LOCUS       sequence-300781-        7318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a G alpha subunit (GNA12) 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 7318)
  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 7318)
  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-GAlpha12-RLuc8
FEATURES             Location/Qualifiers
     source          1..7318
                     /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             1518..2450
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGAALAFHYAYEHQDRIKAIVHMESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETVLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFLQEDAPDEMGKYIKSFVERVLKNEQ"
     primer_bind     complement(3270..3287)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3276..3500
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3633..3652)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3784..3831
                     /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             3839..4861
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3839..4861
                     /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    4991..5112
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5028..5047)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5082..5101
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5161..5177)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5161..5177)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5174..5196)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5185..5201
                     /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(5209..5215,5216..5233,5234..5239))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5254..5275
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5392..5409)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5563..6151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5643..5662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6322..7113,7114..7182))
                     /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     6945..6964
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7183..7287)
                     /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 atgtcagggg ttgtacgaac cctcagccgg tgtctgttgc
     1141 cggctgaagc ggggggtgcc cgggaaagac gagccggctc tggcgctaga gatgcggaac
     1201 gggaagcacg acgaaggtcc agggatatag atgccctctt ggcacgagaa aggagggctg
     1261 tacgccggtt ggttaagata ctccttcttg gtgcaggtga gagcggtaaa agtacgttcc
     1321 tcaaacagat gaggataata cacggtaggg aatttgacca aaaagcgctc cttgagttta
     1381 gggacacaat cttcgataac atactcaaag gttcaagggt tcttgttgat gcacgagata
     1441 agcttggcat tccttggcag tattctgaaa atgaggagca tgggatgttc ctgatggcct
     1501 ctggtggtgg tggatccatg gcttccaagg tgtacgaccc cgagcaacgc aaacgcatga
     1561 tcactgggcc tcagtggtgg gctcgctgca agcaaatgaa cgtgctggac tccttcatca
     1621 actactatga ttccgagaag cacgccgaga acgccgtgat ttttctgcat ggtaacgcta
     1681 cctccagcta cctgtggagg cacgtcgtgc ctcacatcga gcccgtggct agatgcatca
     1741 tccctgatct gatcggaatg ggtaagtccg gcaagagcgg gaatggctca tatcgcctcc
     1801 tggatcacta caagtacctc accgcttggt tcgagctgct gaaccttcca aagaaaatca
     1861 tctttgtggg ccacgactgg ggggctgctc tggcctttca ctacgcctac gagcaccaag
     1921 acaggatcaa ggccatcgtc catatggaga gtgtcgtgga cgtgatcgag tcctgggacg
     1981 agtggcctga catcgaggag gatatcgccc tgatcaagag cgaagagggc gagaaaatgg
     2041 tgcttgagaa taacttcttc gtcgagaccg tgctcccaag caagatcatg cggaaactgg
     2101 agcctgagga gttcgctgcc tacctggagc cattcaagga gaagggcgag gttagacggc
     2161 ctaccctctc ctggcctcgc gagatccctc tcgttaaggg aggcaagccc gacgtcgtcc
     2221 agattgtccg caactacaac gcctaccttc gggccagcga cgatctgcct aagctgttca
     2281 tcgagtccga ccctgggttc ttttccaacg ctattgtcga gggagctaag aagttcccta
     2341 acaccgagtt cgtgaaggtg aagggcctcc acttcctcca ggaggacgct ccagatgaaa
     2401 tgggtaagta catcaagagc ttcgtggagc gcgtgctgaa gaacgagcag agcggaggag
     2461 gcggcagttt cgagaacaag gcggggctgc ctgtggagcc ggccaccttc cagctgtacg
     2521 tcccggcctt gagcgccctg tggcgagaca gcggcattcg ggaagcgttt tctcggcggt
     2581 ctgagttcca actgggagaa tcagtgaaat acttcctgga taacttggac cgaatcggcc
     2641 aattgaacta tttcccctct aagcaagata ttcttttggc tagaaaggct acaaagggta
     2701 tcgtcgagca cgattttgtg attaagaaga ttcccttcaa gatggtagat gtagggggcc
     2761 aaaggagcca gcgacaaaaa tggtttcagt gctttgatgg aataacatct atacttttta
     2821 tggtatcctc cagtgagtac gaccaagtcc tcatggaaga ccgacgaacc aatcggctgg
     2881 tagagtcaat gaatatattt gaaactatcg tcaataataa gctgtttttc aatgtctcta
     2941 tcatactgtt tcttaataag atggatcttc tcgttgagaa agttaaaacc gtctctatca
     3001 aaaaacactt cccggatttt cgcggtgatc cgcacaggct tgaggacgtc cagcgatatt
     3061 tggtgcagtg ttttgatagg aagaggcgca atcgctccaa gccactcttc catcatttca
     3121 caacagctat agacacggag aatgttcggt tcgtgttcca tgctgttaag gacacaatcc
     3181 tccaggagaa cctgaaagac atcatgttgc aatgacagca cagtggcggc cgctcgagtc
     3241 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     3301 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3361 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3421 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3481 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     3541 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3601 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3661 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3721 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac
     3781 ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa
     3841 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     3901 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     3961 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     4021 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     4081 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     4141 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     4201 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     4261 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     4321 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     4381 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     4441 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     4501 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     4561 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     4621 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     4681 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     4741 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     4801 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     4861 gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4921 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4981 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5041 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5101 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     5161 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5221 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5281 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5341 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5401 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5461 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5521 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5581 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5641 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5701 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5761 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5821 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5881 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5941 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6001 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6061 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6121 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6181 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6241 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6301 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6361 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6421 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6481 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6541 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6601 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6661 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6721 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6781 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6841 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6901 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6961 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7021 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7081 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7141 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7201 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7261 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
