LOCUS       sequence-465877-        7220 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Galactose iduced expression of Gcn4 R++ W+ 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 2024.11.21.624739
REFERENCE   2  (bases 1 to 7220)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.11.21.624739"
COMMENT     Sequence Label: pAG415Gal_Gcn4 R3W
FEATURES             Location/Qualifiers
     source          1..7220
                     /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             3597..3653
                     /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             3690..3779
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(3790..3814)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3848..4095
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3864..3882)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4113..4132)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4114..4132)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4142..4159)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4142..4158)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4151..4173)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4299..4754
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4386..4405)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4596..4617
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5055..5462
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5463..6557
                     /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(7051..7070)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7170..7192
                     /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 tgagcacccg tagcaccccg atgtttgaat
     3361 atgaaaatct ggaagataac agcaaagaat ggaccagcct gtttgattgg gatattccgg
     3421 ttaccacacg tgatgttagc ctggcagata aagcaattcg tagcggtagc ggttcaggta
     3481 gtggtagtcc ggaatccagt gatcctgctg ctctaaaacg tgctagaaac actgaagccg
     3541 ccaggcgttc tcgtgcgaga aagttgcaaa gaatgaaaca acttgaagac aaggttgaag
     3601 aattgctttc gaaaaattat cacttggaaa atgaggttgc cagattaaag aaattagttg
     3661 gcgaacgcac cggtggtgga tccggtggtt acccatacga tgttcctgac tatgcgggct
     3721 atccctatga cgtcccggac tatgcaggat cctatccgta tgacgttcca gattacgctg
     3781 ctcagtaaga cccagctttc ttgtacaaag tggtgatggg ctgcaggaat tcgatatcaa
     3841 gctcgagtca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct
     3901 ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata
     3961 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga
     4021 cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg
     4081 aaggctttaa tttgcggccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     4141 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     4201 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     4261 gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta gcggcgcatt
     4321 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4381 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4441 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4501 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4561 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4621 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4681 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4741 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4801 cgcatatcga ccggtcgagg agaacttcta gtatatctac atacctaata ttattgcctt
     4861 attaaaaatg gaatcccaac aattacatca aaatccacat tctcttcaaa atcaattgtc
     4921 ctgtacttcc ttgttcatgt gtgttcaaaa acgttatatt tataggataa ttatactcta
     4981 tttctcaaca agtaattggt tgtttggccg agcggtctaa ggcgcctgat tcaagaaata
     5041 tcttgaccgc agttaactgt gggaatactc aggtatcgta agatgcaaga gttcgaatct
     5101 cttagcaacc attatttttt tcctcaacat aacgagaaca cacaggggcg ctatcgcaca
     5161 gaatcaaatt cgatgactgg aaattttttg ttaatttcag aggtcgcctg acgcatatac
     5221 ctttttcaac tgaaaaattg ggagaaaaag gaaaggtgag agcgccggaa ccggcttttc
     5281 atatagaata gagaagcgtt catgactaaa tgcttgcatc acaatacttg aagttgacaa
     5341 tattatttaa ggacctattg ttttttccaa taggtggtta gcaatcgtct tactttctaa
     5401 cttttcttac cttttacatt tcagcaatat atatatatat atttcaagga tataccattc
     5461 taatgtctgc ccctaagaag atcgtcgttt tgccaggtga ccacgttggt caagaaatca
     5521 cagccgaagc cattaaggtt cttaaagcta tttctgatgt tcgttccaat gtcaagttcg
     5581 atttcgaaaa tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtt ccacttccag
     5641 atgaggcgct ggaagcctcc aagaaggctg atgccgtttt gttaggtgct gtgggtggtc
     5701 ctaaatgggg taccggtagt gttagacctg aacaaggttt actaaaaatc cgtaaagaac
     5761 ttcaattgta cgccaactta agaccatgta actttgcatc cgactctctt ttagacttat
     5821 ctccaatcaa gccacaattt gctaaaggta ctgacttcgt tgttgtcaga gaattagtgg
     5881 gaggtattta ctttggtaag agaaaggaag acgatggtga tggtgtcgct tgggatagtg
     5941 aacaatacac cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc atggccctac
     6001 aacatgagcc accattgcct atttggtcct tggataaagc taatgttttg gcctcttcaa
     6061 gattatggag aaaaactgtg gaggaaacca tcaagaacga attccctaca ttgaaggttc
     6121 aacatcaatt gattgattct gccgccatga tcctagttaa gaacccaacc cacctaaatg
     6181 gtattataat caccagcaac atgtttggtg atatcatctc cgatgaagcc tccgttatcc
     6241 caggttcctt gggtttgttg ccatctgcgt ccttggcctc tttgccagac aagaacaccg
     6301 catttggttt gtacgaacca tgccacggtt ctgctccaga tttgccaaag aataaggtca
     6361 accctatcgc cactatcttg tctgctgcaa tgatgttgaa attgtcattg aacttgcctg
     6421 aagaaggtaa ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt atcagaactg
     6481 gtgatttagg tggttccaac agtaccaccg aagtcggtga tgctgtcgcc gaagaagtta
     6541 agaaaatcct tgcttaaaaa gattctcttt ttttatgata tttgtacata aactttataa
     6601 atgaaattca taatagaaac gacacgaaat tacaaaatgg aatatgttca tagggtagac
     6661 gaaactatat acgcaatcta catacattta tcaagaagga gaaaaaggag gatgtaaagg
     6721 aatacaggta agcaaattga tactaatggc tcaacgtgat aaggaaaaag aattgcactt
     6781 taacattaat attgacaagg aggagggcac cacacaaaaa gttaggtgta acagaaaatc
     6841 atgaaactat gattcctaat ttatatattg gaggattttc tctaaaaaaa aaaaaataca
     6901 acaaataaaa aacactcaat gacctgacca tttgatggag tttaagtcaa taccttcttg
     6961 aaccatttcc cataatggtg aaagttccct caagaatttt actctgtcag aaacggcctt
     7021 aacgacgtag tcgatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     7081 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     7141 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     7201 gtcatcaccg aaacgcgcga
//
