# Patent

## Title
Microginin producing proteins and nucleic acids encoding a microginin gene cluster as well as methods for creating novel microginins

## Abstract

The invention provides for nucleic acid molecules enabling the synthesis of microginin and microginin analogues. The invention also provides for methods for identifying microginins as well creating microginins which may not be found in nature.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application is a 35 U.S.C. 371 National Phase Entry Application from PCT/EP2006/011563, filed Dec. 1, 2006, which claims the benefit of European Patent Application No. 05026396.1 filed on Dec. 2, 2005, the disclosure of which is incorporated herein in its entirety by reference.
TECHNICAL FIELD
The present invention relates to the fields of chemistry, biology, biochemistry, molecular biology. The invention provides for novel nucleic acid molecules enabling the synthesis of microginin and microginin analogues. Microginin finds an application in therapeutics. The invention thus extends into the field of mammalian therapeutics and drug development.
INTRODUCTION
Cyanobacteria and Microginin
Cyanbacteria are gram-negative bacteria. Due to their ability to perform photosynthesis they were long thought to belong to the plant kingdom and were formerly classified as blue-green algae. Cyanbacteria have adapted to almost all ecological niches. Most of strains known up to date are found in fresh water lakes and oceans. In the last few years cyanobacteria have been recognised as a source for biologically active natural compounds.
Cyanobacteria are a group of microscopic organisms somewhere “in between” algae and bacteria and they are found in freshwater and marine areas throughout the world. Scientifically, they are considered to be bacteria, but because they can perform photosynthesis, they also used to be classified as “blue-green algae”.
Cyanobacterial peptides (cyanopeptides) are among the most ubiquitously found potentially hazardous natural products in surface waters used by humans. Though these substances are natural in origin, eutrophication (i.e. excessive loading with fertilising nutrients) has caused massive cyanobacterial proliferation throughout Europe. Thus, cyanopeptides now occur with unnatural frequency and concentration.
A large group among the diverse cyanopeptides are the oligopeptides (peptides with a molecular weight of <2 KD). But while specific cyanopeptides—e.g. microcystins and nodularins—are well studied and recognised as being causative for many animal poisonings and human illness, a substantial and increasing body of evidence points toward a decisive role of other potentially toxic cyanopeptides in the causation of both acute and chronic human illnesses.
Freshwater and marine cyanobacteria are known to produce a variety of bioactive compounds, among them potent hepatotoxins and neurotoxins. Many of the toxic species of cyanobacteria tend to massive proliferation in eutrophicated water bodies and thus have been the cause for considerable hazards for animal and human health. One of the most widespread bloom-forming cyanobacteria is the genus Microcystis, a well-known producer of the hepatotoxic peptide microcystin. Microcystins are a group of closely related cyclic heptapeptides sharing the common structure. So far, more than 80 derivatives of microcystins have been identified, varying largely by the degree of methylation, peptide sequence, and toxicity.
The traditional botanical code describes the genus Microcystis as a coccal, unicellular cyanobacterium that grows as mucilaginous colonies of irregularly arranged cells (under natural conditions, while strain cultures usually grow as single cells). According to this tradition, morphological criteria such as size of the individual cells, colony morphology, and mucilage characteristics are used for species delimitation within Microcystis (i.e., morphospecies). Microcystin-producing strains as well as strains that do not synthesize microcystin have been reported for all species within the genus Microcystis. However, whereas most field samples and strains of Microcystis aeruginosa and Microcystis viridis studied to date were found to contain microcystins, strains of M. wesenbergii, M. novaceckii, and M. ichthyoblabe have only sporadically been reported to contain microcystins.
Beside microcystins, various other linear and cyclic oligopeptides such as anabaenopeptins, aeruginosins, microginins and cyanopeptolins are found within the genus Microcystis (Namikoshi, M., and K. L. Rinehart. 1996. Bioactive compounds produced by cyanobacteria. J. Ind. Microbiol. 17:373-384.).
Similar to microcystins, these peptides possess unusual amino acids like 3-amino-6-hydroxy-2-piperidone (Ahp) in cyanopeptolins, 2-carboxy-6-hydroxyoctahydroindol (Choi) in aeruginosin-type molecules or 3-amino-2 hydroxy-decanoic acid (Ahda) in microginins and numerous structural variants also exist within these groups. These peptides show diverse bioactivities, frequently protease inhibition (Namikoshi, M., and K. L. Rinehart. 1996. Bioactive compounds produced by cyanobacteria. J. Ind. Microbiol. 17:373-384).
The occurrence of both microcystins and other oligopeptides such as anabaenopeptins, microginins and cyanopeptolins in natural Microcystis populations was recently demonstrated. It is well known that the species and genotype composition in natural Microcystis populations is heterogeneous, and both microcystin- and non-microcystin-containing strains have been isolated from the same sample. Just as strains producing microginin and strains not producing microginin have been found. These results suggest a considerable diversity of genotypes with different oligopeptide patterns in natural Microcystis populations.
By typing single Microcystis colonies, it was possible in 1999 to show for the first time that the actual peptide diversity in a natural population of this genus is extremely high. Many of the substances detected belong to well-known groups of cyanobacterial peptides like microcystins, anabaenopeptins, microginins, cyanopeptolins, and aeruginosins, of which many have been discovered in Microcystis spp. In addition, numerous unknown components have been detected in such colonies. However, the origin of these unknown components has yet to be investigated, since besides the observed epiphytic cyanobacteria and algae, heterotrophic bacteria are also known to be present in Microcystis colonies. Chemical screening of cyanobacterial samples (both from field samples and from culture strains) has demonstrated a wide variety of substances: e.g. an almost monospecific bloom of Planktothrix agardhii contained as many as 255 different substances, most of which were oligopeptides.
Thus, it may be concluded, that the situation with respect to the assignment of the capability of microginin production to certain species and strains, i.e. also a true understanding of the genotypes and species involved as well as their evolution has to date, not been possible. In fact PEPCY a research project supported by the European Commission concluded that present information shows that one species or “morphotype” (i.e. individuals with the same morphological characteristics) may comprise a range of genotypes that encode for different “chemotypes” (i.e. morphologically indistinguishable individuals containing different cyanopeptides).
ACE Inhibitors and Microginin
ACE catalyses the conversion of angiotensin I into angiotensin II within the mammalian renin-angiotensin system, leading to arterial stenosis, which in turn causes an increase of blood pressure. ACE inhibitors counteract this process and therefore play a role in human medicine as blood pressure lowering agents. Microginin is an important drug candidate for ACE inhibition. So far only 30 structural variants of microginin are known, making clinical development difficult.
Microginins are characterized by a decanoic acid derivate, 3-amino-2-hydroxy-decanoic acid (Ahda) at the N-terminus and a predominance of two tyrosine units at the C-terminus. They vary in length from 4 to 6 amino acids with the variability occurring at the C-terminal end (Microginins, zinc metalloprotease inhibitors from the cyanobacterium Microcystis aeruginosa, 2000, Tetrahedron 56:8643-8656). In the past it has only been possible by means of synthesis of 3-amino-2-hydroxy-decanoic acid to chemically generate microginin variants (J Org. Chem. 1999 Apr. 16; 64(8):2852-2859. Acylnitrene Route to Vicinal Amino Alcohols. Application to the Synthesis of (−)-Bestatin and Analogues. Bergmeier S C, Stanchina D M.) Alternatively cyanobacterial strains were screened for microginin activity, which was tedious and time consuming. It has so far not been possible to screen for strains efficiently due to the lack of species understanding and a methodology of efficiently distinguishing microginin producers from non-producers (see above). Further it was not possible to easily and efficiently alter and thus develop microginins in order to provide for a variety of lead compounds from which better ACE-inhibitors may be developed.
BRIEF DESCRIPTION OF THE INVENTION
From Microcystis aeruginosa a cluster of genes, spanning about 30 kbps has been isolated encoding a hybrid synthetase composed of non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS) and tailoring enzyme which as the inventors show is responsible for the biosynthesis of microginin. The strain from which this nucleic acid was first isolated by G. C. Kürzinger from Lake Pehlitz 1977].
The inventors provide for a biological system enabling not only the production of micoginins, the heterologous expression of microginin, but also a system for modifying microginin and thus developing so far unknown variants of microginin. The invention further provides for nucleic acids and methods for identifying strains which have the ability to produce microginin.
In particular the invention relates to one or more nucleic acids encoding a microginin synthetase enzyme complex with the following activities: an adenylation domain (A*) wherein, the adenylation domain comprises a peptide sequence according to SEQ ID NO. 1, an acyl carrier protein (ACP), an elongation module (EM) of polyketide synthases (PKS) comprising the following activities: (i) ketoacylsynthase (KS), (ii) acyl transferase (AT) (iii) acyl carrier protein (ACP2), an aminotransferase (AMT), three to five elongation modules (EM) of non-ribosomal peptide synthetases (NRPS) comprising the following activities: (i) condensation domain (C), (ii) adenylation domain (A), (iii) thiolation domain (T) and a thioesterase (TE).
DETAILED DESCRIPTION OF THE INVENTION
As outlined above the invention in particular relates to one or more nucleic acids encoding a microginin synthetase enzyme complex with the following activities: an adenylation domain (A*) wherein, the adenylation domain comprises a peptide sequence according to SEQ ID NO. 1, an acyl carrier protein (ACP), an elongation module (EM) of polyketide synthases (PKS) comprising the following activities: (i) ketoacylsynthase (KS), (ii) acyl transferase (AT) (iii) acyl carrier protein (ACP 2), an aminotransferase (AMT), three to five elongation modules (EM) of non-ribosomal peptide synthetases (NRPS) comprising the following activities: (i) condensation domain (C), (ii) adenylation domain (A), (iii) thiolation domain (T) and a thioesterase (TE).
The inventors have found that microginin is the product of non-ribosomal synthesis. It is important to understand that microginin as previously identified in nature may also in part have been the product of ribosomal synthesis and further processed via various enzymatic reactions.
It is important to note that the nucleic acid claimed herein, i.e. a microginin synthetase enzyme complex may also be present in organisms other organisms than Microcystis sp., such as Nostoc, Anabaena, Plankthotrix or Oscillatoria. The term microginin shall thus not limit the invention to such nucleic acids producing synthetase enzyme complexes resulting in peptides officially termed “microginin”.
Herein, an adenylation domain (A*) is understood to activate octanoic acid as an acyl adenylate and an acyl carrier protein (ACP) is understood to bind the octanoic acid adenylate as a thioester.
An elongation module (EM) of polyketide synthases (PKS) is also known e.g. from the Jamaicamide synthetase gene cluster isolated from Lyngbya majuscula (Chem. Biol. Vol. 11, 2004 pp 817-833. Structure and Biosynthesis of the Jamaicamides, new mixed polyketide-peptide neurotoxin from the marine cyanobacterium Lyngbya majuscula) herein comprises at least the following activities: (i) ketoacylsynthase (KS), (ii) acyl transferase (AT) and (iii) acyl carrier protein (ACP2). The AT is responsible for the recognition of malonyl-CoA, the KS is responsible for the Claisen-type-condensation of the activated octanoic acid adenylate with malonyl-CoA and the ACP2 is responsible for binding of the resulting decanoic acid. An aminotransferase (AMT) performs the β-amination of the decanoic acid.
The nucleic acid according to the invention may have three to five elongation modules (EM) of non-ribosomal peptide synthetases (NRPS) comprising at least the following activities: (i) condensation domain (C), (ii) adenylation domain (A), (iii) thiolation domain (T). The A is responsible for the activation of carboxyl groups of amino acids, the T is responsible for the binding and the transport of the activated intermediate, the C is responsible for the condensation of the activated amino acids with the growing peptide chain.
Finally the nucleic acid according to the invention shall contain a thioesterase (TE) activity which performs the clevage of the final product from the synthetase complex.
One may envision that the nucleic acid according to the invention is present in a vector or a bacterial chromosome, in which case one may envision that the portions designated above while being in one cell need not all, be in, or on, one molecule. It is essential to the invention however, that a cell meant to produce microginin synthetase enzyme complex contains the activities designated above in order to produce an enzyme complex according to the invention which in turn may produce a microginin. Thus, the invention also encompasses derivatives of the nucleic acid molecule as outlined above having the function of a microginin synthetase enzyme complex.
The molecule is characterized by a special adenylation domain (A*) which is unusual in that it is not similar to known adenlyation domains found in other molecules encoding non-ribosomal enzyme complexes such as the microcystin synthetase gene cluster (Chem. Biol. Vol. 7 2000, pp 753-764: Structural organisation of microcystin synthesis in Microcystis aeruginosa PCC 7806: In integrated peptide-polyketide-synthetase system) Molecules encompassed herein are those which carry this adenylation domain (A*) as depicted in SEQ ID NO. 1 and at least an ACP whereby this ACP may stem from another known non-ribosomal enzyme complex, at least one EM of PKS whereby this EM may stem from another known non-ribosomal enzyme complex comprising at least the following activities: (i) KS, (ii) AT (iii) ACP, an AMT whereby this AMT may stem from another known non-ribosomal enzyme complex three to five EMs comprising at least the following activities: (i) C, (ii) A, (iii) T whereby these EMs may stem from another known non-ribosomal enzyme complex and a TE whereby this TE may stem from another known non-ribosomal enzyme complex. Chimeras whereby parts of the above are on one or more vectors and or integrated in chromosomes are equally encompassed by the invention as long as all the components are in one cell.
The invention also pertains to isolated nucleic acid molecules encoding a microginin synthetase enzyme complex comprising an adenylation domain which is 85% identical to SEQ ID NO. 1, more preferred 90% identical to SEQ ID NO. 1 most preferred 95% identical to SEQ ID NO. 1. Sequence identity herein is in percent of total sequence of the adenylation domains when aligned with conventional nucleotide alignment software, such as the best fit and or pileup programs of the GCG package
The invention also pertains to a microginin synthetase enzyme protein complex with the following activities: an adenylation domain (A*) wherein, the adenylation domain comprises a peptide sequence according to SEQ ID NO. 1, an acyl carrier protein (ACP), an elongation module (EM) of polyketide synthases (PKS) comprising the following activities: (i) ketoacylsynthase (KS), (ii) acyl transferase (AT) (iii) acyl carrier protein (ACP 2), an aminotransferase (AMT), three to five elongation modules (EM) of non-ribosomal peptide synthetases (NRPS) comprising the following activities: (i) condensation domain (C), (ii) adenylation domain (A), (iii) thiolation domain (T) and a thioesterase (TE).
The invention in particular also relates to a nucleic acid molecule encoding an adenylation domain (A*) wherein, the adenylation domain comprises a peptide sequence according to SEQ ID NO. 1.
The invention in particular also relates to a peptide molecule, an adenylation domain (A*) wherein, the molecule comprises a peptide sequence according to SEQ ID NO. 1.
The invention in particular also relates to a nucleic acid molecule encoding an adenylation domain (A*) wherein, the molecule comprises a nucleic acid sequence according to SEQ ID NO. 25.
In a preferred embodiment of the invention the nucleic acid additionally and optionally comprises sequences encoding the following activities or domains: a monooxygenase (MO), an integrated N-methyltransferase domain (MT) within one or more elongation modules (EM) of NRPS, a non-integrated N-methyltrasferase (MT), a modifying activity (MA) wherein, said MA is selected from the group comprising the following activities: halogenase, sulfatase, glycosylase, racemase, O-methyltransferase and C-methyltransferase, two or more peptide repeat spacer sequences (SP) consisting of one or more repeats of being either glycine rich or proline and leucine rich, located adjacently upstream and downstream of the MO and/or another MA.
Herein MO is an enzyme catalyzing the hydroxylation of the decanoic acid, an integrated N-methyltransferase domain (MT) within one or more elongation modules (EM) of NRPS catalyses the methylation of the amide bond by the respective module and a non-integrated N-methyltrasferase (MT) catalyzes the methylation of an amino group of the microginin. The term modifying enzyme stands for numerous enzymes such enzymes may add groups or create bonds, in a preferred embodiment MA is selected from the group comprising the following activities: halogenase, sulfatase, glycosylase, racemase, O-methyltransferase and C-methyltransferase.
Nucleic acids encoding two or more peptide repeat spacer sequences (SP) consisting of one or more repeats being either glycine rich or proline and leucine rich have astonishingly been found by the inventors to aid in integration of novel MAs into existing microginin synthetase enzyme complexes. By means of placing such SPs adjacently to MAs the inventors are able to create microginin synthetase enzyme complexes (MSEC) comprising activities previously not found in MSECs. This in turn allows for the creation of novel microginins with potentially novel therapeutic properties. Thus the invention relates to nucleic acids encoding two or more peptide repeat spacer sequences (SP) consisting of one or more repeats being either glycine rich or proline and leucine rich may be positioned adjacently to a MA such as but not limited to a halogenase, a sulfatase, a glycosylase, a racemase, an O-methyltransferase or a C-methyltransferase. These SPs aid in ensuring that the “foreign” activity “works” in the enzyme complex. The inventors have found, that this is due to the lack of secondary structures in the SP peptide chains.
The nucleic acid according to the invention in a preferred embodiment optionally comprises the following sequences, nucleic acid sequences encoding protein sequences as follows:
An adenylation domain (A*) according to SEQ ID NO. 1, an acyl carrier protein (ACP) according to SEQ ID NO. 2, an elongation module of polyketide synthases responsible for the activation and the condensation of malonyl-Co A: (i) ketoacylsynthase domain (KS) according to SEQ ID NO. 3, (ii) acyl transferase domain (AT) according to SEQ ID NO. 4, an acyl carrier protein domain (ACP 2) according to SEQ ID NO. 5, an aminotransferase (AMT) according to SEQ ID NO. 6, an elongation modules of non-ribosomal peptide synthetases responsible for the activation and condensation of alanin: (i) condensation domain (C) according to SEQ ID NO. 7, (ii) adenylation domain (A) according to SEQ ID NO. 8, (iii) thiolation domains (T) according to SEQ ID NO. 9, an elongation modules of non-ribosomal peptide synthetases responsible for the activation and condensation of leucin: (i) condensation domain (C2) according to SEQ ID NO. 10, (ii) adenylation domain (A 2) according to SEQ ID NO. 11, (iii) thiolation domain (T 2) according to SEQ ID NO. 12, an elongation modules of non-ribosomal peptide synthetases responsible for the activation and condensation of tyrosine 1: (i) condensation domain (C 3) according to SEQ ID NO. 13, (ii) adenylation domain (A 3) according to SEQ ID NO. 14 (iii) thiolation domain (T 3) according to SEQ ID NO. 15, an elongation modules of non-ribosomal peptide synthetases responsible for the activation and condensation of tyrosine 2: (i) condensation domain (C4) according to SEQ ID NO. 16, (ii) adenylation domain (A 4) according to SEQ ID NO. 17, (iii) thiolation domain (T 4) according to SEQ ID NO. 18, a thioesterase (TE) according to SEQ ID NO. 19, a monooxygenase (MO) according to SEQ ID NO. 20, two or more peptide repeat spacer sequences (SP1/SP2) according to SEQ ID NO. 21 and 22, an integrated N-methyltransferase domain (MT) within the elongation module (EM) of the NRPS responsible for the activation and condensation of leucin according to SEQ ID 23 and a non-integrated N-methyltrasferase (MT 2) according to SEQ ID NO. 24.
As outlined above, the minimal requirement according to the invention is a nucleic acid encoding a microginin synthetase enzyme complex with the following activities: an adenylation domain (A*) wherein, the adenylation domain comprises a peptide sequence according to SEQ ID NO. 1, an ACP according to SEQ ID NO. 2, an elongation module (EM) of polyketide synthases (PKS) comprising the following activities: (i) ketoacylsynthase (KS) according to SEQ ID NO. 3, (ii) acyl transferase (AT) according to SEQ ID NO 4, (iii) acyl carrier protein (ACP 2) according to SEQ ID NO. 5, an aminotransferase (AMT) according to SEQ ID NO. 6, three to five elongation modules (EM) of non-ribosomal peptide synthetases (NRPS) comprising the following activities: (i) condensation domain (C) according to SEQ ID NO. 7, (ii) adenylation domain (A) according to SEQ ID NO. 8, (iii) thiolation domain (T) according to SEQ ID NO. 9 and a thioesterase (TE) according to SEQ ID NO. 10. A molecule comprising the above sequences is preferred herein.
The invention explicitly also relates to analogs hereto, additionally comprising, e.g. other activities and/or spacer regions both transcribed and non-transcribed.
It is apparent to those skilled in the art, that amino acids may be exchanged maintaining the enzymatic activity required. Thus, the invention also relates to molecules with sequences which are not identical to those outlined above however, altered only in so far as the enzymatic activity desired is retained.
The nucleic acid according to the invention may contain nucleic acids selected from the group comprising: an adenylation domain (A*) according to SEQ ID NO. 25, an acyl carrier protein (ACP) according to SEQ ID NO. 26, an elongation module of polyketide synthases encoding for the activation and the condensation of malonyl-Co A: (i) ketoacylsynthase domain (KS) according to SEQ ID NO. 27, (ii) acyl transferase domain (AT) according to SEQ ID NO. 28, (iii) acyl carrier protein domain (ACP 2) according to SEQ ID NO. 29, an aminotransferase (AMT) according to SEQ ID NO. 30, an elongation modules of non-ribosomal peptide synthetases encoding for the activation and condensation of alanin: (i) condensation domain (c) according to SEQ ID NO. 31, (ii) adenylation domain (A) according to SEQ ID NO. 32, (iii) thiolation domain (T) according to SEQ ID NO. 33, an elongation modules of non-ribosomal peptide synthetases encoding for the activation and condensation of leucin: (i) condensation domain (C 2) according to SEQ ID NO. 34, (ii) adenylation domain (A 2) according to SEQ ID NO. 35, (iii) thiolation domain (T 2) according to SEQ ID NO. 36, elongation modules of non-ribosomal peptide synthetases encoding for the activation and condensation of tyrosine 1: (i) condensation domains (C3) according to SEQ ID NO. 37, (ii) adenylation domains (A 3) according to SEQ ID NO. 38, (iii) thiolation domains (T 3) according to SEQ ID NO. 39, elongation modules of non-ribosomal peptide synthetases encoding for the activation and condensation of tyrosine 2: (i) condensation domains (C4) according to SEQ ID NO. 40, (ii) adenylation domains (A 4) according to SEQ ID NO. 41, (iii) thiolation domains (T 4) according to SEQ ID NO. 42, a thioesterase (TE) according to SEQ ID NO. 43, a monooxygenase (MO) according to SEQ ID NO. 44, two or more peptide repeat spacer sequences (SP1/2) according to SEQ ID NO. 45 and 46, an integrated N-methyltransferase domain (MT) within the elongation module (EM) of the NRPS encoding for the activation and condensation of leucin according to SEQ ID 47 and a non-integrated N-methyltrasferase (MT 2) according to SEQ ID NO. 48.
As outlined above, the minimal requirement according to the invention is a nucleic acid encoding a microginin synthetase enzyme complex with the following activities: an adenylation domain (A*) wherein, the adenylation domain is a nucleic acid sequence according to SEQ ID NO. 25, an ACP with a nucleic acid sequence according to SEQ ID NO. 26, an elongation module (EM) of polyketide synthases (PKS) comprising the following activities: (i) ketoacylsynthase (KS) with a nucleic acid sequence according to SEQ ID NO. 27, (ii) acyl transferase (AT) with a nucleic acid sequence according to SEQ ID NO 28, (iii) acyl carrier protein (ACP 2) with a nucleic acid sequence according to SEQ ID NO. 29, an aminotransferase (AMT) with a nucleic acid sequence according to SEQ ID NO. 30, three to five elongation modules (EM) of non-ribosomal peptide synthetases (NRPS) comprising the following activities: (i) condensation domain (C) with a nucleic acid sequence according to SEQ ID NO. 31, (ii) adenylation domain (A) with a nucleic acid sequence according to SEQ ID NO. 32, (iii) thiolation domain (T) with a nucleic acid sequence according to SEQ ID NO. 33 and a thioesterase (TE) with a nucleic acid sequence according to SEQ ID NO. 43. A molecule comprising the above sequences is preferred herein.
The invention also relates to nucleic acid molecules with sequences which are not identical to those outlined above however, altered only in so far as the enzymatic activity desired is retained. I particular one skilled in the art will know that positions in nucleic acid triplets may “wobble” and these positions may thus be altered with no influence on the peptide sequence. Further multiple amino acids are encoded by more than one DNA triplet. One skilled in the art will know that one may alter such triplets maintaining the amino acid sequence. Thus said sequences are equally encompassed by the invention.
The invention also pertains to isolated nucleic acid molecules encoding a microginin synthetase enzyme complex comprising an adenylation domain which is 85% identical to SEQ ID NO. 25, more preferred 90% identical to SEQ ID NO. 1 most preferred 95% identical to SEQ ID NO. 1. Sequence identity herein is in percent of total sequence of the adenylation domains when aligned with a conventional amino acid alignment software such as the best fit and or pileup programs of the GCG package.
In a preferred embodiment the one or more nucleic acids according to the invention are organized in sequence parts encoding the microginin synthetase enzyme complex in an upstream to downstream manner as depicted in FIG. 1. In a particularly preferred embodiment the activities and domains are arranged as shown and on one molecule.
The nucleic acid molecule may be part of a vector. Such vectors are in particular, bacterial artificial chromosomes (BAC), Cosmids or Fosmids, and Lambda vectors. Preferred plasmid vectors which are able to replicate autonomously in cyanobacteria are derived from the pVZ vectors. Preferred fosmid vectors which are able to replicate autonomously in cyanobacteria are derived from the pCC1FOSTM and pCC2FOSTM vectors (Epicentre Biotechnologies). The integration of the nucleic acid according to the invention into the vector is a procedure known to those skilled in the art (Molecular Cloning: A Laboratory manual, 1989, Cold Spring Harbour Labaratory Press) or in the manuals of manufactures of kits for creation of genomic libraries (e.g. Epicenter Biotechnologies).
In a preferred embodiment the invention concerns a microorganism transformed with a nucleic acid according to the invention. The nucleic acid according to the invention may integrated into the chromosome of the host organism or may present on a separate vector (see also examples). It is preferred that the phototrophic cyanobacterial host organism is selected for the group comprising: Synechocystis sp., Synechococcus sp., Anabaena sp., Nostoc sp., Spirulina sp., Microcystis sp . . . . Cells are cultured as follows:
Media: Bg 11 (for cultivation of cyanobacteria)
Aeration: air containing 0.3-3.0% carbon dioxide
Light intensity: 40-100 μE/m2*s (diameter of illuminated culture vessels of photobioreactor d=4-12 cm)
Cell density at harvest: OD7501-2
And if the host is Microcystis aeruginosa: 
Light quality: Additional red light illumination with 25 μE/m2*s for 24-48 hours before harvesting.
It is preferred that the heterotrophic host organism is selected for the group comprising: E. coli and Bacillus sp. due to a more suitable GC content and codon usage than other heterotrophic bacteria.
In case of using E. coli for the heterologues expression of the microginin synthetase a phosphopanthetein transferase (Ppt) has to be co-expressed in order to enable the synthesis of microginin. The co-expression of the Ppt from a microginin producing strain would be preferred. Other Ppt's with a broad specificity even from heterotophic organisms like Bacillus sp. are also suitable.
In one embodiment of the invention the invention relates to a method of producing a microginin, comprising culturing a cell under conditions under which the cell will produce microginin, wherein said cell comprises a nucleic acid encoding a recombinant microginin, according to the invention, and wherein said cell does not produce the microginin in the absence of said nucleic acid.
The inventors have identified nucleic acid sequences which for the first time make it possible to detect nucleic acids encoding a microginin synthetase enzyme complex. This has been extremely difficult, due to the fact that other gene clusters which encode non-ribosomal protein producing complexes share sequence similarity with the present cluster claimed herein. Such primers or probes according to the invention are selected from the group of, a) nucleic acid according to SEQ ID NO. 49 (Primer A), b) nucleic acid according to SEQ ID NO. 50 (Primer B), c) nucleic acid according to SEQ ID NO. 51 (Primer C), d) nucleic acid according to SEQ ID NO. 52 (Primer D), e) nucleic acid according to SEQ ID NO. 53 (Primer E), f) nucleic acid according to SEQ ID NO. 54 (Primer F), g) nucleic acid according to SEQ ID NO. 55 (Primer G), h) nucleic acid according to SEQ ID NO. 56 (Primer H), i) nucleic acid according to SEQ ID NO. 57 (Primer I) and j) nucleic acid according to SEQ ID NO. 58 (Primer J). It is known to one skilled in the art that such primers or probes may be altered slightly and still accomplishes the task of specifically detecting the desired target sequence. Such alterations in sequence are equally encompassed by the invention. The primers or probes according to the invention may be applied in hybridization reactions and/or amplification reactions. Such reactions are known to one skilled in the art.
The invention also concerns a method for detecting a microginin synthetase gene cluster in a sample wherein, one or more of the nucleic acids according to the invention are, applied in an amplification and/or a hybridization reaction.
In a preferred embodiment of the method according to the invention primers D and F or H and J or E and I or E and A are added to a PCR reaction mixture comprising a sample and wherein, presence of an amplification product represents presence of microginin synthetase gene cluster and absence of an amplification product represents absence of a microginin synthetase gene cluster. As can be seen from the examples (example 3 below), certain combinations are preferred. Samples may be isolated DNA, prokaryotic cells stemming from plates or liquid cultures.
When performing an amplification reaction with primers D and F the most preferred amplification conditions are as follows: a) denaturing, b) 48° C. annealing and c) elongation (product size: 675 bp). These temperatures may vary a bit in the range of 2-8 degrees C.
When performing an amplification reaction with primers H and J the most preferred amplification conditions are as follows: a) denaturing, b) 54° C. annealing and c) elongation (product size: 1174 bp). These temperatures may vary a bit in the range of 2-8 degrees C.
When performing an amplification reaction with primers E and I the most preferred amplification conditions are as follows: a) denaturing, b) 56° C. annealing and c) elongation (product size: 1279 bp). These temperatures may vary a bit in the range of 2-8 degrees C.
When performing an amplification reaction with primers E and A the most preferred amplification conditions are as follows: a) denaturing, b) 57° C. annealing and c) elongation (product size: 621 bp). These temperatures may vary a bit in the range of 2-8 degrees C. Molarity is most commonly 0.2-1.0 μM for the primers. Buffers and other reagents depending on polymerase used.
When performing hybridisation reactions the above nucleic acids are usually labeled. Such labels may be radioactive or non-radioactive, such as fluorescent. The nucleic acid primers or probes may be applied, e.g. for the screening of libraries.
The invention also relates to antibodies against a peptide according to SEQ ID NO. 1 (A*).
The creation of such antibodies is known to one skilled in the art. The antibodies may be polyclonal or monoclonal. Such antibodies may be labeled or non-labeled, they may also be altered in other form, such as humanized.
The inventors have astonishingly found that newly identified peptide repeat spacer sequences (SP) may be placed adjacently to MAs I in order to create novel hybrid gene clusters. These SPs act by spacing the novel activity or domain so that it is functionally active in the microginin synthetase enzyme complex.
The invention thus, further relates to nucleic acids encoding a peptide repeat spacer sequence (SP) wherein, the peptide sequence comprises at least 4 glycin amino acids per single repeat unit (SRU) or, at least 5 proline and/or leucin amino acids per SRU. A SRU within the SP is between 7 and 15 amino acids in length and, the SP comprises between 2 and 10 SRUs.
The invention further relates to peptides of a peptide repeat spacer sequence (SP) wherein, the peptide sequence comprises at least 4 glycin amino acids or, at least 5 proline and/or leucin amino acids, the single repeat unit (SRU) within the SP is between 7 and 15 amino acids in length and, the SP comprises between 2 and 10 SRU. In a preferred embodiment of the invention the SRU is between 9 and 13 amino acids in length in a particularly preferred embodiment the SRU is eleven amino acids in length. In a preferred embodiment the SP comprises between 3 and 9 SRU.
In a preferred embodiment the nucleic acid encoding the peptide repeat spacer sequence (SP) according to the invention, encodes a peptide SRU as shown in SEQ ID NO. 20 or SEQ ID NO. 21. In a further embodiment the peptide repeat spacer sequence (SP) according to the invention, comprises or contains a sequence as shown in SEQ ID NO. 20 or SEQ ID NO. 21. In a further embodiment the nucleic acid according to the invention has a sequence as laid down in SEQ ID NO. 43 or SEQ ID NO. 44.
Not only by means of the above mentioned SPs but in particular because of these the inventors are able to create enzyme complexes resulting in microginin variants which may not be found in nature. This is an essential aspect of the present invention. The invention provides for, for the first time a simple method of producing recombinant microginin variants comprising, modifying the nucleic acid according to the invention in vitro or in vivo, growing a recombinant cell comprising said recombinantly modified nucleic acid encoding a microginin synthetase under conditions which lead to synthesis of a microginin and, recovering the synthesized microginin.
In a preferred embodiment of said method according to the invention, said modifying of said nucleic acid may be an action selected from the group of one or more of the following actions: a) inactivation of one or more of the MTs present, b) substitution of one or more of the MTs present with a halogenase, a sulfatase, a glycosylase, a racemase, an O-methyltransferase or a C-methyltransferase, c) inactivation of the MO, d) substitution of the MO with a halogenase, a sulfatase, a glycosylase, a racemase, an O-methyltransferase or a C-methyltransferase, e) inactivation of the AMT, f) substitution of the AMT with a halogenase, a sulfatase, a glycosylase, a racemase, an O-methyltransferase or a C-methyltransferase, g) inactivation of the PKS module, h) substitution of the entire PKS module with an alternative PKS module and/or substitution of one or more of the domains (KS, AT, ACP) therein, i) inactivation of the A* domain, j) substitution of the A* domain with alternative A domains, k) inactivation of one or more of the NRPS modules and 1) substitution of one or more of the NRPS modules with alternative NRPS modules and/or substitution of one or more of the domains (C, A, T) therein.
Halogenases, sulfatases, glycosylases, racemases, O-methyltransferases or C-methyltransferases are known from prokaryotes. These enzymes are encoded by genes of the secondary metabolism in particular NRPS/PKS systems.
Alternative PKS-systems, entire modules as well as single domains (KS, AT, ACP) are found in cyanobacteria as well as Actinomycetes, Myxobacteria, Bacillus among the bacteria.
Alternative NRPS-systems, entire modules as well as single domains (C, A, T) are found in cyanobacteria as well as Actinomycetes, Myxobacteria, Bacillus among the bacteria.
In a preferred embodiment the above are from cyanobacteria.
It is important to note, that said inactivation and/or substitution may done in many ways, e.g. inactivation may imply deleting the complete activity or domain, or may imply inactivation by means of a single nucleotide exchange.
The methods are known to those skilled in the art and comprise basic molecular biological methods such as DNA isolation, restriction digestion, ligation, transformation, amplification etc.
In a preferred embodiment said alternative modules or domains which are used for substitution of the original module or domain, additionally may comprise one or more SP nucleic acids according to the invention located adjacently upstream of the module or domain used for substitution and one or more SP nucleic acids according the invention located adjacently downstream of the module or domain used for substitution. Thus, in this embodiment of the invention a construct is made comprising the domain which is to be entered into the original nucleic acid according to the invention, further comprising one or more SPs located adjacently in an upstream and downstream manner. This construct is then ligated into the original microginin synthetase encoding nucleic acid. The resultant construct is then brought into a host by means of transformation for either a) integration into the host chromosome or b) with a self-replicating vector.
The polypeptides, i.e. proteins can be any of those described above but with not more than 10 (e.g., not more than: 10, nine, eight, seven, six, five, four, three, two, or one) conservative substitutions. Conservative substitutions are known in the art and typically include substitution of, e.g. one polar amino acid with another polar amino acid and one acidic amino acid with another acidic amino acid. Accordingly, conservative substitutions preferably include substitutions within the following groups of amino acids: glycine, alanine, valine, proline, isoleucine, and leucine (non polar, aliphatic side chain); aspartic acid and glutamic acid (negatively charged side chain); asparagine, glutamine, methionine, cysteine, serine and threonine (polar uncharged side chain); lysine, histidine and arginine; and phenylalanine, tryptophane and tyrosine (aromatic side chain); and lysine, arginine an histidine (positively charged side chain). It is well known in the art how to determine the effect of a given substitution, e.g. on pK1 etc. All that is required of a polypeptide having one or more conservative substitutions is that it has at least 50% (e.g., at least: 55%; 60%; 65%, 70%; 75%; 80%; 85%; 90%; 95%; 98%; 99%; 99.5%; or 100% or more) of the ability of the unaltered protein according to the invention.
In preferred embodiments the polynucleotides, i.e. nucleic acids of the present invention also comprise nucleic acid molecules which are at least 85%, preferably 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to those claimed herein.
The determination of percent identity between two sequences is accomplished using the mathematical algorithm of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410. BLAST nucleotide searches are performed with the BLASTN program, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acids according to the invention. BLAST protein searches are performed with the BLASTP program, score=50, wordlength=3, to obtain amino acid sequences homologous to the EPO variant polypeptide, respectively. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used.



FIGURES
FIG. 1 depicts the structure of microginin.
FIG. 2 depicts the microginin synthetase gene cluster and the biosynthetic pathway of microginin.



EXAMPLES
Example 1
Method for Detecting Gene Clusters According to the Invention
Strains carrying a gene cluster encoding a microginin synthetase complex can be distinguished from strains not carrying such a gene cluster performing a PCR reaction using RedTaq ReadyMix PCR Reaction Mix with MgCl2 (Sigma) and primer pairs and the corresponding annealing temperatures as described. In particular the PCR conditions are as follows: an initial denaturation for 1 minutes at 95° C., followed by 30 cycles of denaturation at 95° C. for 30 seconds, elongation at said annealing temperatures for 30 seconds and extension at 72° C. for 1 kb of product size.
Example 2
Method for Optimised Cultivation of Microginin Producing Microcystis spp
Strains. Media: Bg 11 (for cultivation of cyanobacteria)
Aeration: air containing 0.3-3.0% carbon dioxide
Light intensity: 40-100 pE/m2*s (diameter of illuminated culture vessels of photobioreactor d=4-12 cm)
Light quality: Additional red light illumination with 25 μE/m2*S for 24-48 hours before harvesting.
Cell density at harvest: OD750nm 1-2
Tables









TABLE 1







SEQ ID
MTINYGDLQEPFNKFSTLVELLRYRASSQPERLAYIFLRDGEIE



NO. 1 
EARLTYGELDQKARAIAAYLQSLEAEGERGLLLYPPGLDFISAF


A*
FGCLYAGVVAIPAYPPRRNQNLLRLQAIIADSQARFTFTNAALF



PSLKNQWAKDPELGAMEWIVTDEIDHHLREDWLEPTLEKNSLAF



LQYTSGSTGTPKGVMVSHHNLLINSADLDRGWGHDQDSVMVTWL



PTFHDMGLIYGVIQPLYKGFLCYMMSPASFMERPLRWLQALSDK



KATHSAAPNFAYDLCVRKIPPEKRATLDLSHWCMALNGAEPVRA



EVLKKFAEAFQVSGFKATALCPGYGLAEATLKVTAVSYDSPPYF



YPVQANALEKNKIVGATETDTNVQTLVGCGWTTIDTQIVIVNPE



TLKPCSPEIVGEIWVSGSTIAQGYWGKPQETQETFQAYLADTGA



GPFLRTGDLGFIKDGELFITGRLKEIILIRGRNNYPQDIELTVQ



NSHPALRPSCGAAFTVENKGEEKLVVVQEVERTWLRKVDIDEVK



RAIRKAVVQEYDLQVYAIALIRTGSLPKTSSGKIQRRSCRAKFL



EGSLEILG


 


SEQ ID
MSTEIPNDKKQPTLTKIQNWLVAYMTEMMEVDEDEIDLSVPFDE


NO. 2
YGLDSSMAVALIADLEDWLRRDLHRTLIYDYPTLEKLAKQVSEP


ACP


 


SEQ ID
MEPIAIIGLACRFPGADNPEAFWQLMRNGVDAIADIPPERWDIE


NO. 3
RFYDPTPATAKKMYSRQGGFLKNVDQFDPQFFRISPLEATYLDP


KS
QQRLLLEVTWEALENAAIVPETLAGSQSGVFIGISDVDYHRLAY



QSPTNLTAYVGTGNSTSIAANRLSYLFDLRGPSLAVDTACSSSL



VAVHLACQSLQSQESNLCLVGGVNLILSPETTVVFSQARMIAPD



SRCKTFDARADGYVRSEGCGVVVLKRLRDAIQDGDRILAVIEGS



AVNQDGLSNGLTAPNGPAQQAVIRQALANAQVKPAQISYVEAHG



TGTELGDPIEVKSLKAVLGEKRSLDQTCWLGSVKTNIGHLEAAA



GMAGLIKVVLCLQHQEIPPNLHFQTLNPYISLADTAFAIPTQAQ



PWRTKPPKSGENGVERRLAGLSSFGFGGTNSHVIL


 


SEQ ID
VFLFAGQGSQYVGMGRQLYETQPIFRQTLDRCAEILRPHLDQPL


NO. 4
LEILYPADPEAETASFYLEQTAYTQPTLFAFEYALAQLWRSWGI


AT
EPAAVIGHSVGEYVAATVAGALSLEEGLTLIAKRAKLMQSLPKN



GTMIAVFAAEERVKAVIEPYRTDVAIAAVNGPENFVISGKAPII



AEIIIHLTAAGIEVRLPLKVSHAFHSHLLEPILDSLEQEAAAIS



YQPLQIPLVANLTGEVLPEGATIEARYWRNHARNPVQFYGSIQT



LIEQKFSLFLEVSPKPTLSRLGQQCCPERSTTWLFSLALPPQEE



EQSLLNSLAILYDSQGAE


 


SEQ ID
ITLQTLVGNLLQLSPADVNVHTPFLEMGADSIVMVEAVRRIENT


NO. 5
YNVKIAMRQLFEELSTLDALATYL


ACP 2


 


SEQ ID
KEMLYPIVAQRSQGSRIWDVDGNEYIDMTMGQGVTLFGHQPDFI


NO. 6
MSALQSQLTEGIHLNPRSPIVGEVAALICELTGAERACFCNSGT


AMT
EAVMAAIRIARATTGRSKIALFEGSYHGHADGTLFRNQIIDNQL



HSFPLALGVPPSLSSDVVVLDYGSAEALNYLQTQGQDLAAVLVE



PIQSGNPLLQPQQFLQSLRQITSQMGIALIFDEMITGFRSHPGG



AQALFGVQADIATYGKVVAGGMPIGVIAGKAHYLDSIDGGMWRY



GDKSYPGVDRTFFGGTFNQHPLAMVAARAVLTHLKEQGPGLQQQ



LTERTAALADTLNHYFQAEEVPIKIEQFSSFFRFALSGNLDLLF



YHMVEKGIYVWEWRKHFLSTAHTEADLAQFVQAVKDSITELR


 


SEQ ID
GGDQVPLTEAQRQLWILAQLGDNGSVAYNQSVTLQLSGPLNPVA


NO. 7
MNQAIQQISDRHEALRTKINAQGDSQEILPQVEINCPILDFSLD


C
QASAQQQAEQWLKEESEKPFDLSQGSLVRWHLLKLEPELHLLVL



TAHHIISDGWSMGVILRELGELYSAKCQGVTANLKTPKQFRELI



EWQSQPSQGEELKKQQAYWLATLADPPVLNLPTDKPRPALPSYQ



ANRRSLTLDSQFTEKLKQFSRKQGCTLLMTLLSVYNILVHRLTG



QDDILVGLPASGRGLLDSEGMVGYCTHFLPIRSQLA


 


SEQ ID
TYSELNCRANQLALHYLQKLGVGPEVLVGILVERSLEMIVGLLG


NO. 8
ILKAGGAYVPLDPDYPPERLQFMLEDSQFFLLLTQQHLLESFAQ


A
SSETATPKIICLDSDYQIISQAKNINPENSVTTSNLAYVIYTSG



STGKPKGVMNNHVAISNKLLWVQDTYPLTTEDCILQKTPFSFDV



SVWELFWPLLNGARLVFAKPNGHKDASYLVNLIQEQQVTTLHFV



SSMLQLFLTEKDVEKCNSLKRVICSGEALSLELQERFFARLVCE



LHNLYGPTEAAIHVTFWQCQSDSNLKTVPIGRPIANIQIYILDS



HLQPVPIGVIGELHIGGVGLARGYLNRPELTAEKFIANPFASLD



PPLTPLDKGGDESYKTFKKGGEQPSRLYKTGDLARYLPDGKIEY



LGRIDNQVKIRGFRIELGEIEAVLLSHPQVREAVV


 


SEQ ID
EAIAAIFGQVLKLEKVGIYDNFFEIGGNSLQATQVISRLRESFA


NO. 9
LELPLRRLFEQPTVADLALAV


T


 


SEQ ID
PRDGQLPLSFAQSRLWFLYQLEGATGTYNMTGALSLSGPLQVEA


NO. 10
LKQALRTIIQRHEPLRTSFQSVDGVPVQVINPYPVWELAMVDLT


C2
GKETEAEKLAYQESQTPFDLTNSPLLRVTLLKLQPEKHILLINM



HHIISDGWSIGVFVRELSHLYRAFVAGKEPTLPILPIQYADFAV



WQREWLQGKVLAAQLEYWKRQLADAPPLLELPTDRPRPAIQTFQ



GKTERFELDRKLTQELKALSQQSGCTLFMTLLAAFGVVLSRYSG



QTDIVIGSAIANRNRQDIEGLIGFFVNTLALRLDLS


 


SEQ ID
TYGELNHRANQLAHYLQSLGVTKEQIVGVYLERSLEMAIGFLGI


NO. 11
LKAGAAYLPIDPEYPSVRTQFILEDTQLSLLLTQAELAEKLPQT


A2
QNKIICLDRDWPEITSQPQTNLDLKIEPNNLAYCIYTSGSTGQP



KGVLISHQALLNLIFWHQQAFEIGPLHKATQVAGIAFDATVWEL



WPYLTTGACINLVPQNILLSPTDLRDWLLNREITMSFVPTPLAE



KLLSLDWPNHSCLKTLLLGGDKLHFYPAASLPFQVINNYGPTEN



TVVATSGLVKSSSSHHFGTPTIGRPIANVQIYLLDQNLQPVPIG



VPGELHLGGAGLAQGYLNRPELTAEKFIANPFDPPLTPLDKGGE



EPSKLYKTGDLARYLPDGNVEFLGRIDNQVKIRGFRIETGEIEA



VLSQYFLLAESVV


 


SEQ ID
AQLTQIWSEVLGLERIGVKDNFFELGGHSLLATQVLSRINSAFG


NO. 12
LDLSVQIMFESPTIAGIAGYI


T2


 


SEQ ID
ARDGHLPLSFAQQRLWFLHYLSPDSRSYNTLEILQIDGNLNLTV


NO. 13
LEQSLGELINRHEIFRTTFPTVSGEPIQKIALPSRFQLKVDNYQ


C3
DLDENEQSAKIQQVAELEAGQAFDLTVGPLIQFKLLQLSPQKSV



LLLKMHHIIYDGWSFGILIRELSALYEAFLKNLANPLPALSIQY



ADFAVWQRQYLSGEVLDKQLNYWQEQLATVSPVLTLPTDRPRPA



IQTFQGGVERFQLDQNVTQGLKKLGQDQVATLFMTLLAGFGVLL



SRYSGQSDLMVGSPIANRNQAAIEPLIGFFANTLALRINLS


 


SEQ ID
TYTELNHRANQLAHYLQTLGVGAEVLVGISLERSLEMIIGLLGI


NO. 14
LKVGGAYLPLDPDYPTERLQLMLEDSQVPFLITHSSLLAKLPPS


A3
QATLICLDHIQEQISQYSPDNLQCQLTPANLANVIYTSGSTGKP



KGVMVEHKGLVNLALAQIQSFAVNHNSRVLQFASFSFDACISEI



LMTFGSGATLYLAQKDALLPGQPLIERLVKNGITHVTLPPSALV



VLPQEPLRNLETLIVAGEACSLDLVKQWSIDRNFFNAYGPTEAS



VCATIGQCYQDDLKVTIGKAIANVQIYILDAFLQPVPVGVSGEL



YIGGVGVARGYLNRPELTQEKFIANPFSNDPDSRLYKTGDLARY



LPDGNIEYLGRIDNQVKIRGFRIELGEIEAVLSQCPDVQNTAV


 


SEQ ID
EILAQIWGQVLKIERVSREDNFFELGGHSLLATQVMSRLRETFQ


NO. 15
VELPLRSLFTAPTIAELALTI


T3


 


SEQ ID
NDSANLPLSFAQQRLWFLDQLEPNSAFYHVGGAVRLEGTLNITA


NO. 16
LEQSLKEIINRHEALRTNFITIDGQATQIIHPTINWRLSVVDCQ


C4
NLTDTQSLEIAEAEKPFNLAQDCLFRATLFVRSPLEYHLLVTMH



HIVSDGWSIGVFFQELTHLYAVYNQGLPSSLTPIKIQYADFAVW



QRNWLQGEILSNQLNYWREQLANAPAFLPLPTDRPRPAIQTFIG



SHQEFKLSQPLSQKLNQLSQKHGVTLFMTLLAAFATLLYRYTGQ



ADILVGSPIANRNRKEIEGLIGFFVNTLVLRLSLD


 


SEQ ID
TYAELNHQANQLVHYLQTLGIGPEVLVAISVERSLEMIIGLLAI


NO. 17
LKACGAYLPLAPDYPTERLQFMLEDSQASFLITHSSLLEKLPSS


A4
QATLICLDHIQEQISQYSPDNLQSELTPSNLANVIYTSGSTGKP



KGVMVEHRGLVNLASSQIQSFAVKNNSRVLQFASFSFDACISEI



LMTFGSGATLYLAQKNDLLPGQPLMERLEKNKITHVTLPPSALA



VLPKKPLPNLQTLIVAGEACPLDLVKQWSVGRNFFNAYGPTETS



VCATIGQCYQDDLKVTIGKAIANVQIYILDAFLQPVPIGVPGEL



YIGGVGVARGYLNRPELTAERFIPNPFDPPLTPLKKGGDKSYET



FKKGEEQPSKLYKTGDLARYLPDGNIEYLGRIDNQVKIRGFRIE



LGEIEAVLSQCPDVQNTAV


 


SEQ ID
LQLAQIWSEILGINNIGIQENFFELGGHSLLAVSLINRIEQKLD


NO. 18
KRLPLTSLFQNGTIASLAQLL


T4


 


SEQ ID
TPFFAVHPIGGNVLCYADLARNLGTKQPFYGLQSLGLSELEKTV


NO. 19
ASIEEMAMIYIEAIQTVQASGPYYLGGWSMGGVIAFEIAQQLLT


TE
QGQEVALLALIDSYSPSLLNSVNREKNSANSLTEEFNEDINIAY



SFIRDLASIFNQEISFSGSELAHFTSDELLDKFITWSQETNLLP



SDFGKQQVKTWFKVFQINHQALSSYSPKTYLGRSVFLGAEDSSI



KNPGWHQ


 


SEQ ID
FSLYYFGSYEAEFNPNKYNLLFEGAKFGDRAGFTALWIPERHFH


NO. 20
AFGGFSPNPSVLAAALARETKQIQLRSGSVVLPLHNSIRVAEEW


MO
AVVDNLSQGRVGIAFASGWHPQDFVLAYQSFGQHRELMFQEIET



VQKLWRGEAITVPDGKGQRVEVKTYPQPMQSQLPSWITIVNNPD



TYIRAGAIGANILTNLMGQSVEDLARNIALYRQSLAEHGYDPAS



GTVTVLLHTFVGKDLEQVREQARQPFGQYLTSSVGLLQNMVKSQ



GMKVDFEQLRDEDRDFLLASAYKRYTETSALIGTPESCRQIIDH



LQSIGVDEVACFIDFGVDEQTVLANLPYLQSLKDLYQ


 


SEQ ID
IDPPLTPLDKGIDPPLTPLDKGIDPPLTPLDKG


NO. 21


SP 1


 


SEQ ID
PYQGGLGGDQSPYQGGLGGDQSPYQGGLGGDQSPYQGGLGGDQS


NO. 22
PYQGGLGGDQSPYQGELGGDQSPYQGGLGGDQV


SP 2


 


SEQ ID
PASEMREWVENTVSRILAFQPERGLEIGCGTGLLLSRVAKHCLE


NO. 23
YWATDYSQGAIQYVERVCNAVEGLEQVKLRCQMADNFEGIALHQ


MT
FDTVVLNSIIQYFPSVDYLLQVLEGAINVIGERGQIFVGDVRSL



PLLEPYHAAVQLAQASDSKTVEQWQQQVRQSVAGEEELVIDPTL



FLALKQHFPQISWVEIQPKRGVAHNELTQFRYDVTLHLETINNQ



ALLSGNPTVITWLNWQLDQLSLTQIKDKLLTDKPELWGIRGIPN



QRVEEALKIWEWVENAPDVETVEQLKKLLKQQVDTGINPEQVWQ



LAESLGYTAHLSWWESSQDGSFDVIFQRNSEAEDSKKLTLSKLA



FWDEKPFKIKPWSDYTNNPLRGKLVQKLIP


 


SEQ ID
MTNYGKSMSHYYDLVVGHKGYNKDYATEVEFIHNLVETYTTEAK


NO. 24
SILYLGCGTGYHAALLAQKGYSVHGVDLSAEMLEQAKTRIEDET


MT 2
IASNLSFSQGNICEIRLNRQFNVVLALFHVVNYQTTNQNLLATF



ATVKNHLKAGGIFICDVSYGSYVLGEFKSRPTASILRLEDNSNG



NEVTYISELNFLTHENIVEVTHNLWVTNQENQLLENSRETHLQR



YLFKPEVELLADACELTVLDAMPWLEQRPLTNIPCPSVCFVIGH



KTTHSA


 


SEQ ID
ATGACTATTAACTATGGTGATCTGCAAGAACCCTTTAATAAATT


NO. 25
CTCAACCCTAGTTGAATTACTCCGTTATCGGGCAAGCAGTCAAC


A*
CGGAACGCCTCGCCTATATTTTTCTGCGAGACGGAGAAATCGAA


nucl
GAAGCTCGTTTAACCTATGGGGAACTGGATCAAAAGGCTAGGGC


acid
GATC



GCCGCTTATCTACAATCCTTAGAAGCCGAGGGCGAAAGGGGTTT



ACTGCTCTATCCCCCAGGACTAGATTTTATTTCAGCTTTTTTTG



GTTGTTTATATGCGGGAGTCGTTGCCATTCCCGCCTATCCACCC



CGACGGAATCAAAACCTTTTGCGTTTACAGGCGATTATTGCCGA



TTCTCAAGCCCGATTTACCTTCACCAATGCCGCTCTATTTCCCA



GTTTAAAAAACCAATGGGCTAAAGACCCTGAATTAGGAGCAATG



GAATGGATTGTTACCGATGAAATTGACCATCACCTCAGGGAGGA



TTGGCTAGAACCAACCCTCGAAAAAAACAGTCTCGCTTTTCTAC



AATACACCTCTGGTTCAACGGGAACTCCAAAGGGAGTAATGGTC



AGTCACCATAATTTGTTGATTAATTCAGCCGATTTAGATCGTGG



TTGGGGCCATGATCAAGATAGCGTAATGGTCACTTGGCTACCGA



CCTTCCATGATATGGGTCTGATTTATGGGGTTATTCAGCCTTTG



TACAAAGGATTTCTTTGTTACATGATGTCCCCTGCCAGCTTTAT



GGAACGACCGTTACGTTGGTTACAGGCCCTTTCTGATAAAAAAG



CAACCCATAGTGCGGCCCCCAACTTTGCCTACGATCTTTGTGTG



CGGAAAATTCCCCCTGAAAAACGGGCTAGGTTAGACTTAAGCCA



TTGGTGCATGGCCTTAAATGGGGCCGAACCCGTCAGAGCGGAGG



TACTTAAAAAGTTTGCGGAGGCTTTTCAAGTTTCTGGTTTCAAA



GCCACAGCCCTTTGTCCTGGCTACGGTTTAGCAGAAGCCACCCT



GAAAGTTACGGCGGTTAGTTATGACAGTCCCCCTTACTTTTATC



CCGTTCAGGCTAATGCTTTAGAAAAAAATAAGATTGTGGGAGCC



ACTGAAACCGATACCAATGTGCAGACCCTCGTGGGC



TGCGGCTGGACAACGATTGATACTCAAATCGTCATTGTCAATCC



TGAAACCCTGAAACCTTGCTCCCCTGAAATTGTCGGCGAAATTT



GGGTATCAGGTTCAACAATCGCCCAAGGCTATTGGGGAAAACCT



CAAGAGACTCAGGAAACCTTTCAAGCTTATTTGGCAGATACAGG



AGCC



GGGCCTTTTCTGCGAACAGGAGACTTGGGCTTCATTAAAGATGG



TGAATTGTTTATCACAGGTCGGCTCAAGGAAATTATTCTGATTC



GAGGACGCAATAATTATCCCCAGGATATTGAATTAACCGTCCAA



AATAGTCATCCCGCTCTGCGTCCCAGTTGTGGGGCTGCTTTTAC



CGTTGAAAATAAGGGCGAAGAAAAGCTCGTGGTCGTTCAGGAAG



TGGAGCGCACCTGGCTCCGT



AAGGTAGATATAGATGAGGTAAAAAGAGCCATTCGTAAAGCTGT



TGTCCAGGAATATGAT



TTACAGGTTTATGCGATCGCGCTGATCAGGACTGGCAGTTTACC



AAAAACCTCTAGCGGTAAAATTCAGCGTCGTAGCTGTCGGGCCA



AATTTTTAGAGGGAAGCCTGGAAATTTTGGGCTAA


 


SEQ ID
ATGTCCACAGAAATCCCAAACGACAAAAAACAACCGACCCTAAC


NO. 26
GAAAATTCAAAACTGG


ACP
TTAGTGGCTTACATGACAGAGATGATGGAAGTGGACGAAGATGA


nucl
GATTGATCTGAGCGTTCCCTTTGATGAATATGGTCTCGATTCTT


acid
CTATGGCAGTTGCTTTGATCGCTGATCTAGAGGATTGGTTACGA



CGAGATTTACATCGCACCCTGATCTACGATTATCCAACTCTAGA



AAAGTTGGCTAAACAGGTTAGTGAACCCTGA


 


SEQ ID
ATGGAACCCATCGCAATTATTGGTCTTGCTTGCCGCTTTCCAGG


NO. 27
GGCTGACAATCCAGAAGCTTTCTGGCAACTCATGCGAAATGGGG


KS
TGGATGCGATCGCCGATATTCCTCCTGAACGTTGGGATATTGAG


nucl
CGTTTCTACGATCCCACACCTGCCACTGCCAAGAAGATGTATAG


acid
TCGCCAGGGCGGTTTTCTAAAAAATGTCGATCAATTTGACCCTC



AATTTTTCCGAATTTCTCCCCTAGAAGCCACCTATCTAGATCCT



CAACAAAGACTGCTACTGGAAGTCACCTGGGAAGCCTTAGAAAA



TGCTGCCATTGTGCCTGAAACCTTAGCTGGTAGCCAATCAGGGG



TTTTTATTGGTATCAGTGATGTGGATTATCATCGTTTGGCTTAT



CAAAGTCCTACTAACTTGACCGCCTATGTGGGTACAGGCAACAG



CACCAGTATTGCGGCTAACCGTTTATCATATCTGTTTGATTTGC



GTGGCCCCAGTTTGGCCGTAGATACCGCTTGCTCTTCTTCCCTC



GTCGCCGTTCACTTGGCCTGTCAGAGTTTGCAAAGTCAAGAATC



GAACCTCTGCTTAGTGGGGGGAGTTAATCTCATTTTGTCGCCAG



AGACAACCGTTGTTTTTTCCCAAGCGAGAATGATCGCCCCCGAC



AGTCGTTGTAAAACCTTTGACGCGAGGGCCGATGGTTATGTGCG



CTCGGAAGGCTGTGGAGTAGTCGTACTTAAACGTCTTAGGGATG



CCATTCAGGACGGCGATCGCATTTTAGCAGTGATTGAAGGTTCC



GCGGTGAATCAGGATGGTTTAAGTAATGGACTCACGGCCCCTAA



TGGCCCTGCTCAACAGGCGGTGATTCGTCAGGCCCTGGCAAATG



CCCAGGTAAAACCGGCCCAG



ATTAGCTATGTCGAAGCCCATGGCACGGGGACAGAATTGGGGGA



TCCGATCGAAGTTAAA



TCTCTGAAAGCGGTTTTGGGTGAAAAGCGATCGCTCGATCAAAC



CTGTTGGCTCGGTTCTGTGAAAACCAACATTGGTCATTTAGAAG



CGGCGGCGGGAATGGCGGGTCTGATTAAAGTC



GTTCTCTGCCTACAACACCAAGAAATTCCCCCTAATCTCCACTT



TCAAACCCTTAATCCCTATATTTCCCTAGCTGACACAGCTTTTG



CGATTCCCACTCAGGCTCAACCCTGGCGGACCAAACCCCCTAAG



TCTGGTGAAAACGGTGTCGAACGACGTTTAGCAGGACTCAGTTC



CTTTGGGTTTGGGGGGACAAATTCCCATGTGATTCTC


 


SEQ ID
GTTTTTCTATTTGCCGGTCAAGGTTCTCAATATGTAGGTATGGG


NO. 28
TCGTCAACTGTACGAAACCCAACCCATCTTTCGCCAAACCTTGG


AT
ATCGCTGTGCTGAAATCCTGCGACCCCATTTAGATCAACCCCTC


nucl
TTAGAAATTCTTTATCCTGCTGACCCAGAAGCCGAAACAGCGAG


acid
TTTTTACCTAGAGCAGACTGCCTATACCCAACCCACTTTATTCG



CATTCGAGTATGCCCTAGCACAGTTATGGCGTTCCTGGGGAATA



GAACCGGCGGCAGTAATTGGTCACAGTGTCGGTGAATATGTGGC



GGCCACCGTTGCCGGAGCCTTAAGTCTAGAAGAAGGATTAACGC



TAATTGCCAAACGGGCAAAACTGATGCAGTCTCTCCCCAAGAAT



GGGACAATGATGGCCGTTTTTGCCGCAGAAGAGCGGGTTAAAGC



TGTTATTGAGCCTTATAGGACTGATGTAGCGATCGCTGCTGTTA



ATGGACCAGAAAATTTTGTTATTTCAGGAAAAGCGCCGATTATT



GCTGAGATTATCATTCATTTAACGGCAGCAGGAATAGAAGTTCG



TCCTCTCAAAGTTTCCCATGCTTTTCACTCGCACCTGTTGGAGC



CAATTTTAGATTCCTTAGAACAGGAAGCTGCTGCTATTTCCTAC



CAACCCCTGCAAATTCCCTTAGTTGCTAATTTAACGGGGGAAGT



TCTACCAGAAGGAGCAACGATTGAGGCTCGTTACTGGCGAAATC



ATGCACGCAACCCTGTACAATTTTATGGGAGTATCCAAACGCTG



ATCGAGCAGAAATTCAGTCTTTTTTTAGAAGTTAGCCCTAAACC



GACTTTATCTCGATTGGGTCAACAATGTTGTCCAGAAAGATCGA



CCACTTGGCTATTTTCCCTCGCCCCTCCTCAAGAAGAAGAACAA



AGCCTACTAAATAGTTTGGCGATTCTCTATGATTCCCAAGGAGC



CGAA


 


SEQ ID
ATCACATTGCAAACCCTAGTGGGAAATTTACTGCAATTGTCCCC


NO. 29
TGCTGATGTCAATGTTCATACACCTTTCCTGGAGATGGGGGCAG


ACP 2
ATTCCATTGTCATGGTTGAGGCGGTCAGACGGATTGAGAATACC


nucl
TATAACGTTAAAATTGCTATGCGTCAGTTATTTGAGGAGTTATC


acid
TACTTTAGATGCTTTAGCTACTTATTTA


 


SEQ ID
AAAGAGATGCTTTATCCCATTGTGGCCCAACGTTCTCAAGGATC


NO. 30
AAGAATTTGGGATGTGGACGGTAATGAATATATTGATATGACGA


AMT
TGGGGCAAGGGGTAACGCTGTTTGGGCATCAA


nucl
CCAGACTTCATTATGTCGGCCCTACAAAGCCAACTCACTGAAGG


acid
CATTCATCTCAATCCGCGATCGCCAATTGTGGGAGAAGTGGCCG



CCTTAATTTGTGAACTAACAGGAGCCGAACGA



GCTTGTTTTTGCAACTCTGGAACCGAAGCCGTAATGGCCGCTAT



TCGTATCGCCAGGGCAACAACAGGTCGGAGTAAAATTGCCCTCT



TTGAAGGCTCCTATCATGGACATGCGGACGGAACCCTTTTTAGG



AACCAAATTATTGATAACCAACTCCACTCTTTTCCCCTAGCTCT



AGGCGTTCCCCCCAGCCTTAGTTCCGATGTGGTGGTATTGGACT



ATGGCAGTGCGGAAGCTCTGAACTATTTACAAACCCAGGGGCAG



GATTTAGCGGCGGTCTTAGTAGAACCAATTCAAAGTGGCAATCC



TCTACTCCAACCCCAACAATTTCTCCAAAGTCTGCGACAAATTA



CCAGTCAAATGGGCATTGCCCTGATTTTTGATGAAATGATTACG



GGTTTTCGATCGCACCCAGGGGGAGCGCAAGCTTTATTTGGAGT



ACAGGCGGATATTGCCACCTATGGCAAAGTAGTTGCGGGAGGAA



TGCCCATTGGAGTTATTGCAGGTAAGGCCCATTATCTGGACAGC



ATTGACGGGGGAATGTGGCGTTATGGCGATAAATCCTATCCTGG



GGTGGACAGAACCTTTTTTGGGGGAACCTTTAATCAGCATCCGT



TAGCAATGGTAGCGGCTAGGGCTGTCCTGACCCATTTAAAGGAG



CAGGGGCCAGGTCTGCAACAACAATTAACTGAACGCACTGCGGC



CTTAGCCGATACACTG



AATCATTATTTTCAAGCCGAAGAAGTTCCTATTAAAATCGAACA



GTTTAGTTCTTTCTTCCGGTTTGCCCTCTCTGGCAATTTGGATT



TACTTTTCTATCACATGGTAGAAAAAGGTATTTATGTCTGGGAA



TGGCGTAAACATTTTCTTTCAACCGCCCATACGGAAGCCGATCT



TGCCCAATTTGTCCAAGCGGTTAAGGATAGCATCACAGAATTGC



GT


 


SEQ ID
GGGGGGGATCAAGTCCCTCTCACCGAAGCCCAACGACAACTGTG


NO. 31
GATTTTGGCTCAATTAGGAGACAACGGCTCTGTGGCCTATAACC


C nucl
AATCAGTGACATTGCAATTAAGTGGCCCATTAAATCCCGTCGCA


acid
ATGAATCAAGCTATTCAACAAATCAGCGATCGCCATGAAGCGTT



ACGAACCAAAATTAATGCCCAGGGAGATAGTCAAGAAATCCTGC



CCCAGGTCGAAATTAACTGC



CCTATCTTAGACTTCAGTCTTGACCAAGCTTCGGCCCAACAGCA



AGCAGAACAATGGTTAAAGGAAGAAAGTGAAAAACCCTTTGATT



TGAGCCAGGGTTCTCTCGTGCGTTGGCATCTACTCAAATTAGAA



CCAGAATTACATTTGTTAGTATTAACGGCCCATCACATTATCAG



TGACGGTTGGTCAATGGGGGTAATCCTTCGGGAATTAGGAGAGT



TATATTCAGCCAAATGTCAGGGTGTTACGGCTAATCTTAAAACC



CCAAAACAGTTTCGAGAATTGATTGAATGGCAAAGCCAGCCAAG



CCAAGGGGAAGAACTGAAAAAACAGCAAGCCTATTGGTTAGCAA



CCCTTGCC



GATCCCCCTGTTTTGAATTTACCCACTGACAAACCTCGTCCAGC



TTTACCCAGTTACCAAGCTAATCGTCGAAGTCTAACTTTAGATA



GCCAATTTACAGAAAAACTAAAGCAATTTAGTCGTAAACAGGGC



TGTACCTTGCTGATGACCCTGTTATCGGTTTATAACATTCTCGT



TCATCGTTTGACGGGACAGGATGATATTCTGGTGGGTCTGCCAG



CCTCTGGACGGGGGCTTTTAGATAGTGAAGGTATGGTGGGTTAT



TGCACCCATTTTTTACCAATTCGCAGTCAATTAGCA


 


SEQ ID
ACTTACAGTGAATTAAATTGTCGAGCCAATCAGTTAGCACATTA


NO. 32
TTTACAAAAATTAGGAGTTGGGCCAGAGGTCTTAGTCGGTATTT


A nucl
TGGTCGAACGTTCTTTAGAAATGATTGTCGGATTGTTAGGGATT


acid
CTCAAGGCTGGGGGAGCCTATGTACCTCTTGATCCTGACTATCC



CCCTGAACGTCTTCAATTTATGTTAGAAGATAGTCAATTTTTTC



TCCTCTTAACCCAACAGCATTTACTGGAATCTTTTGCTCAGTCT



TCAGAAACGGCTACTCCCAAGATTATTTGTTTGGATAGCGACTA



CCAAATTATTTCCCAGGCAAAGAATATTAATCCCGAAAATTCAG



TCACAACGAGTAATCTTGCCTATGTAATTTATACCTCTGGTTCG



ACAGGTAAACCGAAGGGCGTGATGAATAATCATGTTGCTATTAG



TAATAAATTGTTATGGGTACAAGACACTTATCCTCTAACCACAG



AAGACTGTATTTTACAAAAAACTCCCTTTAGTTTTGATGTTTCA



GTGTGGGAATTATTCTGGCCCCTACTAAACGGAGCGCGTTTGGT



TTTTGCCAAGCCGAATGGCCATAAAGATGCCAGTTACTTAGTCA



ATCTGATTCAAGAGCAACAAGTAACAACGCTACATTTTGTGTCT



TCTATGCTACAGCTTTTTCTGACAGAAAAAGACGTAGAAAAATG



TAATAGTCTTAAACGAGTCATTTGTAGTGGTGAAGCCCTTTCTT



TAGAGCTTCAAGAACGTTTTTTTGCTCGTTTAGTCTGTGAATTA



CACAATCTTTATGGACCGACAGAAGCCGCTATTCATGTCACATT



TTGGCAATGTCAATCAGATAGCAATTTGAAAACAGTACCCATTG



GTCGGCCGATCGCTAATATCCAAATTTACATTTTAGACTCTCAT



TTCAGCCAGTACCTATTGGAGTAATCGGAGAATTGCACATTGGT



GGGGTTGGTTTGGCGCGGGGTTATTTAAACAGGCCTGAGTTAAC



GGCGGAGAAATTTATTGCAAATCCGTTTGCTTCCCTTGATCCCC



CCCTAACCCCCCTTGATAAGGGGGGAGATGAGAGCTATAAAACT



TTTAAAAAGGGGGGAGAGCAACCATCAAGATTGTATAAA



ACGGGAGATTTAGCTCGTTATTTACCCGATGGCAAGATTGAGTA



TCTAGGGCGCATTGATAATCAGGTAAAAATTCGCGGTTTCCGGA



TTGAATTGGGGGAAATTGAAGCGGTTTTGCTATCCCATCCCCAG



GTACGAGAAGCGGTCGTT


 


SEQ ID
GAGGCGATCGCCGCTATTTTTGGTCAAGTTTTAAAACTGGAAAA


NO. 33
AGTGGGAATTTATGATAACTTTTTTGAGATCGGCGGTAATTCTT


T nucl
TGCAAGCCACTCAAGTTATTTCACGCTTACGAGAAAGTTTTGCC


acid
CTAGAGTTGCCCTTGCGTCGCCTGTTTGAACAACCGACTGTGGC



GGATTTGGCTTTAGCCGTA


 


SEQ ID
CCTCGTGATGGCCAATTACCCCTCTCCTTTGCCCAGTCGCGACT


NO. 34
CTGGTTCTTGTATCAATTAGAAGGAGCCACGGGAACCTATAACA


C2
TGACAGGGGCCTTGAGTTTAAGCGGGCCTCTTCAGGTCGAAGCC


nucl
CTCAAACAAGCCCTAAGAACTATCATTCAACGCCATGAGCCATT


acid
GCGTACCAGTTTCCAATCGGTTGACGGGGTTCCAGTGCAGGTGA



TTAATCCCTATCCTGTTTGGGAATTAGCGATGGTTGATTTGACA



GGAAAGGAGACAGAAGCAGAAAAATTGGCCTATCAG



GAATCCCAAACCCCGTTTGATTTGACCAATAGTCCTTTGTTGAG



GGTAACGCTCCTCAAATTACAGCCAGAAAAGCATATTTTATTAA



TTAATATGCACCATATTATTTCCGATGGCTGGTCAATCGGTGTT



TTTGTTCGTGAATTGTCCCATCTCTATAGGGCTTTTGTGGCGGG



TAAAGAACCAACTTTACCGATTTTACCAATTCAGTATGCGGATT



TTGCCGTTTGGCAGCGAGAGTGGTTACAGGGTAAGGTTTTAGCG



GCTCAATTGGAATATTGGAAGCGACAATTGGCAGATGCTCCTCC



TCTGCTGGAACTGCCCACTGATCGCCCTCGTCCCGCAATCCAAA



CCTTTCAAGGCAAGACAGAAAGATTTGAGCTAGATAGGAAACTG



ACCCAAGAATTAAAGGCATTAAGT



CAACAGTCGGGTTGTACTTTATTTATGACTTTGTTGGCCGCTTT



TGGGGTGGTTTTATCCCGTTATAGTGGCCAGACTGATATCGTCA



TTGGTTCGGCGATCGCCAACCGTAATCGCCAAGACATTGAGGGG



TTAATTGGCTTTTTTGTTAACACTTTGGCGTTGAGGTTAGATTT



ATCA


 


SEQ ID
ACCTATGGAGAATTAAACCATCGCGCCAATCAATTAGCTCACTA


NO. 35
TCTTCAGTCGTTAGGAGTCACCAAAGAACAAATCGTCGGGGTTT


A2
ATCTGGAACGTTCCCTTGAAATGGCGATCGGATTTTTAGGTATT


nucl
CTCAAAGCAGGAGCCGCCTATCTCCCCATTGATCCTGAATATCC


acid
CTCAGTACGCACCCAATTTATTCTCGAAGATACCCAACTTTCGC



TTCTCTTAACTCAGGCAGAACTGGCAGAAAAACTGCCCCAGACT



CAAAACAAAATTATCTGTCTAGATCGGGACTGGCCA



GAAATTACCTCCCAACCCCAGACAAACCTAGACCTAAAGATAGA



ACCTAATAACCTAGCC



TATTGCATCTATACTTCTGGTTCCACAGGACAACCCAAAGGAGT



ACTGATTTCCCATCAAGCCCTACTCAACTTAATTTTCTGGCATC



AACAAGCGTTTGAGATTGGCCCCTTACATAAAGCGACCCAAGTG



GCAGGCATTGCTTTCGATGCAACGGTTTGGGAATTGTGGCCCTA



TCTGACCACAGGAGCCTGTATTAATCTGGTTCCCCAAAATATTC



TGCTCTCACCGACGGATTTACGGGATTGGTTGCTTAACCGAGAA



ATTACCATGAGTTTTGTGCCAACTCCTTTAGCTGAAAAATTATT



ATCCTTGGATTGGCCTAACCATTCTTGTCTAAAAACCCTGTTAC



TGGGAGGTGACAAACTTCATTTTTATCCTGCTGCGTCCCTTCCC



TTTCAGGTCATTAACAACTATGGCCCAACGGAAAATACAGTGGT



TGCGACCTCTGGACTGGTCAAATCATCTTCATCTCATCACTTTG



GAACTCCGACTATTGGTCGTCCCATTGCCAACGTCCAAATCTAT



TTATTAGACCAAAACCTACAACCTGTCCCCATTGGTGTACCAGG



AGAATTACATTTAGGTGGGGCGGGTTTAGCGCAGGGCTATCTCA



ATCGTCCTGAGTTAACGGCTGAAAAATTTATTGCCAATCCCTTT



GATCCCCCCCTAACCCCCCTTGATAAGGGGGGAGAAGAACCCTC



AAAACTCTATAAAACG



GGAGACTTAGCCCGTTATTTACCCGATGGCAATGTAGAATTTTT



GGGACGTATTGACAATCAGGTAAAAATTCGGGGTTTTCGCATCG



AAACTGGGGAAATCGAAGCCGTTTTAAGTCAATATTTCCTATTA



GCTGAAAGTGTAGTC


 


SEQ ID
GCTCAACTGACTCAAATTTGGAGTGAAGTTTTGGGACTGGAACG


NO. 36
CATTGGCGTTAAGGACAACTTTTTTGAATTGGGAGGACATTCTC


T2
TTTTGGCTACCCAGGTTTTATCAAGAATTAATTCAGCCTTTGGA


nucl
CTTGATCTTTCTGTGCAAATTATGTTTGAATCACCAACGATCGC


acid
GGGCATTGCGGGTTATATT


 


SEQ ID
GCTAGAGACGGTCATTTACCCCTGTCTTTTGCTCAACAACGTTT


NO. 37
ATGGTTTTTACATTATCTTTCCCCTGATAGTCGTTCCTACAATA


C3
CCCTGGAAATATTGCAAATTGATGGGAATCTCAATCTGACTGTG


nucl
CTAGAGCAGAGTTTGGGGGAATTAATTAACCGCCATGAAATTTT


acid
TAGAACAACATTCCCCACTGTTTCAGGGGAACCGATTCAGAAAA



TTGCACTTCCTAGTCGTTTTCAGTTAAAAGTTGATAATTATCAA



GATTTAGACGAAAATGAACAATCAGCTAAAATTCAACAAGTAGC



AGAATTGGAAGCAGGACAAGCTTTTGATTTAACGGTGGGGCCAC



TGATTCAGTTTAAGCTATTGCAATTGAGTCCCCAGAAGTCGGTG



CTGCTGTTGAAAATGCACCATATTATCTATGATGGCTGGTCTTT



TGGGATTCTGATTCGGGAATTATCGGCTCTATACGAAGCATTTT



TAAAGAACTTAGCCAATCCTCTCCCTGCGTTGTCTATTCAGTAT



GCAGATTTTGCGGTTTGGCAACGTCAATATCTCTCAGGTGAGGT



CTTAGATAAACAACTCAATTATTGGCAAGAACAGTTAGCAACAG



TCTCTCCTGTTCTTACTTTACCAACGGATAGACCCCGTCCGGCG



ATACAAACTTTTCAGGGAGGAGTTGAGCGTTTTCAACTGGATCA



AAATGTCACTCAAGGTCTTAAAAAGTTAGGTCAAGATCAGGTTG



CAACCCTGTTTATGACGTTGTTGGCCGGTTTCGGCGTTTTGCTA



TCTCGTTATAGTGGTCAATCTGATCTGATGGTGGGTTCTCCGAT



CGCTAATCGTAATCAAGCAGCGATCGAACCTTTAATTGGCTTTT



TTGCTAACACTTTGGCTTTAAGAATTAATTTATCA


 


SEQ ID
ACATACACTGAATTAAACCATCGCGCTAATCAGTTAGCCCATTA


NO. 38
TTTACAAACTTTAGGCGTGGGAGCAGAAGTCTTAGTCGGTATTT


A3
CCCTAGAACGTTCTTTAGAGATGATTATCGGCTTATTAGGGATT


nucl
CTCAAGGTAGGTGGTGCTTATCTTCCTCTTGATCCAGACTATCC


acid
CACTGAGCGTCTTCAGTTGATGTTAGAAGACAGTCAAGTTCCTT



TTTTGATTACCCACAGTTCTTTATTAGCAAAATTGCCTCCCTCT



CAAGCAACTCTGATTTGTTTAGATCATATCCAAGAGCAGATTTC



TCAATATTCTCCAGATAATCTTCAATGTCAGTTAACTCCTGCCA



ATTTAGCTAACGTTATTTATACCTCTGGCTCTACGGGTAAGCCT



AAAGGGGTGATGGTTGAACATAAAGGTTTAGTTAACTTAGCTCT



TGCTCAAATTCAATCTTTTGCAGTCAACCATAACAGTCGTGTGC



TGCAATTTGCTTCTTTTAGTTTTGATGCTTGTATTTCAGAAATT



TTGATGACCTTTGGTTCTGGAGCGACGCTTTATCTTGCACAAAA



AGATGCTTTATTGCCAGGTCAGCCATTAATTGAACGGTTAGTAA



AGAATGGAATTACTCATGTGACTTTGCCGCCTTCAGCTTTAGTG



GTTTTACCCCAGGAACCGTTACGCAACTTAGAAACCTTAATTGT



GGCGGGTGAGGCTTGTTCTCTTGATTTAGTGAAACAATGGTCAA



TCGATAGAAACTTTTTCAATGCCTATGGGCCAACGGAAGCGAGT



GTTTGTGCCACTATTGGACAATGTTATCAAGATGATTTAAAGGT



GACGATTGGTAAGGCGATCGCCAATGTCCAAATTTATATTTTAG



ATGCCTTTTTACAGCCGGTGCCGGTGGGAGTGTCAGGAGAGTTA



TACATTGGTGGAGTTGGGGTGGCAAGGGGCTATTTAAATCGTCC



TGAATTAACCCAAGAAAAATTTATTGCTAATCCTTTTAGTAACG



ACCCAGATTCTCGGCTCTATAAAACTGGCGACTTAGCGCGTTAT



TTACCCGATGGTAATATTGAATATTTAGGACGCATTGACAATCA



GGTAAAAATTCGCGGTTTTCGCATTGAGTTAGGAGAAATTGAAG



CGGTTCTGAGTCAATGTCCCGATGTGCAAAATACGGCGGTG


 


SEQ ID
GAAATTCTGGCTCAAATATGGGGGCAAGTTCTCAAGATAGAAAG


NO. 39
AGTCAGCAGAGAAGAT


T3
AATTTCTTTGAATTGGGGGGGCATTCCCTTTTAGCTACCCAGGT


nucl
AATGTCCCGTCTGCGTGAAACTTTTCAAGTCGAATTACCTTTGC


acid
GTAGTCTCTTTACCGCTCCCACTATTGCTGAATTGGCCCTAACA



ATT


 


SEQ ID
AACGACAGTGCTAACCTCCCGTTATCTTTTGCTCAACAACGTTT


NO. 40
ATGGTTTCTGGATCAATTAGAACCTAACAGCGCCTTTTATCATG


C4
TAGGGGGAGCCGTAAGACTAGAAGGAACATTAAATATTACTGCC


nucl
TTAGAGCAAAGCTTAAAAGAAATTATTAATCGTCATGAAGCTTT


acid
ACGCACAAATTTTATAACGATTGATGGTCAAGCCACTCAAATTA



TTCACCCTACTATTAATTGGCGATTGTCTGTTGTTGATTGTCAA



AATTTAACCGACACTCAATCTCTGGAAATTGCGGAAGCTGAAAA



GCCCTTTAATCTTGCTCAAGATTGCTTATTTCGTGCTACTTTAT



TCGTGCGATCACCGCTAGAATATCATCTACTCGTGACCATGCAC



CATATTGTTAGCGATGGCTGGTCAATTGGAGTATTTTTTCAAGA



ACTAACTCATCTTTACGCTGTCTATAATCAGGGTTTACCCTCAT



CTTTAACGCCTATTAAAATACAATATGCTGATTTTGCGGTCTGG



CAACGGAATTGGTTACAAGGTGAAATTTTAAGTAATCAATTGAA



TTATTGGCGCGAACAATTAGCAAATGCTCCTGCTTTTTTACCTT



TACCGACAGATAGACCTAGGCCCGCAATCCAAACTTTTATTGGT



TCTCATCAAGAATTTAAACTTTCTCAGCCATTAAGCCAAAAATT



GAATCAACTAAGTCAGAAGCATGGAGTGACTTTATTTATGACTC



TCCTGGCTGCTTTTGCTACCTTACTTTACCGTTATACAGGACAA



GCAGATATTTTAGTTGGTTCTCCTATTGCTAACCGTAATCGTAA



GGAAATTGAGGGATTAATCGGCTTTTTTGTTAATACATTAGTTC



TGAGATTGAGTTTAGAT


 


SEQ ID
ACCTATGCTGAATTAAATCATCAAGCTAATCAGTTAGTCCATTA


NO. 41
CTTACAAACTTTAGGAATTGGGCCAGAGGTCTTAGTCGCTATTT


A4
CAGTAGAACGTTCTTTAGAAATGATTATCGGCTTATTAGCCATT


nucl
CTCAAGGCGTGTGGTGCTTATCTCCCTCTTGCTCCTGACTATCC


acid
CACTGAGCGTCTTCAGTTCATGTTAGAAGATAGTCAAGCTTCTT



TTTTGATTACCCACAGTTCTTTATTAGAAAAATTGCCTTCTTCT



CAAGCGACTCTAATTTGTTTAGATCACATCCAAGAGCAGATTTC



TCAATATTCTCCCGATAATCTTCAAAGTGAGTTAACTCCTTCCA



ATTTGGCTAACGTTATTTACACCTCTGGCTCTACGGGTAAGCCT



AAAGGGGTGATGGTTGAACATCGGGGCTTAGTTAACTTAGCGAG



TTCTCAAATTCAATCTTTTGCAGTCAAAAATAACAGTCGTGTAC



TGCAATTTGCTTCCTTTAGTTTTGATGCTTGTATTTCAGAAATT



TTGATGACCTTTGGTTCTGGAGCGACTCTTTATCTTGCTCAAAA



AAATGATTTATTGCCAGGTCAGCCATTAATGGAAAGGTTAGAAA



AGAATAAAATTACCCATGTTACTTTACCCCCTTCAGCTTTAGCT



GTTTTACCAAAAAAACCGTTACCCAACTTACAAACTTTAATTGT



GGCGGGTGAGGCTTGTCCTCTGGATTTAGTCAAACAATGGTCAG



TCGGTAGAAACTTTTTCAATGCCTATGGCCCGACAGAAACGAGT



GTTTGTGCCACGATTGGACAATGTTATCAAGATGATTTAAAGGT



CACGATTGGTAAGGCGATCGCTAATGTCCAAATTTATATTTTGG



ATGCCTTTTTACAACCAGTACCCATCGGAGTACCAGGGGAATTA



TACATTGGTGGAGTCGGAGTTGCGAGGGGTTATCTAAATCGTCC



TGAATTAACGGCGGAAAGATTTATTCCTAATCCTTTTGATCCCC



CCCTAACCCCCCTTAAAAAGGGGGGAGATAAGAGCTATGAAACT



TTTAAAAAGGGGGAAGAGCAACCA



TCAAAACTCTATAAAACGGGAGATTTAGCTCGTTATTTACCCGA



TGGCAATATTGAATATTTAGGACGCATTGACAATCAGGTAAAAA



TTCGCGGTTTTCGCATTGAGTTAGGAGAAATTGAAGCGGTTCTG



AGTCAATGTCCCGATGTGCAAAATACGGCGGTG


 


SEQ ID
TTACAATTAGCTCAAATCTGGTCAGAGATTTTAGGCATTAATAA


NO. 42
TATTGGTATTCAGGAAAACTTCTTTGAATTAGGCGGTCATTCTT


T4
TATTAGCAGTCAGTCTGATCAATCGTATTGAACAAAAGTTAGAT


nucl
AAACGTTTACCATTAACCAGTCTTTTTCAAAATGGAACCATAGC


acid
AAGTCTAGCTCAATTACTAG


 


SEQ ID
ACTCCATTTTTTGCTGTTCATCCCATTGGTGGTAATGTGCTATG


NO. 43
TTATGCCGATTTAGCTCGTAATTTAGGAACGAAACAGCCGTTTT


TE
ATGGATTACAATCATTAGGGCTAAGTGAATTAGAAAAAACTGTA


nucl
GCCTCTATTGAAGAAATGGCGATGATTTATATTGAAGCAATACA


acid
AACTGTTCAAGCCTCTGGTCCCTACTATTTAGGAGGTTGGTCAA



TGGGAGGAGTGATAGCTTTTGAAATCGCCCAACAATTATTGACC



CAAGGTCAAGAAGTTGCTTTACTGGCTTTAATAGATAGTTATTC



TCCCAGTTTACTTAATTCAGTTAATAGGGAGAAAAATTCTGCTA



ATTCCCTGACAGAAGAATTTAATGAAGATATCAATATTGCCTAT



TCTTTCATCAGAGACTTAGCAAGTATATTTAATCAAGAAATCTC



TTTCTCTGGGAGTGAACTTGCTCATTTTACATCAGACGAATTAC



TAGACAAGTTTATTACTTGGAGTCAAGAGACGAATCTTTTGCCG



TCAGATTTTGGGAAGCAGCAGGTTAAAACCTGGTTTAAAGTTTT



CCAGATTAATCACCAAGCTTTGAGCAGCTATTCTCCCAAGACGT



ATCTGGGTAGAAGTGTTTTCTTAGGAGCGGAAGACAGTTCTATT



AAAAATCCTGGTTGGCATCAA


 


SEQ ID
AGCGGGTCTCAAGACCAAAAAACGATACAGTTTAGCCTCTACTA


NO. 44
CTTTGGTAGCTATGAAGCGGAATTTAACCCGAATAAATATAACT


MO
TACTGTTTGAAGGAGCTAAATTTGGCGATCGCGCTGGTTTTACG


nucl
GCCCTTTGGATTCCTGAACGTCATTTCCACGCTTTTGGTGGTTT


acid
TTCTCCCAATCCTTCGGTTTTGGCGGCGGCTTTAGCACGGGAAA



CCAAACAGATTCAACTGCGATCAGGCAGTGTGGTTTTACCGCTA



CATAATTCCATCCGAGTCGCCGAAGAATGGGCAGTGGTGGACAA



TCTTTCCCAGGGCCGCGTTGGTATTGCTTTTGCATCGGGTTGGC



ATCCCCAGGATTTTGTCTTGGCTCCCCAGTCCTTTGGCCAACAT



CGGGAATTGATGTTCCAAGAAATTGAAACCGTCCAGAAACTTTG



GCGAGGGGAAGCGATCACCGTGCCAGACGGAAAGGGTCAA



AGGGTAGAGGTTAAAACCTATCCCCAACCGATGCAGTCCCAGTT



ACCCAGCTGGATTACTATTGTCAATAATCCCGATACCTATATCA



GAGCAGGGGCGATCGGTGCTAATATCCTTACCAATCTGATGGGG



CAAAGCGTGGAAGATTTAGCCCGTAATATTGCGCTATATCGTCA



ATCTTTGGCAGAGCATGGTTATGATCCCGCGTCGGGAACGGTGA



CAGTTCTCCTGCATACTTTTGTTGGCAAGGATTTAGAACAAGTT



CGAGAACAGGCTCGCCAACCCTTTGGGCAATACCTCACCTCCTC



TGTCGGACTCTTGCAGAACATGGTCAAGAGCCAGGGCATGAAAG



TGGATTTTGAACAATTAAGAGACGAAGATCGGGACTTTCTCCTC



GCTTCTGCCTATAAACGCTATACAGAAACCAGTGCTTTAATTGG



CACACCCGAATCCTGTCGTCAAATTATTGATCATTTGCAGTCCA



TCGGTGTGGATGAAGTGGCTTGTTTTATTGATTTTGGGGTAGAT



GAACAAACAGTTTTGGCCAATTTACCCTATCTCCAGTCCCTAAA



AGACTTATATCAA


 


SEQ ID
ATTGATCCCCCCCTAACCCCCCTTGATAAGGGGATTGATCCCCC


NO. 45
CCTAACCCCCCTTGATAAGGGGATTGATCCCCCCCTAACCCCCC


SP 1
TTGATAAGGGG


nucl


acid


 


SEQ ID
CCTTATCAAGGGGGGTTAGGGGGGGATCAATCCCCTTATCAAGG


NO. 46
GGGGTTAGGGGGGGAT


SP 2
CAATCCCCTTATCAAGGGGGGTTAGGGGGTGATCAATCCCCTTA


nucl
TCAAGGGGGGTTAGGGGGTGATCAATCCCCTTATCAAGGGGGGT


acid
TAGGGGGGGATCAATCCCCTTATCAAGGAGAG



TTAGGGGGGGATCAATCCCCTTATCAAGGGGGGTTAGGGGGGGA



TCAAGTC


 


SEQ ID
CCTGCTTCAGAAATGCGAGAGTGGGTCGAAAACACTGTTAGTCG


NO. 47
CATCTTGGCTTTCCAACCAGAACGCGGTTTAGAAATTGGTTGTG


MT
GTACAGGTTTGTTACTCTCCAGGGTAGCAAAGCATTGTCTTGAA


nucl
TATTGGGCAACGGATTATTCCCAAGGGGCGATCCAGTATGTTGA


acid
ACGGGTTTGCAATGCCGTTGAAGGTTTAGAACAGGTTAAATTAC



GCTGTCAAATGGCAGATAATTTTGAAGGTATTGCCCTACATCAA



TTTGATACCGTCGTCTTAAATTCGATTATTCAGTATTTTCCCAG



TGTGGATTATCTGTTACAGGTGCTTGAAGGGGCGATCAACGTCA



TTGGCGAGCGAGGTCAGATTTTTGTCGGGGATGTGCGGAGTTTA



CCCCTATTAGAGCCATATCATGCGGCTGTGCAATTAGCCCAAGC



TTCTGACTCGAAAACTGTTGAACAATGGCAACAACAGGTGCGTC



AAAGTGTAGCAGGTGAAGAAGAACTGGTCATTGATCCCACATTG



TTCCTGGCTTTAAAACAACATTTTCCGCAAATTAGCTGGGTAGA



AATTCAACCGAAACGGGGTGTGGCTCACAATGAGTTAACTCAAT



TTCGCTATGATGTCACTCTCCATTTAGAGACTATCAATAATCAA



GCATTATTGAGCGGCAATCCAACGGTAATTACCTGGTTAAATTG



GCAACTTGACCAACTGTCTTTAACACAAATTAAAGATAAATTAT



TAACAGACAAACCTGAATTGTGGGGAATTCGTGGTATTCCTAAT



CAGCGAGTTGAAGAGGCTCTAAAAATTTGGGAATGGGTGGAAAA



TGCCCCTGATGTTGAAACGGTTGAACAACTCAAAAAACTTCTCA



AACAACAAGTAGATACTGGTATTAATCCTGAACAGGTTTGGCAA



TTAGCTGAGTCTCTCGGTTACACCGCTCACCTTAGTTGGTGGGA



AAGTAGTCAAGACGGTTCCTTTGATGTCATTTTTCAGCGGAATT



CAGAAGCGGAGGACTCAAAAAAATTAACCCTTTCAAAACTTGCT



TTCTGGGATGAAAAACCCTTTAAAATAAAGCCCTGGAGTGACTA



TACTAACAACCCTCTGCGCGGTAAGTTAGTCCAAAAATTAATTC



CT


 


SEQ ID
ATGACAAATTATGGCAAATCTATGTCTCATTACTATGATCTAGT


NO. 48
GGTAGGACATAAAGGTTATAACAAAGATTACGCCACTGAAGTAG


MT 2
AATTCATTCACAATTTAGTTGAGACTTACACAACTGAAGCCAAA


nucl
TCTATCCTATACTTGGGCTGTGGTACGGGTTATCATGCCGCTCT


acid
TTTAGCACAGAAAGGGTATTCTGTACATGGTGTTGATCTCAGTG



CTGAAATGTTAGAGCAGGCTAAAACTCGCATTGAAGATGAAACA



ATAGCTTCTAATCTGAGTTTTTCTCAAGGAAATATTTGTGAAAT



CCGTTTAAATCGTCAGTTTAATGTTGTTCTTGCTCTATTTCATG



TGGTTAACTATCAAACGACCAATCAAAATTTACTGGCAACGTTT



GCAACGGTTAAAAACCATTTAAAAGCTGGGGGGATTTTTATTTG



TGATGTGTCCTATGGGTCTTACGTACTGGGGGAATTTAAGAGTC



GGCCTACGGCATCAATATTGCGTTTAGAGGATAATTCCAATGGT



AACGAAGTAACCTATATTAGTGAACTAAATTTTTTAACCCATGA



AAATATAGTGGAAGTTACTCACAATTTATGGGTAACAAATCAAG



AAAATCAACTTCTAGAGAATTCACGGGAAACACATCTTCAGCGC



TATCTTTTCAAGCCTGAAGTTGAATTGTTGGCTGATGCTTGTGA



ACTAACTGTTCTTGATGCGATGCCCTGGCTTGAACAACGTCCTT



TGACAAACATTCCTTGTCCTTCAGTTTGTTTTGTTATTGGGCAT



AAAACAACCCATTCAGCTTAA


 


SEQ ID
CCGACCTGTGATAAACAATTC


NO. 49


Primer


A


 


SEQ ID
CKNCCDGTDATRAANARYTC


NO. 50


Primer


B


 


SEQ ID
TTCAATATCCTGGGGATA


NO. 51


Primer


C


 


SEQ ID
YTCDATRTCYTGNGGRTA


NO. 52


Primer


D


 


SEQ ID
CGTTGGTTACAGGCCCTTTCT


NO. 53


Primer


E


 


SEQ ID
MGNTGGYTNCARGCNYTNWS


NO. 54


Primer


F


 


SEQ ID
TTAGACTTAAGCCATTGG


NO. 55


Primer


G


 


SEQ ID
YTNGAYYTNWSNCAYTGG


NO. 56


Primer


H


 


SEQ ID
CATAGAAGAATCGAGACCATATTC


NO. 57


Primer


I


 


SEQ ID
CATNSWNSWRTCNARNCCRTAYTC


NO. 58


Primer


J


 


SEQ ID
MTTQTASSANALASFNQFLRDVKAIAQPYWYPTVSNKRSFSEVI


NO. 59
RSWGMLSLLIFLIVGLVAVTAFNSFVNRRLIDVIIQEKDASQFA


ABC
STLTVYAIGLICVTLLAGFTKDIRKKIALDWYQWLNTQIVEKYF


Trans-
SNRAYYKINFQSDIDNPDQRLAQEIEPIATNAISFSATFLEKSL


porter
EMLTFLVVVWSISRQIAIPLMFYTIIGNFIAAYLNQELSKINQA



QLQSKADYNYALTHVRTHAESIAFFRGEKEEQNIIQRRFQEVIN



DTKNKINWEKGNEIFSRGYRSVIQFFPFLVLGPLYIKGEIDYGQ



VEQASLASFMFASALGELITEFGTSGRFSSYVERLNEFSNALET



VTKQAENVSTITTIEENHFAFEHVTLETPDYEKVIVEDLSLTVQ



KGEGLLIVGPSGRGKSSLLRAIAGLWNAGTGRLVRPPLEEILFL



PQRPYIILGTLREQLLYPLTNSEMSNTELQAVLQQVNLQNVLNR



VDDFDSEKPWENILSLGEQQRLAFARLLVNSPSFTILDEATSAL



DLTNEGILYEQLQTRKTTFISVGHRESLFNYHQWVLELSADSSW



ELLSVQDYRLKKAGEMFTNASSNNSITPDITIDNGSEPEIVYSL



EGFSHQEMKLLTDLSLSSIRSKASRGKVITAKDGFTYLYDKNPQ



ILKWLR


 


SEQ ID
ATGACAACCCAAACAGCTTCTAGTGCCAATGCCCTTGCTTCCTT


NO. 60
TAACCAATTTTTAAGGGATGTAAAGGCGATCGCCCAACCCTATT


ABC
GGTATCCCACTGTATCAAATAAAAGAAGCTTTTCTGAGGTTATT


Trans-
CGTTCCTGGGGAATGCTATCACTGCTTATCTTTTTGATTGTGGG


porter
ATTAGTCGCCGTCACGGCTTTTAATAGTTTTGTTAATCGTCGTT


Nucl
TAATTGATGTCATTATTCAAGAAAAAGATGCGTCTCAATTTGCC


acid
AGTACATTAACTGTCTATGCGATCGGATTAATCTGTGTAACGCT



GCTGGCAGGGTTCACTAAAGATATTCGCAAAAAAATTGCCCTAG



ATTGGTATCAATGGTTAAACACCCAGATTGTAGAGAAATATTTT



AGTAATCGTGCCTATTATAAAATTAACTTTCAATCTGACATTGA



TAACCCCGATCAACGTCTAGCCCAGGAAATTGAACCGATCGCCA



CAAACGCCATTAGTTTCTCGGCCACTTTTTTGGAAAAAAGTTTG



GAAATGCTAACTTTTTTAGTGGTAGTTTGGTCAATTTCTCGACA



GATTGCTATTCCGCTAATGTTTTACACGATTATCGGTAATTTTA



TTGCCGCCTATCTAAATCAAGAATTAAGCAAGATCAATCAGGCA



CAACTGCAATCAAAAGCAGATTATAACTATGCCTTAACCCATGT



TCGGACTCATGCGGAATCTATTGCTTTTTTTCGGGGAGAAAAAG



AGGAACAAAATATTATTCAGCGACGTTTTCAGGAAGTTATCAAT



GATACGAAAAATAAAATTAACTGGGAAAAAGGGAATGAAATTTT



TAGT



CGGGGCTATCGTTCCGTCATTCAGTTTTTTCCTTTTTTAGTCCT



TGGCCCTTTGTATATTAAAGGAGAAATTGATTATGGACAAGTTG



AGCAAGCTTCATTAGCTAGTTTTATGTTTGCATCGGCCCTGGGA



GAATTAATTACAGAATTTGGTACTTCAGGACGTTTTTCTAGTTA



TGTAGAACGTTTAAATGAATTTTCTAATGCCTTAGAAACTGTGA



CTAAACAAGCCGAGAATGTCAGCACAATTACAACCATAGAAGAA



AATCATTTTGCCTTTGAACACGTCACCCTAGAAACCCCTGACTA



TGAAAAGGTGATTGTTGAGGATTTATCTCTTACTGTTCAAAAAG



GTGAAGGATTATTGATTGTCGGGCCCAGTGGTCGAGGTAAAAGT



TCTTTATTAAGGGCGATCGCCGGTTTATGGAATGCTGGCACTGG



GCGTTTAGTGCGTCCTCCCCTAGAAGAAATTCTCTTTTTGCCCC



AACGTCCCTACATTATTTTGGGAACCTTACGCGAACAATTGCTG



TATCCTCTAACCAATAGTGAGATGAGCAATACCGAACTTCAAGC



AGTATTACAACAAGTCAATTTGCAAAATGTGCTAAATCGGGTGG



ATGACTTTGACTCCGAAAAACCCTGGGAAAACATTCTCTCCCTC



GGTGAACAACAACGCCTAGCCTTTGCTCGATTGTTAGTGAATTC



TCCGAGTTTTACCATTTTAGATGAGGCGACCAGTGCCTTAGATT



TAACAAATGAGGGGATTTTATACGAGCAATTACAAACTCGCAAG



ACAACCTTTATTAGTGTGGGTCATCGAGAAAGTTTGTTTAATTA



CCATCAATGGGTTTTAGAACTTTCTGCTGACTCTAGTTGGGAAC



TCTTAAGCGTTCAAGATTATCGCCTTAAAAAAGCGGGAGAAATG



TTTACTAATGCTTCGAGTAACAATTCCATAACACCCGATATTAC



TATCGATAATGGATCAGAACCAGAAATAGTCTATTCTCTTGAAG



GATTTTCCCATCAGGAAATGAAACTATTAACAGACCTATCACTC



TCTAGCATTCGGAGTAAAGCCAGTCGAGGGAAGGTGATTACAGC



CAAGGATGGTTTTACCTACCTTTATGACAAAAATCCTCAGATAT



TAAAGTGGCTCAGAACTTAA









In one embodiment the entire gene cluster is transformed and expressed in a heterologous system. SEQ ID NO. 61 encompasses the genes of said cluster.










    1-27260
ATGACTATTAACTATGGTGATCTGCAAGAACCCTTTA



 


Microginin-
ATAAATTCTCAACCCTAGTTGAATTACTCCGTTATCG


 


Cluster
GGCAAGCAGTCAACCGGAACGCCTCGCCTATATTTTT


 


    1-1743
CTGCGAGACGGAGAAATCGAAGAAGCTCGTTTAACCT


 


Adenylation-
ATGGGGAACTGGATCAAAAGGCTAGGGCGATCGCCGC


 


Protein (A*)
TTATCTACAATCCTTAGAAGCCGAGGGCGAAAGGGGT


 


 1892-2158
TTACTGCTCTATCCCCCAGGACTAGATTTTATTTCAG


 


Acyl-Carrier-
CTTTTTTTGGTTGTTTATATGCGGGAGTCGTTGCCAT


 


Protein (ACP)
TCCCGCCTATCCACCCCGACGGAATCAAAACCTTTTG


 


 2204-3016
CGTTTACAGGCGATTATTGCCGATTCTCAAGCCCGAT


 


Methyltrans-
TTACCTTCACCAATGCCGCTCTATTTCCCAGTTTAAA


 


ferase (MT)
AAACCAATGGGCTAAAGACCCTGAATTAGGAGCAATG


 


 3464-13123
GAATGGATTGTTACCGATGAAATTGACCATCACCTCA


 


PKS/NRPS (KS-
GGGAGGATTGGCTAGAACCAACCCTCGAAAAAAACAG


 


AT-ACP-AMT-
TCTCGCTTTTCTACAATACACCTCTGGTTCAACGGGA


 


MO-C-A-T)
ACTCCAAAGGGAGTAATGGTCAGTCACCATAATTTGT


 


13120-17832
TGATTAATTCAGCCGATTTAGATCGTGGTTGGGGCCA


 


NRPS 2 (C-A-
TGATCAAGATAGCGTAATGGTCACTTGGCTACCGACC


 


Mt-T)
TTCCATGATATGGGTCTGATTTATGGGGTTATTCAGC


 


17836-25194
CTTTGTACAAAGGATTTCTTTGTTACATGATGTCCCC


 


NRPS 3 (C-A-
TGCCAGCTTTATGGAACGACCGTTACGTTGGTTACAG


 


T-C-A-T)
GCCCTTTCTGATAAAAAAGCAACCCATAGTGCGGCCC


 


25257-27260
CCAACTTTGCCTACGATCTTTGTGTGCGGAAAATTCC


 


ABC-
CCCTGAAAAACGGGCTACGTTAGACTTAAGCCATTGG


 


Transporter
TGCATGGCCTTAAATGGGGCCGAACCCGTCAGAGCGG


 


(ABC)
AGGTACTTAAAAAGTTTGCGGAGGCTTTTCAAGTTTC


 



TGGTTTCAAAGCCACAGCCCTTTGTCCTGGCTACGGT


 



TTAGCAGAAGCCACCCTGAAAGTTACGGCGGTTAGTT


 



ATGACAGTCCCCCTTACTTTTATCCCGTTCAGGCTAA


 



TGCTTTAGAAAAAAATAAGATTGTGGGAGCCACTGAA


 



ACCGATACCAATGTGCAGACCCTCGTGGGCTGCGGCT


 



GGACAACGATTGATACTCAAATCGTCATTGTCAATCC


 



TGAAACCCTGAAACCTTGCTCCCCTGAAATTGTCGGC


 



GAAATTTGGGTATCAGGTTCAACAATCGCCCAAGGCT


 



ATTGGGGAAAACCTCAAGAGACTCAGGAAACCTTTCA


 



AGCTTATTTGGCAGATACAGGAGCCGGGCCTTTTCTG


 



CGAACAGGAGACTTGGGCTTCATTAAAGATGGTGAAT


 



TGTTTATCACAGGTCGGCTCAAGGAAATTATTCTGAT


 



TCGAGGACGCAATAATTATCCCCAGGATATTGAATTA


 



ACCGTCCAAAATAGTCATCCCGCTCTGCGTCCCAGTT


 



GTGGGGCTGCTTTTACCGTTGAAAATAAGGGCGAAGA


 



AAAGCTCGTGGTCGTTCAGGAAGTGGAGCGCACCTGG


 



CTCCGTAAGGTAGATATAGATGAGGTAAAAAGAGCCA


 



TTCGTAAAGCTGTTGTCCAGGAATATGATTTACAGGT


 



TTATGCGATCGCGCTGATCAGGACTGGCAGTTTACCA


 



AAAACCTCTAGCGGTAAAATTCAGCGTCGTAGCTGTC


 



GGGCCAAATTTTTAGAGGGAAGCCTGGAAATTTTGGG


 



CTAAGAAAATTTCTCGATCGGCACTTAATGTGTTAAA


 



TTCGTATGTCGATTGAAACTTCGACCAATTCTTTCTC


 



TCCCCTTAAGTCCATGTCTCTGGATTTGAAAATTCCT


 



TAAACTTTAACTACATTTCTCAAGAAAGCAAATTGAA


 



TCTAATGTCCACAGAAATCCCAAACGACAAAAAACAA


 



CCGACCCTAACGAAAATTCAAAACTGGTTAGTGGCTT


 



ACATGACAGAGATGATGGAAGTGGACGAAGATGAGAT


 



TGATCTGAGCGTTCCCTTTGATGAATATGGTCTCGAT


 



TCTTCTATGGCAGTTGCTTTGATCGCTGATCTAGAGG


 



ATTGGTTACGACGAGATTTACATCGCACCCTGATCTA


 



CGATTATCCAACTCTAGAAAAGTTGGCTAAACAGGTT


 



AGTGAACCCTGACATTTTTATAAAGTTTGTGCTTAAA


 



AATTTTGAGGAAGTTCTAAAATGACAAATTATGGCAA


 



ATCTATGTCTCATTACTATGATCTAGTGGTAGGACAT


 



AAAGGTTATAACAAAGATTACGCCACTGAAGTAGAAT


 



TCATTCACAATTTAGTTGAGACTTACACAACTGAAGC


 



CAAATCTATCCTATACTTGGGCTGTGGTACGGGTTAT


 



CATGCCGCTCTTTTAGCACAGAAAGGGTATTCTGTAC


 



ATGGTGTTGATCTCAGTGCTGAAATGTTAGAGCAGGC


 



TAAAACTCGCATTGAAGATGAAACAATAGCTTCTAAT


 



CTGAGTTTTTCTCAAGGAAATATTTGTGAAATCCGTT


 



TAAATCGTCAGTTTAATGTTGTTCTTGCTCTATTTCA


 



TGTGGTTAACTATCAAACGACCAATCAAAATTTACTG


 



GCAACGTTTGCAACGGTTAAAAACCATTTAAAAGCTG


 



GGGGGATTTTTATTTGTGATGTGTCCTATGGGTCTTA


 



CGTACTGGGGGAATTTAAGAGTCGGCCTACGGCATCA


 



ATATTGCGTTTAGAGGATAATTCCAATGGTAACGAAG


 



TAACCTATATTAGTGAACTAAATTTTTTAACCCATGA


 



AAATATAGTGGAAGTTACTCACAATTTATGGGTAACA


 



AATCAAGAAAATCAACTTCTAGAGAATTCACGGGAAA


 



CACATCTTCAGCGCTATCTTTTCAAGCCTGAAGTTGA


 



ATTGTTGGCTGATGCTTGTGAACTAACTGTTCTTGAT


 



GCGATGCCCTGGCTTGAACAACGTCCTTTGACAAACA


 



TTCCTTGTCCTTCAGTTTGTTTTGTTATTGGGCATAA


 



AACAACCCATTCAGCTTAAATTCTGCTAAAAAAAATC


 



CAACTTACCTTATTCTCTGAAACCACACAAGCCATGA


 



ATACAATTCAAGATGCCAAGACCGAAAATTACTCAAT


 



CTTAAATCAGTCAATTCCAAGACCTCTCAAACTGAGT


 



AATATCCTATTACGATAAGATTTTGCGTTCTCCTTTG


 



TTTGGAATGTCAGCAGAGGAGTCTCTATATTGGCTAG


 



AGAAATGTTTATGTCAAGAGCATCAGGGCTTCGATGT


 



ACAAGTTAAGTATCATCAAAAAATGCTGAAGAATATG


 



TTACGTTTGACCGATAGTTTGGATTATCTATGGCCAG


 



TTAACCGTGAAATGCGGCTCATGAAAGCTGGGGGGTC


 



AATTGAACGGGCGATCACCAATAACATTAAAGCTTTT


 



CTTCAATTTAAAGAAACTGTAACCGTATTAAATTAGA


 



AAAACCGCAGTGAGGAATTTGAATGGAACCCATCGCA


 



ATTATTGGTCTTGCTTGCCGCTTTCCAGGGGCTGACA


 



ATCCAGAAGCTTTCTGGCAACTCATGCGAAATGGGGT


 



GGATGCGATCGCCGATATTCCTCCTGAACGTTGGGAT


 



ATTGAGCGTTTCTACGATCCCACACCTGCCACTGCCA


 



AGAAGATGTATAGTCGCCAGGGCGGTTTTCTAAAAAA


 



TGTCGATCAATTTGACCCTCAATTTTTCCGAATTTCT


 



CCCCTAGAAGCCACCTATCTAGATCCTCAACAAAGAC


 



TGCTACTGGAAGTCACCTGGGAAGCCTTAGAAAATGC


 



TGCCATTGTGCCTGAAACCTTAGCTGGTAGCCAATCA


 



GGGGTTTTTATTGGTATCAGTGATGTGGATTATCATC


 



GTTTGGCTTATCAAAGTCCTACTAACTTGACCGCCTA


 



TGTGGGTACAGGCAACAGCACCAGTATTGCGGCTAAC


 



CGTTTATCATATCTGTTTGATTTGCGTGGCCCCAGTT


 



TGGCCGTAGATACCGCTTGCTCTTCTTCCCTCGTCGC


 



CGTTCACTTGGCCTGTCAGAGTTTGCAAAGTCAAGAA


 



TCGAACCTCTGCTTAGTGGGGGGAGTTAATCTCATTT


 



TGTCGCCAGAGACAACCGTTGTTTTTTCCCAAGCGAG


 



AATGATCGCCCCCGACAGTCGTTGTAAAACCTTTGAC


 



GCGAGGGCCGATGGTTATGTGCGCTCGGAAGGCTGTG


 



GAGTAGTCGTACTTAAACGTCTTAGGGATGCCATTCA


 



GGACGGCGATCGCATTTTAGCAGTGATTGAAGGTTCC


 



GCGGTGAATCAGGATGGTTTAAGTAATGGACTCACGG


 



CCCCTAATGGCCCTGCTCAACAGGCGGTGATTCGTCA


 



GGCCCTGGCAAATGCCCAGGTAAAACCGGCCCAGATT


 



AGCTATGTCGAAGCCCATGGCACGGGGACAGAATTGG


 



GGGATCCGATCGAAGTTAAATCTCTGAAAGCGGTTTT


 



GGGTGAAAAGCGATCGCTCGATCAAACCTGTTGGCTC


 



GGTTCTGTGAAAACCAACATTGGTCATTTAGAAGCGG


 



CGGCGGGAATGGCGGGTCTGATTAAAGTCGTTCTCTG


 



CCTACAACACCAAGAAATTCCCCCTAATCTCCACTTT


 



CAAACCCTTAATCCCTATATTTCCCTAGCTGACACAG


 



CTTTTGCGATTCCCACTCAGGCTCAACCCTGGCGGAC


 



CAAACCCCCTAAGTCTGGTGAAAACGGTGTCGAACGA


 



CGTTTAGCAGGACTCAGTTCCTTTGGGTTTGGGGGGA


 



CAAATTCCCATGTGATTCTCAGCGAAGCCCCTGTCAC


 



CGTTAAAAACAATCAACAAAATGGGCAGAAGTTGATA


 



GAACGTCCCTGGCATTTGCTGACTTTATCTGCCAAGA


 



ATGAAGAAGCCTTAAAAGCCTTAGTCCATTGTTATCA


 



AAAGTATTTAGCTGATCATCATGAAATTCCTCTCGCT


 



GATGTTTGTTTTACGGCCAATAGTCGGCGATCGCACT


 



TTAATCATCGTTTAGGAGTAGTGGCTAGAGATCGCTT


 



AGAAATGTTGCAGAAGTTAGAGAACTTTAGTAACCAA


 



GAAAGGATGAGAGAACCGAAGAGTATTAACAAAAAAG


 



AAAAACCTAAAATTGTTTTTCTATTTGCCGGTCAAGG


 



TTCTCAATATGTAGGTATGGGTCGTCAACTGTACGAA


 



ACCCAACCCATCTTTCGCCAAACCTTGGATCGCTGTG


 



CTGAAATCCTGCGACCCCATTTAGATCAACCCCTCTT


 



AGAAATTCTTTATCCTGCTGACCCAGAAGCCGAAACA


 



GCGAGTTTTTACCTAGAGCAGACTGCCTATACCCAAC


 



CCACTTTATTCGCATTCGAGTATGCCCTAGCACAGTT


 



ATGGCGTTCCTGGGGAATAGAACCGGCGGCAGTAATT


 



GGTCACAGTGTCGGTGAATATGTGGCGGCCACCGTTG


 



CCGGAGCCTTAAGTCTAGAAGAAGGATTAACGCTAAT


 



TGCCAAACGGGCAAAACTGATGCAGTCTCTCCCCAAG


 



AATGGGACAATGATCGCCGTTTTTGCCGCAGAAGAGC


 



GGGTTAAAGCTGTTATTGAGCCTTATAGGACTGATGT


 



AGCGATCGCTGCTGTTAATGGACCAGAAAATTTTGTT


 



ATTTCAGGAAAAGCGCCGATTATTGCTGAGATTATCA


 



TTCATTTAACGGCAGCAGGAATAGAAGTTCGTCCTCT


 



CAAAGTTTCCCATGCTTTTCACTCGCACCTGTTGGAG


 



CCAATTTTAGATTCCTTAGAACAGGAAGCTGCTGCTA


 



TTTCCTACCAACCCCTGCAAATTCCCTTAGTTGCTAA


 



TTTAACGGGGGAAGTTCTACCAGAAGGAGCAACGATT


 



GAGGCTCGTTACTGGCGAAATCATGCACGCAACCCTG


 



TACAATTTTATGGGAGTATCCAAACGCTGATCGAGCA


 



GAAATTCAGTCTTTTTTTAGAAGTTAGCCCTAAACCG


 



ACTTTATCTCGATTGGGTCAACAATGTTGTCCAGAAA


 



GATCGACCACTTGGCTATTTTCCCTCGCCCCTCCTCA


 



AGAAGAAGAACAAAGCCTACTAAATAGTTTGGCGATT


 



CTCTATGATTCCCAAGGAGCCGAAATAAACTGGGAAG


 



GGTTTAATCAAAATTATCCCCACCATTTACTGGCTCT


 



ACCGACCTATCCTTTTCAACGTCAACGCTATTGGCTT


 



GAAACCGGTAAACCGACTTCTGAAGAAACAACCATGA


 



CGACCAATGCCACTAATGTCCAAGCTATCTCCAGCCA


 



TCAAAAACAACAGGAGATTCTAATCACATTGCAAACC


 



CTAGTGGGAAATTTACTGCAATTGTCCCCTGCTGATG


 



TCAATGTTCATACACCTTTCCTGGAGATGGGGGCAGA


 



TTCCATTGTCATGGTTGAGGCGGTCAGACGGATTGAG


 



AATACCTATAACGTTAAAATTGCTATGCGTCAGTTAT


 



TTGAGGAGTTATCTACTTTAGATGCTTTAGCTACTTA


 



TTTAGCTCAAAATCCGGCTACTGATTGCCAAACTGCT


 



CAAATTAATACCGAGGTGTTTTCTGCGCCCATTGCCT


 



GCTCAAATAACCGATCGCCCAATGTCGTGCTGAGTTC


 



TAATACCAACGGCTTTCAACGTCAAACAGCTTCTCCA


 



GGTTTTTCGGCGATCGCCCCCCTTGCAGGAATGGGAG


 



GAGCAGGGGAAATGGGAGGAGTTGAAGTGCCTCAAGT


 



TTCTGTGCCACAAACCAGTGCGGTAACAGCCTCAGGT


 



TCAACCGTTTCTAGTTCTGCCCTGGAAAACATTATGG


 



GTCAACAGTTACAACTGATGGCCAAACAGTTAGAAGT


 



CTTGCAAACGGCCAATTTTGCCCCGACGACTCCCCGA


 



ACCACAGAAAATTCCCCATCTTCCGTCAGTCAAAATA


 



GGTCAAACGGACTTACACAACAGTTAATTCCCCCCCA


 



GCAATTAGCGGCGAACCTAGAGCCAATAGCCAGTCGC


 



ACCCGTCAAACCAGCAATCAAGCTTCTGCTCCTAAAC


 



CGACAGTAACAGCCACTCCCTGGGGGCCGAAAAAACC


 



ACCCACAGGTGGATTCACTCCCCAACAACAGCAACAT


 



CTAGAGGCATTAATTGCTCGCTTTACGGAACGTACCA


 



AAACCTCTAAGCAAATTGTGCAAAGCGATCGCCTGCG


 



TTTAGCAGATAGTCGAGCCTCGGTCGGATTCCGTATG


 



TCTATTAAAGAGATGCTTTATCCCATTGTGGCCCAAC


 



GTTCTCAAGGATCAAGAATTTGGGATGTGGACGGTAA


 



TGAATATATTGATATGACGATGGGGCAAGGGGTAACG


 



CTGTTTGGGCATCAACCAGACTTCATTATGTCGGCCC


 



TACAAAGCCAACTCACTGAAGGCATTCATCTCAATCC


 



GCGATCGCCAATTGTGGGAGAAGTGGCCGCCTTAATT


 



TGTGAACTAACAGGAGCCGAACGAGCTTGTTTTTGCA


 



ACTCTGGAACCGAAGCCGTAATGGCCGCTATTCGTAT


 



CGCCAGGGCAACAACAGGTCGGAGTAAAATTGCCCTC


 



TTTGAAGGCTCCTATCATGGACATGCGGACGGAACCC


 



TTTTTAGGAACCAAATTATTGATAACCAACTCCACTC


 



TTTTCCCCTAGCTCTAGGCGTTCCCCCCAGCCTTAGT


 



TCCGATGTGGTGGTATTGGACTATGGCAGTGCGGAAG


 



CTCTGAACTATTTACAAACCCAGGGGCAGGATTTAGC


 



GGCGGTCTTAGTAGAACCAATTCAAAGTGGCAATCCT


 



CTACTCCAACCCCAACAATTTCTCCAAAGTCTGCGAC


 



AAATTACCAGTCAAATGGGCATTGCCCTGATTTTTGA


 



TGAAATGATTACGGGTTTTCGATCGCACCCAGGGGGA


 



GCGCAAGCTTTATTTGGAGTACAGGCGGATATTGCCA


 



CCTATGGCAAAGTAGTTGCGGGAGGAATGCCCATTGG


 



AGTTATTGCAGGTAAGGCCCATTATCTGGACAGCATT


 



GACGGGGGAATGTGGCGTTATGGCGATAAATCCTATC


 



CTGGGGTGGACAGAACCTTTTTTGGGGGAACCTTTAA


 



TCAGCATCCGTTAGCAATGGTAGCGGCTAGGGCTGTC


 



CTGACCCATTTAAAGGAGCAGGGGCCAGGTCTGCAAC


 



AACAATTAACTGAACGCACTGCGGCCTTAGCCGATAC


 



ACTGAATCATTATTTTCAAGCCGAAGAAGTTCCTATT


 



AAAATCGAACAGTTTAGTTCTTTCTTCCGGTTTGCCC


 



TCTCTGGCAATTTGGATTTACTTTTCTATCACATGGT


 



AGAAAAAGGTATTTATGTCTGGGAATGGCGTAAACAT


 



TTTCTTTCAACCGCCCATACGGAAGCCGATCTTGCCC


 



AATTTGTCCAAGCGGTTAAGGATAGCATCACAGAATT


 



GCGTCAGGGAGGTTTTATCCCCGCAAAAAAGCCTTCC


 



TGGCCAGTGCCAACGCCTCAAATTGATCCCCCCCTAA


 



CCCCCCTTGATAAGGGGATTGATCCCCCCCTAACCCC


 



CCTTGATAAGGGGATTGATCCCCCCCTAACCCCCCTT


 



GATAAGGGGGGAGATGTTGATGTCGCGCTTGATAAGG


 



GAGGAAATTCTCATTCTGTTAGGGACAGTAAGTTAGG


 



GAAAGGGAGCGGGTCTCAAGACCAAAAAACGATACAG


 



TTTAGCCTCTACTACTTTGGTAGCTATGAAGCGGAAT


 



TTAACCCGAATAAATATAACTTACTGTTTGAAGGAGC


 



TAAATTTGGCGATCGCGCTGGTTTTACGGCCCTTTGG


 



ATTCCTGAACGTCATTTCCACGCTTTTGGTGGTTTTT


 



CTCCCAATCCTTCGGTTTTGGCGGCGGCTTTAGCACG


 



GGAAACCAAACAGATTCAACTGCGATCAGGCAGTGTG


 



GTTTTACCGCTACATAATTCCATCCGAGTCGCCGAAG


 



AATGGGCAGTGGTGGACAATCTTTCCCAGGGCCGCGT


 



TGGTATTGCTTTTGCATCGGGTTGGCATCCCCAGGAT


 



TTTGTCTTGGCTCCCCAGTCCTTTGGCCAACATCGGG


 



AATTGATGTTCCAAGAAATTGAAACCGTCCAGAAACT


 



TTGGCGAGGGGAAGCGATCACCGTGCCAGACGGAAAG


 



GGTCAAAGGGTAGAGGTTAAAACCTATCCCCAACCGA


 



TGCAGTCCCAGTTACCCAGCTGGATTACTATTGTCAA


 



TAATCCCGATACCTATATCAGAGCAGGGGCGATCGGT


 



GCTAATATCCTTACCAATCTGATGGGGCAAAGCGTGG


 



AAGATTTAGCCCGTAATATTGCGCTATATCGTCAATC


 



TTTGGCAGAGCATGGTTATGATCCCGCGTCGGGAACG


 



GTGACAGTTCTCCTGCATACTTTTGTTGGCAAGGATT


 



TAGAACAAGTTCGAGAACAGGCTCGCCAACCCTTTGG


 



GCAATACCTCACCTCCTCTGTCGGACTCTTGCAGAAC


 



ATGGTCAAGAGCCAGGGCATGAAAGTGGATTTTGAAC


 



AATTAAGAGACGAAGATCGGGACTTTCTCCTCGCTTC


 



TGCCTATAAACGCTATACAGAAACCAGTGCTTTAATT


 



GGCACACCCGAATCCTGTCGTCAAATTATTGATCATT


 



TGCAGTCCATCGGTGTGGATGAAGTGGCTTGTTTTAT


 



TGATTTTGGGGTAGATGAACAAACAGTTTTGGCCAAT


 



TTACCCTATCTCCAGTCCCTAAAAGACTTATATCAAC


 



CTCATCTCCCCCCTTATCAAGGGGGGTTAGGGGGGGA


 



TCAATCCCCTTATCAAGGGGGGTTAGGGGGGGATCAA


 



TCCCCTTATCAAGGGGGGTTAGGGGGTGATCAATCCC


 



CTTATCAAGGGGGGTTAGGGGGTGATCAATCCCCTTA


 



TCAAGGGGGGTTAGGGGGGGATCAATCCCCTTATCAA


 



GGAGAGTTAGGGGGGGATCAATCCCCTTATCAAGGGG


 



GGTTAGGGGGGGATCAAGTCCCTCTCACCGAAGCCCA


 



ACGACAACTGTGGATTTTGGCTCAATTAGGAGACAAC


 



GGCTCTGTGGCCTATAACCAATCAGTGACATTGCAAT


 



TAAGTGGCCCATTAAATCCCGTCGCAATGAATCAAGC


 



TATTCAACAAATCAGCGATCGCCATGAAGCGTTACGA


 



ACCAAAATTAATGCCCAGGGAGATAGTCAAGAAATCC


 



TGCCCCAGGTCGAAATTAACTGCCCTATCTTAGACTT


 



CAGTCTTGACCAAGCTTCGGCCCAACAGCAAGCAGAA


 



CAATGGTTAAAGGAAGAAAGTGAAAAACCCTTTGATT


 



TGAGCCAGGGTTCTCTCGTGCGTTGGCATCTACTCAA


 



ATTAGAACCAGAATTACATTTGTTAGTATTAACGGCC


 



CATCACATTATCAGTGACGGTTGGTCAATGGGGGTAA


 



TCCTTCGGGAATTAGGAGAGTTATATTCAGCCAAATG


 



TCAGGGTGTTACGGCTAATCTTAAAACCCCAAAACAG


 



TTTCGAGAATTGATTGAATGGCAAAGCCAGCCAAGCC


 



AAGGGGAAGAACTGAAAAAACAGCAAGCCTATTGGTT


 



AGCAACCCTTGCCGATCCCCCTGTTTTGAATTTACCC


 



ACTGACAAACCTCGTCCAGCTTTACCCAGTTACCAAG


 



CTAATCGTCGAAGTCTAACTTTAGATAGCCAATTTAC


 



AGAAAAACTAAAGCAATTTAGTCGTAAACAGGGCTGT


 



ACCTTGCTGATGACCCTGTTATCGGTTTATAACATTC


 



TCGTTCATCGTTTGACGGGACAGGATGATATTCTGGT


 



GGGTCTGCCAGCCTCTGGACGGGGGCTTTTAGATAGT


 



GAAGGTATGGTGGGTTATTGCACCCATTTTTTACCAA


 



TTCGCAGTCAATTAGCAGGTAATCCCACTTTTGCTGA


 



ATATCTCAAACAAATGCGGGGGGTTTTGTTGTCGGCT


 



TATGAACATCAGGACTATCCCTTTGCTCTTTTGCTCA


 



ATCAGTTAGATTTACCGCGTAATACCAGTCGCTCTCC


 



TTTAATTGATGTCAGTTTCAATTTAGAACCAGTTATT


 



AACCTACCCAAAATGAAAGGATTAGAGATTAGTTTGT


 



TGCCTCAAAGTGTAAGTTTTAAGGATCGAGATTTGCA


 



TTGGAATGTGACAGAAATGGGTGGAGAAGCTCTGATT


 



GATTGTGACTACAATACAGACTTATTTAAAGATGAAA


 



CGATTCAGCGTTGGTTAGGCCATTTTCAAACCTTACT


 



TGAGGCAGTTATTAATGATTCGCAACAAAATCTGCGG


 



GAATTACCCTTATTAAGTTCTGCTGAACGACAACAGT


 



TATTAGTGGATTGGAATCAAACCAAGACCGACTATCC


 



CCAAGATCAGTGTATTCATCAATTATTTGAAGCGCAA


 



GTTGAACGGACTCCCGATGCGATTGCGGTGGTATTTG


 



AAACTCAACAATTAACTTACAGTGAATTAAATTGTCG


 



AGCCAATCAGTTAGCACATTATTTACAAAAATTAGGA


 



GTTGGGCCAGAGGTCTTAGTCGGTATTTTGGTCGAAC


 



GTTCTTTAGAAATGATTGTCGGATTGTTAGGGATTCT


 



CAAGGCTGGGGGAGCCTATGTACCTCTTGATCCTGAC


 



TATCCCCCTGAACGTCTTCAATTTATGTTAGAAGATA


 



GTCAATTTTTTCTCCTCTTAACCCAACAGCATTTACT


 



GGAATCTTTTGCTCAGTCTTCAGAAACGGCTACTCCC


 



AAGATTATTTGTTTGGATAGCGACTACCAAATTATTT


 



CCCAGGCAAAGAATATTAATCCCGAAAATTCAGTCAC


 



AACGAGTAATCTTGCCTATGTAATTTATACCTCTGGT


 



TCGACAGGTAAACCGAAGGGCGTGATGAATAATCATG


 



TTGCTATTAGTAATAAATTGTTATGGGTACAAGACAC


 



TTATCCTCTAACCACAGAAGACTGTATTTTACAAAAA


 



ACTCCCTTTAGTTTTGATGTTTCAGTGTGGGAATTAT


 



TCTGGCCCCTACTAAACGGAGCGCGTTTGGTTTTTGC


 



CAAGCCGAATGGCCATAAAGATGCCAGTTACTTAGTC


 



AATCTGATTCAAGAGCAACAAGTAACAACGCTACATT


 



TTGTGTCTTCTATGCTACAGCTTTTTCTGACAGAAAA


 



AGACGTAGAAAAATGTAATAGTCTTAAACGAGTCATT


 



TGTAGTGGTGAAGCCCTTTCTTTAGAGCTTCAAGAAC


 



GTTTTTTTGCTCGTTTAGTCTGTGAATTACACAATCT


 



TTATGGACCGACAGAAGCCGCTATTCATGTCACATTT


 



TGGCAATGTCAATCAGATAGCAATTTGAAAACAGTAC


 



CCATTGGTCGGCCGATCGCTAATATCCAAATTTACAT


 



TTTAGACTCTCATCTTCAGCCAGTACCTATTGGAGTA


 



ATCGGAGAATTGCACATTGGTGGGGTTGGTTTGGCGC


 



GGGGTTATTTAAACAGGCCTGAGTTAACGGCGGAGAA


 



ATTTATTGCAAATCCGTTTGCTTCCCTTGATCCCCCC


 



CTAACCCCCCTTGATAAGGGGGGAGATGAGAGCTATA


 



AAACTTTTAAAAAGGGGGGAGAGCAACCATCAAGATT


 



GTATAAAACGGGAGATTTAGCTCGTTATTTACCCGAT


 



GGCAAGATTGAGTATCTAGGGCGCATTGATAATCAGG


 



TAAAAATTCGCGGTTTCCGGATTGAATTGGGGGAAAT


 



TGAAGCGGTTTTGCTATCCCATCCCCAGGTACGAGAA


 



GCGGTCGTTTTGGTGAGCGAAAGCGATCGCTCTGAAA


 



ATCGGGCTTTGGTCGCTTATATTGTCCCTAATGATCC


 



TGCTTGTACGACTCAATCATTACGAGAGTTTGTTAAA


 



CGGCAGCTTCCTGACTATATGATCCCAGCTTATTGGC


 



TGATCCTTGACAATTTACCGTTAACCAGCAATGGCAA


 



AATTGATCGTCGGGCTTTACCGTTACCTAATCCAGAG


 



TTAAATCGTTCGATAGACTATGTGGCTCCCAAAAATC


 



CTACCCAGGAGGCGATCGCCGCTATTTTTGGTCAAGT


 



TTTAAAACTGGAAAAAGTGGGAATTTATGATAACTTT


 



TTTGAGATCGGCGGTAATTCTTTGCAAGCCACTCAAG


 



TTATTTCACGCTTACGAGAAAGTTTTGCCCTAGAGTT


 



GCCCTTGCGTCGCCTGTTTGAACAACCGACTGTGGCG


 



GATTTGGCTTTAGCCGTAACGGACATTCATGCCACTT


 



TACAAAAATTACAAACCCCTATTGATGATTTATCAGG


 



CGATCGCGAGGAGATTGAACTATGAAATCTATTGAAA


 



CCTTTTTGTCAGATTTAGCCAATCAAGATATTAAACT


 



CTGGATGGACGGCGATCGCCTGCGTTGTAATGCACCC


 



CAGGGCCTATTAACCCCAGAGATTCAAACAGAACTGA


 



AAAACCGTAAAGCAGAAATCATTCACTTTCTCAATCA


 



ACTGGGTTCAGAGGAGCAAATTAATCCTAGAACGATT


 



CTTCCCATTCCTCGTGATGGCCAATTACCCCTCTCCT


 



TTGCCCAGTCGCGACTCTGGTTCTTGTATCAATTAGA


 



AGGAGCCACGGGAACCTATAACATGACAGGGGCCTTG


 



AGTTTAAGCGGGCCTCTTCAGGTCGAAGCCCTCAAAC


 



AAGCCCTAAGAACTATCATTCAACGCCATGAGCCATT


 



GCGTACCAGTTTCCAATCGGTTGACGGGGTTCCAGTG


 



CAGGTGATTAATCCCTATCCTGTTTGGGAATTAGCGA


 



TGGTTGATTTGACAGGAAGGAGACAGAAGCAGAAAAA


 



ATTGGCCTATCAGGAATCCCAAACCCCGTTTGATTTG


 



ACCAATAGTCCTTTGTTGAGGGTAACGCTCCTCAAAT


 



TACAGCCAGAAAAGCATATTTTATTAATTAATATGCA


 



CCATATTATTTCCGATGGCTGGTCAATCGGTGTTTTT


 



GTTCGTGAATTGTCCCATCTCTATAGGGCTTTTGTGG


 



CGGGTAAAGAACCAACTTTACCGATTTTACCAATTCA


 



GTATGCGGATTTTGCCGTTTGGCAGCGAGAGTGGTTA


 



CAGGGTAAGGTTTTAGCGGCTCAATTGGAATATTGGA


 



AGCGACAATTGGCAGATGCTCCTCCTCTGCTGGAACT


 



GCCCACTGATCGCCCTCGTCCCGCAATCCAAACCTTT


 



CAAGGCAAGACAGAAAGATTTGAGCTAGATAGGAAAC


 



TGACCCAAGAATTAAAGGCATTAAGTCAACAGTCGGG


 



TTGTACTTTATTTATGACTTTGTTGGCCGCTTTTGGG


 



GTGGTTTTATCCCGTTATAGTGGCCAGACTGATATCG


 



TCATTGGTTCGGCGATCGCCAACCGTAATCGCCAAGA


 



CATTGAGGGGTTAATTGGCTTTTTTGTTAACACTTTG


 



GCGTTGAGGTTAGATTTATCAGAAAAACCCAGCTTTG


 



CCGCTTTTTTAAAACAAGTACAGGAAGTCACTCAGGA


 



TGCCTATGAGCATCAAGACTTGCCCTTTGAAATGTTA


 



GTGGAAGAATTACAACTAGAGCGCAAATTAGACCGAA


 



ATCCTTTGGTACAGGTGATGTTTGCCCTACAAAATGC


 



GGCCAATGAAACCTGGAATTTACCTGGGTTGACCATT


 



GAAGAAATGTCTTGGGAACTTGAACCTGCCCGTTTTG


 



ACCTAGAGGTTCATTTATCAGAAGTTAACGCCGGCAT


 



AGCTGGATTCTGTTGCTACACCATTGATCTATTTGAT


 



GATGCAACGATCGCCCGTCTATTGGAACATTTTCAGA


 



ATCTTCTCAGGGCAATTATTGTTAATCCTCAAGAATC


 



GGTAAGTTTATTACCCTTGTTGTCAGAACAGGAAGAA


 



AAGCAACTTTTAGTTGATTGGAATCAAACCCAAGCCG


 



ATTATCCCCAAGATAAGCTTGTCCATCAGTTATTTGA


 



AGTTCAAGCAGCCAGTCAGCCAGAAGCGATCGCTCTA


 



ATCTTTGAAAATCAGGTTTTGACCTATGGAGAATTAA


 



ACCATCGCGCCAATCAATTAGCTCACTATCTTCAGTC


 



GTTAGGAGTCACCAAAGAACAAATCGTCGGGGTTTAT


 



CTGGAACGTTCCCTTGAAATGGCGATCGGATTTTTAG


 



GTATTCTCAAAGCAGGAGCCGCCTATCTCCCCATTGA


 



TCCTGAATATCCCTCAGTACGCACCCAATTTATTCTC


 



GAAGATACCCAACTTTCGCTTCTCTTAACTCAGGCAG


 



AACTGGCAGAAAAACTGCCCCAGACTCAAAACAAAAT


 



TATCTGTCTAGATCGGGACTGGCCAGAAATTACCTCC


 



CAACCCCAGACAAACCTAGACCTAAAGATAGAACCTA


 



ATAACCTAGCCTATTGCATCTATACTTCTGGTTCCAC


 



AGGACAACCCAAAGGAGTACTGATTTCCCATCAAGCC


 



CTACTCAACTTAATTTTCTGGCATCAACAAGCGTTTG


 



AGATTGGCCCCTTACATAAAGCGACCCAAGTGGCAGG


 



CATTGCTTTCGATGCAACGGTTTGGGAATTGTGGCCC


 



TATCTGACCACAGGAGCCTGTATTAATCTGGTTCCCC


 



AAAATATTCTGCTCTCACCGACGGATTTACGGGATTG


 



GTTGCTTAACCGAGAAATTACCATGAGTTTTGTGCCA


 



ACTCCTTTAGCTGAAAAATTATTATCCTTGGATTGGC


 



CTAACCATTCTTGTCTAAAAACCCTGTTACTGGGAGG


 



TGACAAACTTCATTTTTATCCTGCTGCGTCCCTTCCC


 



TTTCAGGTCATTAACAACTATGGCCCAACGGAAAATA


 



CAGTGGTTGCGACCTCTGGACTGGTCAAATCATCTTC


 



ATCTCATCACTTTGGAACTCCGACTATTGGTCGTCCC


 



ATTGCCAACGTCCAAATCTATTTATTAGACCAAAACC


 



TACAACCTGTCCCCATTGGTGTACCAGGAGAATTACA


 



TTTAGGTGGGGCGGGTTTAGCGCAGGGCTATCTCAAT


 



CGTCCTGAGTTAACGGCTGAAAAATTTATTGCCAATC


 



CCTTTGATCCCCCCCTAACCCCCCTTGATAAGGGGGG


 



AGAAGAACCCTCAAAACTCTATAAAACGGGAGACTTA


 



GCCCGTTATTTACCCGATGGCAATGTAGAATTTTTGG


 



GACGTATTGACAATCAGGTAAAAATTCGGGGTTTTCG


 



CATCGAAACTGGGGAAATCGAAGCCGTTTTAAGTCAA


 



TATTTCCTATTAGCTGAAAGTGTAGTCGTTGCCAAGG


 



AAGATAATACTGGGGATAAACGCCTCGTGGCTTATTT


 



GGTTCCCGCCTTGCAAAATGAGGCCCTACCAGAGCAA


 



TTAGCCCAATGGCAAAGTGAATACATCAGTGATTGGC


 



AAAGTCTCTATGAAAGAACCTATAGTCAAGGGCAAGA


 



CAGCCTAGCTGATCTCACTTTTAATATCACGGGTTGG


 



AATAGCAGTTATACTCGTCAACCCCTTCCTGCTTCAG


 



AAATGCGAGAGTGGGTCGAAAACACTGTTAGTCGCAT


 



CTTGGCTTTCCAACCAGAACGCGGTTTAGAAATTGGT


 



TGTGGTACAGGTTTGTTACTCTCCAGGGTAGCAAAGC


 



ATTGTCTTGAATATTGGGCAACGGATTATTCCCAAGG


 



GGCGATCCAGTATGTTGAACGGGTTTGCAATGCCGTT


 



GAAGGTTTAGAACAGGTTAAATTACGCTGTCAAATGG


 



CAGATAATTTTGAAGGTATTGCCCTACATCAATTTGA


 



TACCGTCGTCTTAAATTCGATTATTCAGTATTTTCCC


 



AGTGTGGATTATCTGTTACAGGTGCTTGAAGGGGCGA


 



TCAACGTCATTGGCGAGCGAGGTCAGATTTTTGTCGG


 



GGATGTGCGGAGTTTACCCCTATTAGAGCCATATCAT


 



GCGGCTGTGCAATTAGCCCAAGCTTCTGACTCGAAAA


 



CTGTTGAACAATGGCAACAACAGGTGCGTCAAAGTGT


 



AGCAGGTGAAGAAGAACTGGTCATTGATCCCACATTG


 



TTCCTGGCTTTAAAACAACATTTTCCGCAAATTAGCT


 



GGGTAGAAATTCAACCGAAACGGGGTGTGGCTCACAA


 



TGAGTTAACTCAATTTCGCTATGATGTCACTCTCCAT


 



TTAGAGACTATCAATAATCAAGCATTATTGAGCGGCA


 



ATCCAACGGTAATTACCTGGTTAAATTGGCAACTTGA


 



CCAACTGTCTTTAACACAAATTAAAGATAAATTATTA


 



ACAGACAAACCTGAATTGTGGGGAATTCGTGGTATTC


 



CTAATCAGCGAGTTGAAGAGGCTCTAAAAATTTGGGA


 



ATGGGTGGAAAATGCCCCTGATGTTGAAACGGTTGAA


 



CAACTCAAAAAACTTCTCAAACAACAAGTAGATACTG


 



GTATTAATCCTGAACAGGTTTGGCAATTAGCTGAGTC


 



TCTCGGTTACACCGCTCACCTTAGTTGGTGGGAAAGT


 



AGTCAAGACGGTTCCTTTGATGTCATTTTTCAGCGGA


 



ATTCAGAAGCGGAGGACTCAAAAAAATTAACCCTTTC


 



AAAACTTGCTTTCTGGGATGAAAAACCCTTTAAAATA


 



AAGCCCTGGAGTGACTATACTAACAACCCTCTGCGCG


 



GTAAGTTAGTCCAAAAATTAATTCCTAAAGTACGAGA


 



ATTTCTGCAAGAAAAACTACCCAGTTATATGGTTCCC


 



CAGGCGTTTGTGCTGCTTGATTCCCTTCCTTTGACCC


 



CCAATGGTAAGGTGGATCGTAAGGCGTTACCTTCTCC


 



TGATGCGGCGACTCGTGATTTAGCGAACAGTTTTGTC


 



TTACCCCGCAATCCGATTGAAGCTCAACTGACTCAAA


 



TTTGGAGTGAAGTTTTGGGACTGGAACGCATTGGCGT


 



TAAGGACAACTTTTTTGAATTGGGAGGACATTCTCTT


 



TTGGCTACCCAGGTTTTATCAAGAATTAATTCAGCCT


 



TTGGACTTGATCTTTCTGTGCAAATTATGTTTGAATC


 



ACCAACGATCGCGGGCATTGCGGGTTATATTCAAGCG


 



GTAGATTGGGTCGCCCAGGATCAAGCCGATAGCTCGT


 



TAAATCATGAAAATACTGAGGTAGTGGAGTTCTAAGT


 



TATGACGAAAAAGATTGTTGAATTTGTCTGTTATCTA


 



CGGGATTTAGGCATTACTTTAGAAGCTGATGAAAACC


 



GCTTACGCTGTCAGGCTCCCGAAGGAATTTTGACCCC


 



AGCACTCCGTCAAGAAATTGGCGATCACAAACTGGAA


 



TTATTACAATTTTTACAATGGGTCAAACAGTCTAAAA


 



GTACCGCTCATTTGCCTATTAAACCTGTCGCTAGAGA


 



CGGTCATTTACCCCTGTCTTTTGCTCAACAACGTTTA


 



TGGTTTTTACATTATCTTTCCCCTGATAGTCGTTCCT


 



ACAATACCCTGGAAATATTGCAAATTGATGGGAATCT


 



CAATCTGACTGTGCTAGAGCAGAGTTTGGGGGAATTA


 



ATTAACCGCCATGAAATTTTTAGAACAACATTCCCCA


 



CTGTTTCAGGGGAACCGATTCAGAAAATTGCACTTCC


 



TAGTCGTTTTCAGTTAAAAGTTGATAATTATCAAGAT


 



TTAGACGAAAATGAACAATCAGCTAAAATTCAACAAG


 



TAGCAGAATTGGAAGCAGGACAAGCTTTTGATTTAAC


 



GGTGGGGCCACTGATTCAGTTTAAGCTATTGCAATTG


 



AGTCCCCAGAAGTCGGTGCTGCTGTTGAAAATGCACC


 



ATATTATCTATGATGGCTGGTCTTTTGGGATTCTGAT


 



TCGGGAATTATCGGCTCTATACGAAGCATTTTTAAAG


 



AACTTAGCCAATCCTCTCCCTGCGTTGTCTATTCAGT


 



ATGCAGATTTTGCGGTTTGGCAACGTCAATATCTCTC


 



AGGTGAGGTCTTAGATAAACAACTCAATTATTGGCAA


 



GAACAGTTAGCAACAGTCTCTCCTGTTCTTACTTTAC


 



CAACGGATAGACCCCGTCCGGCGATACAAACTTTTCA


 



GGGAGGAGTTGAGCGTTTTCAACTGGATCAAAATGTC


 



ACTCAAGGTCTTAAAAAGTTAGGTCAAGATCAGGTTG


 



CAACCCTGTTTATGACGTTGTTGGCCGGTTTCGGCGT


 



TTTGCTATCTCGTTATAGTGGTCAATCTGATCTGATG


 



GTGGGTTCTCCGATCGCTAATCGTAATCAAGCAGCGA


 



TCGAACCTTTAATTGGCTTTTTTGCTAACACTTTGGC


 



TTTAAGAATTAATTTATCAGAAAATCCCAGTTTTTTA


 



GAATTATTAGAACAAGTTAAACAGACAACTTTAGAGG


 



GTTATGCTCACCAAGACCTACCCTTTGAGATGTTAGT


 



AGAAAAGCTACAACTTGACCGTGATTTGAGCAGAAAT


 



CCTTTAGTACAAGTCATGTTTGCGCTACAAAATACCT


 



CTCAAGATACTTGGAATCTTTCGGGTTTAAGTATTGA


 



AAGTTTATCTTTATCAGTGGAAGAAACTGTCAGATTT


 



GATCTAGAAGTAAACTGCTGGCAAAATTCAGAAGGTT


 



TAGCAATAGATTGGATTTACAGCAGAGATTTATTTGA


 



CACTGCAACAATTGCAAGAATGGGAGAACATTTTCAA


 



AATTTAGTTCAGGCAATCATACTCAATCCAAAAGCTA


 



CAGTTAAAGAACTTCCTTTATTAACACCCAAGGAACG


 



TGAGCAATTATTAATATCTTGGAATAATAGCAAGACT


 



GATTATCCTCAAGAGCAGTGTATTTATCAATTATTTG


 



AAGCACAAGTTGAACGGACTCCAAAGGCGATCGCAGT


 



GGTATTTGAGGAGCAATCATTAACATACACTGAATTA


 



AACCATCGCGCTAATCAGTTAGCCCATTATTTACAAA


 



CTTTAGGCGTGGGAGCAGAAGTCTTAGTCGGTATTTC


 



CCTAGAACGTTCTTTAGAGATGATTATCGGCTTATTA


 



GGGATTCTCAAGGTAGGTGGTGCTTATCTTCCTCTTG


 



ATCCAGACTATCCCACTGAGCGTCTTCAGTTGATGTT


 



AGAAGACAGTCAAGTTCCTTTTTTGATTACCCACAGT


 



TCTTTATTAGCAAAATTGCCTCCCTCTCAAGCAACTC


 



TGATTTGTTTAGATCATATCCAAGAGCAGATTTCTCA


 



ATATTCTCCAGATAATCTTCAATGTCAGTTAACTCCT


 



GCCAATTTAGCTAACGTTATTTATACCTCTGGCTCTA


 



CGGGTAAGCCTAAAGGGGTGATGGTTGAACATAAAGG


 



TTTAGTTAACTTAGCTCTTGCTCAAATTCAATCTTTT


 



GCAGTCAACCATAACAGTCGTGTGCTGCAATTTGCTT


 



CTTTTAGTTTTGATGCTTGTATTTCAGAAATTTTGAT


 



GACCTTTGGTTCTGGAGCGACGCTTTATCTTGCACAA


 



AAAGATGCTTTATTGCCAGGTCAGCCATTAATTGAAC


 



GGTTAGTAAAGAATGGAATTACTCATGTGACTTTGCC


 



GCCTTCAGCTTTAGTGGTTTTACCCCAGGAACCGTTA


 



CGCAACTTAGAAACCTTAATTGTGGCGGGTGAGGCTT


 



GTTCTCTTGATTTAGTGAAACAATGGTCAATCGATAG


 



AAACTTTTTCAATGCCTATGGGCCAACGGAAGCGAGT


 



GTTTGTGCCACTATTGGACAATGTTATCAAGATGATT


 



TAAAGGTGACGATTGGTAAGGCGATCGCCAATGTCCA


 



AATTTATATTTTAGATGCCTTTTTACAGCCGGTGCCG


 



GTGGGAGTGTCAGGAGAGTTATACATTGGTGGAGTTG


 



GGGTGGCAAGGGGCTATTTAAATCGTCCTGAATTAAC


 



CCAAGAAAAATTTATTGCTAATCCTTTTAGTAACGAC


 



CCAGATTCTCGGCTCTATAAAACTGGCGACTTAGCGC


 



GTTATTTACCCGATGGTAATATTGAATATTTAGGACG


 



CATTGACAATCAGGTAAAAATTCGCGGTTTTCGCATT


 



GAGTTAGGAGAAATTGAAGCGGTTCTGAGTCAATGTC


 



CCGATGTGCAAAATACGGCGGTGATTGTCCGCGAAGA


 



TACTCCTGGCGATAAGCGCTTAGTTGCCTATGTGGTT


 



CTTACTTCTGACTCCCAGATAACTACTAGCGAACTGC


 



GTCAATTTTTGGCGAATCAATTACCCGCCTATCTTGT


 



TCCTAATACCTTTGTTATTTTAGATGATTTGCCCCTA


 



ACCCCCAGTGGCAAATGCGATCGCCGTTCCTTACCTA


 



TACCCGAAACACAAGCGTTATCAAATGACTATATTGC


 



CCCTAAATCTCCCACTGAAGAAATTCTGGCTCAAATA


 



TGGGGGCAAGTTCTCAAGATAGAAAGAGTCAGCAGAG


 



AAGATAATTTCTTTGAATTGGGGGGGCATTCCCTTTT


 



AGCTACCCAGGTAATGTCCCGTCTGCGTGAAACTTTT


 



CAAGTCGAATTACCTTTGCGTAGTCTCTTTACCGCTC


 



CCACTATTGCTGAATTGGCCCTAACAATTGAGCAATC


 



TCAGCAAACCATTGCTGCTCCCCCCATCCTAACCAGA


 



AACGACAGTGCTAACCTCCCGTTATCTTTTGCTCAAC


 



AACGTTTATGGTTTCTGGATCAATTAGAACCTAACAG


 



CGCCTTTTATCATGTAGGGGGAGCCGTAAGACTAGAA


 



GGAACATTAAATATTACTGCCTTAGAGCAAAGCTTAA


 



AAGAAATTATTAATCGTCATGAAGCTTTACGCACAAA


 



TTTTATAACGATTGATGGTCAAGCCACTCAAATTATT


 



CACCCTACTATTAATTGGCGATTGTCTGTTGTTGATT


 



GTCAAAATTTAACCGACACTCAATCTCTGGAAATTGC


 



GGAAGCTGAAAAGCCCTTTAATCTTGCTCAAGATTGC


 



TTATTTCGTGCTACTTTATTCGTGCGATCACCGCTAG


 



AATATCATCTACTCGTGACCATGCACCATATTGTTAG


 



CGATGGCTGGTCAATTGGAGTATTTTTTCAAGAACTA


 



ACTCATCTTTACGCTGTCTATAATCAGGGTTTACCCT


 



CATCTTTAACGCCTATTAAAATACAATATGCTGATTT


 



TGCGGTCTGGCAACGGAATTGGTTACAAGGTGAAATT


 



TTAAGTAATCAATTGAATTATTGGCGCGAACAATTAG


 



CAAATGCTCCTGCTTTTTTACCTTTACCGACAGATAG


 



ACCTAGGCCCGCAATCCAAACTTTTATTGGTTCTCAT


 



CAAGAATTTAAACTTTCTCAGCCATTAAGCCAAAAAT


 



TGAATCAACTAAGTCAGAAGCATGGAGTGACTTTATT


 



TATGACTCTCCTGGCTGCTTTTGCTACCTTACTTTAC


 



CGTTATACAGGACAAGCAGATATTTTAGTTGGTTCTC


 



CTATTGCTAACCGTAATCGTAAGGAAATTGAGGGATT


 



AATCGGCTTTTTTGTTAATACATTAGTTCTGAGATTG


 



AGTTTAGATAATGATTTAAGTTTTCAAAATTTGCTAA


 



ACCATGTTAGAGAGGTTTCTTTAGCAGCCTACGCCCA


 



TCAAGATTTACCTTTTGAAATGTTAGTAGAAGCACTA


 



CACCCTCAACGAGATCTCAGTCATACCCCTTTATTTC


 



AGGTAATGTTTGTTTTGCAAAATACACCAGTGGCTGA


 



TCTAGAACTTAAAAATGTAAAGGTTTGTCCTCTACCG


 



ATGGAAAATAAGACTGCTAAATTTGATTTAACCTTAT


 



CAATGGAGAATCTAGAGGAAGGATTGATTGGGGTTTG


 



GGAATATAACACCGATCTATTTAATGGCTCAACCATT


 



GAGCGAATGAGTGGACATTTTGTCACTTTGTTAGAAG


 



ATATTGTTGCCGCTCCAACGAAGTCAGTTTTACGGTT


 



GTCTTTGCTGACGCAAGAGGAAAAACTGCAATTATTG


 



ATTAAAAATCAGGGTGTTCAAGTTGATTATTCTCAAG


 



AGCAGTGCATCCATCAATTATTTGAAGCGCAAGTTGA


 



ACGGACTCCCGATGCGATTGCGGTGGTATTTGAGGAG


 



CAATCATTAACCTATGCTGAATTAAATCATCAAGCTA


 



ATCAGTTAGTCCATTACTTACAAACTTTAGGAATTGG


 



GCCAGAGGTCTTAGTCGCTATTTCAGTAGAACGTTCT


 



TTAGAAATGATTATCGGCTTATTAGCCATTCTCAAGG


 



CGTGTGGTGCTTATCTCCCTCTTGCTCCTGACTATCC


 



CACTGAGCGTCTTCAGTTCATGTTAGAAGATAGTCAA


 



GCTTCTTTTTTGATTACCCACAGTTCTTTATTAGAAA


 



AATTGCCTTCTTCTCAAGCGACTCTAATTTGTTTAGA


 



TCACATCCAAGAGCAGATTTCTCAATATTCTCCCGAT


 



AATCTTCAAAGTGAGTTAACTCCTTCCAATTTGGCTA


 



ACGTTATTTACACCTCTGGCTCTACGGGTAAGCCTAA


 



AGGGGTGATGGTTGAACATCGGGGCTTAGTTAACTTA


 



GCGAGTTCTCAAATTCAATCTTTTGCAGTCAAAAATA


 



ACAGTCGTGTACTGCAATTTGCTTCCTTTAGTTTTGA


 



TGCTTGTATTTCAGAAATTTTGATGACCTTTGGTTCT


 



GGAGCGACTCTTTATCTTGCTCAAAAAAATGATTTAT


 



TGCCAGGTCAGCCATTAATGGAAAGGTTAGAAAAGAA


 



TAAAATTACCCATGTTACTTTACCCCCTTCAGCTTTA


 



GCTGTTTTACCAAAAAAACCGTTACCCAACTTACAAA


 



CTTTAATTGTGGCGGGTGAGGCTTGTCCTCTGGATTT


 



AGTCAAACAATGGTCAGTCGGTAGAAACTTTTTCAAT


 



GCCTATGGCCCGACAGAAACGAGTGTTTGTGCCACGA


 



TTGGACAATGTTATCAAGATGATTTAAAGGTCACGAT


 



TGGTAAGGCGATCGCTAATGTCCAAATTTATATTTTG


 



GATGCCTTTTTACAACCAGTACCCATCGGAGTACCAG


 



GGGAATTATACATTGGTGGAGTCGGAGTTGCGAGGGG


 



TTATCTAAATCGTCCTGAATTAACGGCGGAAAGATTT


 



ATTCCTAATCCTTTTGATCCCCCCCTAACCCCCCTTA


 



AAAAGGGGGGAGATAAGAGCTATGAAACTTTTAAAAA


 



GGGGGAAGAGCAACCATCAAAACTCTATAAAACGGGA


 



GATTTAGCTCGTTATTTACCCGATGGCAATATTGAAT


 



ATTTAGGACGCATTGACAATCAGGTAAAAATTCGCGG


 



TTTTCGCATTGAGTTAGGAGAAATTGAAGCGGTTCTG


 



AGTCAATGTCCCGATGTGCAAAATACGGCGGTGATTG


 



TCCGTGAAGATACTCCTGGCGATAAACGTTTAGTTGC


 



CTATGTGGTTCTTACTTCTGACTCCCAGATAACTACT


 



AGCGAACTGCGTCAATTCTTGGCTAATCAATTACCTG


 



CCTATCTCGTTCCCAATACCTTTGTTATTTTAGATGA


 



TTTGCCCCTAACCCCCAATGGTAAATGCGATCGCCGT


 



TCCTTACCGCTTCCTGATGATCAGACCAGAAAAAATA


 



TTCCTAAAATTGGCCCGCGTAATTTAGTGGAATTACA


 



ATTAGCTCAAATCTGGTCAGAGATTTTAGGCATTAAT


 



AATATTGGTATTCAGGAAAACTTCTTTGAATTAGGCG


 



GTCATTCTTTATTAGCAGTCAGTCTGATCAATCGTAT


 



TGAACAAAAGTTAGATAAACGTTTACCATTAACCAGT


 



CTTTTTCAAAATGGAACCATAGCAAGTCTAGCTCAAT


 



TACTAGCGCAAGAAACAACTCAGCCAGCCTCTTCACC


 



GTTGATTGCTATCCAGTCTCAAGGTGATAAAACTCCA


 



TTTTTTGCTGTTCATCCCATTGGTGGTAATGTGCTAT


 



GTTATGCCGATTTAGCTCGTAATTTAGGAACGAAACA


 



GCCGTTTTATGGATTACAATCATTAGGGCTAAGTGAA


 



TTAGAAAAAACTGTAGCCTCTATTGAAGAAATGGCGA


 



TGATTTATATTGAAGCAATACAAACTGTTCAAGCCTC


 



TGGTCCCTACTATTTAGGAGGTTGGTCAATGGGAGGA


 



GTGATAGCTTTTGAAATCGCCCAACAATTATTGACCC


 



AAGGTCAAGAAGTTGCTTTACTGGCTTTAATAGATAG


 



TTATTCTCCCAGTTTACTTAATTCAGTTAATAGGGAG


 



AAAAATTCTGCTAATTCCCTGACAGAAGAATTTAATG


 



AAGATATCAATATTGCCTATTCTTTCATCAGAGACTT


 



AGCAAGTATATTTAATCAAGAAATCTCTTTCTCTGGG


 



AGTGAACTTGCTCATTTTACATCAGACGAATTACTAG


 



ACAAGTTTATTACTTGGAGTCAAGAGACGAATCTTTT


 



GCCGTCAGATTTTGGGAAGCAGCAGGTTAAAACCTGG


 



TTTAAAGTTTTCCAGATTAATCACCAAGCTTTGAGCA


 



GCTATTCTCCCAAGACGTATCTGGGTAGAAGTGTTTT


 



CTTAGGAGCGGAAGACAGTTCTATTAAAAATCCTGGT


 



TGGCATCAAGTAATCAATGACTTGCAATCTCAATGGA


 



TTAGCGGCGATCACTACGGTTTAATTAAAAATCCAGT


 



CCTCGCTGAAAAACTCAATAGCTACCTAGCCTAAAAC


 



TTTCAAAAAGCCTGATTATTGTTTAAAATGAATGATC


 



GTTCACCGGTCAGAGGACAAGTATGACAACCCAAACA


 



GCTTCTAGTGCCAATGCCCTTGCTTCCTTTAACCAAT


 



TTTTAAGGGATGTAAAGGCGATCGCCCAACCCTATTG


 



GTATCCCACTGTATCAAATAAAAGAAGCTTTTCTGAG


 



GTTATTCGTTCCTGGGGAATGCTATCACTGCTTATCT


 



TTTTGATTGTGGGATTAGTCGCCGTCACGGCTTTTAA


 



TAGTTTTGTTAATCGTCGTTTAATTGATGTCATTATT


 



CAAGAAAAAGATGCGTCTCAATTTGCCAGTACATTAA


 



CTGTCTATGCGATCGGATTAATCTGTGTAACGCTGCT


 



GGCAGGGTTCACTAAAGATATTCGCAAAAAAATTGCC


 



CTAGATTGGTATCAATGGTTAAACACCCAGATTGTAG


 



AGAAATATTTTAGTAATCGTGCCTATTATAAAATTAA


 



CTTTCAATCTGACATTGATAACCCCGATCAACGTCTA


 



GCCCAGGAAATTGAACCGATCGCCACAAACGCCATTA


 



GTTTCTCGGCCACTTTTTTGGAAAAAAGTTTGGAAAT


 



GCTAACTTTTTTAGTGGTAGTTTGGTCAATTTCTCGA


 



CAGATTGCTATTCCGCTAATGTTTTACACGATTATCG


 



GTAATTTTATTGCCGCCTATCTAAATCAAGAATTAAG


 



CAAGATCAATCAGGCACAACTGCAATCAAAAGCAGAT


 



TATAACTATGCCTTAACCCATGTTCGGACTCATGCGG


 



AATCTATTGCTTTTTTTCGGGGAGAAAAAGAGGAACA


 



AAATATTATTCAGCGACGTTTTCAGGAAGTTATCAAT


 



GATACGAAAAATAAAATTAACTGGGAAAAAGGGAATG


 



AAATTTTTAGTCGGGGCTATCGTTCCGTCATTCAGTT


 



TTTTCCTTTTTTAGTCCTTGGCCCTTTGTATATTAAA


 



GGAGAAATTGATTATGGACAAGTTGAGCAAGCTTCAT


 



TAGCTAGTTTTATGTTTGCATCGGCCCTGGGAGAATT


 



AATTACAGAATTTGGTACTTCAGGACGTTTTTCTAGT


 



TATGTAGAACGTTTAAATGAATTTTCTAATGCCTTAG


 



AAACTGTGACTAAACAAGCCGAGAATGTCAGCACAAT


 



TACAACCATAGAAGAAAATCATTTTGCCTTTGAACAC


 



GTCACCCTAGAAACCCCTGACTATGAAAAGGTGATTG


 



TTGAGGATTTATCTCTTACTGTTCAAAAAGGTGAAGG


 



ATTATTGATTGTCGGGCCCAGTGGTCGAGGTAAAAGT


 



TCTTTATTAAGGGCGATCGCCGGTTTATGGAATGCTG


 



GCACTGGGCGTTTAGTGCGTCCTCCCCTAGAAGAAAT


 



TCTCTTTTTGCCCCAACGTCCCTACATTATTTTGGGA


 



ACCTTACGCGAACAATTGCTGTATCCTCTAACCAATA


 



GTGAGATGAGCAATACCGAACTTCAAGCAGTATTACA


 



ACAAGTCAATTTGCAAAATGTGCTAAATCGGGTGGAT


 



GACTTTGACTCCGAAAAACCCTGGGAAAACATTCTCT


 



CCCTCGGTGAACAACAACGCCTAGCCTTTGCTCGATT


 



GTTAGTGAATTCTCCGAGTTTTACCATTTTAGATGAG


 



GCGACCAGTGCCTTAGATTTAACAAATGAGGGGATTT


 



TATACGAGCAATTACAAACTCGCAAGACAACCTTTAT


 



TAGTGTGGGTCATCGAGAAAGTTTGTTTAATTACCAT


 



CAATGGGTTTTAGAACTTTCTGCTGACTCTAGTTGGG


 



AACTCTTAAGCGTTCAAGATTATCGCCTTAAAAAAGC


 



GGGAGAAATGTTTACTAATGCTTCGAGTAACAATTCC


 



ATAACACCCGATATTACTATCGATAATGGATCAGAAC


 



CAGAAATAGTCTATTCTCTTGAAGGATTTTCCCATCA


 



GGAAATGAAACTATTAACAGACCTATCACTCTCTAGC


 



ATTCGGAGTAAAGCCAGTCGAGGGAAGGTGATTACAG


 



CCAAGGATGGTTTTACCTACCTTTATGACAAAAATCC


 



TCAGATATTAAAGTGGCTCAGAACTTAA

## Claims

1. An isolated nucleic acid sequence encoding a peptide comprising a sequence with at least 95% identity to SEQ ID NO. 1.

2. An isolated nucleic acid encoding a microginin synthetase enzyme complex, wherein the isolated nucleic acid encodes the following activities:
a) adenylation domain (A*) wherein, the adenylation domain comprises the nucleic acid sequence of claim 1
b) acyl carrier protein (ACP)
c) elongation module (EM) of polyketide synthases (PKS) comprising the following activities:
i. ketoacylsynthase (KS)
ii. acyl transferase (AT)
iii. acyl carrier protein (ACP 2)

d) aminotransferase (AMT)
e) three to five elongation modules (EM) of non-ribosomal peptide synthetases (NRPS) comprising the following activities:
i. condensation domain (C)
ii. adenylation domain (A)
iii. thiolation domain (T)

f) thioesterase (TE).

3. The isolated nucleic acid according to claim 2, additionally comprising sequences encoding the following activities or domains:
a) a monooxygenase (MO)
b) an integrated N-methyltransferase domain (MT) within one or more elongation modules (EM) of NRPS;
c) a non-integrated N-methyltrasferase (MT),
d) a modifying activity (MA) wherein, said MA is selected from the group comprising the following activities: halogenase, sulfatase, glycosylase, racemase, O-methyltransferase and C-methyltransferase
e) two or more peptide repeat spacer sequences (SP) consisting of one or more repeats of being either glycine rich or proline and leucine rich, located adjacently upstream and downstream of the MO or the MA, or both.

4. The isolated nucleic acid according to claim 2, further comprising at least one nucleic acid sequence encoding at least one protein sequence as follows:
a. adenylation domain (A*) according to SEQ ID NO. 1
b. acyl carrier protein (ACP) according to SEQ ID NO. 2
c. elongation module of polyketide synthases:
i. ketoacylsynthase domain (KS) according to SEQ ID NO. 3
ii. acyl transferase domain (AT) according to SEQ ID NO. 4
iii. acyl carrier protein domain (ACP 2) according to SEQ ID NO. 5

d. aminotransferase (AMT) according to SEQ ID NO. 6
e. elongation modules of non-ribosomal peptide synthetases:
i. condensation domain (C) according to SEQ ID NO. 7
ii. adenylation domain (A) according to SEQ ID NO. 8
iii. thiolation domains (T) according to SEQ ID NO. 9

f. elongation modules of non-ribosomal peptide synthetases responsible for the activation and condensation of leucin:
i. condensation domain (C 2) according to SEQ ID NO. 10
ii. adenylation domain (A 2) according to SEQ ID NO. 11
iii. thiolation domain (T 2) according to SEQ ID NO. 12

g. elongation modules of non-ribosomal peptide synthetases responsible for the activation and condensation of tyrosine 1 :
i. condensation domain (C 3) according to SEQ ID NO. 13
ii. adenylation domain (A 3) according to SEQ ID NO. 14
iii. thiolation domain (T 3) according to SEQ ID NO. 15

h. elongation modules of non-ribosomal peptide synthetases responsible for the activation and condensation of tyrosine 2:
i. condensation domain (C 4) according to SEQ ID NO. 16
ii. adenylation domain (A 4) according to SEQ ID NO. 17
iii. thiolation domain (T 4) according to SEQ ID NO. 18

i. thioesterase (TE) according to SEQ ID NO. 19
j.) two or more peptide repeat spacer sequences (SP1/SP2) according to SEQ ID NO. 21 and 22
l.) an integrated N-methyltransferase domain (MT) within the elongation module (EM) of the NRPS responsible for the activation and condensation of leucin according to SEQ ID 23 and
m.) a non-integrated N-methyltransferase (MT 2) according to SEQ ID NO. 24.

5. The isolated nucleic acid according to claim 2, further comprising at least one nucleic acid sequence as follows:
a) an adenylation domain (A*) according to SEQ ID NO. 25,
b) acyl carrier protein (ACP) according to SEQ ID NO. 26,
c) elongation module of polyketide synthases encoding for the condensation of acetate:
i. ketoacylsynthase domain (KS) according to SEQ ID NO. 27
ii. acyl transferase domain (AT) according to SEQ ID NO. 28
iii. acyl carrier protein domain (ACP 2) according to SEQ ID NO. 29

d) aminotransferase (AMT) according to SEQ ID NO. 30,
e) elongation modules of non-ribosomal peptide synthetases encoding for the activation and condensation of alanin:
i. condensation domain (C) according to SEQ ID NO. 31
ii. adenylation domain (A) according to SEQ ID NO. 32
iii. thiolation domain (T) according to SEQ ID NO. 33

f) elongation modules of non-ribosomal peptide synthetases encoding for the activation and condensation of leucin:
i. condensation domain (C 2) according to SEQ ID NO. 34
ii. adenylation domain (A 2) according to SEQ ID NO. 35
iii. thiolation domain (T 2) according to SEQ ID NO. 36

g) elongation modules of non-ribosomal peptide synthetases encoding for the activation and condensation of tyrosine 1:
i. condensation domains (C 3) according to SEQ ID NO. 37
ii. adenylation domains (A 3) according to SEQ ID NO. 38
iii. thiolation domains (T 3) according to SEQ ID NO. 39

h) elongation modules of non-ribosomal peptide synthetases encoding for the activation and condensation of tyrosine 2:
i. condensation domains (C 4) according to SEQ ID NO. 40
ii. adenylation domains (A 4) according to SEQ ID NO. 41
iii. thiolation domains (T 4) according to SEQ ID NO. 42

i) thioesterase (TE) according to SEQ ID NO. 43
j) monooxygenase (MO) according to SEQ ID NO. 44
k) two or more peptide repeat spacer sequences (SP1/2) according to SEQ ID NO. 45 and 46.
l.) an integrated N-methyltransferase domain (MT) within the elongation module (EM) of the NRPS encoding for the activation and condensation of leucin according to SEQ ID 47 and
m.) a non-integrated N-methyltrasferase (MT 2) according to SEQ ID NO. 48.

6. The isolated nucleic acid according to claim 2 wherein, the sequence parts of the nucleic acid encoding the microginin synthetase enzyme complex activities a) through f) are arranged upstream to downstream, respectively.

7. A vector comprising the nucleic acid of claim 2.

8. A microorganism transformed with the nucleic acid according to claim 2.

9. A vector according to claim 7 wherein, the vector is able to replicate autonomously.

10. A method of producing a microginin, comprising culturing a cell under conditions under which the cell will produce microginin, wherein said cell is transformed with a nucleic acid encoding the a recombinant microginin synthetase enzyme complex, according to claim 2, and wherein said cell does not produce the microginin in the absence of said nucleic acid, and wherein said cell is cultured in the presence of octanoic acid.

11. A microorganism transformed with the vector according to claim 6.