LOCUS       sequence-465796-        7061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose iduced expression of Gcn4 WT 30mer in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7061)
  AUTHORS   Bremer A, Lang WH, Kempen RP, Sweta K, Taylor AB, Borgia MB, Ansari 
            AZ, Mittag T
  TITLE     Reconciling competing models on the roles of condensates and soluble
            complexes in transcription factor function.
  JOURNAL   bioRxiv [Preprint]. 2024 Nov 22:2024.11.21.624739. doi: 
            10.1101/2024.11.21.624739.
  PUBMED    39605529
REFERENCE   2  (bases 1 to 7061)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Nov 22:2024.11.21.624739. doi: 
            10.1101/2024.11.21.624739."
COMMENT     Sequence Label: pAG415Gal_Gcn4 WT 30mer
FEATURES             Location/Qualifiers
     source          1..7061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    70..573
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        610..714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(715..783,784..1575)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(933..952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1746..2334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2235..2254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2488..2505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2622..2643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2658..2663,2664..2681,2682..2688)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2696..2712
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     primer_bind     2701..2723
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2720..2736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2720..2736
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2755..2775
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2799..3240
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2799..2916
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3211..3234
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     3255..3271
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3255..3271
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    3279..3303
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3306..3326
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(3631..3655)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3689..3936
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3705..3723)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3954..3973)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3955..3973)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3983..4000)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3983..3999)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3992..4014)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4140..4595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4227..4246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4437..4458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4896..5303
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5304..6398
                     /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"
     primer_bind     complement(6892..6911)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7011..7033
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
      121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
      181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
      241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
      301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
      361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
      421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
      481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
      541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
      601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1501 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1861 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2101 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2221 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     2641 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
     2761 aaccctcact aaagggaaca aaagctggag ctctagtacg gattagaagc cgccgagcgg
     2821 gtgacagccc tccgaaggaa gactctcctc cgtgcgtcct cgtcttcacc ggtcgcgttc
     2881 ctgaaacgca gatgtgcctc gcgccgcact gctccgaaca ataaagattc tacaatacta
     2941 gcttttatgg ttatgaagag gaaaaattgg cagtaacctg gccccacaaa ccttcaaatg
     3001 aacgaatcaa attaacaacc ataggatgat aatgcgatta gttttttagc cttatttctg
     3061 gggtaattaa tcagcgaagc gatgattttt gatctattaa cagatatata aatgcaaaaa
     3121 ctgcataacc actttaacta atactttcaa cattttcggt ttgtattact tcttattcaa
     3181 atgtaataaa agtatcaaca aaaaattgtt aatatacctc tatactttaa cgtcaaggag
     3241 aaaaaacccc ggattctaga actagtggat cccccatcac aagtttgtac aaaaaagcag
     3301 gcttcgagaa cctgtatttt cagggtagca tgtttgaata tgagaacctt gaggataaca
     3361 gcaaagaatg gaccagcctg tttgataatg atattccggt taccaccgat gatgttagct
     3421 taggtagcgg tagcggttca ggtagtccgg aaagcagcga tccggcagca ctgaaacgtg
     3481 cccgtaatac cgaagcagca cgtcgtagcc gtgcacgtaa actgcagcgt atgaaacagc
     3541 tggaagataa agttgaagaa ctgctgtgca aaaactacca cctggaaaat gaagttgccc
     3601 gtctgaaaaa actggttggt gaacgttaag acccagcttt cttgtacaaa gtggtgatgg
     3661 gctgcaggaa ttcgatatca agctcgagtc atgtaattag ttatgtcacg cttacattca
     3721 cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag
     3781 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt
     3841 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt
     3901 gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccaat tcgccctata
     3961 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     4021 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     4081 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     4141 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4201 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4261 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4321 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4381 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4441 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4501 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4561 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     4621 catctgtgcg gtatttcaca ccgcatatcg accggtcgag gagaacttct agtatatcta
     4681 catacctaat attattgcct tattaaaaat ggaatcccaa caattacatc aaaatccaca
     4741 ttctcttcaa aatcaattgt cctgtacttc cttgttcatg tgtgttcaaa aacgttatat
     4801 ttataggata attatactct atttctcaac aagtaattgg ttgtttggcc gagcggtcta
     4861 aggcgcctga ttcaagaaat atcttgaccg cagttaactg tgggaatact caggtatcgt
     4921 aagatgcaag agttcgaatc tcttagcaac cattattttt ttcctcaaca taacgagaac
     4981 acacaggggc gctatcgcac agaatcaaat tcgatgactg gaaatttttt gttaatttca
     5041 gaggtcgcct gacgcatata cctttttcaa ctgaaaaatt gggagaaaaa ggaaaggtga
     5101 gagcgccgga accggctttt catatagaat agagaagcgt tcatgactaa atgcttgcat
     5161 cacaatactt gaagttgaca atattattta aggacctatt gttttttcca ataggtggtt
     5221 agcaatcgtc ttactttcta acttttctta ccttttacat ttcagcaata tatatatata
     5281 tatttcaagg atataccatt ctaatgtctg cccctaagaa gatcgtcgtt ttgccaggtg
     5341 accacgttgg tcaagaaatc acagccgaag ccattaaggt tcttaaagct atttctgatg
     5401 ttcgttccaa tgtcaagttc gatttcgaaa atcatttaat tggtggtgct gctatcgatg
     5461 ctacaggtgt tccacttcca gatgaggcgc tggaagcctc caagaaggct gatgccgttt
     5521 tgttaggtgc tgtgggtggt cctaaatggg gtaccggtag tgttagacct gaacaaggtt
     5581 tactaaaaat ccgtaaagaa cttcaattgt acgccaactt aagaccatgt aactttgcat
     5641 ccgactctct tttagactta tctccaatca agccacaatt tgctaaaggt actgacttcg
     5701 ttgttgtcag agaattagtg ggaggtattt actttggtaa gagaaaggaa gacgatggtg
     5761 atggtgtcgc ttgggatagt gaacaataca ccgttccaga agtgcaaaga atcacaagaa
     5821 tggccgcttt catggcccta caacatgagc caccattgcc tatttggtcc ttggataaag
     5881 ctaatgtttt ggcctcttca agattatgga gaaaaactgt ggaggaaacc atcaagaacg
     5941 aattccctac attgaaggtt caacatcaat tgattgattc tgccgccatg atcctagtta
     6001 agaacccaac ccacctaaat ggtattataa tcaccagcaa catgtttggt gatatcatct
     6061 ccgatgaagc ctccgttatc ccaggttcct tgggtttgtt gccatctgcg tccttggcct
     6121 ctttgccaga caagaacacc gcatttggtt tgtacgaacc atgccacggt tctgctccag
     6181 atttgccaaa gaataaggtc aaccctatcg ccactatctt gtctgctgca atgatgttga
     6241 aattgtcatt gaacttgcct gaagaaggta aggccattga agatgcagtt aaaaaggttt
     6301 tggatgcagg tatcagaact ggtgatttag gtggttccaa cagtaccacc gaagtcggtg
     6361 atgctgtcgc cgaagaagtt aagaaaatcc ttgcttaaaa agattctctt tttttatgat
     6421 atttgtacat aaactttata aatgaaattc ataatagaaa cgacacgaaa ttacaaaatg
     6481 gaatatgttc atagggtaga cgaaactata tacgcaatct acatacattt atcaagaagg
     6541 agaaaaagga ggatgtaaag gaatacaggt aagcaaattg atactaatgg ctcaacgtga
     6601 taaggaaaaa gaattgcact ttaacattaa tattgacaag gaggagggca ccacacaaaa
     6661 agttaggtgt aacagaaaat catgaaacta tgattcctaa tttatatatt ggaggatttt
     6721 ctctaaaaaa aaaaaaatac aacaaataaa aaacactcaa tgacctgacc atttgatgga
     6781 gtttaagtca ataccttctt gaaccatttc ccataatggt gaaagttccc tcaagaattt
     6841 tactctgtca gaaacggcct taacgacgta gtcgatatgg tgcactctca gtacaatctg
     6901 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     6961 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     7021 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg a
//
