LOCUS       sequence-465898-        6950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose iduced expression of Gcn4 DBD in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6950)
  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 6950)
  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_DBD
FEATURES             Location/Qualifiers
     source          1..6950
                     /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(3520..3544)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3578..3825
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3594..3612)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3843..3862)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3844..3862)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3872..3889)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3872..3888)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3881..3903)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4029..4484
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4116..4135)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4326..4347
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4785..5192
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5193..6287
                     /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(6781..6800)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6900..6922
                     /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 tgagtccgga aagcagcgat ccggcagcac
     3361 tgaaacgtgc ccgtaatacc gaagcagcac gtcgtagccg tgcacgtaaa ctgcagcgta
     3421 tgaaacagtt agaagataag gttgaagaac tgctgtgcaa aaactatcac ctggaaaatg
     3481 aagttgcccg tctgaaaaaa ctggttggtg aacgttaaga cccagctttc ttgtacaaag
     3541 tggtgatggg ctgcaggaat tcgatatcaa gctcgagtca tgtaattagt tatgtcacgc
     3601 ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct
     3661 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata
     3721 tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa
     3781 accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg gtacccaatt
     3841 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     3901 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     3961 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     4021 gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4081 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4141 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4201 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4261 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4321 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4381 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4441 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt
     4501 ctccttacgc atctgtgcgg tatttcacac cgcatatcga ccggtcgagg agaacttcta
     4561 gtatatctac atacctaata ttattgcctt attaaaaatg gaatcccaac aattacatca
     4621 aaatccacat tctcttcaaa atcaattgtc ctgtacttcc ttgttcatgt gtgttcaaaa
     4681 acgttatatt tataggataa ttatactcta tttctcaaca agtaattggt tgtttggccg
     4741 agcggtctaa ggcgcctgat tcaagaaata tcttgaccgc agttaactgt gggaatactc
     4801 aggtatcgta agatgcaaga gttcgaatct cttagcaacc attatttttt tcctcaacat
     4861 aacgagaaca cacaggggcg ctatcgcaca gaatcaaatt cgatgactgg aaattttttg
     4921 ttaatttcag aggtcgcctg acgcatatac ctttttcaac tgaaaaattg ggagaaaaag
     4981 gaaaggtgag agcgccggaa ccggcttttc atatagaata gagaagcgtt catgactaaa
     5041 tgcttgcatc acaatacttg aagttgacaa tattatttaa ggacctattg ttttttccaa
     5101 taggtggtta gcaatcgtct tactttctaa cttttcttac cttttacatt tcagcaatat
     5161 atatatatat atttcaagga tataccattc taatgtctgc ccctaagaag atcgtcgttt
     5221 tgccaggtga ccacgttggt caagaaatca cagccgaagc cattaaggtt cttaaagcta
     5281 tttctgatgt tcgttccaat gtcaagttcg atttcgaaaa tcatttaatt ggtggtgctg
     5341 ctatcgatgc tacaggtgtt ccacttccag atgaggcgct ggaagcctcc aagaaggctg
     5401 atgccgtttt gttaggtgct gtgggtggtc ctaaatgggg taccggtagt gttagacctg
     5461 aacaaggttt actaaaaatc cgtaaagaac ttcaattgta cgccaactta agaccatgta
     5521 actttgcatc cgactctctt ttagacttat ctccaatcaa gccacaattt gctaaaggta
     5581 ctgacttcgt tgttgtcaga gaattagtgg gaggtattta ctttggtaag agaaaggaag
     5641 acgatggtga tggtgtcgct tgggatagtg aacaatacac cgttccagaa gtgcaaagaa
     5701 tcacaagaat ggccgctttc atggccctac aacatgagcc accattgcct atttggtcct
     5761 tggataaagc taatgttttg gcctcttcaa gattatggag aaaaactgtg gaggaaacca
     5821 tcaagaacga attccctaca ttgaaggttc aacatcaatt gattgattct gccgccatga
     5881 tcctagttaa gaacccaacc cacctaaatg gtattataat caccagcaac atgtttggtg
     5941 atatcatctc cgatgaagcc tccgttatcc caggttcctt gggtttgttg ccatctgcgt
     6001 ccttggcctc tttgccagac aagaacaccg catttggttt gtacgaacca tgccacggtt
     6061 ctgctccaga tttgccaaag aataaggtca accctatcgc cactatcttg tctgctgcaa
     6121 tgatgttgaa attgtcattg aacttgcctg aagaaggtaa ggccattgaa gatgcagtta
     6181 aaaaggtttt ggatgcaggt atcagaactg gtgatttagg tggttccaac agtaccaccg
     6241 aagtcggtga tgctgtcgcc gaagaagtta agaaaatcct tgcttaaaaa gattctcttt
     6301 ttttatgata tttgtacata aactttataa atgaaattca taatagaaac gacacgaaat
     6361 tacaaaatgg aatatgttca tagggtagac gaaactatat acgcaatcta catacattta
     6421 tcaagaagga gaaaaaggag gatgtaaagg aatacaggta agcaaattga tactaatggc
     6481 tcaacgtgat aaggaaaaag aattgcactt taacattaat attgacaagg aggagggcac
     6541 cacacaaaaa gttaggtgta acagaaaatc atgaaactat gattcctaat ttatatattg
     6601 gaggattttc tctaaaaaaa aaaaaataca acaaataaaa aacactcaat gacctgacca
     6661 tttgatggag tttaagtcaa taccttcttg aaccatttcc cataatggtg aaagttccct
     6721 caagaatttt actctgtcag aaacggcctt aacgacgtag tcgatatggt gcactctcag
     6781 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     6841 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     6901 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
//
