LOCUS       sequence-326673-        6199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanoluciferase (NanoLuc) targeted to the extracellular surface of 
            the cell membrane for bioluminescence resonance energy transfer 
            (BRET)-based measurement of ligand binding to unmodified receptors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6199)
  AUTHORS   Toth AD, Garger D, Prokop S, Soltesz-Katona E, Varnai P, Balla A, 
            Turu G, Hunyady L
  TITLE     A general method for quantifying ligand binding to unmodified 
            receptors using Gaussia luciferase.
  JOURNAL   J Biol Chem. 2021 Feb 2:100366. doi: 10.1016/j.jbc.2021.100366.
  PUBMED    33545176
REFERENCE   2  (bases 1 to 6199)
  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: "J Biol 
            Chem. 2021 Feb 2:100366. doi: 10.1016/j.jbc.2021.100366."
COMMENT     Sequence Label: NanoLuc-PM
FEATURES             Location/Qualifiers
     source          1..6199
                     /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             783..1292
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     CDS             1362..1508
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     primer_bind     complement(1556..1573)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1562..1786
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1832..2260
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1919..1938)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2129..2150
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2269..2289)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2274..2603
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2454..2589
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2516..2535
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2670..3464
                     /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(2724..2743)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3334..3353
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3638..3759
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3675..3694)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3729..3748
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3808..3824)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3808..3824)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3821..3843)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3832..3848
                     /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(3856..3862,3863..3880,3881..3886))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3901..3922
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4039..4056)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4210..4798)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4290..4309)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4969..5760,5761..5829))
                     /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     5592..5611
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5830..5934)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6009..6028)
                     /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 gctagcgttt aaacttaagc ttaccggtcg ccaccatgaa ctccttctcc acaagcgcct
      721 tcggtccagt tgccttctcc ctgggcctgc tcctggtgtt gcctgctgcc ttccctgccc
      781 cagtcttcac actcgaagat ttcgttgggg actggcgaca gacagccggc tacaacctgg
      841 accaagtcct tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa
      901 ctccgatcca aaggattgtc ctgagcggtg aaaatgggct gaagatcgac atccatgtca
      961 tcatcccgta tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa atttttaagg
     1021 tggtgtaccc tgtggatgat catcacttta aggtgatcct gcactatggc acactggtaa
     1081 tcgacggggt tacgccgaac atgatcgact atttcggacg gccgtatgaa ggcatcgccg
     1141 tgttcgacgg caaaaagatc actgtaacag ggaccctgtg gaacggcaac aaaattatcg
     1201 acgagcgcct gatcaacccc gacggctccc tgctgttccg agtaaccatc aacggagtga
     1261 ccggctggcg gctgtgcgaa cgcattctgg cgggaggcgg atcaggtggc ggatccagat
     1321 ctggtggcgg ttctggtggc ggtagtggtg gcggtagtaa tgctgtgggc caggacacgc
     1381 aggaggtcat cgtggtgcca cactccttgc cctttaaggt ggtggtgatc tcagccatcc
     1441 tggccctggt ggtgctcacc atcatctccc ttatcatcct catcatgctt tggcagaaga
     1501 agccacgtta ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     1561 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1621 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1681 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1741 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     1801 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     1861 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1921 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1981 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2041 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2101 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2161 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2221 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     2281 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2341 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2401 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     2461 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     2521 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     2581 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     2641 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     2701 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     2761 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     2821 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     2881 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     2941 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3001 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3061 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3121 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3181 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3241 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3301 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3361 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3421 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     3481 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     3541 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     3601 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     3661 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3721 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     3781 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3841 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3901 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3961 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4021 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4081 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4141 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4201 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4261 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4321 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4381 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4441 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4501 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4561 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4621 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4681 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4741 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4801 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4861 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4921 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4981 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5041 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5101 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5161 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5221 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5281 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5341 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5401 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5461 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5521 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5581 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5641 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5701 acccactcgt gcacccaact gatcttcagc atcttttact ttaaccagcg tttctgggtg
     5761 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5821 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5881 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5941 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     6001 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     6061 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     6121 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     6181 cgggccagat atacgcgtt
//
