LOCUS       sequence-467809-        5504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Measure the activity of the reduced differentially methylated region
            (rDMR) of Cyr61 with luciferase assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5504)
  AUTHORS   Wu BK, Mei SC, Chen EH, Zheng Y, Pan D
  TITLE     YAP induces an oncogenic transcriptional program through 
            TET1-mediated epigenetic remodeling in liver growth and 
            tumorigenesis.
  JOURNAL   Nat Genet. 2022 Aug;54(8):1202-1213. doi: 
            10.1038/s41588-022-01119-7. Epub 2022 Jul 14.
  PUBMED    35835915
REFERENCE   2  (bases 1 to 5504)
  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/s41588-022-01119-7"; journalName: "Nat Genet"; date: 
            "2022-08"; volume: "54"; issue: "8"; pages: "1202-1213"
COMMENT     Sequence Label: pGL3-Cyr61-rDMR
FEATURES             Location/Qualifiers
     source          1..5504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        542..738
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      589..724
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      768..777
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             774..2426
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(840..858)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     polyA_signal    2467..2588
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2478..2497)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2532..2551
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2836..2853)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3007..3595)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3087..3106)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3766..4557,4558..4626))
                     /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     4389..4408
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4627..4731)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4758..5213
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4845..4864)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5055..5076
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5344..5392
                     /note="synthetic polyadenylation signal"
     misc_feature    5406..5497
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5446..5465
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgagc tcttacgcgt agaactactc taggtgccct acttgggtga cagtgaactg
       61 ttaggaaagt ggctgccctt gcaaatcttt agggaacaat ggtggcatat aagctgtcag
      121 cttggagggc gaagtactga acacgggcat ttgtgaagca gaaaaaggaa gcagaaattg
      181 aatttgttaa cctaaaacaa tgatttattt ttacaaggat tgaaatgaaa gcctaagagt
      241 taccccatta ccccgaacaa gtgcggcact tgtttcttat tttagcttcg gggtgattct
      301 gtgtgtggtg caagtagagg tggggaacct tttagtaacc tctgaccaca gctgcatgga
      361 tgaagtgtat ctaaaaatta gagaagctgc ttcaatattt taccattggc tactagcaga
      421 agcgagggga aattcctggg acctattggg tcgctggttt gttaatagat gagcttgaag
      481 caaagacttg tgttaatcct aataagccag tgaggaattt caaggctcga gatctgcgat
      541 ctgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
      601 actccgccca gttccgccca ttctccgccc catcgctgac taattttttt tatttatgca
      661 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
      721 ggcctaggct tttgcaaaaa gcttggcatt ccggtactgt tggtaaagcc accatggaag
      781 acgccaaaaa cataaagaaa ggcccggcgc cattctatcc gctggaagat ggaaccgctg
      841 gagagcaact gcataaggct atgaagagat acgccctggt tcctggaaca attgctttta
      901 cagatgcaca tatcgaggtg gacatcactt acgctgagta cttcgaaatg tccgttcggt
      961 tggcagaagc tatgaaacga tatgggctga atacaaatca cagaatcgtc gtatgcagtg
     1021 aaaactctct tcaattcttt atgccggtgt tgggcgcgtt atttatcgga gttgcagttg
     1081 cgcccgcgaa cgacatttat aatgaacgtg aattgctcaa cagtatgggc atttcgcagc
     1141 ctaccgtggt gttcgtttcc aaaaaggggt tgcaaaaaat tttgaacgtg caaaaaaagc
     1201 tcccaatcat ccaaaaaatt attatcatgg attctaaaac ggattaccag ggatttcagt
     1261 cgatgtacac gttcgtcaca tctcatctac ctcccggttt taatgaatac gattttgtgc
     1321 cagagtcctt cgatagggac aagacaattg cactgatcat gaactcctct ggatctactg
     1381 gtctgcctaa aggtgtcgct ctgcctcata gaactgcctg cgtgagattc tcgcatgcca
     1441 gagatcctat ttttggcaat caaatcattc cggatactgc gattttaagt gttgttccat
     1501 tccatcacgg ttttggaatg tttactacac tcggatattt gatatgtgga tttcgagtcg
     1561 tcttaatgta tagatttgaa gaagagctgt ttctgaggag ccttcaggat tacaagattc
     1621 aaagtgcgct gctggtgcca accctattct ccttcttcgc caaaagcact ctgattgaca
     1681 aatacgattt atctaattta cacgaaattg cttctggtgg cgctcccctc tctaaggaag
     1741 tcggggaagc ggttgccaag aggttccatc tgccaggtat caggcaagga tatgggctca
     1801 ctgagactac atcagctatt ctgattacac ccgaggggga tgataaaccg ggcgcggtcg
     1861 gtaaagttgt tccatttttt gaagcgaagg ttgtggatct ggataccggg aaaacgctgg
     1921 gcgttaatca aagaggcgaa ctgtgtgtga gaggtcctat gattatgtcc ggttatgtaa
     1981 acaatccgga agcgaccaac gccttgattg acaaggatgg atggctacat tctggagaca
     2041 tagcttactg ggacgaagac gaacacttct tcatcgttga ccgcctgaag tctctgatta
     2101 agtacaaagg ctatcaggtg gctcccgctg aattggaatc catcttgctc caacacccca
     2161 acatcttcga cgcaggtgtc gcaggtcttc ccgacgatga cgccggtgaa cttcccgccg
     2221 ccgttgttgt tttggagcac ggaaagacga tgacggaaaa agagatcgtg gattacgtcg
     2281 ccagtcaagt aacaaccgcg aaaaagttgc gcggaggagt tgtgtttgtg gacgaagtac
     2341 cgaaaggtct taccggaaaa ctcgacgcaa gaaaaatcag agagatcctc ataaaggcca
     2401 agaagggcgg aaagatcgcc gtgtaattct agagtcgggg cggccggccg cttcgagcag
     2461 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     2521 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     2581 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     2641 aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatccgtcga
     2701 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     2761 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     2821 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2881 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2941 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3001 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3061 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3121 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3181 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3241 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3301 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3361 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3421 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3481 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3541 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3601 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3661 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3721 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3781 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3841 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3901 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3961 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4021 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4081 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4141 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4201 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4261 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4321 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4381 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4441 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4501 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4561 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4621 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4681 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4741 ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4801 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4861 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4921 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4981 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5041 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     5101 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     5161 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttgccattc
     5221 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     5281 ccagcccaag ctaccatgat aagtaagtaa tattaaggta cgggaggtac ttggagcggc
     5341 cgcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata
     5401 gtactaacat acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct
     5461 gtccccagtg caagtgcagg tgccagaaca tttctctatc gata
//
