LOCUS       sequence-300681-        7343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a G alpha subunit (GNAS1) 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 7343)
  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 7343)
  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-GAlphasL-RLuc8
FEATURES             Location/Qualifiers
     source          1..7343
                     /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             1527..2459
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGAALAFHYAYEHQDRIKAIVHMESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETVLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFLQEDAPDEMGKYIKSFVERVLKNEQ"
     primer_bind     complement(3295..3312)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3301..3525
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3658..3677)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3809..3856
                     /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             3864..4886
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3864..4886
                     /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    5016..5137
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5053..5072)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5107..5126
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5186..5202)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5186..5202)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5199..5221)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5210..5226
                     /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(5234..5240,5241..5258,5259..5264))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5279..5300
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5417..5434)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5588..6176)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5668..5687)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6347..7138,7139..7207))
                     /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     6970..6989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7208..7312)
                     /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 atgggctgcc tcgggaacag taagaccgag gaccagcgca
     1141 acgaggagaa ggcgcagcgt gaggccaaca aaaagatcga gaagcagctg cagaaggaca
     1201 agcaggtcta ccgggccacg caccgcctgc tgctgctggg tgctggagaa tctggtaaaa
     1261 gcaccattgt gaagcagatg aggatcctgc atgttaatgg gtttaatgga gagggcggcg
     1321 aagaggaccc gcaggctgca aggagcaaca gcgatggtga gaaggcaacc aaagtgcagg
     1381 acatcaaaaa caacctgaaa gaggcgattg aaaccattgt ggccgccatg agcaacctgg
     1441 tgccccccgt ggagctggcc aaccccgaga accagttcag agtggactac attctgagtg
     1501 tgatgaactc tggtggtggt ggatccatgg cttccaaggt gtacgacccc gagcaacgca
     1561 aacgcatgat cactgggcct cagtggtggg ctcgctgcaa gcaaatgaac gtgctggact
     1621 ccttcatcaa ctactatgat tccgagaagc acgccgagaa cgccgtgatt tttctgcatg
     1681 gtaacgctac ctccagctac ctgtggaggc acgtcgtgcc tcacatcgag cccgtggcta
     1741 gatgcatcat ccctgatctg atcggaatgg gtaagtccgg caagagcggg aatggctcat
     1801 atcgcctcct ggatcactac aagtacctca ccgcttggtt cgagctgctg aaccttccaa
     1861 agaaaatcat ctttgtgggc cacgactggg gggctgctct ggcctttcac tacgcctacg
     1921 agcaccaaga caggatcaag gccatcgtcc atatggagag tgtcgtggac gtgatcgagt
     1981 cctgggacga gtggcctgac atcgaggagg atattgccct gatcaagagc gaagagggcg
     2041 agaaaatggt gcttgagaat aacttcttcg tcgagaccgt gctcccaagc aagatcatgc
     2101 ggaaactgga gcctgaggag ttcgctgcct acctggagcc attcaaggag aagggcgagg
     2161 ttagacggcc taccctctcc tggcctcgcg agatccctct cgttaaggga ggcaagcccg
     2221 acgtcgtcca gattgtccgc aactacaacg cctaccttcg ggccagcgac gatctgccta
     2281 agctgttcat cgagtccgac cctgggttct tttccaacgc tattgtcgag ggagctaaga
     2341 agttccctaa caccgagttc gtgaaggtga agggcctcca cttcctccag gaggacgctc
     2401 cagatgaaat gggtaagtac atcaagagct tcgtggagcg cgtgctgaag aacgagcaga
     2461 gcggaggagg cggcagtgtg cctgactttg acttccctcc cgaattctat gagcatgcca
     2521 aggctctgtg ggaggatgaa ggagtgcgtg cctgctacga acgctccaac gagtaccagc
     2581 tgattgactg tgcccagtac ttcctggaca agatcgacgt gatcaagcag gctgactatg
     2641 tgccgagcga tcaggacctg cttcgctgcc gtgtcctgac ttctggaatc tttgagacca
     2701 agttccaggt ggacaaagtc aacttccaca tgtttgacgt gggtggccag cgcgatgaac
     2761 gccgcaagtg gatccagtgc ttcaacgatg tgactgccat catcttcgtg gtggccagca
     2821 gcagctacaa catggtcatc cgggaggaca accagaccaa ccgcctgcag gaggctctga
     2881 acctcttcaa gagcatctgg aacaacagat ggctgcgcac catctctgtg atcctgttcc
     2941 tcaacaagca agatctgctc gctgagaaag tccttgctgg gaaatcgaag attgaggact
     3001 actttccaga atttgctcgc tacactactc ctgaggatgc tactcccgag cccggagagg
     3061 acccacgcgt gacccgggcc aagtacttca ttcgagatga gtttctgagg atcagcactg
     3121 ccagtggaga tgggcgtcac tactgctacc ctcatttcac ctgcgctgtg gacactgaga
     3181 acatccgccg tgtgttcaac gactgccgtg acatcattca gcgcatgcac cttcgtcagt
     3241 acgagctgct ataatagctc gagtctagag ggcccgttta aacccgctga tcagcctcga
     3301 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3361 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3421 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3481 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3541 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     3601 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3661 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3721 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3781 ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag
     3841 aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     3901 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     3961 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     4021 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     4081 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     4141 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     4201 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     4261 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     4321 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     4381 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     4441 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     4501 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     4561 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     4621 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     4681 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     4741 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     4801 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     4861 gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct
     4921 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4981 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5041 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5101 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5161 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5221 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5281 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5341 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5401 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5461 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5521 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5581 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5641 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5701 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5761 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5821 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5881 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5941 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6001 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6061 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6121 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6181 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6241 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6301 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6361 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6421 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6481 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6541 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6601 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6661 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6721 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6781 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6841 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6901 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6961 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7021 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7081 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7141 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7201 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7261 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7321 cccgaaaagt gccacctgac gtc
//
