LOCUS       sequence-244593-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses modified SPARK beta-2AR-NanoLuc transmembrane component in
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6912)
  AUTHORS   Kim CK, Cho KF, Kim MW, Ting AY
  TITLE     Luciferase-LOV BRET enables versatile and specific transcriptional 
            readout of cellular protein-protein interactions.
  JOURNAL   eLife. 2019 Apr 3;8. pii: 43826. doi: 10.7554/eLife.43826.
  PUBMED    30942168
REFERENCE   2  (bases 1 to 6912)
  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: "eLife."; 
            date: "2019-04-3"; volume: "8. pii: 43826. doi"; pages: " 
            10.7554/eLife.43826"
COMMENT     Sequence Label: pAAV-beta-2AR-NNES-NanoLuc-eLOV-TEVcs-Flag-Gal4-V5
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        201..504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        505..708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     659..679
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     753..772
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        753..771
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      785..794
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2066..2575
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     CDS             3017..3037
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             3050..3073
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3074..3511
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             3512..3853
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="activation domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 activation domain
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     CDS             3854..3895
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3933..3950)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3939..4146
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4175..4315
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4175..4304
                     /label=AAV2 ITR
     rep_origin      4390..4845
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4477..4496)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4687..4708
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4862..4881)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4981..5003
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5041..5059)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5127..5231
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5232..5300,5301..6092)
                     /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     complement(5450..5469)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6263..6851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6752..6771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttgtaca tagtaatcaa ttacggggtc attagttcat
      181 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      241 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      301 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      361 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      421 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      481 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      541 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      601 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      661 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      721 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      781 gcttgccacc atggggcaac ccgggaacgg cagcgccttc ttgctggcac ccaatagaag
      841 ccatgcgccg gaccacgacg tcacgcagca gagggacgag gtgtgggtgg tgggcatggg
      901 catcgtcatg tctctcatcg tcctggccat cgtgtttggc aatgtgctgg tcatcacagc
      961 cattgccaag ttcgagcgtc tgcagacggt caccaactac ttcatcactt cactggcctg
     1021 tgctgatctg gtcatgggcc tggcagtggt gccctttggg gccgcccata ttcttatgaa
     1081 aatgtggact tttggcaact tctggtgcga gttttggact tccattgatg tgctgtgcgt
     1141 cacggccagc attgagaccc tgtgcgtgat cgcagtggat cgctactttg ccattacttc
     1201 acctttcaag taccagagcc tgctgaccaa gaataaggcc cgggtgatca ttctgatggt
     1261 gtggattgtg tcaggcctta cctccttctt gcccattcag atgcactggt accgggccac
     1321 ccaccaggaa gccatcaact gctatgccaa tgagacctgc tgtgacttct tcacgaacca
     1381 agcctatgcc attgcctctt ccatcgtgtc cttctacgtt cccctggtga tcatggtctt
     1441 cgtctactcc agggtctttc aggaggccaa aaggcagctc cagaagattg acaaatctga
     1501 gggccgcttc catgtccaga accttagcca ggtggagcag gatgggcgga cggggcatgg
     1561 actccgcaga tcttccaagt tctgcttgaa ggagcacaaa gccctcaaga cgttaggcat
     1621 catcatgggc actttcaccc tctgctggct gcccttcttc atcgttaaca ttgtgcatgt
     1681 gatccaggat aacctcatcc gtaaggaagt ttacatcctc ctaaattgga taggctatgt
     1741 caattctggt ttcaatcccc ttatctactg ccggagccca gatttcagga ttgccttcca
     1801 ggagcttctg tgcctgcgca ggtcttcttt gaaggcctat gggaatggct actccagcaa
     1861 cggcaacaca ggggagcaga gtggatatca cgtggaacag gagaaagaaa ataaactgct
     1921 gtgtgaagac ctcccaggca cggaagactt tgtgggccat caaggtactg tgcctagcga
     1981 taacattgat tcacaaggga ggaattgtag tacaaatgac tcactgctgg agctggccga
     2041 gaagctggcc ggcctggaca tcaacgtctt cacactcgaa gatttcgttg gggactggcg
     2101 acagacagcc ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt ccagtttgtt
     2161 tcagaatctc ggggtgtccg taactccgat ccaaaggatt gtcctgagcg gtgaaaatgg
     2221 gctgaagatc gacatccatg tcatcatccc gtatgaaggt ctgagcggcg accaaatggg
     2281 ccagatcgaa aaaattttta aggtggtgta ccctgtggat gatcatcact ttaaggtgat
     2341 cctgcactat ggcacactgg taatcgacgg ggttacgccg aacatgatcg actatttcgg
     2401 acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa cagggaccct
     2461 gtggaacggc aacaaaatta tcgacgagcg cctgatcaac cccgacggct ccctgctgtt
     2521 ccgagtaacc atcaacggag tgaccggctg gcggctgtgc gaacgcattc tggcgggtgg
     2581 agctagcggc agccgggcga ccaccctgga acgtattgaa aagagctttg tgattaccga
     2641 tccgcgtctg ccggataacc cgattatttt tgtgagcgat agctttctgc agctgaccga
     2701 atatagccgt gaagaaattc tgggccgtaa ctgccgtttt ctgcagggcc cggaaaccga
     2761 tcgtgcgacc gtgcgtaaaa ttcgtgatgc gattgataac cagaccgaag tgaccgtgca
     2821 gctgattaac tataccaaaa gcggcaaaaa attttggaac ctgtttcatc tgcagccgat
     2881 gcgtgatcag aaaggcgatg tgcagtattt tattggcgtg cagctggatg gtaccgaacg
     2941 tgtgcgtgat gcggcggaac gtgaagctgt gatgctggtc aagaaaaccg cggaggaaat
     3001 cgacgaagcc gctaaggaaa atctctactt ccagatgggc ggaggatccg actacaagga
     3061 tgacgacgat aagaagctac tgtcttctat cgaacaagca tgcgatattt gccgacttaa
     3121 aaagctcaag tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga
     3181 gtgtcgctac tctcccaaaa ccaaaaggtc tccgctgact agggcacatc tgacagaagt
     3241 ggaatcaagg ctagaaagac tggaacagct atttctactg atttttcctc gagaagacct
     3301 tgacatgatt ttgaaaatgg attctttaca ggatataaaa gcattgttaa caggattatt
     3361 tgtacaagat aatgtgaata aagatgccgt cacagataga ttggcttcag tggagactga
     3421 tatgcctcta acattgagac agcatagaat aagtgcgaca tcatcatcgg aagagagtag
     3481 taacaaaggt caaagacagt tgactgtatc ggccaatttt aatcaaagtg ggaatattgc
     3541 tgatagctca ttgtccttca ctttcactaa cagtagcaac ggtccgaacc tcataacaac
     3601 tcaaacaaat tctcaagcgc tttcacaacc aattgcctcc tctaacgttc atgataactt
     3661 catgaataat gaaatcacgg ctagtaaaat tgatgatggt aataattcaa aaccactgtc
     3721 acctggttgg acggaccaaa ctgcgtataa cgcgtttgga atcactacag ggatgtttaa
     3781 taccactaca atggatgatg tatataacta tctattcgat gatgaagata ccccaccaaa
     3841 cccaaaaaaa gagggcaagc cgatccctaa ccctctgctg ggcctggaca gcacttagta
     3901 agaatttcct cgagctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     3961 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     4021 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4081 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     4141 tggggaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     4201 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     4261 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     4321 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     4381 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4441 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     4501 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4561 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     4621 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4681 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     4741 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     4801 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     4861 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     4921 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4981 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5041 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5101 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5161 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5221 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5281 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5341 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5401 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5461 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5521 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5581 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5641 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5701 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5761 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5821 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5881 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5941 agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6001 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6061 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6121 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6181 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6241 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6301 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6361 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6421 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6481 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6541 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6601 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6661 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6721 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6781 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6841 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6901 ttgctcacat gt
//
