LOCUS       sequence-488679-        4918 bp    DNA     circular SYN 08-OCT-2025
DEFINITION  Express 3xFlag C-terminal tagged mouse GPR45-Y287C mutant 
            transiently via transfection in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4918)
  AUTHORS   Xun Y, Jiang Y, Xu B, Tang M, Ludwig S, Nakamura K, Mukhopadhyay S, 
            Liu C, Beutler B, Zhang Z
  TITLE     GPR45 modulates Galpha(s) at primary cilia of the paraventricular 
            hypothalamus to control food intake.
  JOURNAL   Science. 2025 Jun 5;388(6751):eadp3989. doi: 
            10.1126/science.adp3989. Epub 2025 Jun 5.
  PUBMED    40472089
REFERENCE   2  (bases 1 to 4918)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2025-06-5"; volume: "388(6751):eadp3989. doi"; pages: " 
            10.1126/science.adp3989. Epub 2025 Jun 5"
COMMENT     Sequence Label: pCMV-Gpr45-Y287C-3xFlag
FEATURES             Location/Qualifiers
     source          1..4918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        27..330
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        331..534
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     485..505
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     531..555
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          672..768
                     /label=SV40 intron
                     /note="modified SV40 intron with splice donor and acceptor 
                     sites"
     regulatory      846..855
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1977..2042
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2057..2191
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2081..2100)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2135..2154
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2302..2318)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2302..2318)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2315..2337)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2326..2342
                     /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(2350..2356,2357..2374,2375..2380))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2395..2416
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2533..2550)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2704..3292)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2784..2803)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3463..4254,4255..4323))
                     /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     4086..4105
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4324..4428)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4496..4514
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4552..4574)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4674..4693
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4887..4909
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4901..4918
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4902..4918
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gagttcgagc ttgcatgcct gcaggtcgtt acataactta cggtaaatgg cccgcctggc
       61 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      121 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      181 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      241 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      301 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      361 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      421 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      481 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
      541 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
      601 cgggaccgat ccagcctccg gactctagag gatccggtac tagaggaact gaaaaaccag
      661 aaagttaact ggtaagttta gtctttttgt cttttatttc aggtcccgga tccggtggtg
      721 gtgcaaatca aagaactgct cctcagtgga tgttgccttt acttctaggc ctgtacggaa
      781 gtgttacttc tgctctaaaa gctgcggaat tgtacccgcg ggcccaggcc cgaattagag
      841 aattcgccac catggcctgt aacagcacac ccatggggac ttacgaacat ctgctgctga
      901 atgtgagcaa cactttggac cctggggaca ccccactgtc tgcaccgctc aggatctcgc
      961 tggcaataat gatgctgctg atgatcgtgg taggattcct tggcaacacg gtggtctgca
     1021 tcatcgtgta ccagaggcca gccatgcgtt cagctatcaa cctgctgctg gccaccttgg
     1081 ccttctccga catcatgctg tctttatgct gcatgccatt cacggccatc accctcatca
     1141 ctgttcgctg gcatttcggg gaccactttt gtcggctttc agctactctc tattggtttt
     1201 ttgtcctaga gggcgtggcc atcctgctca tcattagcgt ggaccggttt ctcatcatcg
     1261 tgcagcgtca ggacaagctg aacccacgca gggctaagat gatcatcgcg gcctcctggg
     1321 tgctgtcttt ctgcatctct gcgccctcct tcactggctg gacgttcatg gaggtgcctg
     1381 ctcgagcccc acagtgcgtg ctaggctaca ctgagttccc agctgaacgc gcctatgtag
     1441 tgacactggt ggtggcagtg ttctttgctc ccttcggcgt catgttgtgc tcctatctgt
     1501 gcatcctcaa tacggtgcgg aagaacgctg tccgtgtgca caaccagtcg gacagcctgg
     1561 acctcagaca gctgaccgga gctggcctga gacgtctcag acggcagcag cagcaggcca
     1621 gcctggacct gagtttcaaa accaaggcct tcaccaccat cctcatcctc ttcgtgggct
     1681 tttcactctg ctggctgcca cactcagtct gcagcctgct gtctgcgttc agccggcggt
     1741 tctattacag cgcctccttc tacaccacca gcacatgcgt cctgtggctc agttacctca
     1801 agtctgtttt caaccccatc gtctactgct ggaggatcaa gaaattccgc gaggcctgca
     1861 tagagttgct tccccacact ttccaaatcc tccctaaagt gcctgagcgg atccagagga
     1921 aaatccagcc aagcaccatc tatgtgtgca acgaaaacca atccgctgtc ctcgaggact
     1981 acaaagacca tgacggtgat tataaagatc atgacatcga ctacaaggat gacgatgaca
     2041 agtaggcggc cgcggggatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     2101 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     2161 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     2221 tcaggttcag ggggaggtgt gggaggtttt ttcggatcct ctagagtcga tctgcaggca
     2281 tgctagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     2341 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     2401 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2461 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2521 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2581 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2641 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2701 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2761 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2821 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2881 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2941 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3001 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3061 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3121 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3181 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3241 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3301 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3361 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3421 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3481 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3541 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3601 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3661 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3721 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3781 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3841 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3901 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3961 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4021 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4081 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4141 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4201 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4261 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4321 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4381 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4441 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     4501 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     4561 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     4621 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     4681 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     4741 gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg
     4801 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     4861 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagt
//
