 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.
-Thy-1 is a highly
expressed cellsurface 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.
 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.
 Water
 Taq 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 buildingblocks for synthesis of new DNA.
Taq polymerase – has optimal activity around 70°C.
 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.
 Amino Acid Sequence

MKSGSGGGSPTSLWGLLFLSAALSLWPTSGEICGPGIDIRNDYQQLKRLENCTVIEGYLHILLISKAEDYRSYRFPKLTVITEYLLLFRVAGLESLGDLFPNLTVIRGWKLFYNYALVI
FEMTNLKDIGLYNLRNITRGAIRIEKNADLCYLSTVDWSLILDAVSNNYIVGNKPPKECGDLCPGTMEEKPMCEKTTINNEYNYRCWTTNRCQKMCPSTCGKRACTENNECCH
PECLGSCSAPDNDTACVACRHYYYAGVCVPACPPNTYRFEGWRCVDRDFCANILSAESSDSEGFVIHDGECMQECPSGFIRNGSQSMYCIPCEGPCPKVCEEEKKTKTIDSVTSA
QMLQGCTIFKGNLLINIRRGNNIASELENFMGLIEVVTGYVKIRHSHALVSLSFLKNLRLILGEEQLEGNYSFYVLDNQNLQQLWDWDHRNLTIKAGKMYFAFNPKLCVSEIYR
MEEVTGTKGRQSKGDINTRNNGERASCESDVLHFTSTTTSKNRIIITWHRYRPPDYRDLISFTVYYKEAPFKNVTEYDGQDACGSNSWNMVDVDLPPNKDVEPGILLHGLKPW
TQYAVYVKAVTLTMVENDHIRGAKSEILYIRTNASVPSIPLDVLSASNSSSQLIVKWNPPSLPNGNLSYYIVRWQRQPQDGYLYRHNYCSKDKIPIRKYADGTIDIEEVTENPKTE
VCGGEKGPCCACPKTEAEKQAEKEEAEYRKVFENFLHNSIFVPRPERKRRDVMQVANTTMSSRSRNTTAADTYNITDPEELETEYPFFESRVDNKERTVISNLRPFTLYRIDIHSC
NHEAEKLGCSASNFVFARTMPAEGADDIPGPVTWEPRPENSIFLKWPEPENPNGLILMYEIKYGSQVEDQRECVSRQEYRKYGGAKLNRLNPGNYTARIQATSLSGNGSWTDP
VFFYVQAKTGYENFIHLIIALPVAVLLIVGGLVIMLYVFHRKRNNSRLGNGVLYASVNPEYFSAADVYVPDEWEVAREKITMSRELGQGSFGMVYEGVAKGVVKDEPETRVAIKT
VNEAASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQPTLVIMELMTRGDLKSYLRSLRPEMENNPVLAPPSLSKMIQMAGEIADGMAYLNANKFVHRDLAARNCMVAE
DFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTYSDVWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKM
RPSFLEIISSIKEEMEPGFREVSFYYSEENKLPEPEELDLEPENMESVPLDPSASSSSLPLPDRHSGHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALPLPQSSTC
 Codon Optimized Sequence

ATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCGAAATTTGCGGCCCGGGCATTGATATTCGCAACGA
TTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTGATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATAT
CTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCTGGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAG
ATATTGGCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTGGATGCGGTGAGCAACAACTATA
TTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAAAAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGC
TGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCGAAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCG
CCATTATTATTATGCGGGCGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGCAGCGATAGCGA
AGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTCCGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAA
AAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCATTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTA
TGGGCCTGATTGAAGTGGTGACCGGCTATGTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCT
TTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCGAAACTGTGCGTGAGCGAAATTTATCGCA
TGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAACGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAAC
CGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTATCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCA
GCAACAGCTGGAACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGAAAGCGGTGACCCTGACC
ATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCCGCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGT
GAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTGCGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCG
CAAATATGCGGATGGCACCATTGATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGGCGGAA
AAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGATGCAGGTGGCGAACACCACCATGAGCAGCCGCAG
CCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATATCCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTT
TACCCTGTATCGCATTGATATTCATAGCTGCAACCATGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGT
GACCTGGGAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGGTGGAAGATCAGCGCGAATGCG
TGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCATTCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTT
TTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTATTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACA
ACAGCCGCCTGGGCAACGGCGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGCCGCGAACTG
GGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACCGTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAAT
TTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGGGCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAA
AGCTATCTGCGCAGCCTGCGCCCGGAAATGGAAAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAA
ATTTGTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCGATTATTATCGCAAAGGCGGCAAAGG
CCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTGGAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCA
GGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGCGGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGC
GCCCGAGCTTTCTGGAAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACTGGATCTGGAACCG
GAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGCGGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGA
GCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACGCGCGCTGCCGCTGCCGCAGAGCAGCACCTGC

Add EcoRI (GAATTC) at 5’ end and BamHI (GGATCC) at 3’ end.

GAATTCATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCG
AAATTTGCGGCCCGGGCATTGATATTCGCAACGATTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTG
ATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCT
GGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTG
GCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTG
GATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAA
AAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCG
AAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGG
CGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGC
AGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTC
CGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCA
TTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTAT
GTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCT
TTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCG
AAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAA
CGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTA
TCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGG
AACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGA
AAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCC
GCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTG
CGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTG
ATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGG
CGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGAT
GCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATAT
CCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCA
TGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGG
GAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGG
TGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCAT
TCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTA
TTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGG
CGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGC
CGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACC
GTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGG
GCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGA
AAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTT
GTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCG
ATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTG
GAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGC
GGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGG
AAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACT
GGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGC
GGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACG
CGCGCTGCCGCTGCCGCAGAGCAGCACCTGCGGATCC

Add EcoRI (GAATTC) at 5’ end and BamHI (GGATCC) at 3’ end.

GAATTCATGAAAAGCGGCAGCGGCGGCGGCAGCCCGACCAGCCTGTGGGGCCTGCTGTTTCTGAGCGCGGCGCTGAGCCTGTGGCCGACCAGCGGCG
AAATTTGCGGCCCGGGCATTGATATTCGCAACGATTATCAGCAGCTGAAACGCCTGGAAAACTGCACCGTGATTGAAGGCTATCTGCATATTCTGCTG
ATTAGCAAAGCGGAAGATTATCGCAGCTATCGCTTTCCGAAACTGACCGTGATTACCGAATATCTGCTGCTGTTTCGCGTGGCGGGCCTGGAAAGCCT
GGGCGATCTGTTTCCGAACCTGACCGTGATTCGCGGCTGGAAACTGTTTTATAACTATGCGCTGGTGATTTTTGAAATGACCAACCTGAAAGATATTG
GCCTGTATAACCTGCGCAACATTACCCGCGGCGCGATTCGCATTGAAAAAAACGCGGATCTGTGCTATCTGAGCACCGTGGATTGGAGCCTGATTCTG
GATGCGGTGAGCAACAACTATATTGTGGGCAACAAACCGCCGAAAGAATGCGGCGATCTGTGCCCGGGCACCATGGAAGAAAAACCGATGTGCGAA
AAAACCACCATTAACAACGAATATAACTATCGCTGCTGGACCACCAACCGCTGCCAGAAAATGTGCCCGAGCACCTGCGGCAAACGCGCGTGCACCG
AAAACAACGAATGCTGCCATCCGGAATGCCTGGGCAGCTGCAGCGCGCCGGATAACGATACCGCGTGCGTGGCGTGCCGCCATTATTATTATGCGGG
CGTGTGCGTGCCGGCGTGCCCGCCGAACACCTATCGCTTTGAAGGCTGGCGCTGCGTGGATCGCGATTTTTGCGCGAACATTCTGAGCGCGGAAAGC
AGCGATAGCGAAGGCTTTGTGATTCATGATGGCGAATGCATGCAGGAATGCCCGAGCGGCTTTATTCGCAACGGCAGCCAGAGCATGTATTGCATTC
CGTGCGAAGGCCCGTGCCCGAAAGTGTGCGAAGAAGAAAAAAAAACCAAAACCATTGATAGCGTGACCAGCGCGCAGATGCTGCAGGGCTGCACCA
TTTTTAAAGGCAACCTGCTGATTAACATTCGCCGCGGCAACAACATTGCGAGCGAACTGGAAAACTTTATGGGCCTGATTGAAGTGGTGACCGGCTAT
GTGAAAATTCGCCATAGCCATGCGCTGGTGAGCCTGAGCTTTCTGAAAAACCTGCGCCTGATTCTGGGCGAAGAACAGCTGGAAGGCAACTATAGCT
TTTATGTGCTGGATAACCAGAACCTGCAGCAGCTGTGGGATTGGGATCATCGCAACCTGACCATTAAAGCGGGCAAAATGTATTTTGCGTTTAACCCG
AAACTGTGCGTGAGCGAAATTTATCGCATGGAAGAAGTGACCGGCACCAAAGGCCGCCAGAGCAAAGGCGATATTAACACCCGCAACAACGGCGAA
CGCGCGAGCTGCGAAAGCGATGTGCTGCATTTTACCAGCACCACCACCAGCAAAAACCGCATTATTATTACCTGGCATCGCTATCGCCCGCCGGATTA
TCGCGATCTGATTAGCTTTACCGTGTATTATAAAGAAGCGCCGTTTAAAAACGTGACCGAATATGATGGCCAGGATGCGTGCGGCAGCAACAGCTGG
AACATGGTGGATGTGGATCTGCCGCCGAACAAAGATGTGGAACCGGGCATTCTGCTGCATGGCCTGAAACCGTGGACCCAGTATGCGGTGTATGTGA
AAGCGGTGACCCTGACCATGGTGGAAAACGATCATATTCGCGGCGCGAAAAGCGAAATTCTGTATATTCGCACCAACGCGAGCGTGCCGAGCATTCC
GCTGGATGTGCTGAGCGCGAGCAACAGCAGCAGCCAGCTGATTGTGAAATGGAACCCGCCGAGCCTGCCGAACGGCAACCTGAGCTATTATATTGTG
CGCTGGCAGCGCCAGCCGCAGGATGGCTATCTGTATCGCCATAACTATTGCAGCAAAGATAAAATTCCGATTCGCAAATATGCGGATGGCACCATTG
ATATTGAAGAAGTGACCGAAAACCCGAAAACCGAAGTGTGCGGCGGCGAAAAAGGCCCGTGCTGCGCGTGCCCGAAAACCGAAGCGGAAAAACAGG
CGGAAAAAGAAGAAGCGGAATATCGCAAAGTGTTTGAAAACTTTCTGCATAACAGCATTTTTGTGCCGCGCCCGGAACGCAAACGCCGCGATGTGAT
GCAGGTGGCGAACACCACCATGAGCAGCCGCAGCCGCAACACCACCGCGGCGGATACCTATAACATTACCGATCCGGAAGAACTGGAAACCGAATAT
CCGTTTTTTGAAAGCCGCGTGGATAACAAAGAACGCACCGTGATTAGCAACCTGCGCCCGTTTACCCTGTATCGCATTGATATTCATAGCTGCAACCA
TGAAGCGGAAAAACTGGGCTGCAGCGCGAGCAACTTTGTGTTTGCGCGCACCATGCCGGCGGAAGGCGCGGATGATATTCCGGGCCCGGTGACCTGG
GAACCGCGCCCGGAAAACAGCATTTTTCTGAAATGGCCGGAACCGGAAAACCCGAACGGCCTGATTCTGATGTATGAAATTAAATATGGCAGCCAGG
TGGAAGATCAGCGCGAATGCGTGAGCCGCCAGGAATATCGCAAATATGGCGGCGCGAAACTGAACCGCCTGAACCCGGGCAACTATACCGCGCGCAT
TCAGGCGACCAGCCTGAGCGGCAACGGCAGCTGGACCGATCCGGTGTTTTTTTATGTGCAGGCGAAAACCGGCTATGAAAACTTTATTCATCTGATTA
TTGCGCTGCCGGTGGCGGTGCTGCTGATTGTGGGCGGCCTGGTGATTATGCTGTATGTGTTTCATCGCAAACGCAACAACAGCCGCCTGGGCAACGG
CGTGCTGTATGCGAGCGTGAACCCGGAATATTTTAGCGCGGCGGATGTGTATGTGCCGGATGAATGGGAAGTGGCGCGCGAAAAAATTACCATGAGC
CGCGAACTGGGCCAGGGCAGCTTTGGCATGGTGTATGAAGGCGTGGCGAAAGGCGTGGTGAAAGATGAACCGGAAACCCGCGTGGCGATTAAAACC
GTGAACGAAGCGGCGAGCATGCGCGAACGCATTGAATTTCTGAACGAAGCGAGCGTGATGAAAGAATTTAACTGCCATCATGTGGTGCGCCTGCTGG
GCGTGGTGAGCCAGGGCCAGCCGACCCTGGTGATTATGGAACTGATGACCCGCGGCGATCTGAAAAGCTATCTGCGCAGCCTGCGCCCGGAAATGGA
AAACAACCCGGTGCTGGCGCCGCCGAGCCTGAGCAAAATGATTCAGATGGCGGGCGAAATTGCGGATGGCATGGCGTATCTGAACGCGAACAAATTT
GTGCATCGCGATCTGGCGGCGCGCAACTGCATGGTGGCGGAAGATTTTACCGTGAAAATTGGCGATTTTGGCATGACCCGCGATATTTATGAAACCG
ATTATTATCGCAAAGGCGGCAAAGGCCTGCTGCCGGTGCGCTGGATGAGCCCGGAAAGCCTGAAAGATGGCGTGTTTACCACCTATAGCGATGTGTG
GAGCTTTGGCGTGGTGCTGTGGGAAATTGCGACCCTGGCGGAACAGCCGTATCAGGGCCTGAGCAACGAACAGGTGCTGCGCTTTGTGATGGAAGGC
GGCCTGCTGGATAAACCGGATAACTGCCCGGATATGCTGTTTGAACTGATGCGCATGTGCTGGCAGTATAACCCGAAAATGCGCCCGAGCTTTCTGG
AAATTATTAGCAGCATTAAAGAAGAAATGGAACCGGGCTTTCGCGAAGTGAGCTTTTATTATAGCGAAGAAAACAAACTGCCGGAACCGGAAGAACT
GGATCTGGAACCGGAAAACATGGAAAGCGTGCCGCTGGATCCGAGCGCGAGCAGCAGCAGCCTGCCGCTGCCGGATCGCCATAGCGGCCATAAAGC
GGAAAACGGCCCGGGCCCGGGCGTGCTGGTGCTGCGCGCGAGCTTTGATGAACGCCAGCCGTATGCGCATATGAACGGCGGCCGCAAAAACGAACG
CGCGCTGCCGCTGCCGCAGAGCAGCACCTGCGGATCC
~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’
~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’
 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.