IGEM:UCSF/2007/Plasmids

From 2007.igem.org

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<u>'''PROJECT 2</u>
<u>'''PROJECT 2</u>
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(1) PspOMI//pSte2-Ste2//XhoI//GFP//XmaI//RR//STOP//BamHI//AdhTerminator
 +
 +
GGGCCCATCCAATATCACCTGACCTTCATCTTTATGTTTTTCCACTGGTGTAATGTACTCTGTTGCATCATCAATAAGCAACTGGGACGCCAA
 +
TAATTCATACCAACAATCTTTTCTTAATGAGTTGTTTACAAACCCCAGAGGCCCTAAACCTATGTACCTCAAAGCTCGGACATCTTTTTTCTC
 +
TAATAACTTTGATATAACTTTTCCTTTTTGTTCTTCTGCCTTTCCACTGTCACAACGAGAAAGTGATTCCAGATATGCGTTATAACCTCCCCT
 +
TGTCAACGCGTCTTTGTCGTGGCTACTCTGATTAGTATGAAATAATGACCTTAATGGCATCGTATTTTGTTAATTGGCTTGTTCTATGTCATT
 +
TGCTAAATTTTCTCTTTATTTCTTTACTTTGCCAATTCAAGATAATCTTCCCTTCCCAGAGAGAAAAAAGGAAAAATTTAGCTATGAAACCTC
 +
AATAAGCTTTTTAATACACCAAAGATTCAAGATAAGAGCATAGAACGAACTGTAGAATAGTCCGGATATGTTATCCAATGCCTGCCAAAATGC
 +
ATTGTCACACGCTGTAGTGCTCGAATAGGTGTTGCAATCCGTCAATATACGTCTTGCTCTGTGGGTAAATGTCTCGTGCATTAAGACAGGCTA
 +
GTATAAACGAGAAGAAGTATCCTGCTTTGCAATGAAACAATAGTATCCGCTAAGAATTTAAGCAGGCCAACGTCCATACTGCTTAGGACCTGT
 +
GCCTGGCAAGTCGCAGATTGAAGTTTTTTCAACCATGTAAATTTCCTAATTGGGTAAGTACATGATGAAACACATATGAAGAAAAAAGCTTTC
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CTACATATTCAAGATTTTTTTCTGTGGGTGGAATACTATTTAAGGAGTGCTATTAGTATCTTATTTGACTTCAAAGCAATACGATACCTTTTC
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TTTTCACCTGCTCTGGCTATAATTATAATTGGTTACTTAAAAATGCACCGTTAAGAACCATATCCAAGAATCAAAAATGTCTGATGCGGCTCC
 +
TTCATTGAGCAATCTATTTTATGATCCAACGTATAATCCTGGTCAAAGCACCATTAACTACACTTCCATATATGGGAATGGATCTACCATCAC
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TTTCGATGAGTTGCAAGGTTTAGTTAACAGTACTGTTACTCAGGCCATTATGTTTGGTGTCAGATGTGGTGCAGCTGCTTTGACTTTGATTGT
 +
CATGTGGATGACATCGAGAAGCAGAAAAACGCCGATTTTCATTATCAACCAAGTTTCATTGTTTTTAATCATTTTGCATTCTGCACTCTATTT
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TAAATATTTACTGTCTAATTACTCTTCAGTGACTTACGCTCTCACCGGATTTCCTCAGTTCATCAGTAGAGGTGACGTTCATGTTTATGGTGC
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TACAAATATAATTCAAGTCCTTCTTGTGGCTTCTATTGAGACTTCACTGGTGTTTCAGATAAAAGTTATTTTCACAGGCGACAACTTCAAAAG
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GATAGGTTTGATGCTGACGTCGATATCTTTCACTTTAGGGATTGCTACAGTTACCATGTATTTTGTAAGCGCTGTTAAAGGTATGATTGTGAC
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TTATAATGATGTTAGTGCCACCCAAGATAAATACTTCAATGCATCCACAATTTTACTTGCATCCTCAATAAACTTTATGTCATTTGTCCTGGT
 +
AGTTAAATTGATTTTAGCTATTAGATCAAGAAGATTCCTTGGTCTCAAGCAGTTCGATAGTTTCCATATTTTACTCATAATGTCATGTCAATC
 +
TTTGTTGGTTCCATCGATAATATTCATCCTCGCATACAGTTTGGAACCAAACCAGGGAACAGATGTCTTGACTACTGTTGCAACATTACTTGC
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TGTATTGTCTTTACCATTATCATCAATGTGGGCCACGGCTGCTAATAATGCATCCAAAACAAACACAATTACTTCAGACTTTACAACATCCAC
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AGATAGGTTTTATCCAGGCACGCTGTCTAGCTTTCAAACTGATAGTATCAACAACGATGCTAAAAGCAGTCTCAGAAGTAGATTATATGACCT
 +
ATATCCTAGAAGGAAGGAAACAACATCGGATAAACATTCGGAAAGAACTTTTGTTTCTGAGACTGCAGATGATATAGAGAAAAATCAGTTTTA
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TCAGTTGCCCACACCTACGAGTTCAAAAAATACTAGGATAGGACCGTTTGCTGATGCAAGTTACAAAGAGGGAGAAGTTGAACCCGTCGACAT
 +
GTACACTCCCGATACGGCAGCTGATGAGGAAGCCAGAAAGTTCTGGACTGAAGATAATAATAATTTACTCGAGTCTAAAGGTGAAGAATTATT
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CACTGGTGTTGTCCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGAAGGTGAAGGTGATGCTACTTA
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CGGTAAATTGACCTTAAAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATGGCCAACCTTAGTCACTACTTTAACTTATGGTGTTCAATG
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TTTTTCTAGATACCCAGATCATATGAAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAGAAAGAACTATTTTTTTCAA
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AGATGACGGTAACTACAAGACCAGAGCTGAAGTCAAGTTTGAAGGTGATACCTTAGTTAATAGAATCGAATTAAAAGGTATTGATTTTAAAGA
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AGATGGTAACATTTTAGGTCACAAATTGGAATACAACTATAACTCTCACAATGTTTACATCATGGCTGACAAACAAAAGAATGGTATCAAAGT
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TAACTTCAAAATTAGACACAACATTGAAGATGGTTCTGTTCAATTAGCTGACCATTATCAACAAAATACTCCAATTGGTGATGGTCCAGTCTT
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GTTACCAGACAACCATTACTTATCCACTCAATCTaggTTATCCAAAGATCCAAACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTAC
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TGCTGCTGGTATTACCCATGGTATGGATGAATTGTACAAACCCGGGggatctggatcgggatctGATCCAGATTTGGAAATTAGAGCTGCTTT
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TTTGAGACAAAGAAATACTGCTTTGAGAACTGAAGTTGCTGAATTGGAACAAGAAGTTCAAAGATTGGAAAATGAAGTTTCTCAATATGAAAC
 +
TAGATATGGTCCATTGGGTGGTGGTAAATAAGGATCCGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAAATAA
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GTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTGGTCAGGTTGCTTTCTCAGGT
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ATAGCATGAGGTCGCTCTTATTGACCACACC
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 +
 +
(2) PspOMI//pGal1//ATG//Xho//[A]//HMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator
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 +
TGTTATTGTGATAGTATCTCAGCGGGATTTGTGTAGATTCACCTACAGCCTTTAAACCAGCAAAGTGAATTACCGAATCAATTTTATATTCTT
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TGAAAACCTTTTCCAGACCTTTTCGGTCACACAAATCAACCTCATAGAAGGGAATGTGATGCTTGGTCAAGACCTCTAACCTGGCTACAGAAT
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CATAAGTTGAATTCGACAGGTTATCAGCAACAACACAGTCATATCCATTCTCAATTAGCTCTACCACAGTGTGTGAACCAATGTATCCAGCAC
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CACCTGTAACCAAAACAATTTTAGAAGTACTTTCACTTTGTAACTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGG
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ATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAA
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AGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGA
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AGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTC
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GCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCT
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TCAAATTAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGAT
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TTTTGATCTATTAACAGATATATAAATGGAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCAGTTTGTATTACTTCTTATTCA
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AATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGCTCGAGGGAGCTgcttct
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gcatcaacttctaaatataattcacactccttggagaatgagtctattaagaggacgtctcgagatggagtcaatcgagatctcactgaggct
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gttcctcgacttccaggagaaacactaatcactgacaaagaagttatttacatatgtcctttcaatggccccattaagggaagagtttacatc
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acaaattatcgtctttatttaagaagtttggaaacggattcttctctaatacttgatgttcctctgggtgtgatctcgagaattgaaaaaatg
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ggaggcgcgacaagtagaggagaaaattcctatggtctagatattacttgtaaagacatgagaaacctgaggttcgctttgaaacaggaaggc
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cacagcagaagagatatgtttgagatcctcacgagatacgcgtttcccctggctcacagtctgccattatttgcatttttaaatgaagaaaag
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tttaacgtggatggatggacagtttacaatccagtggaagaatacaggaggcagggcttgcccaatcaccattggagaataacttttattaat
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aagtgctatgagctctgtgacacttaccctgctcttttggtggttccgtatcgtgcctcagatgatgacctccggagagttgcaacttttagg
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tcccgaaatcgaattccagtgctgtcatggattcatccagaaaataagacggtcattgtgcgttgcagtcagcctcttgtcggtatgagtggg
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aaacgaaataaagatgatgagaaatatctcgatgttatcagggagactaataaacaaatttctaaactcaccatttatgatgcaagacccagc
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gtaaatgcagtggccaacaaggcaacaggaggaggatatgaaagtgatgatgcatatcataacgccgaacttttcttcttagacattcataat
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attcatgttatgcgggaatctttaaaaaaagtgaaggacattgtttatcctaatgtagaagaatctcattggttgtccagtttggagtctact
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cattggttagaacatatcaagctcgttttgacaggagccattcaagtagcagacaaagtttcttcagggaagagttcagtgcttgtgcattgc
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agtgacggatgggacaggactgctcagctgacatccttggccatgctgatgttggatagcttctataggagcattgaagggttcgaaatactg
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gtacaaaaagaatggataagttttggacataaatttgcatctcgaataggtcatggtgataaaaaccacaccgatgctgaccgttctcctatt
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tttctccagtttattgattgtgtgtggcaaatgtcaaaacagttccctacagcttttgaattcaatgaacaatttttgattataattttggat
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catctgtatagttgccgatttggtactttcttattcaactgtgaatctgctcgagaaagacagaaggttacagaaaggactgtttctttatgg
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tcactgataaacagtaataaagaaaaattcaaaaaccccttctatactaaagaaatcaatcgagttttatatccagttgccagtatgcgtcac
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ttggaactctgggtgaattactacattagatggaaccccaggatcaagcaacaacagccgaatccagtggagcagcgttacatggagctctta
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gccttacgcgacgaatacataaagcggcttgaggaactgcagctcgccaactctgccaagctttctgatcccccaacttcaccttccagtcct
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tcgcaaatgatgccccatgtgcaaactcacttcGGTAGTTCCCTAggttccggtagcggttccggttctatcactattagggctgcttttttg
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gaaaaagaaaatactgctttgagaactgaaattgctgaattggaaaaagaagttggtagatgtgaaaatattgtttctaaatatgaaactaga
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tatggtccattgTAATCGCGGATCCGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAAATAAGTGTATACAAAT
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TTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGT
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CGCTCTTATTGACCACACC
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(3) pGal1//ATG//Xho//[A]//WMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator
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As above, but WMTM1 sequence
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(4) pSte5//ATG//Xho//[A]//HMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator
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As above, but Ste5 promoter sequence:
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CACGAAGTGACTGACAGAATACTGACATCAGCTGATTTCTCATAGAGCTGTTTCTCTGATAACACGTTGTTTGAACATCGACAAGATGAAAATC
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TAGAAGTATCAAGTTTCCTTTAAAGGGATATATAACAGATTCTAAAACTGACAGAAATATTTCGAGTGAAGAAGAAGCGTTAAATATTGGATCT
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TTCCGCAGTTCTACTCTGATACATTTTTGAAGTAGGAGAGTCATTTAGAAGGCGTATTGCTCAATAGTAGAAAGCAGGCCTGTGCACATGAATT
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AATTAAAAAATATAAAGGTAGTGATTAGACGACACATGTCCATAGGTAACCTGTCATAATTTTGAACAATTTCCCTTCTTTTCTTTTTTTTTTT
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TGGGTGCGGCGATATGTAGCTTGTTAATTTACACATCATGTACTTTTCTGCATCAAAATATGAAAGGCGATAGTAGCTAAAGAAAATACCGAGA
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ATTTCCTCGAAAAGTTGACGACAAAAGAAAGGCATAAAAAAGTAATTTGAAAATATTTTAAAACTGTTTTAACCCATCTAGCATCCGCGCTAAA
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AAAGGAAGATACAGGATACAGCGGAAACAACTTTTAAATG
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(5) pSte5//ATG//Xho//[A]//WMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator
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As above.
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(6) pCyc//ATG//Xho//[A]//HMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator
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As above, but Cyc promoter sequence:
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ctcgagcagatccgccaggcgtgtatatatagcgtggatggccaggcaactttagtgctgacacatacaggcatatatatatgtgtgcgacgaca
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catgatcatatggcatgcatgtgctctgtatgtatataaaactcttgttttcttcttttctctaaatattctttccttatacattaggacctttg
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cagcataaattactatacttctatagacacacaaacacaaatacacacactaaattaataatg
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(7) pCyc//ATG//Xho//[A]//WMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator
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 +
As above.
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(8) pCyc//ATG//XhoI//GFP//XmaI//(SerGlySerGlySer)ING2 PH domain//STOP//BamHI
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As above, but //(SerGlySerGlySer)ING2 PH domain sequence as follows:
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TCTGGATCAGGTTCAGCTTCTCCAGTTGAATTTGCTATTGATCCTAACGAACCTACATACTGCTTATGTAATCAAGTTTCTTATGGTGAAATGAT
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TGGTTGCGATAACGAGCAGTGTCCAATAGAATGGTTTCATTTCTCTTGCGTTTCTTTAACATATAAACCTAAGGGAAAATGGTACTGTCCTAAAT
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GTAGAGGTGATAATGAGAAAACTATGGATAAGTCTACTGAAAAGACAAAAAAAGATAGAAGATCAAGA

Latest revision as of 20:12, 26 October 2007

PROJECT 1

Expected sequences

Below you have the sequences of the different parts in the right order, as well as the adaptor sequences between parts. You will need to arrange them accordingly to your particular constructs, so that you will have 1-2-3-4-5-6-7-8.


1-Promoter sequence (variable, depends on particular acceptor)


pFig1

TTGAATGATCAACCAAACGCCGATATAGTCACTATTGGTGATAAAATTAAAATGC AGCTATTGAGAACCCATCCTAACAACATTAAATTGTCTATTAATGGAATTGGTAA AGATGCCCCAACTTTCCAAGAATCTGCTTTGATTGCCGATAAGTTATTGAGTGTC ATGAAGGCCGGCACTTACCCAAAGATTTCCATTTTCTACAATGACCCAGTGTCTT CCCTATCTTTTGAACCATCTGAAAAACCGATCTTTAACGCCAAGACCATTGAACA ATCCCCATCATTCGGCAAATTTGAGATCGACACGGACGCAAACGTTCCAAGAGAT TTGTTTGAATATACTTTGGCTAACCAAATGTTGACAGCAATGGCTCAAGGTTATG CTGCTGAAATTTCCGCCAGAAGAAACGCTATGGATAACGCTTCCAAGAATGCCGG TGATATGATCAATCGTTACTCTATCTTGTACAACAGAACAAGACAAGCTGTCATT ACTAATGAACTGGTTGATATTATTACTGGTGCTTCCTCTTTGGGATGATAAAAAA ATCACCCTGCATTGCCTCTTTATTTGACGTTGTTTTTGTAGAACATGAAACGAAT TTTGACTTGATGAGACGAAGTATATATCCAAAGAATACCTTAAATAGAAAAGGAA AGATAATAAATACTAAACACTACTATATATTCAGGTAAAATACAAAAATTATAAC ATTTTTAAAACTTTTTTTTTTTGAAAGTCCTTCTCGCTTTAGGATTTTTTCCCAT TAAGATTATGATGGTTTCATGTATGTGTCAGTTAAAAAAAATATGGCTAAGTAGC AATGAAACGAACCAAGAAAATGAAAACAATATATAGTGCTGTTGAAATAACAAAG ACATTGGTATATATTTGTAAAATGTCTGTTAAATGTTTTTTATCTCAGGTTCTTG CTTGTCTTTGGTAGAAGAAATTATAGTAAACAAACAAACAAACAAACAAAAAAAA AAAAAAAAAA

pAdh

TTAAAACAAGAAGAGGGTTGACTACATCACGATGAGGGGGATCGAAGAAATGATG GTAAATGAAATAGGAAATCAAGGAGCATGAAGGCAAAAGACAAATATAAGGGTCG AACGAAAAATAAAGTGAAAAGTGTTGATATGATGTATTTGGCTTTGCGGCGCCGA AAAAACGAGTTTACGCAATTGCACAATCATGCTGACTCTGTGGCGGACCCGCGCT CTTGCCGGCCCGGCGATAACGCTGGGCGTGAGGCTGTGCCCGGCGGAGTTTTTTG CGCCTGCATTTTCCAAGGTTTACCCTGCGCTAAGGGGCGAGATTGGAGAAGCAAT AAGAATGCCGGTTGGGGTTGCGATGATGACGACCACGACAACTGGTGTCATTATT TAAGTTGCCGAAAGAACCTGAGTGCATTTGCAACATGAGTATACTAGAAGAATGA GCCAAGACTTGCGAGACGCGAGTTTGCCGGTGGTGCGAACAATAGAGCGACCATG ACCTTGAAGGTGAGACGCGCATAACCGCTAGAGTACTTTGAAGAGGAAACAGCAA TAGGGTTGCTACCAGTATAAATAGACAGGTACATACAACACTGGAAATGGTTGTC TGTTTGAGTACGCTTTCAATTCATTTGGGTGTGCACTTTATTATGTTACAATATG GAAGGGAACTTTACACTTCTCCTATGCACATATATTAATTAAAGTCCAATGCTAG TAGAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTTTTCTAAACCGT GGAATATTTCGGATATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCT TTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCC CTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGAC ATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACAT AACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCA CTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCT TTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATG ACAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCA ACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTT TTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTC CTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAAGTA TAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCT TTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATC AACTATCTCATATACA


pCyc

CAGATCCGCCAGGCGTGTATATATAGCGTGGATGGCCAGGCAACTTTAGTGCTGA CACATACAGGCATATATATATGTGTGCGACGACACATGATCATATGGCATGCATG TGCTCTGTATGTATATAAAACTCTTGTTTTCTTCTTTTCTCTAAATATTCTTTCC TTATACATTAGGACCTTTGCAGCATAAATTACTATACTTCTATAGACACACAAAC ACAAATACACACACTAAATTAATA


pSte5

CACGAAGTGACTGACAGAATACTGACATCAGCTGATTTCTCATAGAGCTGTTTCT CTGATAACACGTTGTTTGAACATCGACAAGATGAAAATCTAGAAGTATCAAGTTT CCTTTAAAGGGATATATAACAGATTCTAAAACTGACAGAAATATTTCGAGTGAAG AAGAAGCGTTAAATATTGGATCTTTCCGCAGTTCTACTCTGATACATTTTTGAAG TAGGAGAGTCATTTAGAAGGCGTATTGCTCAATAGTAGAAAGCAGGCCTGTGCAC ATGAATTAATTAAAAAATATAAAGGTAGTGATTAGACGACACATGTCCATAGGTA ACCTGTCATAATTTTGAACAATTTCCCTTCTTTTCTTTTTTTTTTTTGGGTGCGG CGATATGTAGCTTGTTAATTTACACATCATGTACTTTTCTGCATCAAAATATGAA AGGCGATAGTAGCTAAAGAAAATACCGAGAATTTCCTCGAAAAGTTGACGACAAA AGAAAGGCATAAAAAAGTAATTTGAAAATATTTTAAAACTGTTTTAACCCATCTA GCATCCGCGCTAAAAAAGGAAGATACAGGATACAGCGGAAACAACTTTTAA


2-ATG-XhoI-“A” and 2 adaptor bases: ATGCTCGAGGGAGCT


3-mCherry:

GTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACG AGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCC CTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAG CTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGC AGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTG GGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCAC TACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCA CCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG


4-Adaptor bases, followed by “B” and another adaptor base: GGTAGTTCCCTA


5-Effectors

For YopH:

AACTTATCATTAAGCGATCTTCATCGTCAGGTATCTCGATTGGTGCAGCAAGAGAGCGGTGATTGTACCGGGAAATTAAGAGGTAACGT TGCTGCCAATAAAGAAACTACCTTTCAAGGTTTGACCATAGCCAGTGGAGCCAGAGAGTCAGAAAAAGTATTTGCTCAAACTGTACTAA GCCACGTAGCAAATGTTGTTCTAACTCAAGAAGATACCGCTAAGCTATTGCAAAGTACGGTAAAGCATAATTTGAATAATTATGACTTA AGAAGTGTCGGCAATGGTAATAGTGTACTTGTCAGTTTACGTAGTGACCAAATGACACTACAAGACGCCAAAGTGCTGTTGGAGGCCGC ATTGCGACAAGAGTCGGGAGCGAGGGGGCATGTATCATCTCATTCACATTCAGCCCTTCACGCACCGGGAACCCCGGTGCGTGAAGGAC TGCGTTCACATCTAGACCCCAGAACTCCACCGTTGCCACCGCGTGAACGACCACACACTTCTGGCCATCACGGGGCTGGCGAAGCCAGA GCCACCGCACCAAGCACTGTTTCTCCTTATGGCCCAGAAGCGCGCGCAGAACTCAGCAGCCGCCTCACCACATTGCGCAATACGCTGGC GCCAGCAACGAATGATCCGCGTTACTTACAAGCCTGCGGCGGTGAAAAGCTAAACCGATTTAGAGATATTCAATGCTGTCGGCAAACCG CAGTACGCGCCGATCTTAATGCCAATTACATCCAGGTCGGTAACACTCGTACCATAGCGTGCCAGTATCCGCTACAATCTCAACTTGAA AGCCATTTCCGTATGCTGGCAGAAAACCGAACGCCAGTGTTGGCTGTTTTAGCGTCCAGTTCTGAGATAGCCAATCAAAGATTCGGTAT GCCAGATTATTTCCGCCAGAGTGGTACCTATGGCAGTATCACTGTAGAGTCTAAAATGACTCAGCAAGTTGGTCTCGGTGACGGGATTA TGGCAGATATGTATACTTTAACGATTCGTGAAGCGGGTCAAAAAACAATCTCTGTTCCTGTGGTTCATGTTGGCAATTGGCCCGATCAG ACCGCAGTCAGCTCTGAAGTTACCAAGGCACTCGCTTCACTGGTAGATCAAACAGCAGAAACAAAACGCAATATGTATGAAAGCAAAGG AAGTTCAGCGGTAGGAGATGACTCCAAATTACGGCCGGTAATACATTGCCGTGCGGGTGTTGGCCGTACTGCGCAACTGATTGGCGCAA TGTGCATGAATGATAGTCGTAATAGTCAGTTAAGCGTAGAAGATATGGTCAGCCAAATGCGAGTACAAAGAAATGGTATTATGGTACAA AAAGATGAGCAACTTGATGTTCTGATTAAGTTGGCTGAAGGACAAGGGCGACCATTATTAAATAGC

For YopJ:

ATCGGACCAATATCACAAATAAATATCTCCGGTGGCTTATCAGAAAAAGAGACCAGTTCTTTAATCAGTAATGAAGAGCTTAAAAATAT CATAACACAGTTGGAAACTGATATATCGGATGGATCCTGGTTCCATAAAAATTATTCACGTATGGATGTAGAAGTCATGCCCGCATTGG TAATCCAGGCGAACAATAAATATCCGGAAATGAATCTTAATCTTGTTACATCTCCATTGGACCTTTCAATAGAAATAAAAAACGTCATA GAAAATGGAGTTAGATCTTCCCGCTTCATAATTAACATGGGGGAAGGTGGAATACATTTCAGTGTAATTGATTACAAACATATAAATGG GAAAACATCTCTGATATTGTTTGAACCAGCAAACTTTAACAGTATGGGGCCAGCGATGCTGGCAATAAGGACAAAAACGGCTATTGAAC GTTATCAATTACCTGATTGCCATTTCTCCATGGTGGAAATGGATATTCAGCGAAGCTCATCTGAATGTGGTATTTTTAGTTTGGCACTG GCAAAAAAACTTTACATCGAGAGAGATAGCCTGTTGAAAATACATGAAGATAATATAAAAGGTATATTAAGTGATGGTGAAAATCCTTT ACCCCACGATAAGTTGGACCCGTATCTCCCGGTAACTTTTTACAAACATACTCAAGGTAAAAAACGTCTTAATGAATATTTAAATACTA ACCCGCAGGGAGTTGGTACTGTTGTTAACAAAAAAAATGAAACCATCGTTAATAGATTTGATAACAATAAATCCATTGTAGATGGAAAG GATTATCAGTTTCGGTACATAAAAAGAGAATAGCTGAATATAAAACACTTCTCAAAGTA

For OspF:

CCAATTAAAAAGCCTTGTTTGAAATTGAATTTAGATTCTTTGAACGTTGTTAGATCAGAAATTCCACAAATGTTATCTGCTAATGAAAGAT TGAAAAATAATTTTAACATTTTATATAATCAAATAAGACAGTACCCAGCTTATTATTTTAAAGTTGCTTCTAATGTTCCTACTTACTCTGA TATATGTCAATTTTTCTCAGTTATGTATCAAGGTTTTCAGATAGTTAATCATTCTGGTGATGTTTTTATTCATGCTTGTAGAGAAAATCCA CAATCTAAAGGTGATTTTGTTGGTGACAAGTTCCATATTTCTATAGCTAGAGAACAAGTTCCATTGGCATTTCAAATTTTATCTGGTTTGTT ATTTTCTGAGGACTCTCCTATTGACAAATGGAAGATAACTGATATGAATAGAGTTTCTCAACAGTCAAGAGTTGGTATTGGTGCTCAATTTAC TTTGTATGTTAAATCTGATCAAGAATGTTCTCAGTATTCAGCTTTATTGTTGCATAAAATTAGACAATTCATAATGTGCTTGGAATCTAATTT GTTAAGATCTAAGATTGCTCCAGGTGAATATCCTGCATCTGATGTTAGACCAGAGGATTGGAAGTACGTTTCATATAGAAATGAATTGAGATC TGACAGAGATGGTTCTGAAAGACAAGAGCAGATGTTAAGAGAAGAACCTTTTTATAGATTGATGATTGAG


6-Adaptor bases, followed by “C” followed by another adaptor: GGTAGCGAT


7-Zippers: For RR: GGTTCTGGTTCTGGTTCTGGTTCTCTGGAAATCCGTGCGGCGTTCCTGGAAAAAGAAAACACCGCGCTGCGTACCCGTGCGGCGGAACTGCG TAAACGTGTTGGTCGTTGCCGTAACATCGTTTCTAAATACGAAACCCGTTACGGTCCGCTG For EE:

GGTTCCGGTAGCGGTTCCGGTTCTATCACTATTAGGGCTGCTTTTTTGGAAAAAGAAAATACTGCTTTGAGAACTGAAATTGCTGAATTGG AAAAAGAAGTTGGTAGATGTGAAAATATTGTTTCTAAATATGAAACTAGATATGGTCCATTG


8-Final adaptors, followed by “D”, followed by BamHI, 3 STOPS in three different frames, then NotI, the Adh terminator and SacI:


TAATGCGGGATCCTGAGTAAATAAGCGGCCGCGCGAATTTC TTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAAATAAGTGTAT ACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGT AACTCTTTCCTGTGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTT ATTGACCACACCGAGCTC


PROJECT 2


(1) PspOMI//pSte2-Ste2//XhoI//GFP//XmaI//RR//STOP//BamHI//AdhTerminator

GGGCCCATCCAATATCACCTGACCTTCATCTTTATGTTTTTCCACTGGTGTAATGTACTCTGTTGCATCATCAATAAGCAACTGGGACGCCAA TAATTCATACCAACAATCTTTTCTTAATGAGTTGTTTACAAACCCCAGAGGCCCTAAACCTATGTACCTCAAAGCTCGGACATCTTTTTTCTC TAATAACTTTGATATAACTTTTCCTTTTTGTTCTTCTGCCTTTCCACTGTCACAACGAGAAAGTGATTCCAGATATGCGTTATAACCTCCCCT TGTCAACGCGTCTTTGTCGTGGCTACTCTGATTAGTATGAAATAATGACCTTAATGGCATCGTATTTTGTTAATTGGCTTGTTCTATGTCATT TGCTAAATTTTCTCTTTATTTCTTTACTTTGCCAATTCAAGATAATCTTCCCTTCCCAGAGAGAAAAAAGGAAAAATTTAGCTATGAAACCTC AATAAGCTTTTTAATACACCAAAGATTCAAGATAAGAGCATAGAACGAACTGTAGAATAGTCCGGATATGTTATCCAATGCCTGCCAAAATGC ATTGTCACACGCTGTAGTGCTCGAATAGGTGTTGCAATCCGTCAATATACGTCTTGCTCTGTGGGTAAATGTCTCGTGCATTAAGACAGGCTA GTATAAACGAGAAGAAGTATCCTGCTTTGCAATGAAACAATAGTATCCGCTAAGAATTTAAGCAGGCCAACGTCCATACTGCTTAGGACCTGT GCCTGGCAAGTCGCAGATTGAAGTTTTTTCAACCATGTAAATTTCCTAATTGGGTAAGTACATGATGAAACACATATGAAGAAAAAAGCTTTC CTACATATTCAAGATTTTTTTCTGTGGGTGGAATACTATTTAAGGAGTGCTATTAGTATCTTATTTGACTTCAAAGCAATACGATACCTTTTC TTTTCACCTGCTCTGGCTATAATTATAATTGGTTACTTAAAAATGCACCGTTAAGAACCATATCCAAGAATCAAAAATGTCTGATGCGGCTCC TTCATTGAGCAATCTATTTTATGATCCAACGTATAATCCTGGTCAAAGCACCATTAACTACACTTCCATATATGGGAATGGATCTACCATCAC TTTCGATGAGTTGCAAGGTTTAGTTAACAGTACTGTTACTCAGGCCATTATGTTTGGTGTCAGATGTGGTGCAGCTGCTTTGACTTTGATTGT CATGTGGATGACATCGAGAAGCAGAAAAACGCCGATTTTCATTATCAACCAAGTTTCATTGTTTTTAATCATTTTGCATTCTGCACTCTATTT TAAATATTTACTGTCTAATTACTCTTCAGTGACTTACGCTCTCACCGGATTTCCTCAGTTCATCAGTAGAGGTGACGTTCATGTTTATGGTGC TACAAATATAATTCAAGTCCTTCTTGTGGCTTCTATTGAGACTTCACTGGTGTTTCAGATAAAAGTTATTTTCACAGGCGACAACTTCAAAAG GATAGGTTTGATGCTGACGTCGATATCTTTCACTTTAGGGATTGCTACAGTTACCATGTATTTTGTAAGCGCTGTTAAAGGTATGATTGTGAC TTATAATGATGTTAGTGCCACCCAAGATAAATACTTCAATGCATCCACAATTTTACTTGCATCCTCAATAAACTTTATGTCATTTGTCCTGGT AGTTAAATTGATTTTAGCTATTAGATCAAGAAGATTCCTTGGTCTCAAGCAGTTCGATAGTTTCCATATTTTACTCATAATGTCATGTCAATC TTTGTTGGTTCCATCGATAATATTCATCCTCGCATACAGTTTGGAACCAAACCAGGGAACAGATGTCTTGACTACTGTTGCAACATTACTTGC TGTATTGTCTTTACCATTATCATCAATGTGGGCCACGGCTGCTAATAATGCATCCAAAACAAACACAATTACTTCAGACTTTACAACATCCAC AGATAGGTTTTATCCAGGCACGCTGTCTAGCTTTCAAACTGATAGTATCAACAACGATGCTAAAAGCAGTCTCAGAAGTAGATTATATGACCT ATATCCTAGAAGGAAGGAAACAACATCGGATAAACATTCGGAAAGAACTTTTGTTTCTGAGACTGCAGATGATATAGAGAAAAATCAGTTTTA TCAGTTGCCCACACCTACGAGTTCAAAAAATACTAGGATAGGACCGTTTGCTGATGCAAGTTACAAAGAGGGAGAAGTTGAACCCGTCGACAT GTACACTCCCGATACGGCAGCTGATGAGGAAGCCAGAAAGTTCTGGACTGAAGATAATAATAATTTACTCGAGTCTAAAGGTGAAGAATTATT CACTGGTGTTGTCCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGAAGGTGAAGGTGATGCTACTTA CGGTAAATTGACCTTAAAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATGGCCAACCTTAGTCACTACTTTAACTTATGGTGTTCAATG TTTTTCTAGATACCCAGATCATATGAAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAGAAAGAACTATTTTTTTCAA AGATGACGGTAACTACAAGACCAGAGCTGAAGTCAAGTTTGAAGGTGATACCTTAGTTAATAGAATCGAATTAAAAGGTATTGATTTTAAAGA AGATGGTAACATTTTAGGTCACAAATTGGAATACAACTATAACTCTCACAATGTTTACATCATGGCTGACAAACAAAAGAATGGTATCAAAGT TAACTTCAAAATTAGACACAACATTGAAGATGGTTCTGTTCAATTAGCTGACCATTATCAACAAAATACTCCAATTGGTGATGGTCCAGTCTT GTTACCAGACAACCATTACTTATCCACTCAATCTaggTTATCCAAAGATCCAAACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTAC TGCTGCTGGTATTACCCATGGTATGGATGAATTGTACAAACCCGGGggatctggatcgggatctGATCCAGATTTGGAAATTAGAGCTGCTTT TTTGAGACAAAGAAATACTGCTTTGAGAACTGAAGTTGCTGAATTGGAACAAGAAGTTCAAAGATTGGAAAATGAAGTTTCTCAATATGAAAC TAGATATGGTCCATTGGGTGGTGGTAAATAAGGATCCGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAAATAA GTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTGGTCAGGTTGCTTTCTCAGGT ATAGCATGAGGTCGCTCTTATTGACCACACC


(2) PspOMI//pGal1//ATG//Xho//[A]//HMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator

TGTTATTGTGATAGTATCTCAGCGGGATTTGTGTAGATTCACCTACAGCCTTTAAACCAGCAAAGTGAATTACCGAATCAATTTTATATTCTT TGAAAACCTTTTCCAGACCTTTTCGGTCACACAAATCAACCTCATAGAAGGGAATGTGATGCTTGGTCAAGACCTCTAACCTGGCTACAGAAT CATAAGTTGAATTCGACAGGTTATCAGCAACAACACAGTCATATCCATTCTCAATTAGCTCTACCACAGTGTGTGAACCAATGTATCCAGCAC CACCTGTAACCAAAACAATTTTAGAAGTACTTTCACTTTGTAACTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGG ATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAA AGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGA AGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTC GCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCT TCAAATTAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGAT TTTTGATCTATTAACAGATATATAAATGGAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCAGTTTGTATTACTTCTTATTCA AATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGCTCGAGGGAGCTgcttct gcatcaacttctaaatataattcacactccttggagaatgagtctattaagaggacgtctcgagatggagtcaatcgagatctcactgaggct gttcctcgacttccaggagaaacactaatcactgacaaagaagttatttacatatgtcctttcaatggccccattaagggaagagtttacatc acaaattatcgtctttatttaagaagtttggaaacggattcttctctaatacttgatgttcctctgggtgtgatctcgagaattgaaaaaatg ggaggcgcgacaagtagaggagaaaattcctatggtctagatattacttgtaaagacatgagaaacctgaggttcgctttgaaacaggaaggc cacagcagaagagatatgtttgagatcctcacgagatacgcgtttcccctggctcacagtctgccattatttgcatttttaaatgaagaaaag tttaacgtggatggatggacagtttacaatccagtggaagaatacaggaggcagggcttgcccaatcaccattggagaataacttttattaat aagtgctatgagctctgtgacacttaccctgctcttttggtggttccgtatcgtgcctcagatgatgacctccggagagttgcaacttttagg tcccgaaatcgaattccagtgctgtcatggattcatccagaaaataagacggtcattgtgcgttgcagtcagcctcttgtcggtatgagtggg aaacgaaataaagatgatgagaaatatctcgatgttatcagggagactaataaacaaatttctaaactcaccatttatgatgcaagacccagc gtaaatgcagtggccaacaaggcaacaggaggaggatatgaaagtgatgatgcatatcataacgccgaacttttcttcttagacattcataat attcatgttatgcgggaatctttaaaaaaagtgaaggacattgtttatcctaatgtagaagaatctcattggttgtccagtttggagtctact cattggttagaacatatcaagctcgttttgacaggagccattcaagtagcagacaaagtttcttcagggaagagttcagtgcttgtgcattgc agtgacggatgggacaggactgctcagctgacatccttggccatgctgatgttggatagcttctataggagcattgaagggttcgaaatactg gtacaaaaagaatggataagttttggacataaatttgcatctcgaataggtcatggtgataaaaaccacaccgatgctgaccgttctcctatt tttctccagtttattgattgtgtgtggcaaatgtcaaaacagttccctacagcttttgaattcaatgaacaatttttgattataattttggat catctgtatagttgccgatttggtactttcttattcaactgtgaatctgctcgagaaagacagaaggttacagaaaggactgtttctttatgg tcactgataaacagtaataaagaaaaattcaaaaaccccttctatactaaagaaatcaatcgagttttatatccagttgccagtatgcgtcac ttggaactctgggtgaattactacattagatggaaccccaggatcaagcaacaacagccgaatccagtggagcagcgttacatggagctctta gccttacgcgacgaatacataaagcggcttgaggaactgcagctcgccaactctgccaagctttctgatcccccaacttcaccttccagtcct tcgcaaatgatgccccatgtgcaaactcacttcGGTAGTTCCCTAggttccggtagcggttccggttctatcactattagggctgcttttttg gaaaaagaaaatactgctttgagaactgaaattgctgaattggaaaaagaagttggtagatgtgaaaatattgtttctaaatatgaaactaga tatggtccattgTAATCGCGGATCCGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAAATAAGTGTATACAAAT TTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGT CGCTCTTATTGACCACACC

(3) pGal1//ATG//Xho//[A]//WMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator

As above, but WMTM1 sequence

(4) pSte5//ATG//Xho//[A]//HMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator

As above, but Ste5 promoter sequence:

CACGAAGTGACTGACAGAATACTGACATCAGCTGATTTCTCATAGAGCTGTTTCTCTGATAACACGTTGTTTGAACATCGACAAGATGAAAATC TAGAAGTATCAAGTTTCCTTTAAAGGGATATATAACAGATTCTAAAACTGACAGAAATATTTCGAGTGAAGAAGAAGCGTTAAATATTGGATCT TTCCGCAGTTCTACTCTGATACATTTTTGAAGTAGGAGAGTCATTTAGAAGGCGTATTGCTCAATAGTAGAAAGCAGGCCTGTGCACATGAATT AATTAAAAAATATAAAGGTAGTGATTAGACGACACATGTCCATAGGTAACCTGTCATAATTTTGAACAATTTCCCTTCTTTTCTTTTTTTTTTT TGGGTGCGGCGATATGTAGCTTGTTAATTTACACATCATGTACTTTTCTGCATCAAAATATGAAAGGCGATAGTAGCTAAAGAAAATACCGAGA ATTTCCTCGAAAAGTTGACGACAAAAGAAAGGCATAAAAAAGTAATTTGAAAATATTTTAAAACTGTTTTAACCCATCTAGCATCCGCGCTAAA AAAGGAAGATACAGGATACAGCGGAAACAACTTTTAAATG

(5) pSte5//ATG//Xho//[A]//WMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator

As above.

(6) pCyc//ATG//Xho//[A]//HMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator

As above, but Cyc promoter sequence:

ctcgagcagatccgccaggcgtgtatatatagcgtggatggccaggcaactttagtgctgacacatacaggcatatatatatgtgtgcgacgaca catgatcatatggcatgcatgtgctctgtatgtatataaaactcttgttttcttcttttctctaaatattctttccttatacattaggacctttg cagcataaattactatacttctatagacacacaaacacaaatacacacactaaattaataatg

(7) pCyc//ATG//Xho//[A]//WMTM1//[B]//E34(I)//STOP//[D]-BamHI-AdhTerminator

As above.

(8) pCyc//ATG//XhoI//GFP//XmaI//(SerGlySerGlySer)ING2 PH domain//STOP//BamHI

As above, but //(SerGlySerGlySer)ING2 PH domain sequence as follows:

TCTGGATCAGGTTCAGCTTCTCCAGTTGAATTTGCTATTGATCCTAACGAACCTACATACTGCTTATGTAATCAAGTTTCTTATGGTGAAATGAT TGGTTGCGATAACGAGCAGTGTCCAATAGAATGGTTTCATTTCTCTTGCGTTTCTTTAACATATAAACCTAAGGGAAAATGGTACTGTCCTAAAT GTAGAGGTGATAATGAGAAAACTATGGATAAGTCTACTGAAAAGACAAAAAAAGATAGAAGATCAAGA

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