LOCUS       sequence-332862-        8262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a BRET-based activity sensor for the heterotrimeric G 
            protein Gi3. Composed of the subunits G alpha i3 (GNAI3) 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 8262)
  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 8262)
  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: Gi3-CASE
FEATURES             Location/Qualifiers
     source          1..8262
                     /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             3622..4131
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     polyA_signal    4990..5111
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5027..5046)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5081..5100
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5118..5573)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5255..5276)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5467..5486
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5600..5704
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5706..6063
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5706..5897
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5729..5749)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5914..6049
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6098..6892
                     /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             6098..6892
                     /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(6152..6171)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6762..6781
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7080..7099)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7124..7171
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7500..8088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7989..8008
                     /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 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 cgttgagcgc cgaagacaag
     3301 gcggcagtgg agcgaagcaa gatgatcgac cgcaacttac gggaggacgg ggaaaaagcg
     3361 gccaaagaag tgaagctgct gctactcggt gctggagaat ctggtaaaag caccattgtg
     3421 aaacagatga aaatcattca tgaggatggc tattcagagg atgaatgtaa acaatataaa
     3481 gtagttgtct acagcaatac tatacagtcc atcattgcaa tcataagagc catgggacgg
     3541 ctaaagattg actttgggga agctgccagg gcagatgatg cccggcaatt atttgtttta
     3601 gctggcagtg ctacgcgttc tgtcttcaca ctcgaagatt tcgttgggga ctggcgacag
     3661 acagccggct acaacctgga ccaagtcctt gaacagggag gtgtgtccag tttgtttcag
     3721 aatctcgggg tgtccgtaac tccgatccaa aggattgtcc tgagcggtga aaatgggctg
     3781 aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca aatgggccag
     3841 atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa ggtgatcctg
     3901 cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta tttcggacgg
     3961 ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg
     4021 aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct gctgttccga
     4081 gtaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc caccggtgaa
     4141 gaaggagtca tgactccaga actagcagga gtgattaaac ggttatggcg agatggtggg
     4201 gtacaagctt gcttcagcag atccagggaa tatcagctca atgattctgc ttcatattat
     4261 ctaaatgatc tggatagaat atcccagtct aactacattc caactcagca agatgttctt
     4321 cggacgagag tgaagaccac aggcattgta gaaacacatt tcaccttcaa agacctatac
     4381 ttcaagatgt ttgatgtagg tggccaaaga tcagaacgaa aaaagtggat tcactgtttt
     4441 gagggagtga cagcaattat cttctgtgtg gccctcagtg attatgacct tgttctggct
     4501 gaggacgagg agatgaaccg aatgcatgaa agcatgaaac tgtttgacag catttgtaat
     4561 aacaaatggt ttacagaaac ttcaatcatt ctcttcctta acaagaaaga cctttttgag
     4621 gaaaaaataa agaggagtcc gttaactatc tgttatccag aatacacagg ttccaataca
     4681 tatgaagagg cagctgccta tattcaatgc cagtttgaag atctgaacag aagaaaagat
     4741 accaaggaga tctatactca cttcacctgt gccacagaca cgaagaatgt gcagtttgtt
     4801 tttgatgctg ttacagatgt catcattaaa aacaacttaa aggaatgtgg actttattaa
     4861 gcttctagag ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta
     4921 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     4981 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5041 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5101 aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     5161 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5221 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5281 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     5341 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     5401 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5461 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     5521 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     5581 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5641 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5701 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     5761 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     5821 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     5881 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     5941 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     6001 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     6061 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     6121 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     6181 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     6241 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     6301 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     6361 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     6421 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     6481 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     6541 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     6601 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     6661 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     6721 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     6781 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     6841 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     6901 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     6961 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     7021 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     7081 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     7141 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     7201 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     7261 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     7321 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     7381 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     7441 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     7501 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     7561 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     7621 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     7681 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     7741 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     7801 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     7861 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     7921 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7981 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     8041 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     8101 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     8161 ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc
     8221 aattacgggg tcattagttc atagcccata tatggagttc cg
//
