LOCUS       sequence-306397-        7024 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanoluc complementation assay. Expression of G-alpha-i2 protein 
            fused with the LgBiT(LgB)of the nanoluciferase inserted between 
            residues 91 and 92 of G-alpha-i2. Addition of the HA epitope at N 
            terminus of G-alpha-i2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7024)
  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 7024)
  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-i2-LgB91
FEATURES             Location/Qualifiers
     source          1..7024
                     /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(2615..2632)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2621..2845
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2891..3319
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2978..2997)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3188..3209
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3328..3348)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3333..3662
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3513..3648
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3575..3594
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3729..4523
                     /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(3783..3802)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4393..4412
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4697..4818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4734..4753)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4788..4807
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4867..4883)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4867..4883)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4880..4902)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4891..4907
                     /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(4915..4921,4922..4939,4940..4945))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4960..4981
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5098..5115)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5269..5857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5349..5368)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6028..6819,6820..6888))
                     /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     6651..6670
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6889..6993)
                     /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 ggctgcaccg tgagcgccga ggacaaggcg
     1021 gcggccgagc gctctaagat gatcgacaag aacctgcggg aggacggaga gaaggcggcg
     1081 cgggaggtga agttgctgct gttgggtgct ggggagtcag ggaagagcac catcgtcaag
     1141 cagatgaaga tcatccacga ggatggctac tccgaggagg aatgccggca gtaccgggcg
     1201 gttgtctaca gcaacaccat ccagtccatc atggccattg tcaaagccat gggcaacctg
     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 ggcagatcga ctttgccgac
     1801 ccctccagag cggacgacgc caggcagcta tttgcactgt cctgcaccgc cgaggagcaa
     1861 ggcgtgctcc ctgatgacct gtccggcgtc atccggaggc tctgggctga ccatggtgtg
     1921 caggcctgct ttggccgctc aagggaatac cagctcaacg actcagctgc ctactacctg
     1981 aacgacctgg agcgtattgc acagagtgac tacatcccca cacagcaaga tgtgctacgg
     2041 acccgcgtaa agaccacggg gatcgtggag acacacttca ccttcaagga cctacacttc
     2101 aagatgtttg atgtgggtgg tcagcggtct gagcggaaga agtggatcca ctgctttgag
     2161 ggcgtcacag ccatcatctt ctgcgtagcc ttgagcgcct atgacttggt gctagctgag
     2221 gacgaggaga tgaaccgcat gcatgagagc atgaagctat tcgatagcat ctgcaacaac
     2281 aagtggttca cagacacgtc catcatcctc ttcctcaaca agaaggacct gtttgaggag
     2341 aagatcacac acagtcccct gaccatctgc ttccctgagt acacaggggc caacaaatat
     2401 gatgaggcag ccagctacat ccagagtaag tttgaggacc tgaataagcg caaagacacc
     2461 aaggagatct acacgcactt cacgtgcgcc accgacacca agaacgtgca gttcgtgttt
     2521 gacgccgtca ccgatgtcat catcaagaac aacctgaagg actgcggcct cttctgactc
     2581 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     2641 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2701 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2761 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2821 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2881 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2941 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3001 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3061 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3121 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3181 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3241 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3301 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3361 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3421 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3481 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3541 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3601 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3661 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     3721 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3781 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     3841 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     3901 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     3961 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4021 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4081 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4141 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4201 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     4261 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4321 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4381 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4441 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4501 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4561 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4621 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4681 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4741 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4801 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4861 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4921 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4981 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5041 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5101 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5161 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5221 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5281 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5341 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5401 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5461 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5521 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5581 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5641 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5701 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5761 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5821 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5881 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5941 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6001 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6061 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6121 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6181 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6241 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6301 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6361 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6421 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6481 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6541 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6601 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6661 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6721 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6781 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6841 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6901 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6961 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     7021 cgtc
//
