Week 2 DNA Read, Write, & Edit

Here is the reference of the assignment: https://2026a.htgaa.org/2026a/course-pages/weeks/week-02/lab/index.html

Basic Understanding DNA Gel, restriction enzymes, Benchling intro, Twist intro (▶️Recording | 💻Slides)

easy content in Spanish: ¿Cómo hacer EDICIÓN GENÉTICA con CRISPR? https://www.youtube.com/watch?v=UaxrYWCyLdY&t=1s

As a committed listener in distance, I can only make the benching :

First step: https://www.benchling.com/, make an account. Second Step: choose a DNA sequence: https://www.neb.com/en-gb/tools-and-resources/interactive-tools/dna-sequences-and-maps-tool Begin by importing your DNA sequence and use the Digests tool to test the effects of different restriction enzyme(s). Export your final design as a png and compare with your lab results on your Notion page. See the images below for where to find the Digests tool, selecting the “NEB 2-log” ladder in the Virtual Digest tab, and how to have multiple Digests appear in the same Virtual Digest.

Part 0: Basics of Gel Electrophoresis

We did this call nicely online! Thanks to the introduction from Digby Usher. Here are a couple of pics of the experience: The work is saved in the following link: https://benchling.com/s/seq-33Yxt01UY8AwvjfPOIbd?m=slm-XZXh4GbXIU8Dh07RBlDZ Attend or watch all lecture and recitation videos. Optionally watch bootcamp.

Part 1: Benchling

>Lambda_NEB 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

See this week’s lab protocol “Gel Art: Restriction Digests and Gel Electrophoresis” for details. Overview:

  • I did an account at benchling.com
  • I imported the Lambda DNA. and safe as FASTA data and import in the benchling account as -> Import DNA sequence
  • Here you can see the result of the sequence:

I started to explore the digest:

  • I simulated the restriction Enzyme Digestion with the following Enzymes:
  • EcoRI

  • HindIII

Other suggestions for digestion are the following:

  • BamHI
  • KpnI
  • EcoRV
  • SacI
  • SalI

link zum Experiment in Benchling

Part 2: Gel Art - Restriction Digests and Gel Electrophoresis ( just the play with the gel art interface)

Part of the assignment was to create a pattern/image in the style of Paul Vanouse’s Latent Figure Protocol artworks. What was very useful for understanding the second part, at least the preparation for the lab, was Ronan’s website (Ronan’s website, a helpful tool for quickly iterating on designs!

For this interface, I play with the Enzimes: EcoRI, EcoRV,Sall and SacI

Restriction Digest Parameters 37°C for 60 minutes (incubation) 80°C for 20 minutes (heat inactivation) (optional)

DNA Gel Electrophoresis

Hand-Cast Gel Digest 14.7 μL Water 3.3 μL Loading Dye 2 μL Digest

Ladder 6.6 μL Water 3.3 μL Loading Dye 10 μL Ladder (15 ng/μL Stock)

E-Gel

Digest 18 μL Water 2 μL Digest

Ladder 10 μL Water 10 μL Ladder (15 ng/μL Stock)

link

Benchling Sequence

Part 3: DNA Design Challenge

3.1. Choose your protein.

Which protein have you chosen and why? I selected the Protein Titin, which is related to muscle function and movement; I used UniProt to look up information about the protein and found fascinating functions.

Cell color indicative of number of GO terms Aspect Term Molecular Function actin bindingSource:UniProtKB-KW Molecular Function alpha-actinin bindingSource:MGI1 publication Molecular Function axon guidance receptor activitySource:GO_Central Molecular Function structural constituent of muscleSource:ProtInc1 publication Biological Process homophilic cell adhesion via plasma membrane adhesion moleculesSource:GO_Central Biological Process muscle contractionSource:ProtInc1 publication Biological Process synapse organizationSource:GO_Central

And here the sequence:

MFNYERPKHFIQSQNPCGSRLQPPGPETSSFSSQTKQSSIIIQPRQCTEQRFSASSTLSSHITMSSSAFPASPKQHAGSNPGQRVTTTYNQSPASFLSSILPSQPDYNSSKIPSAMDSNYQQSSAGQPINAKPSQTANAKPIPRTPDHEIQGSKEALIQDLERKLKCKDTLLHNGNQRLTYEEKMARRLLGPQNAAAVFQAQDDSGAQDSQQHNSEHARLQVPTSQVRSRSTSRGDVNDQDAIQEKFYPPRFIQVPENMSIDEGRFCRMDFKVSGLPAPDVSWYLNGRTVQSDDLHKMIVSEKGLHSLIFEVVRASDAGAYACVAKNRAGEATFTVQLDVLAKEHKRAPMFIYKPQSKKVLEGDSVKLECQISAIPPPKLFWKRNNEMVQFNTDRISLYQDNTGRVTLLIKDVNKKDAGWYTVSAVNEAGVTTCNTRLDVTARPNQTLPAPKQLRVRPTFSKYLALNGKGLNVKQAFNPEGEFQRLAAQSGLYESEEL

Length: 498 Mass (Da) 55,395 Last updated 2010-11-02 v2 MD5 Checksum B308F50EB1D13D1628B83E5DC42C2866

Other information about the protein structural constituent of muscle

3.2. Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence.

This is the Exercise: The Central Dogma discussed in class and recitation describes the process by which the DNA sequence becomes transcribed and translated into protein. The Central Dogma gives us the framework to work backwards from a given protein sequence and infer the DNA sequence that the protein is derived from. Using one of the tools discussed in class, NCBI or online tools (google “reverse translation tools”), determine the nucleotide sequence that corresponds to the protein sequence you chose above.

Get to the original sequence of “Myotis myotis” from its genome Myotis myotis isolate mMyoMyo1 unplaced genomic scaffold, mMyoMyo1.p scaffold_m19_p_12, whole genome shotgun sequence

LOCUS NW_023416316 74216526 bp DNA linear CON 30-SEP-2020 DEFINITION Myotis myotis isolate mMyoMyo1 unplaced genomic scaffold, mMyoMyo1.p scaffold_m19_p_12, whole genome shotgun sequence. ACCESSION NW_023416316 VERSION NW_023416316.1 DBLINK BioProject: PRJNA665501 BioSample: SAMN14734277 Assembly: GCF_014108235.1 KEYWORDS WGS; RefSeq. SOURCE Myotis myotis ORGANISM Myotis myotis Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Chiroptera; Yangochiroptera; Vespertilionidae; Myotis. REFERENCE 1 (bases 1 to 74216526) AUTHORS Jebb,D., Huang,Z., Pippel,M., Hughes,G.M., Lavrichenko,K., Devanna,P., Winkler,S., Jermiin,L.S., Skirmuntt,E.C., Katzourakis,A., Burkitt-Gray,L., Ray,D.A., Sullivan,K.A.M., Roscito,J.G., Kirilenko,B.M., Davalos,L.M., Corthals,A.P., Power,M.L., Jones,G., Ransome,R.D., Dechmann,D.K.N., Locatelli,A.G., Puechmaille,S.J., Fedrigo,O., Jarvis,E.D., Hiller,M., Vernes,S.C., Myers,E.W. and Teeling,E.C. TITLE Six reference-quality genomes reveal evolution of bat adaptations JOURNAL Nature 583 (7817), 578-584 (2020) PUBMED 32699395 COMMENT REFSEQ INFORMATION: The reference sequence is identical to JABWUV010000012.1. Assembly name: mMyoMyo1.p The genomic sequence for this RefSeq record is from the whole-genome assembly released by the Bat1K on 2020/08/07. The original whole-genome shotgun project has the accession JABWUV000000000.1.

        ##Genome-Assembly-Data-START##
        Assembly Provider      :: Bat1K
        Assembly Date          :: 16-APR-2019
        Assembly Method        :: DAmar v. APRIL-2019; Bionano Solve DLS v.
                                  3.3; Salsa2 HiC v. git commit: e3ae7d8;
                                  GenomicConsensus v. git commit: 038de5c;
                                  longranger align v. 2.2.0; Freebayes v.
                                  1.2.0; HiGlass manual curation v. 1.5
        Assembly Name          :: mMyoMyo1.p
        Genome Representation  :: Full
        Expected Final Version :: No
        Genome Coverage        :: 90.9x
        Sequencing Technology  :: PacBio Sequel CLR; 10X Genomics chromium
                                  linked reads; Bionano Genomics; Phase
                                  Genomics HiC; PacBio Sequel IsoSeq
        ##Genome-Assembly-Data-END##
        
        ##Genome-Annotation-Data-START##
        Annotation Provider         :: NCBI
        Annotation Status           :: Full annotation
        Annotation Name             :: Myotis myotis Annotation Release 100
        Annotation Version          :: 100
        Annotation Pipeline         :: NCBI eukaryotic genome annotation
                                       pipeline
        Annotation Software Version :: 8.5
        Annotation Method           :: Best-placed RefSeq; Gnomon
        Features Annotated          :: Gene; mRNA; CDS; ncRNA
        ##Genome-Annotation-Data-END##

FEATURES Location/Qualifiers source 1..74216526 /organism=“Myotis myotis” /mol_type=“genomic DNA” /isolate=“mMyoMyo1” /db_xref=“taxon:51298” /chromosome=“Unknown” /sex=“female” /tissue_type=“flight muscle” /dev_stage=“adult” /geo_loc_name=“France: Limerzel” /lat_lon=“47.6333 N 2.3500 W” /collection_date=“2015” /collected_by=“Emma Teeling, Sebastien Puechmaille” CONTIG join(JABWUV010000012.1:1..74216526)

More information

3.3. Codon optimization.

Once a nucleotide sequence of your protein is determined, you need to codon optimize your sequence. You may, once again, utilize google for a “codon optimization tool”. In your own words, describe why you need to optimize codon usage. Which organism have you chosen to optimize the codon sequence for and why?

[Example from Codon Optimization Tool | Twist Bioscience while avoiding Type IIs enzyme recognition sites BsaI, BsmBI, and BbsI]

Lysis protein DNA sequence with Codon-Optimization
ATGGAAACCCGCTTTCCGCAGCAGAGCCAGCAGACCCCGGCGAGCACCAACCGCCGCCGCCCGTTCAAACATGAAGATTATCCGTGCCGTCGTCAGCAGCGCAGCAGCACCCTGTATGTGCTGATTTTTCTGGCGATTTTTCTGAGCAAATTCACCAACCAGCTGCTGCTGAGCCTGCTGGAAGCGGTGATTCGCACAGTGACGACCCTGCAGCAGCTGCTGACCTAA