LOCUS       sequence-300680-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a G alpha subunit (GNAS2) 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 7301)
  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 7301)
  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-GAlphasS-RLuc8
FEATURES             Location/Qualifiers
     source          1..7301
                     /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             1485..2417
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGAALAFHYAYEHQDRIKAIVHMESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETVLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFLQEDAPDEMGKYIKSFVERVLKNEQ"
     primer_bind     complement(3253..3270)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3259..3483
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3616..3635)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3767..3814
                     /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             3822..4844
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3822..4844
                     /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    4974..5095
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5011..5030)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5065..5084
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5144..5160)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5144..5160)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5157..5179)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5168..5184
                     /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(5192..5198,5199..5216,5217..5222))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5237..5258
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5375..5392)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5546..6134)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5626..5645)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6305..7096,7097..7165))
                     /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     6928..6947
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7166..7270)
                     /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 gacagtgaga
     1321 aggcaaccaa agtgcaggac atcaaaaaca acctgaaaga ggcgattgaa accattgtgg
     1381 ccgccatgag caacctggtg ccccccgtgg agctggccaa ccccgagaac cagttcagag
     1441 tggactacat tctgagtgtg atgaactctg gtggtggtgg atccatggct tccaaggtgt
     1501 acgaccccga gcaacgcaaa cgcatgatca ctgggcctca gtggtgggct cgctgcaagc
     1561 aaatgaacgt gctggactcc ttcatcaact actatgattc cgagaagcac gccgagaacg
     1621 ccgtgatttt tctgcatggt aacgctacct ccagctacct gtggaggcac gtcgtgcctc
     1681 acatcgagcc cgtggctaga tgcatcatcc ctgatctgat cggaatgggt aagtccggca
     1741 agagcgggaa tggctcatat cgcctcctgg atcactacaa gtacctcacc gcttggttcg
     1801 agctgctgaa ccttccaaag aaaatcatct ttgtgggcca cgactggggg gctgctctgg
     1861 cctttcacta cgcctacgag caccaagaca ggatcaaggc catcgtccat atggagagtg
     1921 tcgtggacgt gatcgagtcc tgggacgagt ggcctgacat cgaggaggat atcgccctga
     1981 tcaagagcga agagggcgag aaaatggtgc ttgagaataa cttcttcgtc gagaccgtgc
     2041 tcccaagcaa gatcatgcgg aaactggagc ctgaggagtt cgctgcctac ctggagccat
     2101 tcaaggagaa gggcgaggtt agacggccta ccctctcctg gcctcgcgag atccctctcg
     2161 ttaagggagg caagcccgac gtcgtccaga ttgtccgcaa ctacaacgcc taccttcggg
     2221 ccagcgacga tctgcctaag ctgttcatcg agtccgaccc tgggttcttt tccaacgcta
     2281 ttgtcgaggg agctaagaag ttccctaaca ccgagttcgt gaaggtgaag ggcctccact
     2341 tcctccagga ggacgctcca gatgaaatgg gtaagtacat caagagcttc gtggagcgcg
     2401 tgctgaagaa cgagcagagc ggaggaggcg gcagtgtgcc tgactttgac ttccctcccg
     2461 aattctatga gcatgccaag gctctgtggg aggatgaagg agtgcgtgcc tgctacgaac
     2521 gctccaacga gtaccagctg attgactgtg cccagtactt cctggacaag atcgacgtga
     2581 tcaagcaggc tgactatgtg ccgagcgatc aggacctgct tcgctgccgt gtcctgactt
     2641 ctggaatctt tgagaccaag ttccaggtgg acaaagtcaa cttccacatg tttgacgtgg
     2701 gtggccagcg cgatgaacgc cgcaagtgga tccagtgctt caacgatgtg actgccatca
     2761 tcttcgtggt ggccagcagc agctacaaca tggtcatccg ggaggacaac cagaccaacc
     2821 gcctgcagga ggctctgaac ctcttcaaga gcatctggaa caacagatgg ctgcgcacca
     2881 tctctgtgat cctgttcctc aacaagcaag atctgctcgc tgagaaagtc cttgctggga
     2941 aatcgaagat tgaggactac tttccagaat ttgctcgcta cactactcct gaggatgcta
     3001 ctcccgagcc cggagaggac ccacgcgtga cccgggccaa gtacttcatt cgagatgagt
     3061 ttctgaggat cagcactgcc agtggagatg ggcgtcacta ctgctaccct catttcacct
     3121 gcgctgtgga cactgagaac atccgccgtg tgttcaacga ctgccgtgac atcattcagc
     3181 gcatgcacct tcgtcagtac gagctgctat aatagctcga gtctagaggg cccgtttaaa
     3241 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3301 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3361 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3421 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3481 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3541 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3601 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3661 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3721 acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc
     3781 gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg
     3841 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     3901 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     3961 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     4021 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     4081 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     4141 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     4201 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     4261 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     4321 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     4381 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     4441 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     4501 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     4561 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     4621 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     4681 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     4741 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     4801 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt
     4861 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4921 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4981 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5041 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5101 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5161 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5221 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5281 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5341 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5401 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5461 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5521 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5581 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5641 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5701 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5761 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5821 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5881 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5941 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6001 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6061 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6121 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6181 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6241 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6301 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6361 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6421 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6481 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6541 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6601 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6661 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6721 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6781 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6841 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6901 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6961 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7021 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7081 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7141 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7201 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7261 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
