LOCUS       sequence-300658-        7223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a G alpha subunit (GNAI3) containing RLuc8 as an optimal 
            component of a BRET2 biosensor for studying heterotrimeric G protein
            dissociation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7223)
  AUTHORS   Olsen RHJ, DiBerto JF, English JG, Glaudin AM, Krumm BE, Slocum ST, 
            Che T, Gavin AC, McCorvy JD, Roth BL, Strachan RT
  TITLE     TRUPATH, an open-source biosensor platform for interrogating the 
            GPCR transducerome.
  JOURNAL   Nat Chem Biol. 2020 May 4. pii: 10.1038/s41589-020-0535-8. doi: 
            10.1038/s41589-020-0535-8.
  PUBMED    32367019
REFERENCE   2  (bases 1 to 7223)
  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: "Nat Chem 
            Biol. 2020 May 4. pii: 10.1038/s41589-020-0535-8. doi: 
            10.1038/s41589-020-0535-8."
COMMENT     Sequence Label: pcDNA5/FRT/TO-GAlphai3-RLuc8
FEATURES             Location/Qualifiers
     source          1..7223
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1413..2345
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGAALAFHYAYEHQDRIKAIVHMESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETVLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFLQEDAPDEMGKYIKSFVERVLKNEQ"
     primer_bind     complement(3175..3192)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3181..3405
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3538..3557)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3689..3736
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3744..4766
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3744..4766
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4896..5017
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4933..4952)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4987..5006
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5066..5082)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5066..5082)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5079..5101)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5090..5106
                     /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(5114..5120,5121..5138,5139..5144))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5159..5180
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5297..5314)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5468..6056)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5548..5567)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6227..7018,7019..7087))
                     /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     6850..6869
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7088..7192)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga
     1021 attctgcaga tatcaccacc gccagctcct ccctggccaa ggacacttca tcgaccggtg
     1081 agaacctgta cttccagcta atgggctgca cgttgagcgc cgaagacaag gcggcagtgg
     1141 agcgaagcaa gatgatcgac cgcaacttac gggaggacgg ggaaaaagcg gccaaagaag
     1201 tgaagctgct gctactcggt gctggagaat ctggtaaaag caccattgtg aaacagatga
     1261 aaatcattca tgaggatggc tattcagagg atgaatgtaa acaatataaa gtagttgtct
     1321 acagcaatac tatacagtcc atcattgcaa tcataagagc catgggacgg ctaaagattg
     1381 actttgggga agcttctggt ggtggtggat ccatggcttc caaggtgtac gaccccgagc
     1441 aacgcaaacg catgatcact gggcctcagt ggtgggctcg ctgcaagcaa atgaacgtgc
     1501 tggactcctt catcaactac tatgattccg agaagcacgc cgagaacgcc gtgatttttc
     1561 tgcatggtaa cgctacctcc agctacctgt ggaggcacgt cgtgcctcac atcgagcccg
     1621 tggctagatg catcatccct gatctgatcg gaatgggtaa gtccggcaag agcgggaatg
     1681 gctcatatcg cctcctggat cactacaagt acctcaccgc ttggttcgag ctgctgaacc
     1741 ttccaaagaa aatcatcttt gtgggccacg actggggggc tgctctggcc tttcactacg
     1801 cctacgagca ccaagacagg atcaaggcca tcgtccatat ggagagtgtc gtggacgtga
     1861 tcgagtcctg ggacgagtgg cctgacatcg aggaggatat cgccctgatc aagagcgaag
     1921 agggcgagaa aatggtgctt gagaataact tcttcgtcga gaccgtgctc ccaagcaaga
     1981 tcatgcggaa actggagcct gaggagttcg ctgcctacct ggagccattc aaggagaagg
     2041 gcgaggttag acggcctacc ctctcctggc ctcgcgagat ccctctcgtt aagggaggca
     2101 agcccgacgt cgtccagatt gtccgcaact acaacgccta ccttcgggcc agcgacgatc
     2161 tgcctaagct gttcatcgag tccgaccctg ggttcttttc caacgctatt gtcgagggag
     2221 ctaagaagtt ccctaacacc gagttcgtga aggtgaaggg cctccacttc ctccaggagg
     2281 acgctccaga tgaaatgggt aagtacatca agagcttcgt ggagcgcgtg ctgaagaacg
     2341 agcagagcgg aggaggcggc agtgccaggg cagatgatgc ccggcaatta tttgttttag
     2401 ctggcagtgc tgaagaagga gtcatgactc cagaactagc aggagtgatt aaacggttat
     2461 ggcgagatgg tggggtacaa gcttgcttca gcagatccag ggaatatcag ctcaatgatt
     2521 ctgcttcata ttatctaaat gatctggata gaatatccca gtctaactac attccaactc
     2581 agcaagatgt tcttcggacg agagtgaaga ccacaggcat tgtagaaaca catttcacct
     2641 tcaaagacct atacttcaag atgtttgatg taggtggcca aagatcagaa cgaaaaaagt
     2701 ggattcactg ttttgaggga gtgacagcaa ttatcttctg tgtggccctc agtgattatg
     2761 accttgttct ggctgaggac gaggagatga accgaatgca tgaaagcatg aaactgtttg
     2821 acagcatttg taataacaaa tggtttacag aaacttcaat cattctcttc cttaacaaga
     2881 aagacctttt tgaggaaaaa ataaagagga gtccgttaac tatctgttat ccagaataca
     2941 caggttccaa tacatatgaa gaggcagctg cctatattca atgccagttt gaagatctga
     3001 acagaagaaa agataccaag gagatctata ctcacttcac ctgtgccaca gacacgaaga
     3061 atgtgcagtt tgtttttgat gctgttacag atgtcatcat taaaaacaac ttaaaggaat
     3121 gtggacttta ttgatagctc gagtctagag ggcccgttta aacccgctga tcagcctcga
     3181 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3241 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3301 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3361 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3421 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3481 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3541 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3601 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3661 ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag
     3721 aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     3781 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     3841 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     3901 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     3961 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     4021 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     4081 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     4141 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     4201 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     4261 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     4321 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     4381 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     4441 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     4501 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     4561 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     4621 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     4681 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     4741 gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct
     4801 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4861 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4921 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4981 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5041 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5101 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5161 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5221 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5281 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5341 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5401 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5461 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5521 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5581 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5641 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5701 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5761 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5821 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5881 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5941 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6001 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6061 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6121 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6181 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6241 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6301 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6361 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6421 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6481 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6541 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6601 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6661 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6721 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6781 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6841 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6901 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6961 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7021 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7081 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7141 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7201 cccgaaaagt gccacctgac gtc
//
