GB2388112A - Activation of transcription in Toxoplasma gondii using tetracycline-controlled transactivators - Google Patents

Abstract

The invention relates to a control system for regulating gene activation in T. gondii. Two transacting factors isolated from T. gondii by transactivator trapping are disclosed. A tetracycline-inducible transactivator is produced by fusing the tetracycline repressor (TetR) to a T. gondii activating domain. Tetracycline derivatives such as anhydrotetracycline can control gene expression in Apicomplexan parasites carrying such transactivators. This control can be used to produce attenuated strains of parasites such as T. gondii, Plasmodium falciparum, P. vivax, P. berghei, P. yoelii, P. knowlesi, Trypanosoma brucei, Entamoeba histolytica and Giardia lambia that can act as vaccines.

Description

If I À VACCINE 23881 12

The present invention relates to nucleic acid constructs that can act as inducible transactivator systems in Apicomplexan parasites which can be used to create 5 attenuated strains of the parasites that can act as vaccines to protect against infection by wild-type parasite. The transactivator system also permits the systematic study of the genes in Apicomplexan parasites.

An inducible control of individual gene expression is a prerequisite to study the 10 function of essential genes. Several strategies and tools associated with DNA transformation have been developed in the human and animal pathogens of Toxoplasma gondii and the Plasmodium species. The currently available methods to study essential gene function are antisense RNA and ribozyme technology (Nakaar et al J. Biol. Chem. 274 5083-5087 (1999); Gardiner et al Mol. Biochem. Parasitol. 110 15 33-41)). The development of a controlled gene expression system would not only permit the generation of conditional knockouts but would also allow the study of mutated forms of endogenous genes and the expression of toxic genes.

In the originally described tetracycline-controlled inducible expression system 20 (Gossen, M. & Bujard, H., Proc. Nat 'l Acad. Sci. USA 89 55475551 (1992), the fusion of the tetracycline repressor (TetR) with the activating domain of the Herpes simplex virion protein 16 (16) has converted the repressor into an efficient tetracycline-controlled transactivator (tTA). In that case, a minimal promoter fused to tetracycline operator (tetO) sequences is activated in cells expressing tTA and 25 becomes silent in the presence of tetracycline. This system is highly efficient in regulating genes in diverse eukaryotic organisms but has not been established in any protozoan parasite In contrast, the TetR system regulates gene expression in a number of protozoan 30 parasites, including Trypanosoma Brunei (Wirtz, E. & Clayton, C., Science 268 1179-

1183 (1995)), Entamacba histolytica (Hamann et al Mol. Biochem. Parasitol 84 83 91 (1997)), Giardia lambda (Sun, C. H. & Tai, J. H., Mol. Biochem. Parasitol. 105 51 CO (2000)), and Toxoplasma gondii (Meissner et al Nucleic Acids Res. 29 el I 5 (2001).

As in bacteria, TetR interferes with initiation of transcription by binding to tetO 5 sequences, placed properly in the vicinity of the promoter region of protozoan genes.

In the presence of tetracycline the repressor ceases to bind to the tetO sequence and thus interference is abolished, rendering the promoter active.

T. gondii exhibits a remarkably high frequency of stable transformation coupled to the 10 preferential integration at random throughout the genome which have previously been exploited to design insertional mutagenesis strategies leading to the cloning of non essential genes and to the identification of developmentally regulated genes by promoter trapping (Donald et al J. BioL Chem. 271 14010-14019 (1996); Knoll, L. J., & Boothroyd, J. C., Mol. Cell. Biol. 18 807-814 (1998)).

An inducible system based on the tet-Repressor has been reported to control gene expression in several protozoan parasites but best optimised and applied predominantly in Trypanosoma brucei. Indeed, the existence of trans-splicing in kinetoplastida offers a unique opportunity to combine this tetracycline-dependent 20 repression with the powerful T7 polymerase transcription (Wirtz et al Mol. Biochem. Parasitol. 99 89-101 (1999)). In contrast, the broadly used and tighter transactivator system, (tTA) composed of tetR-VP16 fusion has not been reported to function in any protozoan parasites. Recent studies to investigate the use of the tetRepressor to control gene expression in T. gondii found that the tTA was totally inactive (Meissner 25 et al Nucleic Acids Res. 29 el 15 (2001)). While the repression system is suitable for the expression of toxic genes and dominant negative mutants, the necessity to treat the parasites continuously and anhydrotetracycline (ATc) during the procedures of selection and cloning render it inappropriate for the generation of conditional knockouts.

! There exists a need therefore to overcome this considerable limitation to the further study of Apicornplexan parasites to enable the application of techniques based on inducible transactivator technology.

5 According to a first aspect of the present invention, there is provided a nucleic acid construct comprising the tetracycline repressor (TetR) operatively linked to a transacting factor of T. gondii.

The TetR is described in Gossen, M. & Bujard, H. (l 992)(Proc. Nat 'l Acad. Sci USA 10 89 5547-5551) and sequence elements are shown in the constructs of Figure 5.

Transacting factors of T. gondii can be TATi-1 or TATi-3 in which the factor comprises a fusion protein of TetR and a T. gondii activating domain having a sequence as shown in Figure 6. Alternatively, additional transacting factors can be identified using the methodology described in the present application. For example, 15 using a library of degenerated oligonucleotides fused to the TetR could lead to the identification of artificial transcriptional activating domains.

The transacting factor may be TATi-l, TATi-3, or an analog, homolog, ortholog, related polypeptide, derivative, fragment or isoform thereof. The fusion protein 20 formed between TetR and the activating domain may be a contiguous fusion of the two peptide sequences, or one or more additional linker amino acids may be inserted between the protein domains. Alternatively, one or more C-terminal residues from TetR may be truncated, N-terminal residues from the T. gondii activating domain.

25 The term "analog" as used herein refers to a polypeptide that possesses a similar or identical function as a transacting factor of T. gondii (TATi) but need not necessarily comprise an amino acid sequence that is similar or identical to the amino acid sequence of the TATi, or possess a structure that is similar or identical to that of the TATi. As used herein, an amino acid sequence of a polypeptide is "similar" to that of 30 a TATi if it satisfies at least one of the following criteria: (a) the polypeptide has an

/ amino acid sequence that is at least 30% (more preferably, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%) identical to the amino acid sequence of the TATi; (b) the polypeptide is encoded by a 5 nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding at least 5 amino acid residues (more preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino 10 acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues) of the SPI; or (c) the polypeptide is encoded by a nucleotide sequence that is at least 30% (more preferably, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 15 95% or at least 99%) identical to the nucleotide sequence encoding the TATi. As used herein, a polypeptide with "similar structure" to that of a TATi refers to a polypeptide that has a similar secondary, tertiary or Quarternary structure as that of the TATi. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, 20 and crystallographic electron microscopy.

Similarity of TATi polypeptides can also be determined functionally by transfecting a suitable host cell with a nucleic acid construct containing DNA encoding the polypeptide and monitoring for transactivating activity as herein described.

The term "TATi fusion protein" as used herein refers to a polypeptide that comprises (i) an amino acid sequence of a TATi, a TATi fragment, a TATi-related polypeptide or a fragment of an TATi-related polypeptide and (ii) an amino acid sequence of a heterologous polypeptide (i.e., a nonTATi, non-TATi fragment or non-TATi- related 30 polypeptide), which will generally be TetR.

( 5 The term "TATi homolog" as used herein refers to a polypeptide that comprises an amino acid sequence similar to that of a TATi but does not necessarily possess a similar or identical function as the TATi.

5 a The term "TATi ortholog" as used herein refers to a non-T. gondii polypeptide that (i) i comprises an amino acid sequence similar to that of a TATi and (ii) possesses a similar or identical function to that of the TATi.

10 The term "TATi-related polypeptide" as used herein refers to a TATi homolog, a TATi analog, an isoform of TATi, a TATi ortholog, or any combination thereof.

The term "derivative" as used herein refers to a polypeptide that comprises an amino acid sequence of a second polypeptide which has been altered by the introduction of

15 amino acid residue substitutions, deletions or additions. The derivative polypeptide possess a similar or identical function as the second polypeptide. 3 The term "fragment" as used herein refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino 20 acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid 25 residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of a second polypeptide. The fragment of an SPI may or may not possess a functional activity of the second polypeptide.

The term "isoform" as used herein refers to variants of a polypeptide that are encoded 30 by the same gene, but that differ in their pi or MW, or both. Such isoforms can differ

r ! 6 in their amino acid composition (e.g. as a result of alternative splicing or limited proteolysis) and in addition, or in the alternative, may arise from differential post-

translational modification (e.g., glycosylation, acylation, phosphorylation). As used herein, the term "isoform" also refers to a protein that exists in only a single form, i.e., 5 it is not expressed as several variants.

The term "modulate" when used herein in reference to expression or activity of a TATi or a TATi-related polypeptide refers to the upregulation or downregulation of the expression or activity of the TATi or a TATi-related polypeptide. Based on the 10 present disclosure, such modulation can be determined by assays known to those of

skill in the art or described herein.

The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can 15 be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences which results in the highest percent identity. The percent identity is detennined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = # of 20 identical positions/total # of positions x 100).

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and 25 Altschul Proc. Natl. Acad Sci. USA (1990) 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul et al, J. Mol. Biol. (1990) 215:40310 have incorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences 30 homologous to a nucleic acid molecules of the invention. BLAST protein searches

can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. (1997) 25:3389-3402. Alternatively, 5 PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id. ). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

10 Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti Comput.

15 Appl. Biosci. (1994) 10:3-5; and FASTA described in Pearson and Lipman Proc. Nail.

Acad. Sci. USA (1988) 85:2141-8. Within PASTA, ktup is a control option that sets the sensitivity and speed of the search.

In the present invention, the transacting factor of T. gondii may be TATi, although it is 20 envisaged that alternative synthetic fonns of the polypeptide could be made by substitution of one or more amino acids in the molecule. The invention therefore extends to the use of a molecule having TATi activity. The skilled person is aware that various amino acids have similar properties. One or more such amino acids of a substance can often be substituted by one or more other such amino acids without 25 eliminating a desired activity of that substance. Thus the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another 30 (since they have larger aliphatic side chains which are hydrophobic). Other amino acids

f 8 which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); Iysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); 5 and cysteine and methionine (amino acids having sulphur containing side chains).

Substitutions of this nature are oRen referred to as "conservative" or "semi-conservative" amino acid substitutions.

Amino acid deletions or insertions may also be made relative to the amino acid sequence 10 of TATi. Thus, for example, amino acids which do not have a substantial effect on the activity of TATi, or at least which do not eliminate such activity, may be deleted. Amino acid insertions relative to the sequence of TATi can also be made. This may be done to alter the properties of a substance of the present invention (e.g. to assist in identification, purification or expression, where the protein is obtained from a recombinant source, 15 including a fusion protein. Such amino acid changes relative to the sequence of TATi from a recombinant source can be made using any suitable technique e.g. by using site directed mutagenesis. The TATi molecule may, of course, be prepared by standard chemical synthetic techniques, e.g. solid phase peptide synthesis, or by preparation of nucleic acid encoding TATi, and subsequently expression of the nucleic acid in a suitable 20 host cell system.

It should be appreciated that amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-natwally occurring amino acids. Whether or not natural or synthetic amino acids are used, it is preferred that 25 only Amino acids are present.

Whatever amino acid changes are made (whether by means of substitution, insertion or deletion), preferred polypeptides of the present invention have at least 50% sequence identity with a polypeptide as defined in a) above more preferably the degree of sequence

! 9 identity is at least 75%. Sequence identities of at least 90% or at least 95% are most preferred. The degree of amino acid sequence identity can be calculated using a program such as 5 "bestfit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) to find the best segment of similarity between any two sequences. The alignment is based on maximising the score achieved using a matrix of amino acid similarities, such as that described by Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed pp 353-358. Where high degrees of sequence identity are present 10 there will be relatively few differences in amino acid sequence.

The nucleic acid encoding the transacting sequence of T. gondii can be a sequence complementary to, or homologous with the nucleic sequence for TATi-1 or TATi-3.

15 A nucleic acid sequence which is complementary to a nucleic acid sequence useful in a method of the present invention is a sequence which hybridises to such a sequence under stringent conditions, or a nucleic acid sequence which is homologous to or would hybridise under stringent conditions to such a sequence but for the degeneracy of the genetic code, or an oligonucleotide sequence specific for any such sequence.

20 The nucleic acid sequences include oligonucleotides composed of nucleotides and also those composed of peptide nucleic acids. Where the nucleic sequence is based on a fragment of the gene encoding TATi, the fragment may be at least any ten consecutive nucleotides from the gene, or for example an oligonucleotide composed of from 20, 30, 40, or 50 nucleotides.

Stringent conditions of hybridization may be characterized by low salt concentrations or high temperature conditions. For example, highly stringent conditions can be defined as being hybndisation to DNA bound to a solid support in 0.5M NaHPO47 7% sodium dodecyl sulfate (SDS), ImM EDTA at 65 C, and washing in O.lxSSC/ 30 0.1%SDS at 68 C (Ausubel et al eds. "Current Protocols in Molecular Biology" 1,

! 10 page 2.10.3, published by Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, (1989)). In some circumstances less stringent conditions may be required. As used in the present application, moderately stringent conditions can be defined as comprising washing in 0. 2xSSC/0.1%SDS at 42 C (Ausubel et al (1989) 5 supra). Hybridisation can also be made more stringent by the addition of increasing amounts of formamide to destabilise the hybrid nucleic acid duplex. Thus particular hybridisation conditions can readily be manipulated, and will generally be selected according to the desired results. In general, convenient hybridization temperatures in the presence of 50% formamide are 42 C for a probe which is 95 to 100% homologous 10 to the target DNA, 37 C for 90 to 95% homology, and 32 C for 70 to 90% homology.

Examples of preferred nucleic acid sequences for use in a method of the present invention are shown in the attached Figures.

15 The nucleic acid constructs of this aspect of the invention can be provided in the form of vectors, suitably expression vectors.

The term "vector" or "expression vector" generally refers to any nucleic acid vector which may be RNA, DNA or cDNA.

The term "expression vector" may include, among others, chromosomal, episomal, and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such 25 as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.

Generally, any vector suitable to maintain, propagate or express nucleic acid to express a polypeptide in a host may be used for expression in this regard.

( In certain embodiments of the invention, the vectors may provide for specific expression. Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cellspecific. Preferred among inducible vectors are vectors that can be induced for expression by environmental factors that 5 are easy to manipulate, such as temperature and nutrient additives. Particularly preferred among inducible vectors are vectors that can be induced for expression by changes in the levels of chemicals, for example, chemical additives such as antibiotics.

A variety of vectors suitable for use in the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well 10 known and employed routinely by those skilled in the art.

Recombinant expression vectors will include, for example, origins of replication, a promoter preferably derived from a highly expressed gene to direct transcription of a structural sequence, and a selectable marker to permit isolation of vector containing 15 cells after exposure to the vector.

Expression vectors may comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation regions, splice donor and acceptor sites, transcriptional termination sequences, and 5'- flanking 20 non-transcribed sequences that are necessary for expression. Preferred expression vectors according to the present invention may be devoid of enhancer elements.

The promoter sequence may be any suitable known promoter, for example the human cytomegalovirus (CMV) promoter, the CMV immediate early promoter, the HSV 25 thymidine kinase promoter, the early and late SV40 promoters or the promoters of retroviral LTR's, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter. The promoter may comprise the minimum sequence required for promoter activity (such as a TATA box without enhancer elements), for example, the minimal sequence of the

CMV promoter (mCMV). The promoter, if present, can be contiguous to the TetR sequence. The expression vectors or vectors of the invention can be derived from a vector devoid of its own promoter and enhancer elements, for example the plasmid vector PGL2.

Enhancers are able to bind to promoter regions situated several thousands of bases away through DNA folding (Rippe et al TIB5 1995; 20: 500-506 (1995)).

The expression vectors may also include selectable markers, such as antibiotic 10 resistance, which enable the vectors to be propagated.

The nucleic acid sequence of the first aspect of the invention may additionally comprise a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("CAT") or DHFR-TS transcription unit. As is 15 well known, introduction into an expression vector of a promoter-containing fragment

at a restriction site upstream of the CAT gene engenders the production of CAT activity, which can be detected by standard CAT assays. The application of reporter genes relates to the phenotype of these genes which can be assayed in a transformed organism and which is used, for example, to analyse the induction and/or repression of 20 gene expression. Reporter genes for use in studies of gene regulation include other well known reporter genes including the lux gene encoding luciferase which can be assayed by a bioluminescence assay, the aids gene encoding pglucuronidase which can be assayed by a histochemical test, the aphlV gene encoding hygromycin phosphotransferase which can be assayed by testing for hygromycin resistance in the 25 transformed organism, the dhfr gene encoding dihydrofolate reductase which can be assayed by testing for methotrexate resistance in the transformed organism, the neo gene encoding neomycin phosphotransferase which can be assayed by testing for kanamycin resistance in the transformed organism and the lack gene encoding p galactosidase which can be assayed by a histochemical test. All of these reporter

genes are obtainable from Scoli except for the lilac gene. Sources of the lux gene include the luminescent bacteria Fibrin harveyii and V.fscheri, the firefly Photinus pyralis and the marine organism Renilla reniformis.

5 The invention can also be described as providing an Apicomplexan tetracycline inducible transactivator (TATi) system, comprising the tetracycline repressor (TetR) and a transacting factor of T. gondii. Alternatively, it can be described as providing a tetracycline-inducible transactivator (TATi) system, comprising the tetracycline repressor (TetR) and a transacting factor of T. gondii for use in Apicomplexan species.

The advantages of the invention extend to the production of live attenuated vaccines suitable to prevent infection by Apicomplexan parasites, the provision of a system to permit the generation of conditional knock-outs of essential gene(s) in such parasites leading to a greater understanding of the parasites metabolism which may allow for 15 the design of new pharmaceutical agents to block or inhibit the function of the essential gene(s). This system allows for the identification of essential genes and for the validation of such genes as drug targets or vaccine candidates.

To unravel the function of essential genes in an Apicomplexan parasite, for example 20 Toxoplasma gondii or Plasmodium species, the present invention has established a tetracycline-inducible transactivator system (TATi), which ectopically controls gene expression. In a mutant T. gondii strain expressing TATi, a second copy of a gene of interest can be introduced into the cell under the control of the Tet promoter, and the function of the native gene disrupted, for example by homologous recombination, or 25 another gene targeted insertion sufficient to prevent normal gene function. The mutant obtained by this procedure is a fully conditional mutant thus enabling study of the gene concemed.

According to a second aspect of the invention, there is provided a host cell 30 transformed with a nucleic acid construct according to the first aspect. The host cell

( 14 can be a bacterium, for example Escherichia colt, a yeast cell, for example Saccharomyces cerevisiae, or Schizasaccharomyces pombe, or a protozoan, for example Toxoplasma gondii or Plasmodium species, for example Plasmodium falciparum, Plasmodium vivax, Plasmodium berghei, Plasmodium yoelii or 5 Plasmodium knowlesi, or Trypanosoma brucei, or Entamacha histolytica, or Giardia lambia. Introduction of an expression vector into the host cell can be effected by calcium

phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic 10 1ipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection of other methods. Such methods are described in many

standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2n Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, FLY. (1989).

IS According to a third aspect of the invention, there is provided a nucleic acid construct according to the first aspect of the invention for use in medicine. Typically, such uses will be for the treatment orprevention of infections caused by a protozoan, for example Toxoplasma gondii or Plasmodium species, for example Plasmodium 20 falciparum, Plasmodium vivax, Plasmodium berghei, Plasmodium yeelii or Plasmodium knowlesi, or Trypanosoma brucei, or Entamaeba histolytica, or Giardia lambia. This aspect of the invention therefore extends to a method of treatment for or prevention of an infection caused by a protozoan, for example Toxoplasma gondii or Plasm odium speci es, for ex amp le Plasm odium falciparum, Plasmodium vivax, 25 Plasmodium berghei, Plasmodium yeelii or Plasmodium kowlesi, or Trypanosoma brucei, or Entamacba histolytica, or Giardia lambda comprising administration of a nucleic acid construct according to the first aspect of the invention.

Malaria is a disease condition caused in animals by the Plasmodium spp parasites 30 characterized by fever in mild fonns and by metabolic acidosis, severe anaemia and

cerebral malaria, in severer forms, and sometimes in the death of the subject infected.

It is also possible for asymptomatic infection to occur in some affected subjects. In humans, the disease is caused by Plasmodium falciparum and to a lesser extent by Plasmodium vivax where the parasites are transmitted by Anopheles spp mosquitoes.

5 Malaria is caused in rodents by Plasmodium berghei, Plasmodium yuelii and in rhesus monkeys by Plasmodium knowlesi.

According to a fourth aspect of the invention, there is provided a vaccine composition comprising a protozoan, for example Toxoplasma gondii or Plasmodium species, for 10 example Plasmodium falciparum, Plasmodium vivax, Plasmodium berghei, Plasmodium yuelii or Plasm odium kn owles i, or Trypanosoma bru cei, or Entam oeba histolytica, or Giardia lambda transfected with a nucleic acid construct according to the first aspect of the invention.

15 According to a fifth aspect of the invention, there is provided the use of a nucleic acid constrict according to the first aspect of the invention in the preparation of a vaccine for use in the treatment or prophylaxis of an infection caused by protozoan, for example Toxoplasma gondii or Plasmodium species, for example Plasmodium falciparum, Plasmodium vicar, Plasmodium berghei, Plasmodium yoelii or 20 Plasmodium hzawlesi, or Trypanosoma brucei, or Entamacba histolytica, or Giardia lambia. According to a sixth aspect of the invention, there is provided a process for the preparation of a nucleic acid construct according to the first aspect of the invention, 25 the process comprising ligating together nucleic acid sequences encoding a tetracycline-controlled transactivator and a transacting factor of T. gondii, optionally including linker or additional sequences.

According to a seventh aspect of the invention, there is provided a process for the 30 preparation of a host cell according to the second aspect of the invention, the process

comprising transfecting a cell with a nucleic acid construct according to the first aspect of the invention.

According to a eighth aspect of the invention, there is provided a process for the 5 preparation of a vaccine composition according to the fourth aspect of the invention, the process comprising transfecting a host cell with a nucleic acid construct according to the first aspect of the invention.

As described above, the nucleic acid constructs, vectors or expression vectors of the invention can be used in medicine, and the invention therefore extends to 10 compositions comprising the nucleic acid construct according to the first aspect and embodiments of the subsequent aspects as appropriate of the invention. Therefore, the nucleic acid constructs, vectors, or expression vectors or systems of the present invention may be employed in combination with a pharmaceutically acceptable carrier or carriers.

Such carriers may include, but are not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, ethanol and combinations thereof.

The nucleic acid construct, expression vector or vectors of the invention may be 20 employed alone or in conjunction with other compounds, such as therapeutic compounds. The pharmaceutical compositions may be administered in any effective, convenient D manner effective for treating a patients disease including, for instance, administration 25 by oral, topical, intravenous, intramuscular, intranasal, or intradermal routes among others. In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.

( The invention also provides a kit of parts comprising a nucleic acid construct, expression vector or vector of the invention as defined above and an administration vehicle including, but not limited to, tablets for oral administration, inhalers for lung administration and injectable solutions for intravenous administration.

Preferred features of the second and subsequent features of the invention are as for the first aspect mutatis mutandis.

The invention will now be further described by way of example with reference to the 10 following Examples which are present for the purposes of illustration only and are not to be construed as being limiting on the invention. In the Examples reference is made to a number of Figures in which: FIGURE 1 shows trapping of a functional transactivator in T. gondii. FIGURE 15 l(a) shows the scheme of the transactivator-TRAP strategy. A linear DNA fragment encoding dbfrts selectable marker and the TetR without a STOP was stably integrated at random into the genome of the recipient strain. Upon integration in a locus where a functional transactivator fusion has been generated, the TetR-fusion activates expression of the selectable marker gene 20 HXGPRT and of reporter gene LacZ. FIGURE l(b) shows the clone Tati-l regulated LacZ expression in ATcdependent manner. Parasites were grown for 48 hours in the presence of and absence of drug, fixed and stained with X Gal to monitor LacZ expression as performed previously (Meissner et al Nucleic Acids Res. 29 ells (2001)). FIGURE l(c) shows the amino acid 25 sequence of the transactivating domain of TATi-1 (lower sequence line, starting "-PTF") fused to tetR (upper sequence line, starting "..HQ"). FIGURE l(d) shows transient transfection of p7TetOS1 LacZ-CAT into RH parasites or in strains expressing TATi or tTA2s. Cells were grown for 48 hours in the presence or absence of ATc before parasite Iysates were prepared to quantify 30 LacZ expression.

FIGURE 2 shows the generation of a conditional knockout for TgMyoA gene.

FlGVRE 2(a) shows modulation of mycMyoA transgene expression in TATi-l parasites. Detection of mycMyoA by IFA, on intracellular parasites incubated 5 in the presence of or absence of ATc for 48 hours, using rnAb anti-myc. MIC4 was detected under the same setting and exposure time with rabbit polyclonals as control. FIGURE 2(b) shows Westem blot analysis of mycMyoA expression. Parasite Iysates were probed with monoclonal anti-myc and polyclonal anti-MyoA. As internal standard, the Iysate was probed with 10 polyclonal anti-MIC4. The upper band corresponds to the inducible mycMyoA. Parasites were grown for 48 hours in the presence of or absence of ATc before Iysates were prepared. FIGURE 2(c) shows detection of a endogenous MyoA and myMyoA genes by analytical PCR on genomic DNA from RH, TATi-1 transformed with T7S4mycMyoA, myoakol and myoako2.

15 Sequence specific primers were used to amplify endogenous TgMyoA (in an intron) and transgenic mycMyoA (in the 5'-UTR). FIGURE 2(d) shows analysis of myoakol by Western blot with anti-MyoA antibodies which reveals that this clone 1acked endogenous TgMyoA. FIGURE 2(e) shows inducibility of mycMyoA expression in clones myoakol and myoako2 which have 20 regulatable mycMyoA. Intracellular parasites were grown for 48 hours in the presence of or absence of ATc before preparation of total cell Iysate. The level of detection of TgMIC4 was used as control for equal loading.

FTGURE 3 shows phenotypic consequences of TgMyoA depletion for parasitic 25 propagation in culture. FIGURE 3(a) shows plaque assay for myoakol and RH-wt. Parasites were continuously grown on HFF-monolayer in the presence of or absence of ATc for 10 days before fixation and staining of the cells with GIEMSA. FIGURE 3(b) shows invasion-assay of myoakol in comparison to RH-wt parasites. 5.105 freshly Iysed parasites grown in the presence or absence 30 of ATc for 48 hours were inoculated on HFF- monolayer for 20 minutes

( 19 followed by a washing step to remove extraccllular parasites. Cells were further incubated for 24 hours before fixation. The number of vacuoles represented successful invasion events and were counted in 40 eye-fields for

each parasite. 100% represents the number of vacuoles in absence of ATc for 5 RH and myoakol respectively. FIGURE 3(c) shows egression assay of myoako. Parasites were grown for 36 hours of HFF-cells in presence of and absence of ATc. Cells were fixed 5 minutes aRer addition of Ca-ionophore A23187 according to Black et al (Mol. Cell. BioL 20 9399-9408 (2000)).and analysed by IFA using anti-SAGl antibodies. FIGURE 3(d) shows 10 quantification of egress in function of incubation times after addition of Ca ionophore A23 187.

FIGURE 4 shows the parasites depleted in TyoA are avirulent in mice and confer protection against new challenge with wild-type parasites. FIGURE 15 4(a) shows tachyzoites from RH or myoakol mutants that were injected i. p.

into BALB/c mice and monitored for more than 30 days. Groups of mice were given 0.2mg/ml ATc in drinking water or normal water. After 11 days, the group of 10 mice infected with myoako and treated with ATc survived and the drug was removed. After 17 days, 5 mice were challenged with RH wildtype 20 parasites and survived the infection. FIGURE 4(b) shows the development/induction of T. gondii-specific T cells after infection with myoakol mutants. At day 21 after infection, spleens were isolated and the development of T. gondii-specific T cells was determined by IFN-yELISPOT.

The mean of triplicates + is shown.

FIGURE 5 shows the plasTnid maps and respective nucleotide sequences for t he vectors referred to in the Examples and Figures 1 to 4.

Figures 5(a) to (d) show the vectors used to conditionally express MyoA or 30 GFP:

FIGURE 5(a) shows pTetO7Sag4-MyoA FIGURE 5(b) shows pTetO7Sagl-MyoA FIGURE 5(c) shows pTetO7Sag4-GFP FIGURE 5(d) shows pTetO7Sagl-GFP Figures 5(e) to (g) show the vectors used to generate the recipient strain for the transactivator screening by random insertion: FIGURE 5(e) shows pTetO7Sagl-HXGPRT FIGURE 5(f) shows pTetO7Sagl-LacZ 10 FIGURE 5(g) shows pTetO7Sag4-LacZ Figure 5(h) shows the vector used for the random integration pTub8TetRsynthetic: FIGURE 5(h) shows Ptub8TetR-GCN5-DHFRTS Figures 5(i) and 5(j) show the vectors used to express a functional transactivator: FIGURE 5(i) shows pTub8TATi-l-HXGPRT FIGURE 5(j) shows pTub8TATi-3-HXGPRT FIGURE 6 shows the nucleic acid sequences and presumed amino acid sequences of TATi-1 and TATi-3.

Example 1: Preparation of tet-ioducible transsctivator construct 25 T. gondii tachyzoites (RH hx'gpff) were grown in human foreskin fibroblasts (HFF) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2mM glutamine and 25 1lg/ml gentamicin. To generate stable transformants, 5.107 freshly released RHhxgprt- parasites were transfected and selected in presence of mycophenolic acid and xanthine (MPA/X) as previously

described (Donald et al J. Biol. Chem. 271 14010-14019 (1996)). The selection based on chloramphenicol or pyrinmethamine resistance was achieved as described earlier (Soldat, D., & lloothroyd, J. C.,.Sc.ience 260 349-352 (1993); Donald, R. G. K., & Roos, D. S., Proc. Nat 'l Ac.ad. Sci. USA 90 11703-11707 (1993)). Homologous 5 recombination was obtained as described previously (Reiss et al J. Cell. Biol. (2001)).

Plasmids The reporter plasmids for the tet-Transactivator system, p7tetOHXGPRT, p7tetOLacZCAT were described previously (Meissner et al Nucleic Acids. Res. 29 10 ells (2001)). For the construction of p7tetOS4mycMyoA the promoter region of p5RT70Tet4mycMyoA (Meissner et al Nucleic Acids. Res. 29 ells (2001)) was exchanged by p7tetOS4 using NsiI/PacI. The resulting plasmid p7tetOS4mycMyoA was stably introduced by contransfection with pDHFRTs using DHFR-selection.

15 The construct pTgMyoA-klcoTCAT was composed of pT230 CAT previously described (Soldati, D. & Boothroyd, J. C., Mol. Cell. BioL 15 87-93 (1995) ) and flanked on both sides with 2.0 and 2.2Kbp and of 5'- and 3'flanking sequences of TgMyoA gene. The vector was linearized at both ends of the flanking sequences to rise the frequency of double homologous recombination.

The plasmid pTRep-DHFRTs was constructed in two steps. The TetR fragment was amplified with the oligonucleotides Rep-4 and Rep-7, which are: Rep-4 S'CGGAATTCCTTTTCGACAAAATGTCGCGCCTGGACAAGAGCAAAGTCATCAACTCTGC-3'

Rep-7 30 5'CCCTTAATTAATGCATACCGUll'CGCACTTCAGCTG-3'

The resulting PCR-fragment encoding TetR without a STOP-codon was cloned in the EcoRI/Pacl sites of p5RT70GFP. In the second step, the TgDHFRTS selectable marker gene was inserted into the Sacll-site.

The plasmid pTTATi-1-HX was constructed using the rescued RT-PCR fragment that was amplified using a poly-T-Primer with a BamHI-restriction site at the 3'-end and the tetR-specific primer Reps. The fragment was digested using EcoRl/BamHI and inserted between the same sites of p5RT70/HX. Recipient strain for the transactivator 10 trapping was established by con-transformation of RHhxgprt- with p7TetOSILacZ CAT and p7TetOHXGPRT. Stable parasites were selected using CAT. Integration of both plasmids was verified by analytical PCR.

Random insertion was used to identify a transactivating domain, which functions as 15 tet-regulatable transactivator due to its fission with the tetR. For this purpose, a construct expressing the tetR but lacking a stop codon was randomly integrated into T. gondii genome using the d ehydro Sol ate redu ctase-thymidyl ate synthase (DHFR-TS) as selectable marker, which was previously shown to exhibit a very high frequency of integration (10-2). The recipient parasitic cell line used for the screening was deficient 20 in HXGPRT gene and was transformed with two vectors expressing LacZ and HXGPRT under the control of a minimal promoter containing 7 tet-operator sequences (7tetO) (Fig. Ia). One of the parasite mutants resistant to mycophenolic acid and expressing LacZ in a tet-dependent fashion was characterized further. The tetR fusion was rescued by RT-PCR, cloned and sequenced. The fusion consisting of a 26 amino 25 acids attached in the C-term of tetR was named TATi-1 (Fig. 1b). A new expression vector for TATi-l, pTTATi-1-HX, was constructed shown to confer tet-dependent LacZ expression when transiently transfected into a parasitic strain containing p7TetOLacZ. A stable line expressing TATi-1 using HXGPRT as selection was generated in RH to establish a recipient strain for the tet-system.

Example 2: Planue, Invasion and Eeression assays Among many vital functions in obligate intracellular parasites, the process of host cell invasion is prerequisite for their survival and replication. Penetration into host cells is an active process dependent on parasite motion. Gliding motility has been shown 5 previously demonstrated to require an intact actin cytoskeleton and to be powered by a myosin motor. The small unconventional myosin A (TgMyoA) is the primary candidate which localises beneath the plasma membrane (Heintzelman, M. B. & Schwartzman, J. D. J. MoL Biol. 271 139-146 (1997); Hettmann er al Mol. Cell. Biol.

11 1385-1400 (2000)) and exhibits all the biochemical and biophysical properties 10 necessary to generate fast movements. Consistent with an essential role for parasite survival, all our attempts to disrupt TgMyoA gene failed so far.

A second copy of TgMyoA under the control of the tet-promoter was introduced into the TATi-1 expressing cell line and monitored the modulation of TgMyoA transgene 15 expression upon treatment of ATc by Western blot and indirect immunofluorescence (Fig. 2a, b). Using this robust inducible tet-system, we were able to disrupt the endogenous TgMyoA gene by homologous recombination with a vector carrying 5' and 3'- TgMyoA flanking sequences respectively and chloramphenicol acetyltransferase as selectable marker. The absence of endogenous copy was 20 determined by genomic PCR and by Western blot (Fig. 2c). The anticipated role of TgMyoA in parasite motion implied that parasites lacking the protein would be impaired in host cell invasion, egression and spreading. The phenotypic consequences of TgMyoA depletion could be best visualised in a plaque assay (Fig. 3a). After inoculation of F monolayers, the mutant parasites were cultivated with ATc over a 25 period of 5-7 days showed an inability to form plaques in the HFF monolayers while non-treated parasites fonned large plaques of lysis. This process was reversible upon removal of ATc (data not shown). The process of host cell penetration was examined specifcally by invasion assay using freshly Iysed parasites previously cultivated in presence or absence of ATc for 72 hours. The invasion rate was detennined 24 hours 30 later by counting the number of vacuoles (Fig. 3b). The number of parasites per

vacuoles was identical for control and conditional myoako parasites, treated or not with ATc, indicating that the depletion in TgMyoA did not affect intracellular growth.

Parasites use the same machinery to penetrate and egress host cells, so we performed an egression assay using the calcium ionophore for a short period of 5 mitt as S previously described, followed by fixation and visualization by IFA using anti-surface antigen 1 antibodies (SAGI) (Fig. 3c). Egression assay was performed as described previously (Black et al Mol. Cell. Biol. 20 9399-9408 (2000)). The regulated exocytosis by the apical organelles called micronemes plays a critical role in gliding motility and invasion. The micronemes secrete complexes of transmembrane and 10 soluble adhesins upon raise in parasite intracellular calcium. These complexes are interacting with host cell receptors and their redistribution toward the posterior pole is driving gliding motion. To exclude any involvement of TgMyoA in microneme exocytosis, that would explain the phenotypes observed, we examined the discharge by micronemes. The secretion assay revealed no alteration of exocytosis upon 15 TgMyoA depletion. (Fig.3d).

Example 3: Murine virulence assay Freshly Iysed tachyzaites from infected HFF monolayers were washed in PBS and counted under the microscope. Tachyzoites were injected intraperitoneally (i.p) in 0.2 20 ml into mice aged 6-8 weeks. The actual p.f.u, in the inoculum was determined by plaque assay. Mice on anydrotetracycline treatment were housed five per cage and were given 1 mg/ml solution instead of normal drinking water.

RH is a type I strain of T. gondii, which typically kills mice with a LD100 of a single 25 infectious organism. The conditional myoako mutant was assessed for virulence in mice. Both wild type and mutant parasites were inoculated intraperitoneally in two groups of mice supplemented or not with ATc in the drinking water. After even days, all the mice infected with the control and the conditional myoako still expressing TgMyoA transgene were dead (Fig 4a.) In contrast, we observed 100% survival in 30 the group of mice infected with myoako but supplemented with ATc 1 I days post

infection. All the mice were serologically positive for T. gondii as monitored by immunoblot (data not shown). At day 21-post infection, two mice were sacrificed and analysed for the presence of T. gondii-specific T-cells in the spleen, as determined by an IFN--specific ELISPOT. The analysis was positive (Fig 4b), indicating that the 5 mice infected by myoako in the presence of ATc have been able to raise a detectable humoral and a cellular response against the parasites. To determine if these mice were protected against subsequent challenge of RH, they were innoculated i.p. with 150 parasites of RH strain at day 17 post infection. All the mice survived the challenge indicating that the myoako induced a protective immunity.

The tet-system described here has been selected in T. gondii and is not active in the Hela cells (data not shown). The activating domain is capable of interacting specifically with the transcription machinery of the parasite and might function as a transactivator in the closely related parasite Plasmodium Jalciparum.

Acknowledgements This work was mainly Funded by the Deutsche Forschungsgemeinschaft (DFG grant SO 366/1-3).

( pl'cl(17-.Sag1bl\'0.\ -> (;. nets IXiA f;C7't:f, 6423 t'I, c-IgtogrgC'crt, g.-t..qt(1cctc-]ine.

MyoA vts c:lorlert int.o trtO'/ S.lq4c;I'P via N,il/F.l( l I Ct C31r(lr9< t-( t 9t.b3r C7r'r'qc) t t.l,'J Ic-c1c7r q71 t 'trJJ t 31 t t lc7rgr. tgc rI t r3. Ir-(-cJc-t..,c t tc3cc:r-1rc R 0 8 I c-t.-t(Ir-q-c-(grtcc tFt c-gr-t t t c t t c:-c t t c-c-t: t t,t cr/ir:l( <Jt.t.C:C3::r-C11C t t t c cc:yt (:'l'c,( t ct;Ia.tt r c;q(7 160 1 6 1 y(:t cc t-t. t: t.c3t tc(-c,. t t t ilcit get t t.-t(-qc7(-l-ctcg'Ic ( ( c ttctI< -t t 9;tt t'c, 3Jcit 9<t t q7t t c.tc-gt.l3t 9 24 U 241 gc3cct.c:qccr tqlLagac-c3(]t.t tt t.(:CICC.Ct t tqlocttcy<.gEc-ca< 9t t r t t t a<t..'r3t c3<1<tc t ct tc3t (.cc.l<Ct 32() :321 gg.l<-'c l.lcitr t c.tcc(-t.tt rt cg<3tot. t l. c t t t tr.a t t t aLItJr4c3.}t t t t gc-c g-t t. t: t.(.-77(-ct:a t tgc3 t. t.-i. I.t...1 4 ()() 4 0] t.q,qct r].lt t tcllt-,l.t<l.t.tt t t<l.c.cr<il. t t t ( tt('(llft.tt.1 t t-t.-tcqt t;ir'<ttt t t c-c-. t tc-qc c att C,,7(r-t 9r1 4 8() 481 c-aart qt. tg3,, t< C3g( rl.lLc3gt c1r qlgcrt C. t tc-<3::t. t t. t.,c-qc-r.31ctc71r J.llqc7cJgq.I tC1 t.gc-t.32l7c7t '6() 5fi I t ricigt t qgOt t. tCt/(( ctlql t t t t r-( clgt ( ac c3t(:3 t t r7ttettllc9dlocJri3c-t <tr.J t (T,t-rIc.Jr 3 t tl t tcr'ac- t: <-. tC t t c, 10 G4 I t ac,71c;.t.- t t ll1t t(;-p-1c:t c-cI.c t t t cc t t. t. t. c.t ctlt.cactg.1 L.JJJ I t rJcIt;<'c, t c:c3.1ct t: tr,-l< t. t t t c'/20 I2 1 t.ct 1tcactgat agyg.tgtgcJt.ltlC't cc3 act t tclc-L t t t ct. c t t ca<. tC31 t.lCIC7Cl.tCJ tgcJ L.t.llc:t c-qtot t t. c.c. t H0() 801 t t t.ct ct at C. acLy.t t 'qgqlg tgyt aaactcc3.tc L t.t:c.-lc-t r t t ctc t atC a(: t.CJ t;q<g. tcJt yc4t 1.tactogact. t. t H80 cJt] 1 cacUt ttototatcachg,t<JggtcJtcJqtaaact(:ytc t.t, t-c Ict ttEct(-t.lt cactg.:t. tc13g-ctqgr<a. c tc <3.t 960 961 qqtcgitccJcJ kl t.c qa ta.tgat- t.tc qccc3ct.c3ag,ic t cttC tagaaac3aug t.q t 4(i.cc-tg t. Lc-.1 t g,lcggacaa. t.J9a.1 t I ()40 1041 gtgatgcgc3t.t.cqcttgatg.lgcTa.aLgttt:'t.lq(tOgtCaacaLlt..tc c ( t tgqcg.ttt.gt t c-atgactgtt( at.gt(;g 1 120 1121 t t ca t cg<3at c.tt t. t ga.ll. tt.:.1 t Cg t c3.,ugot:qc.;t cctqJ t c<3Ctl.lC cI t (-q t<-tq tgttrJ t.lC cq;tc.la t,,tcg t. cc3t c I 2 00 1201 9t t.ctaggl tc-Jc:<lr3tCc tcr3gtlCj.aCc/CJ tcgcaJc:itaC t/C.:l.lC ttJCt t.tcgtot <J t ct.t.c.-.t-tc. NlY; CAT 1 275 1 M H 2

1276 GAG C.AG AA(:; cVrc A'1\3'1'C'C (;Aii GAG C=(' C'lKi(:'l\; CAT CAT rAT CAT (2AT C'A'I'('AT C\T <:X;1' 1335 3 E Q K L I S E E D L L H H H H H H H D G.72

1336 M'C GAC CTC C7-.G AC',C AAC; ACC AC(; T(-r GAG GAG CTri M A AC' C'C ACG CC(i Ci AAG A(.i 1395 '' 23 T e L A S K T T S E E L K T A T A L K 42 1396 ACi TCG 'l'C(2 GAT G'l'C C'AC C7-G G'l(' CAC CAC T(:C C>' AAT G'IX; 'I'AC AAA C;A 1H.l' CAA A'1\' 14',5 43 R S S D V H A V D H S G N V Y K G F Q I 62.

1456 TCX3 AC-G GAC 1'TG GCG CCG TCG GT(3 AAG GAG GAG C'(:G GAC C'TG ATC; 'I'I'T C,CC AA(i 1.' ATI: 1515 63 W T D L A P S V K E E P D L M F A K C I M2

1516 GTC C AC GC'G GGr; ACA GAC AAG GGC, AAC T1'G AC'C TC;C G'r.' CAG ATC GA'l' CC'A CC); c,r;c 1ll: 1575 83 V Q A G T D K G N L T C V Q I D P P G F 102

1576 GAC GAA CCG rc GAA (,TC CC'G CAG GCG AAT GCG TC=. AAC GTA AAC A' CT(3 ATC GAC CCC' 1635 103 D e P F E V P Q A N A W N V N S L I D P 122 1636 AT ACG TAC GCA C,AC ATC GGC ATG TTG CCT CAC AC.G M(: A1 CCT 1 C Gx cq c rc lrc lG95 123 M T Y G D I G M L P H T N I P C V L D F 142

1696 cEc AA(; GTG C\C' ATC AAG AAT CAA ATC TAC ACG ACT (;CG CAC C(2(, CTC' Gl'C (,: ('C 1'/55 143 L K V R F M K N Q I Y T T A D P L V V A 162

1756 A'PC AAtr C'CC Tl' CCX2 CAf' CTC' GGG AAC ACC AC.G C'C GAC TCC, A'IT G-rT CGA TAC AGA (;A(- 1815 163 I N P F R D L G N T T L D W I V R Y R D 182

1816 ACT rc cU CTC TCC AAA CTC C'Cx CCC CAT CHT rrc 1'AC ACC Gf\- CGA C.C GCr; CIY'f;A(' 1875 183 T F D 1 S K L A P R V F Y T A R R A L D 202

1876 AAC Crc' CAC (>CC GTC AAC AAC 1Y\; CAA ACG ATC' A'IE' GTC; 1Y-(' G(. T CAC; TC-I' GG'C' fXG f.KX' 193'J 203 N L n A V N K S Q T I I V S G E S G A G 222 1936 AM ACG (' fX'G ACG MG C.AG A'IT ATC, ACfl l'AT Tl'l' GfT, C'(,()G AAG AC(, fM TCG A'; 199'' 223 K T E A T. K Q I M R Y F A A A K T G S M 212

1996 CAT rl\, C-f3G ATT' CAC, AAC CX] A'IX.' A'l'G GX'G CKG AA1' CCA (;1\, C'l'('f, C,CA 1'1'r (>iA AAT 20'' 293 D L R I Q N A I H A A N P V L E A F G N 262

2056 C',CG AAG AC ATT CGC AA(' AAC AAC TC.G TC'G (:;T 1'C GX>iN CC rrc ATC, CAG C'l'G CLAT GTG 2LIS 263 A K T I R N N N S 5 R F G R F M Q L D V 2X2

2116 (;'I'C'tX'(:iAA ('.;A (.X'' 47- A^,t; l.11'(X.X: 'IY.'C 1,I'('(;lY-() '(.' Tl"i'(."l'(.'("l'l-;(.;^ NG 'l'(;('l;'l' 2F/', 283 G R E G G I K F G S V v A F L I, E K S R 1(3.? ?176 (,Tr C'lV A(I7(-A('; GA(2 C\ C-A(-7(;AC; CC lY--(;'l',W (-A(: A'l(' '11'(' 'I'A(- ('AA A'lt'l('7 AAC; (>X;.!23S 103 V L T D E Q E R S Y H I F Y Q M C K G:3;?;?

2236 (X GAC C'(.; C'Y'G A1.;.AG (2,AC CCX- 'I'IY' CA'I' A'l1': C-'IY; (:(:(; Cl'(' 7Y'(:('(;'I'AC AA(;'l'At.- A'lY' 229', 323 A D A A M K E R F 11 I L P L 5 Y K Y I 14?

229G AAT CC(J 1'P-. 1\X" C"Pt'i GA('<''(-;(.'C'.A GCX; A1Y'<.';Aj' GAC G'IY' CX'l.;(;A(;'Di' CAC' CluN CrlY"'IY:X2 2355 343 N P L C L D A P G I D D v A E}' H E v C 3(,2 235G GA(- 7\'G 'I'IY' C(;G '1('(: A'IY. AA1' C'IX; ACG C;A(;CN('(';AA (;'IX' (1'(:; A(X' G'l't';'lXX; MX: AT(- (; '1U 2415 36 3 E 5 F R S M N L T E D E V A S V W S I V 382

2416 A(;'l' CMA GTt; C-l\; C'IY; c7-r (XX: AAC C;'IX' GA(; C'IY; A(',N CX'G M'(;,\NC; C.A'I' (/XJ (X'X] A'l'(: (.AC 247'.

383 S G V L L L G N V E V T A T K D G G I D 40.?

2476 (,AC (.X:'C GC'G CC A- (-(, a-x, AAG AA(2 TT(-i C,A(i 1;7=r TIY.' A\A AAC;(.'/:C T(:XC (., C'l <"IY' 25:l', 4()3 D A A A I E G K N L E Y p K K A C G L L 422 2516 '1'1(' CTC GA(.' G('C GA(, CGC Al'l' Cf Ct GAG CTG A('(, G1. Al;(;lr Tt'G 'I'AT C'-C;(-,tXj AAT 2',95 423 F L D A E R I R E E L T V K V S Y A G N 442

259G ('A(, GAG A: C(,C <'-' (.\X; T(X, AA(, C.'AG GAA GC ('A l.lAC A1Y, C"l'(' A(, TC2G TC(, cr c 2655 443 Q E I R G R W Q E D G D M L K S S L A 462

2656 AAG C,CG ATC, TAC GAC AAG 'PTCJ 11\ ATC; TGG A'l ArP CX1C (,1 r. AAC C.,C At7: A1YC AAG 2715 46'] K A M Y D K L P M W I I A V L N R S I 4S2

2716 Cl' CCG GX:: GC' 11Y' AAG A'IY' rrc A1Y: GX.' A, C1(' C,AC A'IY: Tl(' CX' IYC C,AA Clr rrc 2775 483 P P G G F K I F M G M L D I F G F E V F 502

27'16 AAG AAC AAC 'IYC C1C; GAG CM, 1-I( TTC All: AAC A- M'(. AA(- C;AA AT C'I'ti CAC; AA(:; AAC 2835 503 K N N S L E Q F F I N I T N E M L Q K N 522

2836 TT' GTC GA(: A'PC GT(2 Tl'C GAC CCC CwA(, AC' AAG C'l\; TAT Ct-iT GAC C,M:, GGT G1\' TC'C TCC 2895 523 F V D I V F D R E S K L Y R D E G v S S 542 2896 AAG GAG 1G ATT' 11( ACC TCG AAC GCA CAA C;TC ATC AAG A1( 1\, ACG CCG AA(; AAC AAC 2955

543 K E L I F T S N A E V I K I L T A N N 562

2956 U.:; GTC C-: Gcr G('(J Cl'C CY\(I GAC C.AG 1C'( (-XC CCT CXA tOC' AC; C CY\C C=A AAG TTC 3015 563 S V L A A L E D Q C L A P G G S D E K F 582

3016 C'TC TC.G ACC TC>2 AAG AAC CG C'PG AAA CiGA ACC ACC AAC TrC AAG CCT GCG AAG G( TCT 3075 583 L S T C N A L K G T T K F P A K V S 602

3076 CCG AAC ATC AAT TTC' CTC ATC 1'G CAC ACT GTC GC,C GAC ATC CAG TAC AAC CCC GAA C) 3135

603 P N I N F L I S H T V G D I Q Y N A E G 622

3136 T'IE' CTC TTC' AAA AAC AAA GAT GTc CT(3 CGA GCA CAA A1(' ATG GAA A'1t' GTG t'AC; CAA AC,C 3195 623 F L F K N K D V L R A E I H E I V Q Q 5 642

3196 APL-i AAC CC'C GIT CTC GC'C CAA CTC TTC GCT a3c ATC GTC ATCi CAG AAGGCyG AAG ATG CCC 3255 643 K N P V V A Q L F A G I V H Z K G K M A 662

3256 A, GGA CAA CTG ATT GGC TCG CAG TrC CTG TC'G CAC Cl'G CAG A(iC CTC ATG CAA cr ATC 3315 663 R G Q L I G S Q F L 5 Q L Q S L E L I 682

3316 AAC ACC ACC CAG CCT CAC 11' Al'r CCJC 13C' ATC AAG CCti AAC GAC ACGi AAG AAG1 CCC C1( 3375 683 N 5 T E P 8 F I R C I K P N D T K K P L 702

3376 GAC Tt-G GTGi C.CG 1'CG AAA ATG crc ATT CAG CTC, CA(' G('G crc TC'C G'TC CC- GAC, c7'"r CTr 3435 703 D W V P S K M L I Q L H A L S V L E A L 722

3436 CAG CTC CG=' CAA CTC' C,CC TAC TCT TAC AGA CGT CCG 11(' AAG GAG I'PC crc rrc CAG 1C 3495 723 Q L R Q L C Y S Y R R P F K E F L F Q F 742

3496 AAG m A1\2 CAC C1' TCG GC'T 1wP GAA AAT <'CA AAT C'l(-i GAC C'(:C AAA C,AA GCT CC(i CPt.i:3555 743 K Y I D L S A S E N P N L D P K E A A L 762

3556 AC'A C1'C crc AAA A() AGTC AAA CTC1 CCC AGC CA G M TAC CA(;-}G AAG AC'^ A'PCi Grr 3615 763 R L L K S S K L P S E E Y Q L G K T V 782

f 3616 TTC CTC AAG CAG ACG GGC GCG AAA GAA CTG ACG CAG ATT CAG AGA GA TGC C-TT TCT TCT 3675

783 r K Q T G A L S Q I - Q R C L S S 802 3676 TCG GAG CCT CTC GTC TCA GTG CTC GG GCG TAC TAC GCT GGC AGA CGC CAC AAG AAG CAG 3735

803 P L V S V L A Y A C R R Q 822

3736 C13 cm AAA AhG ACC CCC TTC ATC ATT CGC GCC CAG GCT CAC ATC OGC AGA CAC CTG GTG 3795 823 L L X T P Z I R A Q A H I R R L V 842

3?96 GAC AAC AAC GTC AC CCC GCG ACT GTT CAG CCG GCG TTC GGA TCC ACT CGA GAT GCA GGC 3855

843 D N N V S P A T V Q P A F G S T R D A G 862

3856 GGT GC1 TAA ttaatcaccOttOtgotcacetOtcaaatcgacaaaggaaacacactCcotgOaDcatgtgccOcatt 3932 863 G A 865

3933 ataaagaaactgagttOtCccgctOtogcetgeaggtgecacatccacaaaaaccggcOgactotaaataggag tOtEtc 4012 4013 ycaDcaaUcagogaaagettatgactOgatccoaatctctgaaeggarOtOtOgcOgacctoOctgatOttgat cgccgt 4092 4093 cOacacacOcOccacaLgOgtcaRtacacaagacagctatcagttotUttagtcqaacOgottaacacaatect tgcccc 4172 4173 ccOgaggOggatccactagtectagaDcoSccOccaccOcOgtOgaDctccagcEtUtgttcoctEtagUgagg Ottaet 4252 4253 tgcOcDcttOgogLaatcatgOtcatagctOttccctgtOtgsaattOttatccOctcacaattccacacaaca tacOag 4332 4333 cCggaagcataaagtOtaaagcctOgOgtgcctaatgagtgagctsactcacattaattgcottgcOctcacgc ccoct 4412 4413 ttccagtogogaaacotOtogtgccaDctgoattaatgaatcggccaacgOgcgOggagagOcOgtttgogtat toggcg 4492 4493 ctchtcc, gcrtcctouctcactgactcDctgcgctcggEcgtCcOgckOcOgogaDcOgLatcagetcactcaaagOcgg 4572 4573 taatacggttatccacagaatcaggggataacOcaggaaagsacarotgaDcaaaaggccaDcaaaagOccagg aaccOt 4652 4653 aaaaaggoogcgetgctOgagtttteccataggctccgececcaLgacoaDcatcacaaaaatcOacDctcaag Ecagag 4732 4733 gtOgOgaaacocOacaggactateaagataccagOcgtttcaccctOgeaDctccctcgtgcgctatcctOtcc cDacec 4812 4813 tgcoOcetaccggatacctOtccoccetcctccatCcgggaagcgtogcOcettctcatagctcaegotOtagO tatctc 4892 4893 agttcggtgtaggecothcoctccaagctogOckOtgtgcacgaacccOccutccaDcccDaccOctgcOccet atccgg 4972 4973 taactateDtcttgagtccaacccogLaagacacoacttatcgecactOgcagcagccactgOtaacaggatta Dcagag 5052 5053 cUaggeaLgLagOcgOtgctacagagtCcrtgaagtggtggCctaactacgOctacactageaggacagtatet Ogtatc 5132 5133 tgoDctctgctgaaDccaghtaccrtcggaaaaagagttggtagatettgatcoggcaaacaaaccaccgctgg tagagg 5212 5213 tgOttteteLguttgcaaDcagoagattaegcgeagaaaaaaaggatctcaagaagatcetttgatothtecta eggOgt 5292 5293 ctgaegctcagtOgaacgaaaactcacgttaegggatettggtcatgagattatcaaaaaggatcttcacctag atCcht 5372 5373 ttaaattaaaaaLgaagttttaaatcaatetaaagtatataLgagtaaaceLggtctgacagetaccaatgcet aatcag 5452 5453 tgaggcacctatctcagOgatctgtotatetcOttcatccatagttgcctgactcOccotOgtotagataacta egatac 5532 5533 gOgagggettaccatctOgccOcagtgctgcaaLgataCcgcgagacccacDctcaCcgOctccagatttatca Dcasta 5612 5613 aaCcaDccagccggaagOgcogaDcOcagaagtOgtcatgcaactttatccDcotccatccagectatteattO ttgcOg 5692 5693 ggaagctagagLaagLagetcgacagttaatagtttgagcaacgttOttgccattgctacagOcatcgtOgtOt cacOct 5772 5773 cgecotUtOgeaLgocetcattcaUctcagOtecccaacgatcaagOcDagttacatgatcccccatuttgtgo aaaaaa 5852 5853 gcogttaDctcceteggtcetccDatagttUtcagaagtaaghtgOccOcagtottatcactcaLgOttatOgc aDcact 5932 5933 geataattctcttactotcatgocatcOgLaegaLgettttctgtgactOgtgagLactcaaccaagecathct gagaat 6012 6013 agtOtaLgcoecgaccoagttgctattgcccggcgtcastacgggataataccgcOccacataDcagaacetta aaagtg 6092 6093 ctcatcattggAaaacottcetaggOgagaaaactctcaaggatcetacogctOttgagatccagtecgatota acocac 6112 6173 teOtgcacOcaactgatcttcaDcatattetacettcaccagagtttctOgOtgaDcaaaaacaggaagOcaaa atgOcg 6252 6253 caaaaaagOgaataagOgagacacOgaastattgaatactcatactcttcchtttEcaatattattgaaDcatt tatcag 6332 6333 ggttattotctcaLgaDcggatacatatttgaaLgLatttagaaaaataaacaaatagOgOtceegOgcacatt tccocg 6412 6413 aaaagtgecac 6423

pTetO7Sa' l-llyoA -> Cenes DNA sequence 6346 bp ctgacgcgocct.. gaaaagUgccac circular pTetO75AGl-FoA MyoA was cloned into pSlTetO7CFP via NsiI/pacI 1 ctgacocDcactgLagcOgcocattaagcDcgOcggOtgtOgtggetacOcOcaDcgtgacOgctacacrtgcc agcOcc 80 81 ctagcocccDctcatetcOcretcetcocUtccetCctcOccacOttcOccOgetetccccOtcaaactctaaa togOgg 160 161 gOtcccettagggetccDatEtagUgcettacggOacctcgaccocaaaaaacEtgattagggUgatOgetcac otagtg 240 241 ggacatcgccctgatagacgotetetegcacettgacgUtggagtccacghtatetaatagtOgactceLgtCc caaact 320 321 ggaacaacactcaacectateteggtctateceteLgatttataagOgatettgccoatUtcOgcctattggtt aaaaaa 400 401 tgagcgatttaacaaaaatetaaegOgaatettaacaaaatattaaGgottacaattCccattcoccatecagO ctgOg 480 481 caactOttgogaagOgcOatcOgtgcOggectetecgctattacgccaDctogcgaaaggOggaLgtgctgeaa ggcOat 560 561 taagttggOtaacoccagggetetcccagtcaegaegttgraaaacOaeggccagtgagcgcgcotaatacOac tcacta 640 641 taggOcOaattOgotaccOaDctagactetcacetttctctatcactgatagggagUgOtaaactegacettca cetetc 720 721 tctatcactgatagOgagtOgLaaactegacttccacttttctctatcactgatagOgagtogLaaactcoact ttcact 800 801 tetctotatcactgatagOgagUgOtaaactogacUtecacetUtctceatcactgatagOgagtOgLaaactc Oacett 880 881 cactetcctctatcactgatagOgagtogLaaactcgacretcacrctectctatcactgatagOgagtggLaa actega 960 961 ggecOacOgLatcoataaDctccaaLgtgeacctotaggaageLgLagtcactgctgatectcactOttctcOg caaggg 1040 1041 cOgacgaccOgageacagEtettotogOcagaDccgttgtgcagotttcogetatCcteggeLgtotcacaLgt atcatt 1120 1121 gtcotgLaaacacacggttOgaLgecggtttcgctgcaccachtcattatttcttctOgethtttgacgagt AT& CAT 1198 1 M 2

1199 GG CAG AAG CTC ATC TCC GAG GAG GAC CTG CTG CAT C'AT CAT CAT CAT CAT CAT GAT G5T 1258

3 E Q K L I S E E D L L H H H D G 22

1259 ACC GAG CTC GCG AGC AA& ACC ACG TCT GAG GAG CTG AAA ACG GCC AC& GCG CTG AAG AAG 1318

23 T E L A S K T T S E L A S A T A L K K 42

1319 AGG TCG TCC GAT GTC CAC GCG GTC GAC CAC TCC GOC AAT GT& TAC AAA GGA TTT CAA ATC 1378

43 R S S D V A V D H S G U V Y K G F Q Z 62

1379 TGG ACG GAC TTG GCG CC& TCG GTO AAG GAG GAG CCG GAC CTG ATG TTT GCC AAG TGC ATC 1438

63 W T D L A P S V K F E P D L M F A K C r 82 1439 GTG CAG GCG GGG ACA GAC AAG GG AAC TTG AOC TGC GTC CAG ATC GAT CCA CCG W C TTC 1498

83 V Q A G T D K G N L S C V Q I D P P G P 102

1499 GAC GAA CCG TTC GAA GTC CCG CAG GCG AAT &CG TGG AAC GTA AAC AGC CTG ATC GAC CCC 1558

103 D E P F E V P Q a N A W. V N S L I D P 122 1559 AT& ACG TAC GGA GAC ATC GGC ATG TTG CCT CAC ACG AAC ATT CCT TGC GTC CTC GAC TTC L618

123 T Y G D I G L P R S N I P C V L D F 142

1619 CTC AAG GIG CGC TTC ATO AAG AAT CAA ATC TAC AC5 ACT GC5 GAC CCG CTC GTC GTC GCC 1678

143 L K V R F H K N Q I Y T T A D P L V V A 162

1679 ATC AAT CCC TTC CGC GAC CTC GGG AAC ACC ACG CTC GAC TGG ATT GTT CGA TAC AGA GAC 1738

163 N e F R D L G T T L D I V R r R D 182 1739 ACT TTC GAC CTC TCC AAA CTC GCG CCC CAT GTT TTC TAC ACC GCC CGA CGC GCG CTC GAC 1798

183 T F D L S L A P V F Y T A R R A L D 202

1799 AAC CTC CAC GCC GTC AAC AAG TCG C ACG ATC ATC GTG TCC GGT GAG TCT GGC GCG GGC 1858

203 N L H A V N S Q T I I V S G E S G A G 222

1859 AAG ACG GAG GCG ACG AAG CAG ATT ATG AGG TAT TTT GCG GCG GCG AAG ACG GGG TCG ATG 1918

223 K T A T K Q I R Y F A A A T G S 242

1919 GAT TTG CGG ATT CAG AAC GCG ATC ATG GCG GCG AAT CCA GTG CTT GAG GCA TTT GGA AAT 1978

243 D R I Q N A I A A N P V E A F G N 262

1979 GCG AAG ACG ATT CGC AAC AAC AAC TCG TCG CGT TTC GoA CGC TTC ATG CAG CTG GAT GTG 2038 263 A K T I R N N N S S R F G R F H Q L D V 282

2039 GGT CGC GAA GGA GGC ATC AAG T!T GCC TCC GTC GTC GCC m CTC CTG GAA AAG TCG CGT 2098 283 G R E G G I K F G s v v A F L L E K S R 302 2099 GTT CTC A CAG GAC GAA CAG GAG CGG TCG TAC CAC ATC TTC TAC CAA ATG T&C AAG GGG 2158

303 V L T Q D E Q E R S Y H I F Y Q M C K G 322

2159 GCG GAC GCG GCG ATG AAG GAG CGC 'TTC CAT ATC CTG COG CTC TCG GAG TAC AAG TAC ATC 2Z18

323 A D A A K E R F I L P L S E Y K Y z 342 2219 AAT CCG TTG TGC CTG GAC GCG CCA GGG ATC GAC GAC GTC GCG GAG TrC CAC GAA GTC 1C 2278 343 N P L C L n A P G I D D V A E P H E V C 362 2279 GAG TCG TTC CGG TCG ATG AAT CTG ACG GAG GAC GAA GTC GCG AGC GTG TCG AGC ATC GTG 2338

363 S F R S N L T E D E V A S v W S I v 382 2339 AGT GGA GTG CTG COG CTT GCC AAC GTC GAG GTG ACA GCG ACG AAG GAT GGG GGG ATC GAC 2398

383 S G V L L L G N V E V T A T D G G Z D 402

2399 GAC GCC GCG GCG ATC GAG GGG AAG AAC TTG GAG GTT TTC AAA AAG GCC TGC GGG CT& crc 2458 403 D A A A I E G K N L E V F K A C G L L 422

2459 TTC crc GAC GCG GAG CGC ATT CGC GAA GAG CTG ACG GTG AAG GTT TCG TAT GCG GGG AAT 2518 423 F L D A E R I R E E L T V K V s Y A G N 442 2519 CAG GAG ATC CGC GGC CGG TGG AAG CAG G M GAC GGA GC ATG CTC AAG TCG TCG CTC GCG 2578

443 Q E I R C R W K Q E D G D L S S L A 462

2579 AAG GCG ATG TAC GAC AAG TT& TTC ATG TGG ATC ATT GCC GTG TTG AAC CGC AGC ATC AAG 2638

4G3 A Y D K L F n W I I A v L N R S I 482 2639 CCT CCG GGC GGC TTC AAG ATC TTC ATG GGC ATG CTC GAC ATC TTC GGC TTC GAA GTC TTC 2698

483 P P c G F K I Y G M L D I F G F E v F 502 2699 AAG AAC AAC TCG CT& GAG CAG TTC TTC ATC AAC ATC ACG AAC GAA ATG CT& CAG AAG AAC 2758

503 K N N S L F Q F F I N I T N E M L Q 6 N 522

2759 TTC GTC GAC ATC GTC TTC GAC CGC GAG AGC AAG CTG TAT COT GAC GAG GGT GIC TCC TCC 2818

523 F V D I V F D R E S L Y R D G V S S 542

2819 AAG GAG TTG ATT TTC ACC TCG AAC GCA GAA GTG ATC AAG ATC TTG ACG GCG AAG AAC AAC 2878 ' '

543 K E L I F T S N A V I K I L T A K N N 562

2879 TCG GTG CTC GCT GCG CTC GAG GAC CAG TOC CTC GCC CCT GGA GGC AGC GAC GAA AAG TTC 2938 ''

563 S V L A A L E D Q C L A P G G S D E K F 582

2939 cEc TCG ACC TOC AAG AAC GCG CTG AAA GGA ACC ACC AAG TTC AAG CCT GCG AAG GTC TCT 2998 '' 583 L S S C K N A L G S T K F K P A V S 602

2999 CCG AAC ATC AAT TTC C'TC ATC TCG CAC ACT GTC GGC GAC ATC CAG TAC AAC GCC GAA GOC 3058 ''

603 P N I N F L I S S V G D I Q Y N A E G 622

3059 TTC CTc TTC M A AAC AAA GAT GTC CTG CaA GCA GAA ATC ATG GAA ATC GT& CAG CAA AGC 3118 623 F L F K N A D V L R A E Z E Z V Q Q S 642

3119 AAG AAC CCC GTT GTC GCG CAA CTC TTC GT GGC ATC GTC ATG GG AAG GGG AAG ATG GCC 3178

643 N P V V A Q L F A G I V E C K A 662

3179 AAG GGA CAA CTG ATT GGG TCG CAG TTC CTC TCG CAG CTG CAG AGC CTC ATG GAA CTT ATC 3238

663 R G Q L I G S Q F L S Q L Q S L E L I 682

3239 AAC AGC ACC GAG CCT CAC TTC ATT CGC T&C ATC AAG CCG AAC GAC ACG AAG AAG CCC CTC' 3298

683 N 5 S P G F I R C Z K P N D S P L 702

3299 GAC TGG GTG CCG TCG AAA ATG CTC ATT CAG CTG CAC GCG CTC TCC GTC CTC GAO GCT CTT 3358

703 D W V P S n L Z Q L H A L S V L E A L 722 3359 CAG CTC CGT CAA CTC GGC TAC TCT TAC AGA W T cca TnC AAG GAG Trc CTC TTC CAG TTC 3418 723 Q L R Q L G Y S Y R R P F K E F L F Q F 742

3419 AAG TTT ATC CAC CTC TCG GCT TCT CAA AAT CCA AAT CTG GAC CCC AAA GAA GCT GCG CTG 3478

743 K F I D L S A S E N P N L D P K A A L 762

3479 AG CTC cDc AA AGC ACC M A CTG CCC AOC GAA GAA TAC CAG CTC GGG AAG ACA AT& GTT 3538 763 R L L K S S K L P S E Y Q L G R S V 782

3539 TTC CTC AAG CAG ACG GGC GCG AAA GAA CTG ACG CAG ATT CAG AGA GA TGC CTT TCT TCT 3598

783 F L Q T G A E L T Q I Q R E C L S S 802

3599 TGG GAG CCT CTC GTC TCA GTG CTC GAG G X TAC TAC GCT GGC AGA CGC CAC AAG AAG CAG 3658

803 W E P L V S V L E A Y r A G R R K Q 822 3659 CTG CTG AAA AAG ACC CCC rc ATC ATT CGC GCC CAG GCT CAC ATC CGC AGA CAC CTG GTG 3718 823 L L K T P I I R A Q A I R R V 842

3719 GAC AAC AAC GTC AGC CCC GCG ACT GTT CAG CCG GCG TTC GGA TCC ACT CGA GAT GCA G&C 3778

843 D N N V S P A T V Q P A G S T R D A G 862

3179 GGT CCT TAA ttaatcaccOttOtgotcacUtotcaaategacaaaggaaacacacUteOtgcagcatOtgcoccatt 3855 863 G A 865

3856 atuaagaaactgagtegetccOctOtOgcrtgcaggUgtcacatccacaaaaaccgOcagactctaaataggag tOtetc 3935 3936 gcaDcaaacagcOaaagettaLgackggOtcagaatctetgaaeggaLgtgtOgcggacctOgchgaLgeLgat oOccOt 4015 4016 cgacacacocgccacaLgOgecaatacacaagacagctatcagttgUtttagtcOaaccggetaacacaattce tgcOcc 4095 4096 coagagggOgatccactagetctagagcOgccgccaccgcgOtOgagctccagOtEttOttcccettagtgagg gttaat 4175 4176 tgcDcOcttOgagLaatcaLggEcatagctOtttcctOtgtgesattgetatccOctcacaatcccacacaaca tacgag 4255 4256 ccOgaaDcataaagtotaaaDcctOgOgtgcctadUgagtgagctaactcacattaattgagttgcDctcactg cccOct 4335 4336 tcccagEcOggaaacaLgecOtgocaDctgcattaargaatoggccaacDcOcOgUgagaggogOteLgcgLat tgOgcg 4415 4416 ctcttccDcetcctcDctcactgactoOctgoOctcggecgEtcggctgcggcOaucOgLatcaDctcactcaa aggagg 4495 4496 taataeggUtatccacagaatcaggOgataacOcaggaaagaacaLgtgageaaaagOccagcaaaagOccagg saccot 4575 4576 aaaaaggcOgcOttgctggcgtetteccatagOctccgcccacctgacgagcatcacaaaaatcOacOctcaag tcagag 4655 4656 gtOgogaaaccogacaggactataaagataccagOcgtteccecchggaaDctccOtcOtgcOctOtcctUtec cgaccc 4735 4736 tgeegcttaccogatacctOtccgcetUtctcccttcgggaagcgtgOcOcthtctcatagctcacDctgtagg tatotc 4815 4816 agEtcOgtOtagOtcgtCcDctccaegatgOgctOtgtgcacgaacecaccgtEcaDcccoaccgctgcgccet atccOg 4895 4896 taactatcOtcrtgagtccaacccgOtaagacaegacttatcgccachUgcaDcagccactogLaacaggatta gcagag 4975 4976 cOaggLatOtagOcOgtgctacagagetottgaagtggtggcetaactacoDctacactagaaggacagLateL gOtatc 5055 5056 tgaDctctgctgaagccagttacctCcOgaaaaagagttggLagctottgatcagacaaacaaaccaccOctgg taDcgg 5135 5136 tggtUthtttgtUtgcaageaDcagattacDcDcagaaaaaaaggatctcaagaagatccettgatctttecta cOgOgt 5215 5216 ckDacOctcagtOgaacOaaaactcacOttaagggatteLggtcaLgagattatcaaaaaggatcrtcacOtag atcatt 5295 5296 ttaaattaaaaaLgaaghthtaaatcaatataaagLatataLgagtaaacetOgectgacagetaccaaLgctt aatcag 5375 5376 tgagOcacctatctcaDcDatctOtctatetagetcatccatagttgceLgactccoagtcOtOtagataacta cOatac 5455 5456 gggagOgchtaccataLggccccagtgotgcaaLgataccgcgagacccacgctcaccgOctccagatttatca Dcaata 5535 5536 aaccaaccaDcOggaegOgccDaDcocagaagtOgtcctgoaactttatccOcctccatccagtctattaattO ttgacg 5615 5616 ggnaDctagagLangLagetcOccagttaatagtttgcgoaacgttOttgccattgctacaggcatcgtOgtOt cacDct 5695 5696 cOtcOtttOgLatOgchtcattcaDctccggUtcccaacgatcaaggcgagetacaLgatcccacatOttgtgc aaaaaa 5775 5776 gcOgttagatcettcggecctccOatcgUtOtcagaagLaagttggccOcagtghtatcactcaLgghtaLgge aDcact 5855 5856 gcataatectcetactotcaLgecatccOtaagatgattthctgtgactggtgagLactcaaccaeOtcatect gagast 5935 5936 agtotatgcOgcDaccoagttgetcttgcccOgcotcaatacgggataataccOcOccacataDcagaacttta aaagtg 6015 6016 ctcatcattggsaaacOtectecOggOcgaaaactctcaaggatcrtacOgetgttgagatccagttagatOta acccac 6095 6096 tcOtgcacccaactgatcetcaDcatcrtttacttCcaccagcgttectgOgtgagcaaaaacaggaagOcaaa atgccg 6175 6176 caaaaaagggaataugOgcoacacagaaarOttgaatactcatactOttccEtettcaatattattgaagcate tatcag 6255 6256 ggetattOtatcaLgagcOgatacatateLgaatOtatttagaaaaataaacaaatagOggEtCcOcDcacatt tCcccg 6335 6336 aaaagUgocac 6346

* TelO7SAC4-GFP -> Ccnes DNA sequence 4556 bp ctgacgcgcoct. gaaaagUgccac circular Response Plasmud for TATI, Ising bradyzoite specific promoter SAG4 + 7 tetO at -70 - no selecection 1 ctgacOcOccUtUtaDcOgcDcattaagcgcggcOggtgtggtggttacOcOcaDcgtgaccDctacacEtgec aDcgcc 80 81 ctaDcOcecgctcctetcDctetatecccrtcctttctegecacgttaDccOgctEtcOcOgEcaaDctctaaa tOgOgg 160 161 gctcccLttagOgtCccOatttagtgetttacOgUacctagaccccaaaaaacrtgattaggOtgaLgOtccac Otagtg 240 241 gOccatogcectgatagaegOttUttagccateLgaegttggagtccacOtEctttaatagggactcttOtcoc aaact 320 321 ggaacaacactcaacoctatctcggtctatEcrtetgatttataagOgatettgecOattccggcctattOgtt aaaaaa 400 401 tgagctgatetaacaaaaatUtaacOcOaetttteacaaaatattaacocUtacaatttccatecoccatCcag octgcg 480 481 caactgttOggeagOgcOatoggtgcOgOcctcetcgctattacgOcaUctOgcOaaagogggaLgtgctgcaa ggogat 560 561 taagetUgOtaacOccagOgttttcccagecacoacgttgLaaaacOacOgccagtgaDcgoocgrastacOac tcacta 640 641 tagOgcoaattOgOtaccOagctcOactetcacrtttctctatcactgatagggagtogtaaactcoactttca cetEtc 720 721 tOtatcactgatagggagtOgtaaactcOacretcachttEctctatcactgatagogagtgOtaaactcOace ttcact 800 801 tttotctatcactgatagOgagtOgLaaactogachttcacrCttctatatcactgatagggagtOgLaaactc Oacttt 880 881 cactettctOtatcactgatagOgagtOgteaactegacttCcacetttctOtatcactgatagogagtggLaa actcga 960 961 gOtcgacOgLatagataaecttacOccOctgagactaactagaaagaagtOtgcaacagttcatgagogacaaa aggaat 1040 1041 gtgatgcgotecDcrtgaagaeggaatotetaaDcacgecaacaetacOcattOgcgaatOtecatgactottc aLgtOg 1120 1121 ttcatoggatcatttgaaaacatcgtgagOctgOtaceLggtcgcaaacgecOtagtotagLacOgacaataac Otogec 1200 1201 gttcaagOggacOcagtectcOgaagaegagtcgcagcatactgeeactgetttcUtctOtcUtcaacc ATG CAT 1275 1 2 1276 AGT AAA GGA GAA GAA CTT TTC ACT GGA GTT GTC CCA ATT CTT GTT CAA TTA GAT GGT GAT 1335

3 S K G L F T G V V P I L V r. L D G D 22 1336 GTT AAT GGG CAC AAA TTT TCT GTC AGT GGA GAG GGT GAA GGT GAT GCA ACA TAC GGA AAA 1395 '

23 V N G H K F S V S G G E G D A T Y G K 42 ' '

1396 CTT ACC CTT M A TTT ATT TGC ACT ACT GGA AAA CTA CCT GTT CCA TGG CCA ACA CTT GTC 1455

43 L S L K F Z C T S C K L P V P W P T L V 62

1456 ACT ACT TTC TCT TAT GGT GTT CAA TGC TTT TCA AGA TAC CCA GAT CAT ATO AAG CGG CAC 1515

63 T T F S r G V Q C r s Y P D K R H 82 ', 1516 GAC ITC TTC AbG AGC GCC ATG CCT GAG GGA TAC GTG CAG GAG AGG ACC ATC TTC TTC AAG 1575 83 D F F K S A P E G r v Q E R T I P F 102 1576 GAC GAC GGG AAC TAC AAG ACA CGT GCT GAA GTC AAG TTT GAG GGA GAC ACC CTC GTC AAC 1635

103 D D G N T R A F V K F E D T L V N 122 1636 AGG ATC GAG CTT AAG GGA ATC GAT TTC gAG GAG GAC GGA AAC ATC CTC GGC CAC AAG TTG 1695 123 R I r. L K G r D F K L D G N I L G a K L 142 1696 GAA TAC AAC TAC AAC TCC CAC MC GTA TAC ATC ATG GCC GAC AAG CAA AAG AAC GGC ATC 1755

143 r. Y N Y N S a N V Y I H A D K Q K N G I 162 1756 AAA GCC AAC TTC AAG ACC CGC CAC AAC ATC GAA GAC GGC GGC GTG CAA CTC GCT GAT CAT 1815

163 A N F K T R G N I E D G G V Q L A D a 182 1816 TAT CAA CAA AAT ACT CCA ATT GGC GAT GGC CCT GTC CTT TTA CCA GAC AAC CAT TAC CTO 1875

183 Y Q Q N S P r G D G P V L L P D N a Y L 202 1876 TCC ACA CAA TCT GCC CTT TCG M A GAT CCC AAC GAA AAG AGA GAC CAC ATG GTC CTT CTT 1935

203 S T Q S A L S K D P N E R D V L L 222

1936 GAG TTT GTA ACA GCT GCT GGG ATT ACA CAT GGC ATG GAT GAA CTA TAC AAA GCT GCA GTT l99S 223 E F V T a A G I T 8 G D L Y A A V 242 1996 AAT TAA tcacogttotgOtcachtctcaaategacaaaggaaacacacrtagtgoageaLgtgccccattataaagaa 2073 Z43 244

2074 actgageLgtcoogctOtOgcetgcaggUgEcacatccacaaaaacOggccgactctaaataggagtOtetcoc aDcaag 2153 2154 cagagaaaghttatgachOgOtccOaatctctgaacggargtgtgOcggacctgOctgatattgatcDccutcO acacac 2233 2239 gcOccacaLgOgtcaatacacaagacaDctatcaytt4ttetagtogeacogattaacacaatEcrtgccOcoc cDagUg 2313 2314 ggatccactagtCctagaDcgOccOccacgcOgtggagctccagetteLgtUcecettagtgagOgttaattgc OcOct 2393 2394 togcOtaatcatOgtcatagctOtttcctgUgtgaaatLgttatccOctcacaathocacacaacatacoaUco ggaaDc 2473 2474 ataaagtgtaaegectgOgOtgccteargagtgagatoactcacattaattgcOttgcOctcactgcccOctte ccagtc 2553 2554 gOgaaacct(3tcstgccagctgcattaatgeatoggccaacgogcgoggagaggcOgtttgcOtattOggOgo totEccg 2633 2634 cetcctcgctcactgactcOctgcgetcogtcOtUcggcgaggcoagagOtatcagotcactcaaaggcgOtaa tarOg 2713 2714 ttatccacagaotcagoggataacgoaggaaagaacaLgtgagcaaaagOccaDcaaaagOccaggaaccUtaa aaDggc 2793 2794 cOcOttgctOgogttttccatagOctccDcecOcctgacgagcatcacaaaaOtogacgctcaagEcagagOtg Ocgaa 2873 2874 acOcOacaggactatesagataccagOcOtetccccctggaagetccctcOtgcOctctcctOtCccOacoctg ococtt 2953 2954 accggatacctOtcaUcotttctccotCcoggaaDcgtggcgctetctcatagctcacoctOtaggtatctcag tccgot 3033 3034 gtagOtacUtEcOctccaagctOggctOtotgcacosacocccagtCcaDcccOaccgctgcOcattatccOgL aactatc 3113 3114 gtottgagEccaacccOgLaagacacgacUtatcoccactggcagcaDccactogLaacaggattagcagagcO agOtat 3193 3194 gtagOcogtgetacagagetcttgaagtOgtOgcctaactacggctacactagaeggacagtatttOgLatctg cOctet 3273 3274 gctgdagccagttaccetcOgaaaaagagttOgLagetcrtgatccgocaaacaaaccaccgctOgLaDcOgto gEtett 3353 3354 ttgtttgcaagcaUcagattaegcOcagaaaaaaaggatctcaagaagatccrttgatcttetctaegOggtaL gacOct 3433 3434 cagtOgaacgaaaactcacgttaegOgatteLgutcargagattatcaaaaaggatcEtcacotagatccettt aaatta 3513 3514 aaaatgaagetttaaatcaatcteaagLatataLgagtaaaceLggtCtgacagttaccaatgottaatcagtg agOcac 3593 3594 ctatctcagcOatckOtctatetogtecatccatagUtgcctgactccccOtogtgLagataactacDatacOg gagOgc 3673 3674 ttaccatchggcOccagtgctgcaetgatacoDcDagacccacgctcaccOgotccagathtatcaDcaataaa ccagcc 3753 3754 aDccOgaagggeegaDcOcageagtOgtcctgcaactttatccgactccatccagtetattaattOttgocgOg eaDcta 3833 3834 gagLaagtagtecOccagttaatagUttgcOcaaegergttgccattgctacaggcatcgtOgtOtcacOctcO teottt 3913 3914 ggtaLggOttcatUcagctcOgOttCccaacOatcaaggogagUtacaLgatcccccaLgeLgtgcaaaaaaDc ogetag 3993 3994 ctccetcgOtcctccOatcOttetcagaagteagEtggacgcagtgttatcactcatOgttaLgocaDcactgo ataatt.4073 4074 ctcrtactOtcaLgccatcagLaagaLgcttttctgtgactggtgagtactcaaccaagtcattetgagaatag tOtatg 4153 4154 cgOcoaccgagEtgctCttgccCgOcOtcaatacOggataatacoDcOccacataucagaactttaaaagtget catcat 4233 4234 tggaaaacOtCcttegOgOcoaaaactatcaaggatettaccgctottgagatccagttegatgtaacccactc Otgeac 4313 4314 ccaachgatottcaDcatcttetacttccaccaDcgtetatgggtgagoaaaaacaggaagocaaaatgccaca aaaaag 4393 4394 ggaataagOgcoacacogaaaLgttgaatactcatactattcctttetcaatattattgaaDcatttatcagog Etattg 4473 4474 tetcaLgagcogatacatatttgaatgLatetageaaaataaacaaatagOggttCcOcOcacatttcocogaa aeDtgc 4553 4554 cac g556

TetO7SACI -Gl. r -> Cenes DNA sequence 4479 bp ctgacgcgccut geesagtgccac circular Response Plasmud vor TATI. muninal SAGl-Promoter nth 7TetO placed at -70 -no selection 1 ctgacOcOccctOtagcggOgcattaascgcOgcgggUgtggUggttacOcOcagcOtgacoDctacactLgec aUcOcc 80 81 ctagcgCCcOctcetthcgetetcetcccttcottEctcgccacgEtegoOgoctttccccatcaagotctaaa toggOg 160 161 gOtccetttaggOteccuatEtagtgcthtacOgoacetcgaccccaaaaaacttgattaggOtgaLggUtcac utagtg 240 241 gOccategecctgatagacOgttttecOccOtetgacgttggagtccaeuttcEttaataguggactcrtOtEc caaact 320 321 ggaacaacactcaaccOtatctOgutctattctUttgatttataagogatettgccOatttogocotattogEt aaaaaa 400 401 tgaDctgatttOacaaaaetttaacOcOaattEtaacaaaatattaacocttacaatetccatecDccatEcag gaLgag 480 481 caachOtiLgOgaaggocOataggtgcagOcctctecgatattaegecagctOgOgaaagOgggargtgctgOa agOcoat 560 561 taagetOgotaacOccagOgUttecccagtcacOacgttgLaaaacoacgoccagtgaDcOcgcOtaetacOac tcacta 640 641 tagggcOaattOgOtacagagetcDacretcactettctotatcactgatagogagtOgLaaactcDacretca cttetc 720 721 tctatcactgatagggagtggtaaactcOactetcacttttotctatcactgatagOgagtgOtsaactcgact ttcact 800 801 tetctotatcachgatagOgagtOgLaaactcOacettcacetttOtctatcactgatagogagtogLaaactc gactet 880 881 cactetectctatcactgatagOgagtOgLaaactcgacttecacetttctctatcactgatagggagtgOtsa actcOa 960 961 gOtegacOgtatagataaDcttcaatgtgcacchotaggaagctgtagtcactgctgatectcactOttctcoo caagOg 1040 1041 cogacOaccggagtacagtttttgtgOgeagaDcOgetgtgcagchtEccgttchtctogg*tOtotcacaLgt Otcatt 1120 1121 gtcOtutaaacacaeggetggaLgtcggettcgctgoaccactCcattattecteckgOtttEttgaegagt ATG CAT 1198 1 11 H 2

1199 AGT A4A GGA GAA GAA CTT TTC ACT GGA GTT GTC CCA ATT CTr GTT GAA TTA GAT oor GAT 1258 3 S R G E L F T G V V P I L V Y. L D G D 22

1259 GTT AAT GGG CAC AAA TTT TCT GTC AGT CGA GAG GGT GAA GGT GAT GCA ACA TAC GGA AAA 1318,

23 V N G H F S V S G E a Y G D A S Y G 42 1319 CTT ACC CT AAA TTT ATT TGC ACT ACT GGA AAA CTA ccr GTT CCA TOG CCA ACA CTT GTC 1378 43 L T L K P I C T T G K L P V P W P T L V 62

1379 ACT ACT TTC TCT TAT GGT GTT CAA TGC TTT TCA AGA TAC CCA GAT CAT ATG AAG CGG CAC 1438

63 T T F S Y G V Q C F S R Y P D H R 82

1439 GAC TTC TTC AAG AGC GCC ATG CCT GAG GGA TAC GTG CAG GAG AGG ACC ATC TTC TTC AYG 1498

83 D F F K S A P Y. G Y V Q F R S I F Y 102

1499 GAC GAC GGG AAC TAC AAG ACA CGT GCT GAA GTC AAG TTT GAG GGA GAC ACC CTC GTC AAC 1558

103 D D G N T a A E V F E G D T L V N 122 1559 AGG ATC GAG CTT AAG GGA ATC GAT TTC AAG GA GC GGA AAC ATC CTC OGC CAC AA3 TTG 1618

123 R I E L a I D F E D G N I L G L 142 1619 GAA TAC AAC TAC AAC TCC CAC AAC GTA TAC ATC ATG GCC GAC AAG CAA AAG AAC GGC ATC 1678

143 E y N Y N 8 H N V Y I A D Q K N a I 162 1679 AAA GCC AAC TTC AAG ACC CGC CAC AAC ATC GAA GAC GGC GGC GTG CAA CTC GCT GAT CAT 1738

163 A N F T R H N I D G a v Q L A D 182 1739 TAT CAA CAA AAT ACT CCA ATT GCC GAT GGC CCT GTC CTT TTA CCA GAC AAC CAT TAC CTG 1798

183 r Q Q N S P I G D G P V L L P D N H Y L 202 1799 TCC ACA CAA TCT GCC CTT TCG AAA GAT CCC AAC GAA AAG AGA GAC CAC ATG GTC CTT CIT 1858

203 S T Q S a L g D P N E R D V L L 222 1859 GAG TTT GTA ACA GCT GCT GGG ATT ACA CAT GGC ATG GAT GAA CTA TAC AAA GCT GCA GTT 1918

223 E F V S A A G I T H G D E L Y A A V 242

1919 AAT TA tcaccgetgtgctcacttatcaaatogacaaaggaaacacacttagtgcagcaLgtgccccattataaagaa 1996

1997 actgagttgtccoOchgtggcttgcagOtOtcacatccacaaaaaccgOcceactctaaataggagtOtetcoc agcaag 2076 2077 cagcqaaagtEtatgachgogtccOaatctctgaacggaLgtgtggcogacctusctgaLgetgatcDcOgtcO acacac 2156 2157 9cOccacatOgOtcaotacacaagacaUctatcagctgttUtagtcOaacoggttaacacaattcUtgcccccc cgag99 2236 2237 ggatccactagttctaga9cgOccgccaccOcOgtggagctccagetEttgtcccetttagtgaggottaattg ogoOct 2316 2317 tOgcotantcatOgtcataDctOtetcctgtOtgaaattgttatccoctcacaateccacacaacatacgagoa ggsaDc 2396 2397 ataaagUgLaaagcCtggogtgOctaatgagtgaDctoactcacattaattgcOttgooctcactgcccgottt ccagtc 2476 2477 gOgaaacotOteotgccagckOcattaatgaatcOgccaaegcgcOgOgagaggeggtttgcgLattOggcgot ottccy 2556 2557 cttcatcDctcactgactcgotgcgctcOgtcOttcggctgcgOcOagcOgtatcaUctcactcaaagOcOgta atacOg 2636 2637 ttatccacagaatcagOggataacOcaggaaagoacaegUgagoaaaDggccagcaaaagOccaggaacogtaa aaagoc 2716 2717 cOcOttgctOgcOtttttccatagOctccOcccccctgaegagcatcacaaaaatcgacoctcaegtcagagOt Ogogaa 2796 2797 accogacaggactataaagataccagOcgUttccccctggaegctccctcgtgcgOtetcctgtecogacOctg ccOcUt 2876 2877 acoggatacctotcCgcOttectccOttcOggaagcgtggcgctttctcataDctcacOctOtagotatctcag teagOt 2956 2957 gLaggecOttcgetccaaDctOggctOtgtgoacyaacccocogttcagccogaccOctgoOcottatccgOta actatc 3036 3037 gtcetgagtccaacoGggLaagacacOacttatcgccactggcagcaDccactOgLaacaggattaDcagaDcg agotat 3116 3117 gtaggcOgUgctacagagetcEtgaagtggUgOcctsactacggctacactagaaggacagLateLggtatctg Ogctot 3196 3197 yOtgaagOcagetacchtcOgaaaaagagttggtagctcEtgatccOgcaaacaaaccaccOctOgtagoggtO getett 3276 3277 ttOtttgcaaDcagcagattacOcgcagaaaaaaaggatatcaagaagatccettgatcetttetacOgOgtct gacDct 3356 3357 cagtOgaacOaaaactcaeottaagggattttggecaLgagattatcaaaaaggatatecacetagatccettt aaatta 3436 3437 aaaaLgaagtettaaatcaatctBaagtatatatgagLaaacttgotctgacagttaccaaLgcttaatcagtg agacac 3516 3517 ctatctcaDcDatatOtctatttegttcatccatagttgcctgactccoOgtcgtOtagateactacgatacog gagOgc 3596 3597 ttaccatctgOcaccagtgotgcaargataccOcOagacecacgotcaccggctccagatttatcagcaataaa ccagcc 3676 3677 aDccOgaaggOccDaucgcagaagtOgtcctgcaactttatcagcctccatccagtctattaattattgccggg aaDcta 3756 3757 gagLaagLagetegCcagttBatagtttgcOcaaegttgttgccattgatacagOcatcOtOgtOtcacgctcu tcottt 3836 3837 gOtatOgcttcattcagotccOgttcccaacDatcaagqcgagttacaLgatcccocatgttOtgcaaaaaa9c Ogtta9 3916 3917 ctccrtcgetcctcogatcOttgtcagaagtaagttggccgcagtgetatcactcaLggttaLgOcagcactgc ataatt 3996 3997 ctcttactotcatgacatccataegaLgcttetctgtgactggUgagtactcaaccaagtcathOtgagaatag Ugtatg 4076 4077 cgOcOaccgagttgctcrtgcCcOgcOtcaatacOggataataCcgcgccacataDcagaacettaaaagtgot catcat 4156 4157 tOgeaaangtechtcggOgcguaaactatcaaggatcttaccgctUttgagatccagtcagaLgLaacccactc Otgcac 4236 4237 ccaactgatcetcaUcatcetttacrttcaCcagcgtttctgggtgaDcaaaaacaggaagOcaaaaLgCcgoa aaaaag 4316 4317 ggaataagogcOacacggeaaLgttgaatactcatactatCcctttttcaatattattgaaDcatttatcaggg ttattg 4396 4397 tctcatgagoggatacatatttgaaLgtatttagaaaaataaacaaatagUggttCcOcOcacatttCcccDaa aagtgc 4476 4477 cac 447

pTetSagl-llGl.RT -> Gencs DNA sequence 4438 bp ctgacgcgcoct... gaaaagtgccac circular 1 ctgacOcOcectOtaDcOgOgeattaaDcocOgOgggtgt9gtggttacgagcaDcOtgaccsctacacrtgec aDcOcc 80 81 ctaDcDceeoctcctttegctetcetcccetcctEtctcgccaegetcOeOgocettccecOtcaaDctotsaa tOgogg 160 161 gctccctttagogtCccDatUtagtgatetacoOcacctcgaccccaaaaaacetgattagOgtgarOgtecac gLagtg 240 241 goccatOgcoetgatagacOgetetheOcOetttgacgttggagtccacOttcthtaatagtOgactcttOtec caaaet 320 321 ggaacaaeactcaacoetatOtcOgtctattcEtttgathtataagogattttgoOgatetcOgcctattOgtt aaaaaa 400 401 tgagctgatttaacaaaaatttaaeocoaattttaaeaaaatatteacDcttacaatetceatteOccatccag getgOg 480 481 caaetottgggaagogogatoggtgcgOgoctOttcgctattacgecaDetoDcOaaagggggaLgtgchOcaa gOcOat 560 561 taagttOgOtaacgocagogEttecccaytcacOaegttgraaaaegaeoDccagtgaDcDcDcOtaetaegac tcacta 640 641 tagOgcgaattOgOtaccOaDctcOactetcacetttotctatcaetgatagggagtggtaaactagaetEtcacttttc 720 721 tctatcaetgatagOgagtOgtaaactogacttccactttCctetateaetgatagOgagtogLaaaetcgaee tceaet 800 801 tttetctatcactgatagggagtOgLaaactcgactttcaettEtOtetatcactgatagOgagtgoteaactc uaeEtt 880 881 cactUtectetateachgatagogagtOgtaaactagacttCcaetettctctateaetgatagggagtOgLaa aetoga 960 961 gOtagacOgtatcDateaUcetcaaLgtgcacctOtaggsagctgLagtcaetgotgatteteachOteetagg caaggg 1040 1041 ecgacgaeoggagLaeagtetetUtOggcagagcegttgtgeagetEtecOttertctegOttgUgeeacaLgt oteatt 1120 1121 gtagtgLaaaeacaeogttgraLgtogghttcgeLgcaccaetteattatteeetchogtetettgaeOagt ATG CAT 1198 1 M 2

1199 GCG TCC AAA CCC ATT GAA GAC TAC GGC AAG GGC AAG GGC CGT ATT GAG CCC ATC TAT ATC 1258

3 A S P I E D Y G K G a R I r P M I 22 1259 CCC GAC AAC ACC TC TAC AAC GCT GAT GAC TTT CTT GTG OOC CCC CAC TGC AAG CCC TAC 1318

23 P D N S P Y N A D D F L V P P C P Y 42

1319 ATT GAC AAA ATC CTC CTC CCT GGT GGA TTG GTC AAG GAC AGA GTT GAG AAG TTG GCG TAT 1378

43 I D I L L P G G L V K D R V E K L A Y 62

1379 GAC ATC CAC AGA ACT TAC TTC GGC GAG GAG TTG CAC ATC ATT TGC ATC CTG AAA GGC TCT 1438

63 D I H R T Y r G r. E L H I I C I L G S 82 1439 CGC GGC TTC TTC AAC CTT CTG ATC GAC TAC CTT GCC ACC ATA CAG AAT GGT CGT GAG TCC 1498

83 R G P F N L L r D Y L A T I Q N G R r s 102 1499 AGC GTG CCC CCC TTC TTC GAG CAC TAT GTC CGC CTG AAG TCC TAC CAG AAC GAC AAC AGC 1558

103 S P P F F E R Y V R L S Y Q N D N g 122 1559 ACA GGC CAG CTC ACC GTC TTG AGC GAC GAC TTG TCA ATC TTT CGC GAC AAG CAC GTT CTC 1618

123 T G Q L T V L S D D L S I Y R D H V 142

1619 ATT GT GAG GAC ATC GTC GAC ACC GGT TTC ACC CTC ACC GAG TTC GGT GAG CGC CTG AAA 1678

143 Z V D I V D S G F S L T r. F G E R L lff2 1679 GCC GTC w r CCC AAG TCG ATG AGA ATC GCC ACC CTC GTC GAG AAG CGC ACA GAT CGC TCC 1738 163 A V G P S R I A S L V E R T D R 8 182

1739 AAC AGC TTG AAG GGC GAC TTC GTC GGC TTC AGC ATT GAA GAC GTC TGG ATC GTT GGT TGC 1798

183 N S L G D F V G F S I F D W I Y G. C 202

1799 TGC TAC GAC TTC M C GAG ATG TTC CGC GAC TTC GAC CAC GTC GCC GTC CTG AGC GC GCC 1858

203 C Y D F N E Y P R D r D 8 V A V L S D A 222 1859 GCT OGC AAA AAG TTC GAG AAG GCT TAA ttaateaeagttgtgeteaettateaaategaeaaeggaaaeae 1929 223 A R K F F A 231

1930 acttcgtgeaaeatgtgaceeattataaagaaaetgagttgteeeOetOtOgettgeaggtuteaeateeaeaa aaaeeg 2009 2010 gocgactOtOaataggagtOttecDcaDcaaDca9cgaaagtttaLgactgOgtccOaatOtctgaacOgaLgt otoDcg 2089 2090 gaeetoOehgatOttgateOeageagaeaeaegcoccacaLgggteaataeacaagaeaDetatcagetgetet agtoga 2169

2170 acagOttaacacaatCcttgococcCcOagOgggatCcactagaDcoOccgecaccgcOgtggaDctccaDcet ttottc 2249 2250 ccretagtgagogttaattgcOcocttg9cgraatcatOgtcataDctOtUtcctOtOtgaaattOttatccOc tcacaa 2329 2330 ttccacacaacatacOaDccggaagoataaagUgLaaagoctggggtgoctHatgagtgagotaactcacatto attgcg 2409 2410 ttgcOctcactgcccgctttccagecgOgaaacctgtcgtgccagotgcattHatgaategoccaacgcOcgOg gagagg 2489 249O cOgthtgcotattogOcDctcteccDcktcOtooctcactgaccgctgcOctoggtcOttcOgchOcOgOgaDc OgLat 2569 2570 cagotcactcaaaggogOtaatacOgttatccacagaatcaggggateacOcaggoaagaacaLgtgagcaaaa ggccag 2649 2650 caaaagOccaggaacogtaaaaegOccOcOttgctggcgtEtteccatagOctccgoccccctgaGgaDcatca caaaaa 2729 2730 tegaegOtcaagecagagOtgOcOaaacccgacaggactateaagataccagOcgUttccccctogaaDctccc tcotgc 2809 2810 gcEcLcctOttcogacoctgccOcttacOggatacotgtccOcatUtctcccUtogOgaaDcstOgcoctetct catagc 2889 2890 tcacOctgLagOtatctcagttegutOtagOtcottcgctccaagergOgctotgtgcacOaacoccccOtCca gcocoa 2969 2970 ccOctgcOcattatccOgLaactatcOtcttgagtccaacooggtaagacacOacrtategocactOgeagcag ocactg 3049 3050 graacaggattagcagagcgagOtaLgLagOcgOtgatacagagEtcttgaagtgOtOgcctBactacOgctac actaga 3129 3130 aggacagtatttOgLatctgaDctctgctgaagccagetaccttcOgaaaaagagttOgtaDctcttgatccoD caaaca 3209 3210 aaccacouctogLaUcgutOgtEtUtetOtttgcangcagcagattacOcOcagaaaaaaaggatotcaegaag atcatt 3289 3290 tgatckEttctacOgggtctgaegctcagtggaacgaaaactcaeottaagggattttOgEcaLgagattatca aaaagg 3369 3370 atcUtcacctagatccttttaasetaaaaatgaagUtttaaatcaatctaaagLatatatgagLaaacttogtc tgacag 3449 3450. ttaccaatgcrtastcagtgaggcacctatUtcagogatctOtctatetcOtCcatccatagttgcctgactcc OcOtcg 3529 3530 tgLagataactacgatacgOgagOgettaccatctggocccagtgctgoaatgatacoOcOagacccacOctca cogOct 3609 3610 ccagatetatcaDcaataaaccagccagccOgaagggaegagcgcagaagtOgtcctgcaactetatcOgOctc catcca 3689 3690 gtctattaattOttgccOggaagctagagLaagtagUtcgccagetaatagtttgcgcaacgttOttgccattg ctacag 3769 3770 gcategtOgtOtcacDctcOtagettgOtatOgcrtcatecagctccggetcccaacoatcaaggcOagttaca tgatcc 3849 3850 CccatgetgtgCaaaaaagagOttaDctccttcOgtcctccgatogeLgtcagaagLaageLgOccOcagtgtt atcact 3929 3930 catOgttatgOcaacactgcataatectcttactOtcatgccatccgraagaLgcUtetctOtgactOgUgagL actcaa 4009 4010 ccaagtcatectgagaatagtOtaLgcgOcOacagagttgctcUtgcccOgcOtcaatacOggataatacoOcO ccacat 4089 4090 aDcagaacrCtaaaeutgctcatcattggaaaacOttctCcggggcoaaaactctcaaggatcttacOgctOtt gagatc 4169 4170 cagttcgaLgtaacOcactogtgcacccaactgatctCcageatattttactetcaccagcotttctOgotgag caaaaa 4249 4250 caggaagocaaaatgccOcaaaaaagggaataagOgagacacggaaatOttgaatactcatactctCcetette caatat 4329 4330 tattgaagoatttatcagggetattOtctcatgaDcggatacatatttgaatOtatttagaaaaataaacaast agOggt 4409 4410 tccgcgcacattEcOccgaaaagtgccac 4438

pTetSagl-LarZ-T CA T -> Cenes vA sequence 8287 hp ctgacgcgccet.. gaaaegtgocac circular 1 ctgacOcgccotOtagcOgcOcattaegcacggcOggtotggtggttacOcDcagogtgaccOctacacEtgcc agcgOc 80 81 ctagogeccgetcc t tegcttCcetcccktccettctcgecacgetcgcoggcettccecgtcaaDctctaaategOgg 160 161 gctccetEtagOgttcogatttagtgotttacOgOacotcgaccccaaaaaacttgattagOgtgatOgtEcac utagtg 240 241 gOccategcOckDatagacgOtEtetcOcOcettgacgttggagtccacgUtctEtaatagtogactcutgUtc caaact 320 321 ggaacaacactcaacOctatctogotctattcEtetgatttataagOgatettgccOatetcOgcctattgOtt aaaaaa 400 401 tgaDctgateteacaaaaatttaaegcOasttttaacaaaatattoacOchtacaatttccattoOccattcag Octgcg 480 481 caactgeLgOgaagggcOatcOgUgogOgcctattcgctattacgccaectOgcOaaaggOggaLgtgetgcaa ggcOat 560 561 taagttogOtaaegccagogttttcccagtcaegacgergtaaaacOaeggOcagtgagcOcOcOtaatacOac tcacta 640 641 tagOgcgaattogOtaccoaDctcgacttEcactettctctatcactgatagOgagtgOtaaactcgacttEca ctEttc 720 721 tctatcactgatagOgagtgOtaaactogactttcacettcctotatcactgatagOgagtogLaaactogacr tecact 800 801 ttCctctatcactgatagggagtogtaaactcOactttcactttcctctatcactgatagggagtgotaaacta gacett 880 881 cacetttctOtatcactgatagOgagtggraaactcgacrttcacttetctetatcactgatagOgagtOgLaa actcDa 960 961 ggtcoaegetatcOataDgctecaaLgtgcacctotaggaagctgLagtcactgctgatectcactOttctcOg oaegOg 1040 1041 ccgacoacOggagLacagtUtttgtOgOcagagcOgttgtgcagcrttccOttcttctcgettOtgEcacarOt Otcatt 1120 1121 gtagtOtaaacacacOgetOtaLgecOgtttcgctgcaccacttcattatEtattctOgttetttgacOagt ATG CAT 11 98 1 H 2

1199 GCC ATG GAG AAG TTA TTA TTC CGA AGT TCC TAT TCT CTA GAA AGT ATA GGA ACT TCA AGC 1258

3 A M Z K L L F R S S Y S L E S z G T S 5 22 1259 TTG GCA CTG GCC GTC GTT TTA CAA CGT CGT GAC TGG GAA AAC CCT GGC GTT ACC CAA CTT 1318

23 L A L A v v L Q R R D W E N P G V T Q L 42 1319 MT CGC CTT GCA GCA CAT CCC CCT TTC GCC AGC TGG CGT AT AGC GAA GAG GCC CGC ACC 1378

43 N R L a A H P P Y A S W R N S X A R T 62,, 1379 GAT COC CCT TCC CAA CAG TTG CGC AGC CTG AAT GGC GAA TGG CGC TTT GCC TOG TTT CCG 1438

63 D R P S Q Q L R S L N G F W R F A W F P 82 ';

1439 GCA CCA GAA GCG GTG CCG GAA AGC TGG CTG GAG TGC GAT CTT CCT GAG GCC GAT ACT GTC 1498

83 A P Y A V P 8 S W L 8 C D L P 8 A D T v 102 1499 GTC GTC CCC TCA AAC TCG CAG ATG CAC GGT TAC GAT GCG CCC ATC TAC ACC AAC GTA ACC 1558

103 V V P S N 1, Q 11 G r D A P I r s N V S 122: 1559 TAT CCC ATT ACG GTC AAT CCG CCG m GTT CCC ACO GAG AAT CCG ACG GGT TGT TAC TCG 1618 123 r P I S V N P P F V P S N P S G C Y S 142 1619 CTC ACA TTP AAT GTT GAT GAA AGC IGG CTA CAG GAA GGC CAG ACO CGA ATT ATT TTT GAT 1678

143 L T r N V D X S W L Q Y. G Q T R I Z F D 162 1679 GGC G,TT AAC TCG GCG TTT CAT CTG TGG TCC AAC GGG CGC TCG GTC GGT TAC GGC CAG GAC 1738

163 G V N S A F 1} L W C N G R W V G Y G Q D 182

1739 AGT CGT TTG CCG TCT GAA TTT GAC CTG AGC GCA TTT TTA CGC GCC GGA GAA AAC CGC CTC 1798

183 s R L P S E F D L S A P L R A G E N R L 202 1799 GCG G m ATG GTG CTG CGT TGG AGT GAC GGC AGT TAT CTG GAA GAT CAG GAT ATG TGG CGG 1858 203 A V n v L R W S D G S Y L E D Q D M 222 1859 ATG AGC GGC ATT TTC CGT GAC GTC TCG TTG CTG C'AT AAA CCG ACT ACA CAA ATC AGC GAT 1918

223 s G I F R D V S L L p S T Q I S D 242 1919 TTC CAT GTT GCC ACT CGC TTT AAT GAT GAT TTC AGC OCC GCT GTA CIG GAG GCT GAA GTT 1978

243 F N V A S R F N D D F S R A V L E A E V 262

1979 CAG ATG TGC GQC GAG TTG CT GAC TAC CTA CGG GTA ACA GTT TCT TTA TGG C'AG GGT GAA 2038

263 Q c G E L R D Y L R V S V S L W Q G E 282 2039 ACC CAG GTC GCC AGC GGC ACC GCG CCT TTC GGC GGT GAA ATT ATC GAT GAG CGT GGT GGT 2098

283 s Q v A S G S A r 4 a E I I D E R G G 302

20gg TAT GCC GAT CGC GTC ACA CTA CGT CTG AAC GTC GAA AAC CCG AAA CTG TGG AcC GCC G^A 2158 303 Y A D R v s L R L N V E N P K L w s A E 322 2159 ATC CCG AAT CTC TAT CGT GCG GTG GTT GAA CTG CAC ACC GCC GAC CGC ACG CTG AlT GP 2218 323 I P N L Y R A V V E L H T A D G T L I E 342

2219 GCA GAA GCC TGC GAT GTC GGT TTC CGC GAG GTG CGG ATT GAA AAT GGT CTG CTG CTG CTG 2278

343 A E A C D v G F R E v R I E N G L L L L 362 2279 AAC GGC AAG CCG TTG CTG ATT CGA GGC GTT AAC CGT CAC GAG CAT CAT CCT CTO CAT GoT 2338 363 N G P L L I R G V N R E H P L H G 382

2339 CAG GTC ATG GAT GAG CAG ACG ATG GTG CAG GAT ATC CTG CTG ATG AAG GG AAC AAC TTT 2398

383 Q v D E Q T M Q D r L L n K Q N N F 402 2399 AAC GCC GTG CGC TGT TCG CAT TAT CCG AAC CAT CCG CTG TGG TAC ACG cm TGC GAC CGC 2458 403 N A V R C S H Y P N H P L W Y T L c D R 422 2459 TAC GGC CTG TAT GTG GTG GAT GAA GCC AAT ATT GAA ACC CAC GGC ATG GTG CCA A.TG AAT 2518

423 Y G L Y v v D E A N I E T R G M V P N 442 2519 CGT CTG ACC GAT GAT CCG CGC TGG CTA CCG GCG ATG AGC GAA CGC GTA ACG CGA ATG GTG 2578

443 R L T D D P R w L P A M S R Y T R V 462 2579 C^G CGC GAT CGT AAT CAC CCG AGT GTG ATC ATC TCG TCG CTG GGG AAT CAA TCA GGC CAC 2638

463 Q R D R N P S V I I W S L G N E S G H 482

2639 GGC GCT AAT CAC GAC GCG CTG TAT CGC TGG ATC AAA TCT GTC GAT CCT TCC CGC CCG GTG 2698

483 G A N H D A L Y R W I K S V D P S a P v 502 2699 CAG TAT GAA GGC GGC GGA GCC GAC ACC ACG GCC ACC GAT ATT ATT TGC CCG ATG TAC GCG 2758

503 Q Y E G G G A D T T A T D I I C P M Y A 522

2759 CGC w1u GAT GAA GAC CAG CCC TTC CCG GCT GTG CCG AAA TGG TCC ATC AAA AAA TGG CTT 2818 523 R V D D Q P F P A V P K W S I R W L 542

2819 TC& CTA CCT GGA GAG ACG CGC CC& CTG ATC CTT TGC GAA TAC GCC CAC GCG ATG GGT AAC 2878

543 S L P G E T a P L I L C E Y A H A G N 562 2879 hGT CTT GGC GGT TTC GCT AAA TAC TGG CAG GCG TTT CGT CAG TAT CCC CGT TTA CAG GGC 2938 563 S L G G F A Y W Q A r R Q Y P R L Q G 582 2939 GGC TTC GTC TGG GAC TGG GTG GAT CAG TCG CTG ATT AAA TAT GAT GAA AAC GGC AAC CCG 2998 '',.

583 G F V W D W V D Q S L r K Y D E N G N P 602 2999 TGG TCG GCT TAC GGC GGT GAT TTT GGC GAT ACG CCG AAC GAT CGC CAG TTC -lr ATG AAC 3058 '' 603 w S A Y G G D F G D T P N D R Q F C M N 622 3059 GGT CTG GTC TTT GCC GAC CGC ACG CCG CAT CCA GCG CTG ACG GAA GCA AAA CAC CAG CAG 3118

623 G L V F A D R T P P A L T r A K H Q Q 642 3119 CAG TTT TTC CAG TTC CGT TTA TCC GsG CAA ACC ATC GAA GTG ACC AGC GAA TAC CTG TTC 3178 643 Q F F Q r R L S G Q I E V T 5 E r L F 662 3179 CGT CAT AGC GAT AAC GAG CTC CTG CAC TGG ATG GTG GCG CTG GAT GGT AAG W G CTG GCA 3238

663 R S D N E L L R W V a L D G K P L A 682 3239 AGC GCT GAA GT& CCT CTG GAT GTC GCT CCA CAA GGT AAA CAG TTG ATT GAA CTG CCT GAA 3298

683 S G E V P L D ' V a P Q G K Q L Z L P E 702 3299 CTA CCG CAG CCG GAG AGC GCC GGG CAA CTC TGG CTC ACA GTA CGC GTA GTG CAA OCG AAC 3358

703 L P Q P E S A G Q L W L S V R V V Q P N 722

3359 GCG ACC GCA T&G TCA GAA GCC GGG CAC ATC AGC GCC TGG CAG CAG T&G CGT CT& GG GAA 3418

723 A T A W S E A G R I S A w Q Q W R L A 742 3419 AAC CTC AGT GTG ACG CTC CCC GCC GCG TCC CAC GCC ATC CCG CAT CTG ACC ACC AGC GAA 3478

743 N L S V T L P A A S R A I P H L S T S 762

3479 ATG GAT TTT TGC ATC GA& CTG GGT AAT AAG CGT T&G CAA m AAC CGC CAG TCA GGC TTT 3538 763 D F C I E L G N R W Q F N R Q S G F 782

3539 CTT TCA CAG ATG TGG ATT GGC GAT AAA AAA CAA CTG CTG ACG CCG CTG OGC GAT CAG TqC 3598 783 L S Q w z C D R Q L L T P L R D Q F 802 3599 ACC CGT GCA CCG CTG GAT AAC GAC ATT GGC GTA AcT GAA GCG ACC CGC ATT GAC CCT AAC 3658 803 T R A P L D N D I G V S E A T R I D P N 822

3659 GCC TaG GTC GAA CGC TGG AAG.GCG GCG GC CAT TAC CAG GCC GAA GCA GCG TTG TTG CAG 3718 82 3 A W V E R W K A A G Y Q A C A A L L Q 842

3719 TGC ACG GCA GAT ACA CTT GCT GAT OCG GTG CTG ATT AQG ACC GCT CAC GCG TGG CAG CAT 3778

843 C T A D T L A D A V L Z T T A N A Q H 862

3779 CG GGG AAA ACC TTA m ATC AGC CGG AAA ACC TAC CGG ATT GAT GGT AGT GCT CAA ATG 3838 863 Q G T L F I S R T Y R I D G S G Q M 882

3839 GCG ATT ACC CTP CAT GTT CAA GTG GCG AGC GAT ACA CG CAT CCG GCG CG ATT GCC CTG 3898

883 A I T V D V V A S D T P H P A R I G L 902

3899 AAC TGC CAG CTG GCG CAG GTA GCA GG CGG GTA AAC T&C CTC GCA TTA GGG CCG CAA GAA 3958

903 N C Q L A Q V A E R V N W L G L G P Q E 922

3959 AAC TAT CCC GAC CYC CTT ACT GCC GCC T&T TTT GAC CGC T&G GAT CTG CCA TTG TCA GRC 4018

923 N Y P D R L T A A C F D R W D L L S D 942

4019 AT& TAT ACC CCC TAC GTC TTC CCG AGC GA A,AC GGT CTG CGC TGC GGG ACG CGC GAA TG 4078

943 r T P Y V F S E N G L R C c T R E L 962 4079 AAT TAT GGC CCA CAC CAG T&O CGC GCC GAC TTC CAG TTC AAC ATC AGC CGC TAC AGT CAA 4138

963 N Y G P R Q W R C D P Q F N I S R Y S Q 982

4139 CAG CAA CT& ATG GAA ACC ACC CAT CGC CAT C'TG CTG CAC GCG GAA GAA CGC ACA TGG CTG 4198

983 Q Q L E T S R R H L L H A E E C T L 1002

4199 AAT ATC GAC GGT TTC CAT ATG GGG ATT GGT CCC GAC GhC TCC T&G AG'C CCG TCA CTA TCG 4258 1003 N I G H 8 G z G G D D S W S P S V S 1022 4259 CCG GAA TTC CAG CTG ACC CCC GGT OC TAC CAT TAC CAC TTG GTC TGG TGT CAA AAA TAA 4318

1023 F Q L S A G R H Y Q L V W C Q 1042

4319 taattaattaatcacOgttOtgctcacetctcaaatcgacaaaggaaacacacrtcOtgcaDcaLgtgecocat tataaa 4398 4399 gnaactgagttOtCcoDctotOgettgcagOtotcacatccacaaaaaccOgccgactOteaataggagUgttc Ogcagc 4478 4479 aaDcaDcOaaagtttatgactOgUtccoaatctctgaacggargtOtOgoggaccggOt. gaLgttgatcocogtcgacg 4558 4559 gLatagataaDcttgatatgcatOtcOgcOtccgUgaaatCctctgatcaagcogagUgatcaccaatcatcgt ctcagc 4638 4639 gggaLgaeDttgcOgcaagUgacOgctoocogtgggcagtcagaLgcogaaegLaactcaggacOgottgOgOt catOgc 4718 4719 agaacaggOgtOgtgcOtgcattgOgtgcggeLggtgatcctggerOgaccOgtggagaLgcgcOcgcacOaag gggaLg 4798 '., 4799 tOtcagaaacatettgtUtOttatckOtgaacttttagaLgtgetaaagOcgOcOaatattagcagagagtcct cceLgt 4878; 4879 tccatectctcetgaatetOgcccetecetEctctttgogagtgUggLagagaacaaDcactegtccOccOtce ctgacg 4958 4959 acOcaaccOgcOcagaagacatccaccaaacogtOttacacaatcaccrtOtOtgaagtecetgcggaaaacta ctcOtt 5038 5039 gOcattetttcetgaattccckettccDacaaaatgcaLgagaaaaaaatcactggatataccaccgttgatat atcaca 5118 5119 atcocatcOtaaegaacattttgaggcatthcagtcagttgctcaatOtacctataaccagaccOtccagctgg atatta 5198 5199 cOgocttettaaagacagLaaagaaaaataagGacaagttttatccOgccettatEcacattOttgCccgCctg aLgast 5278 5279 getcatCcogaatecOgLaLgocastgaaagacogtgagctggtgataegOgatagtOtecacccttOttacac cOthtt 5358 5359 ccatgagcaaactgaaacgtttecatcDctOtggagtgaataCCaegaegatEtcCggOagtetotacacatat attaUc 5438 5439 aegatOtOgOgtOttaegotgaaaacctoOcctatttccataaagOgettattgagaatatotttteeOtctca Dccast 5518 5519 ccctggOtgaghttcaccagetttgatetsaacgtggecaatatOgacaachtcteogcoccogttttcaccaL gggeaa 5598 5599 atattatacDcaagocgacaaggtgeLgaLgccOctggogattcagotecatcaLgCcotatgtgatOgcttCc atOtcg 5678 5679 gcagaeLgcttaatgaattacaacagtactgagatgagtggeagOgcogogchtaattaatcacageLgtgctc acetct 5758 5759 caaatogacaaaggaaacacacetogtgoaDcatgtgccacattataaagaaactgagttothccOctgtoOct tgUagg 5838 5839 tOtcacatccacaaaaacagocogactctaaataggagtgtttcgOagcaaDcagcDaaagtetatgacteggt ccOaat 5918 5919 ctctgaacggatOtgtOgOggacctgOctgaLgttgatcgccgtegacacaegcgccacaLgOgtcaatacaca agacag 5998 5999 ctatcagttOttttagecOaaccggeteacacaatecttgecccacOgagggogatccactagaUcggcageca ccgcgg 6078 6079 togagctccaDctettgtecocettagtgagOgetanttgcDcOcrtOgcOtaetcaLggtcataDctgettcc tgtOtg 6158

6159 aaattgttatccgatcacaatcccacacaacatacgagccggaegcataaagtOtaaagcctOggOtgcataat gagtga 6238 6239 gctaactcacattaattgcOttgcgotcactgaccgcEttccagtcOggaaacctgecgUgccaDctgcattaa Lgaatc 6318 G319 ggocaacOcocOgOgagaggcOgtetgagtattOggagctcrtccOcetcOtcOctcactgacecOctgcDcta gOtcgt 6398 G399 toggctgcggcgaDcgOtatcagctcactcaaagOcggUaatacgOttatccacagastcaggogataaegcag gaaaga 6478 6479 acaLgtgaDcaaaaggccaDcaaaaggecaggaaccgLaaaaaggccDcgttgctOgcOtEtteccataggctc aDcccc 6558 6559 cctgac9agoatcacaaaaatcOacoctcaagtcagaggtggagoaacccOacaggactataaagataccaggo gttUcc 6638 6639 cOctggaaDctcectcOtgcOctetcctgetcOgaccctgcc9crtaccOgatacOtOtcOgccUtCctcccrt coggaa 6718 6719 gcUtggOgcrEtctcatagctcacOctOtagOtatctcagtEcggUgLaggtcOtEcOctccaaDcgOgctUtg tgcac 6798 6799 gaacccoccottcaDcccgaccgct9cOccrtatccg9taactatcOtcrtgagEccaacOcOgLaagacacOa cEtatc 6878 6879 gocactoOcagcagccactOgLaacaggattaDcagagc. gaggLaLgragOcOgtgctacagagUtcUtgaagtogUgoc 6958 6959 ctaactacgOctacactagaeggacagtatttOgLataLgcgctctgctgaDgocagEtaccrtcOgeaaaaga gttOgt 7038 7039 aDctcttgatccOgcaaacaaaccaccDctOgragcggtggEttEtEtOtttgcaagcagOagattacgcgOag uaaaaa 7118 7119 aggatatcaagaagatccettgatcettUctacgOggtctgaegctcagtOgaaegaaaactcacOttaegoga tettOg 7198 7199 tcatgagattatcaaaaaggatctUcacctagatccrcttaaottaaaaatgaagtttteaatcaatcLaaagL atatat 7278 7279 gagLaaacUtgOtctgacagttaccaarOcrtaatcagtgaggcacctatctcaDcOatctgtctatUtcOttc atccat 7358 7359 agEtgc, ctgactccccOtcOtOtagataactacOatacgggagggOttaccataLgOccocagtgctgcaatgataccOc 7438 7439 gagacacacOctcaccgOctccagatttatcaucaataaaccagccaDccggaagggocOaDcocagaegtOge ceLgca 7518 7519 actttatccOcctccatccagectattaattOttgccgggaagctagagraagLagttcDccagtteatagttt gcOcaa 7598 7599 cOttgttgccattgotacaggcatcOtggUgtcacgctcgtcgtttOgeaLggcrtcattcaDctcagoteccc aacgat 7678 7679 caagocOagetacaLgatcccccaLgttgtgcaaaaaagcggttaDctccrtcOgecctcogatogttotcaga agtaag 7758 7759 ttOgccOcagtUttatcactcatOgEtaLgOcaScactgcataatectcrtactOtcatgccatccOtaagaLg crcetc 7838 7839 tgtgactOgtgagractcaaccaagecatectgagaatagtgtaLgcOgOgaccOaghtgctcrtgcocOgcOt caatac 7918 7919 gggataatacOgcOccacatagoagaactetaaaagtgetcatcattogaaaacOtcchtcOgggogaaaactO tcaagg 7998 7999 atcttacOgatOttgagatccagtCcOatOtaacacactcgtgcacccaactgatchtcaDcatctettacete caccag 8078 8079 cOthtaLgOgtgagcaaaaacaggsagOcaaaatgecgcaaaaaagggaataagOgcOacacggaaaLgeLgaa tactca 8158 8159 tactOttcctttttcaatattattgaagoatttatcagggttattOtctcatgagcggatacatateLgaaLgL atetag 8238 8239 aaaaataaacaaataggggttccgcgcacattCcccogaaaagtgccac 8287

pTclO7Sag4-LacZ-TCAT -> Cenes DNA sequence 8364 bp ctgacgcgcect... gnaaagtgecac circular TetO7Sag4-LacZ-CAT 1 ctgacgagacctOtaDcOgcgoattsaDcoOggcgggtgtggtOgetacOcocaDcgtgacOgctacaceLgcc agcDcc 80 81 ctaDcgOccOctCctttagettecrtccctecctEtctcgccacgetcOccgOcttccOcagecaagctataaa tcoggg 160 161 getccetetaggOteccUatetagtgcettacgOcacctcOaccccaaaaaaceLgattagogtgaLggtccac Otagtg 240 241 g9ccategocctgatagacOgttettcgccetetgaeOttOgageccacOteckttastagtggactceLgttc caaact 320 321 ggaacaacactcaaccctatctcggectatecrEttgatttateagggatettgccgatetcgOcOtattogtt aaaaaa 400 401 tgaDctgatttaacaaaaatetaaegagaatettaacaaaatattaacOcrtacaattcccathcOccatecag OctgOg 480 481 caactOttOggaagOgcgatoggtgOggOcctattogctattaegccagctoOcDaaaggOggatOtgctgcaa gOcgat 560 561 taagttoggLaacDccagggetetcccagtcacoacgeLgLaaaacOaegOccagtgagcOcDcOtsatacoac tcacta 640 641 tagOgogaattOgOtaccoagotegacettcacetttotctatcactgatagOgagtOgLaaactOgactetca cetetc 720 721 tctatcactgatagogagtggtaaactogacettcacttttctotatcactgatagggagtggteeactogace tEcact 800 801 tetctOtatcactgatagggagtOgLaaactcOactttcactEtCctatatcachgatagOgagtgOtaaactc Oacett 880 881 cacetetctctatcactgatagogagtogLaaactcgacttCcactettctctatcactgatagogagtggLaa actega 960 961 gOtcgaegotatagataaDcetacOcogckDagactaactagaaagaagtOtgoaacagttcaLgagOgacaaa aggaat 1040 1041 gtgaLgoggetcgchtgnagaeggaatetetaaDcaegtcaacastacgOcttOgcDaetOtecaLgactOttc atgtOg 1120 1121 tecatcOgatcateLgaaaacatcgtgaggotOgLacctggtcgcaaacgtegtagtotagtaccgacaataac gtogec 1200 1201 gttcaagOggaegcagtectaggaagacgagtcgeagcatactgcaactgcetccOtOtgtetecaacc ATG CAT 1275 1 M 2

1276 GCC AT& GAG AAG TTA TTA TTC CGA AGT TCC TAT TCT CTA GAA AGT ATA GGA ACT TCA AGC 1335

3 A M E K L L F R S S Y S L E S I G T S S 22

1336 TTG GCA CTG GCC GTC GTT TTA CAA OGT CGT GAC TCG GAA AAC CCT GGC GTT ACC CAA CTT 1395

23 L A L A V V L Q R R D W r N P G V T Q L 42 1396 AAT CGC CTT GCA GCA CAT CCC CCT TTC GCC AGC TGG CGT AAT AGC GAA GAG GCC CGC ACC 1455

43 N R L A A H P P F A S W R N S E E A R T 62

1456 GAT CGC CCT TCC CAA CAG TTG CGC AGC CTG AAT GGC GAA TGG CGC TTT GCC TCG TTT CCG 1515

63 D R P S Q Q L R S L N a E W R F A W F P 82 1516 GCA CCA GAA GCG GTG CCG GAA AGC TCG CTG GAG T&C GAT CTT CCT GAG GCC GAT ACT GTC 1575

83 A P E A V P E S W L r. C D L P r. A D T V 102 i576 GTC GT CCC.TCA AAC TOG CAG ATG CAC GGT TAC GAT GCG CCC ATC TAC ACC AAC GTA ACC 1635 103 V V P 8 N W Q R C Y D A P I T N V T 122

1636 TAT CCC ATT ACG GTC AAT CCG CCG TTT GTT CCC ACG GAG AAT CCG ACG GGT TGT TAC TCG 1695

123 Y P I T Y N P P r V P T E N P T G C Y S 142 1696 CTC ACA TTT AAT GTT GAT GAA ACC TGG CTA CAG GAA GGC CAC ACG CGA ATT ATT m GAT 1755 143 L T F N V D E S W L Q E G Q T R I I P D 162

1756 GGC GTT AAC TCG GCG TTT CAT CTG TGG TGC AAC GGG CGC TGG GTC GGT TAC GGC CAG GAC 1815

163 G V N S A F H L C N G R W v G Y G Q D 182 1816 ACT CGT TTG CCG TCT GAA TTT GAC CTG AGC GCA TTT TTA CGC GCC CGA GAA AAC CGC CTC 18?5

183 S R L P S E F D L S A F L R A G E N R L 202

1876 GCG GTG ATC GTG CTG CGT TGG AGT GAC GCC AGT.TAT CTG GAA GAT CAG GAT ATG TGG CGC 1935

203 A V V L R W S D G S Y L E D Q D W R 222

1936 ATG AGC GGC ATT TTC CGT GAC GTC TCG TTG CTG CAT AAA CCG ACT ACA CAA ATC AGC GAT 1995

223 S G I F R D V s L L P T Q I S D 242 1996 TTC CAT GTT GCC ACT CGC TTT AAT GAT GAT TTC AGC CGC GCT GTA CTG GAG GCT GAA GTT 2055

243 F H V A T R F N D D F S R A V L E A E v 262

( 2056 CAG ATG TCC GGC G.G TTG CGT GAC T^C CTA CGG GTA ACA GTT TCT TTA T&G CAG GGT GAA 2115

263 Q n c G L R D Y L R V T V S L W Q G E 282 2116 ACG CAG GTC GCC AGC GGC ACC GOG CCT TTC GGC GGT GAA ATT ATC GAT GAG CGT GGT G'GT 2115

283 T Q V A s G T a P r G G E I Z D E R C G 302 2176 TAT GCC GAT CGC GTC ACA CTA CGT CTG AAC GTC GAA AAC CCG AAA CTG TGG AGC GCC GhA 2235 303 Y A D V S L R L N V E N P K L W S A E 322

2236 ATC CCG AAT CTC TAT CGT GCG GTG GT1' GAA CTG CAC ACC GCC GAC GGC ACG CTG ATT GAA 2295

323 r p N L Y R A v V E L H S A D G S L I E 342 2296 GCA GAA GCC TGC GAT GTC GGT TTC OGC GAG GTG CGG ATT GAA AAT GGT CTG CTG CTG CTG 2355

343 A E A c D V G F R E v R I E N G L L L L 362 2356 AAC GGC AAG CCG TTG CTG ATT CGA GGC GTT AAC CGT CAC GAG CAT CAT CCT CTG CAT GGT 2415

363 N o K P L L I R G V N R H H H P L H G 382 2416 CAG GTC ATG GAT GAG CAG ACG ATG GTG CAG GAT ATC CTG CTG ATG AAG CAG AAC AAC TTT 2475

383 Q V M D E Q T V Q D I L L n K Q N N F 402 2476 AAC GCC GTG CGC TGT TCG CAT TAT CCG AAC CAT CCG CTG TGG TAC ACG CTG TGC GAC CGC 2535

403 N A V R C S Y P N H P L W T L C D R 422

2536 TAC GGC C1'G TAT GTG GTG GAT G,A GCC AAT ATT GAA ACC CAC GGC ATG GTC CCA ATG AAT 2595

423 Y G L Y V V D E A N I E T H G M V P N 442

2596 CGT CTG ACC GAT GAT CCG CGC T&G CTA CCG GCG ATG AGC GAA CGC GTA ACG CGA ATG GTG 2G55

443 R L T D D P R W 4 P A M S E R V T R v 462 2656 CAG CGC GAT CGT AAT CAC CCG AGT GTG ATC ATC T&G TCG CTG GGG AAT GAA TCA GGC CAC 2715

463 Q R D R N P S v I I W S L G N e s 6 H 482 2716 GGC GCT AAT CAC GAC GCG CTG TAT CGC TGG ATC AAA TCT GTC GAT CCT TCC CGC CCG GTG 2775

483 G A N H D A L Y R W I K S V D P S R P v 502 2776 CAG TAT GAA GGC GGC GA GCC GAC ACC ACG GCC ACC GAT ATT ATT TCC CCG ATG TAC GCG 2835

503 Q Y E G G C A D T T A T D I I C P Y a 522 2836 CGC GTG GAT GAA GAC CAG CCC TTC CCG GCT GTG CCG AAA TGG TCC ATC AAA AAA TGG CTT Z895

523 R V D E D Q P F P A v P R w S I K W L 542 2896 TCG CTA CCT GGA GAG ACG CGC CCG CTG ATC CTT TGC GA TAC GCC CAC GCG ATG GGT AAC 2955

543 S L P G E T R P L I L C r. Y A R A N O N S62 2956 AGT CTT GCC GGT TTC GCT AAA TAC T&G CAG GCG m CGT CAG TAT CCC CUT TTA CAG GGC 3015 563 S L G G F A K Y W Q A r R Q Y P R L Q G 582 3016 GGC TTC GTC TGG GAC TGG GTG aT CAG TCG CTG ATT AAA TAT GAT GAA AAC GGC AAC CCG 3075 583 G F V W D W V D Q S L I Y D E N G N P 602

3076 T&G TCG GCT TAC GGC GGT GAT TTT GGC GAT ACG CCG AAC GAT CGC CAG TTC TGT ATG AAC 3135

603 W S A Y G G D F G D T P N D R Q F C N 622

3136 GGT CTG GTC TTT GCC GAC CGC AQG CCG CAT CCA GCG CTG ACG GAA GCA AAA CAC CAG CAG 3195

623 G L V F A D R T P R P A L S E A H Q Q 642

3196 CAG m TTC CAG TTC CGT TTA TCC GGG CAA ACC ATC GAA GTG ACC AGC GAA TAC CTG TTC 3255 643 Q F F Q F R L S G Q T I E V T S e L F 662 3256 CGT CAT AGC G'AT AAC GAG CTC CTG CAC TGG ATG GTG GCG CTG GAT GGT AAG CCG CTG GCA 3315

663 R R S D N E L L W V A L D G P L A 682

3316 AGC GGT GAA GTG CCT CTG GAT GTC GCT CCA CAA GGT AAA CAG TTG ATT GAA CTG CCT GAA' 3315

683 S G e V P L D V A P Q G Q L I E L P E 702 3376 CTA CCG CAG CCG GAG AGC GCC GGG CAA CTC TGG CTC ACA GTA CGC GTA GTG CAA CCG AAC 3435

703 L P Q P E S A G Q L W L T V R V Q e N 722 3436 GOG ACC GCA TGG TCA GAA GCC GGG CAC ATC AGC GCC TGG CAG CAG TGG CGT CTG GCG GAA 3495

723 A T A W s E A G R I S A W Q Q W R L A E 742 3496 AAC CTC AGT GTG ACG CTC CCC GCC GCG TCC CAC GCC ATC CCG CAT CTG ACC ACC AGC GAA 3555

743 N L S V T L P A A S A I P F L T T s E 762 3556 ATG GAT TTT TGC ATC GAG CTG GG/r AAT AAG CGT TGG CAA TT1. AAC CGC CAG TCA GGC TTT 3615 763 D F C I e L G N R W Q F N R Q S G 782

3616 CTT TCA CAG ATG TGG ATT GGC GAT AAA AAA CAA CTG CTG ACG CCG CTG CGC GAT CAG TTC 3675

783 L s Q H W I C D Q L T P L R D Q F 802 -

3676 ACC CGT GCA CCG CTG GAT AAC GAC ATT GGC GTA AGT GAA GCG ACC CGC ATT GAC CCT AAC 3735

803 T R A p L D N D I G V S A S R I D P N 822 3736 G'CC TGG GTC GAA CGC TGG AAG GCG GGC CAT TAC CAG CCC GAA GCA GOG TTG TTG CAG 3795

823 W V R a A G Y Q A E A A L L Q 842 3796 TGC ACG GCA GAT ACA CTT GCT GAT GCG GTG CTG ATT ACG ACC GCT CAC GCG TGG CAG CAT 3855

843 C S a D S L A D A V L I T T A H A Q H 862 3856 CAG GGG AAA ACC TTA TTT ATC AGC CGG AAA AC TAC CGG ATT GAT GGT AGT GGT CAA ATG 3915

863 Q G L F I s R Y R I D G S G Q 882 3916 GCG ATT ACC GTT GAT GTT GAk GTG GCG AC GAT ACA CCG CAT CCG GCG CGG ATT GGC CTG 3975 883 A I T V D V E v A S D T P P R I G L 902 3976 AAC TGC CAG CTG GCG CAG GTA GCA GAG OEG GTA AAC TGG CTC GGA TTA GGG CCG CAA GAA 4035

903 N C Q L A Q V A E R V N W L G L G P Q E 922

4036 AAC TAT OCC GAC CGC CTT ACT GCC GCC TGT m GAC CGC TGG GAT CTG CCA TTG TCA GAC 4095 923 N Y P D R L T A A C F D R D L P L S D 942

4096 ATG TAT ACC CCG TAC GTC TTC CCG AGC GAA AAC GGT CTG CGC TGC GGG ACG CGC GAA TTG 4155

943 M Y T P Y V F P S N G L R C G T R L 962

4156 AAT TAT GGC CCA CAC CAG TGG CGC GGC GAC TTC CAG TTC AAC ATC AGC CGC TAC AGT CAA 4215

963 N Y G P Q W R G D F Q F N I S R Y S Q 982

4216 CAG CAA CTG ATG GAA ACC ACC CAT CGC CAT CTG CTG CAC GCG GAA GAA GGC ACA TGG CTG 4275

983 Q Q L E T S H R H L L H A E E G T W L 1002

4276 AAT ATC GAC GGT TTC CAT ATG GGG ATT G&T GGC GAC GAC TCC TGG AGC CCG TCA GTA TCG 4335

1003 N S D G F H M G I G G D D S W S P S V S 1022

4336 GCG GAA TTC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT CAA AAA TAA 4395

1023 a E F Q L S A G R Y Y Q V W C Q 1042 4396 taattaattaatcaccgttOtgctcacetOtcaastcgacaaaggaaacacacetcOtgcagcatgtgccccat tataaa 4475 4476 gaaactgagttgtecogOtOtOgcttgoagetOtcacatccacaaaaaccOgacOactctaaataggagtOttc oDcaDc 4555, 4556 aaDcagcOaaagettaLgactegOtccOaatetctgaacggaLgtOtgOcogacctggctgaLgttgataDcco tcOacg 4635....

4636 gLatcDataaDcktgataLgcaLgecaDcOtcagtgsasttataLgatcaagcggagtgatcaccaatcatOgt ctcaDc 4715 4716 gggaLgaeottgcggcaaggOccgoctOgcogtgOgcagtcagaLgccoaacUtaactcaggacOgcttgaDct catcDc 4795, 4796 agaacagOgOtOgtgectgcattagOtgOgOttOgtgatcctgghtOgaccOgtggagatgcOcOcOcaegaeg gagaLg 4875.

4876 tgtcagaaacattttgettOttOtotgtgaacrtttagaLgtgttaaagOcOgcOaatattagcagagagtcct ccttOt 4955 4956 tccattctctattgaatetaDcOctteccthatatttgcgagtgtogtagagaacaegcactagetagccOtca ctgaeg 5035 5036 acOcaaccagcacagaagacatccaccaaacgotOttacacaatcaccttototgaagttattgcggsaaacta ctaget S115 5116ggcatttettcergsattcccEtetCcOacaaaatgoaLgagaaaaaastcactogatataccacagttgatat atceca 5195 5196 atcgcateDtaaagaacattetgagOcatetcagtcagttgctcaaLgLacatataaccagaccettcaDctOg atatta 5275 5276 cgOccttettaaagacOgLaaegaaaastanecacaagetttatcagOcetetathcacatecttgecOgcaLg aLgaat 5355 5356 gctcatccOgaeteccgtaLgOcastgaaagaeggtgageLgOtgataLgggatagtattcacOceeDttacac cOttet 5435 5436 ccargagOaaactgaaacgtettcataDctctggagtgaataccacgacDatttccOgoagetectacacatat ateoDc 5515 5516 aagaLgtOgcOtOttacOgtgsaaacctegoctatteccctaaaggotttattgagaatatgtttttogtetca gccaat 5595 5596 ccchOgetgagettcaccageteLgatttaaaeDtggecaataLggacaacttattcOcaccagttetcaccat ggecaa 5675 5676 atattatacOcaaggogacaagOtgctgaLgaegeLggogathcagOttcatcaLgcOgtOtUtgarOgcttcc atotcg 5755 5756 geagaatgettaaLgaattacaacagLactgagatgagtggeagggogggocttsattastcacogetgtgetc acttct 5835 5836 caaategacaaaggaaacacactecOtgcaDcaLgtgececattataaagaaactgageLgEtcoOctOtoDce tgcagg S91S

5916 tgtcacatccacaaaaaccoDccoactctaaataggagUgttccgcagcaaqcagcOaaagettaLgactOgOt ccOaat 5995 5996 ctatgaaeggaLgtOtoOcOgacchOgctgatgetgatagccytcgacacacgogecacaLgOgEcautacaca egacag 6075 6076 ctatcageLgetttagtcOaacOgOttaacacaatccttgccoccccDagggggatccactagaDcOgcogcca ccoagg 6155 6156 tOgagctccagcUtetOttccctUtagUgagOgttasergcgcgceLggcgtaatcarOgEcataDctUttecc tOtgUg 6235 6236 aaattottatccOctcacaattccacacaacatacgagccggaagcataaagtOtaaaqcctOgOgtgOctaat gagtga 6315 6316 gatsactcacattaattgcgeLgcOctcactgoccgcettccageagOgaaacotOtcOtgccageLgoattaa tgaatc 6395 6396 ggccaacDcOcgOggagaggcogettgogLattOggcgctetcccOcetcctagctcachgactcgOtgcgete gotCgt 6475 6476 toggatscOgcoaDcOgtatcagctcactcaaagoaggraatacgOttatccacagaatcagoggataaegcag gaaaga 65S5 6556 acaDgtgaDcaaaaggccageaaaaggocaggaacageaaaaaggOcgcottgctoDcOtetetccataggetc cDccec 6635 6636 cotgacgagcatcacaaaaatagacDctcaagEcagaggUggcgaaaccOgacaggactataaagataccagOc getccc 6715 6716 cectssaasotcactOstscsOtctcctOteccgacectgecgcttaccOgatacOtOtcoOccketctcccrt cOggaa 6795 679G scOtgsogctetOtcatasctcacOctOtaggLatctcagtccggtOtaggecOttegceccaagOtOggctgt Otgcac 6875 6876 gaaccococuttcagcccgaccgotgcOccetatcoggLaactatogtcttgageccaacOcOgLaagacacOa cEtatc 6955 6956 gocactOgcagoagccactggLaacaggattaDcagagagaggraLgLaggcogUgctacagagetceLgaagt gOtgOc 7035 7036 ctaactaegOctacactagaeggacagLatttOgLatctgcgototgctgaagccagetaccetcOgaaaaega gerOgt 7115 7116 aDctceLgatccOgcaaacaaaccaccOctOgLagcggtggteteteLgtttgcaaDcaDcagattacDcgOag aaaaaa 7195 7196 aggatctcaageagatcOthegatettetctaeggggtctgacgctcagtggaacgaaaactcaeDttaagoga tteLgg 7275 7276 tcargagattatcaaaaaggatcetcacctagatcuttttaaattaaaaaLgaagetttaaatcastctaaagt atatat 7355 7356 gagLaaaceLgOtetgacagetaccaatgcttaatcagtgaggcacctatetcaDcgatctotctateteDtcc atccat 7435 7436 agUtgcctgactcccogEcgUgLagataactacOatacgggagggcttaccatctOgcaccagtgctgcaaLga taccoc 75LS 7516 gagacccacDctcaccgOctccagatUtatcagOaataaaccagccagccggaagOgcogaDcocagaagtogt cctgoa 7595 7596 acrttatccOcetccatccagtctattaattghtgccgggaagetagageaagragteoDccaghtaatagter OcOcaa 7675 7676 cgeLgttgacattgctacagOcatcOtOgtOtcacgctcgtogtttOgLaLggcetcathcagctcogOtcccc aacOat 7755 7756 caagOcOagttacaLgatcccacatghEgtgcaaaaaagcggttaDctcchtcOgecctccOatcgttOtcaga agLaag 7835 7836 ttgOccocagtOttatcactcaLggttaLggcaDcactgcatastcatcetactOtcargccatccOtaagatg athtec 7915 7916 totgachOgtgagLactcaaccaagEcatectgagaatagtgLaLgcOgcOacOgagetgctceLgcccggagt caatac 7995 7996 gggatastaccDcgccacataDcagaacteteaaagtgotcatcattOgsaaacotectecOggoogaaaactc tcaagg 8075 8076 atcttacooctghegagatccagetcOaLgLaacOcactcgtgcacccaactgatcttcagcatcetttacket caccag 8155 8156 cUtttctUgOtgageaaaaacaggaaggcaaaatgccgoaaaaaagggaataagOgcOacacggaaatgetgaa tactca 8235 8236 tactctccatttetcaatattattgaaecatttatcagggttattgtOtcaLgaDcggatacatatttgaatOt atttag 8315 8316 aaaaataaacaasta m gttccgcgcacatteccccgaaaegtgocac 8364

pTub8TelR-GCN5-DllFRTS -> Genes rXLA sequence 8398 bp ctgacOcgecet.. gaaaegtgccac circular Pusion between TetR and GCNS -- NsiI-is at the 3?end ot TetR! TRAP was isolated via NsiI/BamHI 1 ctgacocOccctotaDcggoOcattaaDcocOgOgOgtgtggtggttacOcOcagcargacOgctacacttgcc aDcgac 80 81 ctagogcccOctcetttcOcretcrtccettcetttatcgccacgttcOcOggcEttccccgtcaaDctctaaa tegggg 160 161 gctcectttagogteccoatttagtgetttacgOcacctcgaccecaaaaaacUtgattagggtga tgg LtcacgLagtg 240 241 goccatcgecctgatagacogettttcOcocrttgacgttggagtccacOttathtaatagLygactOttOtec caaact 320 321 ggaacaacactcaacoctatctcggtetatectettgatttataagOgathttgccDatttOggectattOght aaaaaa 400 401 tgagetgatttaacaaaaatetaacgogaetettsacaaaatattaaegcetacaatttccatccOccatecag Octgag 480 481 caactgttOggeagOgcOatagOtgogOgcctOttcgctattacgccaDctOgcoaaagggggatOtgatgcaa ggcOat 560 561 taaghegogtHaegOcagOgetetcccagtcacgaegttgLaaaacOacOgccagtgaUcDcOcgLaatacOac tcacta 640 641 tagoOcDaattggOtacoggOcecccOctcgacgOtatcgataagcttaaccacaaacOttgagacocOtOtcc caacca 720 721 cOcaccOtgacacoogtOttccaaccacgcaccOtgagaegcgtgttOtsaccacOcaccotgagacgagtOte ctaacc 800 801 aegcaccctgagacgcotOtccaegcEtgcctgcattgggtgoggUtogtgatcctOgUtggacogOtogagat gcDcOc 880 881 gcaegaaggOgaLgtOtcagaaacattttgettgtectotgtgsacttttagatOtottaaaggaggceastat tagcag 960 961 agagtcctccetgtcOcatectctattgaatttcOccctttccttctcEttgcgagtotogtagagaacaagoa ctcget 1040 1041 cOcOgeccaLgacDacgcaacccgOgOagsagacatccaccaaacOgtgetacacaatcaccttOtOtgeaget attgcg 1120 1121 gaaaactactcgttggcattttttettgaattcatetetegacaaa ATC TCG CGC CTG GAC AAG AGC AAA 1190 1 S R L D S X 8

1191 GTC ATC AAC TCT GCT CTG GAA TTA CTC AAT GAA GTC GGT ATC GAA GGC CTG ACG ACA AGG 1250

9 v I N S A L E L L N E V G Z E G L S T R 28 1251 AAA CTC GCT CAA AAG CTG GGA GTT GAG CAG CCT ACC CTG TAC TGG CAC GTG AAG AAC AAG 1310

29 K L A Q L G V F Q P T L Y W H V R 48

1311 CGG GCC CTG CTC GAT GCC CTG GCA ATC GAG ATG CTG GAC AGG CAT CAT ACC CAC TTC TGC 1370

49 R A L L D A L A I F M L D R R R T R F C 68 -

1371 CCC CTG GAA GGC GAG TCA TGG CAA GAC m CTG CGG AAC AAC GCC AAG TCA TTC OGC TGT 1430 69 P L E G E S W Q D P L R N N A S F R C 88

1431 GCT CTC CTC TCA CAT CGC GAC GGG GCT AAA GTG CAT CTC GGC ACC CGC CCA ACA GAG AAA 1490

89 A L L S R R D G a X L G T R P T E 108 1491 CAG TAC GAA ACC CTG GAA AAT CAG CTC GCG TTC CTG TGT CAG CAA GGC TTC TCC CTG GAG 1550

109 Q Y F S L E N Q L A F L C Q Q G F S L E 128

1551 AAC GCA CTG TAC GCT CTG TCC GCC GTG GGC CAC TTT ACA CTG GGC TGC GTA TTG GAG GAT 1610

129 N a L A L S A V G R P T L G C V L E D 148 1611 CAG GAG CAT CAA GTA GCA AAA GAG GAA AGA GAG ACA CCT ACC ACC GAT TCT ATG CCC CCA 1670

149 Q F R Q V A E F R E T P r D S P P 168 1671 CTT CTG AGA CAA GCA ATT GAG CTG TPC GAC CAT CAG GGA GCC GAA CCT GCC TTC CTT TTC 1730

169 L L R Q A.I F L F D R Q G A P P A P L F 188

1731 GCC CTG GAA CTA ATC ATA TGT GCC CTG GAG AAA CAG CTG AAG TGC GAA AGC GGT ATG Q T 1790

189 G L P L I I C G L E Q L C E S G 208

1791 AAA GGC GCT OCA ACA GGT CTG GGG CTC GCG TCT TTC TTC GGG AAG TCG TTC ATT TTT CAC 1850

209 G A P T G L G L A S F F G S F I P R 228

1851 ACA TTG CAT GCG GCG TTG CCC GCC CTG TTG GAG GAA CTC GCC AAC ACC GTC GTG GGG ACG 1910

z2g r L R A A L P A L L F L A N T V V G T 248 1911 GAG CTG CGA CGC TTC GTT CTC GCC CTC GCA GCC GCC GTC GGC CTC T'CC AGT TOG Q T GCA 1970

249 F L R R F V L A L A A A V G L s S S R A 268 1971 GAG GAG CTT CTC CAC CGG GCC GTC GCT GTT CGT TCC AGT CGC CTC GAA TCG ATC TTG CCG 2030

269 F F L L R R A V A V R S S R L P S l L P 288

2031 TOO GAG ACA GGC TTG GGG TTT CTG CAC CGC GAC GCG GGA CGA GGG CGC' GAA G4G GPA CTc 2090 289 S E T G L G F L R R D A G G A R E E E L 308

2091 GSA ATC ATC AGC TTC TGC TGC GG ACG AAC GAC AGG CAA CCG TlU CAC ATG CGG CAC TTG 2150 309 G I I S F C C V N D R Q P L H M R H L 328

2151 GTG ACG GTG AAA AAC ATC TTC TCT CGA CAG CTC CCG AAG ATG CCG CGA GAG TAC ATC GTC 2210

329 V T V N F S R Q L P K P R Y I V 348

2211 CGG CTC GTT TTT GAC CGA GCC CAC TTC ACC TTC TGT CTC TGC AhG CAA GGC CGC CTC ATC 2270 349 R L v F D R A R F Y C L C K Q G R V I 368 2271 GGA GGA GTC TGC TTC CGC CCC TAC TTC CGC GAA AAA TTC GCG GAA ATT GCT TTC CTC GCG 2330

369 G G v C P R P Y F R E K F A I A F L A 388 2331 GTG ACA TCT ACT GAG CAG GTC AAG GGT TAC GGG ACG CGT CTC ATG AAT CAT CTC AAG GAA 2390

389 V T S T e Q V K G Y G T R L M N L K E 408 2391 CAT GTG AAG AAA TCT GGA ATC GAA TAT TTC CTC ACC TAC GCA GAC AAC TTT GCA GTG GGG 2450

409 H V K K S G Z E Y F L T Y A D N F A V G 428

2451 TAT TTC CGT AAG CAG GGC TTC AGC AGC AAG ATA ACG ATG CCG CGA GAC CGT TGG TTG G5C 2510

429 r F R K Q G F S S I T M P R D R W L G 448 2511 TAC ATC AAG GAC TAC GAC GGC GGT ACG TTG ATG GAG TGT CGT CTC AGC ACC CGA ATA AAT 2570

449 Y I R D Y D G G T L E C R L S T R Z N 468

2571 TAC -lv AAA CTT TCG CAG CTC CTC GCC CTA CAG AAA CTC GCA GTG AAG CGA CGC ATT GAG 2630 469 Y L K L S Q L L A L Q K L A V K R R I 488

2631 CAA TCC GCG CCT TCA GTC GTC TGT CCT TCT CTC TCT TTC TGG AAG GAA AAT CCA CGT CAG 2690

489 Q S A P S V V C P S L S Y W K N P G Q 508

2691 C1G TTG ATG CCG TOG GCC ATT CCG GGC TTG GCC GAA CTA AAC AAG AAT GGC GAG CTG TCT 2750

509 L L M P S A r P G L A Y. L N N G Y. L S 528 2751 CTG CTG CTG TCT TCG GGG CGC GTC GGG GCC GCG CCC CAA GCG TCA GGG GCC CTT CCC GGC 2810

529 L L L S S G R V G A A P Q G S G A L P C 548

2811 GGG CGC ACG GGC GCC TTG GGC TCC AAA AAG CGC CCT TTC GGG CGC GCG GGC TTC GCG AAG 2870

* 549 G R T G A L G S K G P F G R A G F A K 568

2871 GGC GAA AAG GGC CTG OGC GCT GCG TCA CTC AAG GCG CAG ATT GCG G W CTT CTG TCA ACT 2930

569 G E K G L R A A S L K A Q I A A L S T 588

2931 CTG GAA AAG CAT TCT TCC TCT TOG CCC TTC CGG CGA CCT GTC TCG GTC AGC GAG GCC CCC 2990

589 L Y. X S- S S W P F R R P V S V S Y. A P 608

2991 GAC TAC TAC GAG GTC GTG CGA AGA CCG ATC GAC ATC AGC ACC ATG AAA AAA CGA AAT CGA 3050

609 D Y Y e v v R R P I D I S S R N R 6Z8 3051 AAT GGG GAC TAC AGA ACG AAG GAG GCG TTC CAG GAA GAT CTG CTG CTG ATG TTC GAC AAC 3110

629 N 6 D Y R S E A F Q D L L M F D N 648

3111 TGT CGC GTG TAC AAT TCG CCC GAC ACA ATT TAC TAC AAG TAC GCA GAC GAG CTC CAG GCC 3170

649 C R V N S P D S r Y K Y A D L Q A 668 3171 TTC ATC TGG CCC AA3 GTC GAG GCT CTC GOG AGT TTT TAA ttaatcaccgttgtgetcacttctcaaa 3237 669 F I p E A L G S F 681 3238 tagacaaaggaaacacacthegtgeagcatgtgccccattataaagsaactgagttOtecoDctOtgOcttgoa ggtotc 3317 3318 acatccacaaaaacOgOcagactataastaggagtgtttageagoaaDcaDcoaaeotetatgactOgOtccga atctat 3397 3398 gaacOgatototOgoggacctOgatgaLgeLgatcgccgtcgacacacOcDccacatogutcaatacacaagac agctat 3477 3478 cageLghtttagtcgaaccogttsacacaattceLgaccecccgagogggatccactagetctagagOggccgc tOtaga 3557 3558 actagUggatagatcoccOggOctgoaggeatccatceLgcaagtgoatagsaggaaagetOtctgeeOtcgtO gocaga 3637 3638 caDcaacageccagcactctauagacatacagaacgatsaegcathcacOagtOgatacacOcacatctgcutc aceogc 3717 3718 aactcOctttcgttOtgattgacaaaaagaaaacaaggcgaggtgagackgtotgeaaLgOcacaLgaagagtc atcoct 3797 3798 tttcttegataaaggacacaggggtetatOgcacoccatcgtcagctctctcogacccuaggOactatccctga tccctc 3877 3878 cOaaaagagaggsaaacOagagacOgOcaguttatgrathtccgctagacaDccatctccatctogatCcgEcc Otgcgg 3957

3958 gacstasUccaegacctcaaaatcOtagOcOgtgaaetcgtagattecceegaLgcgetccttOttgaggatOt ccacaa 4037 4038 tOggsaacogectegOtectatcoDcagotgotctettaaagectogacaLgOttcOtOtagacaLgOgtottc cccaLg 4117 4118 aeOtgaatgeactccetagUttttagottgOagaegtgtgesaccaLgagcgecaaaaDcDaataggeagcgaL getgaa 4197 4198 gOggacOcogagOccOacatcgcacDacOgetgatacatgatgcacOacaDctcotcarogtegEtcacOtage actggc 4277 4278 acaacaagtgacaaggogoca9coccatEteotccagogetgcaggateccagocagtcatgagOatgcOacga tctgEt 4357 4358 ggattcgtectcaDcatctggatcacattctEcaDchggEcgacgOcctgecetOtgragtOtOtOtgcatOto tttgLa 4437 4438 tgccgcOccOaagtOtctccactogsagocgtagcccgggccgatOtotccDacOtctcOgtOgOggagattgc Ocgaat 4517 4518 cgaggaactcOcOtUtcacattcttOtcccagatctCcacgcecetctcagaaegaLgOtetgogetcOtgtcD ccOcOa 45g7 4598 aLgaaccacaDcaactatecOaggaccochtCccagaacacaegetttOtggtgagaagtogaaaggcctgatc cagcDa 4677 4678 gLaDcgcatagtgcagccOaatttggagaLgacaccaacgeccgttoggEcatccattotccetccattOttaa taatgt 4757 4758 cOgeaaLgagatcaagOtactOgeatCcrtcaLggcctataaagtgaacaLgcogaacggcccgaatcagttcc UtEtgc 4837 4838 tcgOgttettcccggtcrtetecOtccatccacoccaacacaggggcaatOgcLgaggccoaagaaggagcctg Oaaccc 4917 -

4918 gtgcacgogagttgtctccctogtagacgtcaaggagctcattgcgttgctcOgttccgcagtoOctgcOtcOt cagtct 4997 4998 tccEtOtcttctogagaaccacaaagtcOtaggOtacceagttgtOtgagaagotettggaaatgaagaLgogE cDatac 5077 5078 gtcDctEcattgecctcatctottccDaDctccOgacaaaagggaacOaacacagactegOcaggaachgcaDc ctgcOc 5157 5158 aDcageLgatttOtttgaaagaatgtcatetccOgggaacgcagggaagaaaaeOtcOcacggaaactcgcgoO ctacac 5237 5238 gcgUgatOtacagOtgagagOcaacDcccagagacagagctgcctOgtacagtcccOctcetcccacgacaaaa atctog 5317 5318 togacagaatcceLgLactotcectccagaagOctgagagctgotoggagtgaegcacagactcggacgcOctg OtgOcc 5397 5398 ttcagcrtgagOcrtctcogcOgcastatcEtctEctEtgagggaagaggeaacOacgaLgtecaatctotcca coagOg 5477 5478 gtctaaacettcOagOcatgctetcccaggtEttccgtcocatgacaacOgOgetgaatctottgccOactgat Ogagag 5557 5558 ggaagtccagagecOcccotatttgeaaatEtcOtgggaagccacccOttcagOcOachogottOteaggOcOt cUtett 5637 5638 tgtcacacOagaaaagtgteLgaaatctOtOgEcaagtggggccaegggagOcOgttOttgatgOcgaLgcocc tceLgg 5717 '. -

5718 gOgtcatcgcgacgaccagacacacagOtttCtgcatattcacagacacgacaacoccocotagaDcagaaacO cactac 5797...

5798 taaaDcgaaacetcaccagtcOctgctgcactcagageagtgctccOcactgccgtOtOgLaaaatgaaaegOt ectacg 5877 5878 agacaegcOtOtccOgatcgacaagcOaaggatctgcacaceLggtctogatOtcgaacaaegcacOgaggaga gaegga 5957! -

5958 aagtgcttacatagaacaegottatcaaacccgagaaaaagaaaegaacagaagaaaaeggaaacOtcogcata ctteta 6037 6038 aagsaLgaagttcecogatEttcccaaaaeLgOcgtcattttcgcgeacggoagecagataacaggtOtagogg ctgOcc 6117 -

6118 accaacagagacggagcOgeOgacaggacOctactgggactgogeacaDcaDcaagateggatcttcceogOtO gagOtc 6197 6198 caDcEtetOttccatttagtgagOgeteattgaDcgcktggogteatcatgotcataDctgetecetotOtgaa attOtt 6277 6278 atccoctcacaethocacacaacatacoaDccogaagcatesagtgtaaaDcaLggOgtgactastgagtgaUc taactc 6357 6358 acattaattgogttgOgetcactgoccactttccagtcgggaaacctOtcOtgocagctgcatteatgeatcOg ccaaeg 6437 6438 cOcgoggagaggcOgettgcgeattUgocgetettcagcttcetcectcachgactegeegaDctOgOtagtte gOctgc 6517 6518 ggcgaDcogLaecaDctcactcaaaggcgOtBataeggttatccacagnatcagOggataacgcaggaaagaac atOtga 6597 6598 geaaaagOccaDcaaaDgOccaggaaccUtaaaaaggocgagttgctOgcOtttetccatagoctccOcccccc tgacga 6677 6678 goatcacaaaaatcOaegctcaagtcagagotoDcgaaaceogacaggactataaagataccageeOtetccoc ctggaa 6757 6758 gctccctcOtgcDctctcctottcogacoctgcOgcetacoggatacctOtcOgactttctcccetagOgeaDc OtOgcg 6837 6838 ctttctcataDctcacDctOtaggtatctcagttcggtgLaggtcgtEcDctccaegctoggctOtUtgcacOa acccoc 6917 6918 cgEtcaDcccgacoDctgcDcettatcoggLaactategtceLgagEccaacccOgLaagacacOacetataDc cacteg 6997 6998 caDcaDccactgotaacaggattaDcagaDcOagOtatgLaggcggtgctacagagetottgaagtogtoOcct aactac 7077

7078 gOctacactagaaggacagLatUtggtatctgcgctctgctgaagCcagt tacctCcggaaaaagageLggLagotcetg 7157 7158 atccggcaaacaaaccaccOctOgLagcOgtogetettttgEttgOaaDcagcagattacgaDcagaaaaaaag gatctc 7237 7238 aagaagatccettgatckttCctaeggggtctgacgctcagtggaaegaaaactcacg ttsagggatettggtcaLgaga 7317 7318 ttatcaaaaaggatctecacatagatccrEttaaatteaaaargaagetetaaatcaatotaaeOtatataLga gLaaac 7397 7398 ttOgteLgacagttaccaatgettaatcagtgagocacctatetcaDcoatctOtctatttcgtccatccatag ttgOct 747? 7478 gactceccgtcgtotagataactacoataUgOgagggottaccatctOgCcccagtgctgcaaLgataccOcga gaccca 1557 7558 cOctcaccOgetccagatttatcagcaataaaccagccagcoggsagOgccDaDcOcagaagtggtcctgOaac rttatc 7637 7638 cDcctccatccagtctattaettOttgccOggaaDctagagraagLagtCcgccagetaOtagUttgOgcaacU ttOttg 7717 7718 ccattgotacagOcatcotOgtOtcacDctagteOtttggtaLggottcatEcaDctcogOttcccaacOatca agocDa 7797 7798 gttacaLgatcccOcaLghtOtgcaaaaaagcOgttagotcatccgOtcctcagatcottOtcagaagLaagtt Ogocgc 7877 7878 agUgttatcactcaLggetatOgOaDcactgcataatUctcrtactOtcaLgccatccOtaagaLgctUtectg tgactg 7957 7958 gtgagractcaaccaagtcatEctgagastagUgLargcggogaccgagetgctottgccogOcgtcaatacog gateat 8037 8038 accOcoccacataDcagaacrttaaauDtgctcatcattggaaaacOtecttcogoDcoaaaactOtcaaggat crtacc 8117 8 1 1 8 gctOttyagatccagetcgatgLaacccactcgtgcacccaactgatctccaDcatcEtttactttcaccagcO tUtOtg 8197 8198 ggtgaDcaaaaacaggaegOcaaaaLgccocaaaaaagggaataagOgogacacggaaeLgEtgaatactcata ctOtcc 8277 8278 cttttCcaatattattgaaDcatttatcagOgttattgtctcargagoggatacatatttgaatgtatttagOa aaataa 8357 8358 acaaataggggteccgogcacattccocogaaaagtgccac 8398

pTub8TATi-l-llXCPRT -> Genes DNA sequence 6216 bp ctgacgogccot gaaaagtgccac circular RT-PCR-Fragment (Primer PolyT Rep4) were cloned into pGZH after Sequencing cloning into pTUB8-Rep Yia NdeI/BamHI 1 ctgacococcctgtaUcOgoOcaLtaaDcOcOgcogytgtggtggUtacOcOcaScOtgacOgctacacttgec aDcocc 80 81 ctaDcOcecgctcetttagctEtcetccUteccrttctagacacgEtcoccggcrctcOcOgecaaDctctaaa tcogog 160 161 gctcccettagogteccOatttagtgctetaegOcacctegaccccaaaaaacetgattagogtgatOgtccac Otagtg 240 241 gOccatcOccctgatagaeggtetttcOccOtttgacghtggagtccaeOttcEttaatagtggactattgtec caaact 320 321 ggaacaacactcaaccctatatcggtctathetEttgatttataagggattttgccgatetcggcctattOgtt aaaaaa 400 401 tgaDctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcetacaatEtccattagccattcag Octgcg 480 481 caactOttgogaugOgcgatcgOtgcOgOcctettcgotattacgOcaDctggcDaaagogOgatOtgchgOaa ggcOat 560 561 taagttOgOtaaegccagOgttUtcccagtcacDacgttgLaaaacgaegOccagtgaDcOcocgLaatacgac tcacta 640 641 taggOcDaattggetacogOgOcccccctcOacOgtatagataagettaaccacaaaccetgagacOcgtOtec caacca 720 721 cDcaccOtgacacDcotOttccaaccacOcaccctgagaegagtgtcctaaccacDcacOctgagacgcgtgte ctaacc 800 801 aegcacceLgagacgcgtOtecaegcttgcctgoattgggtgcggttOgtgatcctOgttOgaccogtOgagat gcOcOc 880 881 gcacoaagOggatototcagaaaCattttatttgttctctgtgaacttttagaLgtOttaaaggcOgcgaetat taDcag 960 961 agagecctcattottccattctctcEtgaathtcoccotttccttctcettgagagtgtggtagagsacaagca ctogtt 1040 1041 cOccOtcactgacOacgoaacccOcocagaagacatccaccaaacOgtottacacaatcaccttOtOtgsagtt cttgcg 1120 1121 gaaaactactagetggcatttttCcttgaatCccttetcagacaaa ATG TCG CGC CTG GAC AAG AGC AAA 1190 1 s R L D S 8 1191 GTC ATC AAC TCT GCT CTG GAA TTA CTC AAT GAA GTC GGT ATC GAA GGC CTC ACG ACA AGC 1250

9 V I N S A L E L L N E V a I E G L T T R 28 1251 AAA CTC GCT CAA AAG CTG GGA GTT GAG CAG CCT ACC CTG TAC TGG CAC CTG AAG AAC AAG 1310

29 K L A Q K L G V Y Q P T L Y V N R 48

1311 CGG GCC CTG CTC GAT GCC CTG GCA ATC GAG ATG CTG GAC AGG CAT CAT ACC CAC TTC TGC 1370

49 R A L L D A L A I Y. L D R H S F C 68

1371 CCC CTG GAA GCC GAG TCA TW CAA GAC m CTG CGG AAC AAC GCC AAG TCA TTC CGC TGT 1430 69 P L E G E S W Q D Y L R N N A S F R C 88

1431 GCT CTC CTC TCA CAT CGC GAC GGG GCT AAA GTG CAT CTC GGC ACC CGC CCA ACA GAG AAA 1490

89 A L L S 8 R D a A K V 8 L G T R r T 108 1491 CAG TAC GAA ACC CTG GAA AAT CAG CTC GCG TTC CTG TGT CAG CAA GGC TTC TCC CTG GAG 1550

109 Q Y T L Y N Q L A F L C Q Q G F S L E 128

AAC GCA CTC TAC GCT CTG TCC GCiC GTG GGC CAC TTT ACA CTG GGC TCC GTA TTO GAG GAT 1610 129 N A L Y A L 5 A a n F T L a c v L E D 148 1611 CAG CAG CAT CAA CTA GCA AAA GAG GAA AGA GAG ACA CCT ACC ACC GAT TCT ATG CCC CCA 1670

149 Q F 8 Q V A K E E R F T P T T D 8 M P P 168

1671 CTT CTO AGA CAA GCA ATT GAG CTG TrC GC CAT CAG GGA GCC GAA CCT GCC TTC CTT TTC 1730 169 L L R Q A I L F D Q G A Y r A F L F 188 1731 GGC CTG GAA CTA ATC ATA TGT GGC CTG GAG AAA CCC ACG TTC TTT AAT AGT GGA CTC TTC 1790

189 G L F L Z Z C G L Y. P T F F N S G L L 208

1791 TTC CAA ACT GGA ACA ACA CTC AAC CCT ATC TCG GTC TAT TCT TTT GAT TTA TAA gggatett 1852 209 F Q S C T T L N P I S V - Y S F D L 226

1853 gOcOatetegocctattOgttaaaaaaLgagttgatttaccaaaaatttaacgcDaatettaacaaatattaac gatta 1932 1933 caatttaggtOgcccttetegoggaaaLgtgcOcggaacccctatttOthtatEtttetaaatacattcaaata tgtatc 2012 2013 cOctcaLgaDccaataacactgatesaLgetEcaataatatcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaag 2092

( 2093 gatccactagtectagagcOgCcgCcaccOcOgtOtcactgtagcctgccagaacacttOtcaaccOactgtge ccacat 2172 2173 ttttatgcocactgactOgcatgaatgOccagaggoaggeatcagcaagtcacOtaggccaacOcOtgcOcaga aacoct 2252 2253 caagOctcgattotgOgtOggggeLggtaDcatUttatcgacctaaacaagOtttacacttagotggtgcggte ttactg 2332 2333 atctOgacOgattcagcgOtcOcagattategatotgcaaatggtutacacttagotgtcgcOgcttatetagt taagOg 2412 2413 aDcttcOtOgtcOgaDcOtaacaagtcaacagagacgLatogccaatcOttcOcOgtgaegagEcgaaactgac agOaca 2492 2493 tcOtagOgaaactgagaggOtgctcctEtctctccgEcgthtgcgctgcaccatcotgoaagtgcatagaagga aagttg 2572 2573 tctgOtOtcOtgOgcagacaUcaacagtccaucactctagoggcatacagnacoataarOcatEcaUgagtOga tacacg 2652 2653 cacatctgcgEcacocOcaactOgOtetegtectgattgacaaaaagaaaacaaggogagOtgagactgtOtga aarOcc 2732 2733 acatgaagagLcatcacttttcUtcOataaaggacacaggggtctctugcaccacctcOtcaDctctctccDac ccOagg 2812 2813 cactctcOctgatcCctcoguaaagagaggeaaaUgagagacgggcaDcLtctatagoOctatgoagggettac rtatcg 2892 2893 aacrCtttgcoagcOgogtcgotcaggacOgcgacgtggtegaagtcOcggaacatctcuttgeagecOtagoa gcaacc 2972 2973 aacOatccagacOtcrtcaaLgctgaaUccOacgaagtcgcocUtcaegcgUtOgaDcOatetOtgoucetotc Daega 3052 3053 gogtoOcOattctcatcgacEtOggaccgacOgcettcaggcgctcaccOaactcOgtgagOgtgaaaccOgtO tOgacg 3132 3133 arOtcctcaacaatcagaacOtgcUtOtcgcOaaagattgacaagtcOtcOctcaagacgOtgaDctOgcctOt gctOtt 3212 3213 gtogntctOgLaggacktcagOcOgacatagUgetcgaegaagggOgOcaegctggactcacOaccactOtacu tctOta 3292 3293 tOgtOgcaaggtagtegatcagaagOttgaagaaDccgcgagagcctetcaggatgcaaaLgatgtgcaactcO tcgccg 3372 3373 aagtaagttctgtOgatOtcatacOccaacetctcaactetgtccttgaccaatccaccagOgaggaggatttt atcaat 3452 3453 gtagOgcUtgcagUgOgguggoacaagaaagtcatcagcgttgLagaaggtOttOtcOgOgatatacatOggct caatac 3532 3533 ggcccrtgoccttgcogLagtcttcaaLggOtUtOgacgccatettOgatctgacaaegcoccOtagagcagaa acgcac 3612 3613 tactaaagcOaaacttcacOcOtccctgctgcactcagagcagtgctccOcactgccgtOtOgLaaaaLgaaaa gOttot 3692 3693 aegagacacgcgtctcOggatcgacaaDcgaaggatctgcacacotOgtctegaLgtOgeacaaaDcaeggagg agagac 3772 3773 ggaaagtgcttacatogaacaegOttatcaaacccgagaaaaagaaacDaacagaagaaaaaggaaacctccgc atactt 3852 3853 ttaaagaatgaagetccOcoatttheccaaaaaLggcgtcatttCcgcgcacgOcagecagataacagOtOtaD cggctg 3932 3933 cccaccaacagagacOgcocgOccOacaggacOctactgggactgcgaacaDcaDcaagateggatcttcOgcg gtogag 4012 '.;' 4013 ctccagOttttgttcocettagtgagggtteattgcgcgcttggcgtaatcatOgtcatagctOttecctatot gaaatt 4092 4Q93 gttatccOctcacaattccacacaacatacOagccggaagcataaagtOtesaUcctgggOtgCctastgagtg agctaa 4172 4173 ctcacattaseLgcOttgcOctcactgCCcOchthccagtogggsaacctgteOtgOcaDctgcattastgast eggcca 4252 4253 acOcgagOggagaggoggtetgcgLattOggcgetattCcgcttCctcOctcactgactauctgaDctcogtcO ttcggc 4332 4333 tgcgOcOagcgOtatcaDctcactcaaagOcggtsatacggttatccacagastcagOggataacocaggaaag aacaLg 4412 4413 tgagcaaaaggccagcaaaegOccaggaaccOteaaaaggaegcgttgaLgOcutttetccatagoctccOcac cactga 4492 4493 ceagcatcacaaaaatcDaegetcaagecagagOtggogaaacccOacaggactataaagataccagOcatttc eccctg 4572 4573 geagctccOtogtgcgctatcctOttcOgacactgccgcEtacaggatacctotccOcctttctcocttcggga aDcOtg 4652 4653 goOcettctcatagOtcacDctOtagotatctcagttcggtgtaggtcgttcOctccaaDctOggctOtOtgca Ugnacc 4732 4733 coccgetcagccogaccOctgcOcOttatccOgtaactatogEcttgageccaaCCcgOtaagacacgacttat cOccac 4812 4813 tggoagcaDccactOgLaacaggattagcagagcDaggLatgtaggcOgUgctacagagetcttgaagtOgtgO cctaac 4892 4893 tacggctacactagaaggacagLatttOgLatctgagctctgotgaagCcaghtaccttcogaaaaagagttgo tagctc 4972 4973 ttgatccOgcaaacaaaccaccOctOgtaDcogtOgttttthtgtttgcaaDcagOagattacocDcagaaaaa aaggat 5052

5053 ctcaagaagatccettgatcrCtectacOgogechgaegctcagtOgaacgaaaactcacgetaagggattttO gtcaLg 5132 5133 agattatcaaaaaggatcttcacatagatCcetttaaattaaaaaLgaagtthtaaatcaatctaaagtatata LgagLa 5Z12 5213 aacttOgectgacagttaccaatgcttaateagtgaggcaCctatctcaDcgatctOtatattccOtecatCca tageLg 5292 52g3 cetgactccccOtogtOtagataactacOatacOggagggcttaccatotgOccOcagtgctgcaaLgataccO cgagac 5372 5373 ccacgatcaccoDctccagatetatcaDcaataaaccagccagcoggaayggccoaDcOcagaagtggtcUtgc aacttt 5452 5453 atCcgOctccatccagtOtattaattuttgccgOgaagetagagLaagtagetcgOcagttaatagettgOgoa aeOttg 5532 5533 ttgacattgetacagOcateDtOgtotcaegOtcgtcghttggtatgOcetcatccagotcOggttcccaacoa tcaeoa 5612 5613 cOagttacatgatcocacatOttgtgcaaaaaaDcggttagctccttcggtcOtccoatogttOtcageagLaa gttOgc 5692 5693 cOcagtOttatcactcaLgOttatOgcaDcactgcatoatCctcttactgtcatgccatccgtaagatgcette ctgtga 5772 5773 ctOgtgagLactcaaccaagecatectgagaatagtgLaLgcgycOacogagtegctcttgOccggcOtcaata cgggat 5852 5853 aatacOgcOccacatagOagHactttaaaagUgetcatcattggaaaacgtcctCcggOgcOaaaactotcaeg gatcrt 5932 5933 acOgctOttgagatccagtEcOatgLaacceactagtgoacecaactgatcEtcagcatcttttactttcacca DcOttt 6012 6013 ctOgotgagcaaaaacaggaaggcaaaatgccOcaaaaaagggaataagOgcOacacggaaatOttgaatactc atactc 6092 6093 ttcatettCcaaLattattgaagcatttatcagOgLtattgtotcargaDcOgatacatatetgeatotateta gaaaaa 6172 6173 taaacaaataggggtCccgogeacattCccocgaaaagtgecac 6216 , . ..

pTub8TA1 i-3-ll'CPRT -> Genes DNA sequence 6392 bp ctgacgogcoct.. gaaaagtgacac circular 1 ctgacDcgccotgLagcggoOcattaascOcOgogggUgtggtggEtacOcgcagcOtgaccOctacacrtgoc agoOcc 80 8I ctaDcOcooOctccrtEcoctetOtCcOcrtcotEtotcgccacgttcoccogottecccogtcaagotctaaa teggog 160 161 gctccctetaggOttccOathtagtgcettacggeacetegaccccaaaaaacUtgattagogUgatogttcac Otagtg 240 241 gOccatcOcoctgatagacOgtttetcgccotetgaegttggagtccaeOtccttteatagtOgactottgEtc caaact 320 321 ggaacaacactcaacoctatctcggectatccrettgattataagOgattttgccOatttcOgcctattOgtta aaaaa 400 401 tgaDctgathtaacaaaaatttaacgOgaattttaacaaaatattBacOcetacaattcccattcOccatccag Octgcg 480 481 caactOttoggaagOgagatcOgtgcgoDcetOteegetaCtacgccaDctoDcOaaagggOgaLgUgctgcaa ggcgat 560 561 taagttOgOtaacgccagOgttetcccagtcacgacgttgtaaaacOacOgccagUgagcOcoOgtaatacgac tcacta 640 641 tagOgagaattgggLaccgggacocecctcOacgOtatcgateagcUtaaccacaaaccUtgagacgcotOtUc caacca 720 721 cOcacccgacacgogtOttccaaccaegoaccotgagacgcgtgEtotaaccacOcaccctgagacDcgtotEc taacc 800 801 acOcacoctgagacOcOtOttcaegcttgcctgcattgggtgcggttggtgatcctOgttggacOggtOgagat gcOcOc 880 881 gcacgaaggggaLgtOtcagaaacatthtgtetOtectctgtgaacetetagaLgUgttaaagOcgOcgaatat taDcag 960 961 agagecctcattottccattctctceLgasEttOgccattCcotcotaterOcoagUgtgOtagagaacaaDca ctcOtt 1040 1091 cOccgtcactgacgacgoaacccOcOcagaagacatccaccaaaeggtOttacacaatcaccrtOtOtgaaget attgcg 1120 1121 gaaaactactagttggeattttttcttgaethechtthtcgacaaa ATG TCG CGC CTG GAC AAG AGC AAA 1190 l S R L D R S R 8 1191 GTC ATC AAC TCT GCT CTG GAA TTA CTC AAT GAA GTC GGT ATC GAA GGC CT A W ACA AGG 1250

9 V I N S A L E L 1 N V G I G L T T R 28

1251 AAA CTC GCT CAA AAG CTG GGA GTT GAG CAG CCT ACC CTG TAC TGG CAC GTG AAG AAC AAG 1310

29 R L A Q K L G V Q P S L Y W V N R 48

1311 CGG GCC CTG CTC GAT GCC CTG GCA ATC GAG ATG CG GAC AGG CAT CAT ACC CAC TTC TGC 1370

49 R A L L D A L A I E M L D R H H T H F C 68

1371 CCC CTG GAA GGC GAG TCA TGG CAA GAC TTT CTG CGG AAC AAC GCC AAG TCA TTC CGC TGT 1430

69 P L G F S W Q D F L R N N A S F R C 88

1431 GCT CTC CTC TCA CAT CGC GAC GGG GCT AAA GTG CAT CTC GGC ACC CGC CCA ACA GAG AAA 1490

89 A L L S R D G A V a L G T R P T 108 1491 CAG TAC GAA ACC CTG GAA AAT CAG CTC GCG TTC CTG TGT CAG CAA GGC TTC TCC CTG GAG lSSO 109 Q Y E S L a Q L A F L C Q Q G F S L F 128 1551 AAC GCA CTG TAC GCT CTG TCC GCC GTG GGC CAC TTT ACA CTG GGC TCC GTA TTG GAG GAT 1610

129 N A L Y A L S A V G a F T L C V L F D 148 1611 CAG GAG CAT CAA GTA GCA AAA GAG GAA AGA GAG ACA CCT ACC ACC GAT TCT ATG CCC CCA 1670

149 Q E Q V A R F T P T T D S M P P 168

1671 CTT CT& AGA CAA GCA ATT GAG CTG TTC GAC CAT CAG GA GCC GAA CCT GCC TTC CTT TTC 1730

169 L L R Q A I F L D Q G, A E P A F L F 188

1731 GSC CTG GAA CTA ATC ATA TGT oGC CTG GAG AAA CAG CTC ACT CTT GTT CCA AAC TGG AAC 1790 189 G L F L I I C G L F Q L T L V P N W N 208

1791 AAC ACT CAA CCC TAT CTC OGT CTA TTC TTT TCA tUtataagggattttgCcgatUtcggcctattggtt 1859 209 N T Q P L G L F F 219

1860 aaaaaaLgaDctgatetaacaaaaatetaacDcgaattttaacaaaatattaacOcttacaatetagotOgcac etetOg 1939 1940 gOgaaaLgtgoDcOgaacccOtatttOtttathtEtctaaatacatCcaaetatotatCcgOtcaLgagacaat aaccct 2019 2020 gataaatgottcaataatattgaaaaaggaagagLatgagLattcaacatetccgtOtcDccottatEcccett tttgcg 2099 2100 gcattttgcctCcotOtetttgetcacccagaaacgct99tgaaagraaaagatgctgaagatcageLgOgtgc accDag 2179 2180 tgOgttacatOgaactOgatctcaacagOggtaagatcottgagagttetcccacccaaaaacacccecaaaca caaaaa 2259

2260 aaaacaaaggatccactagttctagaDcOgccgccacogcggtgtcactOtagcctgccagaacaceLgtcaac cOactg 2339 2340 totccacattEttaLgcgOactgactgOcaLgaatggecagaggeagOcatcaDcaagtcacOtagOccaacDc OtgaDc 2419 2420 agaaacscLcaegOctegattOtOgotOgOgottogragcatUttatcgacctaaacaagOtttacacrtaggt Ogtgcg 2499 2500 gtettactgatctggaeggatEcaDcOgecDcagattatcgatetgcaaetOgtOtacacttaggtutegOgOc rtattt 2579 2580 agetaagOgaDctecotOgtcggaDcOtOacaagEcaacagagacotatcgccaatcotecOcOgLgaagagtc Oaaact 2659 2660 gacascacatcOtagogaaackgagaggOtgetccrtccLctccgeeOtttgagctgeaccatcctgcaagtgc atagaa 2739 2740 ggaaagEtotctgctotcotOgOcagacagcaacagtccagcactetaDcOgcatacagaacOataaegcatcc aegagt 2819 2820 ggatacacgcacatctgcotcaccoDcaactcgetetcgtUctgattgacaaaaagaaaacaagOcOaggtgag actOtg 2899 Z900tgaaatgccacaLgeagagEcatccetttecttegataaaggacacagogOtctctOgcaccccctcgtcaDct ctctcc 2979 2980 gaccogagocactctccOtgatcoctccOaaaagagaggeaaaegagagacOgOcaDcEtaLgtagoDctaLgc agOgUt 30S9 3060 tacetCtcOaactetttgcgaDcOgogtcOctcaggaeggcgaegtOgtagaagecocggaacatatcgLtgaa gtogta 3139 3140 gcaDcaaccaacOatccagaeOtOttcaatgotgaagcogaegaagtcDcccthcaaDctOttOgaDcOatctO tgcOct 3219 3220 tctogatcOagOgtggcgattOtcatagacrtgOgaccgacggcettcaggogctcaccoaactcggtgagogt gaaaccg 3299 3300 gtgtcgacOatOtcatcaacaatcagaacOtgOttgecgcgaaagattgacaagecgecOctcaagacOgtgaD ctoocc 3379 3380 tOtgctOttOteOttctOgLaggactccagOcOgacatagtgctcoaagaagOgOgOcacoctggactcacDac cactgt 3459 3460 acrtctOtaLgOtogoaagOtagecoatcagaagOttgaagaagccgcgagaDcatttcaggatgcaaaLgaLg tgcaac 3539 3540 tcatcDccgaagtaagtectOtOgatOtcatacOccaacrtctcaactctotcOttgaccaatccaccagOgag gaggat 3619 3620 teLgtcaaLgLagOgcttgeagtOgoggUgcacaagaaagecatcaDcOttOtagaagOtottOtcggggatat acarOg 3699 3700 gotcaataegOcOcttgcocetgccotagecetcaaLgggtttggacOccathttOgatctgacaacgccccOt agaDca 3779 3780 gaaaegcactactaaascDaaacetcacccotcOchgetgoactcagaDcagtgotccgcactgccOtOtogLa aaaLga 3859 3860 aaagOttctacgagacacOcgtctcoggatcOacaegcgaeggatctgeacacctogtctogatotcgaacaaa DcacOg 3939 3940 aggagagaeggaaagtgcrtacatcDaacacggUtatcaaacccgagaaaaagaaacgaacagaagaaaaagga aacctc 4019 4020 cocatacUtttaaegsatgaagteccOcoattetcccaaaaaLggcgtcatttcOgcDcacoDcagtcagataa caggtg 4099, 4100 taDcgOckocccAccaacagagacOgcOcOgecOacaggacgctactOggactgcDaacaDcaDcaagatcOga tctecc 4179 4180 gogOtOgagctccagottttOttcectttagtgagggetaattgcgagcttOgagtaatcaLgOtcataDchOt ttcctg 4259 4260 tutgaaattottatcaDctcacaathccacacaacatacgagcaggaaDcataaagtOtaaagcctggggtgOc tastga 4339 4340 gtgagCtaaCtCaCAtteattgagttgcgctcactgaccgatttccagtagOgeaacctgteUtgccaDctgoa ttaatg 4419 4420 aatcOgccaacDcDcOgggagagacogettgcgtattgggagetcthcoDcttcotcDctcactgactcgctga Dctogg 4499 4500 tagetogOctgcOgagaDcggLatcaDctcactcaaeggaggtaatacggttatccacagaatcaggogatsac ocagga 4579 4580 aegaacatotgagcaaaaggecaDcaaaaggccaggaaccgtaaaaagOccOcottgctOgcottthtccatag Octccg 4659 4660 cceccotgacgaDcatcacaaaastcDacOctcaagtcagaggtggagaaaccogacaggactateaegatacc agocOt 4739 4740 ttccocaLggaagctccatagtgcOctctcctotCcogacechgeaDcttacaggatacctgtccocctetctc ocrtcg 4819 4820 ggaaacOtOgcgctttatcataDctcacDctOtaggLatotcagtecOgtOtaggecgetcgotccaegctogg aeotOt 4899 4900 goacgaacacacOgttcaDcccOaccOctgOgcCttatccggLaactateutcttgagtccaacccOgLaagac aegact 4979 4980 tategccactOgeaDcaDccactgotaacaggattageagagcgagotaLgragOcgOtgctacagagttchtg aagtog 5059 5060 tOgcetaactacoDctacactageaggacagLatttggtatctgcDctaLgchgaagacagttacetecogaaa aagagt 5139 5140 tegLagctcttgatcoggeeaacaaaccaccgct99tagaggtggtttttttotttgOaaDcageagattaego Ocagaa 5219 5220 aaaaaggatctcaagaagatcetttgatcttetctacggggectgaegetcagtogaaegaaaactcaeOttea gogatt 5299 5300 ttOgtcatgagattatcaaaaaggatOtecacotagatcctettaaattaaaaaLgaageteteaatcaetcta aagtat 5379

5380 ataLgagtaaacttggectgacaghtaCcaaLgattaatcagtgagOcacctatctcagcgatctOtOtatEtc Ottcat 5459 5460 ccatagetgcctgactCCccOtagtOtagataactaegatacgggagOgcttaccatckggcOccagtgctgca atgata 5539 5540 ccgOgagacecaegctcaCcOgctccagatttatcagoaataaaccaDccagccOgaagOgaegagcgcagang tOgecc 5619 5620 tgeaacettatccOcctCcatCcagectattaattgttgccgggaaDctagagtaagtaghtcOccagttaata gtEtgc 5699 5700 gcaacgttUttgccattgetacagocatcOtOgUgtcacgctcgLcgEttgOtaLggettcattcayOtccOge tcOcaa 5779 5780 cgatcaagOcoagUtacargatcccccaLgUtOtgcaaaaaegogottagotccetOgOtcctccOategUtOt cagaag 5859 5860 tHagttogccgcagtOttatcactcaLgOttatggcagcactgoatoatcOtcttactgtcatgOcatcOgLaa gatgot 5939 5940 ttectOtgactOgtgagtactcaaccaagtcatEctgagaatagtotaLgogacOaccDagttgctcetgOcog ocgtca 6019 6020 ataUgOgataataccOcOccacataDcagaactttaaaagtgctcatcattogaaaacgtecttOgogOcgaaa actetc 6099 6100 aaggatottaccOctgeLgagatccagtecOaLgLaacocactogtgOacccaactgatctCcagoatcetEta ctetca 6179 6180 ccagcgtetctogOtgaDcaaaaacaggaagOcaaaetgccgoaaaaaagugaataagouagacaeggaaaLge Lgaata 6259 6260 ctcatactcrtccttEttcaatattattgeaDcatttatcaggghtattotctcatgaucOgatacatatetga ntgtat 6339 6340 ttagaaaaetaaacaaataggggEtccgogoacatEtccocgaaaagtgccac 6392 , i

Claims (1)

1. L.- CLAIMS

   1. A nucleic acid construct comprising the tetracycline repressor (TetR) operatively linked to a transacting factor of T. gondii.

   5 2. A nucleic acid construct as claimed in claim 1, in which the transacting factor of T. gondii teas the nucleic acid sequence of the TATi-1 or TATi-3 activating domain as shown in Figure 6, or a sequence complementary or homologous thereto.

   3. A transcriptional activator of T. gondii comprising the amino acid sequence of 10 TATi-1 or TATi-3 as shown in Figure 6, or an analog, bomolog, ortholog, related polypeptide, derivative, fragment or isofonn thereof.

   4. A hancacting factor of T. gondii having an amino acid sequence of the TATi-1 or TATi-3 activating domain as shown in Figure 6, or an analo& homolog, ortholog, 15 related polypeptide, derivative, fragment or isoform thereof.

   5. A vector comprising a nucleic acid construct as defined in claim 1 or claim 2.

   6. An expression vector comprising a nucleic acid constrict as defined in claim 1 20 or claim 2 7. An Apicomplexan tetracycline-inducible transactivator (TATi) system, comprising the tetracycline repressor (TetR) and a transacting factor of T. gondii.

   25 8. A tetracycline-inducible transactivator (TATi) system, comprising the tetracycline repressor (TetR) and a transacting factor of T. gondii for use in Apicomplexan species.

   9. A host cell transformed with a nucleic acid construct as defined in claim 1 or 30 claim 2, or a vector as claimed in claim 4, or an expression vector as claimed in claim

   J 10. A host cell as claimed in claim 9, which is an Apicomplexan host cell.

   5 11. A host cell as claimed in claim 10, in which the Apicomplexan cell is selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium berghei, Plasmodium yoelii, Plasmodium knowlesi, rypanosoma brucei, Entamaeba histolytica, and Giardia lambia.

   10 12. A nucleic acid consbuct as defined in claim 1 or claim 2 for use in medicine.

   13. A host cell as defined in any one of claims 9 to 11 for use in medicine.

   14. A method of treatment for or prevention of an infection caused by a protozoan, 15 selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium berghei, Plasmodium Relit Plasmodium owlesi, rypanosoma Brunei, Entamaeba histolytica and Giardia lambia, comprising administration to a subject of a nucleic acid construct as defined in claim 1 or claim 2, or a host cell as defined in any one of claims 9 to 11.

   15. A method of treatment as claimed in claim 14, in which the protozoan is Toxoplasma gondii.

   16. A method of treatment as claimed in claim 14, in which the protozoan is a 25 Plasmodium species.

   17. A vaccine composition comprising a protozoan selected from the group consisting of Toxoplasma Radii, Plasmodium falciparum, Plasmodium vivax, Plasmodium berghei, Plasmodium yoelii, Plasmodium howlesi, Trypanosoma brucei, 30 Entamacha histolytica and Giardia lambda transfected with a nucleic acid construct as

   se defined in claim 1 or claim 2.

   18. The use of a nucleic acid construct as defined in claim I or claim 2 in the preparation of a vaccine for use in the treatment or prophylaxis of an infection caused 5 by a protozoan selected Tom the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vrvax, Plasmodium berghei, Plasmodium yoelii, Plasmodium knowlesi Trypanosoma brucei, Entamaeba histolytica and Giardia lambia.

   19. A kit of parts comprising a host cell as defined in any one of clauns 9 to 11 and 10 an administration vehicle comprising tablets for oral administration, inhalers for lung administration and injectable solutions for intravenous admisbation.