Biology 373, Molecular Biology LAB Time and Location of Class: Materials: Instructor: Dr. Kurt Toenjes Course Introduction and Objectives

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1 Biology 373, Molecular Biology LAB Time and Location of Class: T 2:00-5:30 or W 2:00-5:30 in SCI 123 Materials: Molecular Biology of the Cell, Fifth edition, Lab handouts Instructor: Dr. Kurt Toenjes, Room 133 Science Hall Phone: ktoenjes@msubillings.edu Office Hours: MWF 9:00-10:00 and by appointment Course Introduction and Objectives: The overall goal of this course is to provide you with a molecular research lab experience. To accomplish this we are going to spend the semester investigating the amino acids involved in determining the wavelength of light emitted from a fluorescent protein. The experimental organisms will be the yeast Candida albicans and the bacteria E. coli. You will become proficient in a number of common techniques and approaches used to investigate the molecular basis of biological questions. These may include but are not limited to: Bioinformatics, DNA isolation, RFLP analysis, Site directed mutagenesis, PCR, DNA electrophoresis, DNA sequencing, Transformation, Southern Blot analysis, and Microscopy. Assessment: You will be assessed based on your attitude and cooperation with the other lab members (25 pts), your lab notebook (25 pts), a written lab report (100 pts), and on a oral final exam (50 pts). The written lab report will be based on your work throughout the semester and will consist of an Abstract, Introduction, a brief Methods section, a Results section and a Discussion section. The final exam will be based on material covered during the entire lab course. No extra credit will be given. Grading: The standards for awarding your grades will be no higher than: A= 90%-100%, B= 80%-89%, C= 70%-79%, D= 60%-69%, and F= 0%-59% Minuses MAY be used for your benefit at our discretion to adjust for the performance of the class. For example, if you earn 69% of the total points possible in the course you MAY be awarded a C- rather than a D. Academic Honesty: You are expected to meet the highest standards of academic honesty and ethics. All work you turn in must be entirely the result of your own effort and represent your understanding of the subject matter. Any cheating or assisting someone else in cheating will result in you receiving 0 pts for the assignment. Attendance: This is mandatory. If you miss class you cannot progress with your own project. Since your project is unique to you, this will put you at a serious disadvantage. If you know you cannot make a lab, make arrangements to attend the other lab section. Lab Etiquette and Safety: During the class you need to respect the rights of your classmates, keep all lab areas CLEAN and stay focused on the experiment you are doing. There is absolutely no eating, drinking, gum chewing, cell phone use or open

2 toed shoes. Full length attire and lab coats are recommended. Remember you it is up to you to keep your classmates safe and up to them to keep you safe. Important Dates: 4/22 and 4/23 ORAL Final Exam 5/1 Lab Report and Lab Notebooks are Due.

3 The goal You are going to change the spectrum of fluorescence for GFP during course of the semester and integrate the modified GFP, CFP and YFP into the genome of Canadia albicans. Two different versions of GFP can be created by making several amino acid substitutions in the protein. Amino acid substitutions change the active site of the molecule and alter the light emitted by the protein. CFP will be created by incorporating the F64L, S65T, Y66W, N146I, M153T, and V163A mutations into GFP by using the QuikChange kit in a two-step mutagenesis scheme. YFP will be created by incorporating the S65G, V68L, S72A, and T203Y mutations in GFP by also using the QuikChange kit in a two-step mutagenesis. In order to accomplish this you first need to design a series of primers for use in a site-directed mutagenesis protocol. Today s exercise is to initiate the design of the primers. Tuesdays lab will design the CFP primers. Wednesday s lab will design the YFP primers. 1. Before anything else is done a plasmid map needed. This will show you what sequences contain the GFP gene and identify the restriction sites that are present and absent from the plasmid. Get the plasmid sequence from bottom of this handout. Use the NEB site (see below), paste in the sequence into the window, check the circular box (plasmids are circular DNA elements) and click submit to determine the location of the open reading frames on the plasmid sequence ad the unique restriction sites. Print out a copy of the plasmid map for reference. This contains a map of where restriction enzyme that cut this plasmid once will bind. a Click the ORF A open reading frame. This will open a second window containing the translated plasmid sequence that corresponds to that ORF (orf A). Click the bottom button to BLAST this sequence at the NCBI database and determine what proteins it corresponds to. The sequences with the lowest E value have the greatest similarity to that open reading frame. Make a note as to the name of the closest related sequence to ORF by returning to the NEB web site, click edit, and next to the GENE: type in the info. Now continue identifying the rest of the ORFs. I suggest you make a note of each ORFs ID in your lab notebook just in case you lose contact with this site before you are done. Now print the final map. 3. Going back to the circular sequence page, bottom right there should be a box with the title LIST. Click the 0 cutters link. This will give you a list of enzymes that cut your sequence 0 times. Now under display click the two cutters and print this map as well. This gives you all enzymes that cut this sequence twice. You already have the single cutters from the original map. 4. Take the GFP sequence below and use the site to create a translation of the GFP gene. To accomplish this, paste the sequence into the window and click the translate button. Now click the text output button. Select

4 the text containing the translation and paste it into a word document. Use #10 currier font to keep it aligned and save this and print it out. ATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGTCCCAATTTTGGTTG AATTAGATGGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGAAG GTGAAGGTGATGCTACTTACGGTAAATTGACCTTAAAATTTATTTGTA CTACTGGTAAATTGCCAGTTCCATGGCCAACCTTAGTCACTACTTTCGG TTATGGTGTTCAATGTTTTGCTAGATACCCAGATCATATGAAACAACA TGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAGAAAGAAC TATTTTTTTCAAAGATGACGGTAACTACAAGACCAGAGCTGAAGTCAA GTTTGAAGGTGATACCTTAGTTAATAGAATCGAATTAAAAGGTATTGA TTTTAAAGAAGATGGTAACATTTTAGGTCACAAATTGGAATACAACTA TAACTCTCACAATGTTTACATCATGGCTGACAAACAAAAGAATGGTAT CAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGTTCTGTTCA ATTAGCTGACCATTATCAACAAAATACTCCAATTGGTGATGGTCCAGT CTTGTTACCAGACAACCATTACTTATCCACTCAATCTGCCTTATCCAAA GATCCAAACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTACT GCTGCTGGTATTACCCATGGTATGGATGAATTGTACAAATAA 5. We now need to locate the amino acids that we will change in our mutagenesis scheme. Identify and highlight the following amino acids and their corresponding codons: a. CFP -> F64, S65, Y66, N146, M153, and V163 b. YFP -> S65, V68, S72, and T Use the genetic code table to figure out which substitutions are possible to change each of the amino acids in GFP to the amino acid required for the color shift to either CFP or YFP. Remember that in c. albicans the Leucine codon CUG is decoded as serine so avoid it. a. CFP -> F64L, S65T, Y66W, N146I, M153T, and V163A b. YFP -> S65G, V68L, S72A, and T203Y 7. Design PCR primers for site-directed mutagenesis. For site directed mutagenesis we nee to include the mutations we want to make with the intervening sequence, plus nt on either side. These primers should also end in a GG, GC, CG, or CC at the 3 and 5 end in order to increase binding strength. 8. Finally we what to either include a new restriction site into this region or destroy an existing one so that we can use restriction analysis to detect our successful mutagenesis products. Use the restriction maps above to try to include such a mutation.

5 QuickTime and a TIFF (LZW) decompressor are needed to see this picture. Plasmid: CACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCAC GAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTT TCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTG GCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCAT ACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCAT CTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGA GTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGA GCTAACCGCTTTTTTTCACAACATGGGGGATCATGTAACTCGCCTTGATCGTT GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA TGCCTGTAGCAATGGCAACAACGTTGCGCAACAGAAAAGCATCTTACGGATG GCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC TGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCT TTTTTTCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGA GCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCA ATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTC CCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTT CTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGG TGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCC TCCCGTATCGTAGTTATCTACACGACGGGCAGTCAGGCAACTATGGATGAAC GAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTA ATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCC

6 CTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCC TTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCT ACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAG CGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACG ACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGC TTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGGAACGCCTGGTATCTTTATAG TCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTC AGGGGGGCCGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTC CTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGAT TCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAG CCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCC AATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGG CACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATG TGAGTTACCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCT CCTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT ATGACCATGATTACGCCAAGCTCGGAATTAACCTGCAGGACCACCTTTGATT GTAAATAGTAATAATTACCACCCTTATCTAATTATTTATTTAACTTATTTATTT ATTTATTATACATATATACAAATCTAATAAAGTGAAAATCTCCCCCTTCACAC TTCACATATGTTAGGCGTCATCCTGTGCTCCCGAGAACCAGTACCAGTACATC GCTGTTTCGTTCGAGACTTGAGGTCTAGTTTTATACGTGAAGAGGTCAATGCC GCCGAGAGTAAAGCCACATTTTGCGTACAAATTGCAGGCAGGTACATTGTTC GTTTGTGTCTCTAATCGTATGCCAAGGAGCTGTCTGCTTAGTGCCCACTTTTTC GCAAATTCGATGAGACTGTGCGCGACTCCTTTGCCTCGGTGCGTGTGCGACAC AACAATGTGTTCGATAGAGGCTAGATCGTTCCATGTTGAGTTGAGTTCAATCT TCCCGACAAGCTCTTGGTCGATGAATGCGCCATAGCAAGCAGAGTCTTCATC AGAGTCATCATCCGAGATGTAATCCTTCCGGTAGGGGCTCACACTTCTGGTAG ATAGTTCAAAGCCTTGGTCGGATAGGTGCACATCGAACACTTCACGAACAAT GAAATGGTTCTCAGCATCCAATGTTTCCGCCACCTGCTCAGGGATCACCGAA ATTTTCATATGAGAACCGTTATCGATAACTAAAGCAGCAACTTCTTCTATAAA AATGGGTTAGTATGACAGTCATTTAAATAAGGAATTTTTCAGTTGGCTTGGTT TCAATTCAATGTTCGTTTTTTTTTTTTCTTGCTGTGTTTGTGTTTGTGTTGTTTA TAGTTGTGTGCACTGAGCGTCGAAAAAAAAAATTCATAGTGAGCCGGGAAAT CTGTATAGCCCAGATAACAACACAAGTCCAAACTAGAAACTCGTCAAACACC AAAAGCAATGTTGAATCAATTGCCTTGCACAAGTACACGTAGGAAAACATAA AACATTGCAATTTTGAATATTGAGCCTTTTGTCGTAACATTGATTGATAGGAT TACTCACCGAATGGTTTTGAAACCACTGCCGACAGATCAATCAATCAATCAA AAAACGTGAACTTTGAAAAAGGGGAAGAACAGATACATTGAAGTTAGCCATT TCCATTGATCGTCACAACATATCTGATAAATTACTTTCAAAATTATAAGCTGA TGTGTGTGTATTATTAATGTGACAGTAACATCCCAAACGAGAAATATTATGTC GACAACAAAAAAGTTTGATCTGAATTGAAAATGAAGTTTTCCCACCCTACCC ATTTGTCATATTGAAACCAATCAACTGATTAATCAATCAATTAGAATTGAAGC

7 TAAACTAAAACATACCACCGTCCATTTTGAATGATTATATTTTTTTAATATTA ATATCGAGATAATGTTTCTAAGAAAGAAAGAAAACCAGGAGTGAAAATTAG AAAAGGAAAGGAAAGGAAAAAAAGAAAAATCTGAAAATATATAAAAAAAA ATTGTTTCGTTGGCAATAAATCTTGGTGAGAACAGCGACCGAAAGCAAATAA GAACAAAATATGAGTGTATTACGTTGAACAACTAATTAACGTGTGTGTATGG ATCTTTTTTTCTTTTTTCTCTTTAACCGACTATAAACAACAAACATTTTTGGGC AGTGCACACACTACTTAATATACACAGCATAAATTACACGATTAGAAACAAA TTAGCTTATTAAAATAACCTAATCAAACCGAATATTTTATGGTATTATGAGTA AACTATATAATATAAATAGCACACACCCACAACAACAACAAAGGAAAACTA AAAGGTTTTTTCTTTTTGAAAAGATCGTTTTCTTTATTATTCTCTAGTTTTGAC GCTCGACATTTTATGATGGAATGAATGGGATGAATCATCAAACAAGAGAAAA TACCCGTGACGAAAATAATAAAATAAGTTCCTCTGATACAGAAGATGAAAAC AACAACAACAAGATATAGAAATGCCTTGGGTGGCTATTTTATAGTCTTAACTT TTTAATGTATATTTGTTTTGTTTTTTTACATAATAATACTTTATAAAAGCTAAG CTAAATTCAAGTAAAATTTCAATCTCTCAAATAAAACATTTTTCTCTTTTTCTT AAATTTAGTTTTATATATTTATAAAATATACAAAGATTTTTTTAAAAAAGTAA CAAGTTATATATGTAATAACAAAAAGAAGAATAACAAGAATACAAAACCAG ATTTCCAGATTTCCAGAATTTCACTCTTATATGCGTCTATTTATGTAGGATGA AAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTA TGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTG GATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATGCAT AGTACCGAGAAACTAGTGCGAAGTAGTGATCAGGTATTGCTGTTATCTGATG AGTATACGTTGTCCTGGCCACGGCAGAAGCACGCTTATCGCTCCAATTTCCCA CAACATTAGTCAACTCCGTTAGGCCCTTCATTGAAAGAAATGAGGTCATCAA ATGTCTTCCAATGTGAGATTTTGGGCCATTTTTTATAGCAAAGATTGAATAAG GCGCATTTTTCTTCAAAGCTTTATTGTACGATCTGACTAAGTTATCTTTTAATA ATTGGTATTCCTGTTTATTGCTTGAAGAATTGCCGGTCCTATTTACTCGTTTTA GGACTGGTTCAGAATTCCTCAAAAATTCATCCAAATATACAAGTGGATCGAT CCTACCCCTTGCGCTAAAGAAGTATATGTGCCTACTAACGCTTGTCTTTGTCT CTGTCACTAAACACTGGATTATTACTCCCAAATACTTATTTTGGACTAATTTA AATGATTTCGGATCAACGTTCTTAATATCGCTGAATCTTCCACAATTGATGAA AGTAGCTAGGAAGAGGAATTGGTATAAAGTTTTTGTTTTTGTAAATCTCGAAG TATACTCAAACGAATTTAGTATTTTCTCAGTGATCTCCCAGATGCTTTCACCCT CACTTAGAAGTGCTTTAAGCATTTTTTTACTGTGGCTATTTCCCTTATCTGCTT CTTCCGATGATTCGAACTGTAATTGCAAACTACTTACAATATCAGTGATATCA GATTGATGTTTTTGTCCATAGTAAGGAATAATTGTAAATTCCCAAGCAGGAAT CAATTTCTTTAATGAGGCTTCCAAAATTGTTGCTTTTTGCGTCTTGTATTTAAA CTGGAGTGATTTATTGACAATATCGAAACTCAACGAATTGCTTATGATAGTAT TATAGCTCATGAATGTGGCTCTCTTGATTGCTGTTCCGTTATGTGTAATCATCC AACATAAATAGGTTAGTTCAGCAGCACATAATGCTATTTTCTCACCTGAAGGT CTTTCAAACCTTTCCACAAACTGACGAACAAGCACCTTAGGTGGTGTTTTACA TAATATATCAAATTGTGGCATGTCGACGATTATTAGTTAAACCACTGCAAAA AGTTGGGGAAAATTTTGCCCATTTTTATACCGTGTCTTCGTCTATCGCCTCCCC CACTCCCCAATCTTTGAATTATTCCGAAATATTCAGCGAACGGGGTGTACACA AAAACTAACATTCTCAACTGCATAATTTGAAAAATGGCGTGGGACAAGAAAA AAAAAAAATTCTCAACCATAGCAATCATGGAATACGGTAAATTTGTGTTGTT

8 CGGTGACTCCATCACCCAGTTTAGTTGTACCCAGTATGGCTTTCATCCAGCAT TACAGAATGTGTATATCCGAAAATTGGATGTTATTAACCGTGGTTTCAGTGGC TACAACTCAGAGCACGCTAGACAAATTCTTCCAAAAATTTTAGAGTCGGAAA CCAATATCAAATTGATGACAATATTTTTTGGAACTAACGATGCATACGACTAC ATCAATGAAATCCAGACAGTCGAGTTAGACAGATATAAAGATAATTTAAGTG TAATGGTACAGATGGTACTAGACAAAAATATCAAACCAATCATTATTGGATC CGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCTCGAGTTTGAATGATTA TATTTTTTTAATATTAATATCGAGATAATGTTTCTAAGAAAGAAAGAAAACCA GGAGTGAAAATTAGAAAAGGAAAGGAAAGGAAAAAAAGAAAAATCTGAAA ATATATAAAAAAAAATTGTTTCGTTGGCAATAAATCTTGGTGAGAACAGCGA CCGAAAGCAAATAAGAACAAAATATGAGTGTATTACGTTGAACAACTAATTA ACGTGTGTGTATGGATCTTTTTTTCTTTTTTCTCTTTAACCGACTATAAACAAC AAACATTTTTGGGCAGTGCACACACTACTTAATATACACAGCATAAATTACA CGATTAGAAACAAATTAGCTTATTAAAATAACCTAATCAAACCGAATATTTT ATGGTATTATGAGTAAACTATATAATATAAATAGCACACACCCACAACAACA ACAAAGGAAAACTAAAAGGTTTTTTCTTTTTGAAAAGATCGTTTTCTTTATTA TTCTCTAGTTTTGACGCTGAAATATTCGTTGTACTGTTGCAAATTTGTATTTGG TCTGCCTTATCTTCTTTTCTACACTTTTTACTTATGAGCTTTATAATTCACAGA TAAGTTTCAGGTTTGCCAGATATACTTGCTTCACTGGTTACTAAACTCTCGGT GTACAAATGACTTGCTTATGTAAGAGGCTACTTTTCTAGTTTTGCAGTCATTC ATTTAAAAACAATTGATCCTGAAGGCTCTAAAGACATAGTAGGTTTACTTTGA CTGTATATTTCCGTGGCATGTTTAAAGGCCCCTTGGCCATAGGATATTGGTGG CGGCACAGTAAGCATTAAAATTGTTGTTTCGCGGAATTACAACATTTCTTCTT TTACATAATAATGTCACTCCTTTTTGATTGGCATAGCCGAGATCTTTTTTTAAC ACAGACTTTAAGCTGTTTTTTGCCACCTGAGAACAGTTTCTATTTTGGTCCAA AAGAAATATTAGACTAAGTTTATTATAAACGAGGCTGGTGATCTTAATAGAA GCTTTATTAAAATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGTCCCAATT TTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGA AGGTGAAGGTGATGCTACTTACGGTAAATTGACCTTAAAATTTATTTGTACTA CTGGTAAATTGCCAGTTCCATGGCCAACCTTAGTCACTACTTTCGGTTATGGT GTTCAATGTTTTGCTAGATACCCAGATCATATGAAACAACATGACTTTTTCAA GTCTGCCATGCCAGAAGGTTATGTTCAAGAAAGAACTATTTTTTTCAAAGATG ACGGTAACTACAAGACCAGAGCTGAAGTCAAGTTTGAAGGTGATACCTTAGT TAATAGAATCGAATTAAAAGGTATTGATTTTAAAGAAGATGGTAACATTTTA GGTCACAAATTGGAATACAACTATAACTCTCACAATGTTTACATCATGGCTGA CAAACAAAAGAATGGTATCAAAGTTAACTTCAAAATTAGACACAACATTGAA GATGGTTCTGTTCAATTAGCTGACCATTATCAACAAAATACTCCAATTGGTGA TGGTCCAGTCTTGTTACCAGACAACCATTACTTATCCACTCAATCTGCCTTAT CCAAAGATCCAAACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTAC TGCTGCTGGTATTACCCATGGTATGGATGAATTGTACAAATAAGCTAGCGTCC CTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAA TTTCGCGTAGATCCAACTCAAGTACAACATGGCTGTCGGTAAAAACAAGAGA TTGTCCAAAGGAAAGAAAGGATTAAAAAAGAAAGTCGTTGACCCATTCACCA GAAAAGATTGGTTTGACATCAAAGCTCCAACCACTTTTGAAAACAGAAATGT TGGTAAAACTTTGATCAACAGATCTACCGGTTTAAAGAATGCCGCTGATGGC TTGAAAGGTAGAGTTTTCGAAGTTTGTTTGGCCGACTTACAAGGTTCCGAAGA

9 CCACTCTTACAGAAAAATCAAATTGAGAGTTGATGAAGTCCAAGGTAAAAAC TTGTTGACCAACTTCCATGAGGCCTCCATGGAGGCCTCATGGAGGCCTCATGG AGGCCTCATGGAGGCCTCATGGTTTGGATTTCACTTCTGACAAATTAAGATCA TTGGTCAGAAAATGGCAATCATTAGTTGAAGCTAATGTCACTGTTAAAACTTC CGACGATTACGTTTTGAGAGTTTTTGCCATTGCTTTCACCAAAAGACAACCAA ACCAAATCAAGAAAACTACTTACGCTCAATCCTCTAAATTGAGAGAAGTCAG AAAGAAGATGATTGAAATTATGCAAAGAGAAGTTTCCAACTGTACTTTAGCT CAATTAACTTCCAAATTGATTCCAGAAGTCATTGGCCGTGAAATTGAAAAAT CCACCCAAACCATTTTCCCATTACAAAATGTCCACATCAGAAAAGTCAAATT GTTGAAACAACCAAAATTCGACTTGGGTTCATTATTGGCTTTGCACGGTGAAG GTTCAACCGAAGAAAAAGGTAAGAAAGTTTCTTCTGGTTTCAAAGATGTTGT TTTAGAATCTGTTTAAGGATAATGATCTGGGGGATCCAAGCTTGAAGTTCCTA TTCTCTAGAAAGTATAGGAACTTCGGCCGCCACCGCGGTGGAGCTCCAATTC GCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTC GTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCC CCCCTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCC CAACAGTTGCGCAGCCTGAATGGCGAATGGCGCGACGCGCCCTGTAGCGGCG CATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGC CAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTT CGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGAT TTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCA CGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTC CACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTA TCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGT TAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT AACGCTTACAATTTAG