LOCUS       sequence-427260-        8800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of Gal4-DBD NFAT5 AroLITE A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8800)
  AUTHORS   Naderi J, Magalhaes AP, Kibar G, Stik G, Zhang Y, Mackowiak SD, 
            Wieler HM, Rossi F, Buschow R, Christou-Kent M, Alcoverro-Bertran M,
            Graf T, Vingron M, Hnisz D
  TITLE     An activity-specificity trade-off encoded in human transcription 
            factors.
  JOURNAL   Nat Cell Biol. 2024 Aug;26(8):1309-1321. doi: 
            10.1038/s41556-024-01411-0. Epub 2024 Jul 5.
  PUBMED    38969762
REFERENCE   2  (bases 1 to 8800)
  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"; doi: 
            "10.1038/s41556-024-01411-0"; journalName: "Nat Cell Biol"; date: 
            "2024-08"; volume: "26"; issue: "8"; pages: "1309-1321"
COMMENT     Sequence Label: pGAL4-DBD-NFAT5-AroLITE_A
FEATURES             Location/Qualifiers
     source          1..8800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             710..1150
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     promoter        complement(4119..4137)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4120..4137)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4157..4174)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4163..4387
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4433..4861
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4520..4539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4730..4751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4870..4890)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4875..5204
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5055..5190
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5117..5136
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5271..6065
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5325..5344)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5935..5954
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6239..6360
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6276..6295)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6330..6349
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6409..6425)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6409..6425)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6422..6444)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6433..6449
                     /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)."
     promoter        complement(join(6457..6463,6464..6481,6482..6487))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6502..6523
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6640..6657)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6811..7399)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6891..6910)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7570..8361,8362..8430))
                     /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     8193..8212
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8431..8535)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8610..8629)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatccac tagtaacggc cgccagtgtg ctggaattca tgaagctact
      721 gtcttctatc gaacaagcat gcgatatttg ccgacttaaa aagctcaagt gctccaaaga
      781 aaaaccgaag tgcgccaagt gtctgaagaa caactgggag tgtcgctact ctcccaaaac
      841 caaaaggtct ccgctgacta gggcacatct gacagaagtg gaatcaaggc tagaaagact
      901 ggaacagcta tttctactga tttttcctcg agaagacctt gacatgattt tgaaaatgga
      961 ttctttacag gatataaaag cattgttaac aggattattt gtacaagata atgtgaataa
     1021 agatgccgtc acagatagat tggcttcagt ggagactgat atgcctctaa cattgagaca
     1081 gcatagaata agtgcgacat catcatcgga agagagtagt aacaaaggtc aaagacagtt
     1141 gactgtatcg gcgatcacca tggtgaagaa ggaaatatct agtccagcaa gaccttgctc
     1201 tgcagaagag gccatgaaag caatgaaaac tactggatgt aatttagata aggtaaatat
     1261 tatccctaat gccctgatga ctccactcat accaagcagt atgattaaga gtgaagatgt
     1321 tactccaatg gaagtaacag cagaaaaaag atcttccact attgcaaaga ctacaaagtc
     1381 tgttggatca actcagcaaa cattagaaaa catctcaaac atagcaggaa atggctctgc
     1441 atcatcacca tcatcttccc acctaccttc tgaaaatgaa aaacagcagc agattcagcc
     1501 caaggcagca aacccagaga ccctgacaac tattcaaacc caggacatct cacagcctgg
     1561 tactgcacca gcagtttctg cttctagtca gctgcccaac agcgatgcac tattgcagca
     1621 ggctacacag gcacagacaa gagaaactca gtctagagag atattacagt cagatggtac
     1681 agtggttaat ttgtcacaac tgactgaggc atcacaacaa cagcagcagt caccactaca
     1741 agaacaagca cagactttac agcagcagat ttcatcaaat attgcaccat caccaaatag
     1801 tgtgagtcag cttcagaata ctattcagca gctgcaagca gggagtgcaa caggcagtac
     1861 tgctagtggc agcagtggaa gtgttgactt ggtccaacaa gttttagagg cacagcagca
     1921 gttatcttca gttttagcat ctgctccaga tggtaatgag aatgttcaag agcagcttag
     1981 tgcagatatt gcacaacaag tcagtcaaat tcagagtggt gtaagccctg gaatggcatc
     2041 ctcaacagag ccaacagtcc ataccagacc agataattta ttacctggaa gagctgaaag
     2101 tgttcatcca cagtctgaaa acacgttatc taatcaacag cagcagcagc agcagcaaca
     2161 gcaagtgatg gaatcttcag ccgcaatggt gatggagatg caacagagta tctgccaggc
     2221 agctgcccag attcagtcag agttagcacc ttcaactgct tcagcaaatg gaaaccttca
     2281 gcaatcgcca gttgcacagc agacttctca catgatgagt gcattgtcta ccaatgagga
     2341 tatgcaaatg cagtgtgaat tggcatcttc tcctcctgca gtttctggaa atgaaacttc
     2401 tacaactacc acacagcagg ttgcaacccc tggcactacc atggcacaga catcaagttc
     2461 aggagatgga gaagaaactg gaacacaagc aaaacagatt cagaacagtg tcgcacagac
     2521 catggtccaa atgcaacata gtggggacaa tcaacctcaa gttaaccttg catcatccac
     2581 aaaaagtatg atgagtgttc agaatagtgg tacccaacaa caaggtaatg gtttagcaca
     2641 gcaagggaat gagatgatgt cacttcaatc tggaaatgca ttgcagcagt cttctcattc
     2701 acaggcccaa cttgcacatc ctcaaaatcc tattgccgat gctcagaacc tttcccagga
     2761 aactcaaggt tctctcgcac atagtccaaa tcctattgtc cacagtcaga cttctacaac
     2821 ctcctctgaa caaatgcagc ctccaatggc acactctcaa agtaccattg ctgtgttaca
     2881 gggctcttca gttcctcaag accagcagtc aaccaacata gcactttccc agagtcccat
     2941 gaataatctt cagactaaca cagtagccca agaagcagca gcagcagcac cgaactcaat
     3001 ttctccactt cagtcaacat caaacagtga acaacaagct gctgcacaac agcaagctcc
     3061 aatatcacac atccagactc ctatgctttc ccaagaacag gcacaacccc cgcagcaggg
     3121 tttagcacag cctcaggtgg ccctgggctc ccttccacct aatccaatgc ctcaaagcca
     3181 acaaggaacc atggcacagt cacagcactc aatagttgcc atgcagagta actctccatc
     3241 ccaggaacag cagcagcagc agcaacagca gcagcaacag agcattttag caagtaatca
     3301 gaataccatg gctacaatgg cgtctccaaa gcaaccacca ccaaacatga tagcaaaccc
     3361 aaatcaaaat ccaatggcta atcaggagca acagaaccag tcaattgcac accaacaaag
     3421 taacatggcc ccaatgaatc aagagcaaca gcccatgcaa gcacagagtc agtccacagt
     3481 ttcctcactt cagaacccag gtcctaccca gtcggaatca tcacagaccc ccttggcaca
     3541 tagctctcct cagattcagt tggtacaagg gtcacctagt tctcaagagc agcaagtaac
     3601 tctcgcatta tctccagcat ccatgtctgc cttgcagacc agtataaatc aacaagatat
     3661 gcaacagtct cctcttgcat cccctcagaa caacatgcct ggaattcaag gagccacatc
     3721 ttcgcctcaa ccacaggcta ctttagcaca caacacagca ggaggcacaa tgaaccaact
     3781 gcagaattct cctggctcat ctcagcagac atcaggaatg gcattagcag gcattcaaaa
     3841 taactgtagt cagcttttaa cctctggacc agctacattg cctgatcagt tgatggccat
     3901 aagtcagcca ggccaaccac aaaacgaggg ccagccacct gtgacaacac ttctttctca
     3961 gcaaatgcca gagaattctc cactggcatc ctctataaac accaaccaga acatcgaaaa
     4021 gattgatttg cttgtttcat tgcaaaacca agggaacaac ttgactggct ccgcagtacc
     4081 ggatccgacg tcggtcgcca ccgtctagag ggccctattc tatagtgtca cctaaatgct
     4141 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     4201 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4261 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4321 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     4381 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     4441 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4501 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4561 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4621 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     4681 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4741 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     4801 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4861 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     4921 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     4981 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     5041 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     5101 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     5161 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     5221 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     5281 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     5341 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     5401 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     5461 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     5521 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     5581 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     5641 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     5701 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     5761 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     5821 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     5881 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     5941 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     6001 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     6061 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     6121 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     6181 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     6241 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     6301 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     6361 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     6421 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     6481 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6541 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6601 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6661 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6721 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6781 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6841 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6901 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6961 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     7021 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     7081 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     7141 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     7201 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     7261 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     7321 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     7381 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     7441 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     7501 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7561 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7621 ttcatccata gttgcctgac ttcccgtcgt gtagataact acgatacggg agggcttacc
     7681 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7741 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7801 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7861 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7921 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7981 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     8041 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     8101 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     8161 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     8221 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     8281 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     8341 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     8401 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     8461 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     8521 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     8581 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     8641 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     8701 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     8761 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
//
