ATCTCACCAA TCATTACGAT TGCTTCAGTC TTAGGATCGT TCTGGAACAT TTCTAGTACG

64501 TCGATGAAGT TAGTACCTGG GATTGGGTCA CCACCGATAC CAACACAAGT TGATTGACCG

64561 AAACCAGCGT CAGTAGTTTG CTTAACTGCT TCGTAAGTAA GAGTACCTGA ACGAGATACG

64621 ATACCTACTT TACCAGGTAA GTGGATGTGA CCAGGCATGA TACCAATCTT ACACTCACCC

64681 GGAGTGATAA CACCTGGGCA GTTAGGACCG ATCATACGAA CGCCCGTTTC TTCTAGCTTC

64741 ACTTTAACAT CAACCATATC TAGTGTAGGG ATGCCTTCAG TGATACAAAC GATTAGCTTA

64801 ATGCCACCGT CGATAGCTTC TAAGATAGCG TCTTTACAGA ATGCAGCTGG TACGTAGATT

64861 ACTGTCGCAG TTGCGCCAGT TGCTTCTACA GCTTCACGTA CAGTGTTGAA TACTGGAAGA

64921 CCAAGGTGAG TTTGACCACC TTTACCAGGT GAAACACCAC CAACCATTTG CGTACCGTAC

64981 TGGATAGCTT GTTCTGAGTG GAAAGTACCC TGACCACCAG TGAAACCCTG ACAGATTACT

65041 TTAGTATCTT TATTAATTAG TACAGACATT ATTTGCCCTC CGCAGCAGCA ACTACTTTCT

65101 CTGCAGCATC AGTTAGTGAT TCAGCAGCGA TGATGTCAAG ACCAGAGTTA GCTAGTACTT

65161 CACGGCCAGC TTCAGCGTTA GTACCTTCAA GACGTACAAC TACAGGTACG CTTACACCAA

65221 CTTCTTTAAC TGCACCAATG ATACCTTCAG CGATCATGTC ACAACGAACG ATACCACCGA

65281 AGATGTTAAC TAGCACTGCT TTCACGTTGT CATCTGAAAG GATGATCTTG AATGCTTCAG

65341 ATACACGCTC TTTAGTCGCG CCGCCACCAA CGTCTAGGAA GTTAGCTGGC TTACCGCCGT

65401 GTAGGTTTAC GATGTCCATT GTACCCATCG CTAGGCCTGC ACCGTTAACC ATACAACCAA

65461 CGTTACCGTC TAGAGCAACG TAGTTTAACT CGAAGCTTGC AGCGTGAGCT TCACGTGCAT

65521 CTTCTTGTGA AGGATCGTGG AATTCACGGA TCTTAGGCTG ACGGAATAGC GCGTTGCCAT

65581 CAACACCAAT CTTGCCGTCT AGGCAATGTA GGTTGTTTTC GTCAGTGATT ACTAGAGGGT

65641 TGATCTCTAG AAGTGCGAAA TCGTGATCGA TGAACATGTT CGCAAGACCT AGGAAGATCT

65701 TTGTGAACTG TTTGATCTGT GCTGGGTTAA GACCAAGCTT GAAGCCTAGC TCACGACCTT

65761 GGTATGCCTG AGGGCCTACT AGTGGATCGA TTTCAGCTTT GTGAATTAGT TCTGGCGTTT

65821 CTTCAGCAAC TTGCTCGATT TCAACACCAC CTTCAGTTGA AGCCATGAAC ACGATTTTAC 65881 GTGAAGCACG GTCAACAACA GCACCAAGGT ATAGCTCATT TGCAATATCA GTGCAGCTTT

65941 CAACTAGGAT CTTAGCAACA GGCTGACCTT TTTCGTCAGT CTGGTAAGTT ACTAGGTTTT

66001 TACCTAACCA GTTTTCAGCA AATGCGCGGA TCTCGTCTTT GCTATCAGCT AGCTTAACAC

66061 CGCCAGCTTT ACCACGGCCA CCCGCGTGTA CTTGACATTT AACGACCCAC TTGTCGCCAC

66121 CAATTTTACC AGCAGCTTCA ACTGCTTCCT GAGGAGTGTC ACAAGCGTAA CCCTCAGACA

66181 CAGGTAAACC ATATTCGGCA AAAAGTTGTT TTGCCTGATA CTCATGCAAA TTCATGATGC

66241 TTTATCCAAT TTTTTATACT AAAAATGCTG AATATTTATG CAAATAATCA GCTATCCCAA

66301 ATTGCGCACA TAGTATAGAT CCCACGGCGT TTTAATAAAA CCCCAGTTAG ACCAAAGGCG

66361 CAAAAAAAAA GCTGTATGTT GCTTAATTGC ACGCATACAG CTTAAGATTT AATCTTTTGA

66421 TTAAACGTCT AGAAGTAGAC GTGTTGGATC TTCTAATAAT TCTTTGATTG TTACTAGGAA

66481 ACCAACTGAT TCTTTACCAT CGATTTGACG GTGGTCATAA GAAAGCGCTA GGTACATCAT

66541 AGGTAGAATT TCTACCTTAC CGTTTACAGC CATTGGACGC TCTTGGATCT TGTGCATACC

66601 AAGAATTGAA GACTGAGGTA GGTTGATGAT AGGCGTAGAA AGTAGTGAAC CGAATACACC

66661 ACCGTTTGTG ATTGTGAAGT TACCACCAGT CATATCATCA ACTGTTAGTT TACCATCACG

66721 ACCTTTAAGC GCTAGCTCAC GGATACCTTT TTCGATTTCA GCAACAGATA GCTTGTCACA

66781 GTCACGAAGT ACTGGTGTTA CTAGACCACG AGGTGTAGAA ACAGCGATGC TGATGTCGAA

66841 GTAGTTGTGA TAAACGATAT CATCACCGTC GATTGATGCA TTTACTTCAG GGAAACGCTT

66901 AAGTGCTTCT GTTACTGCTT TCACGTAGAA AGACATGAAA CCAAGACGAA TACCGTGACG

66961 CTCTTCAAAT ACATCTTTGT ACTGCTTACG AAGGTCCATG ATTGGCTTCA TGTTTACTTC

67021 GTTGAACGTA GTAAGCATTG CTGTTGAATT CTTTGCTTCA AGAAGACGGT TAGCAATTGT

67081 CTTACGAAGA CGTGTCATTG GAACACGCTT CTGCGTACGA TCACCTACTG GTGCTGCTGG

67141 CGCTGCCGCA GCTGCTTTAG CTGGTGCTGA TGCTGGCTTA GCTGCTGGCG CTTTAAGGAA

67201 TGCGTCAACA TCTTCTTTGG TGATGCGACC ACCTTTACCA GAGCCTTTGA TCTGAGAAGC

67261 GTCTAGGCCT TTTTCTGCAA TCAAGCGACG AACTGAAGGC GTAAGCACAT CAGCGTTTTC

67321 GTCAGACGTT GCTGGCGCTG CGTCAGAAGT TGCTGACGCT GCAGCTGGTG CACCACCTGC

67381 AGAAAGCTTA CCAATAACCT GCTCACCAAG TACTGTATCG CCTTCAGCGT GCAAGTGTTC

67441 ACCCATTACA CCGTCTTCAG GTGCAACAAC TTCTAAAACA ACTTTGTCTG TTTCGATGTC

67501 AACTAGGTTT TGATCACGGC TTACTGCCTC ACCTGGTTGA ACGTGCCAAG TTGCGATTGT

67561 AGCGTCTGCT ACTGACTCTG GAAGTACTGG TACTTTAATG TCCACTTCTT TACCTTCTGC

67621 TGCTGGCGCC GCTGCCGCTG GCGCTGCTGG TTGTTCATTT GATTGAGTTG GTGCCGCACC

67681 CGCAGCACCG ATTTGTGCAA TTACTTGCTC ACCTAATACT GTCGCGCCTT CTTCTTCAGA

67741 GATAGCAACA ATTACACCAT CTTCAGGTGC GACAACTTCT AAAACGACTT TATCCGTTTC

67801 GATGTCAACT AGGTTTTGGT CACGGCTTAC CGTATCACCA ACGCTTACAT GCCATGTCGC

67861 AACGGTCGCG TCTGCAACTG ACTCAGGAAG AACAGGCACC TTAATTTCGG TTGTCATCTC

67921 TTATTCCTTA TTTTTTAATT GTTAATGCGT CAGCAATCAA CGCGTTTTGT TCTTTAGTGT

67981 GGGTAGACAT ATATCCACAT GCCGGTGCAG CTGATGCCTT ACGGCCAGCA TATGTTAGGT

68041 TTGCACCTGC TGGGATTGCT TCCCAGAAAT GGTGTTGAGA ACAGTACCAA GCACCTTGGT

68101 TCTGTGGCTC TTCCTGACAC CATACGAAGT CTTTAACATG CTGGTAACGA GCCATGATCT

68161 CATCCATCTC TTTATGAGGG AATGGATATA ACTGTTCAAC ACGTACAATA GCAATATTGT

68221 TTAGTTCAAG CTTACGACGC TCTTGAAGTA GTTCGTAGTA AACCTTACCA CTACAGAATA

68281 CAACACGCTC TACGTTTTCC GGCTTAATCT CATCGATTTC ATCGATCATA TTGTGGAAAA

68341 CACCATCAGA CAGTTCTTCA AGACTAGAGA CTGCTAATGG GTGACGAAGC AATGATTTTG

68401 GTGTCATTAC AATCAATGGA CGACGTAAAG GACGTACTGA TTGACGGCGT AACATTGCAT

68461 AAACCTGCGC TGGCGTAGTT GGTACACATA CTTGCATGTT GTGGTCAGCA CAAAGCTGAA

68521 GGTAACGCTC CATACGTGAA GAACTGTGCT CTGGACCTTG ACCCTCGTAA CCGTGTGGAA

68581 GCAATAGAGT CAAGCCACAC AAACGGCCCC ACTTCTGCTC ACCCGAACTT AAGAATTGGT

68641 CAAATACAAC CTGTGCACCG TTCGCGAAGT CACCAAATTG TGCTTCCCAA AGAACCAGTG

68701 AAGTAGGCTC TGCAGTTGCG TATCCATATT CGAATGCTAA AACAGCTTCT TCAGAAAGTA

68761 CTGAGTCATA AACCTCAAAA GTACCCTGCT CTGGCCTGAT GTTTTGCAAT GGTAAGTAAG

68821 TAGACGCATC GTTTTGGTTA TGAACAACAG CGTGGCGGTG GAAGAAAGTA CCACGGCCAG

68881 AGTCCTGACC AGTTAGACGA ATGTCAGTGC CTTGATCAAC CATAGTCGCG TATGCAAGTG

68941 TCTCAGCCAT ACCCCAATCG AGAGGCTTAT CACCTTTAGC CATCGCTTTA CGGTCATCGT

69001 ATATTTTCTT AACACGAGAC TGTGCTTTGT GATCTTCTGG GTAGCTAGCA ACCTTTTCAC

69061 CAAGCTCTTT AAGCTTATCA ACAGAAACTT GCGTCTCATA AGGCGTATCC CAGTCATGAC

69121 CAACGTATTT CGACCACTCT GATGAATGCT TCGTTTCTGG TTGGATTTCA GAAACCACAC

69181 ATACACCATT GTCTAAGCCG TTTCGATAGT CATCAGCAAG CTGTTTTGCT TCTTGTGCTG 69241 ACAATACCCC TTCAGCGACA AGCTTGTCAG AATATAACTG ACGCGGTACT GGGTGCTTTT 69301 TGATCTTTTG ATACATTAGA GGCTGAGTTG CATTTGGCTC ATCAGCTTCA TTGTGACCGT 69361 GGCGACGGTA ACACACTAAA TCAATAACCA CATCACGCTT AAACTTGTTA CGGAAATCAA 69421 GCGCAATTTG TGTTACGAAT GCAACTGCTT CAGGATCGTC AGAGTTTACG TGGAAGATTG 69481 GTGCCTGAAC CATCTTAGCT ATGTCAGTAC AGTATTCTGT TGAACGCGTA TCTTCTGGTT 69541 TAGATGTTGT AAAACCAACT TGGTTGTTTA CAACGATACG AACAGTACCA CCAACACCGT 69601 ATGCACGTGT CTTTGATAAG TTAAATGTCT CTTGTACAAC ACCTTGGCCT GCAATTGCCG 69661 AGTCACCATG GATGGTGATT GGTAGTGCTT TAGAGCCACT TGCGCAATCT AAACGGTCAA 69721 GACGTGCTCT TACCGAGCCC ATTACCACTG GGTTAACGAT TTCTAAGTGA GACGGGTTAA 69781 AGGCCAATGC CATGTGAACA TCGCCGCCTT GTGTCGCGAA ATCAGAAGAG AAGCCCATGT 69841 GATATTTAAC ATCACCAGAA CCTGCAGACT CGCCATATTT ACCAGCAAAT TCATCGAATA 69901 ACTCTTGAGG GTTCTTACCA AGGACGTTAA CTAATACGTT AAGACGACCA CGGTGAGCCA 69961 TACCGATAAC AACTTCTTCT TGGCCACTTT CACCAGCTCT GTGTACCAGC TCTTTCAGCA 70021 TTGGTACAAG TGCATCGCCA CCTTCTAGTG AGAAACGTTT TGCACCTGGG AACTTAGCAC 70081 CAAGATATTT TTCAAGACCA TCTGCAGCGA TTAAACCTTG CAATAAGCGT AGTTTGTTTT 70141 CTTTGCTAAA TTGAGGCTTA GAGAAACCGG CTTCTAAGCG TTGTTGTAAC CAGCGCTTTT 70201 CTTCGGTTGA TGTGATGTGC ATGTACTCTG CACCAACTGA ACCACAGTAG GTAGACTTTA 70261 ACGCAGCGTA CAAGTCTTTT AATTTCATTG TCTCTTTGCC ACAGGCAAAA GAGCCAACGT 70321 TGAATTCTTT ATCGTGATCC ACATCATCTA AATCGTGGTA AGCCAACTCG AGTTCTCTCA 70381 CTCGGTCACG TTGCCATAAA CCCAACGGGT CTAGATTGGC ATTTTGGTGG CCCCTAAATC 70441 TAAATGCATT AATAAGCTGC AACACACGCA CTTGTTTTGC ATCTGCCGCA CCTTCTGCAG 70501 AAACTACTAC TTCTCTGTGT TTGTTCTTAG CAAGCTCAGC AAATTGTGCT CTTACATCGG 70561 AATGTTTAAT ATCAACATCA ACACCTTCTA CTTTAGGCAG TTGATCAAAC ACTTCTCGCC 70621 ATTCTTCTGG CACTGAAGCC GCATCATCAA GATACGCCTC ATATAAATCT TCTACATATG 70681 CAACGTTACC GCCGTATAAG TGAGAAGATT CCAGCCATGC TTTCATCACA CCTTCGTGCA

70741 TTTATTAGCC CTTTTCTCTA GCGCAGAAAC TTAAACTTAA ACGAAAACAA GATGGCATGC 70801 TCAGCATGCC ATCTTAAGAT AATAATATGC TTTAAACCGA ACGGTTTAAT AGCATAGATT 70861 TAATGTGTCC AATCGCTTTG GTTGGATTCA AACCTTTCGG ACAAACGCTA ACACAGTTCA 70921 TGATACTGTG ACAACGGAAT ACGCTGAACG CGTCATCAAG ACCAGCTAAA CGCTCTTCAG 70981 TTGCTGTATC GCGGCTATCT GCCAAGAAGC GATACGCATG AAGAAGGCCC GCAGGACCGA 71041 TAAATTTATC TGGGTTCCAC CAGAATGATG GGCATGAAGT AGAACAACAT GCACATAAAA 71101 TACACTCATA AAGACCATCT AGCTTTTCAC GGTCTTCAAC AGATTGAAGA CGCTCTTCAC 71161 CGCCAGTTGG CTTATCATTG ATCAAGTAAG GTTTTACTTT CTCGTATTGA GTGTAGAACT 71221 GACTCATGTC AATTACCAAG TCACGAATTA CTGGCAAGCC AGGAAGTGGA CGTACGATAA 71281 TTTTACCTTT GCCATTCTTT TGTAGAGCAG ACAATGGAGT AATACATGCA AGGCCATTTT 71341 TACCATTCAT GTTTAGACCA TCAGAACCAC AAACACCTTC ACGACATGAA CGACGGAAAG 71401 AAAGTGTTGA GTCCTGCTCT TTTAACATAA GTAGTGCGTC AAGCACCATC ATGTCACGAC 71461 CTTCTTCAAC CTCAAGTTTG TATTCCTGCA TACGAGGTGC AGTATCAACA TCTGGGTTGT 71521 AACGATAAAC AGATAATTCT AAAGTTGCTG TTGCCATCGT CTAACTCCTA GTACGTACGA 71581 GCTTTAGGTG GGAATGCTTC ACGCGTAGTC GGCGCAAAGT TAACATCACG CTTTGTCATT 71641 GACTCACTTT CTGGGTGATA CATTGAGTGG CATAGCCAGT TCTCGTCATC ACGCTCTGGG 71701 AAGTCAAATC TTGAGTGCGC ACCACGGCTT TCCGTACGGA AGTTAGCAGC AACTGCTGTT 71761 GAGTATGCTG TTTCCATCAA GTTATCTAGT TCAAGACACT CAATACGTTG CGTGTTGAAT 71821 TCAGTTGACT TGTCATCAAG ACGCGCATAC TGAAGACGTT CACGGATTTC TTTAAGCTGC 71881 TTAAGGCCAT CAGCCATTGC ATCACCTTCA CGGAATACCG AGAAGTTAAG CTGCATACAC 71941 TCTTGCAGAT CTTTCTTGAT TTGAACTGGG TCTTCACCCT TACCAACTTC AGAAGTTTCC 72001 CAACGATTGT AACGAGCAAA CGCTGCATCA ACATCGTCTT TAGACGCTTC ACCAGTTGAC 72061 TCAAAGTCTT TCAAGTAAGA ACCTAGGAAG TTACCTGCTG CACGACCGAA TACAACTAAG 72121 TCAAGTAGCG AGTTACCACC TAGACGGTTT GCACCATGTA CTGATACACA AGCAATCTCA 72181 CCTACTGCGA ATAGACCTTC AACGATACGT TCATTACCGT TGTTGTCTAC GTCAAGAACT 72241 TGACCGTTTA CATTTGTAGG TACACCACCC ATCATGTAGT GACATGTTGG GATTACTGGA 72301 ATTGGCTCTT TAGCTGGGTC TACGTGTGCG AATGTTTTTG ATAGGTCACA AACGCCTGGT 72361 AGACGAAGGT TAAGCATCTC TTCACCTAAG TGGTCAAGTT TCAGCTTAAT GTGAATACCC 72421 CATGGGCCTT CACAACCACG ACCTTCGCGG ATCTCAGTCA TCATTGCACG TGCAACAACG 72481 TCACGACCAG CAAGGTCTTT AGCATTAGGG GCATAACGTT CCATGAAACG TTCGCCATCT 72541 TTATTCAGAA GGTAACCACC TTCACCACGA CAACCTTCTG TTACTAGCGT ACCAGCACCT 72601 GCGATGCCTG TCGGGTGGAA CTGCCACATC TCCATGTCTT GCATGCCGAT GCCAGCGCGT 72661 ACTGCCATAC CAACACCGTC ACCAGTGTTA ATGTGTGCAT TTGTTGTTGA TGCATAGATA 72721 CGACCAGCAC CACCAGTTGC AAGTACAACA GCTTTAGACT TGAAGTAAAC AACTTCACCA 72781 GTCTCGATCT CAATCGCTGT AACACCAACA ACGTCGCCTT TGTCATTTTT AACTAGGTCA 72841 AGGGCATACC ACTCAGAGAA AACGTTTGTT TTATTTTTAA CGTTTTGTTG GTATAGGCAA 72901 TGAAGAAGTG CGTGACCAGT ACGGTCAGCA GCTGCTGCTG TACGTGCAGC CTGCTCGCCG 72961 CCAAAATGCT TTGACTGACC ACCAAAAGGA CGCTGATAAA CACGGCCATT TTCAAAACGA 73021 GAAAATGGTA AGCCCATGTT TTCTAATTCA GTAATAGCTT CAGGACCCGT TTTAGTCATA 73081 TATTCGATAG CGTCTTGGTC ACCGATATAA TCAGAACCTT TAACAGTATC GTACATGTGC 73141 CATTCCCAGT TATCTTCATG AGAGTTGCCA AGTGCTACCG TAATACCACC TTGCGCAGAT 73201 ACTGTGTGAG AACGAGTTGG GAATACTTTA GAGATAAGAG CACATGTCTT GCCTGACTCA 73261 GAAATAGCAA GTGCAGCACG CATACCCGCA CCGCCGGCTC CAACAACTAC AGCATCAAAT 73321 TCACGGACAT TATATTTCAC TTAAACACCC CACAGAACAA ACAAACCAAC AGCAACATAT 73381 GCTAGTGCCA TTAGATTTAA TACAAAGCCT AAAACTGAAC GCATTGTTGA ACACTTGACG 73441 TAGTCAGTAA GAACTTGCCA AAGACCAATA CGAGTATGAA CCATTATACA AACTAGTGTA 73501 ATAATAGTTG CAGCTTTCAT AGCTAAGTTG TCGAACAACC CAGTCCATGC TTCATACGTT 73561 AATTCAGGAG TTAACGCTAA GTAGCCCACG ATAAATACAG CGTATGCTGC AATAATTAAT 73621 GCTGTTGCGC GAAGTGATAC ATAATCTTGT ACACCATCAC GTTTCAGAGT TGCTTGATTT 73681 AAGACCATAT CCACACTCCA CCAAGAATCG CAACGATTAC CCAAATGGCG ATTGCAATTT 73741 TAGCACTTGC ATTGCCCGAC TCTAACTCTT CCCAGTGGCC CATGTCTTGA ATCATGTGAC 73801 GGATACCGCC AATGATGTGG TAAGACAACA CAGCCAAGGT GCCCCACGCA ATGAATTTTG 73861 CAACAAAGCC AGTCATAAGC TCTTTTACAA ATTCAAAACC TTCAGGAGAA GAGAGAGATT 73921 CAGACCACGC CCAAATGACA AATGTCAGCG CGAAGAACAA CGCGACACCA GTGACACGAT 73981 GTAAGATCGA CGCCTTTGCC GTTGCTGGCA TAGATATAGT CGTAAGATCT AGATTTACAG 74041 GTCTTTGCTT TTTCACAGTT ACTTGCCCAT CTTTGCTCGA TAAGAGCTTC AT //

Bacterial Growth Conditions

Any set of known growth conditions can be used to practice embodiments as provided herein, for example, as described in US 2016-0237398 Al, or WO/2015/058179; exemplary growth conditions and parameters are described in Example 1, below.

Making and Assembling Products of Manufacture

In alternative embodiments, products of manufacture as provided herein are comprised of recombinantly generated or substantially isolated components: : (a) a recombinant bacterial Contractile Injection System (CIS) or a Metamorphosis Associated Contractile structure (MAC) formed or configured to comprise a tube having an inner core, (b) a Metamorphosis-Inducing Factor 1 (Mifl) protein positioned in the inner core of the tube of the CIS or MAC, (c) a chaperone 605 protein non-covalently associated with the Mifl protein positioned in the inner core of the tube of the CIS or MAC, and (d) a proteinaceous cargo, or a heterologous protein or peptide, or compound, non-covalently associated or covalently associated or linked to the Mifl . In alternative embodiments, CIS and MACs, including Mifl and chaperone 605 protein, and payloads, as used in products of manufacture as provided herein are produced (synthesized) and fully assembled in vivo by bacteria such as P. luteoviolacea. In alternative embodiments, the bacteria also produce, synthesize or manufacture the payload to be delivered, and the CIS or MAC is assembled in vivo with (or including) the payload loaded or assembled in the inner core or tube of the MAC or CIS. In alternative embodiments, products of manufacture as provided herein, including CIS and MACs, Mifl, chaperone 605 protein, and payloads, are produced (synthesized) and fully assembled as described in the art for example, in Ericson et al, "A contractile injection system stimulates tubeworm metamorphosis by translocating a proteinaceous effector." Elife 8 (2019): e46845.

Translocation mechanisms of effectors via the spike complex of a CIS have been well characterized; for example, in alternative embodiments, CIS and MACs, Mifl, chaperone 605 protein and payloads as used in products of manufacture as provided herein are produced (synthesized) and fully assembled using protocols and components as described for example, by Quentin et al., 2018, Nat Microbiol 3: 1142- 1152; and/or Shneider et al, 2013, PAAR-repeat proteins sharpen and diversify the Type VI secretion system spike. Nature 500:350-353; additional guidance for alternative pathways for loading effectors into the inner tube lumen can be found for example in Heymann JB, et al, 2013, Three-dimensional structure of the toxindelivery particle antifeeding prophage of serratia entomophila. J Biol Chem 288:25276-25284; and/or Sana TG, et al, 2016, Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut. Proc Natl Acad Sci 113:E5044-E5051, and/or Silverman JM, et al, 2013, Haemolysin Co-regulated Protein is an Exported Receptor and Chaperone of Type VI Secretion Substrates. Mol Cell 51, describing how effectors were found to interact with the inner tube protein (hep) and are released post-firing by tube dissociation in the target cytoplasm.

Our results directly showed the previously hypothesized possibility of effector delivery via the tube lumen of a CIS (Heymann et al., 2013; Sana et al., 2016; Shneider et al., 2013; Silverman et al., 2013). Interestingly, the comparison of MACs with a different class of CIS, namely the Type Six Secretion System (T6SS), reveals significant differences. The T6SS effectors that are thought to be delivered by the T6SS tube lumen show protein-protein interactions between the T6SS effector and the T6SS tube protein (Hep) (Sana et al., 2016; Silverman et al., 2013). By contrast, we did not detect such interactions between Mifl and MAC tube protein. One possible explanation could be that the biophysical characteristics of the T6SS tube and the MAC tube are different. While the T6SS tube is inherently unstable and disassembles soon after contraction, see for example, Szwedziak P, et al, 2019, Bidirectional contraction of a type six secretion system. Nat Commun 10: 1565, inner tubes of MACs and other extracellular CISs (and contractile phages) can be readily detected by electron microscopy and therefore seem to be much more stable. Given our observation that expelled MAC tubes were always empty, this poses the question of how the effectors exit such a stable tube after contraction. We hypothesize that this could be the very reason for weak or entirely absent interactions between Mifl and MAC tube, as well as for the low-density region that was seen in subtomogram averages separating Mifl and MAC tube (Fig. 2B). Another mechanistic consequence of low affinity between Mifl and tube could be the requirement of an assembly factor, i.e. JF50 12605, that allows for efficient targeting of Mifl to the tube.

In one alternative embodiment, an exemplary CIS or MAC purification scheme comprises:

P. luteoviolacea was grown in 50 ml SWT media in 250 ml flasks at 30°C for 6 hours or overnight (12-14 h). Cells were centrifuged for 30 minutes at 4000 g and 4°C and resuspended in 5 ml cold extraction buffer (20 mM Tris, pH 7.5, IM NaCl). Cultures were centrifuged for 30 minutes at 4000 g and 4 °C and the supernatant was isolated and centrifuged for 30 minutes at 7000 g and 4°C. The pellet comprising the isolated CIS or MAC was resuspended in 20-100 pl cold extraction buffer and stored at 4°C for further use.

In alternative embodiments, all, several of any one of the components of a product of manufacture as provided herein is heterologous to the assembling bacteria, where optionally the bacteria gains the ability to produce or internally synthesize the component by insertion of one or more recombinant nucleic acid(s) that encode for that component or components. Formulations

In alternative embodiments, provided are formulations, including pharmaceutical formulations, comprising products of manufacture as provided herein for delivering a proteinaceous cargo, a protein or peptide, a drug or a marker, to or into a cell such as a eukaryotic cell, wherein optionally the delivery of the formulation or composition with the eukaryotic cell is in vitro, ex vivo, or in vivo. For example, in alternative embodiments, substantially purified or isolated bacterial CIS or MACs, or the recombinant bacterial CIS or MACs, or liposomes or lipid- comprising nanoparticles incorporating or expressing on their outer surface the substantially purified or isolated bacterial CIS or MACs, or the recombinant bacterial CIS or MACs, as provided herein, are formulated in sterile saline or buffered formulations. In alternative embodiments, formulations as provided herein comprise water, saline, a pharmaceutically acceptable preservative, a carrier, a buffer, a diluent, an adjuvant or a combination thereof.

In alternative embodiments formulations as provided herein are administered orally or rectally, or are formulated as a liquid, a food, a gel, a candy, an ice, a lozenge, a tablet, pill or capsule, or a suppository or as an enema formulation, or for any form of intra-rectal or intra-colonic administration.

In alternative embodiments, formulations are provided herein are administered or are delivered in vivo by any effective means appropriated for a particular treatment. For example, depending on the specific agent to be administered with (by carried by) a CIS or MAC-comprising formulations as provided herein, a suitable means can include oral, rectal, vaginal, nasal, pulmonary administration, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) infusion into the bloodstream. For parenteral administration, CIS or MAC-comprising formulations as provided herein can be formulated in a variety of ways. Aqueous solutions of the modulators can be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art. In alternative embodiments, CIS or MAC-comprising formulations as provided herein are administered encapsulated in liposomes (see below). In alternative embodiments, depending upon solubility, compositions are present both in an aqueous layer and in a lipidic layer, for example, a liposomic suspension. In alternative embodiments, a hydrophobic layer comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such a di acetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.

In alternative embodiments, formulations are provided herein are formulated in any way and can be administered in a variety of unit dosage forms depending upon a desired result, for example, a condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, for example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co., Easton PA (“Remington’s”).

For example, in alternative embodiments, CIS or MAC-comprising formulations as provided herein are formulated in a buffer, in a saline solution, in a powder, an emulsion, in a vesicle, in a liposome, in a nanoparticle, in a nanolipoparticle and the like. In alternative embodiments, the compositions can be formulated in any way and can be applied in a variety of concentrations and forms depending on the desired in vivo, in vitro or ex vivo conditions, a desired in vivo, in vitro or ex vivo method of administration and the like. Details on techniques for in vivo, in vitro or ex vivo formulations and administrations are well described in the scientific and patent literature. Formulations and/or carriers used to practice embodiments as provided herein can be in forms such as tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vivo, in vitro or ex vivo applications.

In practicing embodiments as provided herein, product of manufacture, or CIS or MAC-comprising, formulations as provided herein can comprise a solution of compositions disposed in or dissolved in a pharmaceutically acceptable carrier, for example, acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any fixed oil can be employed including synthetic mono- or diglycerides, or fatty acids such as oleic acid. In one embodiment, solutions and formulations used to practice embodiments as provided herein are sterile and can be manufactured to be generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well known sterilization techniques.

Product of manufacture, or CIS or MAC-comprising formulations, as provided herein can comprise auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities and the like, in accordance with the particular mode of in vivo, in vitro or ex vivo administration selected and the desired results.

Product of manufacture, or CIS or MAC-comprising formulations, as provided herein can be delivered by the use of liposomes. In alternative embodiments, by using liposomes, particularly where the liposome surface carries ligands specific for target cells or organs, or are otherwise preferentially directed to a specific tissue or organ type, one can focus the delivery of the CIS or MAC-comprising formulations, and thus an active agent, to or into a target cells in an in vivo, in vitro or ex vivo application.

Product of manufacture, or CIS or MAC-comprising formulations, can be directly administered, for example, under sterile conditions, to an individual (for example, a patient) to be treated. The modulators can be administered alone or as the active ingredient of a pharmaceutical composition. Compositions and formulations as provided herein can be combined with or used in association with other therapeutic agents. For example, an individual may be treated concurrently with conventional therapeutic agents.

Nanoparticles, Nanolipoparticles and Liposomes

Provided are nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising product of manufacture, or CIS or MAC-comprising formulations, as provided herein. Provided are multilayered liposomes comprising compounds used to practice embodiments as provided herein, for example, as described in Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice embodiments as provided herein.

Liposomes can be made using any method, for example, as described in Park, et al., U.S. Pat. Pub. No. 20070042031, including the method of producing a liposome by encapsulating an active agent (for example, CIS or MAC-comprising formulations as provided herein), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.

In one embodiment, liposome compositions used to practice embodiments as provided herein comprise a substituted ammonium and/or polyanions, for example, for targeting delivery of a compound as provided herein, or a compound used to practice methods as provided herein, to a desired cell type or organ, for example, brain, as described for example, in U.S. Pat. Pub. No. 20070110798.

Provided are nanoparticles comprising compounds as provided herein, for example, used to practice methods as provided herein in the form of active agentcontaining nanoparticles (for example, a secondary nanoparticle), as described, for example, in U.S. Pat. Pub. No. 20070077286. In one embodiment, provided are nanoparticles comprising a fat-soluble active agent used to practice embodiments as provided herein, or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt.

In one embodiment, solid lipid suspensions can be used to formulate and to deliver compositions used to practice embodiments as provided herein to mammalian cells in vivo, in vitro or ex vivo, as described, for example, in U.S. Pat. Pub. No. 20050136121.

Delivery vehicles

In alternative embodiments, any delivery vehicle can be used to practice the methods as provided herein, for example, to deliver products of manufacture, or CIS or MAC-comprising formulations, as provided herein, to mammalian cells, for example, in vivo, in vitro or ex vivo. For example, delivery vehicles comprising polycations, cationic polymers and/or cationic peptides, such as polyethyleneimine derivatives, can be used for example as described, for example, in U.S. Pat. Pub. No. 20060083737.

In one embodiment, a dried polypeptide-surfactant complex is used to formulate CIS or MAC-comprising formulations as provided herein, for example as described, for example, in U.S. Pat. Pub. No. 20040151766.

In one embodiment, compounds and compositions as provided herein, or a compound used to practice methods as provided herein, can be applied to cells using vehicles with cell membrane-permeant peptide conjugates, for example, as described in U.S. Patent Nos. 7,306,783; 6,589,503. In one aspect, the composition to be delivered is conjugated to a cell membrane-permeant peptide. In one embodiment, the composition to be delivered and/or the delivery vehicle are conjugated to a transport-mediating peptide, for example, as described in U.S. Patent No. 5,846,743, describing transport-mediating peptides that are highly basic and bind to polyphosphoinositides.

In one embodiment, electro-permeabilization is used as a primary or adjunctive means to deliver the composition to a cell, for example, using any electroporation system as described for example in U.S. Patent Nos. 7,109,034; 6,261,815; 5,874,268.

Dosaging

In alternative embodiments, product of manufacture, or CIS or MAC- comprising formulations, as provided herein, including pharmaceutical compositions, are administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a subject, for example, a human in need thereof, in an amount of the agent sufficient to cure, alleviate or partially arrest the clinical manifestations and/or its complications (a “therapeutically effective amount”).

The amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient’s physical status, age and the like. Dosage levels may range from about 0.01 mg per kilogram to about 100 mg per kilogram of body weight. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents’ rate of absorption, bioavailability, metabolism, clearance, and the like (see, for example, Hidalgo- Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51 :337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; the latest Remington’s, supra). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods as provided herein are correct and appropriate.

Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and/or Detailed Description sections.

As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of’, “substantially all of’ or “majority of’ encompass at least about 90%, 95%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition. For example, in alternative embodiments, a substantially purified or isolated bacterial CIS or MACs is at least about 90%, 95%, 97%, 98%, 99% or 99.5%, or more pure, or is between about 85% and 99.5% pure, or having no more than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% non-bacterial CIS or MAC elements.

The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court.

Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims. The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.

EXAMPLES

EXAMPLE 1 : Bacteria Stimulate Tubeworm Development by Injecting a Protein Toxin

This example describes exemplary compositions and methods for practicing embodiments as provided herein.

Contractile Injection Systems (CIS) are nanometer-scale syringe-like machines that bear homology to the contractile tails of bacteriophage. The CIS structure contains a contractile sheath and rigid inner tube, that upon contraction can penetrate membranes and injecting a protein cargo. Pseudoalter omonas luteoviolacea is a marine bacterium that produces metamorphosis associated contractile injection systems (MACs) a type of extracellular CIS which is required for the stimulation of metamorphosis of a marine invertebrate Hydroides elegans. This contractile injection system is made up of an array of tails held together in a hexagonal lattice and contain approximately 100 contractile tails. This protein complex contains an effector protein in its inner tube and upon contraction injects this protein into the host cells. This protein is both necessary and sufficient to induce the tubeworm metamorphosis. How this protein functions and the requirements for loading into the complex were previously unknown.

Here we show that Mifl requires interaction with the chaperone 605 protein for Mifl to be loaded within the complex and that this loading is required for metamorphosis. Furthermore, we show Mifl is a toxin, and this toxin activity is found in the C-terminal portion of the protein. We identified Mifl is a membrane associated protein and possesses lipase activity. Through fragmentation analysis we identify a portion of the Mifl protein associates with membrane lipid and this region is required for lipase activity. We show that the protein N and C terminus work cooperatively to maintain this function. Our data shows the loading requirements for the effector Mifl protein and its function as a lipase toxin. These data suggest a role for lipid cleavage in the initiation of tube worm metamorphosis and provides context for the findings previously published showing how lipids and PKC signaling can stimulate tubeworm metamorphosis. Understanding the role that bacteria contribute to animal development and the mechanisms by which they are capable of interacting furthers our understanding of animal-bacteria interactions and their role in animal development.

We have shown that the Mifl protein effector loaded within the inner tube lumen of the MACs structure stimulates the metamorphosis of Hydroides larvae.

Here we show the regions required for Mifl loading into the MACs complex and its effect on Hydroides metamorphosis. To determine how these regions might be used for loading of the protein, we further looked into their ability to associate with the 605-chaperone protein which is also required for Mifl loading into the complex. We then look at the protein folding prediction using ALPHAF0LD2™ to predict a possible function for Mifl once injected into the host cells. Our findings show that the Mifl protein appears to be a porin? and we further validate this finding by determining lipid binding and association with the membrane. Furthermore, we identify that Mifl purification is associated with lipase activity and the C terminal region retains some of this activity. The A portion of the protein appears to possess a lipase chaperone motif and may contribute to the C-terminal function. Our results show that the N and C term domains are required for 605 chaperone binding, subsequent protein complex loading and metamorphosis.

Results and Discussion

Structural prediction of Mifl identifies three domains and suggests membrane association

To determine the structure of Mifl the protein sequence was analyzed for homology and predicted domains via BLAST™, HMMR™, and PHYRE2™ (Creative Commons Attribution-2.0); however, no confident predictions were given (FIG. 8). The sequence appeared to share little homology with known protein structures, only sharing some homology to domain of unknown function 4157, a suspected metallopeptidase but lacking the conserved motif of (HExxH) (see FIG. 9B). To circumvent this issue the program ALPHAFOLD2™ (DeepMind, London, UK), a protein 3d structure prediction software was used to predict the entire protein structure. The structure showed two alpha helical globular domains at the N and C- terminus of the proteins, with a large beta pleated sheet portion in the middle of the protein (figure 1, A). The structure resembled many known membrane transporter proteins which have a beta-pleated sheets that circularize and allow hydrophilic components to pass through the membrane ⁴⁰ .

This observation was searched against the protein data base (PDB) or structural classification of proteins (SCOP) database using 3D-Blast, a search engine using known crystal structures to identify homologs, we found the top predicted structures which hit our predicted structure listed in (Table 1 and table SI). The hits matched to the center beta-pleated sheets portion of the protein and the hits all resembled some form of membrane associated protein complexes such as transporter proteins, membrane porins, and other membrane bound complexes.

Figure 1. Mifl alpha fold prediction. (A) ALPHAF0LD2™ prediction of the effector protein Mifl . (B) Predicted IDDT local superposition-free score for each residue 1-943. (B) predicted alignment error of predicted residues vs scored residues. (C) Sequence coverage of predicted residues. (E,F) Negative staining Transmission electron microscopy of purified Mifl. Scalebar = lOOnm.

Membrane depolarization has been a proposed method of metamorphosis induction due to high concentrations of K ⁺ inducing metamorphosis ⁴¹ . It is also possible for the protein to have multiple functions as the ALPHAF0LD2™ model appears to predict 3 separate domains as seen by the separate nodes seen in the sequence coverage of predicted residues, which can be useful in identifying domains (figure 1, C). To determine if these domains may contain independent activity, we ran these domains independently through ALPHAFOLD™ to determine if 3D-Blast would predict structures other than the membrane associated complexes found during the entire protein search. The First alpha helical domain (amino acids 1-200) search revealed a homology to a known protein lipase chaperone structure (sup figure x, A) (pdb 2ES4). The central region containing the beta-pleated sheets was searched independently (amino acids 200-760) and results mirrored those of the full-length protein. The domain which exists in the C-terminus (amino acid 760-943) appeared to hit a few different structures but interestingly the C-term appeared to hit against a 14- 3-3 zeta (pdb 1IB1). These Findings together suggest that Mifl is a membrane associated protein and likely has distinct functions associated with three different domains found on the protein. The N and C terminal domain may contain protein binding domains or enzymatic activity and the Central beta-pleated sheet domain may anchor the protein in the membrane and potentially facilitate transport of molecules.

Table 1. 3D-Blast hits using ALPHAF0LD2™ predicted model of Mifl (l-943aa)

12 Inqg: 605 177 7.00E-44 29.9 28.4 Transport protein 13 2ysu: 530 175 3.00E-43 32. 1 30.8 Transport protein/hydro lase

14 2iah: 454 175 3.00E-43 34.4 25.1 Membrane protein

15 3efm: 466 169 1.00E-41 33.9 23.8 Membrane protein

16 Ikmo: 597 168 3.00E-41 29.8 27.5 Membrane protein

17 3fhh: 603 167 7.00E-41 29.7 24 Membrane protein

18 lpo3: 563 167 7.00E-41 30.4 23.1 Membrane protein

19 i I kmp: 526 166 1.00E-40 32.9 24.9 Membrane protein

20 2b5m: 706 165 2.00E-40 26.9 23.2 DNA binding protein/ protein binding

Mifl requires the N and C terminus for loading into the MACs complex and the entire Mifl protein is required for the induction of metamorphosis.

To begin to understand how the Mifl protein is loaded into the MACs complex, the protein was knocked out in 200 amino acid pieces across the 943 total amino acids and observed its ability to fill the inner lumen of the protein complex (figure 2, A). Previously we have shown that the 605 protein is required for Mifl filling of the inner tube complex ³⁷ . Using cryo-electron microscopy we observed the ratio of filled versus empty tubes after growing these knockout strains and extracted MACs. Our results show the N-200aa and C-200aa portions of the protein complex were required for filling the protein complex (figure 2, B). Interestingly, both portions of the protein were predicted to contain globular alpha-helical domains and long (approx. 20aa) portions of unfolded residues. We predict that these long-unfolded portions of the protein contain signal sequences necessary for the loading of Mifl into the inner tube complex ⁴² . These data give insight into required portions of the protein which are required for proper protein loading.

To further understand the portions of the protein required for function the A200aa knockouts were tested for metamorphosis. Adding MACs containing 200aa knockouts to Hydroides our results showed that the entire protein has portions which are required for metamorphosis and none of the 200aa knockouts proved to be functional. This is predictable as the complexity of the MACs complex likely limits the modifications of the effector proteins in both the loading, ejection into the host cell, and the function once it enters the host.

Figure 2: The N and C-term domains are required for protein loading into the MACs inner tube complex. (A) Two hundred amino acid residues were systematically removed from Mifl in order to determine their role in Mifl effector loading. (B) The deletion mutants were then subject to cryo-electron microscopy to assess the filled versus empty status of the various deletions. These were plotted as a percentage of filled vs unfilled MACs structures (n=x). (C-G) Representative images from each knockout to determine the filled versus empty status. (D) Metamorphosis assays of extracted MACs complexes with the various mutants were tested and assessed for their ability to induce metamorphosis (the average of 3 biological replicates was plotted with the average of 4 technical replicates each).

Association of the 605 protein with the linker regions between domains suggest stabilization of unfolded residues is required for loading into the MACs complex

To understand the interaction that takes place between Mifl and the loading chaperone protein 605 the two proteins were co-expressed within an expression host E. coll BL21 each with a different protein tag (S-tag 605 and Hisx6-Mifl). We have previously shown these two proteins interact ⁴³ but did not know how where 605 binds to Mifl. To determine the binding interaction portion of Mifl and 605 we broke the protein down into thirds (fragment A, B, and C) with two larger portions covering two thirds of the protein (fragment D and E) overlapping the smaller thirds in case the intersections are required for interaction (Figure 3, A). The reciprocal pulldowns show that the 605 protein tightly binds to the D fragment of Mifl which includes fragment A and B but spans the junction across them. This result is interesting because this junction falls at the 333aa which is after the portion of the protein that is required for loading. Our current hypothesis is the large unfolded connected region which exits between the l-200aa alpha-helical region and the 200-760aa beta-pleated sheet region makes the protein wobbly. This wobble effects the ability for the protein to tightly pack into the small inner diameter of the MACs tube and therefore must be stabilized. By stabilizing the wobble portion of the protein, the protein can linearize and successfully pack into the MACs.

Figure 3, Mifl amino acid residues are required for binding with the MACs loading protein 1 (Mlpl), (a)Western blot showing the presence of Mlpl tagged with a S-tag. Ni ²⁺ agarose pull-down using Mifl or Mifl fragments was washed of unbound protein and the resultant preparation was blotted for the presence of Mlpl. Total lysate was used for comparison of pull-down protein versus total expressed protein, (b and c) The reciprocal S-tag was also used as bait and the Mifl or Mifl fragments were blotted by 6XHis tag antibody.

Mifl contains two toxin domains located at the N and C-term of the protein.

Besides the successful loading of the Mifl protein into the MACs complex the protein also needs to perform some function once delivered to the Hydroides host. We noticed that during expression of the Mifl fragments that certain portions of the Mifl protein appeared to contain a toxin domain and were highly toxic to the E. coli. To assess the toxicity of Mifl, we expressed recombinant Mifl in E. coli BL21 PlysE from the IPTG inducible T7 promoter. The A-E fragment regions of the protein were cloned and expressed. The cells were plated on a media containing 0. ImM IPTG to drive expression of the fragments and serial diluted to determine death after expression. Our results show that the E and A fragment but not the full length protein lead to increased cell death (figure 4, A-B). These results suggest that protein may contain a toxin anti -toxin domain within the protein itself. This gives us insight into the folds that are predicted in each of these domains with the A fragment containing the lipase chaperone domain and the E fragment contain certain toxin domains. It is possible that the N and C termini of the protein interact during expression and prevent cell death from occurring, however, when one of the two termini are missing the protein becomes toxic.

After identifying the toxic regions located in the N and C terminus of the protein, we further broke those pieces into 3 more overlapping proteins Al -A3 and C1-C3. We Identified that the portion of the protein in the N-term likely requires more than the 150aa acid pieces that we broke the protein down into and may require up to the entire 338aa piece of the N-terminus. However, for The C-terminus we were able to identify the 150 aa Cl fragment as the portion of the protein found in the C- term responsible for its toxin phenotype.

We further confirmed these findings by looking for cell death via propidium iodide staining. These same E. coli constructs were expressed and then stained with the membrane impermeable nuclear stain propidium iodide. This will identify cells which have died and/or have compromised cellular membranes. Our results confirm the death assays performed by serial dilution on the IPTG plates.

Mifl binds to glycerophosphoinositol membrane lipids

After identifying the disruption of the cellular membrane via propidium iodide staining, our next aim was to determine whether Mifl was capable of binding to membrane lipids. Since Mifl usually acts in the eukaryotic membranes of the Hydroides we screened the known membrane components which exist in eukaryotes. This was performed using membranes spotted with a range of membrane phospholipids and performed a far western blot to identify specific membrane binding interactions. If the protein successfully binds to the membranes, we can determine the relative association and preference based on spot intensity. Two commercially available membrane strips were used to assess binding, the first membrane strip showed that Mifl was capable of binding to Phosphatidylinositol-4-phosphate (Ptdins(4)P) with high affinity and to a lesser degree Phosphatidic acid (PA) (figure 5, A). The second membrane with a variety of phosphatidylinositol isomers, showed Mifl has high affinity for Ptdins(3,5)P over Ptdins(3)P or Ptdins(4)P (figure 5, B). Finally, To determine the binding region of Mifl for the various phospholipids, the first, middle, and last third of the protein (fragment A, B, and C) were tested for binding. Our results show that interestingly all fragments were able to associate with phosphatidyl serine (PS), which was not seen with the full protein, it is unclear why the fragments but not the full protein was able to associate with this membrane lipid. Fragments A and B were only shown to bind to PS, however, fragment C was able to bind to the membrane lipids, PS, Ptdins(4,5)P, Ptdins(3)P, Ptdin(3,4)P, Ptdins(4)P and Ptdins(5)P in that order of preference (figure x, C). Our results showed that full length mifl binds preferentially binds Ptdins(3,5)P > Ptdins(3)P > Ptdins(4)P > Ptdins(5)P > PA, in that order of preference.

Figure 5, Mifl binds membrane lipids and possesses lipase activity. (A) Lipid spotted membrane with various membrane lipids. (B) Far western using purified Mifl protein and Mifl specific antibody shows binding to both PI3P and PA. (C) Lipid cleavage assay with purified Mifl protein or chaperone (12605) protein, incubated for 1 hour with decanoic acid-PNPP substrate. Cleavage and PnPP (4 -nitrophenyl phosphate) release occurs if acyl-ester linkage is hydrolyzed. (D) PLD specific lipid cleavage assay with phosphatidylcholine substrate to assess enzymatic cleavage site of lipases by presence of choline release. Data are represented as the mean ± SD of n = 12 technical replicates across three independent biological replicates. Significance is indicated as a comparison between the two conditions indicated by the line above (***p < 0.0001; ***p < 0.001).

Association of bacterial proteins with inositol phospholipids is unique due to most bacteria do not make inositol glycolipids. These data suggest a role for this protein specifically in eukaryotes and potentially targeted to specific compartments within the eukaryotic host. The finding of the toxin domain within the E and Cl fragments illuminate the possibility of an enzymatic activity which resides within these fragments and acts non-specifically. The ability to bind to specific lipids and have toxin activity need not be mutually exclusive. The association with membrane lipids and toxin activity suggest a role for this protein as a phospholipase. Since Mifl is associated with inositol phospholipids which make up a tiny fraction of the total membrane lipids it is possible that Mifl is a lipase that acts as a signaling molecule producing lipid second messengers. As mentioned previously DAG is upregulated in Hydroides only when Mifl is present. However, an equally likely alternative possibility exits, it is possible that Mifl associates with the membrane and creates a membrane ion gradient via pore formation. This could lead to the downstream activation of a membrane phospholipid which cleaves membrane lipids and produces DAG. To determine whether Mifl may act as a lipase we performed lipase assays with Mifl . Mifl posesses esterase activity and is able to cleave PLA1 and PLD phospholipid type cleavages

After determining Mifl’s association with membrane lipids, we wanted to test for enzymatic activity. Since membrane binding is a feature that all lipases possess. We then tested for enzymatic activity of the protein by looking at the proteins ability to cleave acyl-ester linkages which is a common function across most lipases. We determined through cleavage of a synthetic lipid decanoic acid-PNPP fusion and through the cleavage of TWEEN-20™, Mifl possesses the ability to cleave acyl ester linkages (figure 6, A and B).

These results prompted us to look further into the specific activity and cleavage products of the protein/ Since there are many different potential enzymatic cleavage sites of phospholipases, we tested all the common cleavage types. Mifl was assayed for specificity to Phospholipase A2 (PLA2), Phospholipase C (PLC), and Phospholipase D (PLD) activity. We saw no activity from the PLA2 specific, or PLC specific assays, however we did see some increased activity in the PLD assay (Figure x, D). PLA1 activity has previously been measured using the tween-20 and PNPP- decanoic acid assays which are a form of PLA1 type cleavages. This result further validates our binding experiments which show that MIF1 can strongly bind to PIPs and weakly binds to PA, which would be the resultant product from a PLD type cleavage. These data suggest a role for Mifl as a phospholipase in the membrane and a potential activator of lipid second messengers. It is unclear whether the PLD activity or the PLA1 activity are required for metamorphosis by Mifl and the products produced (decanoic acid and phosphatidic acid) by either of these activities were not successful in stimulating metamorphosis (see FIG. 11).

Figure 6, Mifl possesses lipase activity. (A) Lipid cleavage assay with purified Mifl protein or chaperone (12605) protein, or a GFP control protein incubated with Tween-20 in the presence of Ca ²⁺ . (B) Purified proteins incubated for 1 hour with decanoic acid-PNPP substrate. Cleavage and PnPP (4 -nitrophenyl phosphate) release occurs if acyl-ester linkage is hydrolyzed. (C) PLD specific lipid cleavage assay with phosphatidylcholine substrate to assess enzymatic cleavage site of lipases by presence of choline release. (D) Phospholipase A2 specific cleavage assay with Mifl, Buffer, or a control protein GH1. (E) Phospholipase C specific cleavage assay with Buffer, Mifl or 605 control protein. Data are represented as the mean ± SD of n = 12 technical replicates across three independent biological replicates. Significance is indicated as a comparison between the two conditions indicated by the line above (***p < 0.0001; ***p < 0.001).

Mifl three identified domains work cooperatively to cleave lipids

To determine the portion of the protein which contains the lipase activity we analyzed each of the Mifl expression fragments for lipase activity via PNPP-decanoic acid cleavage assy. Our results show that the E and C fragment which contain the C- terminal portion of the protein possessed the most activity (figure 7, b). However, we did identify some activity associated with just the A fragment of the protein. To better understand how each of the fragments contribute to the Mifl lipase activity we combinatorial combined these fragments together. Each of the fragments were expressed in a separate E. coli and purified. After purification the protein concentrations were normalized and added together such that the total protein concentration was kept constant. After combining the fragments together, the lipase assay was repeated to determine how the protein works together. Our findings show that the Mifl protein acts cooperatively with each of the fragments contributing to the lipase function. Where the A+B+C fragment retained the most activity followed by the A+C fragment and then by the A+B and B+C, finally the C and A fragment alone possessed some activity while the B fragment alone contained no lipase activity (figure 7, C).

These data suggest that the Mifl protein requires all the protein for wild-type activity and no isolated domain exists which contains the lipase activity. These data corroborate the findings showing any of the 200aa deletions in the protein resulted in a loss of function for the metamorphic activity of this protein. The complexity of these findings obscures the potential targeted KO of a domain or single residue which could be responsible for the activity. Likely many portions of the protein contribute to these functions.

Many Type 6 Secretion System effectors exert their effect on target cells by lipase activity. Many of these lipases are cytotoxic lipases that are used as toxin proteins and delivered to induce cell death in the target cells usually bacteria antagonists. However, there are examples of lipases which produce very specific signaling to occur within the host cell. One example is the MARTX toxins from vibrio cholerae, these are large proteins which self-cleave into smaller functional proteins. The MARTX toxin has been shown to bind PIPs and through PLA1 cleavage inhibit endosomal trafficking and autophagy ⁴⁴ . These proteins are not generic toxics like Phosphatidylcholine specific PLDs which completely degrade the major component of the cellular membrane, but instead act on low abundance lipids and specific signaling pathways within the cell. MIF1 likely acts through a different signal cascade than the MARTX toxin since is not toxic to Hydroides larvae, however, since the lipids Mifl targets are found on endosomes and lysosomes PI(3,5)P, PI(4)P, and PI(3)P, the target lipids and subcellular localization may be shared. The protein may also be cleaved and facilitate its own activity or act as an anti-toxin within the host cell. Our data illuminates the complexity of these large effector toxins and their potential role in multiple different signaling processes.

Conclusion:

Our finding show that the Mifl protein requires the N and C terminus for loading into the MACS complex. The 605 chaperone previously characterized to facilitate loading binds to an unfolded portion of the protein stabilizing what appears to be a wobble region. Systematic knock out analysis of 200 bp within the Mifl protein shows that the entire protein is required for function as a metamorphosis inducing protein. Through expression of the protein and Far-western blot analysis on membranes impregnated with various eukaryotic phospholipids, we identify that Mifl has the ability to bind to inositol phospholipids and phosphatidic acid. Through fragment expression breaking the Mifl protein into 5 different fragments we characterize the protein has ability to bind Lipids through its C-terminus. By further analysis of the fragments, we identified a toxin domain located in the C-terminus of the protein, and the l-200aa N-terminus as well. These fragments were further characterized to possess lipase activity and likely to cleave both PLA1 and PLD type lipid linkages. When combining the fragments, the protein appears to cooperatively increase lipase activity suggesting interactions between the entire protein are required for wild-type levels of activity. Our findings demonstrate the role of a bacterial lipase effector and its functional abilities, which furthers our understanding the complex nature of bacteria-animal signaling interactions that take place. FIG. 10 schematically illustrates the alignment of Mifl and A. coli hemolysin E pore forming toxin via PHYRE2™ (Protein Homology/ Analog Y Recognition Engine) (Creative Commons Attribution-2.0);

SEQ ID NO:6 illustrates the query sequence:

EDEAKRKLPEVARKTVYSHLSPMQKEDVSERYTHLLKLISQQNKFTPTSGTYI WTSKVWNEAVQRKSFWIFEMSKSKAKVADKLDKYHKTHSILLLAKLEEIASD YRKN

SEQ ID NO: 7 illustrates the template sequence:

SQAASVLVGDIKTLLMDSQDKYFEATQTVYEWCGVATQLLAYILKDILIKVL DDGITKLNEAQKSKLLALDSQLTNDFSEK s

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A number of embodiments of the invention have been described.

Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.