LOCUS       sequence-465871-        7778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose iduced expression of Gcn4 DBDGFP in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7778)
  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 7778)
  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_DBDGFP
FEATURES             Location/Qualifiers
     source          1..7778
                     /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             3444..3500
                     /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             3537..3563
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3579..4289
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(3606..3634)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4229..4249
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             4311..4337
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(4348..4372)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      4406..4653
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4422..4440)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4671..4690)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4672..4690)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4700..4717)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4700..4716)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4709..4731)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4857..5312
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4944..4963)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5154..5175
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5613..6020
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             6021..7115
                     /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(7609..7628)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7728..7750
                     /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 cagggtcata tgtctccgga atccagtgat cctgctgctc
     3361 taaaacgtgc tagaaacact gaagccgcca ggcgttctcg tgcgagaaag ttgcaaagaa
     3421 tgaaacaact tgaagacaag gttgaagaat tgctttcgaa aaattatcac ttggaaaatg
     3481 aggttgccag attaaagaaa ttagttggcg aacgcaccgg tggtggatcc ggtggttacc
     3541 catacgatgt tcctgactat gcgggctatc cctatgacag taaaggagaa gaacttttca
     3601 ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg
     3661 tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca
     3721 ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttcact tatggtgttc
     3781 aatgcttttc aagataccca gatcatatga aacggcatga ctttttcaag agtgccatgc
     3841 ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac tacaagacac
     3901 gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta aaaggtattg
     3961 attttaaaga agatggaaac attcttggac acaaattgga atacaactat aactcacaca
     4021 atgtatacat catggcagac aaacaaaaga atggaatcaa agttaacttc aaaattagac
     4081 acaacattga agatggaagc gttcaactag cagaccatta tcaacaaaat actccaattg
     4141 gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct gccctttcga
     4201 aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga
     4261 ttacacatgg catggatgaa ctatacaaag tcccggacta tgcaggatcc tatccgtatg
     4321 acgttccaga ttacgctgct cagtaagacc cagctttctt gtacaaagtg gtgatgggct
     4381 gcaggaattc gatatcaagc tcgagtcatg taattagtta tgtcacgctt acattcacgc
     4441 cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc
     4501 cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt
     4561 cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag
     4621 aaggttttgg gacgctcgaa ggctttaatt tgcggccggt acccaattcg ccctatagtg
     4681 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     4741 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     4801 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggacgc
     4861 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4921 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4981 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5041 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     5101 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5161 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     5221 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5281 gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat
     5341 ctgtgcggta tttcacaccg catatcgacc ggtcgaggag aacttctagt atatctacat
     5401 acctaatatt attgccttat taaaaatgga atcccaacaa ttacatcaaa atccacattc
     5461 tcttcaaaat caattgtcct gtacttcctt gttcatgtgt gttcaaaaac gttatattta
     5521 taggataatt atactctatt tctcaacaag taattggttg tttggccgag cggtctaagg
     5581 cgcctgattc aagaaatatc ttgaccgcag ttaactgtgg gaatactcag gtatcgtaag
     5641 atgcaagagt tcgaatctct tagcaaccat tatttttttc ctcaacataa cgagaacaca
     5701 caggggcgct atcgcacaga atcaaattcg atgactggaa attttttgtt aatttcagag
     5761 gtcgcctgac gcatatacct ttttcaactg aaaaattggg agaaaaagga aaggtgagag
     5821 cgccggaacc ggcttttcat atagaataga gaagcgttca tgactaaatg cttgcatcac
     5881 aatacttgaa gttgacaata ttatttaagg acctattgtt ttttccaata ggtggttagc
     5941 aatcgtctta ctttctaact tttcttacct tttacatttc agcaatatat atatatatat
     6001 ttcaaggata taccattcta atgtctgccc ctaagaagat cgtcgttttg ccaggtgacc
     6061 acgttggtca agaaatcaca gccgaagcca ttaaggttct taaagctatt tctgatgttc
     6121 gttccaatgt caagttcgat ttcgaaaatc atttaattgg tggtgctgct atcgatgcta
     6181 caggtgttcc acttccagat gaggcgctgg aagcctccaa gaaggctgat gccgttttgt
     6241 taggtgctgt gggtggtcct aaatggggta ccggtagtgt tagacctgaa caaggtttac
     6301 taaaaatccg taaagaactt caattgtacg ccaacttaag accatgtaac tttgcatccg
     6361 actctctttt agacttatct ccaatcaagc cacaatttgc taaaggtact gacttcgttg
     6421 ttgtcagaga attagtggga ggtatttact ttggtaagag aaaggaagac gatggtgatg
     6481 gtgtcgcttg ggatagtgaa caatacaccg ttccagaagt gcaaagaatc acaagaatgg
     6541 ccgctttcat ggccctacaa catgagccac cattgcctat ttggtccttg gataaagcta
     6601 atgttttggc ctcttcaaga ttatggagaa aaactgtgga ggaaaccatc aagaacgaat
     6661 tccctacatt gaaggttcaa catcaattga ttgattctgc cgccatgatc ctagttaaga
     6721 acccaaccca cctaaatggt attataatca ccagcaacat gtttggtgat atcatctccg
     6781 atgaagcctc cgttatccca ggttccttgg gtttgttgcc atctgcgtcc ttggcctctt
     6841 tgccagacaa gaacaccgca tttggtttgt acgaaccatg ccacggttct gctccagatt
     6901 tgccaaagaa taaggtcaac cctatcgcca ctatcttgtc tgctgcaatg atgttgaaat
     6961 tgtcattgaa cttgcctgaa gaaggtaagg ccattgaaga tgcagttaaa aaggttttgg
     7021 atgcaggtat cagaactggt gatttaggtg gttccaacag taccaccgaa gtcggtgatg
     7081 ctgtcgccga agaagttaag aaaatccttg cttaaaaaga ttctcttttt ttatgatatt
     7141 tgtacataaa ctttataaat gaaattcata atagaaacga cacgaaatta caaaatggaa
     7201 tatgttcata gggtagacga aactatatac gcaatctaca tacatttatc aagaaggaga
     7261 aaaaggagga tgtaaaggaa tacaggtaag caaattgata ctaatggctc aacgtgataa
     7321 ggaaaaagaa ttgcacttta acattaatat tgacaaggag gagggcacca cacaaaaagt
     7381 taggtgtaac agaaaatcat gaaactatga ttcctaattt atatattgga ggattttctc
     7441 taaaaaaaaa aaaatacaac aaataaaaaa cactcaatga cctgaccatt tgatggagtt
     7501 taagtcaata ccttcttgaa ccatttccca taatggtgaa agttccctca agaattttac
     7561 tctgtcagaa acggccttaa cgacgtagtc gatatggtgc actctcagta caatctgctc
     7621 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     7681 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     7741 gtgtcagagg ttttcaccgt catcaccgaa acgcgcga
//
