LOCUS       sequence-465976-        7220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose iduced expression of Gcn4 WTSLFD+ in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7220)
  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 7220)
  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 WTSLFD
FEATURES             Location/Qualifiers
     source          1..7220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(9..27)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /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(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2234..2253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /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     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2754..2774
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2798..3239
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2798..2915
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3210..3233
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     3254..3270
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3254..3270
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    3278..3302
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3305..3325
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3596..3652
                     /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             3689..3778
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(3789..3813)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3847..4094
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3863..3881)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4112..4131)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4113..4131)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4141..4158)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4141..4157)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4150..4172)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4298..4753
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4385..4404)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4595..4616
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5054..5461
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5462..6556
                     /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(7050..7069)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7169..7191
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
       61 ttaggacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     2761 accctcacta aagggaacaa aagctggagc tctagtacgg attagaagcc gccgagcggg
     2821 tgacagccct ccgaaggaag actctcctcc gtgcgtcctc gtcttcaccg gtcgcgttcc
     2881 tgaaacgcag atgtgcctcg cgccgcactg ctccgaacaa taaagattct acaatactag
     2941 cttttatggt tatgaagagg aaaaattggc agtaacctgg ccccacaaac cttcaaatga
     3001 acgaatcaaa ttaacaacca taggatgata atgcgattag ttttttagcc ttatttctgg
     3061 ggtaattaat cagcgaagcg atgatttttg atctattaac agatatataa atgcaaaaac
     3121 tgcataacca ctttaactaa tactttcaac attttcggtt tgtattactt cttattcaaa
     3181 tgtaataaaa gtatcaacaa aaaattgtta atatacctct atactttaac gtcaaggaga
     3241 aaaaaccccg gattctagaa ctagtggatc ccccatcaca agtttgtaca aaaaagcagg
     3301 cttcgagaac ctgtattttc agggtcatat gagcaccgat agcaccccga tgtggaccag
     3361 cctgtttgac gaagataaca gcaaagaatg gaccagcctg tttgataatg atattccggt
     3421 taccaccgat gatgttagcc tggcagataa agcaattgaa agcggtagcg gttcaggtag
     3481 cggtagtccg gaatccagtg atcctgctgc tctaaaacgt gctagaaaca ctgaagccgc
     3541 caggcgttct cgtgcgagaa agttgcaaag aatgaaacaa cttgaagaca aggttgaaga
     3601 attgctttcg aaaaattatc acttggaaaa tgaggttgcc agattaaaga aattagttgg
     3661 cgaacgcacc ggtggtggat ccggtggtta cccatacgat gttcctgact atgcgggcta
     3721 tccctatgac gtcccggact atgcaggatc ctatccgtat gacgttccag attacgctgc
     3781 tcagtaagac ccagctttct tgtacaaagt ggtgatgggc tgcaggaatt cgatatcaag
     3841 ctcgagtcat gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc
     3901 taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag
     3961 ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac
     4021 gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga
     4081 aggctttaat ttgcggccgg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     4141 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     4201 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     4261 cccttcccaa cagttgcgca gcctgaatgg cgaatggacg cgccctgtag cggcgcatta
     4321 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     4381 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4441 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4501 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4561 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4621 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     4681 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4741 acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4801 gcatatcgac cggtcgagga gaacttctag tatatctaca tacctaatat tattgcctta
     4861 ttaaaaatgg aatcccaaca attacatcaa aatccacatt ctcttcaaaa tcaattgtcc
     4921 tgtacttcct tgttcatgtg tgttcaaaaa cgttatattt ataggataat tatactctat
     4981 ttctcaacaa gtaattggtt gtttggccga gcggtctaag gcgcctgatt caagaaatat
     5041 cttgaccgca gttaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc
     5101 ttagcaacca ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag
     5161 aatcaaattc gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc
     5221 tttttcaact gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca
     5281 tatagaatag agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat
     5341 attatttaag gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac
     5401 ttttcttacc ttttacattt cagcaatata tatatatata tttcaaggat ataccattct
     5461 aatgtctgcc cctaagaaga tcgtcgtttt gccaggtgac cacgttggtc aagaaatcac
     5521 agccgaagcc attaaggttc ttaaagctat ttctgatgtt cgttccaatg tcaagttcga
     5581 tttcgaaaat catttaattg gtggtgctgc tatcgatgct acaggtgttc cacttccaga
     5641 tgaggcgctg gaagcctcca agaaggctga tgccgttttg ttaggtgctg tgggtggtcc
     5701 taaatggggt accggtagtg ttagacctga acaaggttta ctaaaaatcc gtaaagaact
     5761 tcaattgtac gccaacttaa gaccatgtaa ctttgcatcc gactctcttt tagacttatc
     5821 tccaatcaag ccacaatttg ctaaaggtac tgacttcgtt gttgtcagag aattagtggg
     5881 aggtatttac tttggtaaga gaaaggaaga cgatggtgat ggtgtcgctt gggatagtga
     5941 acaatacacc gttccagaag tgcaaagaat cacaagaatg gccgctttca tggccctaca
     6001 acatgagcca ccattgccta tttggtcctt ggataaagct aatgttttgg cctcttcaag
     6061 attatggaga aaaactgtgg aggaaaccat caagaacgaa ttccctacat tgaaggttca
     6121 acatcaattg attgattctg ccgccatgat cctagttaag aacccaaccc acctaaatgg
     6181 tattataatc accagcaaca tgtttggtga tatcatctcc gatgaagcct ccgttatccc
     6241 aggttccttg ggtttgttgc catctgcgtc cttggcctct ttgccagaca agaacaccgc
     6301 atttggtttg tacgaaccat gccacggttc tgctccagat ttgccaaaga ataaggtcaa
     6361 ccctatcgcc actatcttgt ctgctgcaat gatgttgaaa ttgtcattga acttgcctga
     6421 agaaggtaag gccattgaag atgcagttaa aaaggttttg gatgcaggta tcagaactgg
     6481 tgatttaggt ggttccaaca gtaccaccga agtcggtgat gctgtcgccg aagaagttaa
     6541 gaaaatcctt gcttaaaaag attctctttt tttatgatat ttgtacataa actttataaa
     6601 tgaaattcat aatagaaacg acacgaaatt acaaaatgga atatgttcat agggtagacg
     6661 aaactatata cgcaatctac atacatttat caagaaggag aaaaaggagg atgtaaagga
     6721 atacaggtaa gcaaattgat actaatggct caacgtgata aggaaaaaga attgcacttt
     6781 aacattaata ttgacaagga ggagggcacc acacaaaaag ttaggtgtaa cagaaaatca
     6841 tgaaactatg attcctaatt tatatattgg aggattttct ctaaaaaaaa aaaaatacaa
     6901 caaataaaaa acactcaatg acctgaccat ttgatggagt ttaagtcaat accttcttga
     6961 accatttccc ataatggtga aagttccctc aagaatttta ctctgtcaga aacggcctta
     7021 acgacgtagt cgatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     7081 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     7141 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     7201 tcatcaccga aacgcgcgag
//
