LOCUS       sequence-311190-        8113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BRET biosensor for detection of G-alpha-i-GTP in mammalian 
            expression vector pcDNA3.1(+).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8113)
  AUTHORS   Maziarz M, Park JC, Leyme A, Marivin A, Garcia-Lopez A, Patel PP, 
            Garcia-Marcos M
  TITLE     Revealing the Activity of Trimeric G-proteins in Live Cells with a 
            Versatile Biosensor Design.
  JOURNAL   Cell. 2020 Jun 27. pii: S0092-8674(20)30752-2. doi: 
            10.1016/j.cell.2020.06.020.
  PUBMED    32634377
REFERENCE   2  (bases 1 to 8113)
  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: "Cell. 2020 
            Jun 27. pii: S0092-8674(20)30752-2. doi: 
            10.1016/j.cell.2020.06.020."
COMMENT     Sequence Label: pcDNA3.1-G-alpha-i*-BERKY3
FEATURES             Location/Qualifiers
     source          1..8113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             742..1254
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1279..1299
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2020..2271
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     CDS             2752..3003
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     CDS             3481..3732
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     CDS             3823..3852
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3887..3904)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3893..4117
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4163..4591
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4250..4269)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4460..4481
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4600..4620)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4605..4934
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4785..4920
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4847..4866
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4982..5029
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5048..5422
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    5552..5673
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5589..5608)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5643..5662
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5722..5738)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5722..5738)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5735..5757)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5746..5762
                     /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(5770..5776,5777..5794,5795..5800))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5815..5836
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5953..5970)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6124..6712)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6204..6223)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6883..7674,7675..7743))
                     /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     7506..7525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7744..7848)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7923..7942)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcatgg gatgtataaa atcaaaaggg aaagacagca agcttggatc aggcggtagc
      721 gggggatcag gcggtagcgg gatggtcttc acactcgaag atttcgttgg ggactggcga
      781 cagacagccg gctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgttt
      841 cagaatctcg gggtgtccgt aactccgatc caaaggattg tcctgagcgg tgaaaatggg
      901 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcggcga ccaaatgggc
      961 cagatcgaaa aaatttttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc
     1021 ctgcactatg gcacactggt aatcgacggg gttacgccga acatgatcga ctatttcgga
     1081 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg
     1141 tggaacggca acaaaattat cgacgagcgc ctgatcaacc ccgacggctc cctgctgttc
     1201 cgagtaacca tcaacggagt gaccggctgg cggctgtgcg aacgcattct ggcggaattc
     1261 ggatctggtg gttcaggcga aaacctgtat tttcagggcg gaagcggaga agaggaggaa
     1321 aagaaaaaac aacaggagga ggaggcggag aggctgagga ggatacaaga ggaaatggaa
     1381 aaagaaagaa agcggcgaga ggaagatgag cagcgacgcc gcaaggagga ggaagagaga
     1441 aggatgaaat tggagatgga ggcaaaacgc aaacaagaag aggaagaacg gaagaaacgg
     1501 gaggatgatg aaaagcggaa gaaaaaggga tctggagggt cagggggcgc gcccatggtt
     1561 tcaaagggag aagagttgtt cacaggggtt gtaccgattc ttgtagaact tgacggagat
     1621 gtcaacgggc acaagtttag tgtaagtgga gaaggcgagg gcgatgcgac ctatggaaaa
     1681 ctgaccctta aatttatttg tactacgggt aaattgcccg ttccctggcc gacattggta
     1741 actacattcg gatatggttt gatgtgcttt gcaaggtatc cggatcacat gaagcagcat
     1801 gatttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1861 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1921 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1981 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     2041 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     2101 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2161 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2221 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggcgcgccc
     2281 ggaggaatgg tttcaaaggg agaagagttg ttcacagggg ttgtaccgat tcttgtagaa
     2341 cttgacggag atgtcaacgg gcacaagttt agtgtaagtg gagaaggcga gggcgatgcg
     2401 acctatggaa aactgaccct taaatttatt tgtactacgg gtaaattgcc cgttccctgg
     2461 ccgacattgg taactacatt cggatatggt ttgatgtgct ttgcaaggta tccggatcac
     2521 atgaagcagc atgatttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     2581 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     2641 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     2701 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     2761 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     2821 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     2881 aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     2941 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     3001 aagggatccg gaggaatggt ttcaaaggga gaagagttgt tcacaggggt tgtaccgatt
     3061 cttgtagaac ttgacggaga tgtcaacggg cacaagttta gtgtaagtgg agaaggcgag
     3121 ggcgatgcga cctatggaaa actgaccctt aaatttattt gtactacggg taaattgccc
     3181 gttccctggc cgacattggt aactacattc ggatatggtt tgatgtgctt tgcaaggtat
     3241 ccggatcaca tgaagcagca tgatttcttc aagtccgcca tgcccgaagg ctacgtccag
     3301 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     3361 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     3421 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     3481 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     3541 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     3601 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag
     3661 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     3721 gagctgtaca agggatccgg tggcaccggt ggatcaggct cttctcgagg ttactaccat
     3781 ggtatttggg tgggtgaaga aggtcgactt tctcgaggtg gagaacaaaa acttatttct
     3841 gaagaagatc tgtgatctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     3901 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3961 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     4021 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     4081 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     4141 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4201 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     4261 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4321 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     4381 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4441 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     4501 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4561 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     4621 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4681 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4741 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4801 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4861 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4921 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg
     4981 tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa
     5041 ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg
     5101 agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt
     5161 cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt
     5221 gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg
     5281 gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg
     5341 cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt
     5401 cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg ccgccttcta
     5461 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     5521 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     5581 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     5641 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     5701 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5761 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5821 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5881 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5941 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6001 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6061 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6121 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6181 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6241 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6301 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6361 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6421 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6481 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6541 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6601 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6661 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6721 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6781 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6841 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6901 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6961 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7021 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     7081 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7141 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     7201 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7261 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7321 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7381 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7441 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7501 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7561 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7621 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7681 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7741 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7801 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7861 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     7921 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     7981 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     8041 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     8101 agatatacgc gtt
//
