LOCUS       sequence-332861-        8259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a BRET-based activity sensor for the heterotrimeric G 
            protein Gi2. Composed of the subunits G alpha i2 (GNAI2) 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 8259)
  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 8259)
  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: Gi2-CASE
FEATURES             Location/Qualifiers
     source          1..8259
                     /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             3616..4125
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     polyA_signal    4987..5108
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5024..5043)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5078..5097
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5115..5570)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5252..5273)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5464..5483
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5597..5701
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5703..6060
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5703..5894
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5726..5746)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5911..6046
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6095..6889
                     /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             6095..6889
                     /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(6149..6168)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6759..6778
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7077..7096)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7121..7168
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7497..8085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7986..8005
                     /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 ccgtgagcgc cgaggacaag
     3301 gcggcggccg agcgctctaa gatgatcgac aagaacctgc gggaggacgg agagaaggcg
     3361 gcgcgggagg tgaagttgct gctgttgggt gctggggagt cagggaagag caccatcgtc
     3421 aagcagatga agatcatcca cgaggatggc tactccgagg aggaatgccg gcagtaccgg
     3481 gcggttgtct acagcaacac catccagtcc atcatggcca ttgtcaaagc catgggcaac
     3541 ctgcagatcg actttgccga cccctccaga gcggacgacg ccaggcagct atttgcactg
     3601 tcctgcacgc gttctgtctt cacactcgaa gatttcgttg gggactggcg acagacagcc
     3661 ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt ccagtttgtt tcagaatctc
     3721 ggggtgtccg taactccgat ccaaaggatt gtcctgagcg gtgaaaatgg gctgaagatc
     3781 gacatccatg tcatcatccc gtatgaaggt ctgagcggcg accaaatggg ccagatcgaa
     3841 aaaattttta aggtggtgta ccctgtggat gatcatcact ttaaggtgat cctgcactat
     3901 ggcacactgg taatcgacgg ggttacgccg aacatgatcg actatttcgg acggccgtat
     3961 gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa cagggaccct gtggaacggc
     4021 aacaaaatta tcgacgagcg cctgatcaac cccgacggct ccctgctgtt ccgagtaacc
     4081 atcaacggag tgaccggctg gcggctgtgc gaacgcattc tggccaccgg tgaggagcaa
     4141 ggcgtgctcc ctgatgacct gtccggcgtc atccggaggc tctgggctga ccatggtgtg
     4201 caggcctgct ttggccgctc aagggaatac cagctcaacg actcagctgc ctactacctg
     4261 aacgacctgg agcgtattgc acagagtgac tacatcccca cacagcaaga tgtgctacgg
     4321 acccgcgtaa agaccacggg gatcgtggag acacacttca ccttcaagga cctacacttc
     4381 aagatgtttg atgtgggtgg tcagcggtct gagcggaaga agtggatcca ctgctttgag
     4441 ggcgtcacag ccatcatctt ctgcgtagcc ttgagcgcct atgacttggt gctagctgag
     4501 gacgaggaga tgaaccgcat gcatgagagc atgaagctat tcgatagcat ctgcaacaac
     4561 aagtggttca cagacacgtc catcatcctc ttcctcaaca agaaggacct gtttgaggag
     4621 aagatcacac acagtcccct gaccatctgc ttccctgagt acacaggggc caacaaatat
     4681 gatgaggcag ccagctacat ccagagtaag tttgaggacc tgaataagcg caaagacacc
     4741 aaggagatct acacgcactt cacgtgcgcc accgacacca agaacgtgca gttcgtgttt
     4801 gacgccgtca ccgatgtcat catcaagaac aacctgaagg actgcggcct cttctgagct
     4861 tctagagggc cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt
     4921 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     4981 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5041 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5101 gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     5161 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     5221 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     5281 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     5341 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     5401 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     5461 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     5521 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     5581 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5641 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5701 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     5761 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     5821 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     5881 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     5941 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     6001 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     6061 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     6121 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     6181 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     6241 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     6301 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     6361 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     6421 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     6481 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     6541 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     6601 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     6661 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     6721 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     6781 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     6841 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     6901 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     6961 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     7021 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     7081 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     7141 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     7201 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     7261 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     7321 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     7381 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     7441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     7501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7621 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     7681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     8041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     8101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     8161 tcccctgatt ctgtggataa ccgtattacc gccatgcatt agttattaat agtaatcaat
     8221 tacggggtca ttagttcata gcccatatat ggagttccg
//
