LOCUS       sequence-306392-        7021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanoluc complementation assay. Expression of G-alpha-i3 protein 
            fused with the LgBiT(LgB)of the nanoluciferase inserted between 
            residues 91 and 92 of G-alpha-i3. Addition of the HA epitope at N 
            terminus of G-alpha-i3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7021)
  AUTHORS   Laschet C, Dupuis N, Hanson J
  TITLE     A dynamic and screening-compatible nanoluciferase-based 
            complementation assay enables profiling of individual GPCR-G protein
            interactions.
  JOURNAL   J Biol Chem. 2019 Mar 15;294(11):4079-4090. doi: 
            10.1074/jbc.RA118.006231. Epub 2018 Dec 28.
  PUBMED    30593506
REFERENCE   2  (bases 1 to 7021)
  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"; doi: 
            "10.1074/jbc.RA118.006231"; journalName: "J Biol Chem"; date: 
            "2019-03-15- 15"; volume: "294"; issue: "11"; pages: "4079-4090"
COMMENT     Sequence Label: pcDNA3.1.G-alpha-i3-LgB91
FEATURES             Location/Qualifiers
     source          1..7021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             964..990
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2612..2629)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2618..2842
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2888..3316
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2975..2994)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3185..3206
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3325..3345)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3330..3659
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3510..3645
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3572..3591
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3726..4520
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3780..3799)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4390..4409
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4694..4815
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4731..4750)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4785..4804
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4864..4880)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4864..4880)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4877..4899)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4888..4904
                     /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(4912..4918,4919..4936,4937..4942))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4957..4978
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5095..5112)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5266..5854)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5346..5365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6025..6816,6817..6885))
                     /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     6648..6667
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6886..6990)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcacc
      961 atgtacccat acgatgttcc agattacgct ggctgcacgt tgagcgccga agacaaggcg
     1021 gcagtggagc gaagcaagat gatcgaccgc aacttacggg aggacgggga aaaagcggcc
     1081 aaagaagtga agctgctgct actcggtgct ggagaatctg gtaaaagcac cattgtgaaa
     1141 cagatgaaaa tcattcatga ggatggctat tcagaggatg aatgtaaaca atataaagta
     1201 gttgtctaca gcaatactat acagtccatc attgcaatca taagagccat gggacggcta
     1261 gatatcagcg gaggcggagg aagcgtcttc acactcgaag atttcgttgg ggactgggaa
     1321 cagacagccg cctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgctg
     1381 cagaatctcg ccgtgtccgt aactccgatc caaaggattg tccggagcgg tgaaaatgcc
     1441 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcgccga ccaaatggcc
     1501 cagatcgaag aggtgtttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc
     1561 ctgccctatg gcacactggt aatcgacggg gttacgccga acatgctgaa ctatttcgga
     1621 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg
     1681 tggaacggca acaaaattat cgacgagcgc ctgatcaccc ccgacggctc catgctgttc
     1741 cgagtaacca tcaacagtag cggaggcgga ggaagcccgc ggaagattga ctttggggaa
     1801 gctgccaggg cagatgatgc ccggcaatta tttgttttag ctggcagtgc tgaagaagga
     1861 gtcatgactc cagaactagc aggagtgatt aaacggttat ggcgagatgg tggggtacaa
     1921 gcttgcttca gcagatccag ggaatatcag ctcaatgatt ctgcttcata ttatctaaat
     1981 gatctggata gaatatccca gtctaactac attccaactc agcaagatgt tcttcggacg
     2041 agagtgaaga ccacaggcat tgtagaaaca catttcacct tcaaagacct atacttcaag
     2101 atgtttgatg taggtggcca aagatcagaa cgaaaaaagt ggattcactg ttttgaggga
     2161 gtgacagcaa ttatcttctg tgtggccctc agtgattatg accttgttct ggctgaggac
     2221 gaggagatga accgaatgca tgaaagcatg aaactgtttg acagcatttg taataacaaa
     2281 tggtttacag aaacttcaat cattctcttc cttaacaaga aagacctttt tgaggaaaaa
     2341 ataaagagga gtccgttaac tatctgttat ccagaataca caggttccaa tacatatgaa
     2401 gaggcagctg cctatattca atgccagttt gaagatctga acagaagaaa agataccaag
     2461 gagatctata ctcacttcac ctgtgccaca gacacgaaga atgtgcagtt tgtttttgat
     2521 gctgttacag atgtcatcat taaaaacaac ttaaaggaat gtggacttta ttgactcgag
     2581 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     2641 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2701 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2761 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2821 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     2881 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2941 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     3001 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3061 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3121 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3181 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3241 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3301 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3361 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3421 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3481 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3541 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     3601 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3661 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     3721 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3781 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3841 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3901 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3961 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     4021 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     4081 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     4141 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     4201 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     4261 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4321 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4381 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4441 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4501 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4561 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4621 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4681 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4741 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4801 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     4861 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4921 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4981 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5041 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5101 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5161 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5221 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5281 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5341 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5401 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5461 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5521 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5581 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5641 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5701 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5761 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5821 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5881 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5941 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6001 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6061 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6121 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6181 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6241 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6301 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6361 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6421 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6481 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6541 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6601 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6661 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6721 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6781 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6841 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6901 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6961 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7021 c
//
