LOCUS       sequence-410605-        5995 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GPCR/G protein BRET biosensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5995)
  AUTHORS   Janicot R, Maziarz M, Park JC, Zhao J, Luebbers A, Green E, 
            Philibert CE, Zhang H, Layne MD, Wu JC, Garcia-Marcos M
  TITLE     Direct interrogation of context-dependent GPCR activity with a 
            universal biosensor platform.
  JOURNAL   Cell. 2024 Feb 16:S0092-8674(24)00065-5. doi: 
            10.1016/j.cell.2024.01.028.
  PUBMED    38412860
REFERENCE   2  (bases 1 to 5995)
  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: "Cell. 2024 
            Feb 16:S0092-8674(24)00065-5. doi: 10.1016/j.cell.2024.01.028."
COMMENT     Sequence Label: pcDNA3.1-mas-KB1691-Nluc
FEATURES             Location/Qualifiers
     source          1..5995
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1335..2129)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1446..1465)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2056..2075
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2196..2525)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2210..2345
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2264..2283)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2510..2530
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2539..2967)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2649..2670)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2861..2880
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3013..3237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3226..3243
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3281..3796)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             complement(3905..3925)
                     /codon_start=1
                     /product="N-myristoylation signal from Src kinase (Pellman 
                     et al., 1985; Kaplan et al., 1988)"
                     /label=myr
                     /translation="MGSSKSK"
     primer_bind     complement(3947..3966)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3948..3966)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4011..4214)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4040..4060)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4215..4594
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4766..4785
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4860..4964
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4965..5033,5034..5825)
                     /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     complement(5183..5202)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     1321 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     1381 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     1441 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     1501 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     1561 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     1621 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     1681 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     1741 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     1801 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     1861 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     1921 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     1981 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     2041 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     2101 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     2161 gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa
     2221 aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa
     2281 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     2341 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     2401 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     2461 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     2521 cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2581 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2641 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2701 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2761 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2821 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2881 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2941 cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt
     3001 ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
     3061 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
     3121 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
     3181 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg
     3241 aggctgatca gcgggtttaa acgggccctc tagactcgag ttacgccaga atgcgttcgc
     3301 acagccgcca gccggtcact ccgttgatgg ttactcggaa cagcagggag ccgtcggggt
     3361 tgatcaggcg ctcgtcgata attttgttgc cgttccacag ggtccctgtt acagtgatct
     3421 ttttgccgtc gaacacggcg atgccttcat acggccgtcc gaaatagtcg atcatgttcg
     3481 gcgtaacccc gtcgattacc agtgtgccat agtgcaggat caccttaaag tgatgatcat
     3541 ccacagggta caccacctta aaaatttttt cgatctggcc catttggtcg ccgctcagac
     3601 cttcatacgg gatgatgaca tggatgtcga tcttcagccc attttcaccg ctcaggacaa
     3661 tcctttggat cggagttacg gacaccccga gattctgaaa caaactggac acacctccct
     3721 gttcaaggac ttggtccagg ttgtagccgg ctgtctgtcg ccagtcccca acgaaatctt
     3781 cgagtgtgaa gaccatggat ccaccacctc cgctgcccca ctcctccagc cactcgctga
     3841 tgccccaggc cagctccctg ctacctcctg atgatccacc aagcttgatt ccggaattcg
     3901 acgtcttgct cttgctactc cccatggtgc tagccagctt gggtctccct atagtgagtc
     3961 gtattaattt cgataagcca gtaagcagtg ggttctctag ttagccagag agctctgctt
     4021 atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta
     4081 cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg
     4141 gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc
     4201 caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
     4261 aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
     4321 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     4381 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
     4441 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     4501 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
     4561 gattactatt aataactagt caataatcaa tgtcaacgcg tatatctggc ccgtacatcg
     4621 cgaagcagcg caaaacgcct aaccctaagc agattcttca tgcaattgtc ggtcaagcct
     4681 tgccttgttg tagcttaaat tttgctcgcg cactactcag cgacctccaa cacacaagca
     4741 gggagcagat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     4801 aggggatcgg gagatctccc gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc
     4861 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4921 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4981 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5041 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5101 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5161 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5221 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5281 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5341 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5401 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5461 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5521 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5581 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5641 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5701 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5761 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5821 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5881 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5941 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
