LOCUS       sequence-311189-        7381 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 7381)
  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 7381)
  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*-BERKY2
FEATURES             Location/Qualifiers
     source          1..7381
                     /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             2749..3000
                     /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             3091..3120
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3155..3172)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3161..3385
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3431..3859
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3518..3537)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3728..3749
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3868..3888)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3873..4202
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4053..4188
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4115..4134
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4250..4297
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4316..4690
                     /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    4820..4941
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4857..4876)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4911..4930
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4990..5006)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4990..5006)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5003..5025)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5014..5030
                     /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(5038..5044,5045..5062,5063..5068))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5083..5104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5221..5238)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5392..5980)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5472..5491)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6151..6942,6943..7011))
                     /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     6774..6793
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7012..7116)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7191..7210)
                     /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 gggatccgga
     2281 ggaatggttt caaagggaga agagttgttc acaggggttg taccgattct tgtagaactt
     2341 gacggagatg tcaacgggca caagtttagt gtaagtggag aaggcgaggg cgatgcgacc
     2401 tatggaaaac tgacccttaa atttatttgt actacgggta aattgcccgt tccctggccg
     2461 acattggtaa ctacattcgg atatggtttg atgtgctttg caaggtatcc ggatcacatg
     2521 aagcagcatg atttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2581 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2641 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2701 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2761 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2821 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2881 cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2941 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3001 ggatccggtg gcaccggtgg atcaggctct tctcgaggtt actaccatgg tatttgggtg
     3061 ggtgaagaag gtcgactttc tcgaggtgga gaacaaaaac ttatttctga agaagatctg
     3121 tgatctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     3181 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3241 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3301 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3361 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     3421 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3481 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3541 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3601 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3661 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3721 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3781 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3841 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3901 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3961 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4021 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4081 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4141 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4201 aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta
     4261 atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc
     4321 caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt
     4381 ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt
     4441 ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac
     4501 cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt
     4561 gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg
     4621 ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga
     4681 gcaggactga cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4741 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4801 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4861 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4921 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     4981 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5041 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5101 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5161 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5221 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5281 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5341 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5401 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5461 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5521 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5581 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5641 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5701 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5761 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5821 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5881 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5941 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6001 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6061 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6121 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6181 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6241 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6301 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6361 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6421 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6481 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6541 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6601 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6661 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6721 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6781 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6841 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6901 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6961 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7021 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7081 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7141 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     7201 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     7261 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     7321 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     7381 t
//
