LOCUS       sequence-381354-        7524 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Dual reporter backbone plasmid for CCRA experiment- LEU2 Marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7524)
  TITLE     A Novel Mode of Half Site Binding in Budding Yeast
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7524)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: TEF2pro_mCherry_YFP
FEATURES             Location/Qualifiers
     source          1..7524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..47)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(332..1426)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1427..1832)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2133..2588)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2270..2291)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2482..2501
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2714..2736
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2728..2745
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2729..2745
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2755..2774
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2755..2773
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    2782..2889
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     2798..2814
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2799..2815
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2849..2865)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2849..2865)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(2990..2992,2993..2995,2996..3709)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3035..3056)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3296..3315)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3643..3664
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      3730..3918
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     rep_origin      complement(4004..4592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4084..4103)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4665..5064
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             5065..5775
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVDGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5210..5228)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5478..5497
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      5776..5963
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     CDS             complement(6053..6868)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6053..6868)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6776..6795
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6860..6879
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     misc_feature    6949..7452
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     7493..7511
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
       61 ttcctaactt tttgtgtggt gccctcctcc ttgtcaatat taatgttaaa gtgcaattct
      121 ttttccttat cacgttgagc cattagtatc aatttgctta cctgtattcc tttacatcct
      181 cctttttctc cttcttgata aatgtatgta gattgcgtat atagtttcgt ctaccctatg
      241 aacatattcc attttgtaat ttcgtgtcgt ttctattatg aatttcattt ataaagttta
      301 tgtacaaata tcataaaaaa agagaatctt tttaagcaag gattttctta acttcttcgg
      361 cgacagcatc accgacttcg gtggtactgt tggaaccacc taaatcacca gttctgatac
      421 ctgcatccaa aaccttttta actgcatctt caatggcctt accttcttca ggcaagttca
      481 atgacaattt caacatcatt gcagcagaca agatagtggc gatagggttg accttattct
      541 ttggcaaatc tggagcagaa ccgtggcatg gttcgtacaa accaaatgcg gtgttcttgt
      601 ctggcaaaga ggccaaggac gcagatggca acaaacccaa ggaacctggg ataacggagg
      661 cttcatcgga gatgatatca ccaaacatgt tgctggtgat tataatacca tttaggtggg
      721 ttgggttctt aactaggatc atggcggcag aatcaatcaa ttgatgttga accttcaatg
      781 tagggaattc gttcttgatg gtttcctcca cagtttttct ccataatctt gaagaggcca
      841 aaacattagc tttatccaag gaccaaatag gcaatggtgg ctcatgttgt agggccatga
      901 aagcggccat tcttgtgatt ctttgcactt ctggaacggt gtattgttca ctatcccaag
      961 cgacaccatc accatcgtct tcctttctct taccaaagta aatacctccc actaattctc
     1021 tgacaacaac gaagtcagta cctttagcaa attgtggctt gattggagat aagtctaaaa
     1081 gagagtcgga tgcaaagtta catggtctta agttggcgta caattgaagt tctttacgga
     1141 tttttagtaa accttgttca ggtctaacac taccggtacc ccatttagga ccacccacag
     1201 cacctaacaa aacggcatca gccttcttgg aggcttccag cgcctcatct ggaagtggaa
     1261 cacctgtagc atcgatagca gcaccaccaa ttaaatgatt ttcgaaatcg aacttgacat
     1321 tggaacgaac atcagaaata gctttaagaa ccttaatggc ttcggctgtg atttcttgac
     1381 caacgtggtc acctggcaaa acgacgatct tcttaggggc agacattaga atggtatatc
     1441 cttgaaatat atatatatat tgctgaaatg taaaaggtaa gaaaagttag aaagtaagac
     1501 gattgctaac cacctattgg aaaaaacaat aggtccttaa ataatattgt caacttcaag
     1561 tattgtgatg caagcattta gtcatgaacg cttctctatt ctatatgaaa agccggttcc
     1621 ggcgctctca cctttccttt ttctcccaat ttttcagttg aaaaaggtat atgcgtcagg
     1681 cgacctctga aattaacaaa aaatttccag tcatcgaatt tgattctgtg cgatagcgcc
     1741 cctgtgtgtt ctcgttatgt tgaggaaaaa aataatggtt gctaagagat tcgaactctt
     1801 gcatcttacg atacctgagt attcccacag ttaactgcgg tcaagatatt tcttgaatca
     1861 ggcgccttag accgctcggc caaacaacca attacttgtt gagaaataga gtataattat
     1921 cctataaata taacgttttt gaacacacat gaacaaggaa gtacaggaca attgattttg
     1981 aagagaatgt ggattttgat gtaattgttg ggattccatt tttaataagg caataatatt
     2041 aggtatgtag atatactaga agttctcctc ggcacttgcc tatgcggtgt gaaataccgc
     2101 acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa
     2161 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2221 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     2281 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     2341 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     2401 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     2461 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     2521 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     2581 gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     2641 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     2701 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
     2761 gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgacgg tatcgataag
     2821 cttgatatcg aattcctgca gcccggggga tccactagtt ctagagcggc cgccaccgcg
     2881 gtggagctca ttggcattat cacataatga attatacatt atataaagta atgtgatttc
     2941 ttcgaagaat atactaaaaa atgagcaggc aagataaacg aaggcaaaga tggtgagcaa
     3001 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     3061 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     3121 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     3181 cttcggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     3241 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     3301 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     3361 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3421 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     3481 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     3541 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta
     3601 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3661 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag gcgcgccact
     3721 tctaaataag cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa
     3781 ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg
     3841 agtaactctt tcctgtaggt caggttgctt tctcaggtat agtatgaggt cgctcttatt
     3901 gaccaacacc tctaccggaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3961 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4021 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4081 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4141 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4201 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4261 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4321 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4381 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4441 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4501 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4561 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4621 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtccatagc ttcaaaatgt
     4681 ttctactcct tttttactct tccagatttt ctcggactcc gcgcatcgcc gtaccacttc
     4741 aaaacaccca agcacagcat actaaatttc ccctctttct tcctctaggg tgtcgttaat
     4801 tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt ttcttcatcg
     4861 aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg aaaatttttt tttttgattt
     4921 ttttctcttt cgatgacctc ccattgatat ttaagttaat aaacggtctt caatttctca
     4981 agtttcagtt tcatttttct tgttctatta caactttttt tacttcttgc tcattagaaa
     5041 gaaagcatag caatctaatc taagatggtg agcaagggcg aggaggataa catggccatc
     5101 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtggacgg ccacgagttc
     5161 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     5221 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     5281 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     5341 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     5401 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     5461 aacttcccct ccgacggccc cgtaatgcag aagaagacta tgggctggga ggcctcctcc
     5521 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     5581 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     5641 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     5701 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     5761 gagctgtaca agtgagcgaa tttcttatga tttataattt ttattattaa ataagttata
     5821 aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta
     5881 ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagta tgaggtcgct
     5941 cttattgacc acacctctac cggcacctag atcccgggga aatgtgcgcg gaacccctat
     6001 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     6061 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     6121 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     6181 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     6241 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     6301 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     6361 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     6421 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     6481 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     6541 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     6601 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     6661 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     6721 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     6781 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     6841 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     6901 acagttttat tgcacatttc cccgaaaagt gccacctggg tccttttcat cacgtgctat
     6961 aaaaataatt ataatttaaa ttttttaata taaatatata aattaaaaat agaaagtaaa
     7021 aaaagaaatt aaagaaaaaa tagtttttgt tttccgaaga tgtaaaagac tctaggggga
     7081 tcgccaacaa atactacctt ttatcttgct cttcctgctc tcaggtatta atgccgaatt
     7141 gtttcatctt gtctgtgtag aagaccacac acgaaaatcc tgtgatttta cattttactt
     7201 atcgttaatc gaatgtatat ctatttaatc tgcttttctt gtctaataaa tatatatgta
     7261 aagtacgctt tttgttgaaa ttttttaaac ctttgtttat ttttttttct tcattccgta
     7321 actcttctac cttctttatt tactttctaa aatccaaata caaaacataa aaataaataa
     7381 acacagagta aattcccaaa ttattccatc attaaaagat acgaggcgcg tgtaagttac
     7441 aggcaagcga tccgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     7501 tatcacgagg ccctttcgtc tcgc
//
