LOCUS       sequence-332858-        8265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a BRET-based activity sensor for the heterotrimeric G 
            protein Gi1. Composed of the subunits G alpha i1 (GNAI1) tagged with
            NanoLuciferase, G beta 1 (GNB1) and Venus-tagged G gamma 2 (GNG2)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8265)
  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 8265)
  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: Gi1-CASE
FEATURES             Location/Qualifiers
     source          1..8265
                     /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(2667..2684)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    2696..3269
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2719..3271
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2863..2880)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3090..3109
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3643..4152
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     polyA_signal    4993..5114
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5030..5049)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5084..5103
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5121..5576)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5258..5279)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5470..5489
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5603..5707
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5709..6066
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5709..5900
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5732..5752)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5917..6052
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6101..6895
                     /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             6101..6895
                     /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(6155..6174)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6765..6784
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7083..7102)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7127..7174
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7503..8091
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7992..8011
                     /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 gccgacaagc agaagaacgg catcaaggcc
     2341 aacttcaaga tccgccacaa catcgagtcc ggactcagat ctatggccag caacaacacc
     2401 gccagcatag cacaagccag gaagctggta gagcagctta agatggaagc caatatcgac
     2461 aggataaagg tgtccaaggc agctgcagat ttgatggcct actgtgaagc acatgccaag
     2521 gaagaccccc tcctgacccc tgttccggct tcagaaaacc cgtttaggga gaagaagttt
     2581 ttctgtgcca tcctttaacc gcggcgtacg aggcctgcat gctccggacc tgcaggttcg
     2641 aagtcgacag atctcaattg gggcccctat agtgtcacct aaataattcc gcccccccct
     2701 ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
     2761 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac
     2821 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
     2881 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa
     2941 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
     3001 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg
     3061 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
     3121 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat
     3181 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg
     3241 tggttttcct ttgaaaaaca cgatgataat atgggctgca cactgagcgc tgaggacaag
     3301 gcggccgtgg agcgcagcaa gatgatcgac cgcaacctcc gggaggacgg agagaaggca
     3361 gcgcgcgagg tcaagctgct gctgctgggt gctggtgaat ccgggaagag cacaattgtg
     3421 aagcagatga aaattatcca cgaggctggc tactcagagg aagagtgtaa gcagtacaaa
     3481 gcagtggtct acagcaacac catccagtcc atcattgcca tcattagagc tatggggaga
     3541 ttgaaaatcg actttggaga cgctgctcgt gcggatgatg ctcgccaact cttcgtgctt
     3601 gctggggctg cagaggaagg ctttatgacc gcgacgcgtt ctgtcttcac actcgaagat
     3661 ttcgttgggg actggcgaca gacagccggc tacaacctgg accaagtcct tgaacaggga
     3721 ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa ctccgatcca aaggattgtc
     3781 ctgagcggtg aaaatgggct gaagatcgac atccatgtca tcatcccgta tgaaggtctg
     3841 agcggcgacc aaatgggcca gatcgaaaaa atttttaagg tggtgtaccc tgtggatgat
     3901 catcacttta aggtgatcct gcactatggc acactggtaa tcgacggggt tacgccgaac
     3961 atgatcgact atttcggacg gccgtatgaa ggcatcgccg tgttcgacgg caaaaagatc
     4021 actgtaacag ggaccctgtg gaacggcaac aaaattatcg acgagcgcct gatcaacccc
     4081 gacggctccc tgctgttccg agtaaccatc aacggagtga ccggctggcg gctgtgcgaa
     4141 cgcattctgg ccaccggtga actcgcaggc gtcataaaga gactgtggaa ggacagcggt
     4201 gtgcaagcct gcttcaacag atcccgggag taccagctga acgattcggc ggcgtactac
     4261 ctgaatgact tggacagaat agcacaacca aattacatcc caacccagca ggatgttctc
     4321 agaactagag tgaaaacgac gggaattgtg gaaacccact ttactttcaa agatcttcat
     4381 tttaaaatgt ttgacgtggg aggccagaga tcagagcgga agaagtggat tcactgcttt
     4441 gaaggcgtga ctgccatcat cttctgtgtg gccctgagtg actatgacct ggttcttgct
     4501 gaggatgaag aaatgaaccg gatgcacgaa agcatgaagc tgttcgatag catatgtaac
     4561 aacaagtggt ttacggacac atccatcatc cttttcctga acaagaagga cctcttcgaa
     4621 gagaagatca aaaagagtcc cctcacgata tgctatccag aatatgcagg ctcaaacaca
     4681 tatgaagagg cggctgcgta tatccagtgt cagtttgaag acctcaataa aaggaaggac
     4741 acaaaggaaa tttacaccca cttcacttgc gccacggata cgaagaatgt gcagtttgtg
     4801 ttcgatgctg taacggacgt catcataaag aataacctaa aagactgtgg tctcttctaa
     4861 gctggatcca ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt
     4921 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     4981 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5041 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5101 atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5161 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5221 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5281 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5341 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5401 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5461 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     5521 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     5581 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5641 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5701 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     5761 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     5821 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     5881 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     5941 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     6001 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     6061 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     6121 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     6181 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     6241 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     6301 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     6361 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     6421 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     6481 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     6541 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     6601 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     6661 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     6721 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     6781 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     6841 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     6901 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     6961 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     7021 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     7081 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     7141 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     7201 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     7261 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     7321 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     7381 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     7441 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7501 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7561 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7621 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     7681 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7741 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     7801 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7861 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7921 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7981 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     8041 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     8101 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     8161 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta
     8221 atcaattacg gggtcattag ttcatagccc atatatggag ttccg
//
