LOCUS       sequence-300657-        7226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a G alpha subunit (GNAI2) 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 7226)
  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 7226)
  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-GAlphai2-RLuc8
FEATURES             Location/Qualifiers
     source          1..7226
                     /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             1389..2321
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGAALAFHYAYEHQDRIKAIVHMESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETVLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFLQEDAPDEMGKYIKSFVERVLKNEQ"
     primer_bind     complement(3178..3195)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3184..3408
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3541..3560)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3692..3739
                     /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             3747..4769
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3747..4769
                     /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    4899..5020
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4936..4955)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4990..5009
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5069..5085)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5069..5085)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5082..5104)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5093..5109
                     /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(5117..5123,5124..5141,5142..5147))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5162..5183
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5300..5317)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5471..6059)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5551..5570)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6230..7021,7022..7090))
                     /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     6853..6872
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7091..7195)
                     /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 ccgtgagcgc cgaggacaag gcggcggccg
     1141 agcgctctaa gatgatcgac aagaacctgc gggaggacgg agagaaggcg gcgcgggagg
     1201 tgaagttgct gctgttgggt gctggggagt cagggaagag caccatcgtc aagcagatga
     1261 agatcatcca cgaggatggc tactccgagg aggaatgccg gcagtaccgg gcggttgtct
     1321 acagcaacac catccagtcc atcatggcca ttgtcaaagc catgggcaac tctggtggtg
     1381 gtggatccat ggcttccaag gtgtacgacc ccgagcaacg caaacgcatg atcactgggc
     1441 ctcagtggtg ggctcgctgc aagcaaatga acgtgctgga ctccttcatc aactactatg
     1501 attccgagaa gcacgccgag aacgccgtga tttttctgca tggtaacgct acctccagct
     1561 acctgtggag gcacgtcgtg cctcacatcg agcccgtggc tagatgcatc atccctgatc
     1621 tgatcggaat gggtaagtcc ggcaagagcg ggaatggctc atatcgcctc ctggatcact
     1681 acaagtacct caccgcttgg ttcgagctgc tgaaccttcc aaagaaaatc atctttgtgg
     1741 gccacgactg gggggctgct ctggcctttc actacgccta cgagcaccaa gacaggatca
     1801 aggccatcgt ccatatggag agtgtcgtgg acgtgatcga gtcctgggac gagtggcctg
     1861 acatcgagga ggatatcgcc ctgatcaaga gcgaagaggg cgagaaaatg gtgcttgaga
     1921 ataacttctt cgtcgagacc gtgctcccaa gcaagatcat gcggaaactg gagcctgagg
     1981 agttcgctgc ctacctggag ccattcaagg agaagggcga ggttagacgg cctaccctct
     2041 cctggcctcg cgagatccct ctcgttaagg gaggcaagcc cgacgtcgtc cagattgtcc
     2101 gcaactacaa cgcctacctt cgggccagcg acgatctgcc taagctgttc atcgagtccg
     2161 accctgggtt cttttccaac gctattgtcg agggagctaa gaagttccct aacaccgagt
     2221 tcgtgaaggt gaagggcctc cacttcctcc aggaggacgc tccagatgaa atgggtaagt
     2281 acatcaagag cttcgtggag cgcgtgctga agaacgagca gagcggagga ggcggcagtc
     2341 tgcagatcga ctttgccgac ccctccagag cggacgacgc caggcagcta tttgcactgt
     2401 cctgcaccgc cgaggagcaa ggcgtgctcc ctgatgacct gtccggcgtc atccggaggc
     2461 tctgggctga ccatggtgtg caggcctgct ttggccgctc aagggaatac cagctcaacg
     2521 actcagctgc ctactacctg aacgacctgg agcgtattgc acagagtgac tacatcccca
     2581 cacagcaaga tgtgctacgg acccgcgtaa agaccacggg gatcgtggag acacacttca
     2641 ccttcaagga cctacacttc aagatgtttg atgtgggtgg tcagcggtct gagcggaaga
     2701 agtggatcca ctgctttgag ggcgtcacag ccatcatctt ctgcgtagcc ttgagcgcct
     2761 atgacttggt gctagctgag gacgaggaga tgaaccgcat gcatgagagc atgaagctat
     2821 tcgatagcat ctgcaacaac aagtggttca cagacacgtc catcatcctc ttcctcaaca
     2881 agaaggacct gtttgaggag aagatcacac acagtcccct gaccatctgc ttccctgagt
     2941 acacaggggc caacaaatat gatgaggcag ccagctacat ccagagtaag tttgaggacc
     3001 tgaataagcg caaagacacc aaggagatct acacgcactt cacgtgcgcc accgacacca
     3061 agaacgtgca gttcgtgttt gacgccgtca ccgatgtcat catcaagaac aacctgaagg
     3121 actgcggcct cttctgatag ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     3181 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3241 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3301 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3361 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3421 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3481 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3541 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3601 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3661 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc
     3721 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga
     3781 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     3841 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     3901 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     3961 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     4021 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     4081 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     4141 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     4201 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     4261 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     4321 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     4381 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     4441 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     4501 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     4561 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     4621 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     4681 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     4741 ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg
     4801 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4861 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4921 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4981 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5041 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5101 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5161 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5221 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5281 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5341 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5401 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5461 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5521 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5581 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5641 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5701 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5761 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5821 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5881 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5941 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6001 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6061 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6121 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6181 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6241 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6301 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6361 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6421 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6481 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6541 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6601 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6661 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6721 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6781 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6841 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6901 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6961 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7021 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7081 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7141 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7201 ttccccgaaa agtgccacct gacgtc
//
