LOCUS       sequence-401594-        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-cyto-KB1691-Nluc
FEATURES             Location/Qualifiers
     source          1..5995
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(51..71)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        56..385
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      236..371
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     298..317
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             452..1246
                     /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(506..525)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1116..1135
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1420..1541
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1457..1476)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1511..1530
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1590..1606)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1590..1606)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1603..1625)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1614..1630
                     /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(1638..1644,1645..1662,1663..1668))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1683..1704
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1821..1838)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1992..2580)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2072..2091)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2751..3542,3543..3611))
                     /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     3374..3393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3612..3716)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3791..3810)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3982..4361
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4362..4565
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4516..4536
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4610..4629
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4610..4628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4780..5295
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(5333..5350)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5339..5563
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(5609..5995,1..42)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5696..5715)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5906..5927
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
       61 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
      121 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
      181 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
      241 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
      301 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
      361 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
      421 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
      481 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
      541 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
      601 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
      661 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
      721 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
      781 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
      841 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
      901 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
      961 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     1021 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     1081 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     1141 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     1201 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     1261 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     1321 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     1381 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     1441 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     1501 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     1561 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     1621 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     1681 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1741 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1801 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1861 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     1921 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     1981 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2041 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2101 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2161 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     2221 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2281 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2341 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     2401 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     2461 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2521 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2581 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2641 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2701 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2761 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2821 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2881 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2941 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3001 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3061 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3121 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3181 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3241 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3301 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3361 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3421 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3481 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3541 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3601 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     3661 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     3721 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     3781 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     3841 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     3901 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     3961 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     4021 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     4081 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     4141 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     4201 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     4261 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     4321 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     4381 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     4441 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     4501 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     4561 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     4621 actataggga gacccaagct ggctagcacc atggcgagta gcaagagcaa gacgtcgaat
     4681 tccggaatca agcttggtgg atcatcagga ggtagcaggg agctggcctg gggcatcagc
     4741 gagtggctgg aggagtgggg cagcggaggt ggtggatcca tggtcttcac actcgaagat
     4801 ttcgttgggg actggcgaca gacagccggc tacaacctgg accaagtcct tgaacaggga
     4861 ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa ctccgatcca aaggattgtc
     4921 ctgagcggtg aaaatgggct gaagatcgac atccatgtca tcatcccgta tgaaggtctg
     4981 agcggcgacc aaatgggcca gatcgaaaaa atttttaagg tggtgtaccc tgtggatgat
     5041 catcacttta aggtgatcct gcactatggc acactggtaa tcgacggggt tacgccgaac
     5101 atgatcgact atttcggacg gccgtatgaa ggcatcgccg tgttcgacgg caaaaagatc
     5161 actgtaacag ggaccctgtg gaacggcaac aaaattatcg acgagcgcct gatcaacccc
     5221 gacggctccc tgctgttccg agtaaccatc aacggagtga ccggctggcg gctgtgcgaa
     5281 cgcattctgg cgtaactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     5341 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     5401 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     5461 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     5521 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     5581 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     5641 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5701 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5761 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5821 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5881 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5941 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctatt
//
