CN108239642A - A kind of relevant long non-coding RNA of Adipocyte Differentiation and its application - Google Patents
A kind of relevant long non-coding RNA of Adipocyte Differentiation and its application Download PDFInfo
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Abstract
The present invention provides it is a kind of with Adipocyte Differentiation relevant long non-coding RNA slincRAD, the RNA by with DNMT1 protein bindings, the signal paths such as PPAR γ is utilized to adjust the differentiation of adipocyte and the development of adipose tissue.Using the siRNA or shRNA for targeting slincRAD, the expression of slincRAD can be inhibited, and then specifically inhibit the differentiation process of fat.The transgenic mice for striking low slincRAD shows better glucose tolerance, also plays the role of to blood lipid metabolism positive.
Description
Technical field
The present invention relates to physiology and biomedical sector, and in particular to a kind of length with Adipocyte Differentiation regulatory function
Chain non-coding RNA and its application.
Background technology
Long-chain non-coding RNA (lncRNA) is that a kind of transcript length is more than 200bp, has relatively long nucleotide chain
Non-coding RNA.LncRNA has the characteristics that type is more, binding mode is mostly more with quantity, therefore the information that it contains is very rich
Richness, the molecular mechanism for participating in expression regulation is also varied, mainly from epigenetics, transcriptional level and post-transcriptional level
Three levels realize the regulation and control to gene expression.LncRNA may mainly have the function of the following aspects：(1) by egg
White encoding gene upstream promoter area transcribes, and interferes the expression of downstream gene；(2) by inhibit rna plymerase i I or
Mediate chromatin reconstructs and histone modification, influences downstream gene expression；(3) pass through the transcript shape with protein coding gene
Into complementary double-strand, and then the shearing of mRNA is interfered, so as to generate different shear patterns；(4) by with protein coding gene
Transcript forms complementary double-strand, and endogenic siRNA, the expression of controlling gene are further generated under Dicer enzyme effects；
(5) by being attached in specific protein, lncRNA transcripts can adjust the activity of corresponding albumen；(6) as structural constituent
Nucleic acid protein complex is formed with protein；(7) by being attached on specific protein, change the cytoplasm positioning of the albumen；(8)
It is transcribed as microRNA, such as precursor molecule of miRNA, piRNA.Just because of lncRNA can be from transcription and post-transcriptional level
The regulation and control of protein coding gene are participated in, these genes include disease and related gene and development related gene etc. occurs, and pass through ginseng
With regulation protein encoding gene, lncRNA can influence intracellular signal transduction approach, can also influence organismal development process
In signal transduction pathway.Report that lncRNA can have an impact the occurrence and development of a variety of diseases at present, including nerveous system
The disease of system, the diseases such as angiocardiopathy and kinds of tumors cancer.
The research of long non-coding RNA is attracted wide attention in recent years, and with the relevant lncRNA of Adipocyte Differentiation
Research is still very limited.The new lncRNA to play an important role in Adipose Differentiation is explored, it is thin not only to disclose regulation and control fat
The important mechanisms of born of the same parents' function will also provide new detection or therapy target, urgently be opened up in the research of fat metabolism relevant disease
Important scientific issues.
Invention content
The present invention is intended to provide with the relevant long non-coding RNA slincRAD of Adipocyte Differentiation function, and will
SlincRAD establishes slincRAD and strikes low mouse model for regulating and controlling the proliferation of adipocyte and differentiation behavior, is used for
The researchs such as the screening of fat metabolism relevant disease drug.
The expression of low slincRAD is struck in cell and mouse, leads to giving expression to now for Adipocyte Differentiation related gene
Drop, adipose tissue development are affected, weight loss.
Present invention firstly provides long-chain non-coding RNA slincRAD prepare promote Adipocyte Differentiation preparation or
Application in drug, the long-chain non-coding RNA sequence are shown in SEQ ID No.300.
Secondly, the present invention provides the inhibitor for inhibiting long-chain non-coding RNA slincRAD expression, including small interference core
Ribosomal ribonucleic acid (siRNA) and Small Hairpin RNA (shRNA), sequence are shown in Table 2 and table 3.
Further, the present invention provides the shRNA as long-chain non-coding RNA slincRAD expression inhibiting agent and
Applications of the siRNA in medicine for inhibiting adipocyte differentiation is prepared.
Again, the present invention provides the nucleic acid oligomer of detection long-chain non-coding RNA slincRAD, selected from following combination：
Detection primer combination 01：
01 forward primer：5’-ATCCATCTCCCCTACCCATC(SEQ ID NO:200)
01 reverse primer：5’-TGCAGAGGACTCACAACTGG(SEQ ID NO:201)
Detection primer combination 08：
08 forward primer：5’-GGTGCAGTTTTGCACTGAGA(SEQ ID NO:202)
08 reverse primer：5’-GATGGAGAGAAAGGAAGGGG(SEQ ID NO:203)
Detection primer combination 16：
16 forward primers：5’-CCTAGTGCCTTTCTGCCTTG(SEQ ID NO:204)
16 reverse primers：5’-TGGGGTGATTGTGTAGGGAT(SEQ ID NO:205)
Detection primer combination 31：
31 forward primers：5’-CAAGTGGTTCCGGATTCTGT(SEQ ID NO:206)
31 reverse primers：5’-CCGTGAAATGAAGTGGTGTG(SEQ ID NO:207)
Detection primer combination 54：
54 forward primers：5’-GCATTTAATTGGTGCTGGCT(SEQ ID NO:208)
54 reverse primers：5’-GACCCTTCTCCTAGCTGCCT(SEQ ID NO:209)
Further, the reagent the present invention provides detection long-chain non-coding RNA slincRAD expressions is preparing fat
Application in fat differentiation accelerator or inhibitor.
Fourth aspect, the present invention provides the transgene mouse model for striking low long non-coding RNA slincRAD, the mouse
The application of the preparation method of model and the mouse model in fat metabolism relevant disease drug screening.The fat metabolism phase
Related disorders include but not limited to metabolic syndrome, obesity, hyperglycemia, hyperlipidemia, diabetes.
SlincRAD can promote mouse 3T3-L1 cell differentiations, this effect may be by the realization of PPAR γ signal paths
's.
Therefore, slincRAD can be used for promoting the differentiation of adipocyte, can be also used for preparing or screens preparation treatment fat
Fat relevant disease, such as the drug of obesity.
Beneficial effects of the present invention：
The present invention provides it is a kind of with Adipocyte Differentiation relevant long non-coding RNA slincRAD, the RNA by with
DNMT1 protein bindings adjust the differentiation of adipocyte and the development of adipose tissue using signal paths such as PPAR γ.Utilize targeting
The siRNA or shRNA of slincRAD, can inhibit the expression of slincRAD, and then specifically inhibit the differentiation process of fat.
The transgenic mice for striking low slincRAD shows better glucose tolerance, also plays the role of to blood lipid metabolism positive.
Description of the drawings
Fig. 1 is the Differentiation Induction in vitro process of 3T3-L1 adipocytes.(A) oil red O stain of Adipocyte Differentiation process
As a result.The 0th day in adipocyte Differentiation Induction in vitro, 14 days and 21 days sample respectively, oil red O stain are carried out, using to fat
The coloration result of fat drop judges the differentiation situation of adipocyte.The 0th day broken up in induction, the ratio of oil red O stain positive cell
Example is 0%；The 14th day of induction differentiation, the ratio of oil red O stain positive cell is 24.2%；The 21st day of induction differentiation, oil
The ratio of red O staining positive cells is 54.9%.(B) during Adipocyte Differentiation PPAR γ and CEBP α expression.Profit
With Fluorescent quantitative PCR (qRT-PCR), the table of PPAR γ and CEBP α during Adipocyte Differentiation are detected
Up to situation.Using the expression of the 0th day each gene as reference, the expression of gene in induction atomization, expression value are calculated
Form of presentation be：Mean+SD.
Fig. 2 is to strike the expression of low slincRAD genes to inhibit the Differentiation Induction in vitro of adipocyte.(A) oil red O stain
As a result carries out the transfection of first time siRNA 2 days before induction is broken up to 3T3-L1 cells；On the day of induction differentiation processing, into
Second of siRNA transfection of row.At the 9th day of induction differentiation, cell is harvested, oil red O stain is carried out, observes lipid within endothelial cells
Generation and accumulation.Control：It is handled, and the cell of untransfected siRNA without induction differentiation.Mock：Only by induction point
Change is handled, the cell of untransfected siRNA.NC-siRNA：By inducing differentiation processing, and it is unrelated to have transfected a sequence
The cell of siRNA.siRNA1-siRNA17：By inducing differentiation processing, the cell of slincRAD specific siRNAs has been transfected.
(B) quantitative analysis results of oil red O stain.Utilize ImageJ softwares (http://imagej.nih.gov/ij/), to oil red O
Coloration result carries out quantitative analysis.In order to eliminate background stainings, the dye number of Control cells is subtracted as dyeing background first
It goes.Then on the basis of the dye number of Mock, the dye number of other cells is normalized；The dye that will then obtain again
Cell number is normalized in color value, to reflect that siRNA handles the influence to cell differentiation.It is carried out using t-tests
Statistical analysis (the * of experimental result：Using NC-siRNA as control, P<0.05).
Fig. 3 is adjustment effects of the slincRAD-shRNA to Adipocyte Differentiation.(A) gene structure of slincRAD, turn
Record this structure and shRNA target positions.(B) slincRAD-shRNA is to the inhibition of target gene.According in embodiment two
Scheme carries out hormone induction processing to wild type, slincRADshRNA-8, slincRADshRNA-9 3T3-L1 cells.Processing
After 6 days, cell is harvested, extracts total serum IgE, utilizes the expression of qRT-PCR detections slincRAD.With a sequence it is unrelated,
Target control groups (CTL, control) of the shRNA of GFP protein gene as experiment.Experimental data is repeated for biology three times
Average value, data coding method is：Average value ± s.e.m.(C) to the inhibiting effect of cytolipin generation.Hormone induction point
Change the 7th day after separation, oil red O stain is carried out to culture cell, analysis inhibits slincRAD gene expressions to cytolipin
It influences.Upper figure：The oil red O stain of culture plate；Middle figure：The local dye image of culture plate；Figure below：With ImageJ softwares to oil red
The quantitative result of O dyeing.Control group (CTL) is wild type 3T3-L1 cells.
Fig. 4 is transgenic mice building process.(A) the composition process of diagram transgenic mice structure.(B) the 11st nest turns base
Because of the genotype identification of mouse.
Fig. 5 is and identifies for building for transgenic mice.(A) pedigree chart of transgenic mice.By two genotype of acquisition
For shRNA8+/- it is first for transgenic mice (number：13,15) and genotype for shRNA9+/- head it is small for transgenosis
Mouse (number：11) it, mates with wild type C57BL/6J female mices.Using number be No. 9 first for transgenic mice into pedestrian
Work is inseminated.20 nests, 143F1 are obtained in total for mouse, wherein transgenic mice 81.The flagging mode of mouse is as follows in figure, such as
9# (M3, F5), which is indicated, 3 male mouse and 5 female mices in the 9th nest mouse.The mouse that number is No. 1-6 is located before birth
Extremely, for studying brephic mouse development and gene expression situations such as.(B) expression of slincRAD genes is in transgenosis
It is lowered in mouse.Using qRT-PCR, the gene expression dose in birth 10 days, 15 days, 30 days mice organs is examined
It surveys.In PCR, compareed using U6RNA as detection.The expression of slincRAD genes is represented as versus wild type gene water
Flat multiple proportion.The average value that experimental data is repeated for biology three times, data coding method：Average value ± s.e.m.(C)
Mouse genetic ideograph.This table statement be every nest mouse hereditary information, such as the 9th nest mouse have the sub- mouse of 3 males and 5 it is female
Property mouse, have 1 in 3 male mices for transgenic mice, 5 female mices are transgenic mice.In the stage of development one
In column, ED15 represents the mouse of the 15th day embryonic period, embryonic phase；Day15 represents the mouse of the 15th day after birth；7Mo represents after birth 7
The mouse of the moon.
Low slincRAD is struck in Fig. 6 expressions influences the development of mouse.(A) after being born, transgenic mice and wild-type mice
Weight compares (*, the value of significant difference).(B) weight distribution map (*, the value of significant difference).(C) transgenic mice
With the body shape figure of wild-type mice.
The development of mouse adipose tissue of low slincRAD Gene Expressions is struck in Fig. 7 expressions.(A) body fat and muscle are surveyed
Amount.Using toy nuclear magnetic resonance spectrometer, we have detected the body body fat at 1 monthly age and 7 monthly age mouse and Lean mass (every group of n >=4).
White column：Wild-type mice；Black column, transgenic mice.(B) the main organs weight (n >=4) of 5 monthly age mouse.White column：
Wild-type mice；Black column, transgenic mice.(C) Histological section's observation of adipose tissue.Upper figure：White adipose tissue
Hematoxylin eosin staining result.White column：Wild-type mice；Black column, transgenic mice.Graphic scale is 50 μm.Under
Figure：The size distribution of adipocyte.Open circles：Wild-type mice；Filled circles：Knock out mice.Data coding method：It is average
Value ± S.E.M., every group of n=3, * p<0.05；***p<0.001. the histotomy observation of (D) 7 monthly age mouse main organs.Figure
Upper engineer's scale is 100 μm.
Fig. 8 shows slincRAD to the regulating and controlling effect of Adipocyte Differentiation key factor.Using qRT-PCR, we are out of office
In the liver organization of raw type and transgenic mice, analyze Adipocyte Differentiation key factor PPAR γ and CD36 and
The expression of slincRAD.Using wild-type mice gene expression dose as reference, the expression (phase of transgenic mice is calculated
For the multiple of wild type gene expression).Data coding method：Average value ± S.E.M., every group of n=10.
Influences of the low slincRAD to mouse blood sugar and blood lipid metabolism is struck in Fig. 9 expressions.(A) 7 monthly age wild types and transgenosis
In mouse, the total triglycerides of blood glucose, blood plasma (TG), T-CHOL (TC), low-density lipoprotein (LDL), high-density lipoprotein
(HDL) comparative study of content.Open tubular column：Wild-type mice；Solid post：Transgenic mice.Data coding method：Average value ±
S.E.M., every group of n=14.*p<0.05；**p<0.01；***p<0.001.(B) sugared tolerance test.Empty circles：Wild type is small
Mouse；Solid circles：Transgenic mice.Data coding method：Average value ± S.E.M., every group of n=14.(C) insulin resistance
Experiment.Empty circles：Wild-type mice；Solid circles：Transgenic mice.Data coding method：Average value ± S.E.M., every group
n≥3。
Figure 10 represents influences of the slincRAD to Adipose Differentiation key factor.During Adipocyte Differentiation, utilize
The expression of qRT-PCR, day by day detection differentiation key factor, draw curve graph.Blue line：Gene expression in wild-type cell
It is horizontal；Red line, slincRAD strike the gene expression dose in low cell strain.Internal reference is β-actin.Data coding method：It is average
Value ± S.E.M., every group of n=3.
Figure 11 represent flow cytomery wild type slincRAD cells and slincRAD strike low profile (shRNA8 and
ShRNA9 strikes low) variation of cell cycle in cell differentiation procedure.
Figure 12 represents the expression pattern analysis of Dnmt1 genes.Using qRT-PCR, Dnmt1 genes are analyzed in adipocyte point
Expression variation during change.Internal reference is β-actin.Data coding method：Average value ± S.E.M., every group of n=3 biology weight
It is multiple.
Figure 13 represents RNA co-immunoprecipitations.Using anti-DNMT1 antibody and Immunoprecipitation, obtain and occur with DNMT1
The cell RNA component of interaction.Followed by qRT-PCR technologies, slincRAD gene transcripts in immune complex are detected
Content.The negative control of experiment is the immune complex of anti-igg antibody capture.Data coding method：Average value ± S.E.M..
Figure 14 is the Wien map analysis that wild type and slincRAD strike promoter methylation level in low cell.
Figure 15 is the Wien map analysis of DEP genes.
Figure 16 is the DEP indexes of the proliferation-associated genes of differential methylation in wild type 3T3-L1 cells.
Figure 17 is Heat-map analyses.In (- 2) day and (+2) day of adipocyte induction differentiation, comparative analysis base
Because of the DEP indexes of promoter methylation level.Red represents the gene of hyper-methylation level；Green represents hypomethylation gene,
This figure is drawn with Heatmap Illustrator.
Figure 18 is the expression variation of the albumen such as western hybridization researchs p21.Studied cell line includes wild type 3T3-L1
Cell (WT), slincRADshRNA-8 (sh8) and slincRADshRNA-9 (sh9) slincRAD low expression cells.During research
Between point for adipocyte Differentiation Induction in vitro (- 2) to (+4) day.
Figure 19 is slincRAD binding mode figures.
Specific embodiment
With reference to specific embodiment and attached drawing, the present invention is further explained.It should be appreciated that these embodiments are only used for
It is bright the present invention and cannot be used for limiting the scope of the invention.Test method without specific conditions in the following example, usually
According to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in or according to the normal condition proposed by manufacturer.
Embodiment one：Experimental animal and material
Experiment is provided with mouse purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., is raised in Peking University's reality
Test animal center.All experimental programs and operation obtain the approval of experimental animal Ethics Committee of Peking University.
DNA oligonucleotides is purchased from Invitrogen Beijing company, and RNA oligonucleotide has purchased from the sharp rich biotechnology in Guangzhou
Limit company.
Embodiment two：The Differentiation Induction in vitro of 3T3-L1 adipocytes
3T3-L1 PECTORAL LIMB SKELETON Differentiation Induction in vitro is a common Adipocyte Differentiation model, in derivant (1-
Methyl -3- isobutyl groups xanthine (IBMX), insulin, dexamethasone) under the action of, it is thin that ripe fat is divided under induction
Born of the same parents.In order to study the differentiation process of cell, we have detected the generation of fat drop and product in cell first with oil red O stain
Tired situation finds that, in induction differentiation latter stage (the 21st day), the fat stains positive is presented in 54.9% cell.In addition, we are also sharp
With using Fluorescent quantitative PCR (qRT-PCR), detect cell differentiation key factor PPAR γ and CEBP α's
Expression.It was found that expression regulation of the two factors in cell differentiation is consistent with document report, show to induce atomization
It is successful.Research approach and experimental result are specific as follows：
1st, the culture of 3T3-L1 PECTORAL LIMB SKELETONs and Differentiation Induction in vitro：Induce the definition of divergaence time point：It is lured with adding in
Differential medium is led, the same day for proceeding by induction differentiation is the 1st day of induction differentiation.Induction breaks up the 1st day the previous day and is
0th day.Induction break up the 1st day one day after for the 2nd day, and the rest may be inferred defines other induction divergaence time nodes.
In the DMEM fluid nutrient mediums containing 10% fetal calf serum, routine culture 3T3-L1 murine preadipocyte cells, 2 to
Change within 3 days liquid 1 time.At the 1st day of induction differentiation, change conventional medium into induction culture medium, carry out Differentiation Induction in vitro.
Inducing culture is that 167nM actrapid monotards (Sigma), 0.5mmol/L 1- methyl -3- are added on the basis of conventional medium
Isobutyl group xanthine (Sigma) and 1 μM of dexamethasone (Sigma).At the 2nd day of induction differentiation, culture medium is changed into and is only contained
The DMEM culture mediums of 167nM insulin.At the 4th day of induction differentiation, differential medium is gained into conventional DMEM culture mediums, after
Continuous culture, until atomization terminates.
2nd, oil red O stain：According to a conventional method, oil red O reagents are configured.In the material time node of atomization：Induction point
Change early period (the 0th day), induction differentiation mid-term (the 14th day) and induction differentiation latter stage (the 21st day), sampling carry out oil red O dyes
Color.
Experimental procedure：1) the culture cell sample obtained to each timing node, first with the phosphate buffer by ice bath
(PBS) it is light and slow to rinse 3 times；2) cell is fixed 2 minutes with 3.7% formaldehyde；3) it is small with the incubation 1 of oil red O reagents at ambient temperature
When, cellular fat is dyed；4) deionized water rinses 3 times；5) it optical microphotograph Microscopic observation and takes pictures；6) ImageJ softwares are utilized
(http://imagej.nih.gov/ij/), not carry out the cell of induction differentiation to control, oil red O stain result is carried out
Quantitative analysis.
Oil red O stain is as a result, as shown in Figure 1A.The 0th day broken up in induction, the ratio of oil red O stain positive cell are
0%；The 14th day of induction differentiation, the ratio of oil red O stain positive cell is 24.2%；The 21st day of induction differentiation, oil red O dyes
The ratio of color positive cell is 54.9%.Ripe adipocyte is rounded, has a large amount of fat drips to assemble, and oil red O stain shows tangerine
It is red.
3rd, the detection of expression of PPAR γ and CEBP α：Using qRT-PCR, PPAR γ and CEBP α are in atomization for analysis
Expression variation, reference gene are β-actin.The primer sequence information of detection gene expression is shown in Table 1.Experimental result such as Figure 1B institutes
Show, experimental procedure is specific as follows：
1) Total RNAs extraction：Induction is obtained respectively to break up the 0th day (induction pre-differentiation stage), the 14th day (induction differentiation mid-term),
The cell sample in the 21st day (induction differentiation latter stage), using Trizol reagents, extracts cell total rna, and quantified.
2) RNA reverse transcriptions：Use Reverse Transcriptase kit (the TIANScript M-MLV, cat of Tiangeng biochemical technology company:
ER104-03 the reverse transcription reaction of RNA samples) is carried out, is specifically carried out according to the method for kit specification, step is as follows：Take 1
Microgram total serum IgE sample adds in the random reverse transcriptase primer of 2 μ L (10 μM) 6 bases, 2 μ L dNTP (10mM), mixing postpositionWater
Bath denaturation 5 minutes；It is taken out after placing 3 minutes on ice, adds in 4 μ 5 × First-Strand of L Buffer, 0.5 μ L RNase
Inhibitor (40U/ μ L), 1 μ L M-MLV (200U/ μ L), total volume are 20 μ L；Of short duration centrifugation, is placed in after mixing
Reverse transcription reaction is carried out in Eppendorf PCR instruments, response parameter is10 minutes；50 minutes；5 minutes；
It is subsequently placed atIt preserves.Wherein, RNase inhibitor are the product (cat#N211) of Promega companies.
3) real-time fluorescence quantitative PCR：Use the HotMaster Taq DNA of Tiangeng biochemical technology company
Polymerase reagents (cat#:ET106-01-01), the expression of gene in sample is detected, experimental procedure is specific as follows：It takes
2 μ L reverse transcription products add in 2.5 μ L 10 × HotMaster Taq Buffer, 0.5 μ L (10 μM) sense primers, 0.5 μ L (10
μM) downstream primer, 1 μ L dNTP mixtures (each 2.5 μM), 1 μ L SYBR Green I (5 ×), 0.2 μ L HotMaster Taq
DNA polymerase (2.5U/ μ L), are eventually adding 17.3 μ L deionized waters, and reaction total volume is 25 μ L.After mixing it is of short duration from
The heart, is placed in Eppendorf PCR instruments and carries out pcr amplification reaction, and response parameter isPre-degeneration 2 minutes,Denaturation 15
Second,Annealing 15 seconds,Extension 30 seconds；Cycle-index is 40 cycles.3 repetitions of each reaction setting.
4) data analysis：To same sample, the expression of wherein target gene and reference gene (β-actin) is detected respectively.
On the basis of the expression quantity of reference gene, the expression of target gene is normalized.Then usually made using this field
Delta delta Ct methods carry out quantitative analysis to the expression quantity of target gene.Specific method and step may refer to text
It offers：Livak KJ and Schmittgen TD.Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T))
Method.Methods.2001Dec；25(4):402-408.
Table 1, RT-PCR primer pair
Embodiment three：Strike inhibiting effect of the low slincRAD to adipocyte Differentiation Induction in vitro
In order to study influence of the expression to Adipocyte Differentiation of slincRAD genes, we obtain 6 by screening
The siRNAs of slincRAD gene specifics, including siRNA1, siRNA9, siRNA12, siRNA14, siRNA15 and siRNA17
(table 2).Using lipofectamine, these siRNA are directed respectively into the 3T3-L1 cells of culture by we, strike it is low wherein
The expression of slincRAD genes；Followed by oil red O stain, whether the generation of identification of cell fat is affected, so as to
Judge whether the Adipocyte Differentiation process of 3T3-L1 is affected.It was found that with the control cell that is handled without siRNA and
The control cell handled through scramble siRNA is compared, and the siRNA of certain gene specifics can significantly inhibit the life of cytolipin
Into with accumulation (Fig. 2)；The lipid content of cell can be reduced by 70% by especially siRNA-17 using siRNA processing cell.It removes
Other than the generation of research cytolipin, we also using real-time fluorescence quantitative PCR, have studied and strike low slincRAD to adipocyte
Break up the influence of key factor PPAR γ expression.Result of study is shown：Low slincRAD bases are struck using siRNA (such as siRNA-14)
The expression of cause not only affects the generation of cytolipin, moreover it is possible to the expression (Fig. 2) of PPAR γ genes in cell is significantly inhibited, this
It is consistent with its inhibiting effect to cell differentiation.Experimental procedure is specific as follows：
1st, 3T3-L1 cell culture：In the DMEM fluid nutrient mediums containing 10% fetal calf serum, routine culture 3T3-L1
Murine preadipocyte cell, changes liquid for 2 to 3 days 1 time.
2nd, siRNA is transfected：Utilize BLOCK-iTTMRNAi Designer (Invitrogen) program designs slincRAD
The siRNA molecule of gene specific, particular sequence are as shown in table 2.
The day before transfection, by 3T3-L1 cell inoculations in 6 well culture plates, the degrees of fusion after making cell adherent reaches 60-
70%.According to the requirement of operation manual, the Lipofectamine of Invitrogen companies is usedTMRNAiMAX transfection reagents into
Row cell transfection assays.It is for each hole of 6 well culture plates, 100pmol siRNA is thin in 400 μ L Opti-MEM (GIBCO)
RNAiMAX/siRNA mixtures are prepared in born of the same parents' culture solution, is stored at room temperature after twenty minutes, RNAiMAX/siRNA mixtures is added in
Into 6 orifice plates；After transfecting 4 hours, add in normal cell culture medium and cultivated.
After continuing culture 2 days, treat that cell length to Fusion Strain, adds in the DMEM culture mediums containing differentiating inducer, starts
Induce atomization.And second of siRNA transfection is carried out on the day of, RNAiMAX/siRNA mixtures are added directly into induction
In differential medium, siRNA transfections are carried out.
3rd, Differentiation Induction in vitro：The definition of 3T3-L1 Differentiation Induction in vitro timing nodes：To add in inductive differentiation medium,
The same day for proceeding by induction differentiation is the 1st day of induction differentiation.The previous day that induction is broken up the 1st day is the 0th day.Induction differentiation
1st day one day after for the 2nd day, and the rest may be inferred defines other induction divergaence time nodes.
In the DMEM fluid nutrient mediums containing 10% fetal calf serum, routine culture 3T3-L1 murine preadipocyte cells, 2 to
Change within 3 days liquid 1 time.At the 1st day of induction differentiation, change conventional medium into induction culture medium, carry out Differentiation Induction in vitro.
Inducing culture is that 167nM actrapid monotards (Sigma), 0.5mmol/L 1- methyl -3- are added on the basis of conventional medium
Isobutyl group xanthine (Sigma) and 1 μM of dexamethasone (Sigma).At the 2nd day of induction differentiation, culture medium is changed into and is only contained
The DMEM culture mediums of 167nM insulin.At the 4th day of induction differentiation, differential medium is gained into conventional DMEM culture mediums, after
Continuous culture, until atomization terminates.
4th, oil red O stain：At the 9th day of adipocyte Differentiation Induction in vitro, cell sample is obtained, carries out oil red O stain
Analysis.Experimental procedure is specifically shown in embodiment two.Experimental result is shown in Fig. 2.
Table 2, siRNA sequence
Example IV：Build slincRAD shRNA transgenic mices
In order to identify slincRAD genes in body function, need in the feelings for striking slincRAD gene expressions in low Mice Body
Under condition, its influence to Adipocyte Differentiation, cytolipin generation and crucial differentiation factor expression is studied.For this purpose, we are sharp
With the specific shRNA of targeting slincRAD, slincRAD shRNA transgenic mices are constructed.It is specific as follows：
The screening of slincRAD specificity shRNA (slincRAD-shRNA).1) design slincRAD specificity shRNA.
Using Photographing On-line tool, we are directed to the sequence of slincRAD genes, devise different shRNA expressed sequences, pass through sieve
Choosing obtains expressed sequence (shRNA-8, shRNA-9, shRNA-15, shRNA-17 of 4 specific shRNA；Fig. 3 A；Sequence
It is shown in Table 3).2) structure of shRNA expression vectors.Utilize the pSD31 retroviral vectors purchased in Invitrogen companies, structure
Build the shRNA expression vectors of slincRAD specificity.According to the described scheme of kit specification, we synthesize double-strand first
ShRNA expression cassettes, be subcloned into the pSD31 retroviral vectors of linearisation, by be sequenced identify, be directed to
The shRNA expression vectors of slincRAD genes.3) cell transfecting and Differentiation Induction in vitro.It is thin using Lipofectamine 2000
ShRNA expression vectors are imported the 3T3-L1 cells of culture by dysuria with lower abdominal colic transfection reagent.The Differentiation Induction in vitro described using embodiment one
Scheme carries out induction differentiation to the cell for importing shRNA.4) identification of activity shRNA.At the 6th day of induction differentiation, harvest was thin
Born of the same parents.According to the method that embodiment one describes, using real-time fluorescence quantitative PCR, the expression of slincRAD genes, detection are detected
The primer sequence of slincRAD gene expressions is shown in Table 4.It was found that slincRAD-shRNA8 and slincRAD-shRNA9 can efficiently press down
The expression of endogenous cellular slincRAD processed.Wherein, the expression of target gene can be reduced to wild by slincRAD-shRNA8
The 1% of type cellular level, and the expression of target gene can be then reduced to horizontal 3% of wild-type cell by slincRAD-shRNA9
(Fig. 3 B).Oil red O stain finds that slincRAD-shRNA8 and slincRAD-shRNA9 are while target gene is inhibited, also
Significantly suppress the generation (Fig. 3 C) of cytolipin.
Table 3, shRNA sequences
shRNA8(SEQ ID No 21)：
5’GTGGAAAGGACGAAACACCCGGCCTATAACCAGTACAGTTAATCAAGAGTTAACTGTACTGGTTATA
GGCTTTTTTGGATCCCGAAT 3’
shRNA 9(SEQ ID No 22)：
5’GTGGAAAGGACGAAACACCCGGCACCAACCTCTTCCAATATCTCAAGAGGATATTGGAAGAGGTTGG
TGCTTTTTTGGATCCCGAAT 3’
shRNA 15(SEQ ID No 23)：
5’GTGGAAAGGACGAAACACCCGGGATTAGTGTGGCAGATTATCAAGAGTAATCTGCCACACTAATCCT
TTTTTGGATCCCGAAT 3’
shRNA 17(SEQ ID No 24)：
5’GTGGAAAGGACGAAACACCCGGGATTAGTGTGGCAGATTATCAAGAGTAATCTGCCACACTAATCCT
TTTTTGGATCCCGAAT 3’
Table 4, slincRAD amplimer sequences
01 detection primer combines：
01 forward primer： 5’-ATCCATCTCCCCTACCCATC (SEQ ID NO:200)
01 reverse primer： 5’-TGCAGAGGACTCACAACTGG (SEQ ID NO:201)
08 detection primer combines：
08 forward primer： 5’-GGTGCAGTTTTGCACTGAGA (SEQ ID NO:202)
08 reverse primer： 5’-GATGGAGAGAAAGGAAGGGG (SEQ ID NO:203)
16 detection primers combine：
16 forward primers： 5’-CCTAGTGCCTTTCTGCCTTG (SEQ ID NO:204)
16 reverse primers： 5’-TGGGGTGATTGTGTAGGGAT (SEQ ID NO:205)
31 detection primers combine：
31 forward primers： 5’-CAAGTGGTTCCGGATTCTGT (SEQ ID NO:206)
31 reverse primers： 5’-CCGTGAAATGAAGTGGTGTG (SEQ ID NO:207)
54 detection primers combine：
54 forward primers： 5’-GCATTTAATTGGTGCTGGCT (SEQ ID NO:208)
54 reverse primers： 5’-GACCCTTCTCCTAGCTGCCT (SEQ ID NO:209)
Build transgenic mice：Acquisition is separately encoded slincRAD-shRNA8 and slincRAD-shRNA9 expression cassettes
DNA fragmentation, the DNA fragmentation length are 285 bases, include U6 promoters, shRNA coded sequences and one section of 6 nucleotide
Termination sequence TTTTTT.DNA fragmentation is imported in the protokaryon of C57BL/6 mouse fertilized eggs using microinjection, it then will fertilization
In ovum transfer to the rat body of false pregnancy, it is made to develop into mouse (Fig. 4 A).In order to identify transgenic mice, we devise two pairs
Primer pair (table 5) independent, that shRNA expression cassettes can be expanded.By expanding the shRNA expression cassettes integrated in mouse genome,
We identify 4 Male Transgenic mices for incorporating slincRAD-shRNA8 and slincRAD-shRNA9 respectively --- and it is first
For transgenic mice (Fig. 4 B, 5A).
5. shRNA expression cassette amplimers pair of table
Building for transgenic mice be：Transgenic mice with wild type female mice is mated, obtains generation mice.Extraction
For the genomic DNA of mouse tail tissue, using specific primer to (table 5), the shRNA expression cassettes wherein integrated are carried out
PCR amplification, the positive generation mice for as incorporating shRNA expression cassettes of amplified signal.Obtain four slincRAD- altogether in this way
ShRNA transgenic mouse lines (Fig. 5 A).The genotype of three mouse system for slincRAD-shRNA8+/-, thus be named as
slincRADshRNA8Mouse, the genotype of mouse system for slincRAD-shRNA9+/-, thus be named as
slincRADshRNA9。
Inhibit the phenomenon that target gene is expressed to detect shRNA, we were in mouse postnatal 10th day, 15 days and 30
My god, mouse is put to death, obtains mouse tissue, utilizes qRT-PCR, expressions (Fig. 5 B) of the detection slincRAD in internal organs.I
Find, compared with control group wild-type mice, other than the brain tissue of the tenth day, expression of the slincRAD in other organs
Amount significantly reduces, be averagely reduced to wild type tissue expression 60% and it is following.
The transgenic mice of sufficient amount in order to obtain, for follow-up study, we are using natural mating and artificial insemination
Method carries out transgenic mice breeding.In artificial insemination breeding, we take out a slincRAD firstshRNA8The one of mouse
Side seminal vesicle, the ripe sperm of separation, is transferred in wild type false pregnancy rat body.In this way, we obtain 143F in total1
For mouse, wherein 81 be slincRADshRNA8Positive mice, 62 are wild-type mice (Fig. 5 C).Obtained generation mice middle-jiao yang, function of the spleen and stomach
The ratio of property mouse and wild-type mice is essentially 1:1, meet Mendel's regulavity of segregation.This shows the table for striking low slincRAD
Up to level, do not produce serious influence to the survival ability of mouse.For convenience, slincRADshRNA8Transgenic mice
It is referred to as slincRAD and strikes low mouse or KD mouse.
Embodiment five：Strike the influence that mouse is developed in low slincRAD expression
In order to study the influence struck low slincRAD and developed to mouse, we send out the body of wild type and transgenic mice
The situation of educating has carried out comparative study, and the period is the 15th day embryonic period, embryonic phase to after being born 7 months.In embryonic development period, transgenosis is small
Mouse and wild-type mice do not have notable difference on slincRAD expressions, the unusual phase in individual morphology and birth weight
Seemingly.After birth, the weight of transgenic mice starts to become significantly lower than wild-type mice, although maintaining identical raising item
Part (Fig. 6 A).5 days after birth, compared with wild type control mice (n=9), the weight of transgenic mice (n=11) is low~
12% (p<0.05).This otherness is always maintained at the manhood (Fig. 6 B) of mouse.Morphologically, transgenic mice is without open country
The abdomen protuberance (Fig. 6 C) of raw type mouse, this phenomenon may be to be affected derived from the normal development of mouse abdominal adipose.
The present embodiment statistics indicate that, strike the expression of low mouse slincRAD genes, the mice embryonic phase do not developed and generates shadow
It rings, but significantly affects the postnatal development process of transgenic mice.Although compared with control mice, the table of slincRAD genes
Being presented in embryonic period, embryonic phase and birth later stage up to level significantly reduces.
Embodiment six：Strike the influence that mouse adipose tissue is developed in low slincRAD gene expressions
On the basis of above-mentioned work, it is presumed that the influence that slincRAD develops mouse, it may be possible to derived from its interference
The normal differentiation of adipose tissue.In order to verify this imagination, we utilize toy NMR imaging equipment Nuclear
Magnetic resonance spectroscopy (EchoMR-700, MchoMRI, Houston TX), under waking state,
The body fat of mouse, muscle and moisture are detected.Concrete operations according to the scheme that equipment manufacturer provides into
Row, sweep spacing are 0.5-3.2 minutes.Every animal carries out 3 independent measurements.
To 1 monthly age mouse the study found that the body fat content of transgenic mice lower than wild-type mice 12%, Lean mass
It is higher than wild-type mice by 9%.To 7 monthly age mouse the study found that the body fat content of transgenic mice is lower than wild-type mice
22%, Lean mass 6% (Fig. 7 A) lower than wild type.Further, we compare the weight of mouse main organs
Research, finds no significant difference (Fig. 7 B).
In order to analyze the influence struck low slincRAD and developed to mouse adipose and its hetero-organization, it is small that we were prepared for for 7 monthly ages
The histotomy of mouse main organs.The organs and tissues of transgenic mice and wild-type mice are obtained first, are infiltrated 4%
It is fixed under the conditions of 4 DEG C to stay overnight in paraformaldehyde solution；Paraffin embedding, with 5 μm of width serial section；Subsequent progress hematoxylin-she
Red colouring and micro- sem observation.We have found that compared with the normal structure of wild-type mice, in the adipose tissue of transgenic mice
Appearance colours deeper small island areas (Fig. 7 C).Ultramicroscopic observation finds that these regions are by smaller adipocyte group
Into.In the transgenic mice at 7 monthly ages, the average-size of adipocyte is 19.5 ± 4.8 μm, and in wild type control mice
In, the size of adipocyte is then 58 ± 10 μm.In September age transgenic mice, the average-size of adipocyte for 21.4 ±
4.6 μm, in wild-type mice, the size of adipocyte is 74.7 ± 14 μm (Fig. 7 D).In addition to adipose tissue, other organs
Institutional framework, which is showed no, to be substantially change (Fig. 7 D).
Embodiment seven：The study on mechanism of slincRAD
In order to illustrate the mechanism of action of slincRAD, we have studied it to PPAR γ and CD36 the two adipocytes point
Change the regulating and controlling effect of key transcription factor.PPAR- γ are a kind of nuclear receptors, adjust nearly all side of fatty acid metabolism
Face, such as transport, synthesis, storage, mobilization, activation and the oxidation of aliphatic acid.CD36 is a kind of B classes scavenger receptor, can be promoted
Adipocyte Differentiation and adipogenic.
Using qRT-PCR, we mouse it is 10th day postnatal, 15 days and 30 days, have detected PPAR in liver respectively
The expression (Fig. 8) of γ, CD36 and slincRAD gene.The 10th day to be born in mouse, PPAR in transgenic mice liver
The expression ratio wild-type mice of γ, CD36 and slincRAD reduce 50%.But with the continuation of growth course, PPAR γ
The level for being restored to wild-type mice the 15th with the expression of CD36, in the water that the 30th day rises above wild-type mice
It is flat, show certain dosage compensation effect.On the one hand prompting slincRAD is sent out by PPAR γ signal paths to these data
Effect is waved, on the other hand also explains why the adjustment effect that slincRAD develops mouse is mainly manifested in embryonic development
Early stage.
Embodiment eight：Strike influences of the low slincRAD to mouse blood sugar, blood lipid metabolism
White adipose tissue be human body energy metabolism in an important link, the excessive accumulation of human body white adipose tissue
Organism metabolism can be caused abnormal.In order to inquire into effects of the slincRAD in terms of energetic supersession, we are with September age wild type and turn
DNA murine is research object, and influence of the low slincRAD gene expressions to mouse blood sugar, blood lipid metabolism is struck in research.
Blood-sugar content detects：It takes a blood sample from mouse tail vein, with the blood-sugar detecting instrument ACCU-CHEK of Roche Holding Ag
Performa Glucometer (Roche, Denmark) detect mouse's blood sugar content, and blood-sugar detecting instrument specification is pressed in concrete operations
It carries out.Lipids contents detect：By mouse fasting 6 hours, tail vein blood detached plasma composition therein, with Roche Holding Ag
Analyzer (Cobas 4000analyzer), the contents level of detection wherein triglycerides, T-CHOL, LDL-C, HDL-C3.
As shown in Figure 9 A, compared with wild type control mice, the plasma triglyceride content of transgenic mice significantly reduces,
HDL-c contents significantly increase.Ascendant trend is presented, but there was no significant difference in total plasma cholesterol (TC) and LDL-c.
In addition, we have also carried out glucose and insulin resistance experiment.Before starting experiment, continuous three days determination experiments
The glycemic baseline of mouse is horizontal.By mouse overnight fasting (about 16 hours) before experiment, then by the dosage of 1.5g/kg weight to
Injection D-Glucose in mouse peritoneal after injection the 0th, 15,30,60,120 and 180 minute, carries out tail vein blood, uses
The blood-sugar detecting instrument detection mouse's blood sugar content of Roche Holding Ag, analyzes the variation of mouse's blood sugar content, draws glucose tolerance curve (figure
9B)。
Before insulin tolerance experiment, by mouse fasting 6 hours, the dosage of 1IU/kg weight, intraperitoneal injection are then pressed
Actrapid monotard, after injection 0,15,30,60 and 120 minutes, carry out tail vein blood, monitor the variation of mouse's blood sugar content,
Draw insulin resistant linearity curve (Fig. 9 C).
Analysis is found, compared with wild type control mice, the fasting blood sugar level of transgenic mice significantly reduces (Fig. 9 A).
In sugar tolerance experiment, the glucose tolerance that transgenic mice is shown will be considerably better than wild-type mice (Fig. 9 B), such as GTT
Shown in curve.There is no notable difference (Fig. 9 C) but in terms of insulin resistance, between transgenic mice and wild-type mice.
Embodiment nine：Strike influences of the low slincRAD to 3T3-L1 cell differentiations
In order to analyze effects of the slincRAD in Adipocyte Differentiation, we utilize the psd31 established in example IV
Retroviral vector and conventional puromycin screen, and construct stable transfection slincRAD-shRNA8 and slincRAD-
The cell strain of shRNA9, is respectively designated as slincRADshRNA-8、slincRADshRNA-9Cell strain.In addition, we are also by a target
To green fluorescent protein and the shRNA unrelated with slincRAD sequences is transferred to 3T3-L1 cells, constructs one as negative right
According to stable cell strain, be named as shGFP 3T3-L1 cell strains.
With reference to the user's manual and convenient technical process of retroviral vector, we produce first expresses containing shRNA
By the virus infected cell of harvest, puromycin screening is carried out to infection cell, establishes shRNA stabilizations for the retrovirus of frame
Transfection cell strain.Experimental procedure approximately as：293T cells are cultivated in T25 culture bottles, make cell growth to almost melting
(sub-confluent) state of conjunction；Using 2000 transfection reagents of Lipofectamine, by 3.25 μ g shRNA slow virus matter
Grain, 2.88 μ g pRSV plasmids, 1.44 μ g PRRE plasmids and 1.44 μ g pMD2G-VSVG plasmids, corotation culture cell.Transfection 6
Culture medium is replaced after hour, virion is harvested after 72 hours, and passes through 0.45 μm of filter filtering.Then, containing 8mg/
In the culture medium of mL polybrene, the virion of acquisition is added to the 3T3-L1 cells cultivated in 6 orifice plates, makes its infection
Cell.It is screened using the culture medium containing 4mg/mL puromycins, screening obtains the cell strain of stable transfection shRNA.
The detection method established using embodiment two, to slincRADshRNA-8、slincRADshRNA-9And wild type 3T3-
L1 cells have carried out induction differentiation.We are named as day (0) day on the day of adding in derivant, the previous day is named as day (- 1)
My god, it is named as day (+1) day one day after, and so on.
In the 4th day day (+4) of induction differentiation, cell, the expression of detection wherein slincRAD genes are harvested.With open country
Raw type cell is reference, it has been found that slincRADshRNA-8And slincRADshRNA-9The expression water of slincRAD genes in cell
Pancake is as low as the 32% of wild type and 55%.
Further, we utilize high intension analyzer (the Columbus Image Data of Perkin Elmer companies
Storage and Analysis System), the lipid content of cell is analyzed.Used lipid analysis reagent
Box is 493/503 kits of Bodipy of Life Technologies companies, concrete operations reference reagent box and high intension point
The user's manual of analyzer.Experimental program is substantially as follows：4% paraformaldehyde of 3T3-L1 cells in different differential periods is consolidated
It is 15 minutes fixed, it is punched 15 minutes with 0.5%Triton X-100 at room temperature.After PBS washings, with 1:1000 Bodipy 493/
503 solution and 1:10000 350/461 buffer solutions of Hoechst dye 45 minutes.
Using Columbus Image Data Storage and Analysis System high intension analyzers, 20
Observation and the record of cell are carried out under times object lens.With Hoechst 33342 and 488 Air conduct measurement Hoechst (core) of Alexa and
The fluorescence intensity of Bodipy 493 (lipid) utilizes the quantity of bright field image record cell.With subsidiary soft of high intension analyzer
Part system analyzes the fluorescent image of acquisition.
It was found that in slincRADshRNA-8And slincRADshRNA-9In cell, the content of cytolipin is reduced to wild
The 27% and 30% of type cytolipin content.
Embodiment ten：Strike influences of the low slincRAD to cellular gene expression
In order to illustrate the mechanism of action of slincRAD, our comparative studies wild type and shRNA stable cell strains
The gene expression of (slincRAD strikes low cell strain).In the 6 day time after differentiation starts, we are to Adipocyte Differentiation correlation
Cell factor expression variation analyzed day by day, including C/EBP α, PPAR γ, pre-adipocyte factor-1
(pref1), CD36 and Fabp4.C/EBP and PPAR γ are the transcription factors of most critical in Adipocyte Differentiation, other genes are then
It plays a role during fatty acid transport.
In 3T3-L1 adipocytes induction atomization, day (- 2) day after there is contact inhibition, slincRAD genes
Expression i.e. start raise (Figure 10, a line being located above).In subsequent entire " contact inhibition phase ", slincRAD
The expression chronic up-regulation of gene, expression peak value appear in day (0) day, that is, add in the same day of differentiating inducer.Induction point
After melting the beginning, the expression of slincRAD gradually declines.With this formation sharp contrast, the expression of C/EBP α and PPAR γ
Up-regulation appears in day (+1) day and day (+2) day after induction differentiation, and the expression of subsequent pref1, CD36, Fabp4 are
Start time up-regulation.
Data above shows that the up-regulated expression of slincRAD genes will prompt the gene significantly earlier than other cell factors
It may play a role in the initial period of differentiation.
Other than wild-type cell, we also studied the expression regulation of gene in slincRAD down-regulated express cell strains.
It was found that striking the expression of low slincRAD genes, expression (Figure 10, positioned at lower section of Adipose Differentiation correlation factor can be significantly inhibited
A line), this forms striking contrast with expression regulation form active in wild-type cell.C/EPB α and PPAR γ's
Expression only has the 1/4-1/2 of wild-type cell, and the expression of pref1, Fabp4 and CD36 are also suppressed significantly or sluggishness.This
The result shows that, the expression that low slincRAD affects Adipocyte Differentiation and cytolipin generation key factor is struck, and most a bit
The reduction that cytolipin is caused to synthesize eventually.
Embodiment 11：Strike the influence of low slincRAD cell cycles
The adjusting of cell cycle is that an importance of cell differentiation regulation and control is.Low slincRAD pairs is struck in order to study
The influence of cell cycle, our comparative studies wild type 3T3-L1 cells, slincRADshRNA-8And slincRADshRNA-9Cell
Atomization, pay close attention to the change of cell cycle behavior.In atomization is induced, segmentum intercalaris when we have selected three
Point represents proliferation period cell day (- 2), growth inhibition phase cell day (0) and terminal differentiation phase cell day (+2) respectively.
Cell cycle detecting system：The flow cytometer FACS of BD Immunocytometry Systems companies
Calibur system (San Jose, CA)；Analysis software：The MODFIT software packages of Verity Software House companies
(Topsham, ME)；All detections are provided with using identical instrument.Before upper machine analysis, cell is suspended in 35mm cultures
In ware, 70% 4 DEG C of ethyl alcohol fixation is overnight；Under condition of ice bath, with propidium iodide dyeing it is slow (BD Biosciences,
San Jose, CA) it dyes 30 minutes；Then upper machine is analyzed, and 10,000 cell is at least counted in each sample.
In cell Proliferation and differential period, the cell cycle behavior that wild type and slincRAD strike low cell is had no without apparent
Difference；Difference between the two mainly appears on the growth inhibition stage.In the cell growth inhibition stage, 31.8% wild type is thin
Born of the same parents are in S-phase, and are in the slincRAD of S-phasesshRNA-8The quantity of cell then increases to 42.1%,
slincRADshRNA-9The quantity of S-phases cells is further increased to 51.1% in cell.Correspondingly, in G2-phase's
The ratio of wild-type cell is 28%, in slincRADshRNA-8In this ratio drop to 18%, and in slincRADshRNA-9In
It is only then 10% (Figure 11).These statistics indicate that, strike low slincRAD and affect cell cycle behavior.Further it has been found that
Between the suppression level of slincRAD genes and the impact effect of cell cycle, there are apparent dose-effect relationships.
Embodiment 12：Strike the influence that low slincRAD acts on cell differentiation commitment
As an importance of long non-coding RNA effect mechanism, people are to apparent during Adipocyte Differentiation
Regulation and control have carried out more systematic research, it is found that methylase DNMT1 played an important role wherein.
On these and our working foundation, it is presumed that DNMT1 may also assist in the letter of slincRAD effects
Number access.In order to verify this hypothesis, we have studied Dnmt1 genes first during adipocyte Differentiation Induction in vitro
Expression variation.It was found that in day (- 1) and day (0) day of induction differentiation initial period, the expression of Dnmt1 genes is earlier above
Phase reduces；Higher level (Figure 12) is adjusted to after adding in differentiating inducer in day (+1) day, on the expression of the gene is unexpected.
, the result shows that similar with slincRAD genes, it is strictly to be adjusted in early days that the expression of Dnmt1 genes breaks up in induction for these
Control.This regulation relationship altogether prompts us, the two genes may be also functionally what is connected each other.
In order to study, there may be interactions between slincRAD gene transcripts and DNMT1, and We conducted RNA to exempt from
Epidemic disease co-precipitation experiment.Research approach referring generally to Keene et al. paper (Keene JD, Komisarow JM,
Friedersdorf MB.RIP-Chip:the isolation and identification of mRNAs,microRNAs
and protein components of ribonucleoprotein complexes from cell extracts.Nat
Protoc.2006；1(1):302-7).In day (+1) day of induction differentiation, by 1 × 108A 3T3-L1 cells with 1% formaldehyde into
Row crosslinking.Cell is washed with phosphate buffer salt (PBS) solution containing 1mM PMSF, adds in lysis buffer, carries out ultrasound
It is broken, make cell cracking and discharge nucleus.In order to remove non-specific binding, 50 μ L Protein Gs are added in into each sample
Dynabeads (Life Technologies) is incubated 1 hour under the conditions of 4 DEG C.It is removing Dynabeads and is being tied with it
After the albumen of conjunction, remaining lysate and the anti-DNMT1 antibody of 5 μ g (Abcam, China) are incubated, incubated under the conditions of 4 DEG C
Night.The 100 coated magnetic beads of μ L Protein Gs are added in, and are incubated 3 hours at 4 DEG C with antibody-cell lysate.Thoroughly after washing, profit
With proteinase K digestion protein, RNA components is made to be discharged into solution.RNA components are precipitated, are dropped with DNase I (NEB) enzymatic treatment
Genomic DNA therein is solved, the RNA components finally obtained are resuspended in 50 deionized waters of the μ L without RNA enzyme.The experiment
Negative control is the sample being incubated with anti-igg antibody (Santa Cruz) and the sample handled without antibody.
The RNA co-immunoprecipitations product of 150ng is subjected to reverse transcription, qRT-PCR amplifications, analysis wherein slincRAD genes
The content of transcript.It is as follows for the primer pair sequence of PCR amplification：
slincRAD-A forward:5'-TCTGAATTGCCCATCTCTCC-3’；
slincRAD-A reverse:5'-CGTGCCTATGTTCCAATATCC-3’；
slincRAD-B forward:5'-GCCACAGCACTAGGGAAGAC-3’；
slincRAD-B reverse:5'-ACAGTCATGCGTGAAAGCAG-3’；
slincRAD-C forward:5'-TACCATGTCGGTCCCATTTT-3’；
slincRAD-C reverse:5'-TGTGCCAAGTCTTCAGGTTG-3’.
In Day (0) day of 3T3-L1 Adipocyte Differentiations, we utilize anti-DNMT1 antibody and RNA co-immunoprecipitation skills
Art, capture and DNMT1 albumen and the RNA components to interact with it.Utilize qRT-PCR, it has been found that right with feminine gender
It is compared according to group, slincRAD gene transcripts are largely enriched in anti-DNMT1 antibody complexes, illustrate slincRAD genes
There is physical interaction (Figure 13) between transcript and DNMT1 albumen.
Physical interaction between slincRAD and DNMT1 prompts us, and slincRAD gene transcripts take part in fat
The commitment process of fat cell differentiation.In order to identify the downstream signaling molecule regulated and controled by slincRAD, we entrust Haikang
Into bioengineering Co., Ltd, gene promoter methylation chip (NimbleGen meCHIP, Arraystar Mouse is utilized
RefSeq Promoter Microarray), comparative study wild type and slincRAD strike gene in low 3T3-L1 cells
Promoter methylation is horizontal, to find the influence of slincRAD gene pairs downstream elements promoter methylations.
Experimental procedure approximately as：In the day (- 2) and day (+2) day of induction differentiation, cell is harvested, uses DNeasy
Blood＆Tissue Kit (Qiagen, CA) extract cell genomic dna.1 μ g genomic DNAs are taken, are ultrasonically treated, with anti-
Mouse 5-methylcytosine monoclonal antibodies (Diagenode) carry out co-immunoprecipitation, and acquisition contains 5-
The genomic fragment of methylcytosine.It is labeled and after purification, with Arraystar Mouse RefSeq Promoter
Microarray is hybridized, and with Agilent Scanner G2505C scanner scanning hybridization hybrid chips, obtains chip hybridization
As a result.The Algorithm Analysis chip data provided using NimbleScan v2.5 (Roche-NimbleGen), using paired t-
Test calculates p value, and the threshold value that methylation is raised or lowered is set as multiple variation >=2.0 and p value≤0.05.
During 3T3-L1 adipocyte Differentiation Induction in vitro, cell is in vegetative state in day (- 2) day, day (+
2) day then enters differentiation state.Therefore, it collects cell respectively at the two time points, the expression of comparative analysis gene and opens
The methylation level of mover, you can understand the commitment of gene expression in atomization.We first to wild-type cell into
It has gone analysis, has found compared with proliferation period cell, the methylation level of 7509 genomic locus in idiophase wild-type cell
The change having occurred, these sites correspond to 2334 murine genes (peak score>2；p<0.05).Wherein, 582 genes
The methylation level of promoter raises in noble cells, under the methylation levels of 584 gene promoters is in noble cells
It adjusts.Further analysis slincRAD is had found when striking low cell, compared with proliferation period cell, 941 genes in idiophase cell
Methylation level raises, and the methylation level of 469 genes is lowered.
In order to visually show the methylation state of gene promoter, we have carried out Vean diagram (Venn to these data
Diagram analyses) analysis.In wild type and slincRAD strike low cell, it has been found that proliferation-associated genes
Expression receives universal inhibition, and breaks up relevant gene and be then generally induced to express.Compared with wild-type cell,
SlincRAD strikes the negligible amounts of repressed proliferation-associated genes in low cell, and the quantity for the differentiation associated gene being induced
Also less than wild-type cell (Figure 14).Wherein.9 proliferation-associated genes in wild-type cell by specific methylation, it is and another
Outer 4 proliferation-associated genes are in slincRAD strikes low cell by specific methylation.For differentiation associated gene, situation
It is exactly the opposite：20 differentiation associated genes are existed in wild-type cell by specific methylation, 94 differentiation associated genes
SlincRAD is struck in low cell by specific methylation.In order to further analyze the state of each methylation sites, we are to hair
The gene that raw difference is adjusted carries out differential enrichment peak (DEP Promoter) and analyzes, thin in wild type
550 DEP sites are found in born of the same parents, 913 DEP sites are found in slincRAD strikes low cell, wherein shared DEP sites are only
It is 49.
Then, We conducted Gene ontology (GO) analyses.The gene that difference adjusting occurs is divided into 18 by GO analyses
A group, including (Figure 15) such as protein metabolism group, lipid-metabolism group, proliferation relevant groups.In view of striking low slincRAD to cell
The influence in period, we have done proliferation-associated genes further classification with reference to following standard.1) in wild type cell differentiation
In, ascendant trend is presented in the methylation level of gene promoter；2) in slincRAD strikes low cell differentiation, promoter methylation
The amplitude that level rises reduces compared with wild-type cell；3) there are DNMT1 binding sites on gene promoter.It is carried out with the first two standard
Screening obtains 12 genes, including cell cycle-regulatory gene CDKN1A (p21).The research work reported shows
The atomization of CDKN1A genes and adipocyte is closely related.It is found when analyzing wild-type cell data, with proliferation period cell
It compares, the methylation level of p21 gene promoters improves 2.64 times (Figure 16) in idiophase cell.But such case exists
SlincRAD, which is struck in low cell, there is not (Figure 17).In addition, we identified in the promoter region of p21 genes it is multiple
DNMT1 binding sites, the commitment access for prompting the gene that may also assist in slincRAD/DNMT1.
Further, hybridized using western, we have detected p21 albumen, cyclin-A, cyclin-B and cyclin-
The expression variation of dependent kinase 1 (CDK1, cdc2).Western hybridization is carried out according to conventional experimental program.Tool
Body, in day (- 2) to day (+4) day of adipocyte Differentiation Induction in vitro, cell is harvested day by day, and lytic cell obtains thin
Born of the same parents' total protein.In the case where adding in protease inhibitors (cocktail protease inhibitor Roche), RIPA is used
Buffer solution (1%NP-40,0.5% dexycholates, 0.1%SDS, 150 mM NaCl and 50mM Tris [pH 8]) processing is thin
Born of the same parents' total protein.After sonicated, with the bicinchoninic acid assays of Pierce companies, it is dense to measure albumen sample
Degree.Then by protein sample loading, SDS-PAGE electrophoretic separation is carried out.Hybridization uses GE Healthcare companies
Polyvinylidene difluoride films, the primary antibody used have the anti-DNMT1 antibody of Abcam companies, Transgene companies
Anti- β-actin antibody and Shanghai CST (Univ-bio) company anti-p21 antibody, anti-cyclin-A antibody, anti-cyclin-
B1 antibody and anti-phosphorylation cdc2 antibody.Secondary antibody is horseradish peroxidase (HRP) conjugation of antibodies, the rabbit HRP antibody of CST companies
With mouse HRP antibody.Hybridized blots imaging is carried out with BIORAD ChemiDoc XRS systems.
We have found that compared with wild-type cell, slincRAD strikes cyclin-A, cyclin-B albumen in low cell
Significant changes do not occur for expression, and then there are significant differences for the level of p21 and phosphorylation cdc2 albumen.In differentiation
Day (0) day, slincRAD strike the expression quantity of p21 albumen and phosphorylation cdc2 albumen in low cell and significantly increase (Figure 18).
Embodiment 13：SlincRAD mechanism of action hypothesis
In summary result of study, it is proposed that long non-coding RNA slincRAD passes through the apparent tune of slincRAD/DNMT1
Access is controlled, the mechanism of action of Adipocyte Differentiation is adjusted, is specifically shown in Figure 19.In the early stage of cell differentiation, slincRAD bases
Because transcript is by the interaction between DNMT1 protein factors, change the shape that methylates of downstream elements (such as p21) promoter
State adjusts their expression.For p21, the phosphorylation water of cdc2 albumen can further be lowered by lowering the gene expression
It is flat, so as to activate cyclin-A/cdc2 and cyclin-B/cdc2 albumen compositions, cell is made to change from active vegetative state
For " growth inhibition " (growth arrest) state.
SEQUENCE LISTING
<110>Peking University
<120>A kind of relevant long non-coding RNA of Adipocyte Differentiation and its application
<130> 2016PKU006
<160> 27
<170> PatentIn version 3.3
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caacuaggcu ucacaaauat t 21
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uauuugugaa gccuaguugt t 21
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gcguggaugu ggagaaagat t 21
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ucuuucucca cauccacgct t 21
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ccuagcagaa acuaaacuut t 21
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aaguuuaguu ucugcuaggt t 21
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ggaagaggau gacuaggaat t 21
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uuccuaguca uccucuucct t 21
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ggauuagugu ggcagauuat t 21
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uaaucugcca cacuaaucct t 21
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ccacuacccg ccaugauuut t 21
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aaaucauggc ggguaguggt t 21
<210> 21
<211> 87
<212> DNA
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<400> 21
gtggaaagga cgaaacaccc ggcctataac cagtacagtt aatcaagagt taactgtact 60
ggttataggc ttttttggat cccgaat 87
<210> 22
<211> 87
<212> DNA
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gtggaaagga cgaaacaccc ggcaccaacc tcttccaata tctcaagagg atattggaag 60
aggttggtgc ttttttggat cccgaat 87
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<212> DNA
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gtggaaagga cgaaacaccc gggattagtg tggcagatta tcaagagtaa tctgccacac 60
taatcctttt ttggatcccg aat 83
<210> 24
<211> 83
<212> DNA
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<400> 24
gtggaaagga cgaaacaccc gggattagtg tggcagatta tcaagagtaa tctgccacac 60
taatcctttt ttggatcccg aat 83
<210> 200
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<400> 200
atccatctcc cctacccatc 20
<210> 201
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tgcagaggac tcacaactgg 20
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ggtgcagttt tgcactgaga 20
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gatggagaga aaggaagggg 20
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cctagtgcct ttctgccttg 20
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tggggtgatt gtgtagggat 20
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caagtggttc cggattctgt 20
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ccgtgaaatg aagtggtgtg 20
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gcatttaatt ggtgctggct 20
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gacccttctc ctagctgcct 20
<210> 300
<211> 102074
<212> DNA
<213> Mus musculus
<400> 300
ttgcaggtct cctcctatgg tcaggtttca cccagtacag aagagagctc cctactttct 60
ccctaaagga gaaagtcttc cactggtggc ctttggatca agatgtagaa ctctcagttc 120
tttctccagc atcaagtctg cctagagcag ccctgcttcc tgctgtgatg ataatggatg 180
aatcctctga aactgtaaac catccccaag taaatgttgt cctttataag catgtcttgg 240
tcatgctgcc tcatcacagc aataagatca aactaagaca gcagatatca ttaattgttg 300
cttatattta tgaaccaaat gcaagacctt ctaggttata tagcccacaa ggttatgtaa 360
acccatctag gaaagtttat gatggtggat agacaataca actagaacag tctctgtccc 420
tattttaact gttcacctga tgaataatgg attcaggtga ctaagagcct tcttgtaggg 480
ttcagcaacc ttctaaaaat atttcattct tcatataaaa agttaaattt tacttggctc 540
acattgtttc ttatttgccc cagtatcagc taaactccta tgatctaata actccagatt 600
ttctttggtt tctatttcat agtactaaaa gttgggcatt ctcataggac ttaagaaata 660
tgggaacatg aatgtttaaa ttttttttct ttttaagaaa aaaaacaatg atgttaaaag 720
agtaggttat ttttacttta atttacctta gatgtgtcta aaatccaaag ttaaattaaa 780
accattagcc ttggagtaat aatttcctca agaaaaaaaa ttctttaaaa ctttattctt 840
tcttgttcat ttattcatgc aagttgtttt atggccacga gaattgtgat gatgttaaca 900
cgaacagtga tgataagcag ctaaaagcac tttgtcacac atgagtgtga ttaataggac 960
tcaggtaaat atgaaacaaa ctgtgtcaac ccttctctca aagctcaggg atcctcatgg 1020
gtgaagaaat gaaaatattg aaagacccaa ctgtagtgga ggactgcctt gaaccagtgt 1080
tttctagtca tgactgggcc attgcacaca cgggtcacaa tggctgtgac tatacacata 1140
aagcctgtac aagatcaaac cagccaaata tccagcacgg actggagagg gcctcacaga 1200
gtcctaccta tacctgaaga gcttttggaa gttaacagtt ggtgagggaa ggagagtcag 1260
atttcttcag ggatgtagca cctggaaggt taccaattcc tcagtggaga ggcccacacc 1320
cacatacata gtaacaacac taaatggtct taatgtttgt aggttgggtt gtttcttaaa 1380
aagtcaacag gggaagaatg gtggttagat tgtgggagga gttggagagg atgaactgag 1440
gagttggggt tgttccatgc tatatacatg aatggaattc tcaaagataa taaaagatgg 1500
aatgagaaat gactatacca atttttgtct ctaactttca catgtatgcc ttggcaccaa 1560
tgcacgtgta aacacacaca cacacacacg cacacacaca cacacaagta cacatacaca 1620
caggtacaag tacccacatg cacacatgca cacaattata tgtgtgctga ggtgggggca 1680
gttttctata tgttaaggga tctgaactca taggaaccac agatatgaaa caggaaatca 1740
aaggaaagtc aagtaaactt gaaaatttca aaagatgaaa tgaagggttg tgcagggctt 1800
gggggaagtg gttcatttta cattgacaag ttgacaggac aattcttatg tcatttgcat 1860
gagataggta cacctattac aatataacct ccttcggaac tacatctgtg ctacacagaa 1920
taaagaagct aaataaacaa cagtagcaaa gaacagaaaa gtgctttaaa aaagatatta 1980
gtagtttgag gtgggggtag ggaaacatat agtaagccaa tttgacccca gagagaggat 2040
ctgtttggcc cctctaatgg ttctaccaac cactaacgtg taccttctcc agtattcagt 2100
cttgccacaa gtccaattat aataatttcc atgagaagca caacagggtt ttagggatga 2160
tgagaatcac actgccatct gattctctgt ttttggctct tagcagtgtt ccaggcacgg 2220
agatgtacaa agtcaattca cttcagcttg aaaatgtcat ccctctgaca ttcaccaaaa 2280
accctccttc ttaccacagt gtttttgctg actgatcaaa ggggaattat tcttattaaa 2340
accaaattta aagtgtcagc cagatagaag ctccagtgcc ttgtggcatc aaagataacg 2400
ctaatgacca atgatacagg agaagctaag tgcaatgcaa attatgcagc aattctcaca 2460
cacagtgagt ttgagcttct tgcctgtgca aggatgggct attcttgagc cactatgcac 2520
aggagactaa atatgttccc tcttcatctc tcctacatgt taaggcaaag gaaggatcct 2580
acgctgacat gtacagtcca aaggagtaga gggaaagtgg acaggtttct ttagaacctt 2640
cgttgtgttc ctcagacaag ctggatagtt cccatttcta tacccttcca tgtcagaaag 2700
acataattat tggagcactg gtctctgtct gtccaagcag ccttggatca tgttctcagt 2760
tattcttcag tatttgcctg gacacagacc tggaggctcc ccaacaaatc tgtgcagtta 2820
atcagtacag gaaagcagtt tgcagaatcc tacaacagag aaagtgcctc ttctggtggg 2880
tatttaacta gtcacaaccc acatctgtgc tgatttgaga gggacagcca atgtgttaac 2940
tatgtgtaaa ttatacatat atgtactctt ctttagaaga gccaaaggag caaataaaga 3000
aaggactttg tcatcttcac ttagattcaa gaaaacaaaa caacaacaaa accagtatgt 3060
cagtaaagaa actggagtat tggtgctgag aagggatgaa gagcagaggt aaatgctggg 3120
ccatgagttg aaagcatcct agggaccccc ttaggcaaga acataaatta aactgctccc 3180
caaggccagc agtcagagat cagtcattct cttaacaaag gatttttgag gttctttatt 3240
tattgtcaag ggtttttcct gggcctattg ttgttgatgt tgttgttact aaaatatatt 3300
ggatggtcct ccctcacacc ttttgatttg agccaaggtt ataaaagtaa tttgaattgt 3360
atatgagccg tttcacatcc tcatgaagcc tttcaatatg acacatttga aagtgaatga 3420
ttgtgagagg gatgttgaga atagtaaagc cttaccagga aatgtgaatt gcattgctga 3480
catttaccca taaatgaatt actcacagac tcatgcatat gcagaaacga ttgaaatttc 3540
tccattagtt aattgttgat caaaatttta ttagaattta ggtacatatc atttgggcat 3600
attattttct taggtgactg tgtatcgtgt aaaataaaat gagtctccag agtaactgaa 3660
aaaacaaaca aacaaacaaa aaaaccaaac aaacaaacaa aaatcactac attttacaat 3720
tatcagataa ggtgtagggt gataaataca acctttacaa catctgggaa accattattt 3780
ctagaaaata aactgtaaac taagcatgat ttcattatgc atggctttaa agctacgaag 3840
aaacttatgg tttcctctgt tcctataagc tattgtcttt catatcgcat gtttctaagg 3900
aagtgacaca caaaacctca ctgtgccttc taggtccatt gtgaaggtag tgtgataata 3960
taatgaagat actctgaagt acttggaagt gtgtgtgtgt gtgtctgtgt gtgtgtctgt 4020
gtttatatag tcatgtaagt tgtagtgaca ttatggcttt gaggaactac agaatgaaca 4080
tctaatgaga tgtttccctc ttatttaaat agaatgccat aaatgttttc caataaagaa 4140
atcagtgaat tattattcct tatgattcag gttatttggg aatactactg aatttttttt 4200
cctcccaact tcattatttt ttaagttgct ttattatgtc atcaagaacc atgattcatt 4260
ggcacatgca ggaaagtgtt gggcctgagc tcagaaacac aatgtcttat tccacttaag 4320
tcaacagctg tccagagcta acaaactaca ttcttagcaa taatttctaa aagccatttt 4380
gttttttttt tctatcttaa tggtgctatt caattctcta ttccatccct aaacaaagag 4440
gggcaatgat gtaagttatc agaagctgca gacaacccat tgatccctcc attagaataa 4500
acagttgagt gctgtctgga cagatgtgag gattgcagac atgtggtaaa agtaactgag 4560
ttgggttttt ctccccttgt ggctttagca cattattgaa ggggagttcc agctgagatc 4620
agctctcaag ttgaattgtg tctatttgtg gttccaaggt catcagtagt cagagagcga 4680
atacagatta atgtagggaa ttgaatttaa ttctcattac cctggaggaa tcactctgaa 4740
tggatgctgt gcccatttgt ctaacagtcc cttctgcgtt cctcctgcac cttatctaca 4800
gtaatacttt caccgaggca agtaaataat ccagaaaacg acatatcgtt gtaaaagacc 4860
acaagtgtgg attgtgcagt tttaccgcca gtgactagaa taatagctgg gatggaacag 4920
atgtggttta aaatcttcta caaatgagtc acctagaaaa acaccttaca ttaacccttt 4980
caaatggtca gtttttgaaa gaagtcagat agaattagag ggtggagctt gtaaagaaac 5040
cgagataaaa tggtgtaatg gagaccatag gttcaagctt gtttggctgg acaatgagtt 5100
ttttttaggt atttgcttac acacaaataa agcaaggatc cggcctcctc cttgctcttg 5160
agggaaatca tgtgtgcctt ttaattacga actttatttc tgcatgcttc cctttggtcc 5220
aaatccatct cccctaccca tctttctctc cagcaggcta tgcagtactt tcctaatgtg 5280
gagcgagtat tcctgtttca tcagttgctc ctgaaaaaaa ggatctggcc tctgttacag 5340
acactttcgg ggagcatcct ctgagctcta cttgggaatc cagttgtgag tcctctgcat 5400
ttcataatca ctgaccaaag ctcgggaagg gctcctttca gccctgtcct tcatttgaag 5460
agaattgtct ttaagcttct gcagaaagtt gagtgatgcc ctactttaaa aggtagaggg 5520
ttgctttatc cagccttaat gccatagtgt gtgcctggtc ctattgtggc attattatgt 5580
tgtgtttggc tgatcttctt gggggaaggg gtacctgggg gagaggaaag gatggtgttg 5640
agagattgca gggagtccgg caaggggaaa ctgagactgg gatgtacaat acgagagaag 5700
gaaaaaaaaa gaataaaaaa taaaaaaata aaaataaata aataaataaa agatgcagac 5760
tgtgtggtta aggtgagatg cttgaaaaac aaacctacca gatcccttcc ctcagatatg 5820
caataaagtt ttgatgatac cgttttgttt tgttgttgtt gttgttgttg ttgttgctgc 5880
tgctaataac aagaacactg agtattggct tgctttcaat tcttttaaag acacttttaa 5940
aatgctgtac aagtgaaagt tggaagcatg aaatattatg tgtgtgtctg tctgtctgtc 6000
tttgtgctac atgcacgtct gtgtatatgt gtatacgggt gtacatgctg aagtgtggag 6060
gccagcagtt aatgatcagt gccttcctat aatgttttcc acattacatt ttgagacagg 6120
tgatcacgcc taatctaagg cttagcagtt cagttacacc agctctccag taagctgcca 6180
agatctgcct cacatcccaa ccccaccgag cactgcagaa ccacttgtgt gctgatttta 6240
tgtgattagc cagattttgt gtgagtgcta tagaacctga ctcagagctg caaagtgaga 6300
gctttatcca tagagacatc ttcccagccc tgataatggt ttttgaaata tcaaaataaa 6360
tatcaaatgt catcctctag ccattctcag acaaattata gaagtgtcaa ttgagtgcta 6420
caaaaattag atacacaaag agggttaaga aaataacaga agtttctcaa ggatgaaagt 6480
caagccacac tctgaactca aaagatgctg aggaagctga aacttgggca ggtgagtctc 6540
ttttataaaa gatttgtgga gtctctgaca aatctctgaa ttctgtgtta aaaaaatgaa 6600
ccaaactatt ttcttgcaaa acaatgtcca tatgttttcc tggagaaatc cgaaatcgct 6660
gagactctgt cataaggaca catttctcct ttgaccagtt ctgggacaga tctgagaatc 6720
ttatgggatg tgttccagta tagaattgca tttttaaaaa ttgataaaaa tggtcttatc 6780
actgcattgg gactttgaag tttcaatatt tatcaagcta gataggcgaa acgcactttc 6840
ctaaaaggaa ggactataac tcggccaaac aaagaaaggt cctgtgtgcg tgttaacact 6900
ttaataagag attacaccaa aggtcacact ggttcaacac attataaaga ggacaaaagt 6960
atgtatactc aaaggaagat aaaagtgctg aaaacattgc taaattaatt accagaccct 7020
taaatgtctt ctagcaccaa gaccatagag agtgaccttt gggttacttt ttttcctgtc 7080
tctcagaaag aaccatttgc atctcaactg gataaaaatc atttgcattc taacaggttt 7140
ctaaaggtta aacttaatta tacagtctgc aatgctatca gtagtaatat aaatcaaacc 7200
aaacggacgc ttcttagaga atttgataag gttagtgaag gatgggtata tggccaccat 7260
cttctctcac caccaacgtt tgcatacttt ttcactgtag aagaaataaa ataagatctg 7320
agaaatccca tctcaatctt gaatgaaaaa aaatgctttt tctgactaga acaagcatcg 7380
caagcaagga tggtgacaag tacagactca gagtcattcc atgtatcacc tgaccccagc 7440
tatttagggg aaggccagaa actaagtctg actgaggact cacgaatgcc cttttgtttt 7500
cccagagtct gactaaatta tggccaggtt tttatagcct cttccttact ggtgttttac 7560
aaaatgaaag ctagggaatg ctccacttgg ggatgaggca ttatgatcat aaacagtggg 7620
cgattatcat gtagaagaat gcgaattgat ccattaaaaa gagtgggata ttgattgttt 7680
tgtagtgtag ctccctataa tggaaacata ataatggaat accttagggg aaggggtctg 7740
gaagaatgaa acaaatatgg aattatttaa cttcactgtt gagaacccaa agtagaagca 7800
ccacctggat ccagcttggc catgaagagg caaatgaagg tcatgaattc tgtgagatag 7860
cagggagatg catatcaaaa aataaaaata aaaataaata ttgggcgaag acctgaaaaa 7920
gcttggaacc ttttcttcag ttaagcaaga gagcacatgg ggtaagtgca tgctggattg 7980
tggaatattt tttgaaaaat gaacactgca tttactctat gctgtgttta agtcttagct 8040
ctacatctta gtagctatgg gaacttagtc aaataacaaa accatttatg cttcagttac 8100
ccaatgatta aaaatatgga ctaaagcact tctgccacaa agcttgtgga gaaaattgaa 8160
tgaatatcat gtatgttcct ggtgggctat cagtttatat ctgccacatg tcttcgaata 8220
cttaaagaca aatacttcaa atacctgagc acattttgtc agtttttctc taattataag 8280
ttccatgtct tccttttttc ctaatatgca cagatcacct ttaagttaca ggcacgatgg 8340
tttgcagact gcatggtatt tatgatttgc tctgagattg gaaagaagaa ggaaaaatgg 8400
tacctattac ccactttcct tgccaagtca tttggcagat gtaatatcag gatagatgta 8460
tcagttccca cgtgcttttg tgatgctatt atcacatgag cagagtcttc aggcaagtag 8520
tggtaaagca ggactgagcc ttgataatat cagttctgga ccaacagttg atttcatttc 8580
tcttttgtaa ctaattcgtt gcagcaaacc accctacagt tgaagtacct cgtttctgtt 8640
gttcagactg tacacagcag cttccactat tgttaatatc cattgcagca gcattgttgt 8700
atcccacaga aatactttct agggatggaa acatctctca gcataacctt cccacatagc 8760
ttttgcaatc tatttctccc atgctgtttc acagaaaccg tccttaactt agaagtgtgt 8820
acattgggta attaatggtt acatgaacgg atgcttcatt taactctcct ctaggtttat 8880
tttcatctct catcaatggt ctaatgcgag gagagattta gtttatttaa aataagtcta 8940
cgatcactgg gtcactggca ttttggagta cagatttttg gacaattaga aaaatgtata 9000
atcaataaaa gaagagtcct tctgtgctac aaggcaagct tgtctagaaa tattgtgtga 9060
tttacctcta aaacgttatc ttttcagctc tttattgatt agtcgtgact tataactgtt 9120
tgaagggagg aaatatatac tccacagata acactgtctc acagcaaagc aaaggtaatt 9180
tgtttctaac agctggtctg tctgggtaaa aaacccatgg gtcaatgcat tagatatcca 9240
aaactataca ttcatgcaag attttgtcat tgtgaggact tacaactaaa gaatagttca 9300
aagagtgttc cagaaaagtc agtaatgaga agagggagta tatatgtcat aacgatgaat 9360
ttagtggaca tttgtgattg tggtgaccag tctatttgtt aaattttctt gattcccatt 9420
ctcctctttc ttgacggctt taattgtgtc tttaaattgt catgtccatc agtcttcatt 9480
gtctttccct gttaggctct gaagagcaca tttgctgaac ttgtcacctt cgatgctggc 9540
ttgtgctttc agacaacagg ctccttgtta tgcagttaga cctataaata gctgggcgtt 9600
gaacctgcta gctagaagat gtctgtactg aacaattctt tttcaggaac attttttaga 9660
aaataagctt ttgggtaagt ttatcatttt cctagtgaac tagagcatat aactgcacag 9720
tgcgttaata taaataaaga agatttgata ttaaagtact gcatgtagaa ttacagtcat 9780
tgtccccatt ttacctcttg gagtatgaga ttagtataga cttgagatag tctatcactt 9840
aaaaagaaaa gtacatttag ctcttgggat gtaatttgaa gagtcattga tacggagaga 9900
gcaaaataac agaacgagtc ctaaaatgag agcatttgaa ttcaacctgc aactctgaat 9960
tgcccatctc tcctgtcccc cctgcactgt cctcttcttg tcccttcatt ttcccctgcc 10020
ctcattctca tctccatctt tgttccaata gatcccctca tgcacccaca gagaagccac 10080
agaaaagtat cagggaagca catgtggaat gggcatccaa cgtaccatgc tattgatttc 10140
ataataaaaa ggatattgga acataggcac gtctattcgt ttacatacta aactggccat 10200
ttctgctgct tctaaagttg tttagtcatg gagacaaaaa ccacgtggct acatattcta 10260
tgataattac catctgacac tttacaggaa acactggctg acctctgata tagattatgc 10320
tagaaactac aggtctttag attttagtta cttatttatc tacttttttt ggtttgaaat 10380
acacaatgga gtacctcatc aaattttgct agcagcatga acaagaaaga aactagacat 10440
ccctggggtg gagtggggac gtgggaaaat acctgatttc agaagcagga taaaaacttt 10500
acaagatcca atgttgaggt actctgtgtg acattgtttc tctgtccttc ctcacctacc 10560
aaagacattc tcagatttcc ggtagtaaac atctaataac attagtaaat gtctagttag 10620
ctgggcagta agtggaaggt ggaacttcct aggtcagaga aaggaactgt gggaagaaga 10680
ggaaagcctc tttcaccaga aaacatgaga gattttggac atgactaagg accggaaagg 10740
tatagctaga atagattagg tgttcaagtg aaatgaacta gttgagagtc tgcccagcta 10800
agacctaagc tttgaaattg tgcttttttg aggaaatagc tggttaagta ataactgtca 10860
cccatataaa attccagcat taatatgcac agaatattaa tcatcacttc caacctgcct 10920
gtaagactcc tgtgaagaag taaagagcat gctacaaaac aagagaaaaa atatatagca 10980
aatataaata tagggatatc aagaagacaa atgaggccga ttatgatttt caaggtagtc 11040
gaagctgaga aaggtaagaa atgcataagc ccagtgatat tgggttctaa ctcaaaatat 11100
aaatacatcc ttatgatcct actgtgaggt tcccatcttc ttcagggtct ttccccaaac 11160
aattccatca gagtccccag cctctgtcca atatttccgt gtggttaccc gcagctgtgg 11220
gtaaagcctc tcagagaata gttatgttag gttcatgtct gtaagcataa catagtatca 11280
ttcctagtgt cagaggctgg tgcttggcca caggatgggg ttcaagttgg aacagtgatt 11340
gaatataaag aaatgactga atgaagaaat aatggagata gcagacacac tggtgaggga 11400
atctgatagc atttacatag ctcatctaac ttctggaagg tgagtcacca ccgtctactt 11460
ctgtctgaac tgtacacagt catttcctta gaatgagcaa agcaggatgg aaaaaaaaac 11520
agtaactttg cagttctaga acccaacaga cacttcctta gccaggcaac taaggctaac 11580
actaggctga caataacgtg taaatttgac ataaggcaaa acttaggctg acagtgccta 11640
cccttgaaat gatgattaag atagctactt tactttgtgg tctttctcca caaaatacat 11700
aattcaggtg ttttgtttgt ttttatttgt tttttttttt ttttttgatt tccatcaaac 11760
attaccccct cttcctgctc ctgccccaca caaagaccct acctctaccc cctcccattc 11820
ttatctgaga gtgtgcccca cctccatgta acctcccata ctcacaccct ggcacatcaa 11880
gtctctccca ggataggcat atcatttctc actgaggcca gaagaggcaa ccctgccggg 11940
aaatggatac cacaatcagg ttacaacttt aggtaggacc tccgctccag ttttgggggg 12000
cccaaaatgg agagtgagct gtctatctgc tacatatgta cccaggtcct tattcaagac 12060
catgtgaatt ctttggtttg tgactccgat tctgagagct tacaaaagtc caaggtagtt 12120
tgctctgctg gccctcctgt gagattacca tccccttcag ggcccttccc ccaactcttc 12180
cataagagtc cccgacctct gtccaatgtg ggtggattgg agtccttatc cctctcctgg 12240
ggatatagct caatggtaaa atatttgcct aggatgcaag agatcctggg ttcaaacgct 12300
agtaaccccc caaaaaaaac ccatttatag cataatcaag tttaattcaa cagaaaaata 12360
ttagataaat cctaattgag aaactctgca aaatacctgc caattactgt tatagcttga 12420
atctgtggta tctcagtgcc tcatctacta caggattgtt ctctggcagg tggtgctatt 12480
aggaggcggt gcctcaagga aggatacagg tcactgggga aattcatcca aaatgtattt 12540
tctcctgggt agtttcccca ttctcttgct ttattcttcc tggttgtcac aaaataagtg 12600
attcacagtc ctattctctt ccttgacact tctgtaacac taatgcttat gtgctatata 12660
acaatgaaca aaatccctga aatgatgaag cacagaaaaa acaaatattt atttctttga 12720
tttttttttc ctttttggta ctttttaaat gggaaacaat gtcaaagtca ttcaaagata 12780
gtaaaggtat ctcaaatcta gcattatggg atacatgaat ggaacagttt tattattagg 12840
gtaaatgtga aacctggata tccctggcac ctgagttctc cgatgtcagt ttaagatata 12900
tgtataatat gttacatgta ttttaattta tgtatttact tatagtacat ataattttaa 12960
ttcaaatact taatgctgtt gattaatgtg actctcatta ctgttttatt attctagaga 13020
aaggatttga tagtatagat ctcagcttaa ctatattcta tagctatgct tgcctaaaac 13080
caaagtgcca gatggtgatg caataaaatt ctaagccaaa atgcagaatt ctggtctttt 13140
tgaagtagaa actgatactt catgattgct gtgtcgctgt gtcaggttcc attgccatca 13200
aagacaagct aagagatccc taactcagcc tgatttcttg tttggcaatc atcgaggagt 13260
cattatctga aaataatata tgaatcttga taaattttag ataataaaat agtgacaata 13320
ttttatcttg ttcattgtaa caaatgtatc ataaaaatat aaaatgctaa taaggaaaag 13380
tggctgtgag gtagatctaa attattattt tttgtgtggc tgcaaagtta caaaacattt 13440
cactggaaac cctttgccgt cttcctattt attactcatg ggctcctttc ctacaagtgc 13500
aagaaaaatg aggtagtcta tttcaagaca ggacaaaaag agtgggaagt cacagtcacg 13560
ctctggctac actggagtcc ttcagaagct ccacataata acaatataga aagaggagag 13620
gctgtgggta aagggtgtag tctactgggg acacagttaa ttcaaggact gtgttttgga 13680
aataactgaa aagcaacaaa agtgctggta caacctctgg gactttatag tgagcaacac 13740
agcgattcaa cagctcattt gtattttgga gggaaatcag cataagaaag agaaaattcc 13800
tgtatttaaa ggggggaaaa cattttaaag cacagtgtat ttccaaagaa attgttactg 13860
cctggtagaa aatgtgtggc tcataaaaca aggacatttc tatgccaaga atagatctaa 13920
aattgacaca tagttcaacc tatgcattgg tgaaatttga aagctttcaa gactcaagga 13980
aaggtgcgta caaagctact gttcattata acatacataa agcaccgatt gtgaaccagt 14040
taagcatact gtagtcattc tgacatggaa actgatactg tttgaggggt gggtcaagtt 14100
ctgtccgtcc atcaggacag ggagtcattg gcatctctgc cctctactgt cagatacaaa 14160
gagatggatc ctcattcaat cctcaggaaa tcacattttc aatctacaga agcataagta 14220
agcaatctaa gggactcact gttacgaata ttattatcca aagatcaata ctttcaaacc 14280
ttctccagca taatagaaca tcatcaaaag tcacccggac tgtgatgtga aaatatattt 14340
aaagaacact gttagtcttc atgtggctca cgatttgggg gaaaaaaagt ggtttcacct 14400
atgtatttat gtattttata atattagacc tgagatatat aagcaaacag atagtttcca 14460
ttgatttaat atgcttgtct caggatagca tgggcacagt tcaattagca atatcctacc 14520
ttgcaaatac ccaatgacct ctacatttgc accaggcttt gttcaaatac tttgaggcca 14580
gaactgtcat gaaagccagt ctgctttatc acagaactag tagtctgata tactatgttg 14640
gtattaatta agtattcatc caaaccaagg tgaaattgct taaataataa tctagaagag 14700
gcaaaccaag gtgaaatcgc ttaaataata atttagaaga ggcaaaggat gcttaccatt 14760
ctcttaagaa aataggaagg cattgaaaac ttaaagcaca aagggctatt tttgtttttt 14820
tgaaactccc aaggtctaca gagtgtttaa atggtagaat agtctggtag gggaacaggc 14880
aggttacaag gtcagtaaag aattggggag taaaataatg aggtcctcag cgagactgtt 14940
ttccctgagg acaccaaaaa gtgggcaaat gtgaggatca tgtaaagaat catttgtacc 15000
tagtgataga ctgagcaaaa gggaagagaa ataaagacaa acaaatgtaa aagttgcctc 15060
aggaatcctg gctactgaac cataatcttt gtcgctgtcc tttcaggtag actagaaaaa 15120
tcaatatgat gtaaactctg acaaggtaga atagaatgca tctattttag aaggttaaga 15180
taccaccgaa acaagagtga aggcacatct gggtgtcaga ataaaacaaa acaaacaaca 15240
acaacaaaaa aaacacaaca aagcagaaac caaaaactct gaaatcatcc actcaggaaa 15300
taatgagaat acaaacaatt acctaaatta gctactttaa aacttggaaa atcaacccaa 15360
atcttaaaac tgcatggaga agaacgatgg ctgaaagtca gcaaagacaa tgagcactgt 15420
gacattttgc ctcacactcc actccctcca tcattgtctt ccgaattcca taaccgctct 15480
gacagtcagc aagttcacaa tatgccagag gaacagctag catagcaaca gacaagaaag 15540
acatcaagtg catttggagg attctaaagg gcccatccag gaaacacatt tcatttttct 15600
cttcctagca gctcccaatt aaggcttagt ctcagggctt gcctttattt gatacaaacg 15660
cagaattctt cctcaatggc tcttacctta gggtatttgg agaaaacagt ggaaatgcac 15720
tatattgcag gtggctgaaa caataaagtg attaaaagct aacagggaat ttaccaaaac 15780
tatttacaag ggaaaatgga aatgacttgg cctcagggga attagacaga tacaatgtat 15840
cactgaacgt tatatcatgc tcctcgtgaa gagctataca catgctgggg atgaccttag 15900
aaaaccccaa tctcgaactg caaccctata ggtggaacaa caatatgaac aaaagaaaac 15960
cccaatctct cacctgtgat ctatactcct gctcttctta aacacggaac aaagactaaa 16020
gatgttttga atgggctaga accctgaaag tatacactac atgtaaatat agcaaagatt 16080
tggcttattg gtttaaggct ggtaaggaaa ctcccattta attattagtc aaatattaag 16140
ctaacagaga ataaatttca tagctgaaca gaaccaagaa cacaaactta caaaaattag 16200
gtgagaaagg tattaaatag gcaaagagaa attttagcaa tgattttttt aaaagtccac 16260
aaatagcaaa agagccaagt tacaaagtat tctttcaaat ctgaagtgtt actacattac 16320
atgattttac atgtcaaatt ttatcagaaa aaaatggaac ccggaggagg aggaggagaa 16380
ctaggagaaa gaggagaaag gggaataaaa aggatgttca cataaaatag tcaataggaa 16440
cttgcatatc gtgtactggg ccataaatgt tcctcaagat tatctctact gcaggaatga 16500
tggtgctctg ggagaatgca aaagctgtag tagcccaggt aaaggaagca aggtcattct 16560
ggataatatt tccctaaaaa gcctggttgg acctgggcta tttcctctct cactttctgt 16620
tgcagatact aggatgctaa gtgtgtgtgt gtggtatata gcatgtattt ggaaataaga 16680
ggacaacttt gagaagttgg ttcttatttc taccttgttc agataaggta tctgttattg 16740
tccctcttat ttttccctag ctggtttgaa agctttcagg caatagttct atctctgctt 16800
cctatctaga ggcatcagaa ctgggatcac aggtgtatac aaccacatct gaaattttat 16860
gcggagcttc acaaatgaag ctcaggtcac aaggcatgta tgatgaatac ttttccccac 16920
taatctatcc ttctgccctc atccaaaaac ctttaaattt atttagttgg agtattttgt 16980
cacagcaaca gaaaggcaat taatgcagaa aattaataag gataataata acaccatgcc 17040
atatgatcca aggtctttcc aacttatttg caggaggtgt ttggggaagc ctggataaga 17100
agactgggga aagtaaggag tatgacagtt taatgggtgg ttttggtctg tgttcaaacc 17160
agaatgatga aaactgtgga caataaaggc tctgctcatg aacttccaga ttgaaataag 17220
gccatacact gggcaccttg aacagaagtc atttgttctg gagtagaatg agttaagtct 17280
gttcatgcct tcagtcttta aagagttaag tttaaggtga cagctagtta ataggatgga 17340
aggtatgtca gggccgcaca gtactcaaga ggtgccacag gaatggctgt ctcctttcag 17400
ttagacctag ggtaagaatc aagagtaaaa aaccatgagt ggaaaaattt gagaatcttt 17460
ctctggccag aagaggattt ctttgaaagt tggagccaag gaagatgtgg tttctaaaga 17520
tcagtatcac tagaaaggag ccagttctgt aactttagct actatgtggc tgatgcaaga 17580
tgattaccag ttagagacct tctttggcta cacaggaaga aagccagaac aacataatgg 17640
ggtcttgctt caaaacagaa agtcaaaagg aggctgggtc ggtaggtcaa gagcagaaca 17700
cttggcaaaa atacacacag tgggccctca ttttagaaaa actcaagcat ttttttcatc 17760
ttgagaacag cttttaaata ggtttagggc tataaaaatg tagacatttc agtttcaatc 17820
tgagataagg acttctggat gccctgctac aatggcacca ctcagggatg tcattcctcc 17880
agatttgtgt ctcggctgca gcctggtgcc aggcagagga gaaacttctc tcccaggaga 17940
agtgattaca catatctgat gatctgccat atgactttgt aggacaaact gagcaggaga 18000
cgatggtggg tcaatgaaga ttaggactgc accttaaata agactgctta gttattgaaa 18060
gctatttaaa actaaaccat attttatttt ataacaccaa agatattagg ggaggaatca 18120
gtggggagga atcattctct tcttgttacg gctacaatga atcaatctcc tttttctgct 18180
tgtcaacaat actttgtaac tgccacactg aggatggagg gctgagccca gttgaatagg 18240
gaatgccaaa acctagcttt gaccccagca tctccagtaa cagcactgct cctgcaatac 18300
tgcttttgca aacacaaagc ctggaagaat tacagtgtca cacatttcct ctcatatttc 18360
aaacacagac aaggaagaag cagcagttgg agctgggtta gagtccctga cacttgcatt 18420
gagtgggcag tgtgtaaagc tgtatgagtg aaattggtgt tggagtggag actccaagaa 18480
actgggcagg ccagtaacat tgaattcttg ccacagctac cagtgacagt atagcagtaa 18540
ggccataaaa gcaggaatag ctatatactt tgtgtggcac tttaaacata ataatgaact 18600
aagtaaccaa taatgcacta ggttcacatt aaggaaaaaa aaatccaagt acttcccctt 18660
cgagttgttt aacaatgatg gagcccaaat ttaaattctc tactttgcac tacctctgtt 18720
ttccaccatc catattggcc tgttgtatga atctaaatct gctcctgcct gaaggctgcc 18780
atgcatttat gtccccgttc ttctgtctat gcctgttact ttctgtctga gcatacttgt 18840
ctctgacaat gttctctttt attaaacagc acataaaata gcaaatgggg aaggaaggag 18900
ggatgcttca tagaatttta caattattaa gccactgact ccaactgatc ccaactgacc 18960
ccaactgact ccagataagg aacagataac taatagtact acaaatatca gtgtttatca 19020
agcaatatgg aggatttatg gtagactttc catttatggt agatattcca ttcataggat 19080
tgctttaaac ctatacttga tatttgctaa cctatatttg atattattaa tgattgtcat 19140
ttatatatat acatatatat atatatatat aatttaaggt taaatttatg tttcatttat 19200
gtttatatag gttgtgaatg ttggttacat gggaaatcct tagacaagta agattggttg 19260
ttttaaagtc agctaaaaca aacaggctga gtactgaata ggattgccat cttaagtgac 19320
ctccccttat gtcatttact ctagcctgga gctccatcca aagtacaagc tcctttgaat 19380
gcaagtgaaa gctttatcat atggcatgaa catagctttg gaatgcccat gatactgtga 19440
tcactatgta atcaatccta gtgtcagaaa gagacgtgaa gatttaatat ttatatatat 19500
ttgcttcttg gtttgatcaa attccttcat gctgtttgtt tttcttcctt cttgaaatag 19560
gcatatttac tttacaaaat tatatcgtgg aatgctttgt ttttctgcga tctcacagat 19620
aagtttgctt tgtgtctcag aggagaggtt gactagagtt tttggcccaa ggctagacat 19680
atcatcacaa tagatcttga agctggactg ggtgcagttt tgcactgaga aagacatgac 19740
cctcttgggg ggtcaatgtt ggatgattta aatgtaaatg tctccagaag ctcatgtgtt 19800
gaaaggctgg tacccaggaa atgcaactct tgagaggcag ttggacgctt tagcggggag 19860
cacagggcta tgaggagctg tcaaacacta tgtaagacat tgaccccttc ctttctctcc 19920
atctgtactg tgtaaggtga acagtgttct ttaacagttt ttctcatcat cagaagctgt 19980
gctaagtatg agtggaaact tctgaaagtg gaagcgaaaa taaacatttt cttcttcatt 20040
ctactttttt aaagattata aaacacaact ttctctcttc cctttccttc tgccaagccc 20100
ttctatagaa tcccttttct ctcttttata tatatatgtc ctctttgtat taattgtatt 20160
cacacacaca cacacacaca cacacacaca cacacacaca cacatatcct atccagtctt 20220
tacaatgtag gacagacaaa aaataatgag acaaggatgc aagcagccat tgctagcctg 20280
atctatgatt taaaagattt caaactaaaa ctaatcagaa gagataaagt gaggcatttt 20340
attctaatca agaaaacagg taacagttca ccaaaaacac attagtatcc ttaacataca 20400
tgtgacaaac tctagagtat gcaattcatt taaaacaaaa acctactgcc aggtttaaag 20460
acatcagtta acaactactc attattagta ggtgattttt ttaaattagg tatttatttc 20520
agttacattt ccaatgctat cccaaaagtc ccccataccc tcccctcccc attcccctac 20580
ccacccactc ctttcaacaa tggatacagt aaccagacag gtggaaggca aggaacaaaa 20640
tgaaacacta aaaggaatac ttttagaaca aagatagaaa tataaacctc aaaccaattg 20700
aagactcaaa aatgatgaca aaacgttgga cttaatggac acatgcagac tacttcaagt 20760
agtagcacaa tatacattta tttcaagtaa aaagcaaatt tccatcagta tagattgtac 20820
tttatactat caaataattc tctgtatagc tatggattga tatactgatt ttgcatatct 20880
acctaaacat aattgaaaag caataggaaa atacaggcaa atgaggagac atacaattag 20940
aaaaatgtta aatagcacac tcctcagcaa ctgttgacac aaatgaaatc ccaaaggaaa 21000
ttattttgag acaccagaaa acaaaaaaca cacaacataa ggaaacatat gagattcaga 21060
caaaatgttc gtcagtgcta cacttgcatt cataaaatcc ctgttggaaa agcacaagcg 21120
tctcaagtga agagcctatc tttttacttt tggaaacaat acagaaagtt aaaactaaaa 21180
taaccaggga aaaacctaca gaaaggaaat taagagtgca aactcaggag agaatagaaa 21240
agcagtactc caaagcaatg aatgaagttg ttggttcttt gaaactatca acaaaacatg 21300
taactttagc cagatgaact agaattctga agagtcaaat gactgaagtc aaaattaaca 21360
aggagcattc taccacttcc ctctcaaaaa taaaaataga tacataacaa atactaaaag 21420
cattgagata ttaaattaaa tgacatacat acaaatccta ggaatataaa aattattaaa 21480
atagacatag tataagataa aaataaatag aaaatatgaa tggacatgta acgagccccc 21540
caccccccca aaaaaaccct tgaattaacc ataagattca tttgcattag taaccatttt 21600
cattatcaag tgtaaccaac atcttgacat tacaacacgt ctcagtcttt ttgctattat 21660
tgtgataaaa taccctggag gaaaatactt taagacagat tttaacaatc ttacaccttc 21720
aggtaacaac agtccatcca tcactaaagg tgaatcaaga aggaagtctc agcagacaat 21780
cacatccgta cacagaagcc cagagggagt gagggtgggt gtgcctgtca gtcagctcac 21840
tttctctagt cttgtatgag attcccagtt ccagggagag atgctgttca cctttaggct 21900
gaggctgtcc atgtcaatta acatcataag gctaatccct tgcagacatt cccatagccc 21960
agtttgagaa tccctgatca agactcgtat cataggtgat tctagaccat gtcaagttga 22020
cagttaaaac caacatcata catgactttt tcatctacat ctgtgctagg ttatgcttgg 22080
taacttactg tcctgggaca cctgcagctt gccatagact aactacagtg tagacattcc 22140
tggtaaagct agacattttt acttatgaat cgtgaatgca aagacatgca aggatacata 22200
gtaatgaatc ctgaagctct tccctattaa gtaacaacag cctcctaaat tgttctacat 22260
attcattgaa agttctctca ttatcagagc cgcctcatca gaaggattta acaaaggacc 22320
ctaacattcc ctactgaaat acggagggaa cagaaaaatg aaaacaatct tgaaaaagaa 22380
tgaagatgaa catattgtcc tttctgactt tgaaatttac tgcacaactg tggtaatcaa 22440
atgctgggat gctttttgta taaagcagaa aatacaggta agtggagtag aaattctagc 22500
aggaaaaagt aaattttgga cttacgttca atttattttc aacaagactg ctaggacaat 22560
tcaatgcaaa gaaaattttt ttgaacaaat agtgcatgaa aactcagtgt ccaatctttt 22620
ctgtctacca caatgacaac tgcaaaggta cattaataag tacagatatt ggagtcctgt 22680
atgttgtgag ggagaaatta aactcacctt gaaacggttc ttcaatgctt caaagtattt 22740
ggtgtagagg tcatacagaa aaaaatgatg tggaaagaac tcttccaatc accttggtat 22800
cacacttaag tgttcttaaa ccagagtcct gtatgtgaca acggaactta taaatctctg 22860
agaggaatgt tcaggagtaa atctttgtga cctttggtta tacaacagtc tcttagacat 22920
aatatcaaat tcatgaacag gaaaagaaag aaattaaagg aattcatatg ggctcagaac 22980
accccaagac caagttctca gcacaaagga gatattgtct gccccagagg gatatagaac 23040
agggaataag agagaaagac agtagaagca tgacaaggga gaaggggaaa ggaacaagga 23100
gagggagagg tcaggggtat ttgtcccttg gggacaaagg actgcctttg gatagagagg 23160
agacagactc aacccaaagg caaatggtgg tttataacag taaaaggaga aaccccatgt 23220
taggatgagg tgctaaattt caattaagca tgttaattag gagagtcaaa ggggctttcg 23280
atgagtggac tataatgctt tgatagctga actttggtag tcaatcttag gaagaggaag 23340
tagccaaata agggaataga ccttggcagc tagctctcgg gatgtaatct aaaggctttt 23400
agcaaggcag agggaacagg ggagaaaagc aaggcctgcc aaggccattt ttgccatgct 23460
gaggctgaca agagaccctt ctgaaaaaca aagagtaaat ttaactacat caaaaaaaaa 23520
tgtgtttcaa aagcaaccac caagaaaata aaaagagtgt gtgtgtgggg ggggggggga 23580
gcgttagaaa attctgtaag aaaactgtat tttgatatgt aaagaaactt ttatagacaa 23640
cagaagatga gattcatatt tatagaactt gaagtgattg gatatatatt caaagaagat 23700
acacaagtaa tcaatacaca caggaaaaga tgctcaatgt cacttgttac taaaaggtac 23760
aaataggaag ggctacgtgg taccacacca tcaccagtca gtgaataaaa taagcagaag 23820
acagcacttg ccagtgctgg ctaggaggtg aaaaactgtg atttcatatg ttgctgatga 23880
gctatgaagt aaaatggtat aactcatcaa gttgtgttac aacacaatga tgtcactctg 23940
ttttatggtc aagggaagta aagatgcatc cacagaagaa ttcatacatg gatagtcata 24000
gaagcaggct tgtgacaaca aagactccgg aaactataaa tatccatcaa ctgaacagta 24060
gattgaaata atgacatatc catacaaagg agtatgtttg gtctcactaa ttattcagaa 24120
tagtgaatct aggtagagag attgattatg ggccagggtc tattgacata agaagggggt 24180
gctgactgga atgagctccc ctctgggaat aatgaaacat ctaaacgtag agacagtaaa 24240
aatcctataa ctttatttgt gcttaaaatt agacactgga catttcaaat atttgatttt 24300
gagttaataa ttatcaataa acatcatgaa aataagaaac cacaataaac ttcaatgcat 24360
tatgcttata ggacagatga taattccaga tacaaattag aaaactggtc aatttccaaa 24420
tgttattaag tagaaatgtt cagaataaaa ctataaagta caaattttcg aagtagggaa 24480
gacacagtca cagagtccag agactgtaaa agcaaatagc tgcaaacaaa cttctgatac 24540
gctatgctta tgctcatggt cctcaccggc ttatagctat cattttagag aacaaaaaca 24600
tatgccaaaa gttttttttt tttgagacca gacgagagat attactgaga taaaaggagc 24660
cagctgagat gaaagtctgt ggtcctttgc tgtttctgaa catctcaaac ttttcaaaga 24720
catttggaaa ccagaacaaa gcttcaagac agagcaaaaa ctaaagggag ccattatctg 24780
taatagaaat tgccaaaaat aaagtggaag tactatgtga gcccttgggt ctatcctctg 24840
aaaactcttg gaaatttttc atgggaacct aaatagaggc acaatttaat gaaaaggaaa 24900
cattacttgt tttcatgtgt gtgtatggag tgctcaaacc tccacttggt tcttagagta 24960
attacgtata catgtgagaa cagctaaaga aaatatttgt gctaattttc acaagaaata 25020
tagtggaaca tcaaataaat ctaacagcgg tgtcacaaat ctaattatgg cctcattgtt 25080
tcagttagat tactttgttg ccgttagaat tttaatcaaa gtattgtttg tataaatttg 25140
aaaactcttg gcaattgaca agcttgagca atcatatctg acaaactaac caaaaagata 25200
aaataatggg gtcaaatgaa gtggcttctg aaaagtgtgt tcaaacctgt atcatccata 25260
gttgacattt cattgaacgt gaattcaact gagggatgca cctctggatg tgttgatgag 25320
cttgtttcca taaatgagag gagaagagag aagaagaaac tgagaggaga agagtccccc 25380
agagtgggca gccttttcca tcagtagacc agatataaag aggaccaaag aaaaaggaac 25440
gtagggttct tggcttgcca tcttcacttc tttatggcaa gcactggctt taggtatttt 25500
gctatttttg ttttgttttg ttttgttttg tttttctgtt gctgagataa taaccatcac 25560
ccgaagtgac agaaacccaa agagaagcat agaggatgga gtaagtatat tccataaagc 25620
cagcagcatc tgctcagggg tggtgctgcc cacagtgacc aagctagctc agtcagtcaa 25680
ggggctctcc aggggggtag caaggattaa aaagaaaaag acagttagac aacactgtag 25740
catgacccca gcctgttctg aggctgaagc aggcttattt ttctcagtct gcttttatac 25800
cattttaatt acatgcaaga aataaggtca gttctaggtt aaggaacaag caaggcaata 25860
acaatgtccg gtgatcagtt tctaggggct tatcaggata accaaggtat ctttttttgt 25920
cccagcccaa tgtcaaattg ctgccaagtt tctagaagcg gcccccaaag ctctccacaa 25980
cacagttgta tcggccccac tctatgagtt agcgatcagt aaaacgcccc catagatatg 26040
ctcataggtc agtctcattg aaaggagtcc tcagttaaag gttccctctt tccagatgtc 26100
tctagtttgt agcaagttga caaaataata aacgcagctc agcgtctact tctgctactg 26160
gtgccaccat catcactgac accaggaccc tgcttcttca acctcctaag acactttgga 26220
gtccaggagc tcttgaggaa tctacaggtc attagtgccg agttaggatt ggaaagatac 26280
tcagcttcat cggctgagca aggaccgggt tcttcgcctt actgctggga aaacactcac 26340
ccttaagact actcagcctg gatcctgtat ttcaacctaa aatctccctt ttcacagtaa 26400
tattgacctt gtttggttcg ttttctttag agtgcccaga caatacagtg cgccagtaga 26460
tttttaaatg taattttcta aaagatacga tggggaagga taaggaaaat aagatgagct 26520
tggggatgta actccgcaga gaatatgttc ctgtttccct gatcgcccac atcctgcctt 26580
tcttatcttt aactatagaa tgttatcttc ctcgcagata ctcaagaatg gttaaataaa 26640
acgatactct gaaacattcc ttgtgtagct ggcacacact gtaagtcctc agtaactatg 26700
gtgcttgtcg cttctatcaa taatgaatct gattttataa gataacaatg taacccagga 26760
taccaaagga acattgctgt gatattttaa agcgttctaa tatttattag gggttttaat 26820
actctttagc tgttataggg tcttgtctta ctcattagca ccagtaaaaa tgatgagaaa 26880
ataacaattt gtatttaaat atgtggcaag aaggtcacct tctgagaaac aagtttttcc 26940
tcttcatcct gtttaaagta tggaggaaag attgacccca gagtggctgc gtgacaacct 27000
ggcagaatgg gaaaaataca ccagacgtct tcctgagctc cctcctgcac atacagaaat 27060
gggagaggaa agagtggtta cttttcaaat cgctgtagtt gtcacgcaaa tgagtgtact 27120
gtacatactt gagtgcacac aaatacttca tatttagcta ggatgtgctt ctttcttatc 27180
aatctacaga gtaggtaggc ttcagattca taccagaggc atacaggaca acaaactttc 27240
cttaattatt ttaaaatttc ccttccagat cagcaatgtt ataaacttgg tgtgttaagg 27300
ctgctactat ggaatctctg gggttgtagt actgagaggc atcagatgga gactggttat 27360
gccagtaata taatacgggt accattactc tttcagcagg aaaagagaaa catgaccctt 27420
acaaaaactc taatatatac attgatacat acatagatac atagaaagat ttatatatgt 27480
gtgtacatat atatgtatat atgtatatta catacacata tgtgtgtgta tacacatgta 27540
tgcatacaca ttcacacaca ctgacataga ccttggtagc tccgcagcaa tatagaggaa 27600
tcactccttt ggagatttca catccataat atatgaccag ataaaatgta ggagctaaag 27660
agatttgcag cagcacacag ttaatccatc tcagggaagt agagccaccc taccagtata 27720
atttaatttt ggactggatt gagaatctaa cctgaattca ggtgcatttc atacataaaa 27780
ccatgggaca ggttcctgtt ctgtaaggtc tcgaaatagt actgaaaaga tcaggtgcaa 27840
gcagcctagg gcatctctcc attgtttcta ggaatagtgt tgacatcgat ctactcaagc 27900
aaaatttaaa tacacgccca gtaagcacca cctactgtgt gctgggcaac gtgtatctcc 27960
tttattacct ctttccaaga aacatcatat ttgtgtacct caagtttaca gtggttggct 28020
tactacaata gagtggagtc aattcttcag gcataagaaa tgttttaaaa ttctcatcta 28080
tcatttgtgc accattagga attctatcgt catcataaag ttgtgaggaa tagcaaacac 28140
atctattgca tggcaaatac tgcttcagat cttttacatc atctcatgtg atgccccccc 28200
ccaaaacgac ttcttggttg caatcacatg tataggtgca atcacactgt agtttcacag 28260
atgtggaaat gaagcaagag atagcaaatt gtttggctaa tattacaagg ccactaaatt 28320
atggaccagg gactcacaaa aaagcatgct gaattcaaac taaggactct gttaaagcta 28380
cactttcact gagtgatggg taaattatta gccagggatt taactttgct tttacctaaa 28440
cttattgtag tgtttctcaa attttaatgt attcatagat tcttatacaa aggtgtacat 28500
gttcaactca agtttgattg gcagccttag gggatttcaa cagcaatttt gccttactaa 28560
cttgagaaat taaacctcct gtgaagcaat cttaccctaa tacacagaat tgcatgcagg 28620
ctgcaatacc agtgctcctt agcgatgcaa ttgtatatgt taatcacaga tgaattacta 28680
actcatcggt ggtcagacat tttagtaaaa ttaatctgca taaggcatat ctgctcattt 28740
gctttgtctt tcctgttccc tttcttcaca ctggttattc atcactcatt catatccatg 28800
gactaacttt gtgaaccaaa caatacaaca cctgtacatg gttctgttaa tgagcaccat 28860
gctcactttt gctgagcaac aaccttttat agcactcagg attgtattaa ctcaaaggaa 28920
aaaccaaaca gaaaggacta agaggaaaca gcaacagaaa atgaaaaaca gtcacaatga 28980
cagccttgga gagatcagag aaggaagagc aacataatga gcaggggaca aaataagtct 29040
tggtgtttat aaaaacgcat ttcacactga gagactgcac actcactcct gagcaggaag 29100
cccagagcag tggcaagaag tctctgctgt gcggtctgca gctgctgagg aggctgggta 29160
ggcaccacct acctatcagc caacacgcta gggcagagcc cggtgcctct gaaggaaaac 29220
ctccgctggg cagcaatcac cacgggaatg tttcagacat gaccgagaca gaatgaagct 29280
agttggaggt ccccataata catacacaca tataaaatcc acaaaccaat cttagcaggc 29340
agttcctcaa ttgagggtct cattttccaa gtaacactga cttgtgtcaa atggacaaaa 29400
actaagcagc caaaggctta ccaaagtcca ttagagagtc aggaagaagt ggtcaggtgg 29460
acctgctggt ctgtgacaca tctacctact ttcaaagata caagagagct tctctccttt 29520
ttcacctatg cggccatacc acagcagcaa ccgcattcca gggttcaaaa tgacgtgcaa 29580
gaaaatgggc agctgtctta gttccagtta caaatcaaag gagagagcaa ctaatggtct 29640
catttttaag tagcatatat tcattgtaaa aactatgaga tttgattatg actttttctt 29700
tttctttttt ctttttctgg tttttcggtg attatgacat ttttatacac aaatatcata 29760
tactttgttt tgttttgttg attttacgag acaaggtttc tctgtgtagt cctggctgtc 29820
ctggaactca ctatgtagat cagactggcc tcaaactcag agatccacct ctctctgact 29880
cctgagtgct gaaattaaag gcgtgcacca ccatgcccag cctaaattgt ttttaatatc 29940
tagattccac atatgatatg agcaaaaaca gaatatttga ttttcaaatc tggctttttt 30000
ttttttttat taatgtgacc tacaactgtt ctcctgcaaa tgacataatt ttgttcttcc 30060
ttatggccaa ataaaactcc tttgtgttca ttatattttt ctttatcctt tcttttttaa 30120
cattaatttt ttaaagaatt atttatttat tttatgttta tgtagctgtc tccacacaca 30180
ccggaatagg gcatcagatc ccattacaga cggttatgag ccaccatgtg gttgctggga 30240
atcgaactca ggacctctgg aagagcagtc agtgctctta accatggagc catctctcca 30300
gccctttctt tatcctttca tctgttgagg gatacttagg ctgatgccat aacttagcta 30360
ttgtgagtag tgatgtactg gggtcccctc cgctatgacc ttgccagtat ttgttgttat 30420
ttgacttctt gatgacagcc attctaatgg gtatgaaata gaattcctat gtaattttaa 30480
agctctcatt agtttatctg gggaagatgg agtgtactca gaggcaggtc atggacccga 30540
ggagctggct cacccacttc accctgtgga ttttaggggt cgaactcagt ctgttagcct 30600
cagcggcggc aagtgtcttt acctaccatg ataacttgcc gacctcccag aatgtaattt 30660
taatttgcaa ttccctagtg gctaaagata ttgagcacat tttttcacat atttagatac 30720
tacttgtata tcctttaatt ctatgaattc tttctgtatt ctaaatatta attccaagta 30780
agataaaaga actaggaagg tttgccgccc attctgtcag ctgcctcttc atccacactt 30840
ggctctttca tttacggcac aaaagctttt ttattttatt caagcctata tgccactctt 30900
gcttttattc tgaggatgag agtccttttc agaaagtctt tttgaaccct gtatctcgaa 30960
gtatttccct ctagtatttt tgtaggttca gctcatactt aaatgaaggc ctctaatagt 31020
ttggaattga ctttcctata aggtataaga caaggatgta attgtcctct tctgtaggtg 31080
aatattcatt ttttcccaga acggttggtt tggtgacaaa tctgtctttt ctcccctgta 31140
gggtttttgg catctttgtc agagtcagat ggctgttgga tttgcatctt tatcccattt 31200
tcccccttca ggagtggtgt gcatcaattc aaggacctgg tcacccggtg cctgtctaca 31260
gggagccaga gctccaatct agtataatag tatccagtaa aaaaaaaaat ttaaaaaaat 31320
atatgtgtat atatatatat gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtatac 31380
atatcctcca ctgagaccca gaatatggca cacaagtaag tgttggctcc tccaggggag 31440
ggttgcagaa atctgtttct accctcagtt tctatttctg aggtcaccca ctccacagaa 31500
gtctaatttt ttcattgcag atgtctttct cctccttggt tatgtttatt cctagggacc 31560
ttttcttttc cttttttaca tttggaggga ggtatgtgtg gcaattttaa atagaattgt 31620
tttctaactt ctttctcaca agtttattat tggcaaagaa gaagaggaag aaggagaaga 31680
aggaggagaa ggaggagaag gggaagaaga agaagagaag gaggaagagg aggaagaaga 31740
ggaagaggag gcggcagcag cagctacaga ttttggagct ggccagttag cactgagcct 31800
gacaatggat ctaaaagggg agcaggggat ctgacacctt cacatagacc tctattcagg 31860
caaaacacca gtgcacacag aatacaaaat aaataagttt taaaaatagg agaatgactt 31920
attgttgctg ggtgtgatgg cacatacctt aatcctaaca cttgagaggc agaagtaaat 31980
ggacctctct gagtttgagg ccagtctggt ctacataaag agctccagta tagccagggt 32040
gacatagact ctctatgtac atcatagaaa gaccttgttt caaaaggggg gaggggtggc 32100
ttttgcttct ttcttgatct tgctcttcct ttcttgccac ctcacctctt tcccattccc 32160
tcccccatca tgtgctcatg gccagcctct atctctacac tctctttctc tctgtgcctt 32220
tctctgcctc tcctaccctg ttgactcaac gtcccatgcc ctgaataaac tctattctat 32280
acttgctctt tgtttctgtt tttattctgc tatcatctaa taagatagta aaaattacac 32340
acgcctctaa cccaagcaca tgggaggcag agacaggcag ttctctggaa gtttcaggcc 32400
aacaaggcca cacagtgaga acatgtctcc aaaagcgcgc gtgcgcgcgc gcacacacac 32460
acacacacac acacacaaaa tgaaaaaaga cattattctt ttgtatttgt taaaacttgc 32520
tttctcgcct agaatgttgt atatttatga taaagttcca tgagattctg agaagaattt 32580
gtaaccaata gctgttagaa tattctgtgc gtgtctgtta aatccctttg atctgtgggg 32640
cggtttagct gagggctatt catttttaac gggatgattt agatgtagag atgaggtatt 32700
gtcgtctccc actatcactg aatctggacc cgactgtcct tttatatcca gtagtgatca 32760
ttttatgaat ttggccatac caacatttgg tgtatacata tttataatca cgtcctatgg 32820
ttcagccagg tttttactcc cagatcctca aagttgcaac cattctctgt ctacagagca 32880
gctcagtttc aaacaataga agcccacccc agacagcatc tgactgttgt actccagagc 32940
aaagggtttg cttgccacac aggagaagcc actatgacaa caaaccacct cccaagatgt 33000
aagtcgcctg ttgtccttcg gctaggaatg tcagtctctt ctccagcaac tcctgactca 33060
ccttgtgttt ggctttggct ttccctccct tcctaccttt ctgtttttct ctgcttttca 33120
ccagccatca tttcatttct tttgtaattc cagatgtttc tgtctttctt tggaataaag 33180
tttaatcttt gttacttaca ctcttcttct tcttcttctt cttcttcttc ttcttcttct 33240
tcttcttctt cttcctcttc ttcttcttcc tcttcttcct cttcctcttc ttccttttct 33300
ttcttcttta aaaaacactc ttctttcttt aaaagattat tgtattagtc agggttttac 33360
cgctgtgaac agacaccatg accaaggtaa gtcttattaa aaaaaaaaaa aaaacaaaaa 33420
catataattg gggctggctt acaggttcag aggttcagtc tattatcatc aaggtgggag 33480
catggcagca tccaggcagg catggagcag gcagagctga gagttctatg tcttcatcca 33540
aaggctgcta gtggaagact gacttccagg cacctagtat gagagtatta agcccacacc 33600
cacaatgaca cacttactcc aaccgagtca cacctattcc aacaagacca cacctccaaa 33660
tggtgccact ccctggtcca agaatataca aaccatcaca attatttatt tatttagtgc 33720
atatgagcaa ctgtagctgt cctcagacac accagaagag ggcatcagat ctcattacag 33780
atggttgtga gctaccatgt ggttggttgc tgggaattga actcaggtcc tctggaagag 33840
cagctagtgc tcttaaccac tgagccatct cttcagcccc tacttacact cttctaattc 33900
ttaggagttc ctccttaggc taccttcctg acaggcacgt tcacaccacc ttgtaatgct 33960
cttccatgtt ggtcaaactg ggacagttcc tgaaactaaa gtaaactggt ttccagaact 34020
tccagaccca agacaacctt gaattataac tttatttttt gtttttttca aggcagggtt 34080
tctctgtgta gccctggctg tcctggaact cactctgtag accaggctgg ctttgaactc 34140
aagagataca tctgcctctg caccccaggt gtgagcatta aacgtgtgtg ctaccattgt 34200
ctggtgaatg ataactttat ttccacctag tgcctttctg ccttgtaact tctggcacct 34260
aggctaggtc acaaggtggg caaacgtcct cccttcaaat ctgtaggagc aataaactcc 34320
tcttcattcc tcggaactga agggaattgg aggctctttc cctccaggcc cctgtcatgc 34380
tgtgcatggc tgctgtctct tctaatgagg ctcctcttta accaggtttg cactctgggc 34440
ttcaaggccc atcatgactg cctacagctg tgatgggtgt gttcatctaa tccctacaca 34500
atcaccccac tcaacacttc tgtgtctttt cacaaggata cctaaggttg ggacccaggc 34560
cagtcagaga tacacagtaa ggccccgtct tagacaaaac caagccaaac caaccaacca 34620
accaaccaac caaccaaaaa ttccttggtt attttctgat gccatattta tagtccacaa 34680
acctttactg tgattattca ccattatagt gtcattccat gtattttaca taatctacta 34740
taaattcaat gtattgttct taggtctcac aggtcatcca aatatccaga aatattaaac 34800
tgatatccag atcttaaact ggactgtaga attacagtgg ttagataaaa gcctgaattc 34860
ctcaggaatt catgaaactt gttcctatct gccaaaagga gtcattcctg ttacctctgg 34920
aggaagggac aaatggctct ctgtgacacc actcagcatt tcaaggctaa tgctggcctc 34980
taggctccct cgccagcaca agcctcttaa ggtgactctt tcacatagtc atattattgt 35040
accacctgga agagtctaaa ggaaagagag agagagagag agagagagag agagagagag 35100
agagactggg aatggagaaa tggctcagtg gttaagattc aatttccaac aaccacatag 35160
tggctcacaa ccacctgtaa tgggatccaa tgccctcttc tggtgtgtct gaagagaaaa 35220
acagtgtact tatatacata aaataaataa atctaaaaaa acaaaaggaa catggcaagg 35280
gttaggaaag caggagagaa tagtggagat agcaagataa caagaaaagg agactacaat 35340
gtgtgtgtgt gcgtgtgcgt gtgcgtgtgc gtgtgcgtgt gcgtgtgcgt gtgcgtgtgt 35400
gtgtgtgtgt gtgtgtgtgt gtgtgcatgc gcgcgcgccc gcgtgcgcat gtgtgaactg 35460
ttgttgggaa tctcaggtag gagcggttgc catgattatt aacccagctg ctgggctgac 35520
tcccgcagct cccgtgtttg ctgtgagaat ttcttccccg agaaggtata gacaatattt 35580
acccttcttc accaacaagc caaagtacaa ttcactgatg ctcagcctga tgttaatggg 35640
tttacataca gaccatgagg gagggcttaa tggcagaagt gtgagggacc ctaaagtaga 35700
ggcattggaa agtcctgacc ggatccccat agctctttgg gcccctttaa atcaacctcc 35760
ggaacttacg tacggcggta cagtggtcct cacgttcccc aacgctgccc ctctttaata 35820
cagttcctca catggtggtg accccacctg taaaattatt ttcaccgcta cttcataact 35880
gtaatttttc tactgttatg aatcatgatg tgaatatctg ataggcagga tctctgatat 35940
tcgatcccca ctgaagggtg cagcccccaa agcagtcagg gcccaaaggt tgagaagcac 36000
ggtggcacct cctgagaccc taaggcagaa gaatcacatt caaatagaat aggagtggct 36060
ggagtcccgg gtgagagtcc attgggccat cccctccccc ttgcctgagg ggatgcaaat 36120
agtccgtggg tcactgctgg taaggctcta acaagcaggc acagctgatc tgagggagtg 36180
ctcagaggaa cactctgtcc acagcaggga catagtgtga gctgcagatg ctcaagcagt 36240
tataaatact attctgtaga aaactcgttt aattgttgta tgtgacagac gcggtaataa 36300
actggccaca atggctgttc cacgatgggg ggtaggcatt gtctacaaga gagagaaaat 36360
atccagctga gaagagatgg gggataagcg atctgggatc ggccagggcg atctgggaga 36420
gaaggaagga cagctggaga gggaggagga gaaggggatg ggggagggag gagggaggag 36480
ggacttggtg cactttgggt aaattaatag gacgcaatct ggtggtttca atgaaaatgg 36540
cccccagagg tgtgtcacta ttaggaggtg tggccttgat ggaacaggtg tagccttgtt 36600
ggaggaagtg tgtcagtggt gtgggcttta aggtctcaga agttaagaca agcctactgt 36660
gtctctctct tcctgctgcc tagacatcca gatgtagaac tcttagcttc ctctccagca 36720
ccaggtcagc tcatgcttct caccatgacg ataacggact aagcctctga actgaaagcc 36780
agccccaaat aatgtttttg cctttataag agttgccacg atcatggtat ctcttcacag 36840
caatgaaata ctaaaacaaa accatattgt atattttctc cctcaacata tataattact 36900
aaaaccacat agaaatgata aaaaaaaaag gggggggggg ctggagagat ggctcagtgg 36960
ttaagagcac caactattct tccagaagtc ctgagttcca ttcccagcaa ccacatggtg 37020
gctcacaacc atctgtaatg gggtctgatg ccctcttctg gtgtgtctga agacagctac 37080
agtgtattta tatataataa ataaataagt ctttgggaaa aaataaatga taaaagaatt 37140
tgttcacttt actattctgt aagtatttta ctgtaatgtt agcttggtct ggaccactat 37200
gtcaataaga ttccattatg actcagggag atctggtgtg caaagatgtg ggttacacct 37260
tgctttgagg tactcctctg ctgctcttcc tggagggcgg gggttgtttt gttttcattt 37320
atttattttt gaacgcaggt ttcactgtgt tttccagact gatttctttc tttctttctt 37380
tttttattag atattttctt taatgctatc ccaaaagtac cctataccct ccctccaccc 37440
tgcttcccta cccactcact cctgcttctt ggccctggca ttcccctgta ctggggcata 37500
taaagtttgc aagaccaagg ggcctctctt cccaatgatg gccaactagg ccatcttctg 37560
ctatacatgc aggtagagac acgagatatc cattgttttt aataactgag tagtactctg 37620
ctgtgtaaat gtactacatt ttctgttccg gactgatttt taactcttgg gctgaagtaa 37680
tcttctctca gccaacgacc taaggcgaca ggcaggagtc gccacactca cttcccagct 37740
gtgctgcttt aagcaagagc tgcttaaccg cagttgacct gtttgaacgg gggggggggg 37800
gggggggggg ggaaataata gagagataaa aacacctccc tcaagtggag tatttattgg 37860
tgacaagggg atggaaaaga gccaacaatt attgagtaca aagcaattta taccggtccc 37920
tgaggtaagt gcttcgcaag catcaactca aattacaaac tatgtgttca gagaactgat 37980
aacccttgag cagagtgtgg atgttgcttt gaaaatccct cggaagttat gtcagccaaa 38040
agctggcagg aagcaaacgg tcctgtccgt ggggtgaact agaggagact ttgacgatga 38100
cgtaaccact taggaagatc tatgtcatgt ctaaaagaaa gtaaacggaa gatgacgaaa 38160
cgctgcagga cgagcaacca ccgccaagga gcctgcaggg acaaagagtg tcctcaagtc 38220
atcaagtaac tgtgagggga gaaagtgggc ggagggcggg ggggggggag cgaggggggg 38280
gggttgcaag gcccgggtca catgacccgg ccgtgcggag aacctaaggg agtaaaattc 38340
ctgcatcact ctgctcacca gagcacatct atagctgaag agaggagctc gacacgctta 38400
gaattctgct aagagcatac tgagaatatg tttttttgct gtgttgttgt tgtcgaagta 38460
gttacgttct ttttcttttt cggttttcga gaccgtttct ccgtgtagcc tttgctgtcc 38520
tggaactcac tctgtagacc aggctgacct tgaactccca gagattcacc tgcctctgcc 38580
tcccgagtgg tgggcgtaaa ggcgtgcacc acctctacct cgcctggttc agagactatt 38640
tttaccgttt taataaatag agataaaagt tttgatcccc tcaatccctt ttactgcctt 38700
gaatgttaca caacagagtt ataacaaaac atgtacacaa acaccaatat gctgaagatg 38760
gcagaataaa tagaactaag ctatacactg gttgttacag acataatata cagccttcac 38820
atcaataaag acaggggtga gaatctttaa tcctagcaca tgggaggcag aggcaggtga 38880
atctctgtga gttcaaggcc agcctggtct acagagtgag ttctaggaca gccagggcta 38940
cacagagaaa ccttagctcc aaaaaacaaa acacacactt aacaggttcc tggggggcgg 39000
ggggggggcg ctcagtggca cactgttgtt cccagtaact tagggcattg gtgcaagagg 39060
ctcccacagc cagcctaggt agcatgccag tgcttcattt caacactaaa ggggagagga 39120
ctgggtacaa aaaaaggtgt agcaagcaca taccaggtaa aatggaaata gctcagctat 39180
accaacagta agtcaaagag aatttaggag aaaaaagtca taaataagaa agaagccatc 39240
ggtgtatctt tctcaaaaaa ctgtttaaac acatgaaaaa acacagccac agtgtactca 39300
tataagtaaa ataaataatt ctttttaaaa gaaagtcatt gtgtggcaat aactggtgtt 39360
agcatgtgta tcttcctgag taattccctt tatgcctcca cccacgggaa ataactcttt 39420
atctatattc tataggagac taatatgcaa taattataga gtatatgtat cctaaaaagc 39480
aaaaggaagg atggcattct aactaatact tcctatctct ccttccaggt actttattgg 39540
atctgaataa atgtgccatg tgttttttgg caaaattgta tcctagccag atatataacc 39600
ccatgttcat aaccccagcc cttaagaagt ggagacagga agacagcaag cttgtcgtta 39660
gcttcagcca catagagaaa ccctatctta aactccccag aacactaaca agaacactaa 39720
aaggagcaat ggactcagaa ggacacgtct tgtgtgttct ctcttaatct gagtgtggga 39780
tgtgtggaag taggaagaag ctgggagtgc cagggacagg ccagaaggtg atggaggttg 39840
ttatgctcat agtatttgcc tgcctgagag aactccagcc ctattatttt gtatacttaa 39900
tatccaataa taaaatgttt ctaaatgaca tcattataag tatcttaaaa aaactcaagt 39960
agatcaagag cagtgaccat cgaatgatcc aggatgcgac tgagttggag aaggcacagc 40020
gggctcggtt cagcagcagc atcacctctg tgacattcag aagagcaaca taatcaccac 40080
tttggaggtc ggtcacctta caggaacagc tccataaaat atagaaatag gtaaggaatc 40140
atgtaggcta tcgggtttcc gtacaggatg tggagggcgg agcaggagaa actgggaaga 40200
ggaggagtgg ggggggggga ggggggggag cagcttaggg tggagccctg tcctggagcg 40260
catctgtata cagtaggaaa tgagacagtt ggcagataca atagatctaa aggtgttcga 40320
aaagaagcct caaactcagc cggttgtggg agtgcgtctg tctcactgga cacaaccaga 40380
aagagttaaa ctgaacggaa attagtctgt gtgacattcc aggtgggata acagaaatgt 40440
actccactac cagagcagac ttcgttgtcg ttgcagcaat tacaccgtgg gatttgagga 40500
ctgtaacaaa tcagaggctg ctctgcactt cgtagtcttc ttctaaacag taagcagtac 40560
tagctgcagc agcagcaaaa gacacccaga cacatgcggg accacgtggg tgtgcacact 40620
ctgatggagt gacaccctac tacgcaacaa cctggaacct cgatgctttt atggtggcca 40680
gcacagaccg gaaaagttag cttgcttctg catgggaaca agtctcaggc tgaaaaccgc 40740
tgaaagaagg aagctagttt tttcaaacac atgatccatg gtttgttaac aattgttgga 40800
tacattgtta ccatttgaac ttggaccaaa ggaagggttt gacatccctc tcaaccttac 40860
cctgggtgtg agggcagggc tttgccatgc ctgtgggtct ggtgggcctg agtagttggt 40920
tctagacatg gctgtgacgt tggttcagga cttcaccact attattagta ttattattat 40980
tactattatt attaatttat ttaatatatg tgtgtacact gtagctgtct tcggacacac 41040
cagaagagag ctggaaggac tgaatggtgg attgtttggg cccagaagtt tgagataagc 41100
ctaggtcaca cactgaaact taccttaaaa aatttttttt tcttcctatt tctccttttt 41160
aacttaggga aattggctgg aggctgagga atcatgtgga ccttataagg aaaacagaca 41220
taatgtgctt acttctcgtg gctgcgacac aacccctaag agtaaaccca tgtaaaggag 41280
aaagggcata tttggctcgt gtttccagaa ctttcagtcc atggcacgct ggttcggctg 41340
ctttttggcc tgtgaggagg cagagctgca caaccaggag catcgtggag caaaaccatg 41400
tagctcgtgg tagcagggca gcagagaagg aagggcgggt tgatgcggaa acaggaaacc 41460
atagtattct ctaagtgggc ctcgcctcct acacttcctg cctctcctcc cccatgagat 41520
cgggcttcgc taatccatga acccatcatt ggaccaatca ttgatgatgt ttcagtcctt 41580
agggtgtgct ccctgcccac aggtcccact gaacactgcc gctcagggga caaaaggctc 41640
cgtgaacgta tagggtatat ttaagatgct gtcttagtgt cgctactgct gtgataaaac 41700
accatggcca aagaagcatg gggaataaag gacttacttg gtttaagttt ctatgtctct 41760
ctgtacatca ccgaaggaaa acagggcagg aaactggagg caggagctga tgcagaggcc 41820
acggaggggt gctgctttct gcctcacacc ctgtggcttg ctcagcctgt tctcttagag 41880
aatcaaggac caccagccca gggatagaaa cacccacaat ggggtgagtc ttccccagga 41940
atcactaatt aagaaaatgc ctcatgccta tagcctgatc ttgcaaagac actttctcag 42000
tcgaggctcc ctcctctcta gcttgtgtca agtgacacat aactgggtgg gaagacggaa 42060
acctgcagat attgtggcat atgcctccaa tcccagctct agggacactg ccacagcagc 42120
atggtgaatt aaaggcctac ctgggcataa agtgagcata aagctagcag aggcagtata 42180
agaccctgtt ttaacaacca aaagctatgg tctggattca gaaactggat gagatttgat 42240
tattgttgcc cacaaaaagg aaaaagatat gggaagaaat ggatattcgg gcgaagagaa 42300
tcacaaactg agccaggaac tgtcagttag cactcactgt ccctttcttc aaaaaagcag 42360
gtactgtggt ttatgcctgc agttacactt ggactcctcg ctgaggtcag aggatcactt 42420
gagccctgta agtctgaacc agcctgggca gcatagtgat actccaggtt ggcattttgt 42480
ctaggctctg ccccatagtt acctggcaac acccaggtgt attgggatca ctataaaagg 42540
ggctgcttac cccctccccc ctctctcttc tcttgccttt ttaccctctt ccctttctgt 42600
cccttctctt cttcttcctt cttcccctct ctctctctct ctctctctct ctctctctct 42660
cctatcccct ccccatgccc taaacaaact ctattccata ctatatctgt cctattgctg 42720
gtccctgggg gggggtgggg gggatgtctc agcatggtgg gcccgcagag gcaccccttc 42780
ccctacaccg tactgtgcct ccaccaaaca tacccctggc ttctttttct tttttataaa 42840
acacaacact ccatctttaa aacaaaacaa aacaaaacaa aacaaaacaa aacaaaacaa 42900
aacaaaacaa aacaaaacaa aacatagctt gctgcatcat tgtgggcaaa gaatagcttt 42960
gcttgcaact atgtgattaa tttctgagtt gaactcatgt tcatgacttt ggggtggcat 43020
tcttaaaagg caatagtatt tttttctccc tgtcttcatt ctggtcaaaa tgtgaaccat 43080
cttcttcttg cttttgcaga ggacttgggt ttagttttca gcacccacgc ggtggctcac 43140
aaccatctat agccccagtt ccaggggatc tgatgccctc ttctgacttc cttgggcacc 43200
aggcatgcac gtggtacata tatgtagaca aaacactcat gaacataaaa taaatcaatc 43260
taaaaacatt ttttaaaaaa agtacgagcc atctggtact tggaactgca caggtagaag 43320
gaacttgggt ccctggcacc acggagtact ctaccagagt tttgcataaa tttggtagca 43380
tgaatctcaa ttctttatct taatttgaat aaatggagct taagacattt tatagagagc 43440
ctggtgtggt ggctcacacc tttgagccta acacttggga gacagaggca agtggatctc 43500
tcaaggctgg cctggtttac aaagctatat gagaacctga ctcaaaaaaa attaatatgc 43560
ttcaattttt ttggtgacaa gtgttaaatg tatacagaag tggatacatt gatataaaaa 43620
ttgtgtgtat accaacaagc agctttatcc aattcattta tacttgtaat cacttccctt 43680
tccctcatta gactattatg aaacaaattc caaatatcag ttttttaatt ttaatttatt 43740
tttaactgtt acatgtatgg gtattttgac tccgtgtatg tctacagaac atgtgcatgc 43800
atgatgtcct caaaggctag aaggcataga atgtcctgga actgaagcta tagacaactg 43860
tgagccacca tgtgggtgtc tggattaaac ccatgtcctc tggaagggca gcaaatgttc 43920
ctaactgtcg caccaactct ccagttcaca ttacctcttc tgtgcggcca ttctcaaatg 43980
gagggctcct gattctttga gagaagatga gataactcag ggaaagtgtt tcccttggtt 44040
gaaaacagaa gtaacacaaa acaagggatt aggtaaagta ggcagttgtg gaagatgaaa 44100
gggtgtgggg taggagagta gagtgggtag tgttcccact tctcagactc cattttccac 44160
gctgactttc actgggacaa tcactctcag ctgcaaactc atttgctagg agtcagttat 44220
gtatcagtgg ctgcttgtgc aatttggtta tagctgaggc ccaaggggat gtcttctagg 44280
gtttctggaa aagtttgcca actcctcaga aatggcttgc atttacctgg gcaatagtct 44340
gtgatggaag gtgatggaac tacagccaag acgatcagag tagtgagatg gcaggaaacc 44400
ttgttctctg tgccccccac tctgtggttt gtcctgcaat tctaagccct cttaaagctt 44460
gccctcaatg gatctttgca tgacacgaac caccgttcca gaatttggct tcgttaaatc 44520
atttcctaca aaagaaaaac aattacatgg tgcctccctc ctccaattaa gaatattttc 44580
ttggtcctta ttaggtaaaa caaaacaaaa caacaaacca tatttttgac agcttttcat 44640
aaaacttatc atttccagcc atctctccag cccaaaactt atcatttcta cagattctgg 44700
cttgttctga gggcaatggt ggtgtctgaa gataaggaaa aatagctggt atggaatctg 44760
ttctgtaaag gtgagtatga gtattctgga taaagacatt cagtgtgttg cgtataaatc 44820
ccgctttcct tctgctaata gtccttcgta ggagcatccc gtgaagtctt atttacttaa 44880
tgtacccaac tccaaatagt atctcttaaa gctacatcct tcaaacagtc acatggggga 44940
acagagagat ggctcagccg ttaagaacat tgattgcttt tccagaggac ctagatttga 45000
ttcccagaac ctatatggtt actcacaacc atctgactcc agtcagaggt ggtcctatga 45060
ctccttctgg cctctgaagg taccaggcac gcgtatggta tagtcatgcg tgctggcaaa 45120
acactcacgc acagaagtta aatgaaagca accaaccagc tattggtcca gagggtggag 45180
agcttaaaca gacgcttaga gaagacctcc gtcttcaggg tagtgttgca gtcgtgggcc 45240
agcattgctt gggcgtggtt ttattttaca ttagcaaatc aaacaacttt gttatctaga 45300
ttccaaggta tagtgagtat taatcccaga gatgttacta gtgaagaatg atcactctgg 45360
gggaaaaaaa aaatcccaga aggtagaagg cattcctggc aacaagcgta gatcgccccc 45420
tgctggcaag gactagcaaa ggccagcaca aacggcagaa aatgcggtca agaatgatat 45480
acccagaata ggagagcaga agttacaggg aagtttgtat ttatcatcct aattgtcaga 45540
cagacaaact gggggcttcc atggttggag ggtctagcat gccccaaaga catatgtgta 45600
agtgaaggct gcaggtgact taaagaagca aagactgagt gtgaaagtga agctagtggc 45660
caagtcacaa gccagagtgg tacaagagtc agcttggtag atcccaacct tcctaaagcc 45720
gcgaccctcc tcaagctgtg tcagtgtaga aacagcaacg ctttccaagg tgccggcttt 45780
accaacactt tgtccagaac tagaggaact attcaaaatg tttgaactat tcaaactaaa 45840
caaattcaaa cttgtagaag ttcctggcta ggagtcacct cgtcgctacc aaccccctgg 45900
cttatttgtg agaagattga gagatctgac tttacgacaa ctgcggaacc tcagagatgg 45960
caccatttga aatcaccagc tgctgtcact tggacctcaa gcttttgggc agccctcagg 46020
catcctgttt tgttaaaggc aaacgcaacg ggctcaagcg ttctttcagc cgcgactgta 46080
ataaattgcc tagacataat agacgcttaa tccatgtgtg gaagcaaatg aactaatcat 46140
taaatcaggc gtattggagg cctataattt ggcgaggtct cagaagactg attcacttat 46200
atattcgtct gcttcttgcc ttataattat tatgtcttcc tccgcgggga tttgagaaag 46260
cttgtaatag aacatgcggc aaataggaaa tataaataat aaataaagcc agcatcagga 46320
gacacgtacg ttaggcaaac acaagttagt tgaatggaat ggaatgctca agcaataaga 46380
cagcacctaa ttatctaagc ccagctgcaa attggtacta gattttctgc tgaaagaaac 46440
ggaagcaggg catggttatt ttagtttctt aagatttata ggaaattaac tttaagatag 46500
agttttgatc tgctgttcta ttcttagaac tttatgggga tatggcaaac aaccctggat 46560
actgttctaa tgcaaattct ttttttcttt ttctcttttc tctttctttc tttctttctt 46620
tctttctttc tttctttctt tctttcttct ttccttcctt cctccctccc tccctccctc 46680
cctccctccc tccctccctc cctctccctt ccttccttcc ttccttcctt ccttccttcc 46740
ttccttcctc tctctctctt ttctcttttc ttttcctcga aaattctata gagctttctc 46800
acagctacgt gctagagtat gaagctttct cacagctatg tgctatagta tgaacctcta 46860
gcatagctgg ttctcagcct tcctaatgct gcaacccttt aatacagttc ctcatgctgt 46920
ggtgacccct ttaactataa aattattttc attgctattt cataactgta attttgctac 46980
tgtcatgagt tgtaatgtaa agatctgctg tgcagaatat ctgatatacc gacccctatg 47040
aaagggtcat tcgacactcc ccggggatct ctacccacag gtcgagaact gctgctgtag 47100
catggccttc atagctcact gaaagcaatt tagtagacat caaggaaata ttgctgagtt 47160
ggaaactttc aaagtgtgga cgaaatcaaa gaaatcacct gtacgtatcc cattccatag 47220
ttttctatca gcatcacctt gaaccagaca tttaatatgg gtgagaaaca tcatatactc 47280
tctactaagg cggaagcatg gttgttctat actgtccttt ggtgctaatg tgtgctttag 47340
ctagctggga ttaaaggcat gcaccacccc tgcctggctg taaagtttct tttaggagtt 47400
ttgaaggaaa agtgagttaa gcgtctaacg aaaaatcatc tttgggatgg atgcccagct 47460
acaacttcct gactggaaga cgtctgtgtt agtattctga gtgctcatgg ctatggtggc 47520
cggtggctgg tggctcagta agatagttca tggtcaggaa tgttcatgca actgcagttg 47580
gacagtggct ggaagcaccc aacctggaca cactcactca catgcctcct gccaagctgg 47640
gcagattcag tcagcgggag actggaacag gggagctttc tgggccatta aatatctatg 47700
gacctaacag gctcaggact cagctctaaa gtgctcttaa ccactgagtc atctctccag 47760
cccaacaccg actttcttag tgttaggttc agagcagtcc ccaaatggtg gtgctggaga 47820
caatgtcctt cccttctagc ttgcttatca agatgactgg tactaagtaa acagggggct 47880
aaaggtgggt ttctgtttac caggcatgtt ccttaaacca ccaggaccct ccttaaccac 47940
tccaaaggtc tactacgttg agcaagggca tctaattctt agcctgtatc agctcaatga 48000
cctcatctgt tgtcatataa aacctgcttg ctttctcatc attttgtgtg tctgtccgtc 48060
tgcccccacc ccatggcctt gacaagtttg atccccaata aaactttttt gtacccacag 48120
aagagccgtc tagttgagtg acttcacagt tccacataac agaagaattt atttagacta 48180
tgaataacag tggtgtcaga aaggcttcat ctgttgcttc atttaaactg aggctcctca 48240
ataaatgaaa agggaaggga aggtctccag cttgtctgtt tcagtctggc cttatctgtg 48300
ctcatgtggt tagttgcatc ccagatctcc attacgggct actgcaataa aaactccaac 48360
cccacatcac acccatggaa ttggatttca actctaaatc caaggagttc tgagttcctt 48420
ttatatgtca ttgaaggagc cacaaatgac cagaggtgtg tttgaccgaa ttctgagcgt 48480
ccaggagccc agctccacca gcattcctga gagcacactg gggtcctgca cacacacaca 48540
cacacacaca cacacacaca cacaccaggc tagcagggtg tagctggtta tgatgggaag 48600
tgctctgcaa cccccaaggc ttgtggcaaa tatggcccct cattctctaa acagttcaac 48660
tgtcaaaatt ttaaaatagg tacagagagg gacaatatac atgcaggatt agggttagga 48720
gtccagatga gaggagagag tttccaatcc cgctggggtc tctttggggc tgagagggat 48780
ggcgagtgtc gatgtcccaa aggaaacaca ctgagccagg agtcttgtcg aagcaggaaa 48840
taaactttat ggcatcagcc tcttgcttat ataagttcgg aacataggga gagggatttt 48900
tggtggggta agatgatgta agaggcaggg aaggttgacg gagggtatgg agtgactgac 48960
agggtcaagg gcaaagctct acatcagcaa tggcagggca gaggcagggc cagacaggtc 49020
ttgctgagtc tccccgatac ggaagtgact gagacaggtc tcaaaactca agccaggatt 49080
gggtttgtcc tcaaggccca aaccagggcc aaaatggggg gtccaacaca aaagccacaa 49140
tcctgagtgt tagcgttcat ttcttctagg ctctgcccca cagttacctg gcaacagcca 49200
gatgtgcctg actcactatg aactgggctg cttgccccat cctcactctc ttatcctctt 49260
accctctctg tcccttctct ccccattccc ctccctcctt cctggccggc ctttcctctc 49320
ccttcccttc tctctctcct tcttcctctt cttctccctc tctctgcctt tctctctctc 49380
ttcaactgcc ttcccgatgc actaaataaa ctctattcca tactatatct gtcatatggc 49440
tggtcccaca gggggaaggg atgtctcggc atgggcctgc agaggcacct cctttcccca 49500
tgctatactg tgcctctacc aaacatatcc tggttctttc attctttttt tataaaacac 49560
agtcctgagt aggttggttt gattccatgt gccttcacac acttgtgcca ccagcacaca 49620
gattcactgt gcgtcttctc agtctgtacc ccgcccccac tgggagtgaa ccatcagtct 49680
agtggcagtc accattgatt aggattttag acttctttcc cttctcttct tttccctttt 49740
cttctctttt tctctctctc tctttttttt taattggaga gtggatttta gtttccttgc 49800
ttgggtttgt tttagcaggg tcatatcatg gaaccccagc tgtccttgaa ttcacaaagc 49860
tacagctgcc tctgcctact gactgcttag gggattagaa gtgtttgcca ccaagtctgg 49920
tctactttgt tgtttgtttg tttgtttgtt tgttgagaca gggagagtat actttatgtt 49980
ggtaaactga catgtgccgt gtgagctcgc tgagattctg gttctggact tggggcttct 50040
tcttcctcca ttgcagaacc aaacatgtct atctctaagt attctccaag ggcccctgtg 50100
tggggacggg tggctgcctt aacactgaca gggagaaagc aacagtttaa gagtaaaact 50160
acgtttggcc agccctggcg gtacccacct agcacctcag aactcaggag gcagaggtgg 50220
gcagatccct gtaagttcga ggccagcctg gtctacccag tgaattccag gccagccagg 50280
gctacacagc tagaccctgt ctcaaaaaca aacaaatcaa caatcccccc aataacatta 50340
tgtccttgaa aatatctaga acattgacca tttccaacag aaattactat tgtttatcct 50400
gtgatataaa agtgcctgtg ctgagagaat ccaggtgttc agcccctcat gagaagccca 50460
tctgtggaga caaatgtcaa ccagaatctg ccctaacctc tgcagggctc tgtgagcaca 50520
tgacttgatg atgtcatttc tattcattac tcttctttta ttcataaact ttactcaaat 50580
ggacactctg ctttgcctgg ggattctcca tattaggaac tccaactgac cgcctcaatg 50640
gggcttctca ggtgatgtct gctaattgct ttatcttatg tgtatgtgtg cttgtgtatg 50700
tgtgttcagc ctgcatgtgt gggccacatg cgtgtggtgc ccttaggggc tagaagaggg 50760
cctcagatct cttgggactg gagttacaga tggttgtgag ccaccatgct ggtgctaggt 50820
tacgaaccct gggacttctg atagagcagc cagtgctctt aaccgctgag ccacttctct 50880
agcccctgct acatgctttg agtacgcaat ttgaagattc cttgccgtcc atctggctgt 50940
ctcatacctg ctatgtattc tttcccttta gcttgctata ttcttgacaa actcagttaa 51000
agtggacata tggctatata gatcatccac tggactgtaa agaatacgta gcccacattc 51060
aattcaaact tgctatgtag ccgaagatga cctctgcttc acagatgctg gggtcatagg 51120
tgtgtgctac catgtctggc cttcactgat tagttttgtc tacttctgga tttgtaaaag 51180
tggaattaca tggtatataa atatagattt ttaaaaaatt tttttgtttc ttatactcaa 51240
cttaatatct gtgcaaccaa tgtgttactt gtagcaatgt ttttttgttt tgttttgttt 51300
tgttttcctg cctcattcct gtgtactatc ccaatcttat aaataccctg cagtgcattg 51360
agctattaat ctgtagagaa gcacgaggct cttttttgtc tggctgatac gaaggaaacc 51420
ttgacagacc ctcctcctac ctgccaggcc cacttcacct tctgctttgt ttgtggacag 51480
tcatcccttt gtactttttg tttgtttgtt tttgtttttt ttgagacagg gtttctctgt 51540
atagccctgg ttgtcccttt gtactttcag gctaccttgc ttatatccct gtaagagtct 51600
caaaattgct gaaggaagac cccagactca gatagtatgc aaagacaaac aacatttttt 51660
ctgcagaaac aaccaggggt taaccattct ttaaaatggc gacccaagac aaaggcacgc 51720
agggcttata agaagggaac tgggggaacc tctagaagga ttgggtaatc taaaattgca 51780
ctgttgggtg ctagggggtc acagctggcc agcggtctgc attcttatgt catgaatgtt 51840
tagtcatagg atgagatagg gctgcccagc gcagatggtc catcctgaga taagacattt 51900
ccctacttct gtggttatcc ccaggctact ctttgggagg gggttttatg agcttactgt 51960
ggattttacg gtctgttcct ggagctggtg ccttacgtct tcttttggta atcaggtttg 52020
gtctttaagc ggagacaaat ggagtttatg tcaacctttc aaacccatgc taagaaacaa 52080
gcttagtttc tggccagtca aggtgcgaca cggtcgattg gtgaatttag gactgttgta 52140
tttccactga agtaaaagac agctgagaga ggtccccaaa ccctcccttt cctctcttct 52200
tatctctgga atgaggtttc gtgctagcgt ctttaggtct aactaggagt cttccgagaa 52260
ggctcagtga tcacccactg acagacccgt cactgctgac ctttttcgta gagaatgcgt 52320
ggagaccata tcgcccaggc ttccttgtag tttagtgggg tggagggact gagttttaca 52380
tcagtaagca gaagtccatg gcccgctgtg aatctcatga taatcctctt ctgcatggtt 52440
ccccgagttc tttctttttc tgccttctgg gtggtgacct tcataacaag ctaagatgga 52500
aaagccaacc ttggttctta agggactgtg gagaacggag gcttctcctc gattctcagt 52560
cctccagccc ccagctcacc acctagagct gtgctccctg agagtgaaaa atgaatgttc 52620
attgtgtttg ggcccttaga caatcttggg gttaactgaa tacagctgtc agtcatgctt 52680
acaggtatct ctgagataca ctacttatta aagagttaaa agttattata gtaaaagtaa 52740
tacagatgca aaaaaatagt ttaggacagt ctaaaataca attataaacc ttccacctcc 52800
ctttccacct aactggtagc taggcctaca tgtcctaaca cgttcagcta tatctatctt 52860
aattctcttg ttatttctgc ctaatctctt ctacttacca ttttatttat ttatttattt 52920
atttaatcta gtggcttctg actagactat gtatgatgac gagttcccga ggcgttttgt 52980
aacccagtgt gtttccagag tgctgtaaga gactagtatg gactgagtgg tttaagcagt 53040
aagcattgaa tttttgagtt ctggaacctg gaagtgactg ctgagaagtg tgggggcttt 53100
tctgggactc ctgcttgagg tttgtaataa agacccagag acgtttgtat accataaggc 53160
tgttggcctt tgctggaaca gaacctcagc taccatctga agttaacccc aaacccctgc 53220
cactgtgtcc cttcatgtgg ctctcctgtc ctgtccctcc atccccgctc ccccacccca 53280
agctgccagc tctttattct ccaaaaagcc acatttcaga aagagaagca gtatggtgtg 53340
ggggaagggg gtgcctctgc gggcccatgc taaggcatcc cttctcagtg agggacccta 53400
tatactatac tatatatggt atagtataga atagagttta ttcggagcat ggggagagga 53460
gttgagacac atggaggggt agggagggga gtggaggggg gaagcaggag caagaggaga 53520
gagcaagagc aagaaggcaa gagagcgcgg aggggaagag cagccccttt tatagttgcc 53580
aggtaacctt ggggcagagc ctagactaac aactccgttt gctttttaac atggtaatct 53640
cagagagcca gtcctttgag ctggtgaata aggtacacag agaaacccta tctcgaaaca 53700
aacaaacaac aaaaaagaaa agaaaaaaag aaaaagaaaa tacagagaca cacctatagg 53760
ccacttgacg caatggggcg gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt tcatggattg 53820
gcatcgaccc tgtgtgtagt cctttggttc aaacagaagc agcagagatt tctggagccc 53880
tttcataagg gcactaatgg cattcagaag gctccgttct catgaactgt ttggtttcag 53940
ctcctggaca aggggcagag gtgcatattt tacataacaa agcagacctg gcctccagga 54000
tctcccagaa tccctcagtc cctacctgtc ataccctgcc cccaaccctg aactctccag 54060
ctcagaggct gggctgccct tcccctacag cctcttccct atataatcca gacattttgg 54120
tatctcaagg tctttctctc ttcttctctc tctcttcttt ctctgtacgc aagccctttc 54180
ttcccatccc ccacccggcc ccatagcgac tccccggcct caatccttgg gggcagcctc 54240
gcgataaacc tgccttcaac acactctaat ctggtttgag tcggctcatt tcaccctcgg 54300
agaaatgacc tatcactaac cccttctcaa ggctccaccc cgtaacgcca tcacactgag 54360
gcctgggggc caacctagga attttagtgg ccacgggtat agatcttcaa tcttttaccc 54420
taaggaagtt ttaggcagag aggaaataac gtgtgagaag ggctatcaca ctttgtaatc 54480
acacgaagat gaacagttca atgtgggaca aataaaaacc gggaatcaaa ttagtgtgac 54540
ttttcacttc acactttcct ggcgaggcta ggaagctgcc gagatggagt gatgcttctg 54600
gtatgttggc catgggttca cctctagaaa tgttctcagg tgctcaaggg acagcaggag 54660
taaaggtgga gaaaaagagc tggcattggt aaattacctt ctctttcttt cttccccctg 54720
agggaccagt cacacaccgg tataattctg ggcatggagg ggagttgaaa gggtagtaga 54780
gacagagaaa gttagagaga gagagagaga gagagagaga gagacagaca gacagacaga 54840
cagacagaca gagaggagta gaggctaggg atgggcacgt ggaggggggg aggggatggg 54900
caaaaggacg gagtggagac agaaggcaag agagcaagag agagcagagg gggcaagcag 54960
ccccttttat agtgagtcag gcacacctgg ctgttgccag gtaactatgg ggcagagcct 55020
agacaaaatg ctaacaatgg tgcccccact tttagtgtaa atcttcctag ctctattaac 55080
ctgattaaca tattcattgg aggcacgccc caggatccaa ataatcccag agctccatac 55140
ctggatgtta agctcccaag tggttccgga ttctgtcaag ttgacagtta acacataaca 55200
cttgcctaag gtctcagaga agcaacatgt catcttcaca caccacttca tttcacggcg 55260
ggtgctaatt aatcttttgt ctgcaaaaat tgagatctgt gagctggtta aggtcaaacc 55320
cctctgtggg gtgacttggg caagccaagt gtcttgcaca tccagttttg taaacagata 55380
ttaggttacc aattaagcca ttccacctga gccagcagcc tctcttgctc caacccacag 55440
ggggcaggga gacagcagtg catttgtttc tagggcctgg gaagctatgc tttgaaacca 55500
tattaaaatc atatttgaat tgctgtcagc ttaacgcaga gttggcctga agtcccgaag 55560
tgagaatcga ctgtcccata acagatcaat aaagcgatag ccgcatctat agcccaccag 55620
agtccttgac aaagtgatac aaattttgtg ttagcttaaa aagaatcatt taaacactgt 55680
gcagccttca ggtatcagtc ctgagaagat tgtgggggga cagttcctat atagaaaaaa 55740
caaaaaaaca aaaacagaaa cgcatttctt tgttctctgc atcatgtcca agatgacttc 55800
tgtgttgatt cacgttacta agccaatgac atcatttaaa atccacccac tgaatgttta 55860
ccgaatcctt tctcctgtca gcaactccac agttaggttt gttttgtttt atttcagaca 55920
gcgatgagaa cagaaattcc tattggtgag ggcaggtctc atgactctgt cactgttggg 55980
gataaatcat ggggagaatc tgccttctac tggatgtact accattggtc aatgctgacc 56040
taccaggcta gcagttggga accccactta aaacaataat agatggtgtg gtgagggtgt 56100
tagacattca gtctctgcac ttgggagtca gaggcaagga gatccagagg gagtggcact 56160
attaggaggt gtatccttgt gagagtaggt ttagctttga tggaggaagt gtgtcactgg 56220
tgtgggctat gtgatcccct gtgcttaagc caggcctagt gtggtggcat tatccttcct 56280
gctgcctaca gctacctcac cagcaccagg tctgtctgca tgatatcagg cttcccacca 56340
cgactataat ggactgaacc tctgacctgt aagccagccc caatgaaatg ttttccttta 56400
taagagttgt tgtggtcatg gtgtttcttc acagcaatac aaccccccca caccccccac 56460
acacacaaaa gacactacta caaaccattc tatgaactta gctctgcaag agctaagatt 56520
tttctttaag aatagagatg gatgggctgg agagatggct cagtggctaa gagcactgac 56580
tgctcttcca gagatcttga gttcaaatcc caacaacagc acgatggctc acaaccatct 56640
gtaatgggat ctgatgctct tttctggggg catgaagaca tactcatata cataatgcag 56700
atgtaagtat gtactcatat gcataaaata aatctaaaaa aagtaaagaa tagagatagc 56760
tcagtggtta agaccacttg ttcttgtaga aggccctggt ttgattccaa gcacccacat 56820
gatggctcct agccattcat aacaacatcg gttccaaggt gcacatgata cacagataca 56880
tattgcaaga aaatacacat gcatataaaa taaaataaat ctaaaaaaac aatttaaatg 56940
ccatccttga aaaaaacaaa agaatggggg tgggggtgga ggtggactag agagatggct 57000
atgcagttaa gagcacttgt tgctcttcaa gaggaccctg gtttggttcc cagcacccac 57060
atggcagttt gcaactgtga ctacagctcc agggggaccc attgctctct tctagcttct 57120
tcaggcatca ggcatacatg tggtccacag acatgcatgc tggcaaacac ccatacacac 57180
aagcaacaac aacacagagt caggctggtg aggtgattca gtgtgtaaat ctgaatttac 57240
atacctgtcg ccaagcttgc caccccgagt ctgatcctca gagcaagagc cacatggtag 57300
gagagaactg agtgtcacga gatatattct aacctctgtg tacacaaaat aaattaatat 57360
atttttaaaa aaggagacag tctgtaccct gccccaacac catcagtagg gctcagtcca 57420
gacacagaca attgagttac gtcaacgctg tgtacccagc tagcatctct gttgagacct 57480
tctgaggtgc taattacagt tgggaattaa tcattattct aactgagcca gagtcttatt 57540
ttctactcaa gcttctcatt attaagtttg catgaattgc tgagcactgg tgtaaattac 57600
cattcagcga tgcacattaa acactcagag cggtcggttt ttaccttgca agcctatcgt 57660
gcgaatctgt ttgcaccaaa atcggttaac tgcttattgt aaaattccat ttttctttct 57720
ttctcctagc ttactttcac gatcccagga gttttagcgg gctgtttgtt cttccccttt 57780
cgtgctctga aacctaatgt gactccattg gaagttctgt cctttccaga aagtcaccta 57840
gctcagaaaa aaaagtgtcc ttacagattt ggtgtaagaa acctttgctc aaggaagggt 57900
gctcctgtct tttctgatgc cctcttctgg ggtatctgag ggcagctaca gtgctacagt 57960
gcacttacat ataataaata aatctttggg gggaaaaaat gagcaaggct tatctgaagg 58020
ggctgtgcag ggccaggctc cagcccagaa ggggacagaa tactgcggcg gaggtatttt 58080
agaacttctg cttgtctatt cagagaactc caatccctga cacatactgt ggcctttaat 58140
ctcagcactc aaggggtgga gtcagacaga tctctgagtt ccaggatagt ctgctctaca 58200
gaatgagttc tgggacagcc agggcatctc cattcccctg tatctccatt ctccttgtat 58260
gtatcccggt cgggatggtt ggcttactgt ctttctccat tttgcccagt ctgtgacctc 58320
agcgctcagc tcgtcggtgt acagcatact gagctcaccc gcccagtcca ggaggtcacg 58380
tgcccctacc ctgaccatag gcgtcagctt cctgccttct actccatttc tactccttca 58440
tccgtgcaat gtgatgtcct cacgcttgca ggggtgaagc gtggcacctg gggagggagt 58500
ggtgcttggg gactttctgt caagccatgg caaatgcatt cctctgaatt cttgctgggc 58560
acacacacac acacacacac acacacacac acacacacac acagagagag agagagagag 58620
agagagatca aaccacaaaa tggaatgtgt ctttcttcca ataggctcct cgtgtggtgg 58680
gaatgcggct ggcagggtgt catgtgacct gaagcatcct agcgcttttt aaaatgcaag 58740
cccactttta ttatctgcag cccttcagtg atttcctgtt cctctaactc agagcccaaa 58800
tccccccaaa tagtctctgc actaacgggt tcctcaggct ttctctcagc tccctacgct 58860
gcctcttcct cacactccct ctatctactt aacttctcat ttttagctct atgcttcagg 58920
ccattcattt ccttacagaa gcctccgtgg agttccttcc ggtagggctg gtgttccttt 58980
tccagagcgt ttcctccaac tcagtcatga acagtgttat tattggttat ctgtttctct 59040
aatttgactt gtaaatttca tgagcgtgag ggacacgccc acttcactca tcagtgagag 59100
cctaatagca acccataagg tgcgtggaat gtctgagtac gacagatggt gtgctagtcc 59160
aggaacgaaa aacagaacga gggagaggct ggggagcgcg gagggacccc gggtttgtga 59220
ctccagcaag aattcagaag aatgcatttt ccttggcttg acatgaagtc ccccaagagg 59280
tgaccaggaa aggtgatcca ctcctgtctt acttagcact gggtggggga gtggggggct 59340
aggtagtgga gcttatctgt ccctgcaaat gtggacccat ttcagaggaa ttaaatacac 59400
cctgtgggga attacacagt tggtcaatgg aagatccaag atccgctcct tttacagcaa 59460
gctgttttcc tcatgggtag gaaccaccaa ggttcagact ggtcattgcc agggacgtct 59520
gtactgcccc tgtctgtctg accagaatca gcaacatcta tcttagtctg ttattattca 59580
gtccatcctg gagtccgggt cgtgatggct gtcctggact tagtgcggac gccctcgttt 59640
ccgtgctttg ctcctggtat gtttcacttg ttcctgtagg ctgtcacagt gagaacactg 59700
acacagctgc cttgggaatc cagtggaagc tcagccacgt ggcagagata caggtggcaa 59760
agtgtcgggg caggtggcaa cggagctgag agacgattat ccctgtctta ggaggacttt 59820
agatggagca agggagcacg ttcagagact tgcctgggtg cttccgatgc ttagggtcac 59880
atgacaccct gacggccgca ttcacaccgc aggaggatcc tattagaaga catgttccat 59940
tttgtggttt gatatgtgtg tgtgtgtgtg tgtgtgtgtg tgtgcgcgcg cgcgcgcacg 60000
tgcgtgtgtt agagagaggg gagtgtggta taggaggctt taaggagttg gaaacaaacc 60060
cctgtctaca tagtggtatt ctattgatta agagaaaaat gactcttcat agccttcttg 60120
gttggcttcc attgatccca tctgtaatct ttaattgttt tgtaaagaca taattgattc 60180
aatgacaaaa tctttttcct ttcccccctt ttctttcttt cttttttatt ttttatttta 60240
tttttattta ttttatttta ttttcgtctt ttgagagagg agccactgcg tagcccttgt 60300
tagcctggct ctcattatat gcccaccagt ctggtcttaa attcacagag atctgattac 60360
ctctgctgcc tcctgggcac tggaattcaa aaataggagc tttcagccag gtggcttcag 60420
cctcaggagg cagaagcagg aggatatttt cgagttcaag gtcagcctgg tctacaaagc 60480
aagtttccga acagctaggg ccacacaggg aaaccctgtc ttgaaataca acagcaacaa 60540
caatgaaaag gggctttcat attttttgtc ccctttcaag caattgcaaa tacttgtttt 60600
cattgctagt tttatttatt tacaaaactc tgtgctaagt gacacaactc aggcccgtgc 60660
cttcaagaag tgaatggaca tactgaaaga gagaaagggt ccctgagtgt tcaggctgtg 60720
tgtgctcttc acgtgtcctt ggcttccttt tgtgcattct ggctttggat ttagatgggg 60780
gggggcgggg gatatctgca gtggcacgtg catatgttta gaatcgacct tgtagacatc 60840
aaacacggac tatcaaatat taaatcaatc atgcagctga ggcattttat aaatgaacct 60900
gttactccca cattcaaatt ctgaaagcct ttatacacat gagatggcct gtaagtaaag 60960
cgctgtattc atttatttca agtcatttat ccctaactta agtgtgtcca tcctctctac 61020
cttctccctc cctccctcct tccctccctc cttcctctct ccctccctcc cttccctctc 61080
tccctcctcc ctcccttcct ttttcatttt cattttcttt aaattttgag agaaggcttt 61140
actgtgcagc cttggctttt ctcaaacaag ccatcctcct gtctcattca ttccttgctt 61200
ttgtacgaat tcttgaagta aagaactatc aagttcacca tcgatccact gattctggtt 61260
tttgttttgc tgttgtttgt gaagtgagat cgtatacttc ccccctctct tatttttctt 61320
ttcctgaaag ttactatgtt taagggtcaa aatgtcatag attctcagga atcagagtct 61380
accttggggc tggtctggac agcgcaggtc tggttaagcc ttcgtacttg tccttgaacc 61440
tcagtgaaag cctcttgttg ctgtaggtag gagaatgggg gctcttcatc ttttctttct 61500
ttcattttag attctattta tttattttgt atatatgatg agtacactgt cgctgtcttc 61560
agacacatca gaagagggca ttggatccca tgacagatgg ttgtgaacca ccatgtgctt 61620
actgggaatt gaactcagga tctctggaag agcagtcagt gctcttaatc actaagccat 61680
ctctccagcc ctgccctctt catcctttct tatagtcaga cttcctatgc ctctaaagaa 61740
atgcatctaa agttgcatgc ttctgttgtt ctcactggcc tgctgtagaa tttgaagctt 61800
caggtttccc cgttttccag ctggctgtct gcagtggatg gagaggtgag aaaggcatag 61860
tcttgcttca tcaagcaaac ctgtttgctc tcggagcggt attgcataca cctacataca 61920
cacagatgtg ggcatatatc cctgtgtgtc tttgttgcag taaaagttaa cacttgattt 61980
ttctgtccct ggttcttggt tcacaggttc tggaatagac tgtctgtagt ggaaagagtt 62040
tccttataga tctcttttgg gtctcaggca atgctggaga agaggagaac tgggtagtgt 62100
gagaggggaa aagcctttca gtttctacca tctaacctga agacactttg aaactgcctt 62160
atgacttatg tgtatacata ttcgtgtgtg tgtgtgtgtg tgtatttttg tcattgttgt 62220
ttctttcttt ctttatggtg gtggagacca catcctcaca tatgttaggc aatcactgta 62280
cccttgagcc acattcatat atatttacta aaccagcact gtgcctgtta caaggcacaa 62340
aaccatctga gctgacagga aggcacacag gtacaagcat aattgatttg tgagttgggg 62400
gacagcagag gatactttga aagtgtcagg aaggctcaac ctgccatggt gctgcgtgcc 62460
tgtcgtctca gctaccggga ggctgaggca gggtctcttt agtgcatgga tttgaaggcc 62520
agcccagtga atcccaattt ttctttttat atgtaagtac aaagttgctc tctttagaca 62580
catcagaaga gggcattcga ttccattaca gatggttgtg agccactatg tggttgctgg 62640
taattgaact caggacctct ggaagagcag acaatgtgct taatggctgg caagaaagca 62700
atggagtgct tttcagctgt tctcagccac agctgtgtaa ggaatctcct ggaggcatta 62760
gaaagcatgc acactggggt tacacctcca ggaaaccagt tgtaattggt ccggcatcag 62820
tattttctcc agagctcctc atgcagtagg ctgagtggga agctacggtc caactgatgc 62880
tgctttcagt gtcctgtgca gtcatctggg cttcaaatgt aggatgtcag aaggcatctt 62940
agaacatcga gggacactga aaacagtaat ttccttatta tatgttacag gcgaacactg 63000
tgttattttt tcatgaagaa caaaattatt ctttatccac atgcacaaaa taaatatatg 63060
caatgaaatt tcaaagctgc ttgtttattt cagagttcct ttccttacca tgctaaaaga 63120
tatttctttg ctggtctttg atggcaagta cctttgatcc cagcacttgg gagtcagagg 63180
caggtggatc tctgagtttg aggccagccc ggtctacaga gtgagttcca ggacagccag 63240
ggctacactg agaagttaag tagataaagg gagtgtgagg aaggcggcgc tttaaaaaaa 63300
agaaagaaag gagtgaccat attattaaac atttttttaa aaaagattta ctttattcaa 63360
gtgtatgagt gttttgcctg catgtatgta tgtgtaccaa tgtgtgcctg gtgtcctagg 63420
atgccaaaca aaggtgttag actccctgat actggagttg tggtcagctt tgagctgttg 63480
tatgggtgcc aagaactgaa cccaggcctt ccacaagaga aagaagtgtt cttaaccact 63540
gagacgtcgt gtggctggct catatacctg cctgctgaac ttcattagcc agcaggccac 63600
atgcgcctct ggactctctg tcttgtacag ggattctgga atagatgccc taactcaatg 63660
gctaccaata ttaacctata ctctcccact cacgatggag tgctgaaggt caactgcttt 63720
tctcttcacc ttctgctatg atctgaatgt tggagtctct ccaaatttca tatacggaag 63780
agctgtgggg atggctggtg gctgggaacg cttgctgggc aagcacgagg acctaagttt 63840
gaattccaga acccatgtct ggcatggctc tgtgtggggc tgtgggtata ggggaagatg 63900
gcggagggag agagcccgca tccggccaga gttccagggc tctggacagt tggatgcagg 63960
aggactgctg gatgctttcc acgtggcctc gatgggcatc tggctgtgtg aatccactga 64020
ccctacacag cagagggtgg gcaaggggca gctcctggta ccaggttctc agtctccagc 64080
atcccacact ggatacgaca gaggagctga gaactgaata ggggctctgg ggcggcactc 64140
gccccggaaa tacgggaaga gagggcagga ggggagggga gcactgggtg gttcccatgc 64200
aggcgagagt ccttagtctg gaccttgatg aacagagaca gtctatggtt ttagagcttt 64260
actgtagaaa gacagggggg aaggggagaa ggtagaaaga gagagagaga gagaccagcc 64320
atggccacgt agagagaggg ggagaagaga aaaggagcta gagagtaaga aaggtgaagg 64380
cttagagaga gaggtaaaag cttagagagt aagagagtga ggaggggcca aacagcccct 64440
cttatcttgc tgttgctagg taactgtggg gaggagtcta gccagaaggt cagaagcttg 64500
ggatgttgtc tatgtgactg atatccatat gcccctcctg tgggggctgt gggggtggta 64560
acttcgacag cagccaggcg ttcaggagac atgatcaaat gcctcccatt ccatgtaggt 64620
agaaatcacc accacagggt ttcaccaggg ttcaagacct cagctcaact ggagaccagg 64680
gtgcctgccc cacagctctg catgcctgtt accccagcat tgtgggaaga taggaagagg 64740
ttgatacaag catagcctga ggctcactgg tcagccagct tagccaaaat ggtgaggctc 64800
tttccaagag ggaccttgtc tcaaggcaac tgtgatggtt tggatgaaaa tggttcccag 64860
aggttcatag agagtggcac tattagaagg tgtggccttg ttgaagtgtc ttctcttctt 64920
cttgttcttc ttccttcctt ccttccttcc gacttagact tggttgttta taggaataaa 64980
tatacattat agaatacaaa tcaacttcta ctctttataa atataactct ttagcagcag 65040
gaacagagca agatatttta tatccatctc atatctctcc tcgatcaacc tcaacactgg 65100
atgacagttc agggccccaa gattcaggct gaggattaag gttagaactg aaaatggagg 65160
agtgagaaca caagtagaga aggagatagt gcattccatc agcaggaaaa gtatctaagt 65220
ataacagtta cagtattggg gggggggatg gaaacggtta tataaaccgc ccgtggctct 65280
gagtacctga aatctcagca ctcaagggat agagttggaa ggctgattga gtctgaggcc 65340
agcttggcta cgcagtggaa acttgcctca aagaggggtg gtcagaagaa aagaaagaaa 65400
atcttcattt atttttatta cttaagttac tataagtata ctttcaaaaa cataatatat 65460
tggttgcatt agtatatcaa ttacacaata taaatgtgtt ttctgtttcc aaataataga 65520
gaacttggag actttttggc tgaatcattt tccctttaac aggcttgctc ttcccctccc 65580
atggcttaaa tcacattggt aatatatttt cccacaattc ttttctttgg ggcctcctaa 65640
agaataaaac caagaaggcc aattcaatga agttctctta tctggaaatg caaatatttt 65700
cgaaagatag aagtatgtga cgtggggggg gggagtacaa cttagggggc catcaaaata 65760
aagagaattt tccaacaaaa gaaaaaaaaa atgttgccaa tatttaaagt agccctagct 65820
aaattgaaaa gacccccccc cttgaaaaat ctcctttcat atactctccc atttctaaat 65880
ataaattgtt ggcaaataat tatgaggaat aataacacat catttagttt cctgccagcg 65940
aggtctggcg gagtagaagc gcactgaacc ttaggagcac gcagttttaa aaaacgaaca 66000
attggttcga gatgttggga atagaggtta ccatggcaac cgcatctcac agctctgtgt 66060
gaggccctat tgtgttggtg tttgagtggc agacactggt ggctgtgatt tgcactccag 66120
gctaggttaa ggtacagata ggaagttttc cttgggaaag ttcagggagg gtaggttctt 66180
cgacatctgc agaggaagac tgtgatgttt tcagagccat gacagtgcat cagcagtagc 66240
agtagctaat agcgatattc agccttcagc ttggacaaaa caatcctgaa gtctagaatc 66300
tctgcaaaag caactgcaca gatgggtaat ggctaaatgg gcttgtatct agtcctcgta 66360
cattcagtgg tctagctagg tctcctcagc tccaagaaca gacctctgag ctctgctatg 66420
tccttgcagt cacaaacaac ttatgcacta cagggtgtct ctgttttggt cactgaatcc 66480
agaacccgaa tcagagtcta gtacacagtg ggattccacc catggcattc cccacaccat 66540
aacaacaccc cattatgtgc tagactgtac aagttctagt ttctagaata caggctctga 66600
atccagtgac cagaacagac actctgtagc aaataaactc cttctagtgt atttcaatag 66660
ccccatagag ctgcaatttc ggctacaaga agctgacata gattacttgc aagttcaagg 66720
cctgccagag ccctgtagtg agtttaaagc cagcctgggc aatttactga gcccttgact 66780
aaaaacttaa gtatttatat gggtggttaa ctcagtggta gagtgctttc ctagatatta 66840
agggcctgtc ttagttaggg ttttactgct gtgaacagac accatgacca aggcaagtct 66900
tataaaaaag caacatttaa ttggggctgg cttacaggtt cagaggttca gtccattatc 66960
atcaaggtgg gagcatggca gtatccaggc agtcatggct caggaggagc tgagagttct 67020
atgtcttcat ccaaaggctg ctagtggaag actgacttcc aggcaactag ggtgaggatc 67080
ttatacccac atccacagtg acacacccat tccaaccagg tcacacctat tccaacaaga 67140
ccacaccttc agatggtgcc actccctgga aggaaggaag gaaggaaggg agaaggagaa 67200
ggagagagac agagacagag ggccttttgt gagtggcgtt gaatgagcac gcagtgccat 67260
gaaggcgttg ggcacacttt gggagtacaa gtcggtgggg ttctgcttgt cagccctcaa 67320
atgccacaca tcaccactgt accaaatgca aacctttgca cccacccttg tgttggccaa 67380
attcccgctt tgtgtacttt gtgtcacagc tggagaagtc atctggggag attgtgtact 67440
gcgggcaggt gtttgaaaag tcacccctgc gtgtgaagaa ctttggcatc tggctgcgct 67500
ccgactccca cagtggcaca cacaacaagt acagagggta cctaccagga tctgaccacc 67560
gctggtgcag tcacacggtg ctaccgcgac atgggggccc ggataccatg cccgaactca 67620
ctccatccag atcataaagg tggaagagat tgcagttctc aagtatttcc actactccaa 67680
gagcaaattc ccattgcccc atgcagcgcc agcacaagcc acgcttcacc accaagaagc 67740
ccaacacctt cccaaaaaaa atgaggagaa ggaggaagag gaggaaggag aagggagagg 67800
aggaggagga ggaggaagag ggaggaggag gagggaggca gggagggaag tatttatgtc 67860
aaaaatcaaa aggaggcgag acagtcatat atctgtggaa gaatattctg gacagaacag 67920
cttgtgcaaa gaccctaagg tagaaacatg cttttcccgt tcaaagagca ttaagaaaac 67980
taaactagag tgactggaag gccacgaggt aacagagatc acagtaattt gcaaggacaa 68040
gataatttag gctgctttaa cctggatcta gaatgttctc cacagcctgt gtcccactga 68100
atcatcttgt cagcctcaaa gctatacttg tttgttcttt aaatatttgg cacagacagc 68160
tccactgcac tgggcaggtc cagcctgagg ctctgcagga gtggaaaata aggagtgaat 68220
tgaaaaaggc agaagagctg aggagagggc agctgagagg ggttggcagt agaagcgttt 68280
gctgtacaac ctgataagct gggtttgatc ccggctcaca cagaggaagg tgagaatgga 68340
ttcccacaag tctccacaca gataacatgg cacacagatg ccccatcttt cctcctcctc 68400
cttctacaca cacacacaca ggccatttaa agtgtttttt gtttgtttgt tttttcgaga 68460
cagggtttct ctgtatagcc ctggctgtcc tggaactcac tctgtagacc aggctggcct 68520
cgaactcaga aatccacctg cctctgcctc ccgagtgctg ggattaaagg cgtgcgccac 68580
cacgcccggc tcatttaaag tgttttaatt cctttgccat cttgagagcc caggctgtcc 68640
ctgggacatg cctgtttgca aacacattta tattttctgt tctttttctt ccagacagtt 68700
catccgttat taggctttta aggaatactt agtaagttag actgtgggca aacaaaaatc 68760
aataaaacgc tattcttgcc gaggcattaa gtcttgtggg agaaagaaac atttaaccag 68820
caaagacaaa aagtcaaatt cagcaggctc tgggagtctc ggagagctag cctctggctc 68880
cgttagggac agtggtcaca gatggattaa gacaagatcg gaagattaag atcggaagta 68940
aggttacgct gagaacatga ccagtaagac tcaattcaca gaggatagga ttagccacta 69000
ggcaaacgtg attaatttgt tgcctttaag tatgtaaaag cgctttttct ctccacaggc 69060
ataaattcaa acttgccgtg atctcccgtt attcccgcat agatttccag ccttgcctcg 69120
ggctagtggt tggttagctc tttaaagttt gagaatcacg tgggctaata tatgaatgat 69180
ttcagcagtc tccactagaa ccaattctag ccattcccct ccgttaaaga cgtcttacag 69240
gctggctccc tgcgagactc tgcctatagg gggcgctcga gggaggccga agaggtcagg 69300
gccgccgcag ttgcagagcg cagacttgtc ccacaaaccg cagccccctt tctagtaaca 69360
gtggagtccg gtgtgcagtt ttggcagcac tgggttcgcc gtgcccgatg cagagaacga 69420
gcatcagcag atttctaatt taagcaattc cggcctcttc tctttctcct ctccgagcgc 69480
tgtgggtggc agctgctccc caccttcgct catttgagtt aactttaagg cagggacgtt 69540
tgccaagagt tggagaggcg cagggcaggt gagggagcat tgctctcatc tgctgggtga 69600
gctgaaatgc tttgaaataa acaccctact atacacagga taggctccta caagggattc 69660
catccagtgt agcaccaata gcagggccgg gaaaccctgg tttacagtaa ccccttcagt 69720
tagagatgac atgtactaca aagccatcat tggtatacta ttaagactat gcagagacaa 69780
aacacctttg aaaaggaagg aaggaaggaa gaaaccactt gtggtggcgc acgcctttta 69840
tctcaacact cgggaggcag aggcaggagg atttctgtga gttccaggcc agcctggtct 69900
acatagagtt ccaggacagg cagagctgtt gaaaaaaaaa aaatctttgt attagatctt 69960
tctgtcaagt aatgttgtgg ttctgattaa atcctgctga ttcaactttt tcagagttac 70020
taaaagcagt taggctgggt gtgtgatgtt tttttattta ttttcaaggt taaaaaatat 70080
aaaatttata agattaaatg aggaattaac tagaggaaac ttaaaatttc atgttttaag 70140
tcaacgaatg aattgtaaag gatttatgat taactcgtca ttacaaaatc ttttttggtc 70200
tttaatccta gcacttggaa ggcagaggca gatggatctc tgtgagttca aggccagcct 70260
gggctacatg gtgacttcca ggactacata gaaagcccct gtctcaacac aaaaccaaag 70320
ccaaaaatca acacaacaca acacaacaca acacaacaca aacaccaaga ctggatacag 70380
tacataaata aggagtaatg gaattgttta tggcatcatg attgttggcg tagaacctaa 70440
gggggtgggg ggtttggagg actgctgtcc tcacagcaac ctcatcaacg ggattttata 70500
caatctatat tcatttatta tttaaaaata gcttattaaa aaatcctgct ctataatata 70560
ctgccccata aacgctgctc aacggaaacc tcaggctagc ggttggcagg tgcattctcg 70620
ttgccctttt ttgtttagcc aatgttgcag aaatgtacta cttgggggtg agggtgggga 70680
gagtcagctt tggtgaagac agaaccttgt ttgcatatgg tgctggcagc accgcccaag 70740
gacagcttga ggagacctag gctcccccct gtgccagagt ccctcggtct tgctactgag 70800
ttcgctccct taggcattcg cctcctggaa ctccagacga gttcttggtc atcttttgcc 70860
atgattttac caatcctcag agcagcaaag gtatttctta gaaagtccca ggtaacgggt 70920
gacctgaacc tggagccaat aacttgaaga ggttcctaat gtgtccagcc ctgttccagg 70980
caacctgcgt gtatttcttc atctaattcg tccaatagtg ccatgaagta ggtccttgct 71040
agtgttaatt cagcactgaa gaaaccaagc aaggctgggg tgaggggttg gggttggggt 71100
tgggggagct ggcagagtgc ttgctaaatt tgtgcaaagt ccacagctca attccctatt 71160
ctccagaaag caggtatggt cgcacgtgcc ggtaatccca gcacgcagga ggatccgaag 71220
atccaggcca tcctcagcta tatagaaagt ccaaggccag cctggggtac ttgagaccct 71280
ggctcaacac agatgagtaa gttaattaat aataaaaaag tttaaagaaa ataaaagaaa 71340
tcaaaatgca gaatgttcaa gtagcttgta tgtgctatca gacaagaatt tcaacaagct 71400
gtctaaatcg agggatcctt taaactaata tttaagaata gcccatacag tgtgttcaga 71460
cgcctcttat ctgtttaaag agccggctca aaccagcctg gagacagaat attttcaaac 71520
gtgaaccatc tggttgttac aagtcaaacc acaggaagcc tgcattagag ttggctctcc 71580
ggttgactgt gcccagcctc tctctctctg gtttccgctt ctctttgagt gttgacttca 71640
ttctctcaaa taatggcggt gcttcagact tgttttcagt gtgcttatta aagagtaccc 71700
tcaggatcag ctgcaagact gggcagaaga ggaagtcaag ccacgatgct ctcaaccagg 71760
gtcagctcag taagagcttc agaattctgc gctaacaaag agacagagta aataaaaatg 71820
acccgaaggc cttcccgtgg cccggaatct gacctcagca gtgacagtat atgactttgt 71880
tgattgtgct gacctctatc attgaaatgt cccgagggac tgaagagcac gcataccaat 71940
gagagaaaaa tgtcactgcg acgagggccg gaatcttgcg actccatcag ggagaattct 72000
ccctttcatg gtggctccca gatctcgttt agcctgtaag ctaacaagaa gaagaggcgg 72060
tcaacgcaca cgtggtggcc atcacgacaa acgggatgac ctaagagaca aacgattaga 72120
cggcagcgcc accgtgtggt agtccacgtc ctcaccgcaa gctgaggtcg gaagagggtg 72180
tctctgtgat caggaaacgt gcagtttttg tgacatacaa gcaaagggga agggcaggtc 72240
cgcgtcccca cactggattc atattttctg gatttgttga tgccctcccc cagttctgat 72300
cttgctggcc tctgtactgc ctgccttacc tatctgaact gaccgtgtac ctgtgagcat 72360
ccagtgtaat atgtttattc cacacagatt aatttcaagt cctcggggca gttttccgtc 72420
tgctctgtgc atagtgcaca aggctaatac agttacaaga gcccagaatg tggcgccggc 72480
ccagctctct ggtgatgctg ctgcacgcac acacgcggca ctatgctgac accttccttt 72540
tctagatctc ttgaataatc cacgttgttc tgctctgcat cggcctcctt tagatactcc 72600
aggaactgct aaacactcgc ttctctctga ggacacgtct agttcttgag ctccttgctt 72660
cagtttaaaa ttgaactacc tggctcaccc actctgcgtt ttcacaagat gcctggttac 72720
ctaccattta gtccaggttc tgccgttagg ggtcccacgc tctgtttagc ctttgagcgg 72780
caggtctaac actggatcaa atctccttgc tgtggctggc ctttggccat ctgtcccaga 72840
agggcaagcc ttgttctccc aaactccctc tgtcctctct ggattcttcc gtatcgccag 72900
catctcaagt agaagaaccc aaactcttgc tgaagcaaac ctccacccat tccagaacaa 72960
agccagggcg gtcctcaagc cagccaggtt tccccggcta tgggcatctg ttttccatgg 73020
catggtggct gcaccaagaa tgaggtcctc tctccgttaa agggcacacc cttcagagag 73080
gacctggagc acaggactct cctgtgtcaa cagcaagtgt ctgggatgtg atgcactcag 73140
tcagcacttt ggtccattgt tgagtagaca gagcacttcc ggggaacatc gtggagggat 73200
aggggacaat tacactgaga gcattaatgc attcaagttt tacagtaggg ttgttacttc 73260
tatccacagg tttctaagca tatctctctg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 73320
tgtgtgtgtg tgtgtgtgtg tgtgtaaatg tatgcattcc acggcaattg tctattgagt 73380
ttgctcctgg attacctgac tcctctcatc acctccaggt ctaattctta gcaacacgag 73440
gacgatattg cgccacctac tgtccaaatg tgtcctgccc tttctgcttc aaattctcct 73500
ctcattagac ctaaaatagc gccctcgtcc ccactcccag ccccctctgg ctgcctctca 73560
cggttgggtt tgtaatttcc acttggcctt tacatttcca tccagccccg tgggcagcca 73620
tctctaagcc tggtgtccag ctgagctggc ttttatgcgc tcacagcagc ccttccaggt 73680
gcccacggtg gtggtccaag gatcgcagca aaccggactg cttatgtaac tagagacata 73740
aattggtccc aggagtcctg cagcgagtta ctgaaataac attctcttta tcacaggccc 73800
gtttccacag ggccttttta cggggtgctt atgcaacagc cgccccgtca gcagaagtcc 73860
tggaagcgaa gagcagggtg atatttacag tgaataaatc atcgaattca ttacataaga 73920
acttaaaaat actagtgttt ctcctaagaa cttgggaagt tcagaaaaat ctatacccgg 73980
tcttagcaga aaagtgacgt ttcaaaaatt ccaaagcagt cggttaataa tttcaacaga 74040
agcgaaactc gagcaccgtg ggaaacgtct tcccagcctt gactggcatt gactttcggc 74100
tagtctcttc aatgttaagc aatgtcagcc agttaaataa atgtccacaa acgaagtctt 74160
tgttaaattg aaatttgttt tgctccaaaa tgactgttta cacaagacag agacctatgc 74220
agtggaaata taaaggcata aatgaggaag gaggctacac aggagatttc aactctcttc 74280
gaatagtagg tatatcagag ggcttcatgt tactgtaacg aaatgcctga gacaatcgac 74340
ttacaaagag gaaaggttta tttttgctta tagctctaca agtggtaggt agcctagaac 74400
cagatggcct taatctttgc ggctctgaaa agatgtggca aggccatcct cctacatctg 74460
aaagacactg agatattggg gtcccagagt ccccttcaaa gacatgtttc tagtgaccca 74520
aggaccgaag gttttatgaa acctcacctc ctcacagttt ccccacttct caataatact 74580
ttcctggagt ctaaaccttt aacacagtgg tctttggaaa ctactaccca ttttcaagcc 74640
atagtatgtg tgtgtgtatg cacatatatg catgttggtt tacctttgct aaggcttgaa 74700
cccagggcct tatacatacc agactctgcc accatgctac atcagcagcc tataatttgt 74760
tttattattg ttattatttg cgtatgtatg cttatgttca tgtttacatg agggtacagt 74820
tgtgcatgtg actgcatata ttcctgtgtg tctgtgtctg tgtgtgtctg tgtgtgtgtc 74880
tgtgtgtatc tgtgtgtgta tgaatgtgta tgtctgtgtc tgtgtgtgtg tgtgtatctg 74940
tgtgtctgtg tgtgtatgta tctgtgtgtc tgtgtctgtc tgtgtgtgta tctgtgtgtc 75000
tgtgtgtgta tgtatctgtg tgtctgtctg catgtgtatc tgtgtatctg tgtgtgtgtc 75060
tgtgtgtgtg tgtgtatgtg tgtatgtatg tgtgtatgta tgtgtgtgtc tgtgtctgtc 75120
tgcatgtgta tctgtgtatc tgtgtgtgtg tatgtgtgtc tgtgtgtgta tctgttcatg 75180
tgtctatgtg tgtgtgtctg tctgtctgtg tgtgtatgta tgtgtgtgtc tgtctgcatg 75240
tgtatctgtg tatctgtgtg tgtgtatgtg tgtctgtctg tgtgtgtatc tgttcatgtg 75300
tctatgtgtg tgtgtctgtg tgcatctgtg tgcgtctgtg tgtgtgtagg acagagggca 75360
aacctacttg tcattcctca ggaactatgt tttgagaagg ctctctctct tgaccagaac 75420
acacccatta agctaggttg gttggccagc aagccatggg atctacttgt gtctggttcc 75480
ccagctctgg gattacaaac ctgccaccaa gtctgccttt ttagataggt tcaggcattg 75540
aactaggatc cccgtgctcg tataaccagc attgtacctg ctgagccatc tctccaatcc 75600
tatattttta aaatggtaag tagatatact gggactcatt gtgtttattt attagttatt 75660
tattgattta gtttgagaca aagtcttgcc ctatagttca ggctttccag aaactccata 75720
tttagcccag attggcctca aattacaatc cctttacttc agatttctga atgttggggt 75780
aacaggcagg ttaccaccat cctgccttta tatttttctg tcaaaaagta aattcaaaaa 75840
gaaagcatac ttttggaggg gtagggggct ggggagatgg ctcagtggtt cagaactctg 75900
gctgctcttc caaaggattc aggttcaatt cccagcactc ataggtcacc tcacaactgc 75960
ctgtagttcc agatccaggg gatctgacag cttgacacag atacacatac aggtaaagca 76020
ccaaaatcaa tcaatcaatc aatcaatcaa tcaatctagt gtctctgctc acctaaagta 76080
aatatgaggc catggaagct gctagcctca gggacatata ccttgttcct ccttccattc 76140
aacatccatc acactcgcct gcattttaat tccctgcatg tctttctgtc tctctcagtg 76200
aactatgaaa cttcttcact ttcttcactt tctttctttc tttctttctt tctttctttc 76260
tttctttctt tctttcttct ttctttcttt ctttctttct ttctttcttt ctttctttct 76320
ttcttccttt ctttctttct ttctttcttt ctttctttct ttctttctat gtatatgtgc 76380
atgtttgtat gtgttcatgt gcatcacgtg tgtgcagtac ccgaggaggc cagaagaggg 76440
catcatatcc tcttgaaatg gagttcctat gattggaagt tgtcacatgg gtgctaagac 76500
ccaagcctgg gtcctctgca aaacagcaag tgctcttaac ccctgagccc tctctacagt 76560
cctgggctgt gcactcttgg ggtgggggtg ggagagtctg attatttttc actgtacttt 76620
ccctatcacc caacctaatt ctaaaataaa tatgtgctct gtagacattt gttctgtggg 76680
cagctattga aagcagggcc ccaaggcagc tggccccagc accgtgaaaa tgtcagccta 76740
atgttttctc cttgacttct cttatttcct tcataaaatg aactgcaagg aagtggctca 76800
gttagtagaa ggctgttgtg tcttttgcaa agcactgggt ctcatcccca gagctgcata 76860
aagccgtgca cggaggaggt acacacatgg aaccccagca tttggaaagt gaggtcagga 76920
gggccaggag gtcatggtct tcccagatgc acagtgaggt gggtctgggc tgtatgaaaa 76980
cctgtctcca aaaaaaaccc caaaattctc aagaggctgt ttaagctaga catcatatag 77040
ggaccatggc aatgggtcaa ctgcatctct aattgtctct taggagcttc ttagagacac 77100
actcaggaca agctgcccgg agtctttgtg aagaccatgt gtgaggatca tgctagaaat 77160
gtctcaccct cactgagccc ttaccacagg ccaccaggta ggtattccga tctccactca 77220
ctgtggtgaa ggaaactagg ccccagagag ccagcccata gtctgtgacc gtctccttgc 77280
agtctgagcg ggatgcatcc agtgagactc ctctgtctga ctctgaagct caggtctgtc 77340
cttcttccta cctgctccac accagggcac atcgttttga cctgaaggga ctttgcggtt 77400
gttcatgcaa tctatatttt gttacttcag gactttgcca agagaaacaa agaatgctcg 77460
attatcctct aagtctataa ttgattgaat aaatcttaaa attagccaga atccttttgg 77520
gccatattgg gtgaatgggg cctgctgagc cattctgcct gtccggggca gacactttgg 77580
cttggattca ctctgtatgg gatatttttc cccattcatc tctaagccta gctcattggt 77640
ctgtttcacc cctacctccg ctgccagggc agcccactct gacatttatt atcctcagtg 77700
tctgctggga tcagggcttg ggtgtatagg agttgtctca gctcatcctg agatccaatg 77760
agaaaagagg tgtctttcct cccatttcac ttttaagaaa taagtaactt ctcacggtgg 77820
accccacgtt ggggtccaga aatccacagg aggcgttccc ccctaccccc cacccccacc 77880
ccgcggtccc ataacgtgat gcaatgcttc gcacgctgcg ctcacagtgt gggcttcgcc 77940
ccctgccgct cagccttttc ctggtggtct tcggggcaag cggtggtctg agtttgtcat 78000
tgtttcagaa acagagcctg gctttaagga cctgatcagg cacaactcca gcatctaaat 78060
ccccttccta agctgaaggg atttcctttg tctggagttt taaaaagacc ctttccttaa 78120
aaaaaaaaaa aaaaacctct ttgtggagcg aagcaaggga ttctgttttc aacaactggc 78180
ttgagaacag gcgcacacat cctctactgt tcatcgtcgc tgttactggt aggtttgcag 78240
cttaaaggga gtgggcgaaa cgcgctcaag gctactgctt gctcgttgga gatctcagtg 78300
aacggaatta atcaggtgac atttgcatgg tgcctagaac tttgccacca cagactttgt 78360
ttttttcatt aagaggattt atttcttagg taacacaaat ataaacagtc agtaaataga 78420
tccccaggag caaaatcgga gaagcagtga aaatcctgga gacggacgaa ggagaaaatt 78480
ctggaagctc tgtttatgat cgcctccttt tgggaattga agaaaggaaa gcagatgtca 78540
gacagtggaa ggttttacag agaccggttg atggttccgt catcttgaag acaaggtgaa 78600
gtaaaacgga gatgtaatgt ttagcttgga acgtaaggtg aaccggaagt gatttggtat 78660
tgttgaccaa gacctcacca tgcagattgc cgaatccata cctaacccat accaattaaa 78720
ggataggaca gtgagaaact gtctctgcac ttacgagtgt gcgcatgtgc atgtgaaaat 78780
gacattttat gtgtatgagt gttttgcctg catgtatgtc tatgtaccat gtgtggctgg 78840
ctcccaaaga ggccacaaca tgtcatatcc cctagaactg gagtcacaaa tgatgatgag 78900
ccaccatgtg ggtgctgggc attgaaccca tgtcctctgg atgaacagct agcggtgcta 78960
accactgagc catctctcca gtcccaacta ttagggttct taatgataaa tgtgtattag 79020
ttcctttttt attgtgataa ataccatgac cagggcaagt tatagaagga agaaggaaga 79080
gtttatttgg ggatagggtt ccaggaggag agtctataat gagagggaga cactgtggcc 79140
cagaactgag agacctcatc ttcaaatgaa aacacggagc aggatgaact ggagtggggc 79200
cagactgcaa agttcaaaag tctgcaccca gtgatgtata cttcctccag caggctttgc 79260
ctcatgaaga aaggttccac ttcctcccaa aagagccacc agccaggagc tgaccaagtg 79320
tttaaataag tgaccctgtg cagcaggtgg aggggacagt tctcagtcaa accaccacta 79380
tgtgtgaaag catctctctt tcgatatctt tgggttaagt ccagtctcca gagagagcca 79440
tttatctaca gtcacaaagt ctgtgtgaga caccctgtgt taggagcacc cactttgggc 79500
atactgagga cccactatgg gcagatgtca gagactcaca ataaacaatc tttagcagtt 79560
gttgtatgga atgcagtttt ccaaaaattt ctatagttct atgctaacct attcttgtta 79620
gcattctatc tggactctgc ccccacagtt acctggcaac aaccaggtag gctcctcccc 79680
acagttacct ggcaacagcc aggttgcctg gcccaaccgc tgcttgcccc ctcctctctt 79740
tcttactcct acttctctcc cttgcctctt gctccccctc tccccattcc cttcccactc 79800
tctccaagtg gtcatggtca tggctagcct ctatactctc tctctctctc tctctctctc 79860
tctctctctc tctcctccta ctactactac cctttaaact cccttcccca tgcccttaat 79920
aaactctatt ctatactata ccattgtgtg gctggtccct cagggggaag gaatgccttg 79980
gcatgggtcc gctgaggcac tcctttcccc cacacctaca tagaacatat ctctcctctc 80040
tttatctttt tatagtcaca acaatcctgg gataggattg ccagaaaggg agacaaaacc 80100
tgcccaggcc tgccacaagg gccctactca tagaatgtaa ggagtgcagg ggagagctgc 80160
tgaggtcggt cttctacaga caggaggtct gggctccttg ggagtcagtt ctcagccagg 80220
tcttcctgac tcagtttgtg agtagggatc ccttgttcag catccatagt gttagaccta 80280
gtggggaaac ccctacccaa ttcccaattc ggcgtgcacc caaaaaaata cagccctgtc 80340
tgttctgctt tgttctcggc cccaggccgc gggctcatag acaactcttt tacatgaatc 80400
acaagttaca aaatctcatt ttctttcaat aggaaatggc aagctacagt aaaagtagtg 80460
tagcttcact ccgacctcct acaaccttcg aaagttatgc aattatggct cagttggtaa 80520
aatggctgcc atggatgtag caagcacata aaaagctatg cctggccggg cgtggtgatg 80580
cacaccttta atcccagcac tcaggagaaa gaggcaagct gatttctgag tttgaggcca 80640
gcctggtcta caaagtgagt tccaggacag ccagggctat aaagagaaac cctgtttcaa 80700
acaaacaaaa caacaacaac aaaaaagcta tgcctggcac cgcagtgcct tggtggtaga 80760
caggaagggt catgaccttc ctggcctgcc gattttgcca aatcagtatc ctccaggttc 80820
tgtgagagac cttgtcttaa aaaaaattaa gtgagtacca ggcatggtga tgggtggtac 80880
acaccttcaa tcttagcaca tagacagagg caggcagata cacacacaca cacacacaca 80940
cacagacacc acagagattt ttttattggt aaaaaaagat tctactagaa tcaagtgtct 81000
aaataatagg atttaaccaa ggggatcaaa agggattaga gtgaaacacc aaacccacga 81060
agacaaagaa aatcgaaacc agaaaccact ctctccccac ttcctgctgg ctcatttcca 81120
ggtgcctttc tgtctggtcc cattgtctta cttacttgat aatctccctg ggccaggagg 81180
gtgttgattt agagcacctc ctactccgac tgataacagc ctccaggcct tccatgctct 81240
gtggggtctt cttcagcctc ctaatggccc acccgctgtg ctttctgtta caaatgtgaa 81300
aaccttcatc cccgctaacc taccaagtgg taaggtgtta tttattgaac agataccagg 81360
agcacatgaa gggccggggg cacagctcta aatggttggg gaacctcaga gagccaaaca 81420
gataagaaac ctgctcttag ctgcttgtat cttcctgagg tgggagaggg tgcccagaga 81480
atactcagaa acacaacaag caagtgtaag gtgtacttgg caggtgggct atgggaaagg 81540
tcacagtaca gggatggagg agggcacctg attctcccct gttatcagtg ggaggctgca 81600
gtggagcaaa acacccccag tccagatctg cccagcttct gccctaattt ccagtttgat 81660
ctatctggga ccctttcctt gatctccact gaaaacagga acgatgttaa gtgcagctag 81720
gcacgcatat cctaaagccg gactatttga aacagatttc cagctcagca aaatgctccc 81780
cttggaccag taagctgctc tctgctaata aaatttacct gtgtcacagg ttatggagag 81840
ggtgatgtca cccacggaga gagtgatgtc acccatggag agggtgatgt cacctatgga 81900
gagggtgaag tatgtttagc ataagatgag cagagctgag cgtggcacag cggggcagat 81960
gtcacattaa agacagaatg cccgaactgt ggggtggagg acagcgtctg ctcagaaaac 82020
gatggcaggg tgaggttgtg cgacccattt ctacccttca acagctgtac agcctgctgt 82080
gtgggccccc ctgctcaaag cccgaggcta tgccaacatg aacacaagca tgcctcttct 82140
tgtatccatt ggtgtgtttc cgaaaaccgc tcttggtttt ctgtagtgag gagggtgggg 82200
ggttgtttgt ttaaaacttt ataaacagaa gaatttttta aaaatcttat agtcactgta 82260
atttacatgg tacagggctg gcactgacag gtcccttcaa tgacaagtcc gttcaatgac 82320
agagagtttg tctgaagccc aggcttgggt ctccagaatc tctgcaccaa accaattgtg 82380
cagtacattg atcactactg atggagaaat aggagttacc tttctgtttt caagatctat 82440
ctatctatcc gtctatctat ctatctatct atctatctat ctatctatct atccatgaga 82500
tcttagagtt gaacaacagg aaactatacc agtgttccaa accacctggc aactgtcaga 82560
tgttgccagt cacaaaatcc acttccaata agatgccaga cctgtcagca gccttggttt 82620
gggaggttct tagggacact tgctactggc aatatgctgg caagcctacc atgttcacag 82680
cttgtgtgaa aaatatcacc caggcacttc atgtcctgtt gcccgacgac ctccaactgc 82740
tgtcctgcct ccctcagggg gatggggtta gcagaggata tccactcaca atgagatgtc 82800
tcactttctg cgttcagatg agacaattct cactgttttc tttagtaaga aaatgtttcc 82860
cgagagaatt cttcttctta ccacatcatt gagctctaag agaagttagt atctctcaat 82920
gatggagaca cactcttcaa gatctaggct ggcctggtct acatagtgag ttccctgtca 82980
cccagggtgt tacagtgaga ccctgtcctt taccgaaagc agccttcatt ttaatttggt 83040
gttgcttggc ctgtactgtg gggctgttaa aagccacacc catagcctct gtagaagaaa 83100
tagatggcaa aaacttcagt agaacatcga gggtggggct ggagagatgg ctcagcaggt 83160
aaaagcaatg actgctcttc cagaggtcct gagttcaaat cccagcaacc acatggtggc 83220
tcacaaccac ccgtactgag atctgaagaa cattgagggt ggatgagatt gaccagcttg 83280
tcttaggaag gaaagaaggc tcaggatttt gccaatgacc aagggtttct gccagtctca 83340
tcacctcgga agctgagaat gaaggcatgg aagagaaaat tctgcagggc tgagttaggg 83400
ttccagtgat gtggcacatt acctgagatg cacatattca ataacttttc tctttcacta 83460
cctctgctgg gtggtggact agaggagagg gtgaaccttt ttaaaagtag gctggaaaaa 83520
aaatgtgtgc ctccagggcc tcttcaaggg cacaccccaa tgtcctgtgt tcctttatcc 83580
cccttcctaa ccgttctcaa ccttcctaac acttccactc tttgagacag ctcctcatgc 83640
tagggtgacc cccaaccata aaagtatttt cattgctact tcataactgt aaatttgcta 83700
ctgttatgaa tcatgttgta aatatctgat atgcagatgg tctaaggcga tcctctgaaa 83760
gggtcgtttg acccaggtgg gttgcgagct acaggttgag aacagcagct gtagacacta 83820
cggtcctcca aagggttaac tttggctctc agtttgggtt cagagtccat catagttgga 83880
aggtccagtg cagacgcaag aggcttccgg tcacatttca tccacaggca ggaagtagag 83940
ggccttgaat gctgtcacgt gacttaatcc ttttcgttta gtcaagggtc ccagcccata 84000
gcctacatta ggacgggtct tctcacctca gcccaactta gaaatccctc acaaacatgc 84060
tataggtttg ttttttaggt gattctagtt cctgccaatc tgatgatcga agagggtgtt 84120
tttgtttttt ttttagattt ttatttattt attttaattt tatgtatgag tgggttttgc 84180
ctgttatagc tgcaccacat gcatgtagtg cctctagaat ccagaagagg gcgccagaaa 84240
ccaatgtaac tggaattagg actgagccat cttatggatg ctctgacccc aacctggggc 84300
ctctgcagga acagtaagta ctcttaactg atgagccatc tctccagccc ttttaatgat 84360
cagtattaac cttgacattt tttaaaacat tccatcatct cccaagaaca ccacaggtga 84420
gcgagcaaac ctttagcaaa cggactctta ggggacactt aaggtccaaa tcataacaag 84480
tggctagaga tgattaaaca agaatactgg caaactcaac aagtctgggg agacatagtt 84540
gcaattaccc tgtttgaagc gctcctgttt ctgtgcccag gaacggcttg aaacctggag 84600
gagaaacact ttctcaccag ggggaataag gagctggaag tttcgggacc atcttgcttg 84660
cacacctgct gctcagaatc tgggggagag gcctttgcta agccaagtcg ccaacacaaa 84720
gcttctccaa aggccaacag gaatagtggc attgcaagaa gatgtgtgcc atggtaaact 84780
ctgagtgtgg ccaccccaga gggtcgtgag gcagggccag cttttagtgt attcgttgct 84840
ggagttacag aaaggaaggc ttctaaaggc aaaaatgaga aagatcacac aacctctttt 84900
gttgcaacaa tcctttggcc aggcagacta gtggacaagg gcgttcaaag gtcagaggtc 84960
agtccaaatt taaacagaag acggcggatc aaatgtcctt gcacaatcgt ggtgtagtgt 85020
gtaaggttgt agatgccctt gttcgaggct gtgattctgt cagtctttgg agggcagtcc 85080
tttttatcca ggacttggtg acagtcgcta ctccagggct ttccagcttg acctaagtga 85140
ctctgttttc tctctgacaa cctttacact ttccaacccc accctgcgaa ggcctatacc 85200
tgatacaaga tcccctaagc tgttaccgtt cgttcctacg tcaggacttg ctcttaccct 85260
gttctctctg acctacaatg tgccatctca ggtgaccttc tgtgtcccca tggcctggag 85320
acaacgcatt gtcctgggct cccctgttct acctcctttt ccgcccctgc cactctggca 85380
aattctactg tttattaaag ttcccaccct ggcctgtgaa ctggaagtgg ctgtagattg 85440
tggaaagaaa atgtggtctt gtgtgggagg ttccggcacg gtctagaatt tcagcactct 85500
cagtaattct caccctgagt tgacacaaag actaggtgag tgaagatgcc tagattctac 85560
tgtagctgaa atctgggggg gaagggagga ggcatgaaag gtgggagcac tcctaacagg 85620
gatctgccaa gtaaatgcca ggtgtctgaa cctgtactgt ccagggagcc tgtgtgtctc 85680
ctctggacca ggaactggtc tatctttgag gccactgggg ttccggaatt cagagccttc 85740
tcagaagccc ctatcccacc ccaccccgcc ctccgcgtgt cccggaggca gctcagccac 85800
tttgccggac tccctgccca gctgaacctc aacattcctg atgagaccga ggaaggcgct 85860
aattaaacta atcacgggag agagctgctt cccgctcgtg atccgtttgc gagcacctcc 85920
ataacgaggt tattattatg tgtaagggga gtttggctct aaattttaat ttcctgggac 85980
accagagcca atgccaagga ggagtttatg gaaatgcagc ctgatgccaa aaacaaatta 86040
gatttgttct gccacaggat attttgtgaa gttgtgaagg attggccaca tggggagaga 86100
cttaccttga ctgcttcatc tccctgtcgg tctatgccca ggccttagaa tattctcgat 86160
gtaataaagt cccttaaaat gcagactgat tcctttttta aatccacata cttaatttat 86220
aggctcttgt tccctgccct tctccacagg agaaccttga ttgtcttttt taagctgggt 86280
tttacactgt tttcttctcc tgtgtggttt gggaactatc gaagagaaga acaatgccta 86340
acaaggaaaa acagaaatac tcaattggtg tctgactaat caggttacga ctcagaccag 86400
gctggcctcg aactcagaga cttgtctgtc tctgcctcct gagttagcca ccagtgcgca 86460
ggcccagttt ctttcttcgc tgtccctctc catgctttaa acccctggtg tatatttggg 86520
caacttcaag ataactcata ggtgagttaa aggtcccagt acctttgtgt acgatgttca 86580
caacagtacc ctgtgattgg ccctctgtta gttcagtggt ttaagtcaac cttggaacac 86640
acagagttca ctctgcttct gagagtttgt acaataaaca tgttgtatct ggacacaaaa 86700
ggacaaaaga tgaacaataa gccagggaga agaattccat gctgtgccca caggcaaggt 86760
taaatgttgt tattacacac aacgctctcc tgaggatctc tccttatctc accagcccac 86820
cttcgggagt ttgcttattc aatgagaatc tgtgtaataa accacattgt agttgggcat 86880
gagaagataa aagatgaaca gtaaattggg ggcagcggtc acatgccgag ctcacaggca 86940
aaggcttagt gttacacaca aagctcccct taatatcttc ccttagggtt ccctttacgc 87000
cataaaaacc acccaggagg ctagagaggt ggctcagctc ttccgaacac ttgttgctct 87060
tagagagaac ccagcttcaa tgtccagctt ccacatggtg gctcacagcc atctatgact 87120
ctgtcacaag agatctgttg cccctttcat agtgggtcag gcctacctgg ctgttgccag 87180
gtaactgtgg ggaggagcat acctggctgt tgccaggtaa ctgtggggtg gaatttagac 87240
agaatgctaa cagtggagtc ttggtattct tagtgggaca tggctgagac caggagggag 87300
gatgtcgtgc tgaaggttgg cagacagaga gcataggaaa tagtcatctc tggctggatg 87360
tgggggagtg gataggatag gaatatggga taagcttgct ttgtgggctg aaggcttctt 87420
gaacgttgat attctaaatc aattatactt atctatctac ctatctacct acctacctat 87480
ccatctatcc atcaactatc tatctaccta cctacctatc catctatcca tccatcatct 87540
atctatctat ctatccatcc atcatctatc tacctaccta tccatctatc catccatcat 87600
ctatctatct atccatctat ccatccatca tctatctatc cagctattca tccatcatct 87660
gtctatccat ctatctatat acctacctac ctacctacct acctgtctgt ctgtctatct 87720
atctatctat ctatctatca tctatctatc tatttgtgta tatttgtgtt gtgtcttgtg 87780
tgtgcttggg catgtatgta cacacatgtg aaggtcacag gacaggagtt gattctcttc 87840
ttctaccagg tagaaccagg ggtggggggc tattctcaag tatgggcctt ggtaacaaga 87900
gctttaacct gctcagctat ttattccacc ggtccagaga tcctaagttt aaggtgacta 87960
cttggtatgg gcgaggcttt tcttgagtct gtctggttgc tgggacacag ccgaatcagg 88020
gcatcatatg tcggattgag aagtgtgtcc tttcaaagag tcctctgttg aagccctaac 88080
cacagatatc atcacactgt ttgaagatgg agcctagcag gaggaaacaa aagttgaatg 88140
gtactggaaa gtgcggcctt aatcctggtg tcctcgtaag aaggaacaca agttgtccct 88200
ttctacgcat gtgcagaagg cggtgtgagg gaccagtgag aaactggctg tgtgccaagc 88260
acaagaggaa cctcctgggc gaccatccct cccagtacaa ccttaggctt tctgcctcaa 88320
gaactggtag gccatgaatt tctgttctca aagtcaccta cctgacctgt ggcatttcgt 88380
catgacaacc ctggtggctg aagatcctgg ggaagagttt ttcagggcaa aagtcaccga 88440
agatgcagca gggagtgaca caacacagga tcacacaagg gaagaccaga agatggcagt 88500
cacttagcca aggacagctg ctggtagctg cttccagaga gccagttctg ctgcagtgcc 88560
catgtgtcag ggacagtgct gggatctcag tgttttgcag tagatgtagg ccctcaagga 88620
tctcacggtg ggtaaaggac acacaaacac agtgccacta caacatggtc aacggtcagt 88680
gctgctttaa gaaacaaggc gagttgtcat gtagaggatc tgtctcaggg gaggtggtgt 88740
ttgagatgaa catcaaatct gagacggggt ttgccgggag aagagaactg agagtgtcag 88800
gctggagaga cagctcagcg ggtaagagca ctgactgttc ttccataagt cctgagttca 88860
attcccagca atcacaatga gatctgacgc cctcttctgg tccatctgaa gacagctaca 88920
gtgtacttag atataataat taatacatct ttaagagaga gagagaggga gagagagaga 88980
gagagaaagg aagaaagaga gaaagaactg agggtgtaca caccaagggc ccgtggcagt 89040
tgaggcagga ggacagccac atagcaaatt ccaaactaga gaagactgca ggtcaaaaca 89100
ctgggaagcc caagcgagtg caggtcaggt tgggggtggg gcagagtaga ggaagcaaat 89160
tgtgatcaga atgtgaaatg cctgtggggg accatgcaga atggagctgc tacataatct 89220
ctagacggaa gagaaaatcc aggtagtact gcagagtatg agctaagaca ggaaagccac 89280
ttctaggaag tatatgtggg agagaactca agttgatggc tgagatgaag gggatatttg 89340
ggtgaggtga gacctaacaa tgacttctaa tcttctgctt ggatgcctga gatcggtaca 89400
acacttgtca gcattaggga ctgataggtg agaaacagag actgttggct ggggggaaaa 89460
tgggacacag tggcccaagg ggtgttgatg aattccggtt tagacatgcc cactttgagg 89520
ggcccgtggg gaatgtcagc aggcagcttg aacaccgatt tggagttctg ggagaatgtg 89580
ggtaaagttg aacggtacgt acggtaacgg agaatgtggg taaagttgaa cggttaccgt 89640
acggtaactg aagtcaggca ataaacagag ggagcaaaga gccatcagct atcggctggt 89700
tgtcctatgt ccagttgtat gttcctgtgt gttatccacg gccagcaatg ttgtttaggg 89760
gaaactattg ccaccgtctt tttccaatga atactcggag gcacagagag atattaagag 89820
agtttcccaa gcttacataa ttgacaggtg aatttaaacc tgtgtgatct gtccctcaga 89880
catatttatg ttcaaggggt aaggaaagaa gagcccgagg agaaaccatt tgacctcgga 89940
ccaacaacaa agccaaaggg attcttagaa gccagatgga gggcatcaaa tgctcaaagg 90000
cctgcaaaga taaggggagg ccattaggaa gaagttctgg agacaagctg tggtcagaca 90060
gctaggagac cgcagaggca cacggcatta gcagacaggg agcaaaggca gaaagcaaat 90120
gttaagatga ggctgtggtt tgatataaac catgcaacaa ggttagatag gccccgaggg 90180
aggaaggacg ggtggtgggc tggtactgta gcaagcatat gcatgatgtc atgttggcaa 90240
gtttatctcc agtcgacacc ctcctctcca gtgttgaagg cccagaacca agcttgccag 90300
agcaagggtc tgcaggctcc tgtgagctta tgtttaaaaa accagaatgt tgattaatta 90360
attgtttctg agtaatatac tacaaaggga gccagatgtt cgtggtttgt taatgagaac 90420
attatagtca gtaggcagtg cctcatgata attaaaatgt tgccagggag tgtgagctcc 90480
attattagtt atatttgctg actatctcag gtgtccatga tccatagcca tctttccgtg 90540
ggcagacatg aagtccagtg acagattttc tttcttttct tttcttctct tttctttcct 90600
ttccttttct tctctctctc ttctctttct tttttcttcc ttcctttctt tctttttctt 90660
tttaaagatt tctttatatg taagtacacc atagttgtct tcagacaccc cagaaaaggg 90720
catcagacct cattaaggat ggttgtgagc caccttgtgg ttgctgggat ttgaactcag 90780
gacccagcca ccatgtgctt gctgggattt gaactcagga tccaagggag ggcagtcagt 90840
gctcttaact gctgagccat ctctccagcc ccagattgga ttgtgtttgt atgtatgtgt 90900
tcctgtggat gtgtgagtgc ttaggggtgt gtgtgcacac atgccattgt gcgtgggggt 90960
cagagatcag catcagggat cttctaccaa tcccactcct ccttgctttg agacatggtc 91020
ccttactgac ctcagtgctc acagattggc tagaatggct ttccaacaag ctctagggac 91080
acactgtctc tgtctctcta gggacgggat tcccaggcag gccaccatgc cagggttctt 91140
tcatgggtgc tgcggggatc gaactcacat ccttaggctt ctgcagcgag tgccttgctg 91200
gctgagccat ctcttcagct cacatttgga ttgttctctg gcactttgat gtagtgcttc 91260
tcacacggga tcaacatttt atctatatgc aacttgctat gcccattcac cacttccctt 91320
aggtttcctt tttttttttt cccgttaact aaaaattcac agtaaaatat ttcactataa 91380
atatttcagt aaaatattta ctaatgtata ttttaaacgt aaataaatat tttacaaaga 91440
ttatattatt ttaaaaggaa aaatctgtgc aagaaatgtg aatggaggaa tgggctgata 91500
acttgatatt aatcccagtg gtccgggggt agagacggga ttcccagggc aacctggcta 91560
gttacgatag ccaaattgat gagctgcagg ttcacaggca ccactgcctt actgaggaaa 91620
atggagagcg atcgagaaag tgacccaaaa gtccactgct ggcctccaca catgggcaca 91680
cacatatatg cacacacatg ctgtcacata gcacatggga aaatgggaaa atctgactgt 91740
gtgatatgct cctaaatcat tgtgacatct agcagtgacg tagccgagct cctgagaatg 91800
cctttctcaa acaccatgca atccctccat gccatcagag cctgccactc acagcactgc 91860
tccacaccgg acacaggcag atgtataggg attccttcct tccttccttc cttcctttcc 91920
acctcctctt ctttctttct ttcccctctt ccccatctcc ttcctttcca tgttcccact 91980
aggaaagctt gaatatttat gagttagagg aacagatgaa gcccaacgtt tggtaaaaga 92040
ggcaacatca gatagcgaag gagcggcagt aagactcagc ccagaaagga gggagaatcg 92100
ctctgtgatg gaggacttcg ccatgttgat aaaagaattc tcattttaca tcaaaggtga 92160
aagaaaagaa gtttatttag tgcaccaaaa ggagcccccg agtatgaaag tgacagaatc 92220
accagtgcag ttttggttag gggaaactac ttcaagggca atttcccaga atgcaattcc 92280
cagtatgcaa atgagggtgc ttctgtggta tcagggtgtg gctaattctg tttgctctcg 92340
ttattatgca ttgtgctatt tgggttgaac ggagccatga tttttctgtt ttggagatgg 92400
cttatggacc aggcttgcac tgggaaccca tttccttttg ggcctctcaa tgatgagctt 92460
tttttcctac cccacccccc acaatgcagc aacctcccaa agagaaccat atgcaggcac 92520
tgattggatg cgctatttgt attgcgtgtc cggagaggaa gcaccatctg gagatagaac 92580
tgttgaaccc cagaagaaat aaatttgaca agaaggttaa aaatgccgag gtcaaacgca 92640
agaaagaaat gctgataaat ggtcccagaa caacacagaa gccgtacagg cacagccatg 92700
accagacatt tttttctgat tccaccaatc ttccttgttg ctgaaaatca ctgacatttc 92760
gggggttttg atgcgtttgt cccgattcct tttacttcct tcaacatggc acaaggaccg 92820
tctccaacac tggtcagtaa tccagggcct ggggactttt aagtataaca gctgtgagca 92880
aattcaagtc ttcttattcc tcttttactt ctctgcattc agaggaaaca ctgaccagga 92940
gcaatgtatc acaggctcac agatttgtct atcccaagta ggcttttggg gtcccctttg 93000
aagagaaagt gtggatcctt aactggcaca tcactgacag ggcgaacaca tatttgtacc 93060
taagatactt tctgagaaac aaattattta ttctggatag agggacgtgg taatttcctc 93120
aagtttgata gccattgggg tagtctcatg cagaagacac aactggggga aatgagagag 93180
agagagagag agagagagag agagagagag agagagagag agtaaaggag ccaaaggggc 93240
ttttcatggc ttttcacgta tatccagaag agtaaactct aaggaacctc ttactgacca 93300
ggccaataaa tgtccctgtc ccaaacaaaa ggtgaaagct agctgagaaa tgataccaga 93360
ggttgtacat acacatacat acatatacac acatacacac atacatacat gcacacacat 93420
gcatgcatgc acacacacac acacatacat gcacacacac acacacacac acacacacac 93480
acacacacac acacacacac acacatatgc acacacacag aatcacagga ggccagaccc 93540
tctcctctga ggcctgcaag gttctctgtg agtaatgaag tactctgggc atgctccatg 93600
cctacgtggc atttcagatt ctcctgtggg ccctgctttc tttttggatt cagaggaaac 93660
actgtggtaa gacagaggac tgaggcatgt gaggaggtcg ctcactggag ccctgttagg 93720
cagctttgat gaagcttgta gggtgtcagg ctgtgcagct gagagagtcc gatccttctg 93780
aaactctcag gttctggttc ctccaggacc caggtcaact gaaggaggag aaggaggaag 93840
tcaaagagga aggaaggact gctagtaaat ctgagttgct ggagggattc accacagagc 93900
agggtctttg tccattaggg tttgaggact gaagttctca gtgagttcaa agagaatgtt 93960
tagtgggaag aacacttacg aaagaagagg caggggcttg agcaggagtt ctgaaacaga 94020
gctgaggagg taccagtggt ggtaaggcct gtgtgacacc ctgagagaga cttcagtgta 94080
gaagaaacca ggagagatta agaacccaag aaaaagatca ggctatgtcc atactctgag 94140
tgacttcacg ttgtataaaa caatggattg gtttggtttt ggcttaaggt tttggtttaa 94200
ggttgactcc atcttgagtg tggggaataa tctcactcct ggggggacaa acaccacaat 94260
ctctctggca ctacacatgt gcccagggtg atacatgact tattccccag agtggaaaag 94320
gtgccactct gtgacttaat ggggcaaagt gagattgtag gccaataagg gtgcattaaa 94380
aaaaatcata ttggaaaaac cacactgaag aacttagtgg acaggccaga ccaaggaaag 94440
gtgtaacttc ctcatctggc cccccctaaa atgatggaca cctaacatag caataataat 94500
ggacacacac tgacctatta tccaatacac agtgtgtatg tacaccaagg agggaaagac 94560
agaactctga actgtccctg ggacttcacc ttagctcatc ccccattgct gaccgggcct 94620
attcaacgtt catctgccca tccacctgtt cgcccatctg gacctgcttc tgtttttacc 94680
tagaagctgg actcaattct tcagcatcac tgtgaaccct gactccccca cagcctgtag 94740
caccttcttc cctgcagatg gtttctggtt tgttactgta tcaatataga ccctgcttaa 94800
tttccaccag cctatttcaa aaacctcttg accgctgctg gattctcttg taggggaatg 94860
ggtctttcat atgtggtctg gtctgttttg aaaacttctc agtgccagtc acaagagata 94920
gaaatcagaa caagcagtgt aaagggaccc tttcctgtct cacatcctct ctgtttctgg 94980
gcacactaga gaaccaggaa tgatggactt tctggagaca agagtgttat agctagagaa 95040
agaaagatga attgtccatt tttttaaaac cccaggacat atgtattaat gtctttggtc 95100
tcatagatat gactaaggac ttccatatga gaaggctgtg ttggattatc tgggtttgag 95160
gtcaccatga tcatatgggt tcctagacac ttggaaagaa aagttgtctt agttagggtt 95220
ttactgctgt gaacaggcac catgaccaag gcaactctta taaaggacta catttaattg 95280
gggatggctt acaggttcag tggttcagtc cataatcatc aaggtgggaa catggcagca 95340
tccaggcagg catgatgcag gaagagctga gagttctacg tcttcatcca caggaagcca 95400
ggggcagact ctcctcgggc agctaggaag agggtctcaa agcccacccc cacagtgaca 95460
cacttcctcc aacaaggcca caactacacc aacagggcca cacctcctaa cagtgccact 95520
ccctgggtca agtgtattca aaccaacaca aaattcaaga gcagagaaaa aagaaatatg 95580
aagataaagg gaccagaggt tggagttatg ggattgctgg ctctgaagac agaggaagaa 95640
atctcaaact aggatacatg gcagcttcta gcagttaaga aagtaagtta aatttatcaa 95700
ccaattctct ttggggcctc cagaaagagc tgagttctgg tgacaccttg atttttatct 95760
cagggaagcc tacttggaac ttccagcctc caagaatgta agataataaa attgtagtct 95820
ttaaaacctg tttggtttgg gtgggttgtt tttgttttgt tttgatttgt ttttccagca 95880
aaatcaacta atacagagag aaactgtttg ataagatctc ccacccccac ccccaaggat 95940
ggcattttca aagagtgtga aagagcagtg cattgtgggc agtaaggtac agacacaaga 96000
cctacctctg gccatctgga tatgtatgtg tgcaagacga gcttttccac ttcagagggc 96060
tggattgcaa gggacagaag cctctggggg aatacaaatg tcagctaaag ccaggaagta 96120
gaggaggtga ctcaaggaaa ggacacaggc ctcgagaact attttaattt ttggaaggac 96180
aacaagagtt tcaagagttt caggaataca atgtcaataa gaagacccta ggctgctgga 96240
ccaaatggag taggagagga atagaacatg aagtgtaacc cgatctggaa taacaggcta 96300
cacacattct atgggtctct tactgagaac cctggtgggg gggggggggg ggaaggaggg 96360
tgggtggacg gacaggaagg tcagagatgg ggctaactcg gcttagcagc tttaatcaaa 96420
tgccctttag cttcttcttt gattttaccc atgaattatg atgcatgaga ctcatttctc 96480
ctggccctgg ccaaatttca gtcataaaac ttgttttgtt tttgaaagga ctgagacaaa 96540
tttttatgca gaacagcatg tcaaagtaaa gctaacgagt gagtaattgt caaaacaagt 96600
ctaaaagaat tcacttcctc cctgttataa tacaaaatta tttctggtta atcgcgtctc 96660
actctgaagc ccaagttctc taaaggagtt gctttttttt tttttttggt acagagccga 96720
tgggaagttt gacctcagct gagcaaaaca gccagccggt ccagagatac aatgttgcag 96780
atcaagagtc aaacaatctg gggttatctc actcctttta acagccattt tcttttgacc 96840
tttgtgtctg acccaacatc ctgcgaatgc ggggtggggg tggggtggga aactcttggg 96900
aagtatttct aactttaaca gagatctatg gtctctgcta acccccaaag ctaggaatgt 96960
tacaagatag tatctgaatc ctatagaaac aatctccttg cttagtgtca ggacacctgc 97020
cggatgctcc cgccaacatg cctgctacat gtgttggttt atgaagcttt tgttctgtgg 97080
ctatgacagt tcatgcttct tttcccgtgg tgggagtgta tgtgagggag caggtcattc 97140
cgggtaactt ggacccctgt tcccagccca tgctcactcc tctttagctc agaataaacg 97200
gtttccttat atcctttaaa gcagagccat tattacatgc tgccggcatt actgggctcc 97260
taagtgccat caggtttgag ttacaagaag ctatagagtc caagactttc cctgaggctc 97320
tgatgtatat agaaaaggga cacttccccc ctccccccaa aaaccaggat cttacgtagc 97380
agagctggaa atagcctggg gaacatttcc ttgaggccct gctgtatgga gaaggaacat 97440
agtttctttt ctaaaaccag gaatatgctc ggtagagcca gtaatggcct ggactcagag 97500
tcttgaggga gcaatggctt ttgacactga gaacccagct aaggggatat ggttttcagt 97560
cccaaggctg ttgtgtgttt aatctgtgac cactcttgag atactcacat tccctttgtg 97620
caacattgct tgatgaactt cctgctgact ctgtttcatt gtatggtagc cttctgctaa 97680
ccctgcctca tgtctttcct ggaaatcgta tataagatgc catgcttctt agtaagcttg 97740
cttggcataa gacacggcgt gtgcaagacc ttctgatccc aaacttccct ttcctcttat 97800
cttttggtcc aggaccccat cactaaagaa ccagttgctg gtataattca gggacgcttt 97860
atagcttcat acaagaccat ggtgcttgct ttatttacaa gatggaaaga gggacattct 97920
atatagcatt ctcaatctat gggttgagac tcaattaggg cctgcccact ccaggaaagc 97980
cagtgtttcc aaaacactgg gttctatcct cagacaagaa aacacagatg cttacatgat 98040
aattcataac agtagcaaaa ttacagttat gaagtagcaa cgggaatagt tttatggttg 98100
gggtcactac aacatgagga attatattaa cgggtcgcag cactaggaag gttgagaacc 98160
actactctat agcaatatca gattcaaggt cacaaggcct caagctcttt agccctacta 98220
gatgaggtca gccttgtagg tttgtcaaga ccctcctttg gcattcctaa cacgagacct 98280
gtgacatgag cagtcttggt actttcctac tacactgagc tcctacatca cagacattag 98340
ttagatcttc tggatttcag gtactaggta acaaaatggg ccaaaccaac gggaatgagg 98400
taagagtatt tctgaaggaa aactggagga gacctggcct ttgctatcca gaatttggcc 98460
ttatctagat agatgtctct ccaaagcaaa ggatcgcctg acgtctctta ctgcattaac 98520
tgaacagata agagaaggat ccagggaagg caacacagga agaggtaggt agagtagaaa 98580
tttctagttt ctttggggac tcagttaggt tataatgatg tttttggctg gggcaaacag 98640
gtgaaaggat gttttgttac agcagacatg tgaaaggata cgtgatgttt agaaggaata 98700
taaagatgac cccacagaca gtggaagcta gagcttcact ttgctcccca ctcaatagtc 98760
ttcacacatc cacatgtatt ggttcgcctt acgttgtgtt gctgagcttc actgtggtga 98820
ctttggagag ggagatactc accaaagagc ttcccataag attcctgtgg cttcttgcca 98880
cttctgcttc cgtggacttg aggtgagcct cgtggtttct tctggattga actgatgttg 98940
ctgattcgtg tgtggtgtct gccaagtgga ctggttggca gctgctgctt cgtgtttggt 99000
gtttgctagt agactggaca gagaacaaag aagattggaa tcgtcccaaa gaactcttgc 99060
taaacaggtc ttcttccccc aagtcctaat aacctttctt ttccactgcc tctggtggat 99120
ggtgggctag agaggaggtt gaacctttat taaagtaggt agagaaaagt atatgcctgt 99180
aggtagaaat gcaaacagat cagtcactga caagtcagtg cttttagtct ctttgttagt 99240
tatacccaag ttccatcctt ggtctgggct tgatagcaca cacctttagt cctataactc 99300
aggaaataga ggaaagctct atgagtttga gcctagcgtg gtctacatac tgaattttag 99360
gataaccagg gcgacagagt aagacttggc ctttaaaaga aaagttccat cctgtgctgg 99420
ctagttttat gtcactttga cacaagctag aatcatcaga gaataggaag tttcaactga 99480
gaaaatgctt tcctaagatc aggctgcagg caagcctgca gggaatttca ttaattggtg 99540
atggatgcgg aagggccttg ctcattgtgc atggtaccat ccctgagctg gtgtcctggg 99600
ctctataaga aatctgtctg agaaagtcat gaggagcaaa ccagtaatta gcatcccatg 99660
catcagttcc tgcctcctgg tccctgcctc caggtttctg ccttgcttaa attcctgtct 99720
tgactccctt cagtgatgga gtgtaatgag gaagcataag ccaaataaac cctttccttc 99780
ccatgttctt tttagttaca gtgtttcatt aaagcaataa caaccctaac taagatatac 99840
aactttatat tgtgatacaa ttatgtcctt gatggtcaaa catttcctct cgtaaggtgg 99900
aacaggaggt gggagatact gagacgagtc tccttgtaaa ccagattcca aacttgcaaa 99960
ctaaaagtta ttgctgcaca ctttcgttca ttttgtgtat tccctccatt ggtccctccc 100020
tgagtacaaa gctaattgct tctttaatta agccctcttc cacttttact aaagctgttc 100080
acatattcca catcactttc cgctcttggt ttcccactgg accgaggcgg gcatttccag 100140
gacccagctg gcactgttta tctgcagtgg gtaccaggtg ctgggaggat aaattactgt 100200
ttttcaactc tgtttcttct ccttccagtg gatgcatcta aagtggaacc cagggagaac 100260
ccagggagcc tggctgcaga caccgtggag cagtgagtct caccaaagaa agatctcagc 100320
tggatgacag tctttagagc caaacttcaa attagccgtc ttggaaacgc ctttggttac 100380
tccaaaggga cgtaggtgct aatgggcagg catacacacg agcacaccag tatccctctg 100440
tattgtcacc gtgaggctca gtctcataat aattttgtct ccttgtgcag taacagcgag 100500
gaggttattc tgatttacct gagtgtcttt tttcataaat actgaggtgt attcccaggg 100560
ctggtctatt atctgtctgg atagtggatg ttccaacagg gccagcttca tgagcaacac 100620
atagcactct acacttcaaa gggcccaggc ttgttttaat gtactgacac catcatctcg 100680
aaattattat tttttttgta tttcatatac tgtttgtttg tttggttggt ttggtttggt 100740
tttttgtttt ttttttttgt tgttgttttt tttttgtttt gttttttttt tttttttttt 100800
ttcgagacag ggtttctctg tatagccctg gctgtcctgg aattcacttt gtagaccagg 100860
ctggcctcga acttagaaat ccgcctgcct ctgcctccca agtgctggga ttaaaggctt 100920
gtgccaccac cgctcagccc aaggcaactc ttataaggac agcatttaat tggtgctggc 100980
ttacaggttc agagattcag gctttatcat ccaggcagga gcatggtagc agctaagcag 101040
gcatggttca ggaggagctg agagttctac atcttcatct gaaggctgct agcagaatac 101100
tggcttccag gcagctagga gaagggtctt aaagaccaca cccacagtga cacacctact 101160
ccaacaacac cacacctcct aatagtgcca ctccctgggc caagcatata gaaaccatca 101220
cactgcgtaa acatattttt gtcttaatct ttatatacaa atccttattt attaaaattt 101280
ttatctatca tttttcatgt tttaatatta tgagacataa taatcacgta gatagatctt 101340
ccactgtttt aaaaaataaa attgattggg atggcctgca tagcttttta attgtaaaaa 101400
aatattaata tttaactatt attttgtgat tttaggccag tacagctgga cagaggaggg 101460
tggcaattac tttagaagat agggcagaag ccctgtattt gggtccttaa aaacacattt 101520
tagggggctg attttagtat ttgtttgtgt ttaaagaggt caaacatatc ttgaaaatcc 101580
tttaagtttg tgtgtgtgtg cgtgtgcgtg tgcgtgtgtg tgtgtgtgtg tgtgtgtgtg 101640
tagaaaggca tatacataag taacttttat ctaaagggga aagcctgcct ttaggcagta 101700
aaactcaaca gaacccacct gaacagctcc cttgtggcag cagctgtaat tgccctttag 101760
ttcttgatgt tgttattgtt gaaatctgtc aaccttaaag tgttagcctt ttctgttagt 101820
atgctagtgt tgtatatcat acaaacttta ttaaaattaa gacaaaacaa accattaaaa 101880
tcataacata aacatcattg tagctccagg aagttgagag aagagataaa ctagaggaac 101940
gctttaagtc tcactaagga atgtcagcac cttgatttta gtttttacct tttgtattta 102000
tatctcaggc tgtgttgtcc tccttcttgt cacacagagg gtccttttga cccaggacag 102060
ggattaagga catg
Claims (11)
1. long-chain non-coding RNA slincRAD is preparing the application in promoting or inhibiting the preparation or drug of Adipocyte Differentiation,
The sequence of the long-chain non-coding RNA slincRAD is shown in SEQ ID No 300.
2. a kind of small interference ribonucleic acid molecule for inhibiting slincRAD expression, has the sequence shown in SEQ ID No 1-12.
3. a kind of bobby pin structure ribonucleic acid molecule for inhibiting slincRAD expression, has shown in SEQ ID No 21-24
Sequence.
4. the ribonucleic acid molecule described in Claims 2 or 3, the ribonucleic acid molecule is preparing inhibition Adipocyte Differentiation
Application in preparation or drug.
5. the nucleic acid oligomer molecule of long-chain non-coding RNA slincRAD is detected, selected from following combination：
Detection primer combination 01：
01 forward primer：5’-ATCCATCTCCCCTACCCATC(SEQ ID NO:200)
01 reverse primer：5’-TGCAGAGGACTCACAACTGG(SEQ ID NO:201)
Detection primer combination 08：
08 forward primer：5’-GGTGCAGTTTTGCACTGAGA(SEQ ID NO:202)
08 reverse primer：5’-GATGGAGAGAAAGGAAGGGG(SEQ ID NO:203)
Detection primer combination 16：
16 forward primers：5’-CCTAGTGCCTTTCTGCCTTG(SEQ ID NO:204)
16 reverse primers：5’-TGGGGTGATTGTGTAGGGAT(SEQ ID NO:205)
Detection primer combination 31：
31 forward primers：5’-CAAGTGGTTCCGGATTCTGT(SEQ ID NO:206)
31 reverse primers：5’-CCGTGAAATGAAGTGGTGTG(SEQ ID NO:207)
Detection primer combination 54：
54 forward primers：5’-GCATTTAATTGGTGCTGGCT(SEQ ID NO:208)
54 reverse primers：5’-GACCCTTCTCCTAGCTGCCT(SEQ ID NO:209).
6. detect long-chain non-coding RNA slincRAD expressions reagent, the reagent prepare Adipose Differentiation accelerating agent or
Application in inhibitor.
7. strike the transgenic animals of low slincRAD expression, it is characterised in that genome slincRAD expressions are reduced to wild
Type animal expression it is horizontal 60% and it is following..
8. striking the preparation method of the transgenic animals of low slincRAD expression described in claim 7, include the following steps：
(1) DNA fragmentation of coding slincRAD-shRNA expression cassettes is obtained；
(2) DNA fragmentation is imported in the protokaryon of fertilised non-human eggs using microinjection, then by zygote transplation to the same of false pregnancy
In kind animal body, it is made to develop into adult animal；
(3) identification obtains the transgenic animals that slincRAD-shRNA is integrated in genome；
(4) transgenic animals with wild type animal of the same race are mated, obtains offspring animal；
(5) it is that slincRAD strikes low transgenosis and moves that identification, which obtains and the offspring animal of slincRAD-shRNA is integrated in genome,
Object.
9. the transgenic animals of claim 7 or 8, it is characterised in that animal can be selected from rat, mouse or rhesus macaque.
10. strike application of the transgenic animal model of low slincRAD expression in fat metabolism relevant disease drug screening.
11. fat metabolism relevant disease described in claim 10 is selected from metabolic syndrome, obesity, hyperglycemia, hyperlipidemia, glycosuria
Disease.
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| CN111041052A (en) * | 2019-12-30 | 2020-04-21 | 嘉兴学院 | Application of mir-206 in inhibiting triglyceride generation in pig fat cells and lean pig breeding |
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