LOCUS       sequence-488726-        4834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express HA N-terminal tagged mouse Galphai3 transiently via 
            transfection in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4834)
  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 4834)
  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: "Science."; 
            date: "2025-06-5"; volume: "388(6751):eadp3989. doi"; pages: " 
            10.1126/science.adp3989. Epub 2025 Jun 5"
COMMENT     Sequence Label: pCMV-HA-Gnai3
FEATURES             Location/Qualifiers
     source          1..4834
                     /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"
     CDS             832..858
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1973..2107
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1997..2016)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2051..2070
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2218..2234)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2218..2234)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2231..2253)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2242..2258
                     /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(2266..2272,2273..2290,2291..2296))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2311..2332
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2449..2466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2620..3208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2700..2719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3379..4170,4171..4239))
                     /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     4002..4021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4240..4344)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4412..4430
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4468..4490)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4590..4609
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4803..4825
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4817..4834
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4818..4834
                     /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 ggcccaccat gtacccatac
      841 gatgttccag attacgctct tatggccatg gaggcccgaa ttcggatggg ctgcacgttg
      901 agcgccgagg acaaggcggc ggtggagcgg agcaagatga tcgaccgcaa cttgcgggag
      961 gacggggaga aagcggccaa agaagtgaag ctgctgctgc tcggcgctgg agaatctggt
     1021 aaaagtacca tcgtgaaaca gatgaaaatc attcatgagg acggctattc agaggacgaa
     1081 tgtaaacagt ataaagtagt tgtctacagc aatactattc agtccatcat tgcaatcata
     1141 cgagccatgg gacggttgaa gattgatttt ggggaatctg ccagagcaga tgatgcccga
     1201 cagttatttg ttttagctgg gagtgctgaa gaaggagtca tgacttcaga actagcaggc
     1261 gtgattaaac gtttatggcg agatggcggg gtacaggcat gctttagcag gtccagggaa
     1321 tatcagctca atgattctgc ttcatactac ctaaatgatt tggatagaat atcccagacc
     1381 aactacattc caactcagca agatgttctt cggacaagag tgaagactac aggcattgtg
     1441 gagacccact tcaccttcaa ggaactctac ttcaaaatgt ttgatgtagg tggccaaaga
     1501 tccgaacgaa aaaagtggat tcactgtttt gagggagtga cagcaattat cttttgtgtg
     1561 gctctcagtg attacgacct tgttctggct gaggatgagg aaatgaaccg aatgcatgaa
     1621 agcatgaaat tgtttgacag catttgtaac aacaaatggt ttacagacac ttcaatcatt
     1681 ctctttctta ataagaaaga cctttttgag gaaaaaataa agaggagtcc attaacaatc
     1741 tgttatccag aatacacagg ttccaataca tacgaagagg cagctgctta cattcagtgc
     1801 cagtttgaag atctgaaccg aagaaaagat accaaggagg tctacactca cttcacctgt
     1861 gccacagaca ccaaaaatgt gcagtttgtt tttgatgctg ttacggatgt catcattaaa
     1921 aacaacttaa aggaatgtgg gctttattga ctcgaggtac cgcggccgcg gggatccaga
     1981 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     2041 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     2101 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     2161 ggttttttcg gatcctctag agtcgatctg caggcatgct agcttggcgt aatcatggtc
     2221 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2281 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     2341 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2401 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2461 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2521 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2581 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2641 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2701 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2761 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2821 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2881 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2941 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3001 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3061 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3121 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3181 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3241 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3301 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3361 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3421 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3481 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3541 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3601 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3661 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3721 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3781 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3841 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3901 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3961 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4021 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4081 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4141 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4201 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4261 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4321 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     4381 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     4441 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     4501 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     4561 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     4621 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
     4681 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     4741 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     4801 ttcccagtca cgacgttgta aaacgacggc cagt
//
