LOCUS       sequence-465897-        7103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose iduced expression of Gcn4 F124I in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7103)
  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 7103)
  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 F124I
FEATURES             Location/Qualifiers
     source          1..7103
                     /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(3673..3697)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3731..3978
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3747..3765)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3996..4015)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3997..4015)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4025..4042)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4025..4041)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4034..4056)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4182..4637
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4269..4288)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4479..4500
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4938..5345
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5346..6440
                     /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(6934..6953)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7053..7075
                     /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 tgagcaccga tagcaccccg atgtttgaat
     3361 atgaaaatct ggaagataac agcaaagaat ggaccagcct gattgataat gatattccgg
     3421 ttaccaccga tgatgttagc ctggcagata aagcaattga aagcggtagc ggttcaggta
     3481 gcggtagtcc ggaaagcagc gatccggcag cactgaaacg tgcccgtaat accgaagcag
     3541 cacgtcgtag ccgtgcacgt aaactgcagc gtatgaaaca gttagaagat aaggttgaag
     3601 aactgctgtg caaaaactat cacctggaaa atgaagttgc ccgtctgaaa aaactggttg
     3661 gtgaacgtta agacccagct ttcttgtaca aagtggtgat gggctgcagg aattcgatat
     3721 caagctcgag tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc
     3781 gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt
     3841 atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac
     3901 agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc
     3961 tcgaaggctt taatttgcgg ccggtaccca attcgcccta tagtgagtcg tattacgcgc
     4021 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4081 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     4141 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc
     4201 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4261 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4321 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4381 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4441 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4501 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4561 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4621 attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4681 caccgcatat cgaccggtcg aggagaactt ctagtatatc tacataccta atattattgc
     4741 cttattaaaa atggaatccc aacaattaca tcaaaatcca cattctcttc aaaatcaatt
     4801 gtcctgtact tccttgttca tgtgtgttca aaaacgttat atttatagga taattatact
     4861 ctatttctca acaagtaatt ggttgtttgg ccgagcggtc taaggcgcct gattcaagaa
     4921 atatcttgac cgcagttaac tgtgggaata ctcaggtatc gtaagatgca agagttcgaa
     4981 tctcttagca accattattt ttttcctcaa cataacgaga acacacaggg gcgctatcgc
     5041 acagaatcaa attcgatgac tggaaatttt ttgttaattt cagaggtcgc ctgacgcata
     5101 tacctttttc aactgaaaaa ttgggagaaa aaggaaaggt gagagcgccg gaaccggctt
     5161 ttcatataga atagagaagc gttcatgact aaatgcttgc atcacaatac ttgaagttga
     5221 caatattatt taaggaccta ttgttttttc caataggtgg ttagcaatcg tcttactttc
     5281 taacttttct taccttttac atttcagcaa tatatatata tatatttcaa ggatatacca
     5341 ttctaatgtc tgcccctaag aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa
     5401 tcacagccga agccattaag gttcttaaag ctatttctga tgttcgttcc aatgtcaagt
     5461 tcgatttcga aaatcattta attggtggtg ctgctatcga tgctacaggt gttccacttc
     5521 cagatgaggc gctggaagcc tccaagaagg ctgatgccgt tttgttaggt gctgtgggtg
     5581 gtcctaaatg gggtaccggt agtgttagac ctgaacaagg tttactaaaa atccgtaaag
     5641 aacttcaatt gtacgccaac ttaagaccat gtaactttgc atccgactct cttttagact
     5701 tatctccaat caagccacaa tttgctaaag gtactgactt cgttgttgtc agagaattag
     5761 tgggaggtat ttactttggt aagagaaagg aagacgatgg tgatggtgtc gcttgggata
     5821 gtgaacaata caccgttcca gaagtgcaaa gaatcacaag aatggccgct ttcatggccc
     5881 tacaacatga gccaccattg cctatttggt ccttggataa agctaatgtt ttggcctctt
     5941 caagattatg gagaaaaact gtggaggaaa ccatcaagaa cgaattccct acattgaagg
     6001 ttcaacatca attgattgat tctgccgcca tgatcctagt taagaaccca acccacctaa
     6061 atggtattat aatcaccagc aacatgtttg gtgatatcat ctccgatgaa gcctccgtta
     6121 tcccaggttc cttgggtttg ttgccatctg cgtccttggc ctctttgcca gacaagaaca
     6181 ccgcatttgg tttgtacgaa ccatgccacg gttctgctcc agatttgcca aagaataagg
     6241 tcaaccctat cgccactatc ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc
     6301 ctgaagaagg taaggccatt gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa
     6361 ctggtgattt aggtggttcc aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag
     6421 ttaagaaaat ccttgcttaa aaagattctc tttttttatg atatttgtac ataaacttta
     6481 taaatgaaat tcataataga aacgacacga aattacaaaa tggaatatgt tcatagggta
     6541 gacgaaacta tatacgcaat ctacatacat ttatcaagaa ggagaaaaag gaggatgtaa
     6601 aggaatacag gtaagcaaat tgatactaat ggctcaacgt gataaggaaa aagaattgca
     6661 ctttaacatt aatattgaca aggaggaggg caccacacaa aaagttaggt gtaacagaaa
     6721 atcatgaaac tatgattcct aatttatata ttggaggatt ttctctaaaa aaaaaaaaat
     6781 acaacaaata aaaaacactc aatgacctga ccatttgatg gagtttaagt caataccttc
     6841 ttgaaccatt tcccataatg gtgaaagttc cctcaagaat tttactctgt cagaaacggc
     6901 cttaacgacg tagtcgatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     6961 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     7021 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     7081 accgtcatca ccgaaacgcg cga
//
