LOCUS       40924_6831        9405 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pBM5155, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9405)
  AUTHORS   Alexander WG, Peris D, Pfannenstiel BT, Opulente DA, Kuang M, 
            Hittinger CT.
  TITLE     Efficient engineering of marker-free synthetic allotetraploids of 
            Saccharomyces
  JOURNAL   Fungal Genet. Biol. (2015) In press
  PUBMED    26555931
REFERENCE   2  (bases 1 to 9405)
  AUTHORS   Alexander WG, Peris D, Pfannenstiel B, Opulente DA, Kuang M, 
            Hittinger CT.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-SEP-2015) Laboratory of Genetics, University of 
            Wisconsin-Madison, 425-G Henry Mall, 4350 Genetics Biotechnology 
            Center, Madison, WI 53706, USA
REFERENCE   3  (bases 1 to 9405)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9405)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Fungal 
            Genet. Biol. (2015) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (08-SEP-2015) Laboratory of Genetics, University of 
            Wisconsin-Madison, 425-G Henry Mall, 4350 Genetics Biotechnology 
            Center, Madison, WI 53706, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9405
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(31..1035)
                     /codon_start=1
                     /label=tTA
                     /note="tetracycline-controlled transactivator, comprising a
                     fusion of the tetracycline repressor TetR with the 
                     C-terminal activation domain of herpes simplex virus VP16"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     promoter        complement(1140..1343)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     enhancer        complement(1344..1723)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     terminator      1835..2022
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae
                     alcohol dehydrogenase 1 (ADH1) gene"
     protein_bind    2052..2322
                     /label=tetracycline response element
                     /note="contains seven copies of the tetracycline operator
                     tetO"
     misc_feature    2540..2547
                     /label=NotI site for gap-repair cloning
                     /note="NotI site for gap-repair cloning"
     misc_feature    2585..2608
                     /label=6x glycine linker
                     /note="6x glycine linker"
     CDS             2609..3106
                     /codon_start=1
                     /label=13xMyc
                     /note="13 tandem Myc epitope tags"
                     /translation="EQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLIS
                     EEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLD
                     GEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISE
                     EDL"
     misc_feature    3251..3253
                     /label=in-frame amber stop codon
                     /note="in-frame amber stop codon"
     terminator      3299..3546
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3569..3585)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3593..3609)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(3617..3647)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3662..3683)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3971..4559)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4733..5590)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5591..5695)
                     /label=AmpR promoter
     gene            5903..7022
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     rep_origin      7365..8202
                     /label=ARS1
                     /note="S. cerevisiae autonomously replicating sequence 
                     ARS1/ARS416"
     misc_feature    8338..8630
                     /label=CEN4
                     /note="S. cerevisiae CEN4 centromere"
     primer_bind     9389..9405
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gaattcttat tacgatcctc gcgcccccta cccaccgtac tcgtcaattc caagggcatc
       61 ggtaaacatc tgctcaaact cgaagtcggc catatccaga gcgccgtagg gggcggagtc
      121 gtggggggta aatcccggac ccggggaatc cccgtccccc aacatgtcca gatcgaaatc
      181 gtctagcgcg tcggcatgcg ccatcgccac gtcctcgccg tctaagtgga gctcgtcccc
      241 caggctgaca tcggtcgggg gggccgtcga cagtctgcgc gtgtgtcccg cggggagaaa
      301 ggacaggcgc ggagccgcca gccccgcctc ttcgggggcg tcgtcgtccg ggagatcgag
      361 caggccctcg atggtagacc cgtaattgtt tttcgtacgc gcgcggctgt acgcggaccc
      421 actttcacat ttaagttgtt tttctaatcc gcatatgatc aattcaaggc cgaataagaa
      481 ggctggctct gcaccttggc gatcaaataa ttcgatagct tgtcgtaata atggcggcat
      541 actatcagta gtaggtgttt ccctttcttc tttagcgact tgatgctctt gttcttccaa
      601 tacgcaacct aaagtaaagt gccccacagc gctgagtgca tataatgcat tctctagtga
      661 aaaaccttgt tggcataaaa aggctaattg attttcgaga gtttcatact gtttttctgt
      721 aggccgtgta cctaaatgta cttttgctcc atcgcgatga cttagtaaag cacatctaaa
      781 acttttagcg ttattacgta aaaaatcttg ccagctttcc ccttctaaag ggcaaaagtg
      841 agtatggtgc ctatctaaca tctcaatggg taaggcgtcg agcaaagccc gcttattttt
      901 tacatgccaa tacaatgtag gctgctctac acctagcttc tgggcgagtt tacgggttgt
      961 taaaccttcg attccgaccc cattaagcag ctctaatgcg ccgttaatca ctttactttt
     1021 atctaatcta gacatatgaa ttaattcggg ccgcggaggc tggatcggtc ccggtgtctt
     1081 ctatggaggt caaaacagcg tggatggcgt ctccaggcga tctgacggtt cactaaacga
     1141 gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg
     1201 gagttgttac gacattttgg aaagtcccgt tgattttggt gccaaaacaa actcccattg
     1261 acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc acgcccattg
     1321 atgtactgcc aaaaccgcat caccatggta atagcgatga ctaatacgta gatgtactgc
     1381 caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg
     1441 tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg
     1501 gcagtttagc gtaaatactc cacccattga cgtcaatgga aagtccctat tggcgttact
     1561 atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc
     1621 gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac
     1681 cccgtaattg attactatta ataactagtc aataatcaat gtcaacatgg cggtaatgtt
     1741 ggacatgagc caatataaat gtacatatta tgatatggat acaacgtatg caatgggcca
     1801 agctcctcga gtaattcgcg ccacttctaa ataagcgaat ttcttatgat ttatgatttt
     1861 tattattaaa taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt
     1921 tttaaaacga aaattcttgt tcttgagtaa ctctttcctg taggtcaggt tgctttctca
     1981 ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcagatcaa ttcctcgatc
     2041 gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat
     2101 cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg
     2161 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact
     2221 ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga
     2281 aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagctc
     2341 ggtaccctat ggcatgcatg tgctctgtat gtatataaaa ctcttgtttt cttcttttct
     2401 ctaaatattc tttccttata cattaggtcc tttgtagcat aaattactat acttctatag
     2461 acacgcaaac acaaatacac acactaaatt accggatcaa ttcgggggat ccgtttaaac
     2521 aggcctgtta acatcgatag cggccgctag ggccctgcag cggatccccg ggttaattaa
     2581 cggtggaggt ggaggtggag gtggaggtga acaaaagcta atctccgagg aagacttgaa
     2641 cggtgaacaa aaattaatct cagaagaaga cttgaacgga ctcgacggtg aacaaaagtt
     2701 gatttctgaa gaagatttga acggtgaaca aaagctaatc tccgaggaag acttgaacgg
     2761 tgaacaaaaa ttaatctcag aagaagactt gaacggactc gacggtgaac aaaagttgat
     2821 ttctgaagaa gatttgaacg gtgaacaaaa gctaatctcc gaggaagact tgaacggtga
     2881 acaaaaatta atctcagaag aagacttgaa cggactcgac ggtgaacaaa agttgatttc
     2941 tgaagaagat ttgaacggtg aacaaaagct aatctccgag gaagacttga acggtgaaca
     3001 aaaattaatc tcagaagaag acttgaacgg actcgacggt gaacaaaagt tgatttctga
     3061 agaagatttg aacggtgaac aaaagctaat ctccgaggaa gacttgaacg gtgaacaaaa
     3121 attaatctca gaagaagact tgaacggact cgacggtgaa caaaagttga tttctgaaga
     3181 agatttgaac ggtgaacaaa agctaatctc cgaggaagac ttgaacggtg aacaaaaatt
     3241 aatcaatcac tagtgaattc gcgccacttc taaataagcg aatttcttga gggccgcatc
     3301 atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa
     3361 aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta
     3421 gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac
     3481 gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta
     3541 atttgcggcc aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3601 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3661 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3721 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3781 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3841 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3901 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3961 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4021 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4081 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4141 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4201 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4261 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4321 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4381 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     4441 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4501 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4561 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4621 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4681 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4741 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4801 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4861 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4921 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4981 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5041 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5101 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5161 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5221 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5281 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5341 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5401 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5461 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5521 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5581 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5641 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5701 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     5761 aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt cagctgaagc ttcgtacgct
     5821 gcaggtcgac ggatccccgg gttaattaag gcgcgccaga tctgtttagc ttgccttgtc
     5881 cccgccgggt cacccggcca gcgacatgga ggcccagaat accctccttg acagtcttga
     5941 cgtgcgcagc tcaggggcat gatgtgactg tcgcccgtac atttagccca tacatcccca
     6001 tgtataatca tttgcatcca tacattttga tggccgcacg gcgcgaagca aaaattacgg
     6061 ctcctcgctg cagacctgcg agcagggaaa cgctcccctc acagacgcgt tgaattgtcc
     6121 ccacgccgcg cccctgtaga gaaatataaa aggttaggat ttgccactga ggttcttctt
     6181 tcatatactt ccttttaaaa tcttgctagg atacagttct cacatcacat ccgaacataa
     6241 acaaccatgg gtaccactct tgacgacacg gcttaccggt accgcaccag tgtcccgggg
     6301 gacgccgagg ccatcgaggc actggatggg tccttcacca ccgacaccgt cttccgcgtc
     6361 accgccaccg gggacggctt caccctgcgg gaggtgccgg tggacccgcc cctgaccaag
     6421 gtgttccccg acgacgaatc ggacgacgaa tcggacgacg gggaggacgg cgacccggac
     6481 tcccggacgt tcgtcgcgta cggggacgac ggcgacctgg cgggcttcgt ggtcgtctcg
     6541 tactccggct ggaaccgccg gctgaccgtc gaggacatcg aggtcgcccc ggagcaccgg
     6601 gggcacgggg tcgggcgcgc gttgatgggg ctcgcgacgg agttcgcccg cgagcggggc
     6661 gccgggcacc tctggctgga ggtcaccaac gtcaacgcac cggcgatcca cgcgtaccgg
     6721 cggatggggt tcaccctctg cggcctggac accgccctgt acgacggcac cgcctcggac
     6781 ggcgagcagg cgctctacat gagcatgccc tgcccctaat cagtactgac aataaaaaga
     6841 ttcttgtttt caagaacttg tcatttgtat agttttttta tattgtagtt gttctatttt
     6901 aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga catcatctgc ccagatgcga
     6961 agttaagtgc gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac
     7021 tgctgtcgat tcgatactaa cgccgccatc cagtgtcgaa aacgagctcg aattcatcga
     7081 tgatatcaga tccactagtg gcctatgcaa ttctcatgtt tgacagctta tcatcgatcg
     7141 tccaactgca tggagatgag tcgtggcaag aataccaaga gttcctcggt ttgccagtta
     7201 ttaaaagact cgtatttcca aaagactgca acatactact cagtgcagct tcacagaaac
     7261 ctcattcgtt tattcccttg tttgattcag aagcaggtgg gacaggtgaa cttttggatt
     7321 ggaactcgat ttctgactgg gttggaaggc aagagagccc cgagagctta cattttatgt
     7381 tagctggtgg actgacgcca gaaaatgttg gtgatgcgct tagattaaat ggcgttattg
     7441 gtgttgatgt aagcggaggt gtggagacaa atggtgtaaa agactctaac aaaatagcaa
     7501 atttcgtcaa aaatgctaag aaataggtta ttactgagta gtatttattt aagtattgtt
     7561 tgtgcacttg cctgcaagcc ttttgaaaag caagcataaa agatctaaac ataaaatctg
     7621 taaaataaca agatgtaaag ataatgctaa atcatttggc tttttgattg attgtacagg
     7681 aaaatataca tcgcaggggg ttgactttta ccatttcacc gcaatggaat caaacttgtt
     7741 gaagagaatg ttcacaggcg catacgctac aatgacccga ttcttgctag ccttttctcg
     7801 gtcttgcaaa caaccgccgg cagcttagta tataaataca catgtacata cctctctccg
     7861 tatcctcgta atcattttct tgtatttatc gtcttttcgc tgtaaaaact ttatcacact
     7921 tatctcaaat acacttatta accgctttta ctattatctt ctacgctgac agtaatatca
     7981 aacagtgaca catattaaac acagtggttt ctttgcataa acaccatcag cctcaagtcg
     8041 tcaagtaaag atttcgtgtt catgcagata gataacaatc tatatgttga taattagcgt
     8101 tgcctcatca atgcgagatc cgtttaaccg gaccctagtg cacttacccc acgttcggtc
     8161 cactgtgtgc cgaacatgct ccttcactat tttaacatgt ggaattatcg atgccggtgt
     8221 tattcttgct atgataggcc atcgaatgtt aaagggaaga tttattgttt tgccctttaa
     8281 agacgaattt ggaccgtgaa tgtttaaata attctcatgt ttgacagctt atcatcgaac
     8341 tctaagaggt gatacttatt tactgtaaaa ctgtgacgat aaaaccggaa ggaagaataa
     8401 gaaaactcga actgatctat aatgcctatt ttctgtaaag agtttaagct atgaaagcct
     8461 cggcattttg gccgctccta ggtagtgctt tttttccaag gacaaaacag tttctttttc
     8521 ttgagcaggt tttatgtttc ggtaatcata aacaataaat aaattatttc atttatgttt
     8581 aaaaataaaa aataaaaaag tattttaaat ttttaaaaaa gttgattata agcatgtgac
     8641 cttttgcaag caattaaatt ttgcaatttg tgattttagg caaaagttac aatttctggc
     8701 tcgtgtaata tatgtatgct aaagtgaact tttacaaagt cgatatggac ttagtcaaaa
     8761 gaaattttct taaaaatata tagcactagc caatttagca cttctttatg agatatatta
     8821 tagactttat taagccagat ttgtgtatta tatgtattta cccggcgaat catggacata
     8881 cattctgaaa taggtaatat tctctatggt gagacagcat agataaccta ggatacaagt
     8941 taaaagctag tactgttttg cagtaatttt tttctttttt ataagaatgt taccacctaa
     9001 ataagttata aagtcaatag ttaagtttga tatttgattg taaaataccg taatatattt
     9061 gcatgatcaa aaggctcaat gttgactagc cagcatgtca accactatat tgatcaccga
     9121 tatatggact tccacaccaa ctagtaatat gacaataaat tcaagatatt cttcatgaga
     9181 atggcccagc gatatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     9241 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     9301 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     9361 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagt
//
