LOCUS       sequence-331013-        6462 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 6462)
  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 6462)
  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-GGamma12-GFP2
FEATURES             Location/Qualifiers
     source          1..6462
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(66..654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(146..165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(825..1616,1617..1685))
                     /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     1448..1467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1686..1790)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1865..1884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2056..2435
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2436..2639
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2590..2610
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2684..2703
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2684..2702
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2720..2729
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2726..2728,2729..2731,2732..3442)
                     /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(2771..2792)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3032..3051)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3379..3400
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3705..3722)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3711..3935
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3981..4409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4068..4087)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4278..4299
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4418..4438)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4423..4752
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4603..4738
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4665..4684
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4801..5826
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4801..5826
                     /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    5956..6077
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5993..6012)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6047..6066
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6126..6142)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6126..6142)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6139..6161)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6150..6166
                     /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(6174..6180,6181..6198,6199..6204))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6219..6240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6357..6374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
       61 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      121 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      181 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      241 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      301 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      361 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      421 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      481 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      541 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
      601 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
      661 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
      721 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
      781 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
      841 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
      901 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
      961 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1021 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1081 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1141 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     1201 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     1261 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1321 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     1381 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     1441 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     1501 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     1561 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     1621 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     1681 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     1741 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     1801 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     1861 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     1921 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     1981 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     2041 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     2101 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     2161 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     2221 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     2281 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     2341 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     2401 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     2461 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     2521 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     2581 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     2641 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata
     2701 gggagaccca agctggctag ccaccatggt gagcaagggc gaggagctgt tcaccggggt
     2761 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2821 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2881 caagctgccc gtgccctggc ccaccctcgt gaccaccctg agctacggcg tgcagtgctt
     2941 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     3001 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     3061 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     3121 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaaccccc acaacgtcta
     3181 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     3241 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3301 ccccgtgctg ctgcccgaca accactacct gttcacccag tccgccctga gcaaagaccc
     3361 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3421 cggcatggac gagctgtaca agggatccgc cggtaccatg tccagcaaaa cagcaagcac
     3481 caacaatata gcccaggcaa ggagaactgt gcagcagtta agattagaag cctccattga
     3541 aagaataaag gtttcgaagg catcagcgga cctcatgtcc tactgtgagg aacatgccag
     3601 gagtgaccct ttgctgatag gaataccaac ttcagaaaac cctttcaagg ataaaaaaac
     3661 ttgcatcatc ttatagagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     3721 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3781 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3841 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3901 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3961 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4021 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     4081 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4141 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4201 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4261 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4321 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4381 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     4441 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4501 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4561 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     4621 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4681 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4741 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg
     4801 atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     4861 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     4921 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     4981 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     5041 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     5101 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     5161 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     5221 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     5281 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     5341 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     5401 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     5461 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     5521 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     5581 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     5641 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     5701 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     5761 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     5821 gaatagcacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5881 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5941 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     6001 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     6061 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     6121 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     6181 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     6241 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6301 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6361 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     6421 gcggtaatac ggttatccac agaatcaggg gataacgcag ga
//
