Codon Optimization

Codon Usage Bias: Variable frequency of synonomous codons.

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.

Codon Optimization

Codon Usage of HIV vs. Human

Expression Profile of Synthetic vs. Wildtype gp120

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.

A: Control: Empty vector.

B: Native GFP from Aequorea victoria.

C: Mammalian codon optimized GFP.

D: Mammalian codon optimized GFP + S65T replacement.

Codon Optimization of GFP

E. Coli vs. Human Codon Usage

Codon Optimization: Human to Bacteria

Codon Optimization: Human to Bacteria

Codon Optimization: Human to Bacteria

Codon Optimization: Human to Bacteria

Codon Optimization: Human to Bacteria

Molecular biology technique used to amplify specific nucleotide sequences.

Developed by Kary Mullis in 1983.Takes advantage of Taq polymerase from

thermophile Thermus aquaticus.Taq is a heat-resistant enzyme that can

function in temperatures above 70°C.

Polymerase Chain Reaction

Polymerase Chain Reaction

Polymerase Chain Reaction

WaterTaq Buffer containing divalent and monovalent

cations – 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.Deoxynucleotide triphosphates (dNTPs), the

building-blocks for synthesis of new DNA.Taq polymerase – has optimal activity around

70°C.

Components of PCR

NCBISearch 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”

EncorbioSelect the organism that the gene will be

codon optimized to.Paste the amino acid sequence into the top box.Generate the coding sequence.

Homework Assignment

Amino Acid Sequence MKSGSGGGSPTSLWGLLFLSAALSLWPTSGEICGPGIDIRNDYQQLKRLENCTVIEGYLHILLISKAEDYRSYRFPKLTVITEYLLLFRVAGLESLGDLFPNLTVIRGWKLFYNYALVIFEMTNL

KDIGLYNLRNITRGAIRIEKNADLCYLSTVDWSLILDAVSNNYIVGNKPPKECGDLCPGTMEEKPMCEKTTINNEYNYRCWTTNRCQKMCPSTCGKRACTENNECCHPECLGSCSAPDNDTACVACRHYYYAGVCVPACPPNTYRFEGWRCVDRDFCANILSAESSDSEGFVIHDGECMQECPSGFIRNGSQSMYCIPCEGPCPKVCEEEKKTKTIDSVTSAQMLQGCTIFKGNLLINIRRGNNIASELENFMGLIEVVTGYVKIRHSHALVSLSFLKNLRLILGEEQLEGNYSFYVLDNQNLQQLWDWDHRNLTIKAGKMYFAFNPKLCVSEIYRMEEVTGTKGRQSKGDINTRNNGERASCESDVLHFTSTTTSKNRIIITWHRYRPPDYRDLISFTVYYKEAPFKNVTEYDGQDACGSNSWNMVDVDLPPNKDVEPGILLHGLKPWTQYAVYVKAVTLTMVENDHIRGAKSEILYIRTNASVPSIPLDVLSASNSSSQLIVKWNPPSLPNGNLSYYIVRWQRQPQDGYLYRHNYCSKDKIPIRKYADGTIDIEEVTENPKTEVCGGEKGPCCACPKTEAEKQAEKEEAEYRKVFENFLHNSIFVPRPERKRRDVMQVANTTMSSRSRNTTAADTYNITDPEELETEYPFFESRVDNKERTVISNLRPFTLYRIDIHSCNHEAEKLGCSASNFVFARTMPAEGADDIPGPVTWEPRPENSIFLKWPEPENPNGLILMYEIKYGSQVEDQRECVSRQEYRKYGGAKLNRLNPGNYTARIQATSLSGNGSWTDPVFFYVQAKTGYENFIHLIIALPVAVLLIVGGLVIMLYVFHRKRNNSRLGNGVLYASVNPEYFSAADVYVPDEWEVAREKITMSRELGQGSFGMVYEGVAKGVVKDEPETRVAIKTVNEAASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQPTLVIMELMTRGDLKSYLRSLRPEMENNPVLAPPSLSKMIQMAGEIADGMAYLNANKFVHRDLAARNCMVAEDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTYSDVWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEIISSIKEEMEPGFREVSFYYSEENKLPEPEELDLEPENMESVPLDPSASSSSLPLPDRHSGHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALPLPQSSTC

Codon Optimized Sequence ATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCGGCCCGGGCATTGATATTCGCAACGATTATCAGCA

GCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGGCGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCTTTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGGAACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACTGGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACGCGCGCTGCCGCTGCCGCAGAGCAGCACCTGC

Example: Insulin-like Growth Factor 1 Receptor (IGF1R)

Add EcoRI (GAATTC) at 5’ end and BamHI (GGATCC) at 3’ end. GAATTCATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCG

GCCCGGGCATTGATATTCGCAACGATTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGGCGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCTTTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGGAACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACTGGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACGCGCGCTGCCGCTGCCGCAGAGCAGCACCTGCGGATCC

Example: Insulin-like Growth Factor 1 Receptor (IGF1R)

Add EcoRI (GAATTC) at 5’ end and BamHI (GGATCC) at 3’ end. GAATTCATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCG

GCCCGGGCATTGATATTCGCAACGATTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGGCGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCTTTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGGAACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACTGGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACGCGCGCTGCCGCTGCCGCAGAGCAGCACCTGCGGATCC

Example: Insulin-like Growth Factor 1 Receptor (IGF1R)

~5’ End5’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’OH3’CTTAAGTACTTTTCGCCGTCGCCGCCGCCG5’

Designing Amplification Oligos

~3’ End5’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’

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.

Designing Amplification Oligos