Edited to include the whole 5UTR in the promoter.

01/08/2012

The binding sites for the proposed primers are shown. Note: A commercial promoter was found, different from the one designed.

Selected sequence to be used as promoter (1000bp) Negative strand, 19648443 19649442

TATAGGCGTGAGCCACCGCGCCCGGCCTGTAACCTGTTTTCTTTCTTATCCCCAGTGCCTGAGACACCCCTGGTGCAGAGAGA GCCCCTACAAACATGCTGAATAGCCTGCTCCACCTAATCCCTAAATACTGCACAGTAAACCCTTACGTGGGTGTGACAGGAGT CATTTAAACTGTCTCCTTTCTTTCGGATCTTTGGGATGTTTACAATACTTCCCTGTTATAGAAAACAGTGATGACTTTCTTAT TCGTGAATGTTTAGGAAAGGCTGTAGGACCTTTTCGTCACAGAGAAGTATGTTTGAAAAACTTTTGCCACATATTGCCAAATT GCCCCCAGCAGGGCCATCCCGATTTGCCACTATACTTTCGTGTATTGGTAGCAGGAATCGCCTCCACACCTCTTTGTGGAAAT CTGCAGCTCTCTGAATCCTTGGCGGGCATGGGCGTGCGTCTCCCAGGTGTGGCACGCGTGGTTTACACACCAAGCCTGGTTCA GCTCTGCCAAGAAACCAGCGCTTTTTAGAACAATGGCTTAGCCAGGAGGGTGGTGCTGGGTTTTTCTTTATCGGCTTGGGTGT GGAATCGCGGACTGCGCCCCTCCAATCTGATCTGGAGAGTGGCTGACTGCGTGTGCAGAGCTCTGGGGGTGTTGGTCTGGGAT TGTGCCATCAGCTTTGCAAGCAAGTAAGGGAGCGGAAAAGGCCGGGAAAGGCCCTGCCGCGAGCACGCTGCCAAGAGCCCCCA GCAGCAGTTCGGCTTAGGACTCGGGTTGCGGCGGGTGTCACCTTCTCAGGGGCTAGCAAGGCAGCCAGGGCCCAGGCGTCTGA GTGAGGGGCGGGAGAGGAGGCGAGGCAGAAAGTGGACCTTCCAGCGGAAAGGCCATTTTCCCCAAGGCCGAGCCCAGGGAAGT CCCTTCCTATAGAATTCAGGCAGGGTGGGAGGCAGGGCGCGCTCGTGCCCCTCAGCCAGCTGCAGGTGCTCTCTGTCCCCAGG CGCC

EcoRI site:

Primers: http://www.yeastgenome.org/tmp/sorted.tmp.1163.html These primers are for PCR Forward-Primer TATAGGCGTGAGCCACCG Reverse-Primer GACAGAGAGCACCTGCAGCT Length: 18 Length: 20 Percent GC: 61 Percent GC: 60 Tm: 54 Tm: 54 Self-Anneal: 16 Self-Anneal: 20 End-Anneal: 8 End-Anneal: 12 Offset: 1 Offset: 990 Pair-Anneal: 14 Pair-End-Anneal: 10 Total valid primer pairs: 69 NOTE: The reverse primer does not capture the 5UTR, needs to be changed These primers will amplify a fragment of 990 basepairs The sequence of DNA that will be amplified by these primers is

1 TATAGGCGTG 51 CCCAGTGCCT 101 GAATAGCCTG 151 TGGGTGTGAC 201 GATGTTTACA 251 CGTGAATGTT 301 TTTGAAAAAC 351 CCGATTTGCC 401 CCTCTTTGTG 451 GCGTCTCCCA 501 TCTGCCAAGA 551 GTGCTGGGTT 601 CTCCAATCTG 651 TGTTGGTCTG 701 AAAGGCCGGG 751 GCAGTTCGGC 801 TAGCAAGGCA 851 CGAGGCAGAA 901 AGCCCAGGGA 951 CGCGCTCGTG

AGCCACCGCG GAGACACCCC CTCCACCTAA AGGAGTCATT ATACTTCCCT TAGGAAAGGC TTTTGCCACA ACTATACTTT GAAATCTGCA GGTGTGGCAC AACCAGCGCT TTTCTTTATC ATCTGGAGAG GGATTGTGCC AAAGGCCCTG TTAGGACTCG GCCAGGGCCC AGTGGACCTT AGTCCCTTCC CCCCTCAGCC

CCCGGCCTGT TGGTGCAGAG TCCCTAAATA TAAACTGTCT GTTATAGAAA TGTAGGACCT TATTGCCAAA CGTGTATTGG GCTCTCTGAA GCGTGGTTTA TTTTAGAACA GGCTTGGGTG TGGCTGACTG ATCAGCTTTG CCGCGAGCAC GGTTGCGGCG AGGCGTCTGA CCAGCGGAAA TATAGAATTC AGCTGCAGGT

AACCTGTTTT AGAGCCCCTA CTGCACAGTA CCTTTCTTTC ACAGTGATGA TTTCGTCACA TTGCCCCCAG TAGCAGGAAT TCCTTGGCGG CACACCAAGC ATGGCTTAGC TGGAATCGCG CGTGTGCAGA CAAGCAAGTA GCTGCCAAGA GGTGTCACCT GTGAGGGGCG GGCCATTTTC AGGCAGGGTG GCTCTCTGTC

CTTTCTTATC CAAACATGCT AACCCTTACG GGATCTTTGG CTTTCTTATT GAGAAGTATG CAGGGCCATC CGCCTCCACA GCATGGGCGT CTGGTTCAGC CAGGAGGGTG GACTGCGCCC GCTCTGGGGG AGGGAGCGGA GCCCCCAGCA TCTCAGGGGC GGAGAGGAGG CCCAAGGCCG GGAGGCAGGG

Commercial promoter sequence: Negative strand, 19651550 19652569

TGTGCTATTTTCCACAGCACATTGGAGCGTAGCCGTGTGGGTGGATGTAGGGGAAGGGGG CTCCAGGTGGAGAGAAGCAGGAGGGCCAGAGCACATCCTCAGGACTCGTAGGCCAGGACC GGAACCTTGTGTTTTCCTCGAATGAAATGGGAAGCAGGGACCCCATGCTGGTAAGCAGTC TGCCTGCAGGCGGGTGAGGGTGGCGGGAAGCGCCTGGTGAGAGGGGCCGAGAGTCTGTTC CACACCTGCCTCCTCCCCACAGCGTGGTGCAGAGCAGGAGCCCAGCGAACATTGATCAGA CAGCGTGGGAACGGCTTTTCCAGACTCCTTCCTTCCCAAGCATCTCTGGTTTTCGGTGAC CACACAGACCCTTGGCCCTCTTCTCAGCTATTCAACGAGAGAGCAGATTTATGGATTGGA GACCCCCATCAAAAGTCCCCAGCCTGTGGCTCCCCAGGGTTAGGATCTCTGACTCAATGG GGCCCTGTGCACACTGGGGGATGTGAAGGGCCCGGGGCAGCGGTGGCTGGGGGTGCGTTT CGGGGGAGCTGCATGCTCTCACTGTGGTTCGTGGCTGAGCCCTGCCGATCTTATGTAACC AGAGAACTCAGAGAACCGCATCTGGCAGCGGCAAGTCTGGAAAGCTGGAAGAGCTCCATG CCAGGCTGAATCAATCAGCAGCCCCCACGCCCAGGGCAAACATAGGCTCTTTTGAAGATT GGAGATGTGCCCTGCCCAAGCCTTCAGAAACATTCTCGCATTTCAGAAGTGAACAAAAGC AAACAGCCCGGGACCTAATCCCCAAAACCTGGGCTGTAGGGAGCAGAGGTCCAGAGGACG CCACACCCCACATTTGAATATTTTTTTTTCTTGTCACGCAAACTTCCTGGGCAGGAACTC CAAGGGCTCAATGGACGGGAAGCAGGGTCCTTAAATACGTCCCCTCTTGGCTCTTGCCGT

TCCAGGAGCCCCAGTTACCGGGAGAGGCTGTGTCAAAGGTAATCCTCTTCCCTGCATTCC T

PstI site:

This sequence does not contain the 5 UTR.