LOCUS       sequence-465960-        7103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Galactose iduced expression of Gcn4  L123A 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 L123A
FEATURES             Location/Qualifiers
     source          1..7103
                     /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(3672..3696)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3730..3977
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3746..3764)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3995..4014)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3996..4014)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4024..4041)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4024..4040)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4033..4055)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4181..4636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4268..4287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4478..4499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4937..5344
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5345..6439
                     /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(6933..6952)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7052..7074
                     /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 agggtagcat gagcaccgat agcaccccga tgtttgaata
     3361 tgaaaatctg gaagataaca gcaaagaatg gaccagcgca tttgataatg atattccggt
     3421 taccaccgat gatgttagcc tggcagataa agcaattgaa agcggtagcg gttcaggtag
     3481 cggtagtccg gaaagcagcg atccggcagc actgaaacgt gcccgtaata ccgaagcagc
     3541 acgtcgtagc cgtgcacgta aactgcagcg tatgaaacag ttagaagata aggttgaaga
     3601 actgctgtgc aaaaactatc acctggaaaa tgaagttgcc cgtctgaaaa aactggttgg
     3661 tgaacgttaa gacccagctt tcttgtacaa agtggtgatg ggctgcagga attcgatatc
     3721 aagctcgagt catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg
     3781 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta
     3841 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca
     3901 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct
     3961 cgaaggcttt aatttgcggc cggtacccaa ttcgccctat agtgagtcgt attacgcgcg
     4021 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     4081 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     4141 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca
     4201 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4261 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4321 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4381 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4441 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4501 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     4561 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4621 ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4681 accgcatatc gaccggtcga ggagaacttc tagtatatct acatacctaa tattattgcc
     4741 ttattaaaaa tggaatccca acaattacat caaaatccac attctcttca aaatcaattg
     4801 tcctgtactt ccttgttcat gtgtgttcaa aaacgttata tttataggat aattatactc
     4861 tatttctcaa caagtaattg gttgtttggc cgagcggtct aaggcgcctg attcaagaaa
     4921 tatcttgacc gcagttaact gtgggaatac tcaggtatcg taagatgcaa gagttcgaat
     4981 ctcttagcaa ccattatttt tttcctcaac ataacgagaa cacacagggg cgctatcgca
     5041 cagaatcaaa ttcgatgact ggaaattttt tgttaatttc agaggtcgcc tgacgcatat
     5101 acctttttca actgaaaaat tgggagaaaa aggaaaggtg agagcgccgg aaccggcttt
     5161 tcatatagaa tagagaagcg ttcatgacta aatgcttgca tcacaatact tgaagttgac
     5221 aatattattt aaggacctat tgttttttcc aataggtggt tagcaatcgt cttactttct
     5281 aacttttctt accttttaca tttcagcaat atatatatat atatttcaag gatataccat
     5341 tctaatgtct gcccctaaga agatcgtcgt tttgccaggt gaccacgttg gtcaagaaat
     5401 cacagccgaa gccattaagg ttcttaaagc tatttctgat gttcgttcca atgtcaagtt
     5461 cgatttcgaa aatcatttaa ttggtggtgc tgctatcgat gctacaggtg ttccacttcc
     5521 agatgaggcg ctggaagcct ccaagaaggc tgatgccgtt ttgttaggtg ctgtgggtgg
     5581 tcctaaatgg ggtaccggta gtgttagacc tgaacaaggt ttactaaaaa tccgtaaaga
     5641 acttcaattg tacgccaact taagaccatg taactttgca tccgactctc ttttagactt
     5701 atctccaatc aagccacaat ttgctaaagg tactgacttc gttgttgtca gagaattagt
     5761 gggaggtatt tactttggta agagaaagga agacgatggt gatggtgtcg cttgggatag
     5821 tgaacaatac accgttccag aagtgcaaag aatcacaaga atggccgctt tcatggccct
     5881 acaacatgag ccaccattgc ctatttggtc cttggataaa gctaatgttt tggcctcttc
     5941 aagattatgg agaaaaactg tggaggaaac catcaagaac gaattcccta cattgaaggt
     6001 tcaacatcaa ttgattgatt ctgccgccat gatcctagtt aagaacccaa cccacctaaa
     6061 tggtattata atcaccagca acatgtttgg tgatatcatc tccgatgaag cctccgttat
     6121 cccaggttcc ttgggtttgt tgccatctgc gtccttggcc tctttgccag acaagaacac
     6181 cgcatttggt ttgtacgaac catgccacgg ttctgctcca gatttgccaa agaataaggt
     6241 caaccctatc gccactatct tgtctgctgc aatgatgttg aaattgtcat tgaacttgcc
     6301 tgaagaaggt aaggccattg aagatgcagt taaaaaggtt ttggatgcag gtatcagaac
     6361 tggtgattta ggtggttcca acagtaccac cgaagtcggt gatgctgtcg ccgaagaagt
     6421 taagaaaatc cttgcttaaa aagattctct ttttttatga tatttgtaca taaactttat
     6481 aaatgaaatt cataatagaa acgacacgaa attacaaaat ggaatatgtt catagggtag
     6541 acgaaactat atacgcaatc tacatacatt tatcaagaag gagaaaaagg aggatgtaaa
     6601 ggaatacagg taagcaaatt gatactaatg gctcaacgtg ataaggaaaa agaattgcac
     6661 tttaacatta atattgacaa ggaggagggc accacacaaa aagttaggtg taacagaaaa
     6721 tcatgaaact atgattccta atttatatat tggaggattt tctctaaaaa aaaaaaaata
     6781 caacaaataa aaaacactca atgacctgac catttgatgg agtttaagtc aataccttct
     6841 tgaaccattt cccataatgg tgaaagttcc ctcaagaatt ttactctgtc agaaacggcc
     6901 ttaacgacgt agtcgatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6961 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     7021 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     7081 ccgtcatcac cgaaacgcgc gag
//
