LOCUS       sequence-332868-        8374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a BRET-based activity sensor for the heterotrimeric G 
            protein Gs. Composed of the subunits G alpha s(short isoform) (GNAS)
            tagged with NanoLuciferase, G beta 1 (GNB1) and Venus-tagged G gamma
            1 (GNG1). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8374)
  AUTHORS   Schihada H, Shekhani R, Schulte G
  TITLE     Quantitative assessment of constitutive G protein-coupled receptor 
            activity with BRET-based G protein biosensors.
  JOURNAL   Sci Signal. 2021 Sep 7;14(699):eabf1653. doi: 
            10.1126/scisignal.abf1653. Epub 2021 Sep 7.
  PUBMED    34516756
REFERENCE   2  (bases 1 to 8374)
  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: "Sci 
            Signal."; date: "2021-09-7"; volume: "14(699):eabf1653. doi"; pages:
            " 10.1126/scisignal.abf1653. Epub 2021 Sep 7"
COMMENT     Sequence Label: Gs(short)-CASE
FEATURES             Location/Qualifiers
     source          1..8374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      547..556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1573..1626
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1633..2367
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD
                     HMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     primer_bind     1770..1791
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1894..1915)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2155..2174)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2629..2646)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    2658..3231
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2825..2842)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3052..3071
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3671..4180
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     polyA_signal    5102..5223
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5139..5158)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5193..5212
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5230..5685)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5367..5388)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5579..5598
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5712..5816
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5818..6175
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5818..6009
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5841..5861)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6026..6161
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6210..7004
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             6210..7004
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6264..6283)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6874..6893
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7192..7211)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7236..7283
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7612..8200
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8101..8120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggtcgcca ccatggggga gcttgaccag ttacggcagg aggccgagca acttaagaac
      601 cagattcgag acgccaggaa agcatgtgca gatgcaactc tctctcagat cacaaacaac
      661 atcgacccag tgggaagaat ccaaatgcgc acgaggagga cactgcgggg gcacctggcc
      721 aagatctacg ccatgcactg gggcacagac tccaggcttc tcgtcagtgc ctcgcaggat
      781 ggtaaactta tcatctggga cagctacacc accaacaagg tccacgccat ccctctgcgc
      841 tcctcctggg tcatgacctg tgcatatgcc ccttctggga actatgtggc ctgcggtggc
      901 ctggataaca tttgctccat ttacaatctg aaaactcgtg aggggaacgt gcgcgtgagt
      961 cgtgagctgg caggacacac aggttacctg tcctgctgcc gattcctgga tgacaatcag
     1021 atcgtcacca gctctggaga caccacgtgt gccctgtggg acatcgagac cggccagcag
     1081 acgaccacgt ttaccggaca cactggagat gtcatgagcc tttctcttgc tcctgacacc
     1141 agactgttcg tctctggtgc ttgtgatgct tcagccaaac tctgggatgt gcgagaaggc
     1201 atgtgccggc agaccttcac tggccacgag tctgacatca atgccatttg cttctttcca
     1261 aatggcaatg catttgccac tggctcagac gacgccacct gcaggctgtt tgaccttcgt
     1321 gctgaccagg agctcatgac ttactcccat gacaacatca tctgcgggat cacctctgtc
     1381 tccttctcca agagcgggcg cctcctcctt gctgggtacg acgacttcaa ctgcaacgtc
     1441 tgggatgcac tcaaagccga ccgggcaggt gtcttggctg ggcatgacaa ccgcgtcagc
     1501 tgcctgggcg tgactgacga tggcatggct gtggcgacag ggtcctggga tagcttcctc
     1561 aagatctgga acgagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc
     1621 ggccctaccg gtatggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc
     1681 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg
     1741 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1801 gggatcactc tcggcatgga cgagctgtac aagggtggca gcggtggcat ggtgagcaag
     1861 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1921 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1981 ctgaagctga tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2041 ctgggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     2101 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2161 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2221 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2281 aactacaaca gccacaacgt ctatatcacc gccgacaaac agaagaacgg catcaaggcc
     2341 aacttcaaga tccgccacaa catcgagtcc ggactcagat ctccagtgat caatattgag
     2401 gacctgacag aaaaggacaa attgaagatg gaagttgacc agctcaagaa agaagtgacg
     2461 ctggaaagaa tgctggtgtc caaatgttgt gaagagttca gggattatgt tgaagaaaga
     2521 tctggggaag atccattagt aaagggtatc ccagaggaca aaaatccctt caaggagctc
     2581 aaaggagggt gtgtgatttc ataagtcgac agatctcaat tggggcccct atagtgtcac
     2641 ctaaataatt ccgccccccc ctctccctcc ccccccccta acgttactgg ccgaagccgc
     2701 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     2761 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     2821 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
     2881 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     2941 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     3001 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     3061 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     3121 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
     3181 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata atggatccat
     3241 gggctgcctc ggcaacagta agaccgagga ccagcgcaac gaggagaagg cgcagcgcga
     3301 ggccaacaaa aagatcgaga agcagctgca gaaggacaag caggtctacc gggccacgca
     3361 ccgcctgctg ctgctgggtg ctggagagtc tggcaaaagc accattgtga agcagatgag
     3421 gatcctgcat gttaatgggt ttaatggaga gggcggcgaa gaggacccgc aggctgcaag
     3481 gagcaacagc gatggtgaga aggcaaccaa agtgcaggac atcaaaaaca acctgaaaga
     3541 ggcgattgaa accattgtgg ccgccatgag caacctggtg ccccccgtgg agctggccaa
     3601 ccccgagaac cagttcagag tggactacat cctgagtgtg atgaacagcg gtggaggcgg
     3661 gacgcgttct gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta
     3721 caacctggac caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt
     3781 gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat
     3841 ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat
     3901 ttttaaggtg gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac
     3961 actggtaatc gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg
     4021 catcgccgtg ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa
     4081 aattatcgac gagcgcctga tcaaccccga cggctccctg ctgttccgag taaccatcaa
     4141 cggagtgacc ggctggcggc tgtgcgaacg cattctggcc accggtggtg gcggatccgt
     4201 gcctgacttt gacttccctc ccgaattcta tgagcatgcc aaggctctgt gggaggatga
     4261 aggagtgcgt gcctgctacg aacgctccaa cgagtaccag ctgattgact gtgcccagta
     4321 cttcctggac aagatcgacg tgatcaagca ggctgactat gtgccgagcg atcaggacct
     4381 gcttcgctgc cgtgtcctga cttctggaat ctttgagacc aagttccagg tggacaaagt
     4441 caacttccac atgtttgacg tgggtggcca gcgcgatgaa cgccgcaagt ggatccagtg
     4501 cttcaacgat gtgactgcca tcatcttcgt ggtggccagc agcagctaca acatggtcat
     4561 ccgggaggac aaccagacca accgcctgca ggaggctctg aacctcttca agagcatctg
     4621 gaacaacaga tggctgcgca ccatctctgt gatcctgttc ctcaacaagc aagatctgct
     4681 cgctgagaaa gtccttgctg ggaaatcgaa gattgaggac tactttccag aatttgctcg
     4741 ctacactact cctgaggatg ctactcccga gcccggagag gacccacgcg tgacccgggc
     4801 caagtacttc attcgagatg agtttctgag gatcagcact gccagtggag atgggcgtca
     4861 ctactgctac cctcatttca cctgcgctgt ggacactgag aacatccgcc gtgtgttcaa
     4921 cgactgccgt gacatcattc agcgcatgca ccttcgtcag tacgagctgc tctaatctag
     4981 agggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     5041 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     5101 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5161 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5221 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     5281 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     5341 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     5401 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     5461 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     5521 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     5581 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     5641 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     5701 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5761 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     5821 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     5881 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     5941 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     6001 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     6061 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     6121 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     6181 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     6241 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     6301 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     6361 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     6421 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     6481 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     6541 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     6601 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     6661 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     6721 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     6781 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     6841 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     6901 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     6961 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     7021 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     7081 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     7141 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     7201 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     7261 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     7321 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     7381 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     7441 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     7501 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     7561 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     7621 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     7681 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     7741 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     7801 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     7861 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7921 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7981 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     8041 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     8101 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     8161 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     8221 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     8281 tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg
     8341 ggtcattagt tcatagccca tatatggagt tccg
//
