LOCUS       sequence-465956-        7106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose iduced expression of Gcn4 ATtoG KtoR in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7106)
  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 7106)
  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 SATtoG KtoR
FEATURES             Location/Qualifiers
     source          1..7106
                     /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"
     protein_bind    complement(3675..3699)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3733..3980
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3749..3767)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3998..4017)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3999..4017)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4027..4044)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4027..4043)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4036..4058)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4184..4639
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4271..4290)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4481..4502
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4940..5347
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5348..6442
                     /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(6936..6955)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7055..7077
                     /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 agggtagcca tatgggtggt gatggtggtc cgatgtttga
     3361 atatgaaaat ctggaagata atggtcgcga atggggtggt ctgtttgata atgatattcc
     3421 ggttggcggt gatgatgttg gcctgggtga tcgtggtatt gaaggtggta gcggtagcgg
     3481 ttcaggtagt ccggaaagca gcgatccggc agcactgaaa cgtgcccgta ataccgaagc
     3541 agcacgtcgt agccgtgcac gtaaactgca gcgtatgaaa cagttagaag ataaggttga
     3601 agaactgctg tgcaaaaact atcacctgga aaatgaagtt gcccgtctga aaaagctggt
     3661 tggtgaacgt taagacccag ctttcttgta caaagtggtg atgggctgca ggaattcgat
     3721 atcaagctcg agtcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat
     3781 ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt
     3841 ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt
     3901 acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac
     3961 gctcgaaggc tttaatttgc ggccggtacc caattcgccc tatagtgagt cgtattacgc
     4021 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4081 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4141 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc
     4201 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4261 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4321 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4381 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4441 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4501 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     4561 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4621 atattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4681 cacaccgcat atcgaccggt cgaggagaac ttctagtata tctacatacc taatattatt
     4741 gccttattaa aaatggaatc ccaacaatta catcaaaatc cacattctct tcaaaatcaa
     4801 ttgtcctgta cttccttgtt catgtgtgtt caaaaacgtt atatttatag gataattata
     4861 ctctatttct caacaagtaa ttggttgttt ggccgagcgg tctaaggcgc ctgattcaag
     4921 aaatatcttg accgcagtta actgtgggaa tactcaggta tcgtaagatg caagagttcg
     4981 aatctcttag caaccattat ttttttcctc aacataacga gaacacacag gggcgctatc
     5041 gcacagaatc aaattcgatg actggaaatt ttttgttaat ttcagaggtc gcctgacgca
     5101 tatacctttt tcaactgaaa aattgggaga aaaaggaaag gtgagagcgc cggaaccggc
     5161 ttttcatata gaatagagaa gcgttcatga ctaaatgctt gcatcacaat acttgaagtt
     5221 gacaatatta tttaaggacc tattgttttt tccaataggt ggttagcaat cgtcttactt
     5281 tctaactttt cttacctttt acatttcagc aatatatata tatatatttc aaggatatac
     5341 cattctaatg tctgccccta agaagatcgt cgttttgcca ggtgaccacg ttggtcaaga
     5401 aatcacagcc gaagccatta aggttcttaa agctatttct gatgttcgtt ccaatgtcaa
     5461 gttcgatttc gaaaatcatt taattggtgg tgctgctatc gatgctacag gtgttccact
     5521 tccagatgag gcgctggaag cctccaagaa ggctgatgcc gttttgttag gtgctgtggg
     5581 tggtcctaaa tggggtaccg gtagtgttag acctgaacaa ggtttactaa aaatccgtaa
     5641 agaacttcaa ttgtacgcca acttaagacc atgtaacttt gcatccgact ctcttttaga
     5701 cttatctcca atcaagccac aatttgctaa aggtactgac ttcgttgttg tcagagaatt
     5761 agtgggaggt atttactttg gtaagagaaa ggaagacgat ggtgatggtg tcgcttggga
     5821 tagtgaacaa tacaccgttc cagaagtgca aagaatcaca agaatggccg ctttcatggc
     5881 cctacaacat gagccaccat tgcctatttg gtccttggat aaagctaatg ttttggcctc
     5941 ttcaagatta tggagaaaaa ctgtggagga aaccatcaag aacgaattcc ctacattgaa
     6001 ggttcaacat caattgattg attctgccgc catgatccta gttaagaacc caacccacct
     6061 aaatggtatt ataatcacca gcaacatgtt tggtgatatc atctccgatg aagcctccgt
     6121 tatcccaggt tccttgggtt tgttgccatc tgcgtccttg gcctctttgc cagacaagaa
     6181 caccgcattt ggtttgtacg aaccatgcca cggttctgct ccagatttgc caaagaataa
     6241 ggtcaaccct atcgccacta tcttgtctgc tgcaatgatg ttgaaattgt cattgaactt
     6301 gcctgaagaa ggtaaggcca ttgaagatgc agttaaaaag gttttggatg caggtatcag
     6361 aactggtgat ttaggtggtt ccaacagtac caccgaagtc ggtgatgctg tcgccgaaga
     6421 agttaagaaa atccttgctt aaaaagattc tcttttttta tgatatttgt acataaactt
     6481 tataaatgaa attcataata gaaacgacac gaaattacaa aatggaatat gttcataggg
     6541 tagacgaaac tatatacgca atctacatac atttatcaag aaggagaaaa aggaggatgt
     6601 aaaggaatac aggtaagcaa attgatacta atggctcaac gtgataagga aaaagaattg
     6661 cactttaaca ttaatattga caaggaggag ggcaccacac aaaaagttag gtgtaacaga
     6721 aaatcatgaa actatgattc ctaatttata tattggagga ttttctctaa aaaaaaaaaa
     6781 atacaacaaa taaaaaacac tcaatgacct gaccatttga tggagtttaa gtcaatacct
     6841 tcttgaacca tttcccataa tggtgaaagt tccctcaaga attttactct gtcagaaacg
     6901 gccttaacga cgtagtcgat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     6961 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     7021 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     7081 tcaccgtcat caccgaaacg cgcgag
//
