Codon Optimization

1. Codon Optimization

2. Codon Optimization • Codon Usage Bias: Variable frequency of synonomouscodons. • Codon preferences reflect natural selection in organisms for translational optimization. • Optimal codons allow faster translation rates, thus highly expressed genes are coded by these. • Codon optimization can be used to produce a synthetic version of a foreign gene for more efficient expression.

3. Codon Usage of HIV vs. Human

4. Expression Profile of Synthetic vs. Wildtype gp120

5. HIV Codons Decrease Efficiency of Thy-1 Expression -Thy-1 is a highly expressed cell-surface protein. -Replacing codons of Thy-1 with HIV envelope codons significantly decreases expression.

6. Codon Optimization of GFP • A: Control: Empty vector. • B: Native GFP from Aequoreavictoria. • C: Mammalian codon optimized GFP. • D: Mammalian codon optimized GFP + S65T replacement.

7. E. Coli vs. Human Codon Usage

8. Codon Optimization: Human to Bacteria

9. Codon Optimization: Human to Bacteria

10. Codon Optimization: Human to Bacteria

11. Codon Optimization: Human to Bacteria

12. Codon Optimization: Human to Bacteria

13. Polymerase Chain Reaction • Molecular biology technique used to amplify specific nucleotide sequences. • Developed by Kary Mullis in 1983. • Takes advantage of Taq polymerase from thermophileThermusaquaticus. • Taq is a heat-resistant enzyme that can function in temperatures above 70°C.

14. Polymerase Chain Reaction

15. Polymerase Chain Reaction

16. Components of PCR • Water • Taq Buffer containing divalent and monovalentcations – Provides suitable chemical environment for optimal activity of DNA polymerase (pH, [salt], co-factors,etc.) • DNA template containing target region to be amplified. • Sense and anti-sense primers. • Deoxynucleotidetriphosphates (dNTPs), the building-blocks for synthesis of new DNA. • Taq polymerase – has optimal activity around 70°C.

17. Homework Assignment • NCBI • Search through GENE databases. • Enter the name of the gene of interest. • Search for Homo sapien or E. coli and select the gene. • Scroll down to mRNA and proteins and select. • Copy the “translation” • Encorbio • Select the organism that the gene will be codon optimized to. • Paste the amino acid sequence into the top box. • Generate the coding sequence.

18. Example: Insulin-like Growth Factor 1 Receptor (IGF1R) • Amino Acid Sequence • MKSGSGGGSPTSLWGLLFLSAALSLWPTSGEICGPGIDIRNDYQQLKRLENCTVIEGYLHILLISKAEDYRSYRFPKLTVITEYLLLFRVAGLESLGDLFPNLTVIRGWKLFYNYALVIFEMTNLKDIGLYNLRNITRGAIRIEKNADLCYLSTVDWSLILDAVSNNYIVGNKPPKECGDLCPGTMEEKPMCEKTTINNEYNYRCWTTNRCQKMCPSTCGKRACTENNECCHPECLGSCSAPDNDTACVACRHYYYAGVCVPACPPNTYRFEGWRCVDRDFCANILSAESSDSEGFVIHDGECMQECPSGFIRNGSQSMYCIPCEGPCPKVCEEEKKTKTIDSVTSAQMLQGCTIFKGNLLINIRRGNNIASELENFMGLIEVVTGYVKIRHSHALVSLSFLKNLRLILGEEQLEGNYSFYVLDNQNLQQLWDWDHRNLTIKAGKMYFAFNPKLCVSEIYRMEEVTGTKGRQSKGDINTRNNGERASCESDVLHFTSTTTSKNRIIITWHRYRPPDYRDLISFTVYYKEAPFKNVTEYDGQDACGSNSWNMVDVDLPPNKDVEPGILLHGLKPWTQYAVYVKAVTLTMVENDHIRGAKSEILYIRTNASVPSIPLDVLSASNSSSQLIVKWNPPSLPNGNLSYYIVRWQRQPQDGYLYRHNYCSKDKIPIRKYADGTIDIEEVTENPKTEVCGGEKGPCCACPKTEAEKQAEKEEAEYRKVFENFLHNSIFVPRPERKRRDVMQVANTTMSSRSRNTTAADTYNITDPEELETEYPFFESRVDNKERTVISNLRPFTLYRIDIHSCNHEAEKLGCSASNFVFARTMPAEGADDIPGPVTWEPRPENSIFLKWPEPENPNGLILMYEIKYGSQVEDQRECVSRQEYRKYGGAKLNRLNPGNYTARIQATSLSGNGSWTDPVFFYVQAKTGYENFIHLIIALPVAVLLIVGGLVIMLYVFHRKRNNSRLGNGVLYASVNPEYFSAADVYVPDEWEVAREKITMSRELGQGSFGMVYEGVAKGVVKDEPETRVAIKTVNEAASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQPTLVIMELMTRGDLKSYLRSLRPEMENNPVLAPPSLSKMIQMAGEIADGMAYLNANKFVHRDLAARNCMVAEDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTYSDVWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEIISSIKEEMEPGFREVSFYYSEENKLPEPEELDLEPENMESVPLDPSASSSSLPLPDRHSGHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALPLPQSSTC • Codon Optimized Sequence • ATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCGGCCCGGGCATTGATATTCGCAACGATTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGGCGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCTTTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGGAACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACTGGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACGCGCGCTGCCGCTGCCGCAGAGCAGCACCTGC

19. Example: Insulin-like Growth Factor 1 Receptor (IGF1R) • Add EcoRI (GAATTC) at 5’ end and BamHI (GGATCC) at 3’ end. • GAATTCATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCGGCCCGGGCATTGATATTCGCAACGATTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGGCGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCTTTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGGAACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACTGGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACGCGCGCTGCCGCTGCCGCAGAGCAGCACCTGCGGATCC

20. Example: Insulin-like Growth Factor 1 Receptor (IGF1R) • Add EcoRI (GAATTC) at 5’ end and BamHI (GGATCC) at 3’ end. • GAATTCATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCGGCCCGGGCATTGATATTCGCAACGATTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGGCGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCTTTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGGAACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACTGGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACGCGCGCTGCCGCTGCCGCAGAGCAGCACCTGCGGATCC

21. Designing Amplification Oligos ~5’ End 5’GAATTCATGAAAAGCGGCAGCGGCGGCGGC3’ 3’CTTAAGTACTTTTCGCCGTCGCCGCCGCCG5’ ~*You need an available 3’OH, so for this end you make a primer against the antisense strand. 5’GAATTCATGAAAAGCGGCAGCGGCGGCGGC3’ 5’GAATTCATGAAAAGCGGCAGCGGC3’OH 3’CTTAAGTACTTTTCGCCGTCGCCGCCGCCG5’

22. Designing Amplification Oligos ~3’ End 5’TGCCGCTGCCGCAGAGCAGCACCTGCGGATCC3’ 3’ACGGCGACGGCGTCTCGTCGTGGACGCCTAGG5’ ~Again, you need a 3’OH so this time make a primer against the sense strand. 5’TGCCGCTGCCGCAGAGCAGCACCTGCGGATCC3’ HO3’GGCGTCTCGTCGTGGACGCCTAGG5’ 3’ACGGCGACGGCGTCTCGTCGTGGACGCCTAGG5’

23. Designing Amplification Oligos • Primers need to be anywhere from 18-25 nucleotides. • Don’t include the restriction site or Kozak sequence as part of your 18-25 nucleotides for the primer. • Should contain at least 50% GC composition. • Must flank the entire CODING sequence.