CN106676115B - 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster and its application - Google Patents

2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster and its application Download PDF

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CN106676115B
CN106676115B CN201710054193.2A CN201710054193A CN106676115B CN 106676115 B CN106676115 B CN 106676115B CN 201710054193 A CN201710054193 A CN 201710054193A CN 106676115 B CN106676115 B CN 106676115B
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陈文青
高耀杰
邓子新
徐顾丹
巫攀
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Wuhan University WHU
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Abstract

The present invention relates to clone, sequencing, analysis, functional study and its applications of 2 '-amino of RNA virus natural products -2'-deoxyadenosine biological synthesis gene clusters such as treatment leukaemia 2 '-chloro of the natural products Pentostatin generated of Actinomycesa lmadurae ATCC 39365 and mycoplasma virus, measles virus.Whole gene cluster includes 13 genes altogether: 5 genes relevant to 2 '-chloro Pentostatin synthesis；4 genes relevant to the synthesis of 2 '-amino -2'-deoxyadenosine；4 genes relevant to the two transhipment adjusting.The biosynthesis of 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine can be blocked or improved by the genetic manipulation to above-mentioned biosynthesis gene.Gene and its albumen provided by the present invention can be used for genetic engineering, protein expression, enzymic catalytic reaction of the compound etc., can also be used to find and find to can be used for medicine, the compound of industry or agricultural, gene, albumen.

Description

2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster and It is applied
Technical field
The invention belongs to microbial gene resource and genetic engineering fields, and in particular to the spray of nucleoside antibiotic 2 '-chloro Si Tading (2 '-chloropentostatin, 2 '-Cl PTN) and 2 '-amino -2'-deoxyadenosine (2 '-amino-2 ' - Deoxyadenosine, 2 '-amino dA) biological synthesis gene cluster clone, sequence analysis, functional verification in genosome, External Biochemical Research and its application.
Background technique
1979, American scientist was for the first time from (the Actinomadura sp.ATCC of Actinomycesa lmadurae ATCC 39365 39365) isolated in fermentation liquid 2 '-amino of purines nucleoside antibiotic -2'-deoxyadenosine (J Antibiot (Tokyo), 1979,32,1367-1369；Arch Biochem Biophys,1989,270,374-382).2 '-amino -2'-deoxyadenosine With extensive anti-rna viral activity, and (J in the treatment of the RNA virus such as mycoplasma viruses, morbilli has been applied to it Antibiot(Tokyo),1979,32,1367-1369；Agr Biol Chem Tokyo,1985,49,2711-2717).Due to Adenosine deaminase that 2 '-amino -2'-deoxyadenosine is extremely easy to be widely present in cell (adenosine deaminase, ADA) effect catalysis forms 2 '-amino -2 '-deoxyinosine (2 '-amino dI), and early-stage study person is equally from Actinomycesa lmadurae Isolated strong effective 2 '-chloro of inhibitor Pentostatin (2 '-of adenosine deaminase in 39365 fermentation liquid of ATCC chloropentostatin,2'-Cl PTN).It is equally used as purines nucleoside antibiotic, 2 '-chloro Pentostatins are clinically Treatment (J Antibiot (Tokyo), 1985,38,1344-1349) applied to blood type cancer.
Chemically in structure from the point of view of, analogue of the 2 '-amino -2'-deoxyadenosine as adenosine, with adenosine in structure On difference be that hydroxyl on the position C-2 ' turns ammonia and generates amino；2 '-chloro Pentostatins are then containing a special 1,3- Diaza heptatomic ring and C-2 ' occur chloro.In report early period, is fed and tested by isotope, show 2 '-amino- The amino for the C-2 ' being related in the biosynthesis pathway of 2'-deoxyadenosine is formed since the hydroxyl of C-2 ' turns amino (Arch Biochem Biophys, 1989,270,374-382).It is same to be tested by isotope labelling, it is determined that 2 '-chlorine Biosynthesis for Pentostatin is the position C-6 and N-1 shapes using adenosine as precursor, by the C-1 insertion purine ring of D-ribose At special 1,3- diaza heptatomic ring (Biochemistry, 1984,23,904-907；Biochemistry,1987,26, 5636-5641)；In addition, the initial phase of 2 '-chloro Pentostatin biosynthesis and the biosynthesis pathway of L-Histidine and The biosynthesis pathway of Pentostatin has certain similitude (Arch Biochem Biophys, 1989,270,374-382； Cell Chem Biol,2017,24,DOI:10.1016/j.chembiol.2016.12.012).Although 2 '-chloro spray departments he Fourth and 2 '-amino -2'-deoxyadenosine are clinically widely used and also have in terms of chemical synthesis good research Progress, but over the past decades, the biosynthesis about the two purines nucleoside antibiotics is still known little about it.
Summary of the invention
In order to overcome the deficiencies in the prior art, the present invention is with a kind of Actinomycesa lmadurae (Actinomadura Sp.ATCC 39365) generate treatment leukaemia natural products purines 2 '-chloro of nucleoside antibiotic Pentostatin and have 2 '-amino of natural products purines nucleoside antibiotic -2'-deoxyadenosine of RNA virus resisting is target molecule, to its biosynthesis Gene cluster is cloned, and by sequence analysis, functional verification, external biochemical test, disclosing a gene cluster includes two only The phenomenon that vertical biosynthesis pathway.Whole gene cluster is that the nucleotides sequence comprising 13 genes is classified as SEQ ID in the present invention In NO:1 shown in 10898-25249, wherein it is responsible for the gene of 2 '-chloro Pentostatin biosynthesis, i.e. adaC, adaB, AdaA, adaK, adaL totally 5 genes；It is responsible for the gene of 2 '-amino -2'-deoxyadenosine biosynthesis, i.e. adaF, adaG, AdaJ, adaM totally 4 genes；It is responsible for the gene of 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine transhipment and regulation AdaE, adaD, adaH, adaI totally 4 genes.
The present invention also provides the nucleotide sequence that one encodes cation transporter, the amino acid sequence of coding is SEQ ID NO.2, is named as adaE, and nucleotide sequence is located in SEQ ID NO:1 at 10898-12337 base.
The present invention also provides the nucleotide sequence that one encodes MFS transport protein, the amino acid sequence of coding is SEQ ID NO.3, is named as adaD, and nucleotide sequence is located in SEQ ID NO:1 at 12392-13600 base.
The present invention also provides the nucleotide sequence that one encodes ATP phosphoribosyltransferase, the amino acid sequences of coding It is classified as SEQ ID NO.4, is named as adaC, nucleotide sequence is located in SEQ ID NO:1 at 13672-14559 base.
The present invention also provides the nucleotide sequence of an encoding short-chain dehydrogenase, the amino acid sequence of coding is SEQ ID NO.5, is named as adaB, and nucleotide sequence is located in SEQ ID NO:1 at 14604-15308 base.
The present invention also provides the nucleotide sequence that one encodes SAICAR synzyme, the amino acid sequence of coding is SEQ ID NO.6, is named as adaA, and nucleotide sequence is located in SEQ ID NO:1 at 15295-16014 base.
The present invention also provides the nucleotide sequence of an encoding aminotransferases, the amino acid sequence of coding is SEQ ID NO.7, is named as adaF, and nucleotide sequence is located in SEQ ID NO:1 at 16239-17516 base.
The present invention also provides the nucleotide sequence that one encodes dehydrogenase, the amino acid sequence of coding is SEQ ID NO.8, is named as adaG, and nucleotide sequence is located in SEQ ID NO:1 at 17509-18564 base.
The present invention also provides the nucleotide sequence that one encodes abc transport albumen, the amino acid sequence of coding is SEQ ID NO.9, is named as adaH, and nucleotide sequence is located in SEQ ID NO:1 at 18593-20380 base.
The present invention also provides the nucleotide sequence that one encodes abc transport albumen, the amino acid sequence of coding is SEQ ID NO.10, is named as adaI, and nucleotide sequence is located in SEQ ID NO:1 at 20402-22180 base.
The present invention also provides the nucleotide sequence that one encodes NUDIX hydrolase, the amino acid sequence of coding is SEQ ID NO.11, is named as adaJ, and nucleotide sequence is located in SEQ ID NO:1 at 22177-22662 base.
The present invention also provides the nucleotide sequence that one encodes phosphoriboisomerase A, the amino acid sequences of coding For SEQ ID NO.12, it is named as adaK, nucleotide sequence is located in SEQ ID NO:1 at 22659-23396 base.
The present invention also provides the nucleotide sequence that one encodes ATP phosphoribosyltransferase, the amino acid sequences of coding It is classified as SEQ ID NO.13, is named as adaL, nucleotide sequence is located in SEQ ID NO:1 at 23580-24446 base.
The present invention also provides the nucleotide sequence that one encodes phosphohydrolase, the amino acid sequence of coding is SEQ ID NO.14, is named as adaM, and nucleotide sequence is located in SEQ ID NO:1 at 24455-25249 base.
From clone biological synthetic gene cluster, using microbiology, molecular biology, biochemistry and organic chemistry phase In conjunction with technique study its biosynthesis, by the research to its biosynthesis mechanism disclose a gene cluster include two solely The phenomenon that vertical biosynthesis pathway, has contained a protection and protected machine in this unique biosynthesis pathway System, and the Enzymatic Mechanism of the unique chemical moieties formation including 2 '-chloro Pentostatins.
The principle for using metabolic engineering on this basis, by combination biology to the rational modification of biosynthesis pathway, Explore the newtype drug that stable structure, activity are more preferable and can generate by microorganism bulk fermentation.
The application of of the invention 2 '-chloro Pentostatin and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster, including (but being not limited to):
(1) the present invention also provides generate 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biosynthesis gene The approach for the microbial body that cluster interrupts, at least one of gene includes the nucleotide sequence in SEQ ID NO.1.
(2) the clone DNA comprising nucleotide sequence provided by the present invention or at least partly nucleotide sequence can be used for from More libraries are positioned in Actinomycesa lmadurae ATCC 39365 (Actinomadura sp.ATCC 39365) genomic library Plasmid.These Library plasmids include at least the partial sequence in the present invention.
(3) comprising nucleotide sequence provided by the present invention or at least partly nucleotide sequence can be modified or be mutated. These approach include insertion, displacement or missing, polymerase chain reaction, mistake mediated polymerization enzyme chain reaction, locus specificity Mutation, not homotactic reconnect or is oriented evolutions with the homologous sequence in other sources at the different piece of sequence, or leads to Cross ultraviolet light or chemical reagent mutagenesis etc..
(4) clone gene comprising nucleotide sequence provided by the present invention or at least partly nucleotide sequence can pass through Suitable expression system is expressed in foreign aid host to obtain corresponding enzyme or other higher bioactivity or yield.Outside these Sourcesink master includes streptomycete, pseudomonas, Escherichia coli, bacillus, yeast, plant and animal etc..
(5) albumen required for amino acid sequence provided by the present invention can be used to separate and the system that can be used for antibody It is standby.
(6) polypeptide comprising amino acid sequence provided by the present invention or at least partly sequence may remove or substitute certain Still there is bioactivity even to have new biological activity after a little amino acid, or improves yield or optimize protein dynamics Feature or other properties being dedicated to.
(7) gene or gene cluster comprising nucleotide sequence provided by the present invention or at least partly nucleotide sequence can be with It is expressed in heterologous host and their functions in host metabolism chain is understood by DNA chip technology.
(8) gene or gene cluster comprising nucleotide sequence provided by the present invention or at least partly nucleotide sequence can be with By Genetic Recombination come construction recombination plasmid to obtain new bio route of synthesis, insertion, displacement, missing or mistake can also be passed through Living and then acquisition new bio route of synthesis.
(9) ATP can be catalyzed comprising nucleotide sequence coded albumen provided by the present invention and generate AMP, and can lead to It crosses and is recombinated with the biosynthesis pathway of other natural products or part biological route of synthesis, to obtain new purine nucleosides chemical combination Object.
(10) adenosine and its analogue can be catalyzed comprising nucleotide sequence coded albumen provided by the present invention Inosine and its analogue are synthesized, and can be by synthesizing way with the biosynthesis pathway of other natural products or part biological Diameter recombination, to obtain new purine nucleoside compounds.
Therefore, recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing said gene cluster are also guarantor of the present invention The range of shield.
Above-mentioned protein, above-mentioned gene cluster, above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium It is also the scope of protection of the invention synthesizing the application in 2 '-chloro Pentostatins and/or 2 '-amino -2'-deoxyadenosine.
It is a further object to provide a kind of 2 '-chloro Pentostatins of synthesis or 2 '-amino -2'-deoxyadenosines Method.
Method provided by the invention collects tunning for the above-mentioned recombinant bacterium that ferments to get 2 '-chloro Pentostatins And/or 2 '-amino -2'-deoxyadenosine.
In short, provided by the present invention includes 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biosynthesis phase All genes and Protein Information closed can help the biosynthesis mechanism it is appreciated that purine nucleosides antibiotic, be further Genetic modification provides material and knowledge.Gene and its albumen provided by the present invention can also be used to find and discovery can be used for The compound or gene, albumen of medicine, industry or agricultural.
Detailed description of the invention
The chemical structure of Fig. 1: 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine.
Fig. 2: A) the gene structure figure of 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster. B) 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster interrupt the height of schematic diagram and tunning Differentiate liquid chromatography mass spectrometric combined instrument analysis (LC-MS), wherein WT- wild type, ST- standard items, LG1- mutant strain.C) 2 '-chloros spray Si Tading first mass spectrometric data.
Fig. 3: 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster interrupt schematic diagram and hair The high-resolution liquid chromatography mass spectrometric combined instrument (LC-MS) of ferment product is analyzed.
Fig. 4: 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biosynthesis pathway speculate
A) 2 '-chloro Pentostatin.B) 2 '-amino -2'-deoxyadenosine.
Fig. 5: pyrophosphohydrolase functional analysis
A) AdaJ protein SDS-PAGE is analyzed.Difference divalent metal ion is to AdaJ biochemical reaction when B) using ATP as substrate Activity influence schematic diagram.C) respectively using ATP and AMP as substrate when AdaJ biochemical reaction HPLC analysis.D) with cobalt ions for 2 AdaJ is catalyzed different substrate active schematic diagrames when valence metal ion.
Specific embodiment
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.Provided implementation Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
Experimental method used in the following example is conventional method unless otherwise specified.
Material as used in the following examples, reagent etc., are commercially available unless otherwise specified.1. gram Grand, 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine of analysis biological synthesis gene clusters:
We have extracted the total DNA of Actinomycesa lmadurae ATCC 39365 first, using sequencing technologies to its total DNA into The sequencing of row genome-wide screening, and construct the total genome of Actinomycesa lmadurae ATCC 39365 using pJTU2463b as carrier Library.According to 1984 and Hanvey in 1987 etc. reports, adenosine was the biosynthesis direct precursor of Pentostatin, passed through D- The position C-6 of the C-1 insertion purine ring of ribose formd special 1,3- diaza heptatomic ring with N-1, and according to 2017 The research to Pentostatin biosynthesis pathway such as Chen.With (the S.antibioticus NRRL of antibiosis streptomycete NRRL 3238 3238) the gene penA, penB, penC of coding Pentostatin biosynthesis are as probe in, with Actinomycesa lmadurae ATCC The sequencing result of 39365 total DNAs carries out sequence analyses and comparison, finds the coding albumen homologous with it.The present invention is drawn using a pair Object 39365cluster-idF:ACACCACCTCGATGCTCG, 39365cluster-idR:ACCAGGTTCTCGGTCAGG is to total Genomic library is screened, it is isolated contain penA, the clay of penB, penC homologous gene adaC, adaB, adaA, then Pass through DNA sequence dna and sequence analysis, it is determined that the sticking grain in a covering coloring body 34.2kb region of DNA domain, so that it is determined that 2 '- The Position Approximate of the biological synthesis gene cluster of chloro Pentostatin and 2 '-amino -2'-deoxyadenosine in the genome.The present invention Sequence further is carried out to the sticking grain for containing 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster Analysis, obtained target gene cluster segment is cloned on carrier pJTU2463.The mutation that interrupt targets gene cluster segment obtains Bacterial strain does not generate 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine.DNA sequencing analyzes the chromosomal region of 17kb, Analysis of biological information contains 15 opening code-reading frames.Detailed analysis result is listed in table 1.
Each gene and coding in table 1:2 '-chloro Pentostatin and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster The functional analysis of albumen
aNCBI registration number is provided in bracket
bOpening code-reading frame except 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster
The determination on the biological synthesis gene cluster boundary of 2.2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine:
The generation for not influencing 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine is interrupted on gene orf1；And According to the functional analysis of gene coded protein, the biosynthesis gene of 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine Cluster is determined as gene adaE to adaM, covers the region of chromosome 14.3kb, includes 13 opening code-reading frames.Entire 2 '-chloro The biological synthesis gene cluster of Pentostatin and 2 '-amino -2'-deoxyadenosine totally 13 genes: 5 with 2 '-chloro Pentostatins Synthesize relevant gene；4 genes relevant to the synthesis of 2 '-amino -2'-deoxyadenosine；4 relevant to the two transhipment adjusting Gene.As shown in Figure 2 A.Said gene cluster is interrupted in Chromosome level, obtains mutant strain LG1, its fermentation liquid is carried out High resolution mass spectrum liquid phase combined instrument (HRLC-MS), which is analyzed, determines that mutant strain does not generate 2 '-chloro Pentostatins and 2 '-amino -2 ' - Desoxyadenossine.As shown in Figure 2 B.
The in vivo functionality of 3.2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biosynthesis related genes determines:
Each gene is successively knocked out one by one on 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine gene cluster, and Engagement, which is transferred in host strain CXR14, carries out heterogenous expression, and host strain CXR14 is the large fragment for generating Polyoxin industrial strain The streptomycete of missing, and 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine itself are not generated.Mutant strain fermentation Liquid is tested and analyzed using liquid chromatography mass spectrometric combined instrument (HRLC-MS), determining i.e. adaC, adaB, adaA, and adaK, adaL totally 5 Gene is gene relevant to 2 '-chloro Pentostatin biosynthesis；Totally 4 genes are and 2 '-by adaF, adaG, adaJ, adaM Amino-relevant the gene of 2'-deoxyadenosine biosynthesis, adaE, adaD, totally 4 genes as transhipment and are adjusted by adaH, adaI Gene.As shown in Figure 3.
The biosynthesis of 4.2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine
Certain genes in vivo 2 '-in 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster Have certain correlation with the biosynthesis of L-Histidine and Pentostatin in the biosynthesis of chloro Pentostatin, with dATP and PRPP is initial compounds, and ATP phosphoribosyltransferase AdaC, AdaL are catalyzed dATP formation ribose phosphate dATP (PR-dATP) (compound 1) recycles ribose phosphate AMP cyclization hydrolase HisI, ribose phosphate ATP in L-Histidine biosynthesis pathway Tri- pyrophosphatase HisE, ribose phosphate allomerase AdaK albumen synthesize compound 2, pass through one system of SAICAR synzyme later The catalytic action of column synthesizes compound 5, forms 6- hydroxyl Pentostatin using the catalytic action of a dephosphorylation enzyme AdaM (compound 6) finally has short-chain dehydrogenase AdaB to catalyze and synthesize 6- hydroxyl Pentostatin Pentostatin (compound 7), then Chloro occurs in 2 ' positions under cation transporter AdaE catalysis and generates 2 '-chloro Pentostatins (compound 8).Such as Fig. 4 A institute Show.The biosynthesis of 2 '-amino -2'-deoxyadenosine is to pass through NUDIX water as initial compounds by adenosine triphosphate (ATP) The co-catalysis and adjusting for solving enzyme AdaJ and dehydrogenase A daG form adenosine, and adenosine is again 2 ' under the catalysis of aminopherase AdaF Position hydroxyl turns ammonia and forms 2 '-amino -2'-deoxyadenosine.As shown in Figure 4 B.
The NUDIX hydrolase A daJ's of 5.2 '-amino -2'-deoxyadenosine biosynthesis related genes adaJ coding is external Functional verification:
By adaJ gene using PCR amplification rear clone to expression vector, heterologous overexpression, purifying in Escherichia coli, Purer albumin A daJ is obtained after analyzing by SDS-PAGE.By bioinformatic analysis, it is found that AdaJ is pyrophosphoric acid hydrolysis Enzyme.Using adenosine triphosphate (ATP) as substrate, the external biochemical reaction that AdaJ is catalyzed adenosine triphosphate (ATP) passes through the present invention After Mass Spectrometer Method, analysis finds to form adenosine monophosphate (AMP).AdaJ known to as a result, can be catalyzed adenosine triphosphate (ATP) shape At adenosine monophosphate (AMP), and this reacts the first step as 2 '-amino -2'-deoxyadenosine biosynthesis pathway, shape The biosynthesis of 2 '-amino -2'-deoxyadenosine is begun at adenosine monophosphate (AMP).Followed by different divalent metals from Sub and different reaction substrate finds AdaJ biochemical reaction activity research, when divalent cobalt ions is added in the reaction system The reactivity of AdaJ is maximum.And other than ATP, AdaJ can also be using dATP, GTP as reaction substrate, but cannot Utilize ADP.As shown in Figure 5.
[embodiment 1] 2 '-chloro Pentostatin and 2 '-amino -2'-deoxyadenosine producing strains Actinomycesa lmadurae ATCC The extraction of 39365 total DNAs:
Take 30 μ L Actinomycesa lmadurae ATCC, 39365 spore into the TSB culture medium of 50mL, 30 DEG C, 200rpm, culture 24-36h, until cloudy state is presented in culture medium.39365 bacterium solution of Actinomycesa lmadurae ATCC of 50mL, 4,000rpm, 4 DEG C, Supernatant is removed in 10min centrifugation, collects thallus.Thallus is dissolved in 10.3% sucrose solution of 25mL shaking and mixes washing thalline, 4, 000rpm, 4 DEG C, supernatant is removed in 10min centrifugation；Thallus is dissolved in again to vibrate in the set buffer of 15mL and is mixed, 4,000rpm, 4 DEG C, supernatant is removed in 10min centrifugation, is repeated twice；Thallus is dissolved in the set buffer of 10mL after concussion mixing, is added 50 μ L's Lysozyme soln (100mg/mL) is placed in warm bath 30min in 37 DEG C of water-baths；280 μ L Proteinase K Solution (50mg/ are then added ML), 600 μ L 10%SDS are added after mixing, is mixed by inversion and is placed on 55 DEG C of warm bath 4h, are mixed by inversion every 15min during this, 100 μ L of Proteinase K Solution is added every 30min, until mycelium cracks bleach；The NaCl of 4mL 5M is added later, overturns It mixes, places bacterium solution at room temperature to 37 DEG C or so；The chloroform that 10mL is added is mixed by inversion to milky, and 4,000rpm, 4 DEG C, 10min takes out the isopropanol mixing of 0.6 times of volume of addition after supernatant；After mixing, there is cotton-shaped DNA to be precipitated, by the DNA of precipitation Carefully choose and with 75% ethanol washing twice, be placed in ventilation dry up, be dissolved in suitable ultrapure water.Nucleic acid electrophoresis (40v, 12h) detects the size of DNA, and measures its concentration and the determining purity for proposing total DNA of OD value with Nanodrop 2000.
[embodiment 2] 2 '-chloro Pentostatin and 2 '-amino -2'-deoxyadenosine producing strains Actinomycesa lmadurae genome The foundation in library.
The dosage of Sau 3AI is determined by a series of dilution experiment first, prepares the digestion body that total volume is 500 μ L It is (5 μ L 0.1u/ μ L Sau 3AI, the DNA that 495 μ L 10Xbuffer diluted) solution 1, takes 300 μ l solution 1 and 100 μ L DNA is mixed into solution 2 (Sau 3AI final concentration 0.075u/100 μ L), and 200 μ L solution 1 and 200 μ L DNA is taken to be mixed into solution 3 (Sau 3AI final concentration 0.05u/100 μ L), takes 200 μ L solution 2 and 200 μ L DNA to be mixed into (the Sau 3AI final concentration of solution 4 0.0375u/100 μ L), take 200 μ L solution 4 and 200 μ l DNA to be mixed into (the Sau 3AI final concentration 0.01875u/100 μ of solution 5 L).Above-mentioned solution mixing is all placed on ice, is then taken out and is immediately placed on ice after 37 DEG C of water-bath 1h together, with 12cm long 1% Agarose gel electrophoresis is maker with control DNA and λ mix, 5 μ L, 30v voltage of applied sample amount, after 18h under gel imager Detect digestion quality.According to pre- digestion experimental result select suitable dilution carry out pulsed field gel electrophoresis (pulsed field gel electrophoresis condition: 16 DEG C, electrophoresis time 16h, voltage 6.0v of the temperature of pump, 120 ° of corner, corner time 1s, 6s), by sample loading to preparatory standard It in the low melting-point agarose gel of 1% got ready, is detected under long wave ultraviolet light after pulsed field gel electrophoresis, recycling 48-kb is left The gel of right size is dissolved in 10x β-agarase I reaction buffer, and 65 DEG C of warm bath wait for that gel is completely dissolved, after being cooled to 42 DEG C β-agarase (adding 1 μ L enzyme by 100 μ L volumes) 42 DEG C of warm bath 1h are added, then 65 DEG C of warm bath 15min inactivate enzyme.12, 000rmp, 15min centrifuging and taking supernatant are added the sodium acetate of the 3M of the newest preparation of 1/10 volume and the isopropanol of 2 times of volumes, fill 12,000rpm, 15min centrifugation removal supernatant, 75% ethanol washing twice, are dried at room temperature after dividing mixing to be placed at room temperature for 10min Afterwards plus suitable quantity of water dissolves.Electrophoresis detection recycles fragment masses and carries out dephosphorylation process, is cloned into carrier pJTU2463b.
The processing of pJTU2463b carrier: HpaI single endonuclease digestion is used after extracting plasmid detected through gel electrophoresis, dephosphorylation (prevents From even), then BamHI digestion is used, obtain the DNA fragmentation of 7-kb and 2-kb, the segment of gel recycling 7-kb.
The enzyme of total DNA segment and carrier pJTU2463b connect: being with concentration by the carrier handled well that concentration is 15ng/ μ L The dephosphorylation total DNA segment that 46.2ng/ μ L is recovered to carries out enzyme company, enzyme disjunctor system: 4 μ L, DNA piece of carrier with the ratio of 1:3 13 μ L, T4buffer 2 μ L, T4 ligase (NEB) 1 μ L of section, ultrapure water are mended to 20 μ L systems.After 16 DEG C of warm bath 12h, 70 DEG C of temperature Bathe 10min enzyme inactivation.After carrying out detected through gel electrophoresis to enzyme-linked product, subsequent step is carried out.
Library packaging: taking out 25 μ L packaging proteins out of -80 DEG C refrigerators and 10 μ L enzyme-linked products mix, 30 DEG C of warm bath 25 μ L packaging proteins are added after 90min, continue 30 DEG C of warm bath 90min, and PDB is added and is diluted to 1mL, 25 μ L chloroforms are added.
The preparation of EPI300 competence: drawing single colonie on LB solid plate, chooses single bacterium and drops down onto 5mL LB 37 DEG C overnight Culture, bacterium solution are forwarded in 50mL LB by 1%, and the 1M MgSO of 500 μ L is added437 DEG C are cultivated to OD600=0.85.
Transfection: taking above-mentioned 10 μ L of packaging product that PDB is added to be diluted to 1mL, and diluted 10 μ L of packaging product and 100 μ L is taken to prepare Good EPI300 competence mixes, 37 DEG C of warm bath 20min, is applied to A Bo and draws on LA culture dish, chooses Dan Ke after 37 DEG C of culture 12h It is grand in 96 orifice plates equipped with LB culture medium, after 37 DEG C of culture for 24 hours, isometric 40% glycerol is added and is stored in -80 DEG C.
[embodiment 3] 2 '-chloro Pentostatin and 2 '-amino -2'-deoxyadenosine producing strains Actinomycesa lmadurae ATCC 39365 fermentation conditions, product efficient liquid phase (HPLC) testing conditions.
By the spore inoculating of Actinomycesa lmadurae ATCC 39365 in seed culture medium, 30 DEG C, 220rmp cultivates 48h, It is forwarded in fermentation shake flask according to 4% inoculum concentration, 32 DEG C, 220rmp is cultivated 6 days.Fermentation liquid is collected, by fermentation liquid 9, 000rpm is centrifuged 20min, is extracted repeatedly 3 times after taking supernatant with isometric n-butanol, gained extract liquor is removed through Rotary Evaporators Be dissolved in 1.5mL pure water after going solvent, n-butanol, take supernatant after 12,000rpm high speed centrifugations, cross 0.22 μm of miillpore filter into The detection and analysis of row HPLC.
HPLC testing conditions: the A Xiang Weijia ultrapure water of 0.15% trifluoroacetic acid (TFA), B phase is methanol.It is initially 95% A phase A phase when 30min inside gradient is eluted to 80%, 31min is converted to 10% and this concentration is kept persistently to be eluted to 40min, in 41min, A phase is converted to 95%, is kept to 60min.Flow velocity is 0.5mL/min, Detection wavelength 254nm, column temperature 30 ℃。
[embodiment 4] 2 '-chloro Pentostatin and 2 '-amino -2'-deoxyadenosine producing strains Actinomycesa lmadurae ATCC 39365 and its heterogenous expression engagement transfer method.
The target plasmid that transfer will be engaged first is transformed into E. coli ET12567/pUZ8002 competence, It is verified after growing transformant, positive monoclonal is inoculated in the LB culture solution of 5mL, 37 DEG C are incubated overnight, and bacterium solution is pressed 10% is inoculated in 37 DEG C of culture 3-5h in 5mL LB culture solution.Host streptomycete spore 5 is taken, 000rpm is centrifuged 3min, removes supernatant Twice, 5,000rpm centrifugation 3min remove supernatant to milli-Q water afterwards.The TES of 700 μ L is added, according to receptor streptomycete after mixing Difference adds the 700 pre- germination fluids of μ L 2x spore with different temperature (45 DEG C or 50 DEG C) thermal shock 5min or 10min, and 30 DEG C Cultivate 3-5h.
By cultured Escherichia coli in 4 DEG C, 4,000rpm centrifugation 3min, after going supernatant addition 20mL LB to wash twice Supernatant is removed in centrifugation, and 1mL LB culture medium is added and mixes Bacillus coli cells；By the receptor spore 5 of cultured streptomycete, 000rpm is centrifuged 3min, is washed twice after removing supernatant with 20mL LB, by the above-mentioned Escherichia coli handled well and the spore handled well It mixes, is coated on MS culture dish, ultrapure water of the 1mL containing the appropriate antibiotic for screening is added afterwards for 24 hours to cover, is placed in 30 DEG C culture a couple of days to joint element grow.
In the sticking grain of [embodiment 5] containing 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster The method of PCR-targeting
The sticking grain 3G12 that 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biological synthesis gene cluster will be contained turns Change into E. coli BW25113/pIJ790 competent cell, 30 DEG C of overnight incubations, chooses single bacterium and fall within 5mL LB training It supports in base (containing A Bo drawing and chloramphenicol), 30 DEG C of overnight incubations, bacterium solution is transferred in 50mL LB by 1% and concentration is added simultaneously For the L-arabinose of 1M, 30 DEG C of culture 3h to OD600 are 0.4-0.6.Thallus is collected in 4 DEG C, 4,000rpm are centrifuged 5min, After 10% glycerol washes 3 times plus 200 μ L, 10% glycerol mixes thallus, stand-by with the every pipe packing of 50 μ L.The segment for taking 5 μ L to handle well It mixes, is added in electric revolving cup after electricity turn (electricity turns condition: 200 Ω, 25 μ F, 2.5kV), 37 DEG C of precultures with competent cell 30min is coated on LA culture dish, and 37 DEG C of culture 8h carry out subsequent authentication after growing transformant.
[embodiment 6] is encoded containing 2 '-chloro Pentostatins and 2 '-amino -2'-deoxyadenosine biosynthesis related genes Protein reconstitution, isolates and purifies overexpression
Target gene carries out PCR amplification, and DNA sequencing is verified on correct rear clone to expression vector, and expression plasmid is transformed into In E. coli BL21 (DE)/pLysE, picking positive monoclonal is incubated overnight for 37 DEG C in 5mL LB culture medium, is pressed In 500mL LB, thallus OD is arrived in 37 DEG C of cultures for 1% switching600To 0.5-0.8, IPTG (final concentration 0.1-0.2mM) is added and lures It leads, 18 DEG C of cultures 20h, 6,000rmp centrifugation 15min collect thallus.
The cracking buffer of appropriate (20mL-30mL) is added into the above-mentioned thallus being collected into, uses ultrasound after shaking mixed bacterium Broken instrument ultrasonication Bacillus coli cells, 4 DEG C of 12,000rpm centrifugation 20min take supernatant.Under the conditions of 4 DEG C, supernatant is filled Enter to have in the gravity column of nickel filler, be eluted with the Tris buffer containing various concentration (20mM-200mM) imidazoles, to different dense The lower sample eluted of degree carries out SDS-PAGE analysis, collects purer protein sample.
SEQUENCE LISTING
<110>Wuhan University
<120>2'- chloro Pentostatin and 2'- amino -2'- desoxyadenossine biological synthesis gene cluster and its application
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 36432
<212> DNA
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
tggtcggcgt cccggtcgtc cccgtcgtgt gcgcgagcac gaccgggcgg gcgcggtcgg 60
agacgaaggc ggcgggcatg ccgcgcaggt cggccttgga ggtgggcggg atcgtggaca 120
gcgtcgcggg cgtgatcgag ccggggtcca cgccgttcgt cccgaaccag cgccggtagt 180
aggggctctc ggcggccgcg tggcggaccg tgcgccgcag ccgccgccgg gtcaggtcct 240
cctggagctc ggggtcgctg cccgcgagca tgtcgtcggc gccctcgccc ggggacccga 300
acgtctccag ggtcacgcgc atgtcgtcgg ccagcgcgtt cacgtcccgc aggcggaacc 360
tgcggcccga catcatcgac caggcataac cgagctggcg taccgcggtg ccgaacacgc 420
gtcgtccttc cactcgtggg gggtggcccc ccgggagacc cggtcccccg ggggttcacg 480
cgagggtgat cagccgttca gcacgaagat ggcggtggac agggcgctga cggcgtactt 540
ggcgtaacgg cgagcggtct tgaccatgag gatctccctg gttgctcggg gcgcacgggt 600
ggcgcccgct gagatccaag gatgtgcggg ggcgcaagag agccactaca gggtcactac 660
aagggggcct cgcctcacac cagcgtcagg tagtgggcca gctcggcggc ggagtccgtg 720
gcgatcccga cgtggccgtc cgggcgcacg agcgtgtgcc gcaccccgcc gtacggctcg 780
gccggcgcgt acgcccgcac ctgcgggccg cccggcacgc ggggcgcggg atccggcgac 840
agcagcgtga agtgcgggcc gcgcagcagg tcgaacaggc gcgcccggga gccgtcggcc 900
agccgcagcg gcccgtcggg cacccgttcg ccgcccgcgc gggccagcga gccgccccgg 960
tagccgaccc ggagctggta ggtctcctcg tcccgccggt acgacttccg gtcgtgcagg 1020
tccgtgctga ggccgagcac gcgggcggcg atcgggcggc gctcctcctc gtaggtgtcc 1080
agcagcgacg ccggcccgcc ggtcagcgcg cgggcgagct tccagcccag gttgtaggcg 1140
tcctggacgc cggtgttgag cccctggccg cccgccgggg agtgcacgtg ggcggcgtcc 1200
ccggccagca gcacgcgccc ggcgcggtag gcgtcggcca gccggatgtt gacccggtag 1260
agcgaggacc acagcagctc ggtgacgcgg gccgcgcgca gcatcgagcg ttcggcgaag 1320
agccgctgga agccggccag cgtcaggtcc ggcgtcgcgc ccggcctgag cggcgccatg 1380
aactggaagg cgtcggtgcc ggccatgggg tagagcgcga ccatccccct gcccggccgc 1440
acccaggcgt ggatgtgctc gcggtccagc ccctcgacgc gcacgtcccc gagggccatg 1500
cgctcctcct cgcgggtctc gccggtgaac ccgatgccga gcgccttgcg caccgtgctc 1560
ctgccgccgt cggcggcgac caggtagcgg gcccgcaggg tcgccccggt gccgagccgg 1620
gccgtcacgc cgtgcgcgtc ctgctcgaag ccggtcagct cgacgccgtg ctcgacggcg 1680
ccgcccagct cggccagccg ttcccgcagc accgcctcgg tgcgccactg cgggtgcatc 1740
cgcaccatcg ggtacggcac ctgctcgctc gccgccgccg gccggtgcat gcggccctgg 1800
tagaccggca gccgcccgat cctgacccgg atcggcgggt agggcgtcgt cccggccagg 1860
acccggtcga cgatcccgag gtcgtcgaag atctccagcg tccgcggctg gaggcccttg 1920
cccttggagc ccgccgaggg cgccgccgcc cgctcgacga cgcggtgggc gatcccgcgc 1980
ctggccagct ccacggcgag ggtgaggccg gtgggaccgg cgccgacgat gagcacgtcc 2040
gtcttgtccg tcatggggcg accgtgccag gcgccgctgc cagatctctg tcagcgccct 2100
gccggaggtc acgagcaggg cggacggtag ggcaggtcgt cgaagtagcg ccgccactcg 2160
gcccgggtga gcggcatccc ggcggcctcg cagacgcgcg cggccaccga ctccggccgc 2220
aggtcccaca accgcaggtc gtggccggcg gccgagacga gggtgtggtc gtcggcgtac 2280
aggacggtcc ggaggtcggg gtgggacagc tcggcgatcg ggcggcggga gcgcaggtcc 2340
cacaggcggg tggtgcggga ggcgtccgtg gtggccaggg cggcgccgtc ggggctgaac 2400
ccggcgatca ggacggtgcc gccgtgcccg cgcagggccg ccggacgggg ggcgcggccc 2460
gggtcggcga tcctccagac gcgggcggtc cggtcgtggc cggtgctgac cagcacgtcg 2520
cccttcgggc cgaagcgcac gccggccacc gcgtcggcgt gctcctcgtg gtgggcgacc 2580
tggtcgaggg cgcggcggcg cacgtcccac agccggacgc cgcggtcggc gccgccgctg 2640
gccagcaggc cgccgccggg gctgaagtcg agcgcgccga cgtcgccgaa gccgtcgcgg 2700
gcggccaggc ggcgcggcgg gccggtgacg tcccagagca gcagccggcc gccgtggtcg 2760
ccggtggcca ggacgcgtcc gtcggggctg agcgcggcga ccgcgaggcc gcgcaccggc 2820
agccggacgt gccgggccgg gcggtagggg tcgcggatgt cccacacggt cacggccacg 2880
ccgcgctcga cggtggccag ccggcggccg tcgccgctca gcgccggatc ggcggccggt 2940
ggaacgttcc agcgcgacag cagcacgcgg tggcgggggt cgcggacgtc ccacagcgcg 3000
ccgccgcgcg cgccggtcgt ggccatcagc gtccggtccc ggctcagcac cagcccggtc 3060
accgggtcgg gcaggcgcac cggatggcgc cactggtaga aggcgccggc cgccgacccg 3120
gtggccagcg cctgaccgtc ggcggtgaag gcgaggccga gcacgcggtc ggggtgcggg 3180
aacgaggtga cctccgcgcg ggtggccacc gtccacagcc ggaccgcgcg gtcgtcgccg 3240
gcggcggcca ggacggtgcc gtccgggctg aacgccaggg cgccggcggg gtcgccgtac 3300
cggacgggcc ggtcgcagcg gcgggcgcgc aggaggcaga gccaggtgcc gcggccgggc 3360
gccgaccagg ccagcgcgcc gccgtcgggg ctgatcgcga gcgcggcggg gccgtcctgg 3420
aggccggtcg cgatcgtgtc cgcgggggag gcgggccggg ccgggtccca caggcgcacg 3480
ctgccgtcgt cgtggccggt gaccaccgtg tcgtcgccga cggcgaagcg ggtgacctgg 3540
gcggaggcgc gtaccggagg cagggcgacc gggcggccgg cgagcctcca gcgccggacg 3600
ccgtgcgcgg cggcggcgag caccgtgcgg ccgtccggtg tgaacgccgc cgtcgccgcg 3660
ccgggcaggg cggcgacgtg ccgggggcgg gcggggtcaa cgacgttcca cacctcgacg 3720
ccgtgccggg tggccgcgag caggacgcgg tcaccgccgg agggccggaa ggcgagcgcg 3780
ccgaccggac cggcggcgcg cggcagggtg gccagcgggg tgcggcggcg cgagtcggtg 3840
gcgtcccaca gctcgaccac gccgttgtcg gcgccggtcg cgagcagggc gtggtcctgg 3900
ctgtaggtga tcgcccccag ccggtcgccg gccgagcgca ggcgggtggc gtacggctcg 3960
gcggtgtcga gcagcgtgcc gcgggtgccc ggggtgtcgg ccaggccctg ggcggccacc 4020
gccacctgca tcgccagcgc cgggtccgtg gcccgcagcg cccgcgcctg gacggcggcc 4080
cgctcggcga gcgcggtgac gcgggcggcg ctcgcccggc cgctcttctg ccacgccagc 4140
cccgcgcccg ccccggcgac caccaccagc accaccagcg ccaccaccag cccgcgcacc 4200
ctccgcccga ccggacacgc cctggcgccc ccgccggagc cggaggcggg agccgcgggc 4260
cctccgccag aggaggaagc cgccgactcg ggatcgggag aggcgggaac gggatcctcc 4320
cggtcggagc ggggaagggc gggtctctcc cggtcgggag agggagaagc gggcgcgggg 4380
ccgggggcga gaggcggcgc ggtgacgtgc cggtaggcgg cgtgggtgag cgtgtcgccg 4440
tcgcggcggt gccaggtggc gcgcatcgcg tgggacaggc gcggcagcgc gtcggccgcc 4500
gccggggcgt cgtcgcgtac gcccaggtcg tgcaggatcg tctcggccag ctccggctcc 4560
agggacagcc ccgcgacccg ggccggttcg gtgatcgcgc ggcgcagctc gtcgccggtc 4620
atcgggccga gcggcagcga ccgcgtccgc agcgcctcga ccagtggcgg aaaccccagg 4680
caccgctcgt agcagtcggc ccgcatcccg atcaccaccg gcagcccgcc cgccgcgagc 4740
gcgcacagct cctcgacgta cgcctcccgc ccggaccccg cccgagccgt gaacagctcc 4800
tcgaaccggt cgaccaccag cagcgcgcac tccgccaccg gccgcccccc gcccgggacc 4860
ggcgagccgg gcgtacgcca caacaccccg gcacggcccc ccgccggcga acccgccagg 4920
acggccggca ccagccccgc ggccagcacc gacgtcttgc ccgaccccgc cgccccggcc 4980
agcaccacgg gacgggcggc gcgcggctcc ccgagcagcc ccaccagctc ggacacgacc 5040
cgctcgcgcc cgaagaacca cctggcgtcc ccggtcgtga acggccccgg cggccagggg 5100
cacggatccc gcgcgcaccg gtcccgctcg ctccccagcc ccgcgccgaa cccgtcgtcc 5160
atcccgctcc cgtcactttg tgaagttgat cggggactca gggtacgcgg gcccgccagg 5220
cggcgtacgc cggtcgggaa ggcggcatcg cccggaccgg cgcgaagccg gggacggtca 5280
gcgcttgcgg ccgacgcccg cgcagtagac ggcctcgcgg tcggcgtaca gcgtctcggg 5340
gacgggacgc cagcgcggca gcggcaccac gcccggctcc agcagctcca gcccgtcgaa 5400
gaagctcgtg acctcctcaa gggtgcgcag cgtcatgggc gccccgccgc tctcgttcca 5460
gtggcggacg ccctcttcga gctgcctgcc gaccacgccg tgggagacgg ccaggtggct 5520
gcccggcgcc atcggctcca gcagctcggc gatgatcgag cgggccaggc cggtgtcggg 5580
aaggaagtcc acgacgccga gcagggtcag cccgatcggc cggtcgaagt cgaggtgctc 5640
ggcggcggcg gccaggatct tgccggggtc gcgcaggtcg gcgtcgacga aggcggtcgt 5700
ccccgcgggc gcggcccgca gcagcgcccg cgcgtgggcc aggaccatgg ggtcgttgtc 5760
gacgtacacg acgtggctgt ccggggccac cgcctgggcg acctggtggg tgttgtccgc 5820
cgtggggatg ccggtgccga tgtccaggaa ctgccggatg cccgcctccc cggccatgta 5880
ccgcaccgcg cggccgagga actccctgtt cgtacgggcc agcaccggca ggtcgggcac 5940
ggcggcgagg atctgctcgc tcaccatccg gtcgatctcg tagttgtcct tgccgcccag 6000
ccacaggtcg tagatgcggg cggggctcgg aacatcggtg ttcagcgaac gttcgctcat 6060
cgcgacgcct tccggacgaa aggggctacg ggggagatcg tagccccaag atcccttcaa 6120
ggggactcag agcaggcggc gctggatcgg gtcggccatg gccgtggtga cgatccactc 6180
gacgacggcg gcgtcgggcg ggtcgtcctc gcggccggcg aacagcaggt gggcgctgcc 6240
gatcagggtg agcgccaggg cgtcgacgtc ggcgtcgacc gcgatgcggc ccatctcgcg 6300
ctcggcggcg aggtaggcgg cgatcatggt cgtggcctcg cccaggagcg ggatgcccgc 6360
cgacccggcc tggcgcaggc gggcgcgcag gccgtcgcgg gagatgacga ggctgacgac 6420
ggccacggcg accgagccga acagctccag cagcgcgagg gtgaggttcc ccgcgacggt 6480
gccggtgccg gcgctcgcgc gcagggcggc ggactgggcc tcgacgcggc ggatgcggtc 6540
ggtcaccagc tcggcgagga aggcgtcgaa gtcggcgaag tggcggtgga gcacgccctt 6600
ggcgcagccc gcctcggtgg tgaccgcccg gctggtcaga gcgttggccc cgtcgcggag 6660
caggacgcgc tcggcggcgt cgaagagctg ttcgcgcacg tcgcggaggg ctacgcctgt 6720
gggcaccggg ccggtctcct tccttcgcga atcctgttca gagtagacga gtgggcacgt 6780
gcccattaga gtgggcgcat gcccactcta cccccgagaa cgagccgcac ctgcaccggc 6840
ggatggccga gtccttcggc gccgacgccg accgctacga ccgcacccgc ccccgctacc 6900
ccgaggagat ggtgacccgg atcgccgggt cgagccccgg ccccgacgtg ctggacgtcg 6960
gcatcggcac cggcatcgcg gcccgccagt tccaggcggc cggctgccgg gtgtccggcg 7020
tggacgtgga cccgcgcatg gccgacctgg cccggcgcga cggcatcgag gtggacgtgt 7080
ccgccttcga gtcctgggac ccggccggcc ggacgttcga cgcggtcgtc gccggccaga 7140
cctggcactg gatcgacccg gtcgcgggcg cggccaaggc ggcccaggtg ctgcggcccg 7200
ggggcaggct ggcgctgttc tggaacgtct tccagacgcc ggacctcctg gccgacgcct 7260
tcgccgaggt ctaccgccgc gtgctgcccg acctgccggc gctcggccag gccggccccg 7320
ccgtgggcat gtacacgtcg ctgctcgaca agatcgccga cggggtccgg acggcgggcg 7380
cgttcggcga gcccgagcgg tgggactacg cgtgggagcg cgtctacacc cgggacgagt 7440
ggctggcgca ggtgcccacg cacggcccgg tcaccggatt gccgccggag cggcgccgcg 7500
aactgctggc gggcatcggg gccgcgatcg acgcggtggg cggcgccttc accgtgcact 7560
acgacgtcct ggtgctcacc gccgtccggc gggctgagta gccgttactc atgcggtcat 7620
gcgagcgcgc gcggacgatc ctcctcaggg cgacacccca ggaggaacca ccgtgagcgc 7680
cacacatgac atgagaaccg ggcggaaagg gccggcgcgg tggcgcgggc ggctcacgtt 7740
cgccgcgatc ggcgtgctgg tcgccgcctt gatcccggcg ctggcgacgc cggcgtcggc 7800
ggcggtcccc ggcttctcgc tgaccttctc catcgactat ctccgccaga tcgaggaccc 7860
ggacagcgga ggcgtcggcg acggcgacta ctaccccaag gtcaggatcg ccgacggccc 7920
gctcgagacc ggcccgcgca tcgaggacga cgagttcgag cccctcggcc tgcccgaggc 7980
cccgggcggc tgggtcttca ccaagtccga cctgcccggc gaccagccga ccgtgaacat 8040
caccgtcgcc ctctgggact atgacggcgg cctcaacacc aacgacgacc gcatggacat 8100
cagcccccag aacgacgacc tcgacctcga cctcgtctac gacgtgcgct cgggcaccct 8160
gtcgggcgac ggcgtgcagt tcggcacgcc gtgcgtcgac tcccaggggc gggagaaggg 8220
ccagacgtgc gccgagggcg acggcgacca cggcttcccc agggacaacg acggcaggaa 8280
gaccaggatc ggcttcaccg tctccaccgt gatgcccgac accgaccacg acggcatccc 8340
ggacctcgtc gagctgtccg ggatccgcga caggaacggc ggggtcatcg ccgacctccc 8400
ggcgctcggc gcggacccgt gccgcaagac gatcgtgctg caggccgact acatggccga 8460
cgccaggcac tcccaccggc ccaagccgga cgcgatcacc ctggtcagga acgccttcga 8520
cgccgcgccg gtcaaggcgg agaacccgtg cccgtacccc gggacgcacc gggacggcgt 8580
cgacttcgtc tacctccagg gcgcgcaact gcccgagcag gccgccatgg gcctggccga 8640
cgacagcgac ttccgcgacg cgcgcaccgc cgacttcccc gccgagctgg cccggtacgc 8700
gcactacgcg atcttcgccc acgacctcgt cgtcaccacc gcgaccggga gctcgacgtc 8760
gccgggcacg tcggggcagt gctgcgagcc caccagggac aacaacaagg acttcatcgt 8820
caccttcggc tcgtggcgga cgatgtgcgt ggccgacttc ggcgtggact acggcggcga 8880
cggccggctc cagaccaccc cggcgggcga cgacgtggtg atcggcaccc agatccacgt 8940
gggcccggac cgtacgtgcg acaccaccgg cgcggacccg accgaccggc aggtgctcac 9000
cgtcgggacc ggccgggacg acgccgaggt cggcaccgtc caggaccagg ccgggaccat 9060
catgcacgag ctcgggcacg ccctgggcct gcggcacggc ggcgacgtca acgaccagtg 9120
gaagcccaac tacctcagcg tcatgaacta cttcttccag tcgggcatcc ccaccggccc 9180
gccgccgctg ctggtcaacc agctcaggga ctggaagatc ggcgcgatcc gggtcggcta 9240
ctccagcggc ggcctgcccc cgctcaccga gggcacgctg aacgagtccg ccggcatcgg 9300
cgacggcacc gaccacacct tctggtggac cgacaacctc gccaacccca ccagcacgtt 9360
ccggccgctg cgctcggccg ccggcaacgc gccgatcaac tggaacgaca acaccgcccc 9420
cggcaccggc gccccgctca tcgacacggg gacggtgaac gtggacatca acgccagcgg 9480
cggcagcacg accctgcgcg accacgacga ctgggccgcc gtcaagtacc gggccgcgct 9540
ctcgccggac gccaagggct gggcctgcag cagcttcccc gtctccgcgc ccacgtgcag 9600
cggctcgggc agcggctcgg gcagcggctc gggcgccgag ccggagaagg agctcgactt 9660
caccaccgcc gtacggcagg agatgtcctt cttcaacctc tacgaccccg acatcgtcac 9720
cgccaagagc gtcgacaagc ctgacgccga acccggcgac accctcacct accaggtgaa 9780
gctggacaac gtcggcaccg gcccggcggc ctccgtcggc gtgaccgaca ccccgcccac 9840
cgggccggct cagacccgcc aggttcccta cctgggcgcc ggacggtcca cgaccgagac 9900
gttcacgtac acggtgccgt gcgacacggc cgacgcggcc gtcctgacca acacggccac 9960
cgccaccgcg acggacaccg ccgacggccc cgaggccgac acgggcgaca acatcgccaa 10020
ggccgccacc accgcgcacg cgccgcggct caccctgacc aagaccgccc cggccacggt 10080
gaacgccgga gaggcgatga ccgtcgggct gcgggcggcc aacgtcggca gcggcagggc 10140
caccgacgtc gtcctcaccg acacgctgcc caaggaggtc tactacagca gcgcgctcga 10200
ccgcggcagc ggcccccgtc ccggcacggt cacccgcaac ccggacggca ccaccaccct 10260
gacctggacg ctcggccccc tcgacggcgg cgcggacgtg tccgtcgggt tcacggcccg 10320
gcccggcctg ctgttcacga agggcgccga gctgcccgac acggcggccg ccgcgtaccg 10380
gaacgcgggc ggctgcgtct acgagcccgc cacggcgtcc gccacgacga cggtcaccga 10440
ggtcgccccc actcgcgacc cgcgctcgca cggctactgg aagacgcacc aggaggctcg 10500
caccgccgag ttcctggccc gcgtccaggc caccgaccag cggttcgaca ccgggcagga 10560
cggcgagctg gccgacgccg aggccgtcgc cgcgctctcg gccggcggcc cgcaacccgg 10620
tccggcccgc ttccagctcc tggccacgct gctcgacctg gcggcccggc aggtcaacgc 10680
gagcacccgg ctcgactcgc cgctcgtccg gcggctcggg gtgcgcacgg tcggcgaggc 10740
ggtcaggtac gccttcgcca ccctcgacct gccgcccggc ggcgcggcgg ccgggcgcta 10800
ctccgacacc acctcgatgc tcgacgagat cgtgaacaac cggagcgagg tctactgacc 10860
cccgccagga cgccgccatg agggcgccgc ggccggctca ggccggttgc ggcgtccgcg 10920
gctcggccct ggccggggcg tagaggccga ggcggcgcag ggccggcgcg gtcatcatgg 10980
tggtgatcag ggccatcagc acgagcaccg tgaacgtgcc gggcgagatc agccccatgc 11040
cgagcccggt gctgagcacg acgatctcgg tgacgccgcg ggcgttcatg agcacgccga 11100
gcccgagcgc gaggcgtccc ggcatgccgc ccgcccaggc gatcaccccg gccgcgccca 11160
gcttgccgag gacggcggcc accagcagca ccgccccgcc cagcagcacc accggatgac 11220
cgaacgccag atgcacgtcg gtgcgcagcc cgatcgaggc gaagaacacc ggcagcaaca 11280
tcgcgcggtt gagcgagccg aggcgttcgg gcaccgcgcc gagcacgggc gcgtcgcgag 11340
ggaaggcgac ccccgccagc agggcgccga agatcgcgtg cacgccgatg gcgtcggtcg 11400
ccgccgccag ggcgaagatc aggcccagga ccagcgcgag cgccgccgga gcgggcagcc 11460
ggcgggcggc gtgccgggcc gccagggcgg cgagcgcggg gcgtacgacg agggtcacgg 11520
cgaggaccag ggcggcggcc agccccagcg aggtcagcac gcccgccggg gaaccggcgt 11580
gggccagggc gatcaccgcg gccagcgcgc accaggcgag cacgtcggcc agccccgcgc 11640
acaggatcgc cagcgagccc agccgggtgg cggtcagccc ggcctcctgg aggatccggg 11700
ccagcacggg gaacgcggtg acgctcaacg ccgtcccggc gaacaggacg aacgccgtgc 11760
ccgacgccga cggcccggcg agcgcaccgg cgaacgggac cgccgcgacc gctcccaggg 11820
cgaacggcac ggccgtcatc gccagcgcgg cgacccccac gaccacgcgc tgccggccga 11880
gggcggccgg gtcgaactcc cgccccaccg tgaacatgaa gaccgccagc ccggcctggg 11940
ccagcagttc gagctgcggc gcgacccagc cgggcaggat cagcgcggac accccgggtg 12000
ccagcgagcc cagcgcggac ggcccgagca gcagcccggc caccatctgc cccaccacgg 12060
acggctggcc cgcccggcgc gccaggacgc cgcccagcga ggccagcccg ccgatgaccg 12120
ccacggcgag gaacagccgc aggatcgact gggcgggcgg caccccggcg tgcgcggccg 12180
gcgccgccgc ctgccccggc tgctgcgcgc ccggcccgaa caccagcagc atcgacacgc 12240
ccgccacggc gatcagcagc agcgcgaccg ccaggccccg ggccggggcg ggcagcaggg 12300
cctgttcggt gtcgtcgagc gggtcgtgtc cgcgcatggt cgcaccttcc tgcctactcc 12360
gcgatgggct cccggcccct cggcggccgg ctcagtcggc ggccgggagc ggccgggccg 12420
cccgccggcc gatcagcagc agggcgccga cggcggcgac gatgccgacg gcgatgacgg 12480
ggatcagggc ctggtagccg atctcgccgg ccaccaggcc gaacagcggg ggaccgagca 12540
gggagccgag gctgccggcc tgggcgatga cgccgttgcc gacgtcggcg tggtcgaggc 12600
gttccaggac gagggggagc gcggcgaaga ccagggcgcc caggaagccg ttctccatgg 12660
agatgacgcc cgcgccggtg agcgcgcccc cggcggagcc gccggcgtac aggatccagg 12720
cggccggcac gacgaggaag cccgacaggg ccagcacgcc gacccgtacg cccctgcgca 12780
gcaggacgcc caccgccagc ccgccggcca cgccgagcag cgagatcacc gaggtcagcg 12840
cgcccgcggc ggcggccgag cggccgtact gctcgatgag cagcgtgggc agcagcacga 12900
tcaccgggat ggtcaccagc gcggtcaggc agaaggccgc ggccagcacg cccggccagg 12960
tgagcgcccg cgcccggggc accggcccgg ccgcctgcgc cccgtcggcg cgcggcagcc 13020
gcgcccacgt caccgcggcg atcagcagcg tcagcccggc gatcaggccg gtccagccgc 13080
gccaccccag cgcgtcgccg gccgcgccgc cggccagcgt gctggccccc agcccgaccg 13140
ggacgaacgt cgcccagacc gacagggcgg tgcccctgtc gcgctcgtgg gccaggcgca 13200
ggatcagcgc cgggcaggag atggtgacca ggacgtagcc gacgccctcg acgccgcggg 13260
cgatgagcag ccaggcgaag tcgttcgccg ccatgctggc cgcgcccgcc gccgcgatca 13320
ggaccaggcc ggtgaccagc gaccgcccgg tgccgaagcg gcggacgaga taccccgccg 13380
gcaggccgac caccgccccg acgccgacca ccgccgagat cacccagccc agctccggca 13440
gcgacagccc gagctgggcc gccatcgcgg gcccgaccga ggagaacttg ccgaggctca 13500
tcgcgccgac gacccccccg acgtagacga ggaggatcgt cagccagggg gagcgtcgcg 13560
gggacgcctc cgtgatcttt gccgccgacc gggggtccat gagtctcctt cgcactgcgc 13620
tccgcgggcc agggttccca cgtccgatct tcgccatgcc ggcggcgccg atcaacccgc 13680
ttcccccgtg gcgaagatgc cgatgcccga ctcggtgacg ttgaccgccc cggccgacag 13740
cagtgcgtcg atcgtctcgg gcacctcccg cctgggcagg acgccctgga ggacgaccat 13800
ggtcccgtcc gcgaggtcgg cgcccgcgcg ccacgactgg cccggcatcg cgggcaccac 13860
ctcggccagc cggtcggcgg gggcacggac cgtcagcagc acgtgggccg ggcccaccca 13920
ggcggcgcgc agcagcaggg cgacgttcct gatcacgccg cgcctggccg ggtcggcgta 13980
ggcggccggg ttggcgacga gctgcgcgcc gcaggtccgg acggtctcga tgacgcgcag 14040
gtcgttctgg cgcagcgagt ggccggtctc cacgacgtcc atcatggcgt cggccagctc 14100
ggggatcttg gcctcggtcg cgccgtagga cacgaccacc tcggcccgcc gtcccgcctc 14160
gcggaagaag cggcggccga tgttcgggta ctcggtggcc accctgaccc cgtcgtcgag 14220
gtcggcggcg ctgcgggcgg ggtggccgga cggcaccgcg agcaccagcc gccacggccg 14280
ctcggtgttc ctggagtagt cgaacgactc caccacctcg acctcggccc cggtctcctc 14340
cacccagtcg gcgccggtca gcccgaggtc gaaggtgccg tccccgacga ccagggggat 14400
ctcgcgcggc ttgtagaagg ccacgtgcga cacgccggag tggtccaccg tgccgcggta 14460
ggagcggtcc gagcgccgcc gtacgggcag ccccgcggtc gcgaagagct ccagggcccg 14520
ccgctccagc gaacccttcg gcagggcgag cgagatcatg gtttcctcct gacgggcacg 14580
gcgggacggc gggggcgcga gcatcacacg tgcagcagcc ggtcgatgcc gacgacgggc 14640
ggctcgggca ggcaggacgg ccgcacctcg atctcgccca ccacgaggtc gtccggccag 14700
gagtcgacga tggtggccac gcagcgggcc accgaccggg tgctcatctt gtgcggcgag 14760
ccgtcgccgt cgaggttggc gatggcgccc ggcgagacca ggcaggcgcg caccccgttg 14820
cggcgctcct ccagcagcag cgtctcgacc agcgccttca gcccggcctt ggtggcgctg 14880
taggcggccc cgccctcgaa gtaccgcgtc ccggcgtggc tgcccatcgc gacgaacatc 14940
ccgccggcgg cccgcaacgc cggcaggacg gctttgagca ggtggaagca ggcgctcagg 15000
ttgatcgcga ccagccgctc ccagtcctcc gcgcgcagct cggcgatcgg gtcgagcacc 15060
cggtccaccg cgttggacac gcagacgtcg agccggccgg ccgagccgag cacctgggcg 15120
gtcgcctccg cgatctgcgc cgggtccgac aggtcgcacc tgtgctcgcc gagccagtcc 15180
tcacccgcga gcgagcggtt cagggagaag acgcggtagc cgcgctcgcg cagctcctcg 15240
gcgatggccc gcccgctgcc ccggttgctg ccggtgacca gcgcggccgg ggtctcatgt 15300
gatgacatcg aggacgcggc tccactggtt cacgatctcc tcgcccgcca cgccgtcgcg 15360
gaacaggtcc ttgtccagcg gggagccgtc ctggtggcgc aggcgcatgc agtcctggga 15420
gatctcggag atgacggtca gcccgccgtc ggaggtccgg ccgaagatga ggcagaagtc 15480
ccacacctcc agcgggcgca gccaggaccg cagcacgtcg ttgacggcga gggacagctc 15540
ccgcatctcc tccaccggca ggtccagcgc ccgcaggtag tcctcgccga tcggctggtc 15600
ctcggggtcg gtccggtagt cgaacttcac gaccgggcgc ggcagcgggg cgccgtcctc 15660
gaacaggccg gggtacttgc gggtggtcga ccccgtggcc cggttcttca cgatcacctc 15720
gaacggcggc gccgggcagt agtcggccac gtacgagtcg gccgagaccc gttcccggaa 15780
cgcgcacggc acgccggcgt cggccagccg ggccgccgcc ctttcgtaga aatcgagccg 15840
gagcggcccg gtgcgctcga cgatttcatc acggtggtac gtgaaactgc gcaggctggg 15900
gattatctgc accaggcacc gcccgtcggg cagcagccac agacgcttac tgcgcccgac 15960
gatatccggc tctccccggc cggccggagt tcccggctct gatccgttat ccacgccgcc 16020
tccatccgcc gacaatggcc cacacaactt gatattgcgg gcacctcgtt gtcaaggcgg 16080
cgtgaccctg gaaaaaggag cacttgacgt ggctctcgcg gtcttgatat gtcgtccgcc 16140
ctcgccggga gcactggaag aaagagtact tgacgggtcc cggcgaccgg acggtactgt 16200
cttcagaaat tgaaattccg atcttaatta gaatcccaat ggggggagga tcgttgtcgg 16260
aatatgagcg catagctcac tcgaagatca cgcgtaagtt caggtccgcg gggcagcagc 16320
tcttgttctc gcgggggcgg cggggcgagg tgtccgacgc gaacggcgta ccgtacatcg 16380
atttcgtcat gggttatggc ccagtgataa tcggccacgc ggacgcccag ttcaacgaaa 16440
ttctctgcgg ttatctcggc aacggagtca tgctgccggg ctacaccacg ttccaccagg 16500
aatacctcga ccggctgctc ggcgagcggc ccggcgaccg cggcgcgttc ttcaagaccg 16560
cctccgaggc cgtcaccgcc gccttccggc tggccgcgat gcgcaccggc cggctcggta 16620
tcatccgcag cggctacgtc ggctggcacg actcccagat cgccgactcc ctcaagtggc 16680
acgagccgct gcactccccg ctgcgggaca agctgcgcta caccgacggc atgcgtggcg 16740
tcggcgagtc cgagccggtg gccaactggg tggacctgcg cctggaatcc ctcgccgagc 16800
tgctggaacg gcaccgcggc cggctgggct gcttcgtctt cgacgcgtac ctggcctcct 16860
tcaccacggc cgacgtgctg cgccaggccg tcgccatgtg ccgcgaggcc ggcctgctca 16920
ccgtcttcga cgagaccaag accggcggcc ggatctcgcc tctcggctac gaccacgaca 16980
acgccctcgg ctccgacctg atcgtgatcg gcaaggcgct ggccaacggc gccccgctga 17040
gcatcctggc cggcgacgcc gacctgctcg cgctggccga gaaggcccgg ctgagcggca 17100
cgttctccaa ggagatgatc gccgtctacg cggcgctggc cacccgcgac atcctggaga 17160
agcccgtcgg cgactcgccg gacggctgga ccgagctcgg ccggatcggc acgcaggtgg 17220
ccgccgcctt caccgcggcc gccgcggacg ccggggtcga ggccctggtg ggcgcccgtc 17280
cggtgctcgg cggcggcatg ttcgagctcg tctaccacga cgtggagcta ctgggcgaca 17340
aggagcgccg cgaggcgctg ctggccgagc tcgccggggt cggcatcctg ctgctcgaag 17400
ggcacccgtc cttcgtgtgc ctggcccacc gcgacatcga ctggggcgac ctgcgcgacc 17460
gggtgcggca ggcgttcgag gcgtggaccg cccccacggg ggccggccgt ggctgacctc 17520
ctgcgcctcg ccctggtcgg ctgcggccgc cagatgcagc agaacctgtt cccgttcctg 17580
caacgcatcc gcggccacca ggtcgtggcc tgcgtcgacc ccgacctgtc gctggccgcc 17640
gacgtgcagt cgctggccgg cggcgcgacc tgcgtctcct cggtggacga gctcgacctg 17700
gagatggtgg acgccgccgt gctggccgtc ccgccggagc cgtcctacct gctggtccgg 17760
cagctcgccg agcgcggggt ggactgcttc gtcgagaagc cggccgggcc gtccacgccc 17820
gcgctccagg acctggagca cgtggtgcgc cgcagcgggc ggcacgtcca ggtgggcttc 17880
aacttccgct acgccgagac cctgcagcgg ctgcacgagc tgagcgagga gatccgggcc 17940
acgccgtgct cggtgaccat cgacttctac agccgccacc cgtccgcgcc gcagtggggg 18000
gtggacacca ccctggaggc gtggatccgg cacaacgggg tgcacgccat cgacctggcc 18060
cgctggttcg tgccgtcgcc ggtcgtccag gtggacgcgc acgccatcgc cagtgacgcc 18120
gaccggttcc agatcaacct gttcctgcgg caccacgacg gctcgctgtc cacgctgcgc 18180
atgggcaacc acgtcaagcg gttcatggtg ggcgtcaccg tccagggcat ggacggcagc 18240
cggtacagcg cgccgtcgct ggaacgggtg acgctcgaac tgtccgacgg cgtgccccac 18300
ggccaggagc tgcacgccac ccgcaacctg gaccacgggt gggggcgcag cggcttcggg 18360
cccgagttgc aggcgttcgt ggacgcctgc gcgcagcggt cggccgagcc ccagacgggc 18420
ggccgtcccc cggtcaaggg cgtgccgtcg gtctccgacg cgctggccgc ctccgccctg 18480
tgcgaccggg tgatggccga gctcaacacc gccgcgacga acggcttcgg cctgctgacc 18540
gggtccgtcc gggcctcggc ctaggccggg ccgtcgacga ggagggggag gcatgaccga 18600
gcgggcgcga ccggccgcgg gcgggcggcc ggtcgcgtcg ccggatccgg ccgggtcacc 18660
tgatccggcc gcggcgcggg gggtgggccg gtggatcgcc ggtcatctgc gccggcaccg 18720
gccgtcgctg ctgctgttca ccgtggccgc cgcggcggcg agcctgtgct ccacgctcgt 18780
ccccgtccag atcggcgcag cgttcgccga ggccaccggc ccgcgccacg acctcggcgc 18840
ggtcggggtg gcggcgctgg cggccgcgct gctggcggcc ggccggttcg tcgccgacct 18900
gctgtcgaac ggctcgatgg aggtcgtcgc ccagcgcgtc aaacgggacg tgcgcgacga 18960
gctgtaccgc agcctgctga ccaagcgcat ggccttccac gaccagcagc gcatcggcga 19020
catcctggcc cgcgccatca acgacgcgag actggtcgac tacatgctca gccccggggc 19080
ggccaccgcc gccaacggcg tgctggccct gttggtgccg atcctgttca tcgcctcgct 19140
ggacccgcgg ctgctgctgg cccccggcgt gctggtcctg gtgttcgcct acgcgctgcg 19200
ttaccacctg cgccggctct acccgctggt catgcgcacc cgcgagacct tcgccgacct 19260
caacgaacgc ttctccacca cgctgtcggg catcgccacc gtcaaggcag ccacgcagga 19320
ggacttcgag cggcacgccc tgagatcggc cgccgccgcc taccgggacg ccttcgtccg 19380
gcgcggccgg gcgcaggcgg tctacctgcc ggcgctgagc ttcgccctcg ccatggtgac 19440
cggctccctg cactccctct acctctactc gcagggggag ctggcgctgt cccaggtggt 19500
gacctacctc ggctggctgc tgctgttcgc gcagcccgtc accatgtccg agcaggccgt 19560
gccggtcatc caggagggct tcgcggccgc cgcgcgcatg cggcagatca tcgaaggcgc 19620
ccccggcgag cgcgaggaca cccgcggcag cacggccgcc gtcgagggtg cgatcacctt 19680
cgaccgggtg tcgctgcgcc acgagggccg ggacatcctg cgcgaggtct ccttccacct 19740
gcccgccggc cgcaccctcg ccgtcgtcgg ccccaccggc agcggcaaga gcatgctgat 19800
caaactggtc aaccgcatgt acgacgccac cgagggccgg gtgctgatcg acgggcggga 19860
cgtgcgcgag tgggagccgg gcgcgctgcg ccgccagatc ggccacgtgg accaggagat 19920
cttcctgttc tccaagagcg tcctggacaa catcgccttc ggcgccccgc acgccgccgg 19980
gtacgaggac gtcctgcgcg tcgccaagca ggcgtgcgcg gacgagttcg tccaggggat 20040
ggccgacggc tacgccaccg tgctcaacga gggcggcacg acgctgtccg gcgggcagcg 20100
gcagcggctg gccatcgcca gggccctgct gaccgagccg cgcatcctca ccctcgacga 20160
cgcgaccagc gccgtggacg cccgtaccga atcggccatc accgaggcga tcgagcgcgc 20220
gacggcgggc cgcacgacgg tgctggtctc gcaccgcccc ggccagatcc gccgcgccga 20280
cctcatcctg ctgctggacg gcggccgggt cgtcgaccag ggctcgcacg acgaactgat 20340
ggcgcgctgc gcgctctacc gggagatcta cagtggctga cgcccaggat cggcggcgcc 20400
cgtgagcgac ctgttcgcgg gcctgcacgc cgacgcctac gaccgcgtgt acggcaaccg 20460
ccgcctgctg gcccgcatcc tgcggcagtt gcgcgcccac cgcgccggca tgatcggctc 20520
ggccctgctc gtcgcgggct cggtgctgct gaccgcctcc gtgccgatcg tcgtctcccg 20580
cctgatcgac caggccggcg gcggcagcgg ccccggcctg ctgctgtgcc tgttcgtggt 20640
cgcggcggcg gtgctggcct gggcgatgaa ctgggcccgc caggcgatca ccgcgcggct 20700
ggtgggcggc atggtctacc ggctgcagtg cgaggccgcc gacgccgcgc tcgccaagga 20760
cgtcgccttc tacgacgaga actcggtcgg caaggtgctc agccgggtca ccggcgacac 20820
cgagggcttc ggctcggtgc tcaccctcac cctgaacctg atcagccagc tgttcctggt 20880
ggtgctgctc ggcggcgtgg tgttctggat cgaccccggc ctcggcctgg tcatcctggc 20940
ggcgctgcct ttcctgctgg gcaccgccct ggccttccgc cgggtggccc gcacggcggc 21000
ggcggccacg cggcgggtga tggccaaggt caacgccaac gtgcacgaga ccatgctcgg 21060
catcgcggtc gccaagagct tcggccgcga gcgggccgtc cacgacgact tcgacgacgt 21120
caaccggctg tcgtaccgcg tctacgtccg gcagggcctc atctacgcgg tcatcctgcc 21180
ggtcctgacg ctgctggcgg gcctggccac ggcggtcgtg ctgtaccagg gcggccggtt 21240
cgcggcgatc ggcaggctct cggcgggcga gtggtacttc gccctccagg cgctcggcat 21300
gctctggcag ccggtcaccc aggccgcctc gttctggagc ctgttccagc aggggctggc 21360
cgccgccgag cgggtgttcg cgctgatcga cagcgaccac gccgtcgtcc agagcggcaa 21420
cgcgccggtg accgcgctga ccggcgagat cgaggcgcgc ggcctgcact tccggtacgg 21480
ctcgggcgcc gccgtgttcc gcgacttcga cgtgcgcctc gcggcccgcg agacggtggc 21540
ggtcgtcggg cacaccggcg gcggcaagtc cacgctggcc aagctgatcg cccgcgccta 21600
cgacttccag ggcggcgagc tgctggtcga cggccgcgac atccgcggcc tggacctgcg 21660
ggcctaccgg cggcgggtgg gggtcgtccc gcagcacccg ttcatcttcg cgggcacgct 21720
cgccgagaac atcgcctacg gccgccccgg cgccggccgc gacgacgtcg tacgggccgt 21780
cgagcggatc ggccaccgcg tgtgggaccg cagcatgccg atgtccctgg acgaccgcct 21840
ggccgccggc gggcagggcg tgtcggtcgg gcagcggcag ctcatcgccc tggccaggat 21900
gttcgtccgc gagccggaca tcctgctgct ggacgagccg accgccagcg tcgacccgct 21960
gaccgaacgc ggcatccagg acgcgctggc ccggctgtgc gccgggcgca cggtcgtggt 22020
gatcgcccac cggctgtcca ccatccggcg cgccgaccgc gtcctcgtgc tgcacggcgg 22080
cgagatcgcc gagcagggcc ggttcgacga gctgctgcgc cgcgacggcc cgttcgccgc 22140
gctctacgcc acctactacg cgcaccagga gaccgcatga cctgcgcccc ccatccggcg 22200
gagttcgagc cgatccagcg ggaggcgtcc gccacgggcg tgcgctgcgt ggtcggcgcc 22260
gtgctgttca acccgctcgg cgaggtgttc ctgcaacggc gggcggccca cgtgcggctc 22320
ttccccggct gctgggacat cgtcggcggg cacgtcgagc ggggcgagac gctgtgcgcg 22380
gcgctggcca gggagatcga ggaggagacc ggctggcggc tgctgcgggt gggcggcctg 22440
gtggacgtct tcgactggac cggcggcgac ggcggcctgc ggcgcgagat cgacgtgctg 22500
gccacggtcg agggcgacct gacccggccc gccatcgagc aggacaagtt cgacgaggcc 22560
cgctggctgg acggcgacgc cctgcgccgc ctggccgccg agtcgcccgg ctcgggcatg 22620
gtcgagctgg ccctgcgcgc gctggccatg cggccggtct agggctccgg tttgcgtacc 22680
gcgtccaggg cctgtcgcag ggtgaaccgg ccggcgtaca gcgccttgcc caccaccacg 22740
ccctcgacgc ccagcggggt cagggaggag acggcgcgca ggtcgtccag gctggagatc 22800
ccgccggagg ccacgacggg ccggccggtg gcggcgcaga cgtcgcgcag cagcttcagg 22860
ttggggccgc cgagggtgcc gtccctggcg atgtcggtga cgacgtaccg ggcgcagcct 22920
gccgcgtcga ggcgggccag ggtctcgtac agctcgccgg cgtcgcgggt ccagccgtgg 22980
ctgcgtacgg tggtgcccca gacgtccagc tcgacggcca cccggtcacc gtgccggtcg 23040
atgacctccg ccacccacgc cggacgctcc agcgcgccgg tgccgagcac cacccggccg 23100
cagccggtgg ccagcgccgc cgccaggctc gcgtcgtcgc gcacgccgcc gcacagctcg 23160
accgggatgt ccagggcgcc gacgacctcg gcgatccgcg cccggttcga gccggtgccg 23220
aaggcggcgt ccaggtccac caggtgcacc cagtcggcgc cggagtcctg ccaggccagc 23280
gccgcgtcga gcgggtcgcc gtaccaggtc tcggaccccg actcgccgcg cagcaggcgg 23340
acggcccggc cgccgcgcac gtcgacggcg ggcaggaggg tcaggcatgt ggacactcgt 23400
cgtttcctct cccgtgaacc ccccttttcc atcatcgcga acactggtca ggaatcgaag 23460
tcaaccttcg gccaccacca tggttcactt gccgccgccc ggccggacgg ggacgcttgc 23520
cggtgcgcgc cgcacgccca gtcgccgggg gccggtcccc ctgttccgag gaggactgca 23580
tggtctcgct cgcgctgccc aagggcacgt tcctggagcg tcccgtcctc gatctgttcg 23640
ccgcggccgg gctggaggtg cgccggccgt ccgagcgcag ctaccgggcg agcatcgcct 23700
acgacggcgg cgtcgaggtg gccttccaca agccgcgcga gatcccgctc gccgtggaga 23760
gaggcgtctt cgacttcggc gtgaccggca ccgactggat cgaggagacc ggcgcgaagg 23820
tcgagctggt cgaggcgagc gggtgcgtgc cgccctggcg gctggtgctc gccgtcccct 23880
ccgggcatcc cgccgtggac gccgccggcc tgcccgccgg ggcccgggtc gcgaccggct 23940
tcccgaagat ctcccggcag tacttccaga gcgtgccgct gcccgtacgg atcgtcccgt 24000
cgttcggcgc cacggaggcg aaggtccccg agctggccga cgccgtcatc gagaccgacg 24060
gccccggctc cgccctggac gagcacgacc tgcgcgtcgt cgccacgttg cgcacctgct 24120
taccgcaggt cgtcgcgagc cccgccgcct ggcgggacgc ccgcagacgc gccgcgatcc 24180
agcgggtcgc gcggctgctg gcctcggtcg acgggggagc ggcccacgtg ctgctgaccg 24240
tccgcaccac cacgcgcgac ctgccacggg tggccgggtc gatgccggaa cggtcctggc 24300
gggccggcac cggcctgacc gagaacctgg tggtcgtgca ggggctggcg gcccggcgcg 24360
gcctggccga gaccatcggc ggcatcctgg ccgccggcgc gctcgacgtc atcgagtcgc 24420
gcgtcgggaa ggacgtcacg ccgtgagccg ccggatgatc gtgtgcgacc tcgacgggac 24480
gctcctggac tcccgcggcc aggtctccga gcgcaccagg accgccgtgc gccgcgcccg 24540
cgccgccggg cacgtgttcg tcatcgcgac cgcgcggccg gtccgcgaca cccgtccggt 24600
ggccgcggcg ctggaccacg cggccgtcgc cgtctgcggg aacggctcga tcaccttcga 24660
cttcggcagc gaggaggtgg tcgactaccg cccgctggac cggcagccgc tcgccgcgac 24720
gctggcgctg ctgcgcgacc gtttccccgg cgtgcgcctg ggcgccgagt gccgcctcga 24780
actgctcctg gaggacgcct tccacctgcc cgagccgctg gcccgcgacg cccggcgggt 24840
gccccgcctg gagggcgaga tcgaccggca cgacgtcggc aagctcatgg tccagctcga 24900
aggcgccgcc cgccagtact acgagaccgt gcgcgggctg ctgaccggct gcgaggtcac 24960
catctcggcc gacgtgttct gcgaggtcat gcggtccggc gtgaccaagg ccgcggccct 25020
ggagtcgatg gcgtccaggc tcggcctggg cagcgccgac gtgatcgcct tcggcgacat 25080
gccgaacgac ctccccatgc tgacctgggc cggaaccgcg gtggcggtgg ccaacgccca 25140
tcccgccgtg ctcggcgcgg tgggcgaggt gaccgcctcc aacgacgacg acggggtggc 25200
cgcgtggctg gaacggcacg ccatggccga tttctcagag aagtattgac gcttgtctga 25260
cgactattca tgatggtcgc tggagccggg aacgcaccgg ccaggatcgg cctgcggagg 25320
caggccgcca aggcccgcgc cgggcgcggg cgtcccgtct gagaggtgcc ccagtgcccc 25380
gattagccgt tgcccctgtg ttcgtccgcg tcctcgcact cttcgcggcg ttgtgctcgc 25440
tcgccgcctg caccgccgtc agccccgccg ccgagaagaa gggcgccacc tcggtcggcg 25500
tggccttcag ccgcggcggg cgcggcgaca gatccttcaa cgactcggtg ggggccggcg 25560
tcgaccgcgc gaaggccgag ctgggcgtct cggccaagga gctcagcccc aacgccgccg 25620
ggtccgacct ggaggagatc ctgcggctgc tggcccagac cggccacaac ccggtgctgg 25680
ccgtcggctt cttctacgcc cagccgctgg ccaaggtcgc ccccgccttc ccccgcaccc 25740
agttcgtcat catcgacgac gccaccgtga agctgcccaa cgtcaccaac gtggtgttcc 25800
gcgaggagca ggccagctac ctggtcggcg cggcggccgc gctcaagtcc gccagcggga 25860
agatcggctt cgtcggcggg gtgcagacgc cgccgcagga gaagttcctc gccgggtacg 25920
cggccggcgc ccgcgcggtg aaacccggca tccagctctc cagcgtgttc atgtcccagc 25980
cgcccgactt cagcggtttc tcggcccccg acaaggccca ggaggcggcc cgcggcatgt 26040
acgacggcgg cgccgacgtc gtctaccacg ccgccggggc gtccgggctc ggcgtcttcc 26100
aggcggccaa ggcggccggc aagtgggcga tcggcgtcga cgtcgaccag cgcaagacgg 26160
tcgatcccgg gctcgccggg gtgatcctga ccagcgccac caagcagctc gacgtcgcgg 26220
tcctgagcat cgtccgggac gcggtggcgg gccggccgat cgccggggtg cgcgagttcg 26280
ggctcaagga gggcggggtc ggctacgcca ccagcggcgg cgagctcgcc gacatccagc 26340
cgaggctgga ggaactgcgc aagcaaatcg tctcgggtga gatcaaggtg cccgccaggc 26400
cggcggagag ccgatgagcc gggcgcgggc ggcgggccgg gaagcgctgc gcgtgctcgc 26460
cgtcccggcg ctcgcgctgc tgatcgcgct cacggtcgcc accgcgctgc tggccgcggt 26520
gggggccgcg ccggggcgga cgtacctgac gatgctggag ttcggagtcc ggccggactc 26580
gctcgtctcc gcggtgaacc ggtcggccgt ctacttcttc gccgccctcg ccgtggccgt 26640
caccttccgg atgaagctgt tcaacatcgg ggtcgagggc cagtaccacg tggccgcgct 26700
gttcgcggcg gtcgccgggt cggccgtgcg gctgccgggc ccgctgcacc tggcgttcgt 26760
ctgcctggtc gccgtgctgg ccggagccct ctgggccggg atcgccgcgg tgctcaaggt 26820
gtggcgcggg gtctccgagg tgctgtccac gctgctgctg aactacgtcg ccaccgccgt 26880
cgtcgcctac ctgctggccg aggtcttcgc cgcgggcagc acctccggct cgacgcagga 26940
gctgccgccg tcggcccggc tgccgcagct cacgctgccg tcgggcgacc agctcggctc 27000
ggccacgctg ggcgcggtcg tgctgggcgt ggcggtgcac gtgctgctca cccgcacccg 27060
ctacggctac gagctgcggg ccggcggcct gaacccggtg gcggcgcgcg tccagggcat 27120
cgacccgcgc cggatgatcc tcgtcacgat gttgttgtcc ggcggcctgg cgggcctggc 27180
cgggctgccc gacctgctgg gccagaccta ccggtacggc atggacttcc cctccggcgc 27240
gggcttcacc gggatcgccg tggccctgct cggccgcaac acgcccgcgg gcatcgcgct 27300
ggccgccctg ctcttcgggt tcctggaacg gtcggcgctg atcctcgaca tcgagggcat 27360
ccccaaggag atcgtcacga tcatgcaggg cgtcatcgtg ctcagcgtcg tggtggccta 27420
cgaggtggcg cggcggatgg aacgcaggcg ggccgagcgg ctcgtacggg agcaggcgtc 27480
cccggcgctg gagccgtccg tatgagcgcg ggacggtacc ggtggtggcg ggccgcgctc 27540
gtcctgttgc tggcgatgtc ggtggtcagg gtgttcaccg gcgccgacga gctcaccggg 27600
tcgggcgtcg tgggggcggc gctgcggctg gcggtcccga tcgggctcgc cgggctgagc 27660
ggcctgtgga gcgagcgcgc gggcgtcgcc aacctcggca tcgagggcat gatgatcatc 27720
ggcacggtct gcggggcctg ggccgggtac gggttcggcg tctgggccgg ggtgctggcc 27780
ggcacgctcg gcggggtcgc ggccgggctg ctgcacgcgg tcgtcaccgt caccttcggg 27840
gtcgaccagg tggtgtgcgg catcgcgatc aacatcctgg cggtgggcgt ggcccggttc 27900
atgtcggtca tcgccttcag cggcctgccg ggggcggggg ccacccagtc gccgccggtg 27960
agcgggtcga tcggcacggt gagcgtgccc gtcctggcgg gcggcggggg cacgcccgac 28020
ctgctcggcg ggctggagcg cggccggttc ttcctggtgt ccgacctcgc cgggatcgtg 28080
cgggggttcc tgcacgaggt ctcgctgctc accgtggtcg ccgtcgcgct ggtcccgctg 28140
agcttcgtgc tgctctggcg taccgcgttc gggctgcggc tgcggtcggc gggcgagcac 28200
ccggcggcgg cggactcgct cggggtgccg gtgctgcgga tgcggtacta cggcgtgctg 28260
gtctccggcg ggctggccgg gctcggcggg gcctacctgg tcaccgtctc ctcgaccgtc 28320
taccgcgagg gccagaccgg cggccggggc ttcatcgggc tggccgccat gatcttcggc 28380
aactggcggc cgtccggcac ggccgccggg gcgctgctgt tcggttacgc cgacgccctg 28440
caactgcgcc aggccagcgc ggtgcacgtg ctgctgctgc tcggggccgt gctggcctgg 28500
gtcgccgccg tcgcgctcgt ccgtacgcgg ctgcgcctgg ccgcgacgct ggcgctgctc 28560
ggcgccgccg ccttcgcggt cttccagggc accgactcgg tccccaccca gctcgtgtac 28620
gccacgccgt acctcaccac gctgctggtg ctggtcacgg tcgcccaacg gctccgcatg 28680
ccggcctacg tcggcatccc ctaccggcga ggagacgcgt gatgaccatc gaccgggagc 28740
cccgcctgat ggggtacgcc gaccgctggt ccgcggcgcc cggtgagcgg ctggccgtgc 28800
acgcctccgg cccggacggg gacgccgacg ccgccctcgt ggacctcgtc cgggagagcg 28860
acctgacccc ggccgtgccg gtgacgatcg cgccgcggcc gctgcggccc ggctcccggc 28920
tggagacggc ctcggtgccg acgcccgcca ggggcggcct cgccctctgg atccggccga 28980
agcggctcgg gccgcgccag gtcgtggccc gcctgggcgc ctcggaatgg cggctgctgc 29040
tcgcgctcga cgaggagggc aggcccgagg ccaccgtgct gacgccgtcg gggagcatgg 29100
gctgccggct gcgctcggcc gtcccggcgg gggagtggtc actgctggcc gtggcgtggg 29160
aggcggcgcc gctgatgacc gtcctgcatc tgaacgactt ccgcaccccg ggcgtgagcg 29220
ccgggcgggc ccgcctgccg ctgccgtccc cgtcgggcga gctgctgatc ggggcgggcg 29280
acgagcatcc gtacgcgggc cggatcgccc tgcccatgct gaccgagggc ccgctcgcca 29340
tgcccggccg cgttccgctg accgacctga ccaccggtcc cgagctggtc aaacacctcg 29400
gccccgagct gctggccctg tgggacccgt ccgccgcccc cggcgcgccc gtcgtcccgg 29460
acgtcagcgg caacgggcac cacgcgaacg ccgtcaaccg gccgctggcc gggctcgccg 29520
ggccgggcgg cggcgcccgc accgcgctcc aggtcagccc ggaggacctc gacgacgccc 29580
gctggccggt gacgtgcgag ctgaccgtcc cgccggacgc cggctccacc gtgctcggcg 29640
tgcgcctgcg gacccggggg cagtcgtgcg tgctgccggt ggtcgtgcgc ccggacgcga 29700
cgcgcaggag cccggtcgcg gtcgtcctgc ccacgttcac ctacctcgcc tacgccaacc 29760
accggcagtc cagcgaggcc gagtacttcg gcgactaccg gcaggtcacc gaccgcgtga 29820
tcacgctctc cccggtggac gcctacctca acgagcaccg cgagctcggc ctgtccctgt 29880
acgaccagcg gcgcgagggc ggccacgtca cccactcctc cagacgccgc ccggtgctca 29940
acctcacccc cgactaccgc tggtggatga ccggcgcgcc gcgccacttc gccgccgacc 30000
tgctgctgct gcgctggctg cgctcggccg gcttcgcctt cgacgtgctg gccgacgagg 30060
acgtgcacga aggcggcgcc ggcctgctgt ccgcctaccg cgtcgtcctg accggcagcc 30120
accccgagta cgtcaccgcc cccgaacgcg cggccttcgg cgcgtacgtg tccggcggcg 30180
gccggctcat gtacctgggc ggcaacgggt tctactgggt gaccggcgtc gacccccggc 30240
acccgcacgc catcgaggtc cgccgcggcc acgtcggcga ctggtgggac gacgagcccg 30300
gcgaggacgt gctgacctcc accggcgagc ccggcggcct ctggcggcat cgcggcctgg 30360
cgccgcagga gctgctcggc gtcggtttcg ccgcccaggg ctgggggccc gcccccggct 30420
acacccgcct gcccgacagc taccgggagg aggcgtcctg ggtgttcgag ggcgtcggga 30480
ccgacccggt gggctgctgc ggtctcgcgc tcggcggggc ggcgggggac gagatcgacc 30540
gggccgcgcc cgagctcggc accccgccgg acaccctggt gctcgcgacc tccgcgggcg 30600
gccacggcca ccactaccgg ccccctaccg aggaggccgg gctgggcgcg caggccgtac 30660
gcgccgacat gaccctgcga ctgctcggat caggcggcgc ggtgttcgcc gccggatccg 30720
tcaactgggt gaccagcctc gcctgctgtg acggtgacat caggcgaatc acccggaacg 30780
tcctgtcccg attcctgcaa cccgcagctt gtgaggagga gtgacgtgac caccgcgagc 30840
gcgaggcacc tcgactaccc ctatccccgc acgtcggccg agctgaccga cgagatgttg 30900
tccatgcccg cagaggaact ggcggccatc gcccgccatc ctctgaccgt cttccccgac 30960
accaccgagc tctactaccg gctggcccgg gagatggccg acgagttacg cgcccgcaac 31020
atgcgcggcg agcccacccg ctggatcctg cccgtgggcc cgaaggcgca gtacccgatc 31080
ctggcgcgca tctgcaacga ggagcgcatc agctgggccg acgtgttcgc cttccacatg 31140
gacgagttcc tcgactggca gggccggccg gtggacccct cgcacccctt cagcttccgc 31200
ggctattgcg accgcaacct ctaccagctc ctcgaccccg acctgcggcc ccacccgggc 31260
aacgtcgtct tccccgacgt gttcgacccg gacgcgttcg gcacgcgtct gcgcggcgag 31320
ggcggcgccg acacctgtta cgcgggcttc ggctaccggg ggcacctggc cttcaacgag 31380
ccgccgtcca cgcgctggca ccagtacacc gccgcggagt tcgccgcctc gaccacgcgc 31440
gtcgtcccgc tgctcgacga cacgatcgtg gcccactccc accgggtgac cggcggctac 31500
acgcaggcga tcccgcggat ggcgatcacc gtcggcatgt cggagatcct ctccgcccgc 31560
cggctgcacc tgatcaccga cgggggcgcc tggaagcggt acatcgtccg ggtgctgctg 31620
ctcaccaccg agccggacgt gctgctcccg gtgaccttcg cgcaccacca ccccgacgtg 31680
cgcgtgaccg tcgacgccga cagcatcgcg ccctgcgccc tggggctggg ctcgtgaccg 31740
tcgtcgcggt cggggcctac atcgtcgacc tctacatgtt cggcgaccac ctgccggagc 31800
cgggcgagag cgtcaacgcc gccgactacg ccaccgcgca cggcggcaag gccgcgaacg 31860
tggccgtcgc cgcggcccgg ctgggcgcgc ccgcccggtt cgtcggctgc ctcggcgacg 31920
acgccgcccg gccggtggcg ctggccgagc tgcgcgccga gggcatcgag gtcggcgagt 31980
gcaccacggc cgtcggcgtc gcgaccgggc gcagcttcgt ctacgtcgac gcgggcggca 32040
ggcagatggt catgacctgg ccgggggccg ccgacctgct gccgcccgcc caggcggccg 32100
cggtcgccgc cggcctgccc cccgggtccg tgctcgtcct gcagggggag atcccgctgg 32160
agatctccta cgccgccgcg acggcggccc cgccgggcgt gcgcgtcctg ctcaacccga 32220
gcccggcggg accgttcctg gaggggaccg ggaaggagct gctggcccgg gccgacctgc 32280
tggtgctcaa cgagggcgag ctgtccgcgc tggccggcga gacccccggc gacctggacg 32340
acctgcgcgg caggaccagg ggacgcaccg tcgtcgtcac ccgcggcgag cacggcgccg 32400
agatcgccga cgacggcggc actatgctga tcgacgtccc cagcgtgcgc gttgtggaca 32460
ccaccggcgc gggtgacgcc ttcaccggcg ccctggcggc cgccctctgc gccggcgcgg 32520
gcctggtgcg cggcgtccgg ctcgcctgcc aggtggcgac cgtctcggtc acgcgccggt 32580
tctgtgcccc cagctaccca tcggccgccg agctcggcat cgccccgctc cgcggtccgt 32640
ccgtgcagga gagacccgcg tgacacgacg tcatcccacc ctggccatcc agatccgtga 32700
ccggctgcac gccctgatcg tcgagcaggg gctgcgcccc ggtgacaggc tgccgtcgga 32760
gaacgagctc atgaccctgt tcgacgtcgg gcgcaccagc atcagggagg cgttcaagct 32820
cctggagcag gaagggctca tccaggccag gcacggcgac ggccgctacc tcacctcgca 32880
gcccagcctg gaccggccgc tgacccgcct cgaaggcgtc accgagatgc tggccagccg 32940
cgggttcacc gccgacaaca ccgtcctcga cgtcatggcc accgagccgg accgccacca 33000
gcgggagctg ctgcaactgc cgcccggcga ggccatcgtc cggctggaac gcctgcgcag 33060
gcaccgggac gacgcgctgc tgtactcgat cgacctgttc ccgcgctcgc tcatcggccg 33120
ccccctggac gaggtggact ggaccggctc gctgttccag ctcctcaccg agcacggcca 33180
caccatcgcc tacgccgtcg cccaggtccg cgcggtgacc ctgagccacg cccaggccga 33240
gcgcatcggc acccacgagg acggcggcgc ctggctgctg ctcctccaga cccaccacgg 33300
cagcgccgga cggccggtgc tgtattcgca ggactaccac aggggcaccg acttctcctt 33360
ccacctggtg agacgccgcg actgagtgag ccgccgccgc gtaccgaccg accgccgttc 33420
gccgacgcag agaggatcat gcgatgagtg ccatcacgct ggaggccgcc cagccgatcc 33480
cggcctcccc ttccagcctc cgggcgaggt gctccaggct gtccgtcttc tggctcgccg 33540
aggacgaggt ctccttctac gggctcccgc cgctgctgat gaaggtgtcg aacgtggccg 33600
gggtcgcggt cgtgcgcagc tgcgcggagg cccggctgct gctggccggc gaggacttcg 33660
acgtggcggt gatcccgctc gccgtactgc cggacgtgct cggcggacgc ccccgccggg 33720
cccgcccgca gatcctggcc atggtgcgtc agggggagga gctcgccccc tgcgtcgccc 33780
agcggtacgg gctggccggt tgcctgctgt gggacgagat caatgtcgcc ggcttgtcgg 33840
gcaccttcga tcggctgctg cgcggcgacg cgcccgtccc gtccaggggc gcccccgacc 33900
tgctcggccg cctgaccgag cgcgagcaca tggtgctggc gctgctgctg cgcggcatga 33960
gcaaccacca gatcgcccgc tcgatgggca tctccatcca cggcgtcaag cgccacatct 34020
cgaacctgct cgtcaagttc aactgctcca accgcaccga ggtcgccctg gtcgcccagc 34080
gtctcggcct cgaccccgcc tcccgcaacc cgcgccacct gagcagcacc cgcacgtagc 34140
acgtacgacg aaccatcccc gaacggtcat cgagaaacgg agtgacaatg gacgtccctc 34200
tcatggaact cagcggccgc gcccccgtcg tcaggctgca tgacatcgag gcggacatgg 34260
ccgccgccac cgacgccatc aggtcgcagc tgaccggatg gggcttcatg gccgcggagg 34320
tgcccggcat cggcgagcgc gtcgaggcca tgatgaacga gttcgccgcg gcctgccggg 34380
cgaccgggcc gagcctgtcc gactacgcct acgacgtcgt cccgcagctc gccgtcggcg 34440
gcacgcacgg gttcttcccg tacaactcgg agatcccgcg cctggccaac ggcgtgcccg 34500
acccgaagga gttcatccac gtcagcggcg ccatgatcgg cgaccagccg cccggggcgg 34560
gtgacgtgct gcgggccttc ccggcgttcg gcacccgcgc cgccgaggtg ttcgacatcg 34620
ccttccggct gatctcgctc ttcggcgagg tcgtccgggg catgatgccg cccggcacgc 34680
cggagctgga cctctcgcac gacgcgacga acctgcgggt gatccactac cgggacgtcg 34740
gcgaccgcga ggtgctggcc cacgagcact ccggcatcca gatgctcggc ctccagctgc 34800
ccccgtccga ccagggcctg cagtacgtgc tgcacgacgg cacctgggtc gagccggtga 34860
tcgccgggac cgacgtcgtg ctgtgcaaca tcggccggat gctcaccagc gcctccgacg 34920
ggcggttccg gccgtccacg caccgggtgc acaccaagcc gatgccggcc ggctacgagc 34980
gcctgtcgtc ggtgctcttc gcctacccgc agcacaaggc ccgccagtgg aagatggtgg 35040
acggcgagct gatgtcgctg aacgccacct ggggcgactt catcgacagc cgcttccagg 35100
ggctcggcaa gcagtcctga ccacccggcc tcctggcggc cgggcggccg tcaggaggcg 35160
gtggcccgga cggccgcccg gatcacgtcg gcgacctcct tcggccgcga caccgcgacc 35220
gcgtgcgagg cgccggcgac ctccacggcg gtcgcgccgg cccgctcggc cccgaaccgc 35280
tgcacgtcgg ggttgatggc ctggtcggcg tcggcgatga cggcccacga cggcttggcc 35340
cgccaggccg ccgtggccgc cgcctcggtg aacgccccgg ccgccagggg ccgctgcgcc 35400
accgcgagga cccgggtgac ctcggccggc acgtcggcgg cgaagacctg ggggaaggcg 35460
tcggcggcga tggtgaactc gacggcggcg ccgccgtccg gcagcgggta ggccgcctgc 35520
cgcaggctcg cgccgagcgg cggctcgggg aagcggccct ggagctcgcc caggctctcg 35580
ccctcgtcca ggacgtacgc ggcgacgtag acgaggccga cgacgttctc cgccgcgccg 35640
gccacggtga tgatcgcgcc gccgtacgag tggcctgcca gcacgaccgg gccgtcgatc 35700
tgggccacca cggaggcgac gtacgccgcg tccgaggcca ggccgcgcag cgggttcggc 35760
ggggccacca cagggacgcc gtcccgctgc aactcggcga tgacgccgga ccagctcgcc 35820
gcgtcggcga acgcgccgtg cacgaggacg accgtcgggg tggtgctctc cgtcatggga 35880
aaaactcctt ctcggggcaa gaggggaccg gtcggccccg gtagcgatcc aacagccggg 35940
ccgctcgcgc gcacgaggcg gaaacgtact tctgcctgtg gttccgcctc gtctttcccc 36000
aggacggtcg cttggatggg tgacggcccc cctgcttccc accccttctt cctggagcga 36060
tcatgatcaa gcccgtcctg gaacccgccg ctcaggcgtt cgccgaggcc accgccgagc 36120
cgccgtacct cttccagctc ccgccggagg agggccgcaa ggccgtcaac gaggtgcagt 36180
ccggcccggc cgagctgccc gccgtggacg aggagtgggt gacggtcccc ggcggaccca 36240
ccggcgaggt cagggcccgc atcgtgcggc cggccggcag caccggcgac ctgcccgtga 36300
tcatctacat ccacggcgcc ggctgggtct tcggcaacgc ccacacccac gaccggctgg 36360
tccgcgagct ggcgaccggc gccggcgccg ccgtggtctt ccccgagtac gacctgtcgc 36420
ccgagcaccg ct 36432
<210> 2
<211> 479
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365(Actinomadura sp. ATCC 39365)
<400> 1
Met Arg Gly His Asp Pro Leu Asp Asp Thr Glu Gln Ala Leu Leu Pro
1 5 10 15
Ala Pro Ala Arg Gly Leu Ala Val Ala Leu Leu Leu Ile Ala Val Ala
20 25 30
Gly Val Ser Met Leu Leu Val Phe Gly Pro Gly Ala Gln Gln Pro Gly
35 40 45
Gln Ala Ala Ala Pro Ala Ala His Ala Gly Val Pro Pro Ala Gln Ser
50 55 60
Ile Leu Arg Leu Phe Leu Ala Val Ala Val Ile Gly Gly Leu Ala Ser
65 70 75 80
Leu Gly Gly Val Leu Ala Arg Arg Ala Gly Gln Pro Ser Val Val Gly
85 90 95
Gln Met Val Ala Gly Leu Leu Leu Gly Pro Ser Ala Leu Gly Ser Leu
100 105 110
Ala Pro Gly Val Ser Ala Leu Ile Leu Pro Gly Trp Val Ala Pro Gln
115 120 125
Leu Glu Leu Leu Ala Gln Ala Gly Leu Ala Val Phe Met Phe Thr Val
130 135 140
Gly Arg Glu Phe Asp Pro Ala Ala Leu Gly Arg Gln Arg Val Val Val
145 150 155 160
Gly Val Ala Ala Leu Ala Met Thr Ala Val Pro Phe Ala Leu Gly Ala
165 170 175
Val Ala Ala Val Pro Phe Ala Gly Ala Leu Ala Gly Pro Ser Ala Ser
180 185 190
Gly Thr Ala Phe Val Leu Phe Ala Gly Thr Ala Leu Ser Val Thr Ala
195 200 205
Phe Pro Val Leu Ala Arg Ile Leu Gln Glu Ala Gly Leu Thr Ala Thr
210 215 220
Arg Leu Gly Ser Leu Ala Ile Leu Cys Ala Gly Leu Ala Asp Val Leu
225 230 235 240
Ala Trp Cys Ala Leu Ala Ala Val Ile Ala Leu Ala His Ala Gly Ser
245 250 255
Pro Ala Gly Val Leu Thr Ser Leu Gly Leu Ala Ala Ala Leu Val Leu
260 265 270
Ala Val Thr Leu Val Val Arg Pro Ala Leu Ala Ala Leu Ala Ala Arg
275 280 285
His Ala Ala Arg Arg Leu Pro Ala Pro Ala Ala Leu Ala Leu Val Leu
290 295 300
Gly Leu Ile Phe Ala Leu Ala Ala Ala Thr Asp Ala Ile Gly Val His
305 310 315 320
Ala Ile Phe Gly Ala Leu Leu Ala Gly Val Ala Phe Pro Arg Asp Ala
325 330 335
Pro Val Leu Gly Ala Val Pro Glu Arg Leu Gly Ser Leu Asn Arg Ala
340 345 350
Met Leu Leu Pro Val Phe Phe Ala Ser Ile Gly Leu Arg Thr Asp Val
355 360 365
His Leu Ala Phe Gly His Pro Val Val Leu Leu Gly Gly Ala Val Leu
370 375 380
Leu Val Ala Ala Val Leu Gly Lys Leu Gly Ala Ala Gly Val Ile Ala
385 390 395 400
Trp Ala Gly Gly Met Pro Gly Arg Leu Ala Leu Gly Leu Gly Val Leu
405 410 415
Met Asn Ala Arg Gly Val Thr Glu Ile Val Val Leu Ser Thr Gly Leu
420 425 430
Gly Met Gly Leu Ile Ser Pro Gly Thr Phe Thr Val Leu Val Leu Met
435 440 445
Ala Leu Ile Thr Thr Met Met Thr Ala Pro Ala Leu Arg Arg Leu Gly
450 455 460
Leu Tyr Ala Pro Ala Arg Ala Glu Pro Arg Thr Pro Gln Pro Ala
465 470 475
<210> 3
<211> 402
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Met Asp Pro Arg Ser Ala Ala Lys Ile Thr Glu Ala Ser Pro Arg Arg
1 5 10 15
Ser Pro Trp Leu Thr Ile Leu Leu Val Tyr Val Gly Gly Val Val Gly
20 25 30
Ala Met Ser Leu Gly Lys Phe Ser Ser Val Gly Pro Ala Met Ala Ala
35 40 45
Gln Leu Gly Leu Ser Leu Pro Glu Leu Gly Trp Val Ile Ser Ala Val
50 55 60
Val Gly Val Gly Ala Val Val Gly Leu Pro Ala Gly Tyr Leu Val Arg
65 70 75 80
Arg Phe Gly Thr Gly Arg Ser Leu Val Thr Gly Leu Val Leu Ile Ala
85 90 95
Ala Ala Gly Ala Ala Ser Met Ala Ala Asn Asp Phe Ala Trp Leu Leu
100 105 110
Ile Ala Arg Gly Val Glu Gly Val Gly Tyr Val Leu Val Thr Ile Ser
115 120 125
Cys Pro Ala Leu Ile Leu Arg Leu Ala His Glu Arg Asp Arg Gly Thr
130 135 140
Ala Leu Ser Val Trp Ala Thr Phe Val Pro Val Gly Leu Gly Ala Ser
145 150 155 160
Thr Leu Ala Gly Gly Ala Ala Gly Asp Ala Leu Gly Trp Arg Gly Trp
165 170 175
Thr Gly Leu Ile Ala Gly Leu Thr Leu Leu Ile Ala Ala Val Thr Trp
180 185 190
Ala Arg Leu Pro Arg Ala Asp Gly Ala Gln Ala Ala Gly Pro Val Pro
195 200 205
Arg Ala Arg Ala Leu Thr Trp Pro Gly Val Leu Ala Ala Ala Phe Cys
210 215 220
Leu Thr Ala Leu Val Thr Ile Pro Val Ile Val Leu Leu Pro Thr Leu
225 230 235 240
Leu Ile Glu Gln Tyr Gly Arg Ser Ala Ala Ala Ala Gly Ala Leu Thr
245 250 255
Ser Val Ile Ser Leu Leu Gly Val Ala Gly Gly Leu Ala Val Gly Val
260 265 270
Leu Leu Arg Arg Gly Val Arg Val Gly Val Leu Ala Leu Ser Gly Phe
275 280 285
Leu Val Val Pro Ala Ala Trp Ile Leu Tyr Ala Gly Gly Ser Ala Gly
290 295 300
Gly Ala Leu Thr Gly Ala Gly Val Ile Ser Met Glu Asn Gly Phe Leu
305 310 315 320
Gly Ala Leu Val Phe Ala Ala Leu Pro Leu Val Leu Glu Arg Leu Asp
325 330 335
His Ala Asp Val Gly Asn Gly Val Ile Ala Gln Ala Gly Ser Leu Gly
340 345 350
Ser Leu Leu Gly Pro Pro Leu Phe Gly Leu Val Ala Gly Glu Ile Gly
355 360 365
Tyr Gln Ala Leu Ile Pro Val Ile Ala Val Gly Ile Val Ala Ala Val
370 375 380
Gly Ala Leu Leu Leu Ile Gly Arg Arg Ala Ala Arg Pro Leu Pro Ala
385 390 395 400
Ala Asp
402
<210> 4
<211> 295
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Met Ile Ser Leu Ala Leu Pro Lys Gly Ser Leu Glu Arg Arg Ala Leu
1 5 10 15
Glu Leu Phe Ala Thr Ala Gly Leu Pro Val Arg Arg Arg Ser Asp Arg
20 25 30
Ser Tyr Arg Gly Thr Val Asp His Ser Gly Val Ser His Val Ala Phe
35 40 45
Tyr Lys Pro Arg Glu Ile Pro Leu Val Val Gly Asp Gly Thr Phe Asp
50 55 60
Leu Gly Leu Thr Gly Ala Asp Trp Val Glu Glu Thr Gly Ala Glu Val
65 70 75 80
Glu Val Val Glu Ser Phe Asp Tyr Ser Arg Asn Thr Glu Arg Pro Trp
85 90 95
Arg Leu Val Leu Ala Val Pro Ser Gly His Pro Ala Arg Ser Ala Ala
100 105 110
Asp Leu Asp Asp Gly Val Arg Val Ala Thr Glu Tyr Pro Asn Ile Gly
115 120 125
Arg Arg Phe Phe Arg Glu Ala Gly Arg Arg Ala Glu Val Val Val Ser
130 135 140
Tyr Gly Ala Thr Glu Ala Lys Ile Pro Glu Leu Ala Asp Ala Met Met
145 150 155 160
Asp Val Val Glu Thr Gly His Ser Leu Arg Gln Asn Asp Leu Arg Val
165 170 175
Ile Glu Thr Val Arg Thr Cys Gly Ala Gln Leu Val Ala Asn Pro Ala
180 185 190
Ala Tyr Ala Asp Pro Ala Arg Arg Gly Val Ile Arg Asn Val Ala Leu
195 200 205
Leu Leu Arg Ala Ala Trp Val Gly Pro Ala His Val Leu Leu Thr Val
210 215 220
Arg Ala Pro Ala Asp Arg Leu Ala Glu Val Val Pro Ala Met Pro Gly
225 230 235 240
Gln Ser Trp Arg Ala Gly Ala Asp Leu Ala Asp Gly Thr Met Val Val
245 250 255
Leu Gln Gly Val Leu Pro Arg Arg Glu Val Pro Glu Thr Ile Asp Ala
260 265 270
Leu Leu Ser Ala Gly Ala Val Asn Val Thr Glu Ser Gly Ile Gly Ile
275 280 285
Phe Ala Thr Gly Glu Ala Gly
290 295
<210> 5
<211> 234
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Met Ser Ser His Glu Thr Pro Ala Ala Leu Val Thr Gly Ser Asn Arg
1 5 10 15
Gly Ser Gly Arg Ala Ile Ala Glu Glu Leu Arg Glu Arg Gly Tyr Arg
20 25 30
Val Phe Ser Leu Asn Arg Ser Leu Ala Gly Glu Asp Trp Leu Gly Glu
35 40 45
His Arg Cys Asp Leu Ser Asp Pro Ala Gln Ile Ala Glu Ala Thr Ala
50 55 60
Gln Val Leu Gly Ser Ala Gly Arg Leu Asp Val Cys Val Ser Asn Ala
65 70 75 80
Val Asp Arg Val Leu Asp Pro Ile Ala Glu Leu Arg Ala Glu Asp Trp
85 90 95
Glu Arg Leu Val Ala Ile Asn Leu Ser Ala Cys Phe His Leu Leu Lys
100 105 110
Ala Val Leu Pro Ala Leu Arg Ala Ala Gly Gly Met Phe Val Ala Met
115 120 125
Gly Ser His Ala Gly Thr Arg Tyr Phe Glu Gly Gly Ala Ala Tyr Ser
130 135 140
Ala Thr Lys Ala Gly Leu Lys Ala Leu Val Glu Thr Leu Leu Leu Glu
145 150 155 160
Glu Arg Arg Asn Gly Val Arg Ala Cys Leu Val Ser Pro Gly Ala Ile
165 170 175
Ala Asn Leu Asp Gly Asp Gly Ser Pro His Lys Met Ser Thr Arg Ser
180 185 190
Val Ala Arg Cys Val Ala Thr Ile Val Asp Ser Trp Pro Asp Asp Leu
195 200 205
Val Val Gly Glu Ile Glu Val Arg Pro Ser Cys Leu Pro Glu Pro Pro
210 215 220
Val Val Gly Ile Asp Arg Leu Leu His Val
225 230
<210> 6
<211> 239
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Val Asp Asn Gly Ser Glu Pro Gly Thr Pro Ala Gly Arg Gly Glu Pro
1 5 10 15
Asp Ile Val Gly Arg Ser Lys Arg Leu Trp Leu Leu Pro Asp Gly Arg
20 25 30
Cys Leu Val Gln Ile Ile Pro Ser Leu Arg Ser Phe Thr Tyr His Arg
35 40 45
Asp Glu Ile Val Glu Arg Thr Gly Pro Leu Arg Leu Asp Phe Tyr Glu
50 55 60
Arg Ala Ala Ala Arg Leu Ala Asp Ala Gly Val Pro Cys Ala Phe Arg
65 70 75 80
Glu Arg Val Ser Ala Asp Ser Tyr Val Ala Asp Tyr Cys Pro Ala Pro
85 90 95
Pro Phe Glu Val Ile Val Lys Asn Arg Ala Thr Gly Ser Thr Thr Arg
100 105 110
Lys Tyr Pro Gly Leu Phe Glu Asp Gly Ala Pro Leu Pro Arg Pro Val
115 120 125
Val Lys Phe Asp Tyr Arg Thr Asp Pro Glu Asp Gln Pro Ile Gly Glu
130 135 140
Asp Tyr Leu Arg Ala Leu Asp Leu Pro Val Glu Glu Met Arg Glu Leu
145 150 155 160
Ser Leu Ala Val Asn Asp Val Leu Arg Ser Trp Leu Arg Pro Leu Glu
165 170 175
Val Trp Asp Phe Cys Leu Ile Phe Gly Arg Thr Ser Asp Gly Gly Leu
180 185 190
Thr Val Ile Ser Glu Ile Ser Gln Asp Cys Met Arg Leu Arg His Gln
195 200 205
Asp Gly Ser Pro Leu Asp Lys Asp Leu Phe Arg Asp Gly Val Ala Gly
210 215 220
Glu Glu Ile Val Asn Gln Trp Ser Arg Val Leu Asp Val Ile Thr
225 230 235
<210> 7
<211> 425
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Met Gly Gly Gly Ser Leu Ser Glu Tyr Glu Arg Ile Ala His Ser Lys
1 5 10 15
Ile Thr Arg Lys Phe Arg Ser Ala Gly Gln Gln Leu Leu Phe Ser Arg
20 25 30
Gly Arg Arg Gly Glu Val Ser Asp Ala Asn Gly Val Pro Tyr Ile Asp
35 40 45
Phe Val Met Gly Tyr Gly Pro Val Ile Ile Gly His Ala Asp Ala Gln
50 55 60
Phe Asn Glu Ile Leu Cys Gly Tyr Leu Gly Asn Gly Val Met Leu Pro
65 70 75 80
Gly Tyr Thr Thr Phe His Gln Glu Tyr Leu Asp Arg Leu Leu Gly Glu
85 90 95
Arg Pro Gly Asp Arg Gly Ala Phe Phe Lys Thr Ala Ser Glu Ala Val
100 105 110
Thr Ala Ala Phe Arg Leu Ala Ala Met Arg Thr Gly Arg Leu Gly Ile
115 120 125
Ile Arg Ser Gly Tyr Val Gly Trp His Asp Ser Gln Ile Ala Asp Ser
130 135 140
Leu Lys Trp His Glu Pro Leu His Ser Pro Leu Arg Asp Lys Leu Arg
145 150 155 160
Tyr Thr Asp Gly Met Arg Gly Val Gly Glu Ser Glu Pro Val Ala Asn
165 170 175
Trp Val Asp Leu Arg Leu Glu Ser Leu Ala Glu Leu Leu Glu Arg His
180 185 190
Arg Gly Arg Leu Gly Cys Phe Val Phe Asp Ala Tyr Leu Ala Ser Phe
195 200 205
Thr Thr Ala Asp Val Leu Arg Gln Ala Val Ala Met Cys Arg Glu Ala
210 215 220
Gly Leu Leu Thr Val Phe Asp Glu Thr Lys Thr Gly Gly Arg Ile Ser
225 230 235 240
Pro Leu Gly Tyr Asp His Asp Asn Ala Leu Gly Ser Asp Leu Ile Val
245 250 255
Ile Gly Lys Ala Leu Ala Asn Gly Ala Pro Leu Ser Ile Leu Ala Gly
260 265 270
Asp Ala Asp Leu Leu Ala Leu Ala Glu Lys Ala Arg Leu Ser Gly Thr
275 280 285
Phe Ser Lys Glu Met Ile Ala Val Tyr Ala Ala Leu Ala Thr Arg Asp
290 295 300
Ile Leu Glu Lys Pro Val Gly Asp Ser Pro Asp Gly Trp Thr Glu Leu
305 310 315 320
Gly Arg Ile Gly Thr Gln Val Ala Ala Ala Phe Thr Ala Ala Ala Ala
325 330 335
Asp Ala Gly Val Glu Ala Leu Val Gly Ala Arg Pro Val Leu Gly Gly
340 345 350
Gly Met Phe Glu Leu Val Tyr His Asp Val Glu Leu Leu Gly Asp Lys
355 360 365
Glu Arg Arg Glu Ala Leu Leu Ala Glu Leu Ala Gly Val Gly Ile Leu
370 375 380
Leu Leu Glu Gly His Pro Ser Phe Val Cys Leu Ala His Arg Asp Ile
385 390 395 400
Asp Trp Gly Asp Leu Arg Asp Arg Val Arg Gln Ala Phe Glu Ala Trp
405 410 415
Thr Ala Pro Thr Gly Ala Gly Arg Gly
420 425
<210> 8
<211> 351
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Val Ala Asp Leu Leu Arg Leu Ala Leu Val Gly Cys Gly Arg Gln Met
1 5 10 15
Gln Gln Asn Leu Phe Pro Phe Leu Gln Arg Ile Arg Gly His Gln Val
20 25 30
Val Ala Cys Val Asp Pro Asp Leu Ser Leu Ala Ala Asp Val Gln Ser
35 40 45
Leu Ala Gly Gly Ala Thr Cys Val Ser Ser Val Asp Glu Leu Asp Leu
50 55 60
Glu Met Val Asp Ala Ala Val Leu Ala Val Pro Pro Glu Pro Ser Tyr
65 70 75 80
Leu Leu Val Arg Gln Leu Ala Glu Arg Gly Val Asp Cys Phe Val Glu
85 90 95
Lys Pro Ala Gly Pro Ser Thr Pro Ala Leu Gln Asp Leu Glu His Val
100 105 110
Val Arg Arg Ser Gly Arg His Val Gln Val Gly Phe Asn Phe Arg Tyr
115 120 125
Ala Glu Thr Leu Gln Arg Leu His Glu Leu Ser Glu Glu Ile Arg Ala
130 135 140
Thr Pro Cys Ser Val Thr Ile Asp Phe Tyr Ser Arg His Pro Ser Ala
145 150 155 160
Pro Gln Trp Gly Val Asp Thr Thr Leu Glu Ala Trp Ile Arg His Asn
165 170 175
Gly Val His Ala Ile Asp Leu Ala Arg Trp Phe Val Pro Ser Pro Val
180 185 190
Val Gln Val Asp Ala His Ala Ile Ala Ser Asp Ala Asp Arg Phe Gln
195 200 205
Ile Asn Leu Phe Leu Arg His His Asp Gly Ser Leu Ser Thr Leu Arg
210 215 220
Met Gly Asn His Val Lys Arg Phe Met Val Gly Val Thr Val Gln Gly
225 230 235 240
Met Asp Gly Ser Arg Tyr Ser Ala Pro Ser Leu Glu Arg Val Thr Leu
245 250 255
Glu Leu Ser Asp Gly Val Pro His Gly Gln Glu Leu His Ala Thr Arg
260 265 270
Asn Leu Asp His Gly Trp Gly Arg Ser Gly Phe Gly Pro Glu Leu Gln
275 280 285
Ala Phe Val Asp Ala Cys Ala Gln Arg Ser Ala Glu Pro Gln Thr Gly
290 295 300
Gly Arg Pro Pro Val Lys Gly Val Pro Ser Val Ser Asp Ala Leu Ala
305 310 315 320
Ala Ser Ala Leu Cys Asp Arg Val Met Ala Glu Leu Asn Thr Ala Ala
325 330 335
Thr Asn Gly Phe Gly Leu Leu Thr Gly Ser Val Arg Ala Ser Ala
340 345 350
<210> 9
<211> 595
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Met Thr Glu Arg Ala Arg Pro Ala Ala Gly Gly Arg Pro Val Ala Ser
1 5 10 15
Pro Asp Pro Ala Gly Ser Pro Asp Pro Ala Ala Ala Arg Gly Val Gly
20 25 30
Arg Trp Ile Ala Gly His Leu Arg Arg His Arg Pro Ser Leu Leu Leu
35 40 45
Phe Thr Val Ala Ala Ala Ala Ala Ser Leu Cys Ser Thr Leu Val Pro
50 55 60
Val Gln Ile Gly Ala Ala Phe Ala Glu Ala Thr Gly Pro Arg His Asp
65 70 75 80
Leu Gly Ala Val Gly Val Ala Ala Leu Ala Ala Ala Leu Leu Ala Ala
85 90 95
Gly Arg Phe Val Ala Asp Leu Leu Ser Asn Gly Ser Met Glu Val Val
100 105 110
Ala Gln Arg Val Lys Arg Asp Val Arg Asp Glu Leu Tyr Arg Ser Leu
115 120 125
Leu Thr Lys Arg Met Ala Phe His Asp Gln Gln Arg Ile Gly Asp Ile
130 135 140
Leu Ala Arg Ala Ile Asn Asp Ala Arg Leu Val Asp Tyr Met Leu Ser
145 150 155 160
Pro Gly Ala Ala Thr Ala Ala Asn Gly Val Leu Ala Leu Leu Val Pro
165 170 175
Ile Leu Phe Ile Ala Ser Leu Asp Pro Arg Leu Leu Leu Ala Pro Gly
180 185 190
Val Leu Val Leu Val Phe Ala Tyr Ala Leu Arg Tyr His Leu Arg Arg
195 200 205
Leu Tyr Pro Leu Val Met Arg Thr Arg Glu Thr Phe Ala Asp Leu Asn
210 215 220
Glu Arg Phe Ser Thr Thr Leu Ser Gly Ile Ala Thr Val Lys Ala Ala
225 230 235 240
Thr Gln Glu Asp Phe Glu Arg His Ala Leu Arg Ser Ala Ala Ala Ala
245 250 255
Tyr Arg Asp Ala Phe Val Arg Arg Gly Arg Ala Gln Ala Val Tyr Leu
260 265 270
Pro Ala Leu Ser Phe Ala Leu Ala Met Val Thr Gly Ser Leu His Ser
275 280 285
Leu Tyr Leu Tyr Ser Gln Gly Glu Leu Ala Leu Ser Gln Val Val Thr
290 295 300
Tyr Leu Gly Trp Leu Leu Leu Phe Ala Gln Pro Val Thr Met Ser Glu
305 310 315 320
Gln Ala Val Pro Val Ile Gln Glu Gly Phe Ala Ala Ala Ala Arg Met
325 330 335
Arg Gln Ile Ile Glu Gly Ala Pro Gly Glu Arg Glu Asp Thr Arg Gly
340 345 350
Ser Thr Ala Ala Val Glu Gly Ala Ile Thr Phe Asp Arg Val Ser Leu
355 360 365
Arg His Glu Gly Arg Asp Ile Leu Arg Glu Val Ser Phe His Leu Pro
370 375 380
Ala Gly Arg Thr Leu Ala Val Val Gly Pro Thr Gly Ser Gly Lys Ser
385 390 395 400
Met Leu Ile Lys Leu Val Asn Arg Met Tyr Asp Ala Thr Glu Gly Arg
405 410 415
Val Leu Ile Asp Gly Arg Asp Val Arg Glu Trp Glu Pro Gly Ala Leu
420 425 430
Arg Arg Gln Ile Gly His Val Asp Gln Glu Ile Phe Leu Phe Ser Lys
435 440 445
Ser Val Leu Asp Asn Ile Ala Phe Gly Ala Pro His Ala Ala Gly Tyr
450 455 460
Glu Asp Val Leu Arg Val Ala Lys Gln Ala Cys Ala Asp Glu Phe Val
465 470 475 480
Gln Gly Met Ala Asp Gly Tyr Ala Thr Val Leu Asn Glu Gly Gly Thr
485 490 495
Thr Leu Ser Gly Gly Gln Arg Gln Arg Leu Ala Ile Ala Arg Ala Leu
500 505 510
Leu Thr Glu Pro Arg Ile Leu Thr Leu Asp Asp Ala Thr Ser Ala Val
515 520 525
Asp Ala Arg Thr Glu Ser Ala Ile Thr Glu Ala Ile Glu Arg Ala Thr
530 535 540
Ala Gly Arg Thr Thr Val Leu Val Ser His Arg Pro Gly Gln Ile Arg
545 550 555 560
Arg Ala Asp Leu Ile Leu Leu Leu Asp Gly Gly Arg Val Val Asp Gln
565 570 575
Gly Ser His Asp Glu Leu Met Ala Arg Cys Ala Leu Tyr Arg Glu Ile
580 585 590
Tyr Ser Gly
595
<210> 10
<211> 592
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Val Ser Asp Leu Phe Ala Gly Leu His Ala Asp Ala Tyr Asp Arg Val
1 5 10 15
Tyr Gly Asn Arg Arg Leu Leu Ala Arg Ile Leu Arg Gln Leu Arg Ala
20 25 30
His Arg Ala Gly Met Ile Gly Ser Ala Leu Leu Val Ala Gly Ser Val
35 40 45
Leu Leu Thr Ala Ser Val Pro Ile Val Val Ser Arg Leu Ile Asp Gln
50 55 60
Ala Gly Gly Gly Ser Gly Pro Gly Leu Leu Leu Cys Leu Phe Val Val
65 70 75 80
Ala Ala Ala Val Leu Ala Trp Ala Met Asn Trp Ala Arg Gln Ala Ile
85 90 95
Thr Ala Arg Leu Val Gly Gly Met Val Tyr Arg Leu Gln Cys Glu Ala
100 105 110
Ala Asp Ala Ala Leu Ala Lys Asp Val Ala Phe Tyr Asp Glu Asn Ser
115 120 125
Val Gly Lys Val Leu Ser Arg Val Thr Gly Asp Thr Glu Gly Phe Gly
130 135 140
Ser Val Leu Thr Leu Thr Leu Asn Leu Ile Ser Gln Leu Phe Leu Val
145 150 155 160
Val Leu Leu Gly Gly Val Val Phe Trp Ile Asp Pro Gly Leu Gly Leu
165 170 175
Val Ile Leu Ala Ala Leu Pro Phe Leu Leu Gly Thr Ala Leu Ala Phe
180 185 190
Arg Arg Val Ala Arg Thr Ala Ala Ala Ala Thr Arg Arg Val Met Ala
195 200 205
Lys Val Asn Ala Asn Val His Glu Thr Met Leu Gly Ile Ala Val Ala
210 215 220
Lys Ser Phe Gly Arg Glu Arg Ala Val His Asp Asp Phe Asp Asp Val
225 230 235 240
Asn Arg Leu Ser Tyr Arg Val Tyr Val Arg Gln Gly Leu Ile Tyr Ala
245 250 255
Val Ile Leu Pro Val Leu Thr Leu Leu Ala Gly Leu Ala Thr Ala Val
260 265 270
Val Leu Tyr Gln Gly Gly Arg Phe Ala Ala Ile Gly Arg Leu Ser Ala
275 280 285
Gly Glu Trp Tyr Phe Ala Leu Gln Ala Leu Gly Met Leu Trp Gln Pro
290 295 300
Val Thr Gln Ala Ala Ser Phe Trp Ser Leu Phe Gln Gln Gly Leu Ala
305 310 315 320
Ala Ala Glu Arg Val Phe Ala Leu Ile Asp Ser Asp His Ala Val Val
325 330 335
Gln Ser Gly Asn Ala Pro Val Thr Ala Leu Thr Gly Glu Ile Glu Ala
340 345 350
Arg Gly Leu His Phe Arg Tyr Gly Ser Gly Ala Ala Val Phe Arg Asp
355 360 365
Phe Asp Val Arg Leu Ala Ala Arg Glu Thr Val Ala Val Val Gly His
370 375 380
Thr Gly Gly Gly Lys Ser Thr Leu Ala Lys Leu Ile Ala Arg Ala Tyr
385 390 395 400
Asp Phe Gln Gly Gly Glu Leu Leu Val Asp Gly Arg Asp Ile Arg Gly
405 410 415
Leu Asp Leu Arg Ala Tyr Arg Arg Arg Val Gly Val Val Pro Gln His
420 425 430
Pro Phe Ile Phe Ala Gly Thr Leu Ala Glu Asn Ile Ala Tyr Gly Arg
435 440 445
Pro Gly Ala Gly Arg Asp Asp Val Val Arg Ala Val Glu Arg Ile Gly
450 455 460
His Arg Val Trp Asp Arg Ser Met Pro Met Ser Leu Asp Asp Arg Leu
465 470 475 480
Ala Ala Gly Gly Gln Gly Val Ser Val Gly Gln Arg Gln Leu Ile Ala
485 490 495
Leu Ala Arg Met Phe Val Arg Glu Pro Asp Ile Leu Leu Leu Asp Glu
500 505 510
Pro Thr Ala Ser Val Asp Pro Leu Thr Glu Arg Gly Ile Gln Asp Ala
515 520 525
Leu Ala Arg Leu Cys Ala Gly Arg Thr Val Val Val Ile Ala His Arg
530 535 540
Leu Ser Thr Ile Arg Arg Ala Asp Arg Val Leu Val Leu His Gly Gly
545 550 555 560
Glu Ile Ala Glu Gln Gly Arg Phe Asp Glu Leu Leu Arg Arg Asp Gly
565 570 575
Pro Phe Ala Ala Leu Tyr Ala Thr Tyr Tyr Ala His Gln Glu Thr Ala
580 585 590
<210> 11
<211> 161
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Met Thr Cys Ala Pro His Pro Ala Glu Phe Glu Pro Ile Gln Arg Glu
1 5 10 15
Ala Ser Ala Thr Gly Val Arg Cys Val Val Gly Ala Val Leu Phe Asn
20 25 30
Pro Leu Gly Glu Val Phe Leu Gln Arg Arg Ala Ala His Val Arg Leu
35 40 45
Phe Pro Gly Cys Trp Asp Ile Val Gly Gly His Val Glu Arg Gly Glu
50 55 60
Thr Leu Cys Ala Ala Leu Ala Arg Glu Ile Glu Glu Glu Thr Gly Trp
65 70 75 80
Arg Leu Leu Arg Val Gly Gly Leu Val Asp Val Phe Asp Trp Thr Gly
85 90 95
Gly Asp Gly Gly Leu Arg Arg Glu Ile Asp Val Leu Ala Thr Val Glu
100 105 110
Gly Asp Leu Thr Arg Pro Ala Ile Glu Gln Asp Lys Phe Asp Glu Ala
115 120 125
Arg Trp Leu Asp Gly Asp Ala Leu Arg Arg Leu Ala Ala Glu Ser Pro
130 135 140
Gly Ser Gly Met Val Glu Leu Ala Leu Arg Ala Leu Ala Met Arg Pro
145 150 155 160
Val
<210> 12
<211> 245
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Val Ser Thr Cys Leu Thr Leu Leu Pro Ala Val Asp Val Arg Gly Gly
1 5 10 15
Arg Ala Val Arg Leu Leu Arg Gly Glu Ser Gly Ser Glu Thr Trp Tyr
20 25 30
Gly Asp Pro Leu Asp Ala Ala Leu Ala Trp Gln Asp Ser Gly Ala Asp
35 40 45
Trp Val His Leu Val Asp Leu Asp Ala Ala Phe Gly Thr Gly Ser Asn
50 55 60
Arg Ala Arg Ile Ala Glu Val Val Gly Ala Leu Asp Ile Pro Val Glu
65 70 75 80
Leu Cys Gly Gly Val Arg Asp Asp Ala Ser Leu Ala Ala Ala Leu Ala
85 90 95
Thr Gly Cys Gly Arg Val Val Leu Gly Thr Gly Ala Leu Glu Arg Pro
100 105 110
Ala Trp Val Ala Glu Val Ile Asp Arg His Gly Asp Arg Val Ala Val
115 120 125
Glu Leu Asp Val Trp Gly Thr Thr Val Arg Ser His Gly Trp Thr Arg
130 135 140
Asp Ala Gly Glu Leu Tyr Glu Thr Leu Ala Arg Leu Asp Ala Ala Gly
145 150 155 160
Cys Ala Arg Tyr Val Val Thr Asp Ile Ala Arg Asp Gly Thr Leu Gly
165 170 175
Gly Pro Asn Leu Lys Leu Leu Arg Asp Val Cys Ala Ala Thr Gly Arg
180 185 190
Pro Val Val Ala Ser Gly Gly Ile Ser Ser Leu Asp Asp Leu Arg Ala
195 200 205
Val Ser Ser Leu Thr Pro Leu Gly Val Glu Gly Val Val Val Gly Lys
210 215 220
Ala Leu Tyr Ala Gly Arg Phe Thr Leu Arg Gln Ala Leu Asp Ala Val
225 230 235 240
Arg Lys Pro Glu Pro
245
<210> 13
<211> 288
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Met Val Ser Leu Ala Leu Pro Lys Gly Thr Phe Leu Glu Arg Pro Val
1 5 10 15
Leu Asp Leu Phe Ala Ala Ala Gly Leu Glu Val Arg Arg Pro Ser Glu
20 25 30
Arg Ser Tyr Arg Ala Ser Ile Ala Tyr Asp Gly Gly Val Glu Val Ala
35 40 45
Phe His Lys Pro Arg Glu Ile Pro Leu Ala Val Glu Arg Gly Val Phe
50 55 60
Asp Phe Gly Val Thr Gly Thr Asp Trp Ile Glu Glu Thr Gly Ala Lys
65 70 75 80
Val Glu Leu Val Glu Ala Ser Gly Cys Val Pro Pro Trp Arg Leu Val
85 90 95
Leu Ala Val Pro Ser Gly His Pro Ala Val Asp Ala Ala Gly Leu Pro
100 105 110
Ala Gly Ala Arg Val Ala Thr Gly Phe Pro Lys Ile Ser Arg Gln Tyr
115 120 125
Phe Gln Ser Val Pro Leu Pro Val Arg Ile Val Pro Ser Phe Gly Ala
130 135 140
Thr Glu Ala Lys Val Pro Glu Leu Ala Asp Ala Val Ile Glu Thr Asp
145 150 155 160
Gly Pro Gly Ser Ala Leu Asp Glu His Asp Leu Arg Val Val Ala Thr
165 170 175
Leu Arg Thr Cys Leu Pro Gln Val Val Ala Ser Pro Ala Ala Trp Arg
180 185 190
Asp Ala Arg Arg Arg Ala Ala Ile Gln Arg Val Ala Arg Leu Leu Ala
195 200 205
Ser Val Asp Gly Gly Ala Ala His Val Leu Leu Thr Val Arg Thr Thr
210 215 220
Thr Arg Asp Leu Pro Arg Val Ala Gly Ser Met Pro Glu Arg Ser Trp
225 230 235 240
Arg Ala Gly Thr Gly Leu Thr Glu Asn Leu Val Val Val Gln Gly Leu
245 250 255
Ala Ala Arg Arg Gly Leu Ala Glu Thr Ile Gly Gly Ile Leu Ala Ala
260 265 270
Gly Ala Leu Asp Val Ile Glu Ser Arg Val Gly Lys Asp Val Thr Pro
275 280 285
<210> 14
<211> 264
<212> PRT
<213>Actinomycesa lmadurae ATCC 39365 (Actinomadura sp. ATCC 39365)
<400> 1
Met Ile Val Cys Asp Leu Asp Gly Thr Leu Leu Asp Ser Arg Gly Gln
1 5 10 15
Val Ser Glu Arg Thr Arg Thr Ala Val Arg Arg Ala Arg Ala Ala Gly
20 25 30
His Val Phe Val Ile Ala Thr Ala Arg Pro Val Arg Asp Thr Arg Pro
35 40 45
Val Ala Ala Ala Leu Asp His Ala Ala Val Ala Val Cys Gly Asn Gly
50 55 60
Ser Ile Thr Phe Asp Phe Gly Ser Glu Glu Val Val Asp Tyr Arg Pro
65 70 75 80
Leu Asp Arg Gln Pro Leu Ala Ala Thr Leu Ala Leu Leu Arg Asp Arg
85 90 95
Phe Pro Gly Val Arg Leu Gly Ala Glu Cys Arg Leu Glu Leu Leu Leu
100 105 110
Glu Asp Ala Phe His Leu Pro Glu Pro Leu Ala Arg Asp Ala Arg Arg
115 120 125
Val Pro Arg Leu Glu Gly Glu Ile Asp Arg His Asp Val Gly Lys Leu
130 135 140
Met Val Gln Leu Glu Gly Ala Ala Arg Gln Tyr Tyr Glu Thr Val Arg
145 150 155 160
Gly Leu Leu Thr Gly Cys Glu Val Thr Ile Ser Ala Asp Val Phe Cys
165 170 175
Glu Val Met Arg Ser Gly Val Thr Lys Ala Ala Ala Leu Glu Ser Met
180 185 190
Ala Ser Arg Leu Gly Leu Gly Ser Ala Asp Val Ile Ala Phe Gly Asp
195 200 205
Met Pro Asn Asp Leu Pro Met Leu Thr Trp Ala Gly Thr Ala Val Ala
210 215 220
Val Ala Asn Ala His Pro Ala Val Leu Gly Ala Val Gly Glu Val Thr
225 230 235 240
Ala Ser Asn Asp Asp Asp Gly Val Ala Ala Trp Leu Glu Arg His Ala
245 250 255
Met Ala Asp Phe Ser Glu Lys Tyr
260

Claims (7)

1. the biological synthesis gene cluster of one kind 2 '-chloro Pentostatin and 2 '-amino -2'-deoxyadenosine, which is characterized in that institute The nucleotides sequence for the gene cluster stated is classified as in SEQ ID NO:1 shown in 10898-25249, wherein being responsible for 2 '-chloro spray departments The gene of his fourth biosynthesis, i.e.,adaC,adaB,AdaA, adaK,adaLTotally 5 genes；It is responsible for 2 '-amino -2 '-deoxidation gland The gene of glycosides biosynthesis, i.e.,adaF,adaG,adaJ,adaMTotally 4 genes；It is responsible for 2 '-chloro Pentostatins and 2 '-amino- The gene of 2'-deoxyadenosine transhipment and regulationadaE,adaD,adaH,adaITotally 4 genes；Specifically:
adaEAt 10898-12337 base in SEQ ID NO:1, cation transporter is encoded, amino acid sequence is SEQ ID NO.2；
adaDAt 12392-13600 base in SEQ ID NO:1, MFS transport protein, amino acid sequence SEQ are encoded ID NO.3；
adaCAt 13672-14559 base in SEQ ID NO:1, ATP phosphoribosyltransferase, amino acid sequence are encoded It is classified as SEQ ID NO.4；
adaBAt 14604-15308 base in SEQ ID NO:1, encoding short-chain dehydrogenase, amino acid sequence SEQ ID NO.5；
adaAAt 15295-16014 base in SEQ ID NO:1, SAICAR synzyme is encoded, amino acid sequence is SEQ ID NO.6；
adaFAt 16239-17516 base in SEQ ID NO:1, encoding aminotransferases, amino acid sequence is SEQ ID NO.7；
adaGAt 17509-18564 base in SEQ ID NO:1, dehydrogenase is encoded, amino acid sequence is SEQ ID NO.8；
adaHAt 18593-20380 base in SEQ ID NO:1, abc transport albumen, amino acid sequence SEQ are encoded ID NO.9；
adaIAt 20402-22180 base in SEQ ID NO:1, abc transport albumen, amino acid sequence SEQ are encoded ID NO.10；
adaJAt 22177-22662 base in SEQ ID NO:1, NUDIX hydrolase, amino acid sequence SEQ are encoded ID NO.11；
adaKAt 22659-23396 base in SEQ ID NO:1, phosphoriboisomerase A, amino acid sequence are encoded For SEQ ID NO.12；
adaLAt 23580-24446 base in SEQ ID NO:1, ATP phosphoribosyltransferase, amino acid sequence are encoded It is classified as SEQ ID NO.13；
adaMAt 24455-25249 base in SEQ ID NO:1, phosphohydrolase, amino acid sequence SEQ are encoded ID NO.14。
2. a kind of encoding gene of NUDIX hydrolaseadaJ, which is characterized in that in its nucleotide such as SEQ ID NO:1 Shown in 22177-22662 bit base.
3. a kind of encoding gene of NUDIX hydrolase as claimed in claim 2adaJThe NUDIX hydrolase A daJ of coding.
4. application of the NUDIX hydrolase A daJ as claimed in claim 3 in synthesis 2 '-amino -2'-deoxyadenosine.
5. containing the biological synthesis gene cluster of 2 '-chloro Pentostatin and 2 '-amino -2'-deoxyadenosine described in claim 1 Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium.
6. recombinant vector described in claim 5, expression cassette, transgenic cell line or recombinant bacterium synthesize 2 '-chloro spray departments he Application in fourth and/or 2 '-amino -2'-deoxyadenosine.
7. the method for a kind of 2 '-chloro Pentostatins of synthesis and/or 2 '-amino -2'-deoxyadenosine, for claim 5 institute of fermenting The recombinant bacterium stated collects tunning to get 2 '-chloro Pentostatins and/or 2 '-amino -2'-deoxyadenosine.
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