LOCUS       sequence-311180-        6652 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 6652)
  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 6652)
  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*-BERKY1
FEATURES             Location/Qualifiers
     source          1..6652
                     /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"
     primer_bind     complement(1861..1880)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     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"
     primer_bind     2208..2229
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2362..2391
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2426..2443)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2432..2656
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2702..3130
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2789..2808)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2999..3020
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3139..3159)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3144..3473
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3324..3459
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3386..3405
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3521..3568
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3587..3961
                     /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    4091..4212
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4128..4147)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4182..4201
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4261..4277)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4261..4277)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4274..4296)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4285..4301
                     /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(4309..4315,4316..4333,4334..4339))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4354..4375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4492..4509)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4663..5251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4743..4762)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5422..6213,6214..6282))
                     /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     6045..6064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6283..6387)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6462..6481)
                     /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 gggatccggt
     2281 ggcaccggtg gatcaggctc ttctcgaggt tactaccatg gtatttgggt gggtgaagaa
     2341 ggtcgacttt ctcgaggtgg agaacaaaaa cttatttctg aagaagatct gtgatctaga
     2401 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2461 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2521 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2581 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2641 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2701 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2761 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2821 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2881 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2941 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3001 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3061 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3121 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3181 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3241 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3301 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3361 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3421 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3481 cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc
     3541 atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac
     3601 cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga
     3661 ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga
     3721 cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg
     3781 ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa
     3841 cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga
     3901 gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg
     3961 acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4021 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4081 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4141 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4201 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4261 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4321 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4381 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4441 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4501 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4561 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4621 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4681 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4741 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4801 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4861 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4921 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4981 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5041 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5101 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5161 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5221 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5281 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5341 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5401 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5461 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5521 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5581 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5641 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5701 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5761 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5821 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5881 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5941 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6001 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6061 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6121 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6181 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6241 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6301 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6361 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     6421 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     6481 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     6541 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     6601 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg tt
//
