LOCUS       sequence-465872-        7226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose iduced expression of Gcn4 solvvol W+ in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7226)
  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 7226)
  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 solvvol W
FEATURES             Location/Qualifiers
     source          1..7226
                     /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"
     CDS             3603..3659
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             3696..3785
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(3796..3820)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3854..4101
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3870..3888)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4119..4138)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4120..4138)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4148..4165)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4148..4164)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4157..4179)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4305..4760
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4392..4411)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4602..4623
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5061..5468
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5469..6563
                     /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(7057..7076)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7176..7198
                     /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 cagggtagcc atatgagcac caccgaaacc acaccgatgt
     3361 ttgaatatga aaatctggaa gaacagacca aagaatggac cacactgttt gattgggaaa
     3421 ttccggttac caccgaagaa gttaccctgg cagataaagc aattgaaacc ggtagcggta
     3481 gtggtagcgg ttcaccggaa tccagtgatc ctgctgctct aaaacgtgct agaaacactg
     3541 aagccgccag gcgttctcgt gcgagaaagt tgcaaagaat gaaacaactt gaagacaagg
     3601 ttgaagaatt gctttcgaaa aattatcact tggaaaatga ggttgccaga ttaaagaaat
     3661 tagttggcga acgcaccggt ggtggatccg gtggttaccc atacgatgtt cctgactatg
     3721 cgggctatcc ctatgacgtc ccggactatg caggatccta tccgtatgac gttccagatt
     3781 acgctgctca gtaagaccca gctttcttgt acaaagtggt gatgggctgc aggaattcga
     3841 tatcaagctc gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca
     3901 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt
     3961 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg
     4021 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga
     4081 cgctcgaagg ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg
     4141 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     4201 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     4261 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg
     4321 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4381 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4441 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4501 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4561 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4621 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4681 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4741 aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4801 tcacaccgca tatcgaccgg tcgaggagaa cttctagtat atctacatac ctaatattat
     4861 tgccttatta aaaatggaat cccaacaatt acatcaaaat ccacattctc ttcaaaatca
     4921 attgtcctgt acttccttgt tcatgtgtgt tcaaaaacgt tatatttata ggataattat
     4981 actctatttc tcaacaagta attggttgtt tggccgagcg gtctaaggcg cctgattcaa
     5041 gaaatatctt gaccgcagtt aactgtggga atactcaggt atcgtaagat gcaagagttc
     5101 gaatctctta gcaaccatta tttttttcct caacataacg agaacacaca ggggcgctat
     5161 cgcacagaat caaattcgat gactggaaat tttttgttaa tttcagaggt cgcctgacgc
     5221 atataccttt ttcaactgaa aaattgggag aaaaaggaaa ggtgagagcg ccggaaccgg
     5281 cttttcatat agaatagaga agcgttcatg actaaatgct tgcatcacaa tacttgaagt
     5341 tgacaatatt atttaaggac ctattgtttt ttccaatagg tggttagcaa tcgtcttact
     5401 ttctaacttt tcttaccttt tacatttcag caatatatat atatatattt caaggatata
     5461 ccattctaat gtctgcccct aagaagatcg tcgttttgcc aggtgaccac gttggtcaag
     5521 aaatcacagc cgaagccatt aaggttctta aagctatttc tgatgttcgt tccaatgtca
     5581 agttcgattt cgaaaatcat ttaattggtg gtgctgctat cgatgctaca ggtgttccac
     5641 ttccagatga ggcgctggaa gcctccaaga aggctgatgc cgttttgtta ggtgctgtgg
     5701 gtggtcctaa atggggtacc ggtagtgtta gacctgaaca aggtttacta aaaatccgta
     5761 aagaacttca attgtacgcc aacttaagac catgtaactt tgcatccgac tctcttttag
     5821 acttatctcc aatcaagcca caatttgcta aaggtactga cttcgttgtt gtcagagaat
     5881 tagtgggagg tatttacttt ggtaagagaa aggaagacga tggtgatggt gtcgcttggg
     5941 atagtgaaca atacaccgtt ccagaagtgc aaagaatcac aagaatggcc gctttcatgg
     6001 ccctacaaca tgagccacca ttgcctattt ggtccttgga taaagctaat gttttggcct
     6061 cttcaagatt atggagaaaa actgtggagg aaaccatcaa gaacgaattc cctacattga
     6121 aggttcaaca tcaattgatt gattctgccg ccatgatcct agttaagaac ccaacccacc
     6181 taaatggtat tataatcacc agcaacatgt ttggtgatat catctccgat gaagcctccg
     6241 ttatcccagg ttccttgggt ttgttgccat ctgcgtcctt ggcctctttg ccagacaaga
     6301 acaccgcatt tggtttgtac gaaccatgcc acggttctgc tccagatttg ccaaagaata
     6361 aggtcaaccc tatcgccact atcttgtctg ctgcaatgat gttgaaattg tcattgaact
     6421 tgcctgaaga aggtaaggcc attgaagatg cagttaaaaa ggttttggat gcaggtatca
     6481 gaactggtga tttaggtggt tccaacagta ccaccgaagt cggtgatgct gtcgccgaag
     6541 aagttaagaa aatccttgct taaaaagatt ctcttttttt atgatatttg tacataaact
     6601 ttataaatga aattcataat agaaacgaca cgaaattaca aaatggaata tgttcatagg
     6661 gtagacgaaa ctatatacgc aatctacata catttatcaa gaaggagaaa aaggaggatg
     6721 taaaggaata caggtaagca aattgatact aatggctcaa cgtgataagg aaaaagaatt
     6781 gcactttaac attaatattg acaaggagga gggcaccaca caaaaagtta ggtgtaacag
     6841 aaaatcatga aactatgatt cctaatttat atattggagg attttctcta aaaaaaaaaa
     6901 aatacaacaa ataaaaaaca ctcaatgacc tgaccatttg atggagttta agtcaatacc
     6961 ttcttgaacc atttcccata atggtgaaag ttccctcaag aattttactc tgtcagaaac
     7021 ggccttaacg acgtagtcga tatggtgcac tctcagtaca atctgctctg atgccgcata
     7081 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     7141 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     7201 ttcaccgtca tcaccgaaac gcgcga
//
