LOCUS       sequence-488675-        4921 bp    DNA     circular SYN 08-OCT-2025
DEFINITION  Express mouse GPR45-S214P mutant transiently via transfection in 
            mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4921)
  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 4921)
  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-S214P
FEATURES             Location/Qualifiers
     source          1..4921
                     /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             1980..2045
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2060..2194
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2084..2103)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2138..2157
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2305..2321)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2305..2321)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2318..2340)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2329..2345
                     /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(2353..2359,2360..2377,2378..2383))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2398..2419
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2536..2553)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2707..3295)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2787..2806)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3466..4257,4258..4326))
                     /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     4089..4108
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4327..4431)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4499..4517
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4555..4577)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4677..4696
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4890..4912
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4904..4921
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4905..4921
                     /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 ccctatctgt
     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 acagcctgct 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 tagctcgagg
     1981 actacaaaga ccatgacggt gattataaag atcatgacat cgactacaag gatgacgatg
     2041 acaagtaggc ggccgcgggg atccagacat gataagatac attgatgagt ttggacaaac
     2101 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     2161 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     2221 gtttcaggtt cagggggagg tgtgggaggt tttttcggat cctctagagt cgatctgcag
     2281 gcatgctagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     2341 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     2401 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2461 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2521 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2581 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2641 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2701 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2761 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2821 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2881 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2941 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3001 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3061 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3121 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3181 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3241 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3301 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3361 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3421 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3481 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3541 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3601 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3661 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3721 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3781 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3841 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3901 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3961 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4021 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4081 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4141 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4201 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4261 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4321 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4381 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4441 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     4501 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     4561 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     4621 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     4681 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     4741 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
     4801 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     4861 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     4921 t
//
