LOCUS       sequence-306402-        7100 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Nanoluc complementation assay. Expression of G-alpha-s protein fused
            with the LgBiT(LgB)of the nanoluciferase inserted between residues 
            113 and 114 of G-alpha-s. Addition of the HA epitope at N terminus 
            of G-alpha-s..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7100)
  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 7100)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-s-LgB113
FEATURES             Location/Qualifiers
     source          1..7100
                     /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             923..949
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2691..2708)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2697..2921
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2967..3395
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3054..3073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3264..3285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3404..3424)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3409..3738
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3589..3724
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3651..3670
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3805..4599
                     /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(3859..3878)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4469..4488
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4773..4894
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4810..4829)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4864..4883
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4943..4959)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4943..4959)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4956..4978)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4967..4983
                     /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(4991..4997,4998..5015,5016..5021))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5036..5057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5174..5191)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5345..5933)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5425..5444)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6104..6895,6896..6964))
                     /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     6727..6746
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6965..7069)
                     /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 aagcttacca tgtacccata cgatgttcca gattacgctg gctgcctcgg
      961 gaacagtaag accgaggacc agcgcaacga ggagaaggcg cagcgtgagg ccaacaaaaa
     1021 gatcgagaag cagctgcaga aggacaagca ggtctaccgg gccacgcacc gcctgctgct
     1081 gctgggtgct ggagaatctg gtaaaagcac cattgtgaag cagatgagga tcctgcatgt
     1141 taatgggttt aatggagagg gcggcgaaga ggacccgcag gctgcaagga gcaacagcga
     1201 tggtgagaag gcaaccaaag tgcaggacat caaaaacaac ctgaaagagg cgattgaaac
     1261 cattgtggcc gccatgagca acctggatat cagcggaggc ggaggaagcg tcttcacact
     1321 cgaagatttc gttggggact gggaacagac agccgcctac aacctggacc aagtccttga
     1381 acagggaggt gtgtccagtt tgctgcagaa tctcgccgtg tccgtaactc cgatccaaag
     1441 gattgtccgg agcggtgaaa atgccctgaa gatcgacatc catgtcatca tcccgtatga
     1501 aggtctgagc gccgaccaaa tggcccagat cgaagaggtg tttaaggtgg tgtaccctgt
     1561 ggatgatcat cactttaagg tgatcctgcc ctatggcaca ctggtaatcg acggggttac
     1621 gccgaacatg ctgaactatt tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa
     1681 aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg agcgcctgat
     1741 cacccccgac ggctccatgc tgttccgagt aaccatcaac agtagcggag gcggaggaag
     1801 cccgcgggtg ccccccgtgg agctggccaa ccccgagaac cagttcagag tggactacat
     1861 cctgagtgtg atgaacgtgc ctgactttga cttccctccc gaattctatg agcatgccaa
     1921 ggctctgtgg gaggatgaag gagtgcgtgc ctgctacgaa cgctccaacg agtaccagct
     1981 gattgactgt gcccagtact tcctggacaa gatcgacgtg atcaagcagg ctgactatgt
     2041 gccgagcgat caggacctgc ttcgctgccg tgtcctgact tctggaatct ttgagaccaa
     2101 gttccaggtg gacaaagtca acttccacat gtttgacgtg ggtggccagc gcgatgaacg
     2161 ccgcaagtgg atccagtgct tcaacgatgt gactgccatc atcttcgtgg tggccagcag
     2221 cagctacaac atggtcatcc gggaggacaa ccagaccaac cgcctgcagg aggctctgaa
     2281 cctcttcaag agcatctgga acaacagatg gctgcgcacc atctctgtga tcctgttcct
     2341 caacaagcaa gatctgctcg ctgagaaagt ccttgctggg aaatcgaaga ttgaggacta
     2401 ctttccagaa tttgctcgct acactactcc tgaggatgct actcccgagc ccggagagga
     2461 cccacgcgtg acccgggcca agtacttcat tcgagatgag tttctgagga tcagcactgc
     2521 cagtggagat gggcgtcact actgctaccc tcatttcacc tgcgctgtgg acactgagaa
     2581 catccgccgt gtgttcaacg actgccgtga catcattcag cgcatgcacc ttcgtcagta
     2641 cgagctgctc taactcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     2701 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2761 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2821 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2881 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2941 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3001 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3061 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3121 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3181 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3241 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3301 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3361 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3421 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3481 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3541 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3601 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3661 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3721 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     3781 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3841 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3901 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3961 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4021 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4081 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4141 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4201 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4261 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4321 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4381 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4441 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4501 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4561 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4621 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4681 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4741 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4801 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4861 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4921 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4981 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5041 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5101 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5161 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5221 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5281 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5341 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5401 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5461 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5521 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5581 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5641 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5701 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5761 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5821 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5881 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5941 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6001 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6061 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6121 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6181 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6241 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6301 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6361 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6421 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6481 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6541 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6601 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6661 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6721 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6781 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6841 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6901 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6961 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7021 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7081 gaaaagtgcc acctgacgtc
//
