LOCUS       sequence-471736-        5215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Use as a MRE/CRE/SRE reporter for luciferase assays to measure GPCR 
            activation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5215)
  AUTHORS   Jiang Y, Xun Y, Zhang Z
  TITLE     Central regulation of feeding and body weight by ciliary GPR75.
  JOURNAL   J Clin Invest. 2024 Aug 13;134(19):e182121. doi: 10.1172/JCI182121.
  PUBMED    39137039
REFERENCE   2  (bases 1 to 5215)
  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: "J Clin 
            Invest."; date: "2024-08-13"; volume: "134(19):e182121. doi"; pages:
            " 10.1172/JCI182121"
COMMENT     Sequence Label: pGL3-MRE/CRE/SRE
FEATURES             Location/Qualifiers
     source          1..5215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(223..234)
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="NHKV"
     regulatory      479..488
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             485..2137
                     /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(551..569)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     polyA_signal    2178..2299
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2189..2208)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2243..2262
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2547..2564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2718..3306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2798..2817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3477..4268,4269..4337))
                     /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     4100..4119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4338..4442)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4469..4924
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4556..4575)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4766..4787
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5055..5103
                     /note="synthetic polyadenylation signal"
     misc_feature    5117..5208
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5157..5176
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgagc tcttacacag gatgtccata ttaggacatc tgcgtcagca ggtttccacg
       61 gccacgcgta tgctaaagga cgtcacattg cacaatctta aatgctaaag gacgtcacat
      121 tgcacaatct taaatgctaa aggacgtcac attgcacaat cttaactcga gacttcaagc
      181 cctattcatc ccatggccgt catactgtga cgtctttcag agcactttgt gattgctcag
      241 tcctaagtat aagccctata aaatgatggg ctttgaaatg ctggtcaggg tagagtgaga
      301 agcaccagca ggcagtaaca gccaaccctt agccattgct aagggcagag aactggtgga
      361 gcctttctct tactcccagg acttcagcac ctaagacagc tccaaaacaa accagaacag
      421 tcagctccgg gggagcacga ctgggcgaga agcttggcat tccggtactg ttggtaaagc
      481 caccatggaa gacgccaaaa acataaagaa aggcccggcg ccattctatc cgctggaaga
      541 tggaaccgct ggagagcaac tgcataaggc tatgaagaga tacgccctgg ttcctggaac
      601 aattgctttt acagatgcac atatcgaggt ggacatcact tacgctgagt acttcgaaat
      661 gtccgttcgg ttggcagaag ctatgaaacg atatgggctg aatacaaatc acagaatcgt
      721 cgtatgcagt gaaaactctc ttcaattctt tatgccggtg ttgggcgcgt tatttatcgg
      781 agttgcagtt gcgcccgcga acgacattta taatgaacgt gaattgctca acagtatggg
      841 catttcgcag cctaccgtgg tgttcgtttc caaaaagggg ttgcaaaaaa ttttgaacgt
      901 gcaaaaaaag ctcccaatca tccaaaaaat tattatcatg gattctaaaa cggattacca
      961 gggatttcag tcgatgtaca cgttcgtcac atctcatcta cctcccggtt ttaatgaata
     1021 cgattttgtg ccagagtcct tcgataggga caagacaatt gcactgatca tgaactcctc
     1081 tggatctact ggtctgccta aaggtgtcgc tctgcctcat agaactgcct gcgtgagatt
     1141 ctcgcatgcc agagatccta tttttggcaa tcaaatcatt ccggatactg cgattttaag
     1201 tgttgttcca ttccatcacg gttttggaat gtttactaca ctcggatatt tgatatgtgg
     1261 atttcgagtc gtcttaatgt atagatttga agaagagctg tttctgagga gccttcagga
     1321 ttacaagatt caaagtgcgc tgctggtgcc aaccctattc tccttcttcg ccaaaagcac
     1381 tctgattgac aaatacgatt tatctaattt acacgaaatt gcttctggtg gcgctcccct
     1441 ctctaaggaa gtcggggaag cggttgccaa gaggttccat ctgccaggta tcaggcaagg
     1501 atatgggctc actgagacta catcagctat tctgattaca cccgaggggg atgataaacc
     1561 gggcgcggtc ggtaaagttg ttccattttt tgaagcgaag gttgtggatc tggataccgg
     1621 gaaaacgctg ggcgttaatc aaagaggcga actgtgtgtg agaggtccta tgattatgtc
     1681 cggttatgta aacaatccgg aagcgaccaa cgccttgatt gacaaggatg gatggctaca
     1741 ttctggagac atagcttact gggacgaaga cgaacacttc ttcatcgttg accgcctgaa
     1801 gtctctgatt aagtacaaag gctatcaggt ggctcccgct gaattggaat ccatcttgct
     1861 ccaacacccc aacatcttcg acgcaggtgt cgcaggtctt cccgacgatg acgccggtga
     1921 acttcccgcc gccgttgttg ttttggagca cggaaagacg atgacggaaa aagagatcgt
     1981 ggattacgtc gccagtcaag taacaaccgc gaaaaagttg cgcggaggag ttgtgtttgt
     2041 ggacgaagta ccgaaaggtc ttaccggaaa actcgacgca agaaaaatca gagagatcct
     2101 cataaaggcc aagaagggcg gaaagatcgc cgtgtaattc tagagtcggg gcggccggcc
     2161 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     2221 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     2281 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     2341 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa
     2401 ggatccgtcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
     2461 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     2521 aggtgccggc agcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2581 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2641 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2701 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2761 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2821 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2881 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2941 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3001 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3061 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3121 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3181 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3241 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3301 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3361 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3421 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3481 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3541 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3601 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3661 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3721 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3781 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3841 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3901 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3961 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4021 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4081 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4141 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4201 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4261 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4321 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4381 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4441 cgcacatttc cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg
     4501 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4561 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4621 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4681 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4741 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4801 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4861 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     4921 atttgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     4981 tcgctattac gccagcccaa gctaccatga taagtaagta atattaaggt acgggaggta
     5041 cttggagcgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt
     5101 gtgaatcgat agtactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag
     5161 caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttctctat cgata
//
