Clinical Microbiology & Molecular Biology Practices - Assignment Example

NUMBER: SHS80204-2 Clinical Microbiology & Molecular Biology Practicals Practical 2 Week 3. 4th of February Agarose Gel Electrophoresisof DNA Results and Discussion for Portfolio. To be handed in in-class on the 18th of February 2010. Video: preparation Agarose Gel Electrophoresis. Discussion/ Questions. Questions to answer from the .Agarose Gel Electrophoresis Video. 1. Why is the blue dye added to the samples of DNA? To make the solution easier to see for loading also it because it has a high density then the electrophoresis buffer which allows the sample to fall to the bottom of the well. 2. What is the limit of detection of DNA? The maximum limit is between 200-500ng and a minimum of 4-5ng of DNA to each well. 3. What charge does DNA carry at neutral pH and why? The DNA has a negative charge at neutral pH 4. In electrophoresis, in which direction does DNA migrate? The DNA migrate towards the positive charge. 5. Do large or small DNA fragments migrate fastest? Large DNA fragment travels slower than the smaller DNA fragment . 6. In the gel, what does each "band" of DNA consist of? Each band of DNA consist of large population of DNA molecules of the same size . 7. How is the DNA in the gel visualised? The band of DNA allocated in the same place in the gel because of the same size therefore that allows the visualised electrophoresis. 8. What is the stain called used in the visualisation? How does this stain DNA? The dye called ethidium bromide that is used in visualisation and it does this by inserting the dye between the middle blokes. 9. What is the purpose of shining ultra-violet light through the gel? The purpose of shining ultra-violet light through the gel because that makes the DNA reddish to orange colour which makes it easy to detect the DNA. 10. How can the information from the gel be used to find the size (length) of a fragment of DNA of unknown size? Transformator which uses the UV light to measure the distance from the DNA to the wall. Results. Attach the photograph (or photocopy) of your agarose gel here. Make sure you label the lanes and indicate the samples loaded in each. Figure 1. Agarose gel electrophoresis of the DNA samples which were digested with restriction enzymes in a previous practical. For the molecular size standard, E.H, which is in lane 1 if you followed the loading scheme in the schedule, fill in the following Table 1. TABLE 1. The molecular sizes and their migration distance from the wells of the fragments of E.H used as molecular size standards Size of fragment (bp) log.10 of frag. size distance of band from well (mm) 21226 4.326 15 5148/4973 3.696 22 4268 3.630 24 3530 3.547 26 2027 3.306 33 1904 3.279 34 1709 3.232 36 1375 3.138 39 947 2.976 44 831 2.919 46 564 2.751 51 Use this to information construct a standard curve for the gel. The graph should be attached. Using the standard curve, fill in Table 2 below for the bands seen in lanes 3 (EcoRI) and 4 (HindIII) and estimate the sizes of the bands. TABLE 2. Estimations of the sizes of unknown fragments produced by digestion of DNA with the restriction enzymes EcoRI and HindIII. a) DNA digested with the restriction enzyme EcoRI. Band No. Distance from well (mm) log.10 of size size of fragment (bp) 1 (largest) 15 3.825 6683 2 20 3.725 5309 3 22 3.650 4467 4 23 3.625 4217 5 (smallest) 26 3.525 3350 b) DNA with the restriction enzyme HindIII. Band No. Distance from well (mm) log.10 of size size of fragment (bp) 1 (largest) 10 4.075 11885 2 12 3.975 9441 3 15 3.875 7499 4 18 3.8 6310 5 25 3.575 3758 6 27 3.5 3162 7 (smallest 45 2.925 841 Discussion/Questions: 1). What is the total size of the  DNA (genome) based on your digests with EcoRI and HindIII? Based on the digest with EcoRI and HindIII the total size of the  DNA (genome) was found to be 24026 bp and 42896 bp respectively. 2). Why do these two results differ from each other? The possible reasons as observed in the results of the gel separation are the errors in relative separation of distinct bands after restriction digestion. HindIII shows a total of 7 bands and EcoRI shows 5 bands as against 6 fragments which is generally seen due to the presence of five restriction sites.. 3). The known size of the  genome is 48502 bp What does this tell you about the accuracy of the method? The difference in the size of the genome calculated from the two different restriction enzymes shows errors in the method used which depends on agarose concentration, electrophoresis conditions and the quality of the gel preparation. 4). Why are the digested DNA samples more likely to give better estimates of the  genome size than simply using the information in lane 2, the undigested  DNA and the standard curve to obtain an estimate? Digested DNA samples are more likely to give better estimates of the  genome size than the undigested  DNA because smaller fragments resolve better giving distinct bands for individual fragments according to size followed by the calculation of the size of the individual bands from the standard curve which gives a more accurate size. 5). What factors contribute to the errors in this method? The agarose gel electrophoresis method depends on the agarose concentration, electrophoresis conditions anfd the quality of the gel preparation to separate distinct fragments. Errors in the method could be due to distortion of bands produced as a result of a number of factors including migration of samples off the gel, loss of sample from the well due to leakage resulting in blurred bands, smearing of the bands due to loading too much DNA or it could even be due to the fact that agarose was not completely melted before the gel was poured. Errors could also be due to temperature and pH variation as well as cross contamination of samples. 6). How many restriction sites are there in  DNA for EcoRI and HindIII respectively? N.B.  has a linear genome. Generally  DNA has five EcoRI restriction sites and HindIII has six. However according to the results observed  DNA has four EcoRI restriction sites whereas HindIII has six. Literature shows contradictory statements on the number of HindIII digest of  DNA. However it was found that wild type  DNA has six restriction sites for HindIII and standard lambda lab strain like clind 1 ts857Sam7 has seven restriction sites as a result of a single base substitution. 7). Does this agree with the number of bands known to be produced in the digest of  with EcoRI AND HindIII together i.e. the E.H standard, if not why might such a discrepancy occur? This does not agree with the the digest of  with EcoRI AND HindIII together .The linear  DNA genome generally shows six bands when digested with EcoRI and eight bands with HindIII. The discrepancy in the number of bands with EcoRI could be due to the errors in the method that might have resulted in the leakage of the sample resulting in loss of fragments or the smearing of DNA due to overloading of the sample. 8). How does the HaeIII digest of  DNA differ from that of the other two restriction enzymes? HaeIII digest of  DNA differ from the other two restriction digests in the fact that it shows numerous fragments that are not clearly resolved. 9). Why would this pattern with HaeIII be expected? Explain. The restriction site of HaeIII is 5’GGCC3’ which occurs much more frequently in the  DNA than EcoRI and HindIII restriction sites which are six nucleotides in length as against HaeIII restriction site which is only four nucleotides long. 10). What sort of pattern is shown by the digest of ML DNA with EcoRI and why is the pattern so different to that of ? The digest of ML DNA shows a blurry gel image making interpretation difficult. However the pattern is different to that of the EcoRI digest with  showing one, few or no restriction site for EcoRI in ML DNA. 11). Are there any particularly intense bands in the ML DNA digest and if so what do they represent? ML DNA digest shows one intense band which could be the ML DNA itself or a restricted fragment of the circular ML DNA 12). Challenge question. Go to the website called Webcutter at http://rna.lundberg.gu.se/cutter2/index.html This program site will cut a given piece of DNA with any enzyme you choose. Copy and Paste the DNA sequence for the λ bacteriophage into the program and cut it with the following enzymes, EcoRI, HindIII. For each enzyme say how many sites there are, the position of the sites and the sizes of the fragments after cutting. Compare your values from Table 2 above to the predicted values - How close are observed to the predicted sizes? Observed sizes are pretty close in the case of EcoRI except for the loss of a fragment of approximately 22000 bp whereas HindIII shows fragments that are close to the predicted sizes. Do the number of fragments agree? Number of fragments do not agree in the case of EcoRI. HindIII which is observed to be 7 and EcoRI observed to be 5 are predicted to be 7 and 6 respectively. How can you explain a discrepancy in the number of fragments? The discrepancy in the number of fragments could be due to the errors in the method that might have resulted in the leakage of the sample resulting in lose of fragments or the smearing of DNA due to overloading of the sample. The DNA sequence of λ bacteriophage is GGGCGGCGACCTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAAGGCGTTTCCGTTCTTCTTCG TCATAACTTAATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGAAACGACAGGTGCTGAAAGCGAGGC TTTTTGGCCTCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCAACAAAAAGCA GCTGGCTGACATTTTCGGTGCGAGTATCCGTACCATTCAGAACTGGCAGGAACAGGGAATGCCCGTTCTG CGAGGCGGTGGCAAGGGTAATGAGGTGCTTTATGACTCTGCCGCCGTCATAAAATGGTATGCCGAAAGGG ATGCTGAAATTGAGAACGAAAAGCTGCGCCGGGAGGTTGAAGAACTGCGGCAGGCCAGCGAGGCAGATCT CCAGCCAGGAACTATTGAGTACGAACGCCATCGACTTACGCGTGCGCAGGCCGACGCACAGGAACTGAAG AATGCCAGAGACTCCGCTGAAGTGGTGGAAACCGCATTCTGTACTTTCGTGCTGTCGCGGATCGCAGGTG AAATTGCCAGTATTCTCGACGGGCTCCCCCTGTCGGTGCAGCGGCGTTTTCCGGAACTGGAAAACCGACA TGTTGATTTCCTGAAACGGGATATCATCAAAGCCATGAACAAAGCAGCCGCGCTGGATGAACTGATACCG GGGTTGCTGAGTGAATATATCGAACAGTCAGGTTAACAGGCTGCGGCATTTTGTCCGCGCCGGGCTTCGC TCACTGTTCAGGCCGGAGCCACAGACCGCCGTTGAATGGGCGGATGCTAATTACTATCTCCCGAAAGAAT CCGCATACCAGGAAGGGCGCTGGGAAACACTGCCCTTTCAGCGGGCCATCATGAATGCGATGGGCAGCGA CTACATCCGTGAGGTGAATGTGGTGAAGTCTGCCCGTGTCGGTTATTCCAAAATGCTGCTGGGTGTTTAT GCCTACTTTATAGAGCATAAGCAGCGCAACACCCTTATCTGGTTGCCGACGGATGGTGATGCCGAGAACT TTATGAAAACCCACGTTGAGCCGACTATTCGTGATATTCCGTCGCTGCTGGCGCTGGCCCCGTGGTATGG CAAAAAGCACCGGGATAACACGCTCACCATGAAGCGTTTCACTAATGGGCGTGGCTTCTGGTGCCTGGGC GGTAAAGCGGCAAAAAACTACCGTGAAAAGTCGGTGGATGTGGCGGGTTATGATGAACTTGCTGCTTTTG ATGATGATATTGAACAGGAAGGCTCTCCGACGTTCCTGGGTGACAAGCGTATTGAAGGCTCGGTCTGGCC AAAGTCCATCCGTGGCTCCACGCCAAAAGTGAGAGGCACCTGTCAGATTGAGCGTGCAGCCAGTGAATCC CCGCATTTTATGCGTTTTCATGTTGCCTGCCCGCATTGCGGGGAGGAGCAGTATCTTAAATTTGGCGACA AAGAGACGCCGTTTGGCCTCAAATGGACGCCGGATGACCCCTCCAGCGTGTTTTATCTCTGCGAGCATAA TGCCTGCGTCATCCGCCAGCAGGAGCTGGACTTTACTGATGCCCGTTATATCTGCGAAAAGACCGGGATC TGGACCCGTGATGGCATTCTCTGGTTTTCGTCATCCGGTGAAGAGATTGAGCCACCTGACAGTGTGACCT TTCACATCTGGACAGCGTACAGCCCGTTCACCACCTGGGTGCAGATTGTCAAAGACTGGATGAAAACGAA AGGGGATACGGGAAAACGTAAAACCTTCGTAAACACCACGCTCGGTGAGACGTGGGAGGCGAAAATTGGC GAACGTCCGGATGCTGAAGTGATGGCAGAGCGGAAAGAGCATTATTCAGCGCCCGTTCCTGACCGTGTGG CTTACCTGACCGCCGGTATCGACTCCCAGCTGGACCGCTACGAAATGCGCGTATGGGGATGGGGGCCGGG TGAGGAAAGCTGGCTGATTGACCGGCAGATTATTATGGGCCGCCACGACGATGAACAGACGCTGCTGCGT GTGGATGAGGCCATCAATAAAACCTATACCCGCCGGAATGGTGCAGAAATGTCGATATCCCGTATCTGCT GGGATACTGGCGGGATTGACCCGACCATTGTGTATGAACGCTCGAAAAAACATGGGCTGTTCCGGGTGAT CCCCATTAAAGGGGCATCCGTCTACGGAAAGCCGGTGGCCAGCATGCCACGTAAGCGAAACAAAAACGGG GTTTACCTTACCGAAATCGGTACGGATACCGCGAAAGAGCAGATTTATAACCGCTTCACACTGACGCCGG AAGGGGATGAACCGCTTCCCGGTGCCGTTCACTTCCCGAATAACCCGGATATTTTTGATCTGACCGAAGC GCAGCAGCTGACTGCTGAAGAGCAGGTCGAAAAATGGGTGGATGGCAGGAAAAAAATACTGTGGGACAGC AAAAAGCGACGCAATGAGGCACTCGACTGCTTCGTTTATGCGCTGGCGGCGCTGCGCATCAGTATTTCCC GCTGGCAGCTGGATCTCAGTGCGCTGCTGGCGAGCCTGCAGGAAGAGGATGGTGCAGCAACCAACAAGAA AACACTGGCAGATTACGCCCGTGCCTTATCCGGAGAGGATGAATGACGCGACAGGAAGAACTTGCCGCTG CCCGTGCGGCACTGCATGACCTGATGACAGGTAAACGGGTGGCAACAGTACAGAAAGACGGACGAAGGGT GGAGTTTACGGCCACTTCCGTGTCTGACCTGAAAAAATATATTGCAGAGCTGGAAGTGCAGACCGGCATG ACACAGCGACGCAGGGGACCTGCAGGATTTTATGTATGAAAACGCCCACCATTCCCACCCTTCTGGGGCC GGACGGCATGACATCGCTGCGCGAATATGCCGGTTATCACGGCGGTGGCAGCGGATTTGGAGGGCAGTTG CGGTCGTGGAACCCACCGAGTGAAAGTGTGGATGCAGCCCTGTTGCCCAACTTTACCCGTGGCAATGCCC GCGCAGACGATCTGGTACGCAATAACGGCTATGCCGCCAACGCCATCCAGCTGCATCAGGATCATATCGT CGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGT GCCTTTTCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTG AGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTTTAACGGTGAACTGTT CGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAG CGCATCAGCAACCCGAACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGCAGATTAATGACAGCG GTGCGGCGCTGGGATATTACGTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGAT ACCCCGTGAGTTACCCGGCGGGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGACGGGCAGACT CGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGCTCGACACGCTGCAGAACACGCAGC TGCAGAGCGCCATTGTGAAGGCGATGTATGCCGCCACCATTGAGAGTGAGCTGGATACGCAGTCAGCGAT GGATTTTATTCTGGGCGCGAACAGTCAGGAGCAGCGGGAAAGGCTGACCGGCTGGATTGGTGAAATTGCC GCGTATTACGCCGCAGCGCCGGTCCGGCTGGGAGGCGCAAAAGTACCGCACCTGATGCCGGGTGACTCAC TGAACCTGCAGACGGCTCAGGATACGGATAACGGCTACTCCGTGTTTGAGCAGTCACTGCTGCGGTATAT CGCTGCCGGGCTGGGTGTCTCGTATGAGCAGCTTTCCCGGAATTACGCCCAGATGAGCTACTCCACGGCA CGGGCCAGTGCGAACGAGTCGTGGGCGTACTTTATGGGGCGGCGAAAATTCGTCGCATCCCGTCAGGCGA GCCAGATGTTTCTGTGCTGGCTGGAAGAGGCCATCGTTCGCCGCGTGGTGACGTTACCTTCAAAAGCGCG CTTCAGTTTTCAGGAAGCCCGCAGTGCCTGGGGGAACTGCGACTGGATAGGCTCCGGTCGTATGGCCATC GATGGTCTGAAAGAAGTTCAGGAAGCGGTGATGCTGATAGAAGCCGGACTGAGTACCTACGAGAAAGAGT GCGCAAAACGCGGTGACGACTATCAGGAAATTTTTGCCCAGCAGGTCCGTGAAACGATGGAGCGCCGTGC AGCCGGTCTTAAACCGCCCGCCTGGGCGGCTGCAGCATTTGAATCCGGGCTGCGACAATCAACAGAGGAG GAGAAGAGTGACAGCAGAGCTGCGTAATCTCCCGCATATTGCCAGCATGGCCTTTAATGAGCCGCTGATG CTTGAACCCGCCTATGCGCGGGTTTTCTTTTGTGCGCTTGCAGGCCAGCTTGGGATCAGCAGCCTGACGG ATGCGGTGTCCGGCGACAGCCTGACTGCCCAGGAGGCACTCGCGACGCTGGCATTATCCGGTGATGATGA CGGACCACGACAGGCCCGCAGTTATCAGGTCATGAACGGCATCGCCGTGCTGCCGGTGTCCGGCACGCTG GTCAGCCGGACGCGGGCGCTGCAGCCGTACTCGGGGATGACCGGTTACAACGGCATTATCGCCCGTCTGC AACAGGCTGCCAGCGATCCGATGGTGGACGGCATTCTGCTCGATATGGACACGCCCGGCGGGATGGTGGC GGGGGCATTTGACTGCGCTGACATCATCGCCCGTGTGCGTGACATAAAACCGGTATGGGCGCTTGCCAAC GACATGAACTGCAGTGCAGGTCAGTTGCTTGCCAGTGCCGCCTCCCGGCGTCTGGTCACGCAGACCGCCC GGACAGGCTCCATCGGCGTCATGATGGCTCACAGTAATTACGGTGCTGCGCTGGAGAAACAGGGTGTGGA AATCACGCTGATTTACAGCGGCAGCCATAAGGTGGATGGCAACCCCTACAGCCATCTTCCGGATGACGTC CGGGAGACACTGCAGTCCCGGATGGACGCAACCCGCCAGATGTTTGCGCAGAAGGTGTCGGCATATACCG GCCTGTCCGTGCAGGTTGTGCTGGATACCGAGGCTGCAGTGTACAGCGGTCAGGAGGCCATTGATGCCGG ACTGGCTGATGAACTTGTTAACAGCACCGATGCGATCACCGTCATGCGTGATGCACTGGATGCACGTAAA TCCCGTCTCTCAGGAGGGCGAATGACCAAAGAGACTCAATCAACAACTGTTTCAGCCACTGCTTCGCAGG CTGACGTTACTGACGTGGTGCCAGCGACGGAGGGCGAGAACGCCAGCGCGGCGCAGCCGGACGTGAACGC GCAGATCACCGCAGCGGTTGCGGCAGAAAACAGCCGCATTATGGGGATCCTCAACTGTGAGGAGGCTCAC GGACGCGAAGAACAGGCACGCGTGCTGGCAGAAACCCCCGGTATGACCGTGAAAACGGCCCGCCGCATTC TGGCCGCAGCACCACAGAGTGCACAGGCGCGCAGTGACACTGCGCTGGATCGTCTGATGCAGGGGGCACC GGCACCGCTGGCTGCAGGTAACCCGGCATCTGATGCCGTTAACGATTTGCTGAACACACCAGTGTAAGGG ATGTTTATGACGAGCAAAGAAACCTTTACCCATTACCAGCCGCAGGGCAACAGTGACCCGGCTCATACCG CAACCGCGCCCGGCGGATTGAGTGCGAAAGCGCCTGCAATGACCCCGCTGATGCTGGACACCTCCAGCCG TAAGCTGGTTGCGTGGGATGGCACCACCGACGGTGCTGCCGTTGGCATTCTTGCGGTTGCTGCTGACCAG ACCAGCACCACGCTGACGTTCTACAAGTCCGGCACGTTCCGTTATGAGGATGTGCTCTGGCCGGAGGCTG CCAGCGACGAGACGAAAAAACGGACCGCGTTTGCCGGAACGGCAATCAGCATCGTTTAACTTTACCCTTC ATCACTAAAGGCCGCCTGTGCGGCTTTTTTTACGGGATTTTTTTATGTCGATGTACACAACCGCCCAACT GCTGGCGGCAAATGAGCAGAAATTTAAGTTTGATCCGCTGTTTCTGCGTCTCTTTTTCCGTGAGAGCTAT CCCTTCACCACGGAGAAAGTCTATCTCTCACAAATTCCGGGACTGGTAAACATGGCGCTGTACGTTTCGC CGATTGTTTCCGGTGAGGTTATCCGTTCCCGTGGCGGCTCCACCTCTGAATTTACGCCGGGATATGTCAA GCCGAAGCATGAAGTGAATCCGCAGATGACCCTGCGTCGCCTGCCGGATGAAGATCCGCAGAATCTGGCG GACCCGGCTTACCGCCGCCGTCGCATCATCATGCAGAACATGCGTGACGAAGAGCTGGCCATTGCTCAGG TCGAAGAGATGCAGGCAGTTTCTGCCGTGCTTAAGGGCAAATACACCATGACCGGTGAAGCCTTCGATCC GGTTGAGGTGGATATGGGCCGCAGTGAGGAGAATAACATCACGCAGTCCGGCGGCACGGAGTGGAGCAAG CGTGACAAGTCCACGTATGACCCGACCGACGATATCGAAGCCTACGCGCTGAACGCCAGCGGTGTGGTGA ATATCATCGTGTTCGATCCGAAAGGCTGGGCGCTGTTCCGTTCCTTCAAAGCCGTCAAGGAGAAGCTGGA TACCCGTCGTGGCTCTAATTCCGAGCTGGAGACAGCGGTGAAAGACCTGGGCAAAGCGGTGTCCTATAAG GGGATGTATGGCGATGTGGCCATCGTCGTGTATTCCGGACAGTACGTGGAAAACGGCGTCAAAAAGAACT TCCTGCCGGACAACACGATGGTGCTGGGGAACACTCAGGCACGCGGTCTGCGCACCTATGGCTGCATTCA GGATGCGGACGCACAGCGCGAAGGCATTAACGCCTCTGCCCGTTACCCGAAAAACTGGGTGACCACCGGC GATCCGGCGCGTGAGTTCACCATGATTCAGTCAGCACCGCTGATGCTGCTGGCTGACCCTGATGAGTTCG TGTCCGTACAACTGGCGTAATCATGGCCCTTCGGGGCCATTGTTTCTCTGTGGAGGAGTCCATGACGAAA GATGAACTGATTGCCCGTCTCCGCTCGCTGGGTGAACAACTGAACCGTGATGTCAGCCTGACGGGGACGA AAGAAGAACTGGCGCTCCGTGTGGCAGAGCTGAAAGAGGAGCTTGATGACACGGATGAAACTGCCGGTCA GGACACCCCTCTCAGCCGGGAAAATGTGCTGACCGGACATGAAAATGAGGTGGGATCAGCGCAGCCGGAT ACCGTGATTCTGGATACGTCTGAACTGGTCACGGTCGTGGCACTGGTGAAGCTGCATACTGATGCACTTC ACGCCACGCGGGATGAACCTGTGGCATTTGTGCTGCCGGGAACGGCGTTTCGTGTCTCTGCCGGTGTGGC AGCCGAAATGACAGAGCGCGGCCTGGCCAGAATGCAATAACGGGAGGCGCTGTGGCTGATTTCGATAACC TGTTCGATGCTGCCATTGCCCGCGCCGATGAAACGATACGCGGGTACATGGGAACGTCAGCCACCATTAC ATCCGGTGAGCAGTCAGGTGCGGTGATACGTGGTGTTTTTGATGACCCTGAAAATATCAGCTATGCCGGA CAGGGCGTGCGCGTTGAAGGCTCCAGCCCGTCCCTGTTTGTCCGGACTGATGAGGTGCGGCAGCTGCGGC GTGGAGACACGCTGACCATCGGTGAGGAAAATTTCTGGGTAGATCGGGTTTCGCCGGATGATGGCGGAAG TTGTCATCTCTGGCTTGGACGGGGCGTACCGCCTGCCGTTAACCGTCGCCGCTGAAAGGGGGATGTATGG CCATAAAAGGTCTTGAGCAGGCCGTTGAAAACCTCAGCCGTATCAGCAAAACGGCGGTGCCTGGTGCCGC CGCAATGGCCATTAACCGCGTTGCTTCATCCGCGATATCGCAGTCGGCGTCACAGGTTGCCCGTGAGACA AAGGTACGCCGGAAACTGGTAAAGGAAAGGGCCAGGCTGAAAAGGGCCACGGTCAAAAATCCGCAGGCCA GAATCAAAGTTAACCGGGGGGATTTGCCCGTAATCAAGCTGGGTAATGCGCGGGTTGTCCTTTCGCGCCG CAGGCGTCGTAAAAAGGGGCAGCGTTCATCCCTGAAAGGTGGCGGCAGCGTGCTTGTGGTGGGTAACCGT CGTATTCCCGGCGCGTTTATTCAGCAACTGAAAAATGGCCGGTGGCATGTCATGCAGCGTGTGGCTGGGA AAAACCGTTACCCCATTGATGTGGTGAAAATCCCGATGGCGGTGCCGCTGACCACGGCGTTTAAACAAAA TATTGAGCGGATACGGCGTGAACGTCTTCCGAAAGAGCTGGGCTATGCGCTGCAGCATCAACTGAGGATG GTAATAAAGCGATGAAACATACTGAACTCCGTGCAGCCGTACTGGATGCACTGGAGAAGCATGACACCGG GGCGACGTTTTTTGATGGTCGCCCCGCTGTTTTTGATGAGGCGGATTTTCCGGCAGTTGCCGTTTATCTC ACCGGCGCTGAATACACGGGCGAAGAGCTGGACAGCGATACCTGGCAGGCGGAGCTGCATATCGAAGTTT TCCTGCCTGCTCAGGTGCCGGATTCAGAGCTGGATGCGTGGATGGAGTCCCGGATTTATCCGGTGATGAG CGATATCCCGGCACTGTCAGATTTGATCACCAGTATGGTGGCCAGCGGCTATGACTACCGGCGCGACGAT GATGCGGGCTTGTGGAGTTCAGCCGATCTGACTTATGTCATTACCTATGAAATGTGAGGACGCTATGCCT GTACCAAATCCTACAATGCCGGTGAAAGGTGCCGGGACCACCCTGTGGGTTTATAAGGGGAGCGGTGACC CTTACGCGAATCCGCTTTCAGACGTTGACTGGTCGCGTCTGGCAAAAGTTAAAGACCTGACGCCCGGCGA ACTGACCGCTGAGTCCTATGACGACAGCTATCTCGATGATGAAGATGCAGACTGGACTGCGACCGGGCAG GGGCAGAAATCTGCCGGAGATACCAGCTTCACGCTGGCGTGGATGCCCGGAGAGCAGGGGCAGCAGGCGC TGCTGGCGTGGTTTAATGAAGGCGATACCCGTGCCTATAAAATCCGCTTCCCGAACGGCACGGTCGATGT GTTCCGTGGCTGGGTCAGCAGTATCGGTAAGGCGGTGACGGCGAAGGAAGTGATCACCCGCACGGTGAAA GTCACCAATGTGGGACGTCCGTCGATGGCAGAAGATCGCAGCACGGTAACAGCGGCAACCGGCATGACCG TGACGCCTGCCAGCACCTCGGTGGTGAAAGGGCAGAGCACCACGCTGACCGTGGCCTTCCAGCCGGAGGG CGTAACCGACAAGAGCTTTCGTGCGGTGTCTGCGGATAAAACAAAAGCCACCGTGTCGGTCAGTGGTATG ACCATCACCGTGAACGGCGTTGCTGCAGGCAAGGTCAACATTCCGGTTGTATCCGGTAATGGTGAGTTTG CTGCGGTTGCAGAAATTACCGTCACCGCCAGTTAATCCGGAGAGTCAGCGATGTTCCTGAAAACCGAATC ATTTGAACATAACGGTGTGACCGTCACGCTTTCTGAACTGTCAGCCCTGCAGCGCATTGAGCATCTCGCC CTGATGAAACGGCAGGCAGAACAGGCGGAGTCAGACAGCAACCGGAAGTTTACTGTGGAAGACGCCATCA GAACCGGCGCGTTTCTGGTGGCGATGTCCCTGTGGCATAACCATCCGCAGAAGACGCAGATGCCGTCCAT GAATGAAGCCGTTAAACAGATTGAGCAGGAAGTGCTTACCACCTGGCCCACGGAGGCAATTTCTCATGCT GAAAACGTGGTGTACCGGCTGTCTGGTATGTATGAGTTTGTGGTGAATAATGCCCCTGAACAGACAGAGG ACGCCGGGCCCGCAGAGCCTGTTTCTGCGGGAAAGTGTTCGACGGTGAGCTGAGTTTTGCCCTGAAACTG GCGCGTGAGATGGGGCGACCCGACTGGCGTGCCATGCTTGCCGGGATGTCATCCACGGAGTATGCCGACT GGCACCGCTTTTACAGTACCCATTATTTTCATGATGTTCTGCTGGATATGCACTTTTCCGGGCTGACGTA CACCGTGCTCAGCCTGTTTTTCAGCGATCCGGATATGCATCCGCTGGATTTCAGTCTGCTGAACCGGCGC GAGGCTGACGAAGAGCCTGAAGATGATGTGCTGATGCAGAAAGCGGCAGGGCTTGCCGGAGGTGTCCGCT TTGGCCCGGACGGGAATGAAGTTATCCCCGCTTCCCCGGATGTGGCGGACATGACGGAGGATGACGTAAT GCTGATGACAGTATCAGAAGGGATCGCAGGAGGAGTCCGGTATGGCTGAACCGGTAGGCGATCTGGTCGT TGATTTGAGTCTGGATGCGGCCAGATTTGACGAGCAGATGGCCAGAGTCAGGCGTCATTTTTCTGGTACG GAAAGTGATGCGAAAAAAACAGCGGCAGTCGTTGAACAGTCGCTGAGCCGACAGGCGCTGGCTGCACAGA AAGCGGGGATTTCCGTCGGGCAGTATAAAGCCGCCATGCGTATGCTGCCTGCACAGTTCACCGACGTGGC CACGCAGCTTGCAGGCGGGCAAAGTCCGTGGCTGATCCTGCTGCAACAGGGGGGGCAGGTGAAGGACTCC TTCGGCGGGATGATCCCCATGTTCAGGGGGCTTGCCGGTGCGATCACCCTGCCGATGGTGGGGGCCACCT CGCTGGCGGTGGCGACCGGTGCGCTGGCGTATGCCTGGTATCAGGGCAACTCAACCCTGTCCGATTTCAA CAAAACGCTGGTCCTTTCCGGCAATCAGGCGGGACTGACGGCAGATCGTATGCTGGTCCTGTCCAGAGCC GGGCAGGCGGCAGGGCTGACGTTTAACCAGACCAGCGAGTCACTCAGCGCACTGGTTAAGGCGGGGGTAA GCGGTGAGGCTCAGATTGCGTCCATCAGCCAGAGTGTGGCGCGTTTCTCCTCTGCATCCGGCGTGGAGGT GGACAAGGTCGCTGAAGCCTTCGGGAAGCTGACCACAGACCCGACGTCGGGGCTGACGGCGATGGCTCGC CAGTTCCATAACGTGTCGGCGGAGCAGATTGCGTATGTTGCTCAGTTGCAGCGTTCCGGCGATGAAGCCG GGGCATTGCAGGCGGCGAACGAGGCCGCAACGAAAGGGTTTGATGACCAGACCCGCCGCCTGAAAGAGAA CATGGGCACGCTGGAGACCTGGGCAGACAGGACTGCGCGGGCATTCAAATCCATGTGGGATGCGGTGCTG GATATTGGTCGTCCTGATACCGCGCAGGAGATGCTGATTAAGGCAGAGGCTGCGTATAAGAAAGCAGACG ACATCTGGAATCTGCGCAAGGATGATTATTTTGTTAACGATGAAGCGCGGGCGCGTTACTGGGATGATCG TGAAAAGGCCCGTCTTGCGCTTGAAGCCGCCCGAAAGAAGGCTGAGCAGCAGACTCAACAGGACAAAAAT GCGCAGCAGCAGAGCGATACCGAAGCGTCACGGCTGAAATATACCGAAGAGGCGCAGAAGGCTTACGAAC GGCTGCAGACGCCGCTGGAGAAATATACCGCCCGTCAGGAAGAACTGAACAAGGCACTGAAAGACGGGAA AATCCTGCAGGCGGATTACAACACGCTGATGGCGGCGGCGAAAAAGGATTATGAAGCGACGCTGAAAAAG CCGAAACAGTCCAGCGTGAAGGTGTCTGCGGGCGATCGTCAGGAAGACAGTGCTCATGCTGCCCTGCTGA CGCTTCAGGCAGAACTCCGGACGCTGGAGAAGCATGCCGGAGCAAATGAGAAAATCAGCCAGCAGCGCCG GGATTTGTGGAAGGCGGAGAGTCAGTTCGCGGTACTGGAGGAGGCGGCGCAACGTCGCCAGCTGTCTGCA CAGGAGAAATCCCTGCTGGCGCATAAAGATGAGACGCTGGAGTACAAACGCCAGCTGGCTGCACTTGGCG ACAAGGTTACGTATCAGGAGCGCCTGAACGCGCTGGCGCAGCAGGCGGATAAATTCGCACAGCAGCAACG GGCAAAACGGGCCGCCATTGATGCGAAAAGCCGGGGGCTGACTGACCGGCAGGCAGAACGGGAAGCCACG GAACAGCGCCTGAAGGAACAGTATGGCGATAATCCGCTGGCGCTGAATAACGTCATGTCAGAGCAGAAAA AGACCTGGGCGGCTGAAGACCAGCTTCGCGGGAACTGGATGGCAGGCCTGAAGTCCGGCTGGAGTGAGTG GGAAGAGAGCGCCACGGACAGTATGTCGCAGGTAAAAAGTGCAGCCACGCAGACCTTTGATGGTATTGCA CAGAATATGGCGGCGATGCTGACCGGCAGTGAGCAGAACTGGCGCAGCTTCACCCGTTCCGTGCTGTCCA TGATGACAGAAATTCTGCTTAAGCAGGCAATGGTGGGGATTGTCGGGAGTATCGGCAGCGCCATTGGCGG GGCTGTTGGTGGCGGCGCATCCGCGTCAGGCGGTACAGCCATTCAGGCCGCTGCGGCGAAATTCCATTTT GCAACCGGAGGATTTACGGGAACCGGCGGCAAATATGAGCCAGCGGGGATTGTTCACCGTGGTGAGTTTG TCTTCACGAAGGAGGCAACCAGCCGGATTGGCGTGGGGAATCTTTACCGGCTGATGCGCGGCTATGCCAC CGGCGGTTATGTCGGTACACCGGGCAGCATGGCAGACAGCCGGTCGCAGGCGTCCGGGACGTTTGAGCAG AATAACCATGTGGTGATTAACAACGACGGCACGAACGGGCAGATAGGTCCGGCTGCTCTGAAGGCGGTGT ATGACATGGCCCGCAAGGGTGCCCGTGATGAAATTCAGACACAGATGCGTGATGGTGGCCTGTTCTCCGG AGGTGGACGATGAAGACCTTCCGCTGGAAAGTGAAACCCGGTATGGATGTGGCTTCGGTCCCTTCTGTAA GAAAGGTGCGCTTTGGTGATGGCTATTCTCAGCGAGCGCCTGCCGGGCTGAATGCCAACCTGAAAACGTA CAGCGTGACGCTTTCTGTCCCCCGTGAGGAGGCCACGGTACTGGAGTCGTTTCTGGAAGAGCACGGGGGC TGGAAATCCTTTCTGTGGACGCCGCCTTATGAGTGGCGGCAGATAAAGGTGACCTGCGCAAAATGGTCGT CGCGGGTCAGTATGCTGCGTGTTGAGTTCAGCGCAGAGTTTGAACAGGTGGTGAACTGATGCAGGATATC CGGCAGGAAACACTGAATGAATGCACCCGTGCGGAGCAGTCGGCCAGCGTGGTGCTCTGGGAAATCGACC TGACAGAGGTCGGTGGAGAACGTTATTTTTTCTGTAATGAGCAGAACGAAAAAGGTGAGCCGGTCACCTG GCAGGGGCGACAGTATCAGCCGTATCCCATTCAGGGGAGCGGTTTTGAACTGAATGGCAAAGGCACCAGT ACGCGCCCCACGCTGACGGTTTCTAACCTGTACGGTATGGTCACCGGGATGGCGGAAGATATGCAGAGTC TGGTCGGCGGAACGGTGGTCCGGCGTAAGGTTTACGCCCGTTTTCTGGATGCGGTGAACTTCGTCAACGG AAACAGTTACGCCGATCCGGAGCAGGAGGTGATCAGCCGCTGGCGCATTGAGCAGTGCAGCGAACTGAGC GCGGTGAGTGCCTCCTTTGTACTGTCCACGCCGACGGAAACGGATGGCGCTGTTTTTCCGGGACGTATCA TGCTGGCCAACACCTGCACCTGGACCTATCGCGGTGACGAGTGCGGTTATAGCGGTCCGGCTGTCGCGGA TGAATATGACCAGCCAACGTCCGATATCACGAAGGATAAATGCAGCAAATGCCTGAGCGGTTGTAAGTTC CGCAATAACGTCGGCAACTTTGGCGGCTTCCTTTCCATTAACAAACTTTCGCAGTAAATCCCATGACACA GACAGAATCAGCGATTCTGGCGCACGCCCGGCGATGTGCGCCAGCGGAGTCGTGCGGCTTCGTGGTAAGC ACGCCGGAGGGGGAAAGATATTTCCCCTGCGTGAATATCTCCGGTGAGCCGGAGGCTATTTCCGTATGTC GCCGGAAGACTGGCTGCAGGCAGAAATGCAGGGTGAGATTGTGGCGCTGGTCCACAGCCACCCCGGTGGT CTGCCCTGGCTGAGTGAGGCCGACCGGCGGCTGCAGGTGCAGAGTGATTTGCCGTGGTGGCTGGTCTGCC GGGGGACGATTCATAAGTTCCGCTGTGTGCCGCATCTCACCGGGCGGCGCTTTGAGCACGGTGTGACGGA CTGTTACACACTGTTCCGGGATGCTTATCATCTGGCGGGGATTGAGATGCCGGACTTTCATCGTGAGGAT GACTGGTGGCGTAACGGCCAGAATCTCTATCTGGATAATCTGGAGGCGACGGGGCTGTATCAGGTGCCGT TGTCAGCGGCACAGCCGGGCGATGTGCTGCTGTGCTGTTTTGGTTCATCAGTGCCGAATCACGCCGCAAT TTACTGCGGCGACGGCGAGCTGCTGCACCATATTCCTGAACAACTGAGCAAACGAGAGAGGTACACCGAC AAATGGCAGCGACGCACACACTCCCTCTGGCGTCACCGGGCATGGCGCGCATCTGCCTTTACGGGGATTT ACAACGATTTGGTCGCCGCATCGACCTTCGTGTGAAAACGGGGGCTGAAGCCATCCGGGCACTGGCCACA CAGCTCCCGGCGTTTCGTCAGAAACTGAGCGACGGCTGGTATCAGGTACGGATTGCCGGGCGGGACGTCA GCACGTCCGGGTTAACGGCGCAGTTACATGAGACTCTGCCTGATGGCGCTGTAATTCATATTGTTCCCAG AGTCGCCGGGGCCAAGTCAGGTGGCGTATTCCAGATTGTCCTGGGGGCTGCCGCCATTGCCGGATCATTC TTTACCGCCGGAGCCACCCTTGCAGCATGGGGGGCAGCCATTGGGGCCGGTGGTATGACCGGCATCCTGT TTTCTCTCGGTGCCAGTATGGTGCTCGGTGGTGTGGCGCAGATGCTGGCACCGAAAGCCAGAACTCCCCG TATACAGACAACGGATAACGGTAAGCAGAACACCTATTTCTCCTCACTGGATAACATGGTTGCCCAGGGC AATGTTCTGCCTGTTCTGTACGGGGAAATGCGCGTGGGGTCACGCGTGGTTTCTCAGGAGATCAGCACGG CAGACGAAGGGGACGGTGGTCAGGTTGTGGTGATTGGTCGCTGATGCAAAATGTTTTATGTGAAACCGCC TGCGGGCGGTTTTGTCATTTATGGAGCGTGAGGAATGGGTAAAGGAAGCAGTAAGGGGCATACCCCGCGC GAAGCGAAGGACAACCTGAAGTCCACGCAGTTGCTGAGTGTGATCGATGCCATCAGCGAAGGGCCGATTG AAGGTCCGGTGGATGGCTTAAAAAGCGTGCTGCTGAACAGTACGCCGGTGCTGGACACTGAGGGGAATAC CAACATATCCGGTGTCACGGTGGTGTTCCGGGCTGGTGAGCAGGAGCAGACTCCGCCGGAGGGATTTGAA TCCTCCGGCTCCGAGACGGTGCTGGGTACGGAAGTGAAATATGACACGCCGATCACCCGCACCATTACGT CTGCAAACATCGACCGTCTGCGCTTTACCTTCGGTGTACAGGCACTGGTGGAAACCACCTCAAAGGGTGA CAGGAATCCGTCGGAAGTCCGCCTGCTGGTTCAGATACAACGTAACGGTGGCTGGGTGACGGAAAAAGAC ATCACCATTAAGGGCAAAACCACCTCGCAGTATCTGGCCTCGGTGGTGATGGGTAACCTGCCGCCGCGCC CGTTTAATATCCGGATGCGCAGGATGACGCCGGACAGCACCACAGACCAGCTGCAGAACAAAACGCTCTG GTCGTCATACACTGAAATCATCGATGTGAAACAGTGCTACCCGAACACGGCACTGGTCGGCGTGCAGGTG GACTCGGAGCAGTTCGGCAGCCAGCAGGTGAGCCGTAATTATCATCTGCGCGGGCGTATTCTGCAGGTGC CGTCGAACTATAACCCGCAGACGCGGCAATACAGCGGTATCTGGGACGGAACGTTTAAACCGGCATACAG CAACAACATGGCCTGGTGTCTGTGGGATATGCTGACCCATCCGCGCTACGGCATGGGGAAACGTCTTGGT GCGGCGGATGTGGATAAATGGGCGCTGTATGTCATCGGCCAGTACTGCGACCAGTCAGTGCCGGACGGCT TTGGCGGCACGGAGCCGCGCATCACCTGTAATGCGTACCTGACCACACAGCGTAAGGCGTGGGATGTGCT CAGCGATTTCTGCTCGGCGATGCGCTGTATGCCGGTATGGAACGGGCAGACGCTGACGTTCGTGCAGGAC CGACCGTCGGATAAGACGTGGACCTATAACCGCAGTAATGTGGTGATGCCGGATGATGGCGCGCCGTTCC GCTACAGCTTCAGCGCCCTGAAGGACCGCCATAATGCCGTTGAGGTGAACTGGATTGACCCGAACAACGG CTGGGAGACGGCGACAGAGCTTGTTGAAGATACGCAGGCCATTGCCCGTTACGGTCGTAATGTTACGAAG ATGGATGCCTTTGGCTGTACCAGCCGGGGGCAGGCACACCGCGCCGGGCTGTGGCTGATTAAAACAGAAC TGCTGGAAACGCAGACCGTGGATTTCAGCGTCGGCGCAGAAGGGCTTCGCCATGTACCGGGCGATGTTAT TGAAATCTGCGATGATGACTATGCCGGTATCAGCACCGGTGGTCGTGTGCTGGCGGTGAACAGCCAGACC CGGACGCTGACGCTCGACCGTGAAATCACGCTGCCATCCTCCGGTACCGCGCTGATAAGCCTGGTTGACG GAAGTGGCAATCCGGTCAGCGTGGAGGTTCAGTCCGTCACCGACGGCGTGAAGGTAAAAGTGAGCCGTGT TCCTGACGGTGTTGCTGAATACAGCGTATGGGAGCTGAAGCTGCCGACGCTGCGCCAGCGACTGTTCCGC TGCGTGAGTATCCGTGAGAACGACGACGGCACGTATGCCATCACCGCCGTGCAGCATGTGCCGGAAAAAG AGGCCATCGTGGATAACGGGGCGCACTTTGACGGCGAACAGAGTGGCACGGTGAATGGTGTCACGCCGCC AGCGGTGCAGCACCTGACCGCAGAAGTCACTGCAGACAGCGGGGAATATCAGGTGCTGGCGCGATGGGAC ACACCGAAGGTGGTGAAGGGCGTGAGTTTCCTGCTCCGTCTGACCGTAACAGCGGACGACGGCAGTGAGC GGCTGGTCAGCACGGCCCGGACGACGGAAACCACATACCGCTTCACGCAACTGGCGCTGGGGAACTACAG GCTGACAGTCCGGGCGGTAAATGCGTGGGGGCAGCAGGGCGATCCGGCGTCGGTATCGTTCCGGATTGCC GCACCGGCAGCACCGTCGAGGATTGAGCTGACGCCGGGCTATTTTCAGATAACCGCCACGCCGCATCTTG CCGTTTATGACCCGACGGTACAGTTTGAGTTCTGGTTCTCGGAAAAGCAGATTGCGGATATCAGACAGGT TGAAACCAGCACGCGTTATCTTGGTACGGCGCTGTACTGGATAGCCGCCAGTATCAATATCAAACCGGGC CATGATTATTACTTTTATATCCGCAGTGTGAACACCGTTGGCAAATCGGCATTCGTGGAGGCCGTCGGTC GGGCGAGCGATGATGCGGAAGGTTACCTGGATTTTTTCAAAGGCAAGATAACCGAATCCCATCTCGGCAA GGAGCTGCTGGAAAAAGTCGAGCTGACGGAGGATAACGCCAGCAGACTGGAGGAGTTTTCGAAAGAGTGG AAGGATGCCAGTGATAAGTGGAATGCCATGTGGGCTGTCAAAATTGAGCAGACCAAAGACGGCAAACATT ATGTCGCGGGTATTGGCCTCAGCATGGAGGACACGGAGGAAGGCAAACTGAGCCAGTTTCTGGTTGCCGC CAATCGTATCGCATTTATTGACCCGGCAAACGGGAATGAAACGCCGATGTTTGTGGCGCAGGGCAACCAG ATATTCATGAACGACGTGTTCCTGAAGCGCCTGACGGCCCCCACCATTACCAGCGGCGGCAATCCTCCGG CCTTTTCCCTGACACCGGACGGAAAGCTGACCGCTAAAAATGCGGATATCAGTGGCAGTGTGAATGCGAA CTCCGGGACGCTCAGTAATGTGACGATAGCTGAAAACTGTACGATAAACGGTACGCTGAGGGCGGAAAAA ATCGTCGGGGACATTGTAAAGGCGGCGAGCGCGGCTTTTCCGCGCCAGCGTGAAAGCAGTGTGGACTGGC CGTCAGGTACCCGTACTGTCACCGTGACCGATGACCATCCTTTTGATCGCCAGATAGTGGTGCTTCCGCT GACGTTTCGCGGAAGTAAGCGTACTGTCAGCGGCAGGACAACGTATTCGATGTGTTATCTGAAAGTACTG ATGAACGGTGCGGTGATTTATGATGGCGCGGCGAACGAGGCGGTACAGGTGTTCTCCCGTATTGTTGACA TGCCAGCGGGTCGGGGAAACGTGATCCTGACGTTCACGCTTACGTCCACACGGCATTCGGCAGATATTCC GCCGTATACGTTTGCCAGCGATGTGCAGGTTATGGTGATTAAGAAACAGGCGCTGGGCATCAGCGTGGTC TGAGTGTGTTACAGAGGTTCGTCCGGGAACGGGCGTTTTATTATAAAACAGTGAGAGGTGAACGATGCGT AATGTGTGTATTGCCGTTGCTGTCTTTGCCGCACTTGCGGTGACAGTCACTCCGGCCCGTGCGGAAGGTG GACATGGTACGTTTACGGTGGGCTATTTTCAAGTGAAACCGGGTACATTGCCGTCGTTGTCGGGCGGGGA TACCGGTGTGAGTCATCTGAAAGGGATTAACGTGAAGTACCGTTATGAGCTGACGGACAGTGTGGGGGTG ATGGCTTCCCTGGGGTTCGCCGCGTCGAAAAAGAGCAGCACAGTGATGACCGGGGAGGATACGTTTCACT ATGAGAGCCTGCGTGGACGTTATGTGAGCGTGATGGCCGGACCGGTTTTACAAATCAGTAAGCAGGTCAG TGCGTACGCCATGGCCGGAGTGGCTCACAGTCGGTGGTCCGGCAGTACAATGGATTACCGTAAGACGGAA ATCACTCCCGGGTATATGAAAGAGACGACCACTGCCAGGGACGAAAGTGCAATGCGGCATACCTCAGTGG CGTGGAGTGCAGGTATACAGATTAATCCGGCAGCGTCCGTCGTTGTTGATATTGCTTATGAAGGCTCCGG CAGTGGCGACTGGCGTACTGACGGATTCATCGTTGGGGTCGGTTATAAATTCTGATTAGCCAGGTAACAC AGTGTTATGACAGCCCGCCGGAACCGGTGGGCTTTTTTGTGGGGTGAATATGGCAGTAAAGATTTCAGGA GTCCTGAAAGACGGCACAGGAAAACCGGTACAGAACTGCACCATTCAGCTGAAAGCCAGACGTAACAGCA CCACGGTGGTGGTGAACACGGTGGGCTCAGAGAATCCGGATGAAGCCGGGCGTTACAGCATGGATGTGGA GTACGGTCAGTACAGTGTCATCCTGCAGGTTGACGGTTTTCCACCATCGCACGCCGGGACCATCACCGTG TATGAAGATTCACAACCGGGGACGCTGAATGATTTTCTCTGTGCCATGACGGAGGATGATGCCCGGCCGG AGGTGCTGCGTCGTCTTGAACTGATGGTGGAAGAGGTGGCGCGTAACGCGTCCGTGGTGGCACAGAGTAC GGCAGACGCGAAGAAATCAGCCGGCGATGCCAGTGCATCAGCTGCTCAGGTCGCGGCCCTTGTGACTGAT GCAACTGACTCAGCACGCGCCGCCAGCACGTCCGCCGGACAGGCTGCATCGTCAGCTCAGGAAGCGTCCT CCGGCGCAGAAGCGGCATCAGCAAAGGCCACTGAAGCGGAAAAAAGTGCCGCAGCCGCAGAGTCCTCAAA AAACGCGGCGGCCACCAGTGCCGGTGCGGCGAAAACGTCAGAAACGAATGCTGCAGCGTCACAACAATCA GCCGCCACGTCTGCCTCCACCGCGGCCACGAAAGCGTCAGAGGCCGCCACTTCAGCACGAGATGCGGTGG CCTCAAAAGAGGCAGCAAAATCATCAGAAACGAACGCATCATCAAGTGCCGGTCGTGCAGCTTCCTCGGC AACGGCGGCAGAAAATTCTGCCAGGGCGGCAAAAACGTCCGAGACGAATGCCAGGTCATCTGAAACAGCA GCGGAACGGAGCGCCTCTGCCGCGGCAGACGCAAAAACAGCGGCGGCGGGGAGTGCGTCAACGGCATCCA CGAAGGCGACAGAGGCTGCGGGAAGTGCGGTATCAGCATCGCAGAGCAAAAGTGCGGCAGAAGCGGCGGC AATACGTGCAAAAAATTCGGCAAAACGTGCAGAAGATATAGCTTCAGCTGTCGCGCTTGAGGATGCGGAC ACAACGAGAAAGGGGATAGTGCAGCTCAGCAGTGCAACCAACAGCACGTCTGAAACGCTTGCTGCAACGC CAAAGGCGGTTAAGGTGGTAATGGATGAAACGAACAGAAAAGCCCACTGGACAGTCCGGCACTGACCGGA ACGCCAACAGCACCAACCGCGCTCAGGGGAACAAACAATACCCAGATTGCGAACACCGCTTTTGTACTGG CCGCGATTGCAGATGTTATCGACGCGTCACCTGACGCACTGAATACGCTGAATGAACTGGCCGCAGCGCT CGGGAATGATCCAGATTTTGCTACCACCATGACTAACGCGCTTGCGGGTAAACAACCGAAGAATGCGACA CTGACGGCGCTGGCAGGGCTTTCCACGGCGAAAAATAAATTACCGTATTTTGCGGAAAATGATGCCGCCA GCCTGACTGAACTGACTCAGGTTGGCAGGGATATTCTGGCAAAAAATTCCGTTGCAGATGTTCTTGAATA CCTTGGGGCCGGTGAGAATTCGGCCTTTCCGGCAGGTGCGCCGATCCCGTGGCCATCAGATATCGTTCCG TCTGGCTACGTCCTGATGCAGGGGCAGGCGTTTGACAAATCAGCCTACCCAAAACTTGCTGTCGCGTATC CATCGGGTGTGCTTCCTGATATGCGAGGCTGGACAATCAAGGGGAAACCCGCCAGCGGTCGTGCTGTATT GTCTCAGGAACAGGATGGAATTAAGTCGCACACCCACAGTGCCAGTGCATCCGGTACGGATTTGGGGACG AAAACCACATCGTCGTTTGATTACGGGACGAAAACAACAGGCAGTTTCGATTACGGCACCAAATCGACGA ATAACACGGGGGCTCATGCTCACAGTCTGAGCGGTTCAACAGGGGCCGCGGGTGCTCATGCCCACACAAG TGGTTTAAGGATGAACAGTTCTGGCTGGAGTCAGTATGGAACAGCAACCATTACAGGAAGTTTATCCACA GTTAAAGGAACCAGCACACAGGGTATTGCTTATTTATCGAAAACGGACAGTCAGGGCAGCCACAGTCACT CATTGTCCGGTACAGCCGTGAGTGCCGGTGCACATGCGCATACAGTTGGTATTGGTGCGCACCAGCATCC GGTTGTTATCGGTGCTCATGCCCATTCTTTCAGTATTGGTTCACACGGACACACCATCACCGTTAACGCT GCGGGTAACGCGGAAAACACCGTCAAAAACATTGCATTTAACTATATTGTGAGGCTTGCATAATGGCATT CAGAATGAGTGAACAACCACGGACCATAAAAATTTATAATCTGCTGGCCGGAACTAATGAATTTATTGGT GAAGGTGACGCATATATTCCGCCTCATACCGGTCTGCCTGCAAACAGTACCGATATTGCACCGCCAGATA TTCCGGCTGGCTTTGTGGCTGTTTTCAACAGTGATGAGGCATCGTGGCATCTCGTTGAAGACCATCGGGG TAAAACCGTCTATGACGTGGCTTCCGGCGACGCGTTATTTATTTCTGAACTCGGTCCGTTACCGGAAAAT TTTACCTGGTTATCGCCGGGAGGGGAATATCAGAAGTGGAACGGCACAGCCTGGGTGAAGGATACGGAAG CAGAAAAACTGTTCCGGATCCGGGAGGCGGAAGAAACAAAAAAAAGCCTGATGCAGGTAGCCAGTGAGCA TATTGCGCCGCTTCAGGATGCTGCAGATCTGGAAATTGCAACGAAGGAAGAAACCTCGTTGCTGGAAGCC TGGAAGAAGTATCGGGTGTTGCTGAACCGTGTTGATACATCAACTGCACCTGATATTGAGTGGCCTGCTG TCCCTGTTATGGAGTAATCGTTTTGTGATATGCCGCAGAAACGTTGTATGAAATAACGTTCTGCGGTTAG TTAGTATATTGTAAAGCTGAGTATTGGTTTATTTGGCGATTATTATCTTCAGGAGAATAATGGAAGTTCT ATGACTCAATTGTTCATAGTGTTTACATCACCGCCAATTGCTTTTAAGACTGAACGCATGAAATATGGTT TTTCGTCATGTTTTGAGTCTGCTGTTGATATTTCTAAAGTCGGTTTTTTTTCTTCGTTTTCTCTAACTAT TTTCCATGAAATACATTTTTGATTATTATTTGAATCAATTCCAATTACCTGAAGTCTTTCATCTATAATT GGCATTGTATGTATTGGTTTATTGGAGTAGATGCTTGCTTTTCTGAGCCATAGCTCTGATATCCAAATGA AGCCATAGGCATTTGTTATTTTGGCTCTGTCAGCTGCATAACGCCAAAAAATATATTTATCTGCTTGATC TTCAAATGTTGTATTGATTAAATCAATTGGATGGAATTGTTTATCATAAAAAATTAATGTTTGAATGTGA TAACCGTCCTTTAAAAAAGTCGTTTCTGCAAGCTTGGCTGTATAGTCAACTAACTCTTCTGTCGAAGTGA TATTTTTAGGCTTATCTACCAGTTTTAGACGCTCTTTAATATCTTCAGGAATTATTTTATTGTCATATTG TATCATGCTAAATGACAATTTGCTTATGGAGTAATCTTTTAATTTTAAATAAGTTATTCTCCTGGCTTCA TCAAATAAAGAGTCGAATGATGTTGGCGAAATCACATCGTCACCCATTGGATTGTTTATTTGTATGCCAA GAGAGTTACAGCAGTTATACATTCTGCCATAGATTATAGCTAAGGCATGTAATAATTCGTAATCTTTTAG CGTATTAGCGACCCATCGTCTTTCTGATTTAATAATAGATGATTCAGTTAAATATGAAGGTAATTTCTTT TGTGCAAGTCTGACTAACTTTTTTATACCAATGTTTAACATACTTTCATTTGTAATAAACTCAATGTCAT TTTCTTCAATGTAAGATGAAATAAGAGTAGCCTTTGCCTCGCTATACATTTCTAAATCGCCTTGTTTTTC TATCGTATTGCGAGAATTTTTAGCCCAAGCCATTAATGGATCATTTTTCCATTTTTCAATAACATTATTG TTATACCAAATGTCATATCCTATAATCTGGTTTTTGTTTTTTTGAATAATAAATGTTACTGTTCTTGCGG TTTGGAGGAATTGATTCAAATTCAAGCGAAATAATTCAGGGTCAAAATATGTATCAATGCAGCATTTGAG CAAGTGCGATAAATCTTTAAGTCTTCTTTCCCATGGTTTTTTAGTCATAAAACTCTCCATTTTGATAGGT TGCATGCTAGATGCTGATATATTTTAGAGGTGATAAAATTAACTGCTTAACTGTCAATGTAATACAAGTT GTTTGATCTTTGCAATGATTCTTATCAGAAACCATATAGTAAATTAGTTACACAGGAAATTTTTAATATT ATTATTATCATTCATTATGTATTAAAATTAGAGTTGTGGCTTGGCTCTGCTAACACGTTGCTCATAGGAG ATATGGTAGAGCCGCAGACACGTCGTATGCAGGAACGTGCTGCGGCTGGCTGGTGAACTTCCGATAGTGC GGGTGTTGAATGATTTCCAGTTGCTACCGATTTTACATATTTTTTGCATGAGAGAATTTGTACCACCTCC CACCGACCATCTATGACTGTACGCCACTGTCCCTAGGACTGCTATGTGCCGGAGCGGACATTACAAACGT CCTTCTCGGTGCATGCCACTGTTGCCAATGACCTGCCTAGGAATTGGTTAGCAAGTTACTACCGGATTTT GTAAAAACAGCCCTCCTCATATAAAAAGTATTCGTTCACTTCCGATAAGCGTCGTAATTTTCTATCTTTC ATCATATTCTAGATCCCTCTGAAAAAATCTTCCGAGTTTGCTAGGCACTGATACATAACTCTTTTCCAAT AATTGGGGAAGTCATTCAAATCTATAATAGGTTTCAGATTTGCTTCAATAAATTCTGACTGTAGCTGCTG AAACGTTGCGGTTGAACTATATTTCCTTATAACTTTTACGAAAGAGTTTCTTTGAGTAATCACTTCACTC AAGTGCTTCCCTGCCTCCAAACGATACCTGTTAGCAATATTTAATAGCTTGAAATGATGAAGAGCTCTGT GTTTGTCTTCCTGCCTCCAGTTCGCCGGGCATTCAACATAAAAACTGATAGCACCCGGAGTTCCGGAAAC GAAATTTGCATATACCCATTGCTCACGAAAAAAAATGTCCTTGTCGATATAGGGATGAATCGCTTGGTGT ACCTCATCTACTGCGAAAACTTGACCTTTCTCTCCCATATTGCAGTCGCGGCACGATGGAACTAAATTAA TAGGCATCACCGAAAATTCAGGATAATGTGCAATAGGAAGAAAATGATCTATATTTTTTGTCTGTCCTAT ATCACCACAAAATGGACATTTTTCACCTGATGAAACAAGCATGTCATCGTAATATGTTCTAGCGGGTTTG TTTTTATCTCGGAGATTATTTTCATAAAGCTTTTCTAATTTAACCTTTGTCAGGTTACCAACTACTAAGG TTGTAGGCTCAAGAGGGTGTGTCCTGTCGTAGGTAAATAACTGACCTGTCGAGCTTAATATTCTATATTG TTGTTCTTTCTGCAAAAAAGTGGGGAAGTGAGTAATGAAATTATTTCTAACATTTATCTGCATCATACCT TCCGAGCATTTATTAAGCATTTCGCTATAAGTTCTCGCTGGAAGAGGTAGTTTTTTCATTGTACTTTACC TTCATCTCTGTTCATTATCATCGCTTTTAAAACGGTTCGACCTTCTAATCCTATCTGACCATTATAATTT TTTAGAATGGTTTCATAAGAAAGCTCTGAATCAACGGACTGCGATAATAAGTGGTGGTATCCAGAATTTG TCACTTCAAGTAAAAACACCTCACGAGTTAAAACACCTAAGTTCTCACCGAATGTCTCAATATCCGGACG GATAATATTTATTGCTTCTCTTGACCGTAGGACTTTCCACATGCAGGATTTTGGAACCTCTTGCAGTACT ACTGGGGAATGAGTTGCAATTATTGCTACACCATTGCGTGCATCGAGTAAGTCGCTTAATGTTCGTAAAA AAGCAGAGAGCAAAGGTGGATGCAGATGAACCTCTGGTTCATCGAATAAAACTAATGACTTTTCGCCAAC GACATCTACTAATCTTGTGATAGTAAATAAAACAATTGCATGTCCAGAGCTCATTCGAAGCAGATATTTC TGGATATTGTCATAAAACAATTTAGTGAATTTATCATCGTCCACTTGAATCTGTGGTTCATTACGTCTTA ACTCTTCATATTTAGAAATGAGGCTGATGAGTTCCATATTTGAAAAGTTTTCATCACTACTTAGTTTTTT GATAGCTTCAAGCCAGAGTTGTCTTTTTCTATCTACTCTCATACAACCAATAAATGCTGAAATGAATTCT AAGCGGAGATCGCCTAGTGATTTTAAACTATTGCTGGCAGCATTCTTGAGTCCAATATAAAAGTATTGTG TACCTTTTGCTGGGTCAGGTTGTTCTTTAGGAGGAGTAAAAGGATCAAATGCACTAAACGAAACTGAAAC AAGCGATCGAAAATATCCCTTTGGGATTCTTGACTCGATAAGTCTATTATTTTCAGAGAAAAAATATTCA TTGTTTTCTGGGTTGGTGATTGCACCAATCATTCCATTCAAAATTGTTGTTTTACCACACCCATTCCGCC CGATAAAAGCATGAATGTTCGTGCTGGGCATAGAATTAACCGTCACCTCAAAAGGTATAGTTAAATCACT GAATCCGGGAGCACTTTTTCTATTAAATGAAAAGTGGAAATCTGACAATTCTGGCAAACCATTTAACACA CGTGCGAACTGTCCATGAATTTCTGAAAGAGTTACCCCTCTAAGTAATGAGGTGTTAAGGACGCTTTCAT TTTCAATGTCGGCTAATCGATTTGGCCATACTACTAAATCCTGAATAGCTTTAAGAAGGTTATGTTTAAA ACCATCGCTTAATTTGCTGAGATTAACATAGTAGTCAATGCTTTCACCTAAGGAAAAAAACATTTCAGGG AGTTGACTGAATTTTTTATCTATTAATGAATAAGTGCTTACTTCTTCTTTTTGACCTACAAAACCAATTT TAACATTTCCGATATCGCATTTTTCACCATGCTCATCAAAGACAGTAAGATAAAACATTGTAACAAAGGA ATAGTCATTCCAACCATCTGCTCGTAGGAATGCCTTATTTTTTTCTACTGCAGGAATATACCCGCCTCTT TCAATAACACTAAACTCCAACATATAGTAACCCTTAATTTTATTAAAATAACCGCAATTTATTTGGCGGC AACACAGGATCTCTCTTTTAAGTTACTCTCTATTACATACGTTTTCCATCTAAAAATTAGTAGTATTGAA CTTAACGGGGCATCGTATTGTAGTTTTCCATATTTAGCTTTCTGCTTCCTTTTGGATAACCCACTGTTAT TCATGTTGCATGGTGCACTGTTTATACCAACGATATAGTCTATTAATGCATATATAGTATCGCCGAACGA TTAGCTCTTCAGGCTTCTGAAGAAGCGTTTCAAGTACTAATAAGCCGATAGATAGCCACGGACTTCGTAG CCATTTTTCATAAGTGTTAACTTCCGCTCCTCGCTCATAACAGACATTCACTACAGTTATGGCGGAAAGG TATGCATGCTGGGTGTGGGGAAGTCGTGAAAGAAAAGAAGTCAGCTGCGTCGTTTGACATCACTGCTATC TTCTTACTGGTTATGCAGGTCGTAGTGGGTGGCACACAAAGCTTTGCACTGGATTGCGAGGCTTTGTGCT TCTCTGGAGTGCGACAGGTTTGATGACAAAAAATTAGCGCAAGAAGACAAAAATCACCTTGCGCTAATGC TCTGTTACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGACTGCATATGTTGTGTTTTACAGTA TTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGT TCAGCTTTTTTATACTAAGTTGGCATTATAAAAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCA CTATCAGTCAAAATAAAATCATTATTTGATTTCAATTTTGTCCCACTCCCTGCCTCTGTCATCACGATAC TGTGATGCCATGGTGTCCGACTTATGCCCGAGAAGATGTTGAGCAAACTTATCGCTTATCTGCTTCTCAT AGAGTCTTGCAGACAAACTGCGCAACTCGTGAAAGGTAGGCGGATCCCCTTCGAAGGAAAGACCTGATGC TTTTCGTGCGCGCATAAAATACCTTGATACTGTGCCGGATGAAAGCGGTTCGCGACGAGTAGATGCAATT ATGGTTTCTCCGCCAAGAATCTCTTTGCATTTATCAAGTGTTTCCTTCATTGATATTCCGAGAGCATCAA TATGCAATGCTGTTGGGATGGCAATTTTTACGCCTGTTTTGCTTTGCTCGACATAAAGATATCCATCTAC GATATCAGACCACTTCATTTCGCATAAATCACCAACTCGTTGCCCGGTAACAACAGCCAGTTCCATTGCA AGTCTGAGCCAACATGGTGATGATTCTGCTGCTTGATAAATTTTCAGGTATTCGTCAGCCGTAAGTCTTG ATCTCCTTACCTCTGATTTTGCTGCGCGAGTGGCAGCGACATGGTTTGTTGTTATATGGCCTTCAGCTAT TGCCTCTCGGAATGCATCGCTCAGTGTTGATCTGATTAACTTGGCTGACGCCGCCTTGCCCTCGTCTATG TATCCATTGAGCATTGCCGCAATTTCTTTTGTGGTGATGTCTTCAAGTGGAGCATCAGGCAGACCCCTCC TTATTGCTTTAATTTTGCTCATGTAATTTATGAGTGTCTTCTGCTTGATTCCTCTGCTGGCCAGGATTTT TTCGTAGCGATCAAGCCATGAATGTAACGTAACGGAATTATCACTGTTGATTCTCGCTGTCAGAGGCTTG TGTTTGTGTCCTGAAAATAACTCAATGTTGGCCTGTATAGCTTCAGTGATTGCGATTCGCCTGTCTCTGC CTAATCCAAACTCTTTACCCGTCCTTGGGTCCCTGTAGCAGTAATATCCATTGTTTCTTATATAAAGGTT AGGGGGTAAATCCCGGCGCTCATGACTTCGCCTTCTTCCCATTTCTGATCCTCTTCAAAAGGCCACCTGT TACTGGTCGATTTAAGTCAACCTTTACCGCTGATTCGTGGAACAGATACTCTCTTCCATCCTTAACCGGA GGTGGGAATATCCTGCATTCCCGAACCCATCGACGAACTGTTTCAAGGCTTCTTGGACGTCGCTGGCGTG CGTTCCACTCCTGAAGTGTCAAGTACATCGCAAAGTCTCCGCAATTACACGCAAGAAAAAACCGCCATCA GGCGGCTTGGTGTTCTTTCAGTTCTTCAATTCGAATATTGGTTACGTCTGCATGTGCTATCTGCGCCCAT ATCATCCAGTGGTCGTAGCAGTCGTTGATGTTCTCCGCTTCGATAACTCTGTTGAATGGCTCTCCATTCC ATTCTCCTGTGACTCGGAAGTGCATTTATCATCTCCATAAAACAAAACCCGCCGTAGCGAGTTCAGATAA AATAAATCCCCGCGAGTGCGAGGATTGTTATGTAATATTGGGTTTAATCATCTATATGTTTTGTACAGAG AGGGCAAGTATCGTTTCCACCGTACTCGTGATAATAATTTTGCACGGTATCAGTCATTTCTCGCACATTG CAGAATGGGGATTTGTCTTCATTAGACTTATAAACCTTCATGGAATATTTGTATGCCGACTCTATATCTA TACCTTCATCTACATAAACACCTTCGTGATGTCTGCATGGAGACAAGACACCGGATCTGCACAACATTGA TAACGCCCAATCTTTTTGCTCAGACTCTAACTCATTGATACTCATTTATAAACTCCTTGCAATGTATGTC GTTTCAGCTAAACGGTATCAGCAATGTTTATGTAAAGAAACAGTAAGATAATACTCAACCCGATGTTTGA GTACGGTCATCATCTGACACTACAGACTCTGGCATCGCTGTGAAGACGACGCGAAATTCAGCATTTTCAC AAGCGTTATCTTTTACAAAACCGATCTCACTCTCCTTTGATGCGAATGCCAGCGTCAGACATCATATGCA GATACTCACCTGCATCCTGAACCCATTGACCTCCAACCCCGTAATAGCGATGCGTAATGATGTCGATAGT TACTAACGGGTCTTGTTCGATTAACTGCCGCAGAAACTCTTCCAGGTCACCAGTGCAGTGCTTGATAACA GGAGTCTTCCCAGGATGGCGAACAACAAGAAACTGGTTTCCGTCTTCACGGACTTCGTTGCTTTCCAGTT TAGCAATACGCTTACTCCCATCCGAGATAACACCTTCGTAATACTCACGCTGCTCGTTGAGTTTTGATTT TGCTGTTTCAAGCTCAACACGCAGTTTCCCTACTGTTAGCGCAATATCCTCGTTCTCCTGGTCGCGGCGT TTGATGTATTGCTGGTTTCTTTCCCGTTCATCCAGCAGTTCCAGCACAATCGATGGTGTTACCAATTCAT GGAAAAGGTCTGCGTCAAATCCCCAGTCGTCATGCATTGCCTGCTCTGCCGCTTCACGCAGTGCCTGAGA GTTAATTTCGCTCACTTCGAACCTCTCTGTTTACTGATAAGTTCCAGATCCTCCTGGCAACTTGCACAAG TCCGACAACCCTGAACGACCAGGCGTCTTCGTTCATCTATCGGATCGCCACACTCACAACAATGAGTGGC AGATATAGCCTGGTGGTTCAGGCGGCGCATTTTTATTGCTGTGTTGCGCTGTAATTCTTCTATTTCTGAT GCTGAATCAATGATGTCTGCCATCTTTCATTAATCCCTGAACTGTTGGTTAATACGCTTGAGGGTGAATG CGAATAATAAAAAAGGAGCCTGTAGCTCCCTGATGATTTTGCTTTTCATGTTCATCGTTCCTTAAAGACG CCGTTTAACATGCCGATTGCCAGGCTTAAATGAGTCGGTGTGAATCCCATCAGCGTTACCGTTTCGCGGT GCTTCTTCAGTACGCTACGGCAAATGTCATCGACGTTTTTATCCGGAAACTGCTGTCTGGCTTTTTTTGA TTTCAGAATTAGCCTGACGGGCAATGCTGCGAAGGGCGTTTTCCTGCTGAGGTGTCATTGAACAAGTCCC ATGTCGGCAAGCATAAGCACACAGAATATGAAGCCCGCTGCCAGAAAAATGCATTCCGTGGTTGTCATAC CTGGTTTCTCTCATCTGCTTCTGCTTTCGCCACCATCATTTCCAGCTTTTGTGAAAGGGATGCGGCTAAC GTATGAAATTCTTCGTCTGTTTCTACTGGTATTGGCACAAACCTGATTCCAATTTGAGCAAGGCTATGTG CCATCTCGATACTCGTTCTTAACTCAACAGAAGATGCTTTGTGCATACAGCCCCTCGTTTATTATTTATC TCCTCAGCCAGCCGCTGTGCTTTCAGTGGATTTCGGATAACAGAAAGGCCGGGAAATACCCAGCCTCGCT TTGTAACGGAGTAGACGAAAGTGATTGCGCCTACCCGGATATTATCGTGAGGATGCGTCATCGCCATTGC TCCCCAAATACAAAACCAATTTCAGCCAGTGCCTCGTCCATTTTTTCGATGAACTCCGGCACGATCTCGT CAAAACTCGCCATGTACTTTTCATCCCGCTCAATCACGACATAATGCAGGCCTTCACGCTTCATACGCGG GTCATAGTTGGCAAAGTACCAGGCATTTTTTCGCGTCACCCACATGCTGTACTGCACCTGGGCCATGTAA GCTGACTTTATGGCCTCGAAACCACCGAGCCGGAACTTCATGAAATCCCGGGAGGTAAACGGGCATTTCA GTTCAAGGCCGTTGCCGTCACTGCATAAACCATCGGGAGAGCAGGCGGTACGCATACTTTCGTCGCGATA GATGATCGGGGATTCAGTAACATTCACGCCGGAAGTGAATTCAAACAGGGTTCTGGCGTCGTTCTCGTAC TGTTTTCCCCAGGCCAGTGCTTTAGCGTTAACTTCCGGAGCCACACCGGTGCAAACCTCAGCAAGCAGGG TGTGGAAGTAGGACATTTTCATGTCAGGCCACTTCTTTCCGGAGCGGGGTTTTGCTATCACGTTGTGAAC TTCTGAAGCGGTGATGACGCCGAGCCGTAATTTGTGCCACGCATCATCCCCCTGTTCGACAGCTCTCACA TCGATCCCGGTACGCTGCAGGATAATGTCCGGTGTCATGCTGCCACCTTCTGCTCTGCGGCTTTCTGTTT CAGGAATCCAAGAGCTTTTACTGCTTCGGCCTGTGTCAGTTCTGACGATGCACGAATGTCGCGGCGAAAT ATCTGGGAACAGAGCGGCAATAAGTCGTCATCCCATGTTTTATCCAGGGCGATCAGCAGAGTGTTAATCT CCTGCATGGTTTCATCGTTAACCGGAGTGATGTCGCGTTCCGGCTGACGTTCTGCAGTGTATGCAGTATT TTCGACAATGCGCTCGGCTTCATCCTTGTCATAGATACCAGCAAATCCGAAGGCCAGACGGGCACACTGA ATCATGGCTTTATGACGTAACATCCGTTTGGGATGCGACTGCCACGGCCCCGTGATTTCTCTGCCTTCGC GAGTTTTGAATGGTTCGCGGCGGCATTCATCCATCCATTCGGTAACGCAGATCGGATGATTACGGTCCTT GCGGTAAATCCGGCATGTACAGGATTCATTGTCCTGCTCAAAGTCCATGCCATCAAACTGCTGGTTTTCA TTGATGATGCGGGACCAGCCATCAACGCCCACCACCGGAACGATGCCATTCTGCTTATCAGGAAAGGCGT AAATTTCTTTCGTCCACGGATTAAGGCCGTACTGGTTGGCAACGATCAGTAATGCGATGAACTGCGCATC GCTGGCATCACCTTTAAATGCCGTCTGGCGAAGAGTGGTGATCAGTTCCTGTGGGTCGACAGAATCCATG CCGACACGTTCAGCCAGCTTCCCAGCCAGCGTTGCGAGTGCAGTACTCATTCGTTTTATACCTCTGAATC AATATCAACCTGGTGGTGAGCAATGGTTTCAACCATGTACCGGATGTGTTCTGCCATGCGCTCCTGAAAC TCAACATCGTCATCAAACGCACGGGTAATGGATTTTTTGCTGGCCCCGTGGCGTTGCAAATGATCGATGC ATAGCGATTCAAACAGGTGCTGGGGCAGGCCTTTTTCCATGTCGTCTGCCAGTTCTGCCTCTTTCTCTTC ACGGGCGAGCTGCTGGTAGTGACGCGCCCAGCTCTGAGCCTCAAGACGATCCTGAATGTAATAAGCGTTC ATGGCTGAACTCCTGAAATAGCTGTGAAAATATCGCCCGCGAAATGCCGGGCTGATTAGGAAAACAGGAA AGGGGGTTAGTGAATGCTTTTGCTTGATCTCAGTTTCAGTATTAATATCCATTTTTTATAAGCGTCGACG GCTTCACGAAACATCTTTTCATCGCCAATAAAAGTGGCGATAGTGAATTTAGTCTGGATAGCCATAAGTG TTTGATCCATTCTTTGGGACTCCTGGCTGATTAAGTATGTCGATAAGGCGTTTCCATCCGTCACGTAATT TACGGGTGATTCGTTCAAGTAAAGATTCGGAAGGGCAGCCAGCAACAGGCCACCCTGCAATGGCATATTG CATGGTGTGCTCCTTATTTATACATAACGAAAAACGCCTCGAGTGAAGCGTTATTGGTATGCGGTAAAAC CGCACTCAGGCGGCCTTGATAGTCATATCATCTGAATCAAATATTCCTGATGTATCGATATCGGTAATTC TTATTCCTTCGCTACCATCCATTGGAGGCCATCCTTCCTGACCATTTCCATCATTCCAGTCGAACTCACA CACAACACCATATGCATTTAAGTCGCTTGAAATTGCTATAAGCAGAGCATGTTGCGCCAGCATGATTAAT ACAGCATTTAATACAGAGCCGTGTTTATTGAGTCGGTATTCAGAGTCTGACCAGAAATTATTAATCTGGT GAAGTTTTTCCTCTGTCATTACGTCATGGTCGATTTCAATTTCTATTGATGCTTTCCAGTCGTAATCAAT GATGTATTTTTTGATGTTTGACATCTGTTCATATCCTCACAGATAAAAAATCGCCCTCACACTGGAGGGC AAAGAAGATTTCCAATAATCAGAACAAGTCGGCTCCTGTTTAGTTACGAGCGACATTGCTCCGTGTATTC ACTCGTTGGAATGAATACACAGTGCAGTGTTTATTCTGTTATTTATGCCAAAAATAAAGGCCACTATCAG GCAGCTTTGTTGTTCTGTTTACCAAGTTCTCTGGCAATCATTGCCGTCGTTCGTATTGCCCATTTATCGA CATATTTCCCATCTTCCATTACAGGAAACATTTCTTCAGGCTTAACCATGCATTCCGATTGCAGCTTGCA TCCATTGCATCGCTTGAATTGTCCACACCATTGATTTTTATCAATAGTCGTAGTCATACGGATAGTCCTG GTATTGTTCCATCACATCCTGAGGATGCTCTTCGAACTCTTCAAATTCTTCTTCCATATATCACCTTAAA TAGTGGATTGCGGTAGTAAAGATTGTGCCTGTCTTTTAACCACATCAGGCTCGGTGGTTCTCGTGTACCC CTACAGCGAGAAATCGGATAAACTATTACAACCCCTACAGTTTGATGAGTATAGAAATGGATCCACTCGT TATTCTCGGACGAGTGTTCAGTAATGAACCTCTGGAGAGAACCATGTATATGATCGTTATCTGGGTTGGA CTTCTGCTTTTAAGCCCAGATAACTGGCCTGAATATGTTAATGAGAGAATCGGTATTCCTCATGTGTGGC ATGTTTTCGTCTTTGCTCTTGCATTTTCGCTAGCAATTAATGTGCATCGATTATCAGCTATTGCCAGCGC CAGATATAAGCGATTTAAGCTAAGAAAACGCATTAAGATGCAAAACGATAAAGTGCGATCAGTAATTCAA AACCTTACAGAAGAGCAATCTATGGTTTTGTGCGCAGCCCTTAATGAAGGCAGGAAGTATGTGGTTACAT CAAAACAATTCCCATACATTAGTGAGTTGATTGAGCTTGGTGTGTTGAACAAAACTTTTTCCCGATGGAA TGGAAAGCATATATTATTCCCTATTGAGGATATTTACTGGACTGAATTAGTTGCCAGCTATGATCCATAT AATATTGAGATAAAGCCAAGGCCAATATCTAAGTAACTAGATAAGAGGAATCGATTTTCCCTTAATTTTC TGGCGTCCACTGCATGTTATGCCGCGTTCGCCAGGCTTGCTGTACCATGTGCGCTGATTCTTGCGCTCAA TACGTTGCAGGTTGCTTTCAATCTGTTTGTGGTATTCAGCCAGCACTGTAAGGTCTATCGGATTTAGTGC GCTTTCTACTCGTGATTTCGGTTTGCGATTCAGCGAGAGAATAGGGCGGTTAACTGGTTTTGCGCTTACC CCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTT GTGCATCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAAC CCCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACA CACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATG GTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCGCCAGAGAT AATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTT GGTAGGTGAGAGATCTGAATTGCTATGTTTAGTGAGTTGTATCTATTTATTTTTCAATAAATACAATTGG TTATGTGTTTTGGGGGCGATCGTGAGGCAAAGAAAACCCGGCGCTGAGGCCGGGTTATTCTTGTTCTCTG GTCAAATTATATAGTTGGAAAACAAGGATGCATATATGAATGAACGATGCAGAGGCAATGCCGATGGCGA TAGTGGGTATCATGTAGCCGCTTATGCTGGAAAGAAGCAATAACCCGCAGAAAAACAAAGCTCCAAGCTC AACAAAACTAAGGGCATAGACAATAACTACCGATGTCATATACCCATACTCTCTAATCTTGGCCAGTCGG CGCGTTCTGCTTCCGATTAGAAACGTCAAGGCAGCAATCAGGATTGCAATCATGGTTCCTGCATATGATG ACAATGTCGCCCCAAGACCATCTCTATGAGCTGAAAAAGAAACACCAGGAATGTAGTGGCGGAAAAGGAG ATAGCAAATGCTTACGATAACGTAAGGAATTATTACTATGTAAACACCAGGCATGATTCTGTTCCGCATA ATTACTCCTGATAATTAATCCTTAACTTTGCCCACCTGCCTTTTAAAACATTCCAGTATATCACTTTTCA TTCTTGCGTAGCAATATGCCATCTCTTCAGCTATCTCAGCATTGGTGACCTTGTTCAGAGGCGCTGAGAG ATGGCCTTTTTCTGATAGATAATGTTCTGTTAAAATATCTCCGGCCTCATCTTTTGCCCGCAGGCTAATG TCTGAAAATTGAGGTGACGGGTTAAAAATAATATCCTTGGCAACCTTTTTTATATCCCTTTTAAATTTTG GCTTAATGACTATATCCAATGAGTCAAAAAGCTCCCCTTCAATATCTGTTGCCCCTAAGACCTTTAATAT ATCGCCAAATACAGGTAGCTTGGCTTCTACCTTCACCGTTGTTCGGCCGATGAAATGCATATGCATAACA TCGTCTTTGGTGGTTCCCCTCATCAGTGGCTCTATCTGAACGCGCTCTCCACTGCTTAATGACATTCCTT TCCCGATTAAAAAATCTGTCAGATCGGATGTGGTCGGCCCGAAAACAGTTCTGGCAAAACCAATGGTGTC GCCTTCAACAAACAAAAAAGATGGGAATCCCAATGATTCGTCATCTGCGAGGCTGTTCTTAATATCTTCA ACTGAAGCTTTAGAGCGATTTATCTTCTGAACCAGACTCTTGTCATTTGTTTTGGTAAAGAGAAAAGTTT TTCCATCGATTTTATGAATATACAAATAATTGGAGCCAACCTGCAGGTGATGATTATCAGCCAGCAGAGA ATTAAGGAAAACAGACAGGTTTATTGAGCGCTTATCTTTCCCTTTATTTTTGCTGCGGTAAGTCGCATAA AAACCATTCTTCATAATTCAATCCATTTACTATGTTATGTTCTGAGGGGAGTGAAAATTCCCCTAATTCG ATGAAGATTCTTGCTCAATTGTTATCAGCTATGCGCCGACCAGAACACCTTGCCGATCAGCCAAACGTCT CTTCAGGCCACTGACTAGCGATAACTTTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTGGGTA CTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCTATCCCTGATCAGTTTCTTGAAGGTAAACTCATCA CCCCCAAGTCTGGCTATGCAGAAATCACCTGGCTCAACAGCCTGCTCAGGGTCAACGAGAATTAACATTC CGTCAGGAAAGCTTGGCTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACCTCAAGCCAGAATGC AGAATCACTGGCTTTTTTGGTTGTGCTTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGCTCA GGTGAGAACATCCCTGCCTGAACATGAGAAAAAACAGGGTACTCATACTCACTTCTAAGTGACGGCTGCA TACTAACCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGGGCTAAATTCTTCAACGCTAACTTT GAGAATTTTTGCAAGCAATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAATAAAGCACCAACG CCTGACTGCCCCATCCCCATCTTGTCTGCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTTTT CATAAATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGCTCTTGTGTTAATGGTTTCTTTTTTGTGCTCAT ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGT GATAATGGTTGCATGTACTAAGGAGGTTGTATGGAACAACGCATAACCCTGAAAGATTATGCAATGCGCT TTGGGCAAACCAAGACAGCTAAAGATCTCGGCGTATATCAAAGCGCGATCAACAAGGCCATTCATGCAGG CCGAAAGATTTTTTTAACTATAAACGCTGATGGAAGCGTTTATGCGGAAGAGGTAAAGCCCTTCCCGAGT AACAAAAAAACAACAGCATAAATAACCCCGCTCTTACACATTCCAGCCCTGAAAAAGGGCATCAAATTAA ACCACACCTATGGTGTATGCATTTATTTGCATACATTCAATCAATTGTTATCTAAGGAAATACTTACATA TGGTTCGTGCAAACAAACGCAACGAGGCTCTACGAATCGAGAGTGCGTTGCTTAACAAAATCGCAATGCT TGGAACTGAGAAGACAGCGGAAGCTGTGGGCGTTGATAAGTCGCAGATCAGCAGGTGGAAGAGGGACTGG ATTCCAAAGTTCTCAATGCTGCTTGCTGTTCTTGAATGGGGGGTCGTTGACGACGACATGGCTCGATTGG CGCGACAAGTTGCTGCGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCA GATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCATTATGACAAATACAGCAAAAATACTCA ACTTCGGCAGAGGTAACTTTGCCGGACAGGAGCGTAATGTGGCAGATCTCGATGATGGTTACGCCAGACT ATCAAATATGCTGCTTGAGGCTTATTCGGGCGCAGATCTGACCAAGCGACAGTTTAAAGTGCTGCTTGCC ATTCTGCGTAAAACCTATGGGTGGAATAAACCAATGGACAGAATCACCGATTCTCAACTTAGCGAGATTA CAAAGTTACCTGTCAAACGGTGCAATGAAGCCAAGTTAGAACTCGTCAGAATGAATATTATCAAGCAGCA AGGCGGCATGTTTGGACCAAATAAAAACATCTCAGAATGGTGCATCCCTCAAAACGAGGGAAAATCCCCT AAAACGAGGGATAAAACATCCCTCAAATTGGGGGATTGCTATCCCTCAAAACAGGGGGACACAAAAGACA CTATTACAAAAGAAAAAAGAAAAGATTATTCGTCAGAGAATTCTGGCGAATCCTCTGACCAGCCAGAAAA CGACCTTTCTGTGGTGAAACCGGATGCTGCAATTCAGAGCGGCAGCAAGTGGGGGACAGCAGAAGACCTG ACCGCCGCAGAGTGGATGTTTGACATGGTGAAGACTATCGCACCATCAGCCAGAAAACCGAATTTTGCTG GGTGGGCTAACGATATCCGCCTGATGCGTGAACGTGACGGACGTAACCACCGCGACATGTGTGTGCTGTT CCGCTGGGCATGCCAGGACAACTTCTGGTCCGGTAACGTGCTGAGCCCGGCCAAACTCCGCGATAAGTGG ACCCAACTCGAAATCAACCGTAACAAGCAACAGGCAGGCGTGACAGCCAGCAAACCAAAACTCGACCTGA CAAACACAGACTGGATTTACGGGGTGGATCTATGAAAAACATCGCCGCACAGATGGTTAACTTTGACCGT GAGCAGATGCGTCGGATCGCCAACAACATGCCGGAACAGTACGACGAAAAGCCGCAGGTACAGCAGGTAG CGCAGATCATCAACGGTGTGTTCAGCCAGTTACTGGCAACTTTCCCGGCGAGCCTGGCTAACCGTGACCA GAACGAAGTGAACGAAATCCGTCGCCAGTGGGTTCTGGCTTTTCGGGAAAACGGGATCACCACGATGGAA CAGGTTAACGCAGGAATGCGCGTAGCCCGTCGGCAGAATCGACCATTTCTGCCATCACCCGGGCAGTTTG TTGCATGGTGCCGGGAAGAAGCATCCGTTACCGCCGGACTGCCAAACGTCAGCGAGCTGGTTGATATGGT TTACGAGTATTGCCGGAAGCGAGGCCTGTATCCGGATGCGGAGTCTTATCCGTGGAAATCAAACGCGCAC TACTGGCTGGTTACCAACCTGTATCAGAACATGCGGGCCAATGCGCTTACTGATGCGGAATTACGCCGTA AGGCCGCAGATGAGCTTGTCCATATGACTGCGAGAATTAACCGTGGTGAGGCGATCCCTGAACCAGTAAA ACAACTTCCTGTCATGGGCGGTAGACCTCTAAATCGTGCACAGGCTCTGGCGAAGATCGCAGAAATCAAA GCTAAGTTCGGACTGAAAGGAGCAAGTGTATGACGGGCAAAGAGGCAATTATTCATTACCTGGGGACGCA TAATAGCTTCTGTGCGCCGGACGTTGCCGCGCTAACAGGCGCAACAGTAACCAGCATAAATCAGGCCGCG GCTAAAATGGCACGGGCAGGTCTTCTGGTTATCGAAGGTAAGGTCTGGCGAACGGTGTATTACCGGTTTG CTACCAGGGAAGAACGGGAAGGAAAGATGAGCACGAACCTGGTTTTTAAGGAGTGTCGCCAGAGTGCCGC GATGAAACGGGTATTGGCGGTATATGGAGTTAAAAGATGACCATCTACATTACTGAGCTAATAACAGGCC TGCTGGTAATCGCAGGCCTTTTTATTTGGGGGAGAGGGAAGTCATGAAAAAACTAACCTTTGAAATTCGA TCTCCAGCACATCAGCAAAACGCTATTCACGCAGTACAGCAAATCCTTCCAGACCCAACCAAACCAATCG TAGTAACCATTCAGGAACGCAACCGCAGCTTAGACCAAAACAGGAAGCTATGGGCCTGCTTAGGTGACGT CTCTCGTCAGGTTGAATGGCATGGTCGCTGGCTGGATGCAGAAAGCTGGAAGTGTGTGTTTACCGCAGCA TTAAAGCAGCAGGATGTTGTTCCTAACCTTGCCGGGAATGGCTTTGTGGTAATAGGCCAGTCAACCAGCA GGATGCGTGTAGGCGAATTTGCGGAGCTATTAGAGCTTATACAGGCATTCGGTACAGAGCGTGGCGTTAA GTGGTCAGACGAAGCGAGACTGGCTCTGGAGTGGAAAGCGAGATGGGGAGACAGGGCTGCATGATAAATG TCGTTAGTTTCTCCGGTGGCAGGACGTCAGCATATTTGCTCTGGCTAATGGAGCAAAAGCGACGGGCAGG TAAAGACGTGCATTACGTTTTCATGGATACAGGTTGTGAACATCCAATGACATATCGGTTTGTCAGGGAA GTTGTGAAGTTCTGGGATATACCGCTCACCGTATTGCAGGTTGATATCAACCCGGAGCTTGGACAGCCAA ATGGTTATACGGTATGGGAACCAAAGGATATTCAGACGCGAATGCCTGTTCTGAAGCCATTTATCGATAT GGTAAAGAAATATGGCACTCCATACGTCGGCGGCGCGTTCTGCACTGACAGATTAAAACTCGTTCCCTTC ACCAAATACTGTGATGACCATTTCGGGCGAGGGAATTACACCACGTGGATTGGCATCAGAGCTGATGAAC CGAAGCGGCTAAAGCCAAAGCCTGGAATCAGATATCTTGCTGAACTGTCAGACTTTGAGAAGGAAGATAT CCTCGCATGGTGGAAGCAACAACCATTCGATTTGCAAATACCGGAACATCTCGGTAACTGCATATTCTGC ATTAAAAAATCAACGCAAAAAATCGGACTTGCCTGCAAAGATGAGGAGGGATTGCAGCGTGTTTTTAATG AGGTCATCACGGGATCCCATGTGCGTGACGGACATCGGGAAACGCCAAAGGAGATTATGTACCGAGGAAG AATGTCGCTGGACGGTATCGCGAAAATGTATTCAGAAAATGATTATCAAGCCCTGTATCAGGACATGGTA CGAGCTAAAAGATTCGATACCGGCTCTTGTTCTGAGTCATGCGAAATATTTGGAGGGCAGCTTGATTTCG ACTTCGGGAGGGAAGCTGCATGATGCGATGTTATCGGTGCGGTGAATGCAAAGAAGATAACCGCTTCCGA CCAAATCAACCTTACTGGAATCGATGGTGTCTCCGGTGTGAAAGAACACCAACAGGGGTGTTACCACTAC CGCAGGAAAAGGAGGACGTGTGGCGAGACAGCGACGAAGTATCACCGACATAATCTGCGAAAACTGCAAA TACCTTCCAACGAAACGCACCAGAAATAAACCCAAGCCAATCCCAAAAGAATCTGACGTAAAAACCTTCA ACTACACGGCTCACCTGTGGGATATCCGGTGGCTAAGACGTCGTGCGAGGAAAACAAGGTGATTGACCAA AATCGAAGTTACGAACAAGAAAGCGTCGAGCGAGCTTTAACGTGCGCTAACTGCGGTCAGAAGCTGCATG TGCTGGAAGTTCACGTGTGTGAGCACTGCTGCGCAGAACTGATGAGCGATCCGAATAGCTCGATGCACGA GGAAGAAGATGATGGCTAAACCAGCGCGAAGACGATGTAAAAACGATGAATGCCGGGAATGGTTTCACCC TGCATTCGCTAATCAGTGGTGGTGCTCTCCAGAGTGTGGAACCAAGATAGCACTCGAACGACGAAGTAAA GAACGCGAAAAAGCGGAAAAAGCAGCAGAGAAGAAACGACGACGAGAGGAGCAGAAACAGAAAGATAAAC TTAAGATTCGAAAACTCGCCTTAAAGCCCCGCAGTTACTGGATTAAACAAGCCCAACAAGCCGTAAACGC CTTCATCAGAGAAAGAGACCGCGACTTACCATGTATCTCGTGCGGAACGCTCACGTCTGCTCAGTGGGAT GCCGGACATTACCGGACAACTGCTGCGGCACCTCAACTCCGATTTAATGAACGCAATATTCACAAGCAAT GCGTGGTGTGCAACCAGCACAAAAGCGGAAATCTCGTTCCGTATCGCGTCGAACTGATTAGCCGCATCGG GCAGGAAGCAGTAGACGAAATCGAATCAAACCATAACCGCCATCGCTGGACTATCGAAGAGTGCAAGGCG ATCAAGGCAGAGTACCAACAGAAACTCAAAGACCTGCGAAATAGCAGAAGTGAGGCCGCATGACGTTCTC AGTAAAAACCATTCCAGACATGCTCGTTGAAGCATACGGAAATCAGACAGAAGTAGCACGCAGACTGAAA TGTAGTCGCGGTACGGTCAGAAAATACGTTGATGATAAAGACGGGAAAATGCACGCCATCGTCAACGACG TTCTCATGGTTCATCGCGGATGGAGTGAAAGAGATGCGCTATTACGAAAAAATTGATGGCAGCAAATACC GAAATATTTGGGTAGTTGGCGATCTGCACGGATGCTACACGAACCTGATGAACAAACTGGATACGATTGG ATTCGACAACAAAAAAGACCTGCTTATCTCGGTGGGCGATTTGGTTGATCGTGGTGCAGAGAACGTTGAA TGCCTGGAATTAATCACATTCCCCTGGTTCAGAGCTGTACGTGGAAACCATGAGCAAATGATGATTGATG GCTTATCAGAGCGTGGAAACGTTAATCACTGGCTGCTTAATGGCGGTGGCTGGTTCTTTAATCTCGATTA CGACAAAGAAATTCTGGCTAAAGCTCTTGCCCATAAAGCAGATGAACTTCCGTTAATCATCGAACTGGTG AGCAAAGATAAAAAATATGTTATCTGCCACGCCGATTATCCCTTTGACGAATACGAGTTTGGAAAGCCAG TTGATCATCAGCAGGTAATCTGGAACCGCGAACGAATCAGCAACTCACAAAACGGGATCGTGAAAGAAAT CAAAGGCGCGGACACGTTCATCTTTGGTCATACGCCAGCAGTGAAACCACTCAAGTTTGCCAACCAAATG TATATCGATACCGGCGCAGTGTTCTGCGGAAACCTAACATTGATTCAGGTACAGGGAGAAGGCGCATGAG ACTCGAAAGCGTAGCTAAATTTCATTCGCCAAAAAGCCCGATGATGAGCGACTCACCACGGGCCACGGCT TCTGACTCTCTTTCCGGTACTGATGTGATGGCTGCTATGGGGATGGCGCAATCACAAGCCGGATTCGGTA TGGCTGCATTCTGCGGTAAGCACGAACTCAGCCAGAACGACAAACAAAAGGCTATCAACTATCTGATGCA ATTTGCACACAAGGTATCGGGGAAATACCGTGGTGTGGCAAAGCTTGAAGGAAATACTAAGGCAAAGGTA CTGCAAGTGCTCGCAACATTCGCTTATGCGGATTATTGCCGTAGTGCCGCGACGCCGGGGGCAAGATGCA GAGATTGCCATGGTACAGGCCGTGCGGTTGATATTGCCAAAACAGAGCTGTGGGGGAGAGTTGTCGAGAA AGAGTGCGGAAGATGCAAAGGCGTCGGCTATTCAAGGATGCCAGCAAGCGCAGCATATCGCGCTGTGACG ATGCTAATCCCAAACCTTACCCAACCCACCTGGTCACGCACTGTTAAGCCGCTGTATGACGCTCTGGTGG TGCAATGCCACAAAGAAGAGTCAATCGCAGACAACATTTTGAATGCGGTCACACGTTAGCAGCATGATTG CCACGGATGGCAACATATTAACGGCATGATATTGACTTATTGAATAAAATTGGGTAAATTTGACTCAACG ATGGGTTAATTCGCTCGTTGTGGTAGTGAGATGAAAAGAGGCGGCGCTTACTACCGATTCCGCCTAGTTG GTCACTTCGACGTATCGTCTGGAACTCCAACCATCGCAGGCAGAGAGGTCTGCAAAATGCAATCCCGAAA CAGTTCGCAGGTAATAGTTAGAGCCTGCATAACGGTTTCGGGATTTTTTATATCTGCACAACAGGTAAGA GCATTGAGTCGATAATCGTGAAGAGTCGGCGAGCCTGGTTAGCCAGTGCTCTTTCCGTTGTGCTGAATTA AGCGAATACCGGAAGCAGAACCGGATCACCAAATGCGTACAGGCGTCATCGCCGCCCAGCAACAGCACAA CCCAAACTGAGCCGTAGCCACTGTCTGTCCTGAATTCATTAGTAATAGTTACGCTGCGGCCTTTTACACA TGACCTTCGTGAAAGCGGGTGGCAGGAGGTCGCGCTAACAACCTCCTGCCGTTTTGCCCGTGCATATCGG TCACGAACAAATCTGATTACTAAACACAGTAGCCTGGATTTGTTCTATCAGTAATCGACCTTATTCCTAA TTAAATAGAGCAAATCCCCTTATTGGGGGTAAGACATGAAGATGCCAGAAAAACATGACCTGTTGGCCGC CATTCTCGCGGCAAAGGAACAAGGCATCGGGGCAATCCTTGCGTTTGCAATGGCGTACCTTCGCGGCAGA TATAATGGCGGTGCGTTTACAAAAACAGTAATCGACGCAACGATGTGCGCCATTATCGCCTGGTTCATTC GTGACCTTCTCGACTTCGCCGGACTAAGTAGCAATCTCGCTTATATAACGAGCGTGTTTATCGGCTACAT CGGTACTGACTCGATTGGTTCGCTTATCAAACGCTTCGCTGCTAAAAAAGCCGGAGTAGAAGATGGTAGA AATCAATAATCAACGTAAGGCGTTCCTCGATATGCTGGCGTGGTCGGAGGGAACTGATAACGGACGTCAG AAAACCAGAAATCATGGTTATGACGTCATTGTAGGCGGAGAGCTATTTACTGATTACTCCGATCACCCTC GCAAACTTGTCACGCTAAACCCAAAACTCAAATCAACAGGCGCCGGACGCTACCAGCTTCTTTCCCGTTG GTGGGATGCCTACCGCAAGCAGCTTGGCCTGAAAGACTTCTCTCCGAAAAGTCAGGACGCTGTGGCATTG CAGCAGATTAAGGAGCGTGGCGCTTTACCTATGATTGATCGTGGTGATATCCGTCAGGCAATCGACCGTT GCAGCAATATCTGGGCTTCACTGCCGGGCGCTGGTTATGGTCAGTTCGAGCATAAGGCTGACAGCCTGAT TGCAAAATTCAAAGAAGCGGGCGGAACGGTCAGAGAGATTGATGTATGAGCAGAGTCACCGCGATTATCT CCGCTCTGGTTATCTGCATCATCGTCTGCCTGTCATGGGCTGTTAATCATTACCGTGATAACGCCATTAC CTACAAAGCCCAGCGCGACAAAAATGCCAGAGAACTGAAGCTGGCGAACGCGGCAATTACTGACATGCAG ATGCGTCAGCGTGATGTTGCTGCGCTCGATGCAAAATACACGAAGGAGTTAGCTGATGCTAAAGCTGAAA ATGATGCTCTGCGTGATGATGTTGCCGCTGGTCGTCGTCGGTTGCACATCAAAGCAGTCTGTCAGTCAGT GCGTGAAGCCACCACCGCCTCCGGCGTGGATAATGCAGCCTCCCCCCGACTGGCAGACACCGCTGAACGG GATTATTTCACCCTCAGAGAGAGGCTGATCACTATGCAAAAACAACTGGAAGGAACCCAGAAGTATATTA ATGAGCAGTGCAGATAGAGTTGCCCATATCGATGGGCAACTCATGCAATTATTGTGAGCAATACACACGC GCTTCCAGCGGAGTATAAATGCCTAAAGTAATAAAACCGAGCAATCCATTTACGAATGTTTGCTGGGTTT CTGTTTTAACAACATTTTCTGCGCCGCCACAAATTTTGGCTGCATCGACAGTTTTCTTCTGCCCAATTCC AGAAACGAAGAAATGATGGGTGATGGTTTCCTTTGGTGCTACTGCTGCCGGTTTGTTTTGAACAGTAAAC GTCTGTTGAGCACATCCTGTAATAAGCAGGGCCAGCGCAGTAGCGAGTAGCATTTTTTTCATGGTGTTAT TCCCGATGCTTTTTGAAGTTCGCAGAATCGTATGTGTAGAAAATTAAACAAACCCTAAACAATGAGTTGA AATTTCATATTGTTAATATTTATTAATGTATGTCAGGTGCGATGAATCGTCATTGTATTCCCGGATTAAC TATGTCCACAGCCCTGACGGGGAACTTCTCTGCGGGAGTGTCCGGGAATAATTAAAACGATGCACACAGG GTTTAGCGCGTACACGTATTGCATTATGCCAACGCCCCGGTGCTGACACGGAAGAAACCGGACGTTATGA TTTAGCGTGGAAAGATTTGTGTAGTGTTCTGAATGCTCTCAGTAAATAGTAATGAATTATCAAAGGTATA GTAATATCTTTTATGTTCATGGATATTTGTAACCCATCGGAAAACTCCTGCTTTAGCAAGATTTTCCCTG TATTGCTGAAATGTGATTTCTCTTGATTTCAACCTATCATAGGACGTTTCTATAAGATGCGTGTTTCTTG AGAATTTAACATTTACAACCTTTTTAAGTCCTTTTATTAACACGGTGTTATCGTTTTCTAACACGATGTG AATATTATCTGTGGCTAGATAGTAAATATAATGTGAGACGTTGTGACGTTTTAGTTCAGAATAAAACAAT TCACAGTCTAAATCTTTTCGCACTTGATCGAATATTTCTTTAAAAATGGCAACCTGAGCCATTGGTAAAA CCTTCCATGTGATACGAGGGCGCGTAGTTTGCATTATCGTTTTTATCGTTTCAATCTGGTCTGACCTCCT TGTGTTTTGTTGATGATTTATGTCAAATATTAGGAATGTTTTCACTTAATAGTATTGGTTGCGTAACAAA GTGCGGTCCTGCTGGCATTCTGGAGGGAAATACAACCGACAGATGTATGTAAGGCCAACGTGCTCAAATC TTCATACAGAAAGATTTGAAGTAATATTTTAACCGCTAGATGAAGAGCAAGCGCATGGAGCGACAAAATG AATAAAGAACAATCTGCTGATGATCCCTCCGTGGATCTGATTCGTGTAAAAAATATGCTTAATAGCACCA TTTCTATGAGTTACCCTGATGTTGTAATTGCATGTATAGAACATAAGGTGTCTCTGGAAGCATTCAGAGC AATTGAGGCAGCGTTGGTGAAGCACGATAATAATATGAAGGATTATTCCCTGGTGGTTGACTGATCACCA TAACTGCTAATCATTCAAACTATTTAGTCTGTGACAGAGCCAACACGCAGTCTGTCACTGTCAGGAAAGT GGTAAAACTGCAACTCAATTACTGCAATGCCCTCGTAATTAAGTGAATTTACAATATCGTCCTGTTCGGA GGGAAGAACGCGGGATGTTCATTCTTCATCACTTTTAATTGATGTATATGCTCTCTTTTCTGACGTTAGT CTCCGACGGCAGGCTTCAATGACCCAGGCTGAGAAATTCCCGGACCCTTTTTGCTCAAGAGCGATGTTAA TTTGTTCAATCATTTGGTTAGGAAAGCGGATGTTGCGGGTTGTTGTTCTGCGGGTTCTGTTCTTCGTTGA CATGAGGTTGCCCCGTATTCAGTGTCGCTGATTTGTATTGTCTGAAGTTGTTTTTACGTTAAGTTGATGC AGATCAATTAATACGATACCTGCGTCATAATTGATTATTTGACGTGGTTTGATGGCCTCCACGCACGTTG TGATATGTAGATGATAATCATTATCACTTTACGGGTCCTTTCCGGTGATCCGACAGGTTACG lambda DNA 48502 base pairs Graphic map | Table by enzyme name | Table by site position Table by Enzyme Name Enzyme No. Positions Recognition name cuts of sites sequence EcoRI 5 21226 26104 31747 39168 44972 g/aattc More info HindIII 6 23130 25157 27479 36895 37459 a/agctt More info 44141 Every enzyme analyzed cuts this sequence. Table by Site Position Cut Enzyme No. Positions Recognition site name cuts of other sites sequence 21226 EcoRI 5 26104 31747 39168 g/aattc More info 44972 23130 HindIII 6 25157 27479 36895 a/agctt More info 37459 44141 25157 HindIII 6 23130 27479 36895 a/agctt More info 37459 44141 26104 EcoRI 5 21226 31747 39168 g/aattc More info 44972 27479 HindIII 6 23130 25157 36895 a/agctt More info 37459 44141 31747 EcoRI 5 21226 26104 39168 g/aattc More info 44972 36895 HindIII 6 23130 25157 27479 a/agctt More info 37459 44141 37459 HindIII 6 23130 25157 27479 a/agctt More info 36895 44141 39168 EcoRI 5 21226 26104 31747 g/aattc More info 44972 44141 HindIII 6 23130 25157 27479 a/agctt More info 36895 37459 44972 EcoRI 5 21226 26104 31747 g/aattc More info 39168 References: 1. Alberts, B. et al(2008) Molecular Biology of the Cell: Reference Edition, 5th Edition, Garland Science Taylor & Francis group LIC, Oxford, UK 2. Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, Vol. 2, John Wiley and Sons, NY. 3. Brown, T.A. Gene Cloning and DNA Analysis An Introduction, 4th Edition Blackwell Publishing 4. Lewin, B. (2004)  Genes VIII Pearson Prentice Hall, Upper Saddle River, NJ 5. Pálinkášová B. (2007) Investigations into the Lambda / HindIII Restriction Fragment Insertion Frequencies Using pUC18 as a Cloning Vector, diplomovka.sme. sk 6. Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 7. Tait, R. C. (2000) Introductory Experiments in Recombinant DNA, Curr. Issues Mol. BIol. 2(3): 71-85 Fig. 1. General scheme for agarose gel electrophoresis. Loading wells Agarose gel Place gel in tank, submerged with buffer and load DNA sample. Run sample, apply voltage for a suitable time Remove gel and visualise DNA under u.v. light The sizes are as follows:- Read More