LOCUS       sequence-408989-        6453 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a G gamma subunit containing GFP2 as an ""non-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 6453)
  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 6453)
  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: pcDNA3.1-GGamma5-GFP2
FEATURES             Location/Qualifiers
     source          1..6453
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(47..838,839..907))
                     /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     670..689
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(908..1012)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1087..1106)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1278..1657
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1658..1861
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1812..1832
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1906..1925
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1906..1924
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1942..1951
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1948..1950,1951..1953,1954..2664)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNPHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLFTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1993..2014)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2254..2273)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2601..2622
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2918..2935)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2924..3148
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3194..3622
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3281..3300)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3491..3512
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3631..3651)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3636..3965
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3816..3951
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3878..3897
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4014..5039
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4014..5039
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    5169..5290
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5206..5225)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5260..5279
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5339..5355)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5339..5355)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5352..5374)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5363..5379
                     /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(5387..5393,5394..5411,5412..5417))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5432..5453
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5570..5587)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5741..6329)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5821..5840)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
       61 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
      121 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
      181 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
      241 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
      301 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
      361 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
      421 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
      481 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
      541 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
      601 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
      661 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
      721 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
      781 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
      841 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
      901 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
      961 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     1021 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     1081 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     1141 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     1201 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     1261 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     1321 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     1381 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     1441 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     1501 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     1561 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     1621 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     1681 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     1741 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     1801 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     1861 tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta
     1921 tagggagacc caagctggct agccaccatg gtgagcaagg gcgaggagct gttcaccggg
     1981 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     2041 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     2101 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgagctacgg cgtgcagtgc
     2161 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2221 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2281 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2341 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaaccc ccacaacgtc
     2401 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2461 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2521 ggccccgtgc tgctgcccga caaccactac ctgttcaccc agtccgccct gagcaaagac
     2581 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2641 ctcggcatgg acgagctgta caagggatcc gccggtacca tgtctggttc atccagcgtc
     2701 gcggctatga aaaaagtggt tcaacagctc cggctggagg ccgggctcaa tcgggtgaag
     2761 gtttcccagg cagctgcaga tttgaaacag ttctgtctgc agaatgctca acatgaccct
     2821 ctgctgactg gagtatcttc aagtacaaat cccttcaggc cccagaaagt ctgttccttt
     2881 ttgtagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     2941 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3001 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3061 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3121 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3181 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3241 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3301 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3361 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3421 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3481 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3541 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3601 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3661 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3721 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3781 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3841 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3901 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3961 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgatgaaaa
     4021 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     4081 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     4141 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     4201 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     4261 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     4321 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     4381 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     4441 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     4501 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     4561 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     4621 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     4681 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     4741 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     4801 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     4861 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     4921 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     4981 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     5041 acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5101 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5161 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5221 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5281 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5341 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5401 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5461 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5521 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5581 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5641 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5701 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5761 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5821 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5881 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5941 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6001 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6061 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6121 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6181 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6241 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6301 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6361 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6421 tcttcaccta gatcctttta aattaaaaat gaa
//
