LOCUS       sequence-326679-        5549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanoluciferase (NanoLuc) targeted to the extracellular surface of 
            the cell membrane and intracellularly labeled with monomeric Venus 
            fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5549)
  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 5549)
  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-Venus
FEATURES             Location/Qualifiers
     source          1..5549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             348..857
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     CDS             927..1073
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     regulatory      1125..1134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1131..1133,1134..1136,1137..1850)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1176..1197)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1437..1456)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1784..1805
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1973..2094
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2010..2029)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2064..2083
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2101..2556)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2238..2259)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2450..2469
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2583..2687
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2689..3046
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2689..2880
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2712..2732)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2897..3032
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3081..3875
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3081..3875
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3135..3154)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3745..3764
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4063..4082)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4107..4154
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4483..5071
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4972..4991
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5246..5549
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 actcagatct cgaggccacc atgaactcct tctccacaag cgccttcggt ccagttgcct
      301 tctccctggg cctgctcctg gtgttgcctg ctgccttccc tgccccagtc ttcacactcg
      361 aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac
      421 agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga
      481 ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag
      541 gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg
      601 atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc
      661 cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa
      721 agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca
      781 accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt
      841 gcgaacgcat tctggcggga ggcggatcag gtggcggatc cagatctggt ggcggttctg
      901 gtggcggtag tggtggcggt agtaatgctg tgggccagga cacgcaggag gtcatcgtgg
      961 tgccacactc cttgcccttt aaggtggtgg tgatctcagc catcctggcc ctggtggtgc
     1021 tcaccatcat ctcccttatc atcctcatca tgctttggca gaagaagcca cgtaagcttc
     1081 gaattctgca gtcgacggta ccgcgggccc gggatccacc ggtcgccacc atggtgagca
     1141 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1201 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1261 ccctgaagct gatctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1321 ccctgggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact
     1381 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     1441 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1501 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1561 acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg
     1621 ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc gaccactacc
     1681 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct
     1741 accagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1801 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg
     1861 actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     1921 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     1981 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2041 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt
     2101 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     2161 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     2221 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     2281 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     2341 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     2401 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     2461 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     2521 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg
     2581 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2641 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa
     2701 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     2761 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     2821 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     2881 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     2941 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     3001 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga
     3061 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3121 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3181 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3241 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
     3301 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3361 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3421 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3481 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     3541 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     3601 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     3661 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     3721 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     3781 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     3841 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt ttgaaatgac
     3901 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     3961 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4021 tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac
     4081 aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt
     4141 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
     4201 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
     4261 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
     4321 ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4381 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4441 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4501 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4561 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4621 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4681 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4741 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4801 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4861 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4921 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4981 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5041 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5101 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5161 ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag
     5221 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     5281 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     5341 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     5401 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     5461 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     5521 tctacgtatt agtcatcgct attaccatg
//
