LOCUS       sequence-221595-        3693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single pathway reporter; wnt promoter-driven expression of 
            intracellular NanoLuc; MultiSite Gateway entry vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3693)
  AUTHORS   Maier J, Elmenofi S, Taschauer A, Anton M, Sami H, Ogris M
  TITLE     Luminescent and fluorescent triple reporter plasmid constructs for 
            Wnt, Hedgehog and Notch pathway.
  JOURNAL   PLoS One. 2019 Dec 20;14(12):e0226570. doi: 
            10.1371/journal.pone.0226570. eCollection 2019.
  PUBMED    31860685
REFERENCE   2  (bases 1 to 3693)
  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: "PLoS One.";
            date: "2019-12-20"; volume: "14(12):e0226570. doi"; pages: " 
            10.1371/journal.pone.0226570. eCollection 2019"
COMMENT     Sequence Label: pMuLE_ENTR_TOP-NLuc1.1_L5-L4
FEATURES             Location/Qualifiers
     source          1..3693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        55..86
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      142..151
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             148..663
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    712..833
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(723..742)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     777..796
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(1023..1118)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1149..1168)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1150..1168)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1173..1189)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1173..1189)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1291..1310)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1302..2111
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1369..1388)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2258..2846
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2747..2766
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3000..3017
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3176..3203)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3295..3381)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3430..3452
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3444..3461
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3445..3461
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3478..3573
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 ggtaagatca aaggggcgcg cccgcgtgct agcccgggct cgagatctag actctagagg
       61 gtatataatg gaagctcgaa ttccagcttg gcattccggt actgttggta aaaagcttgg
      121 caatccggta ctgttggtaa agccaccatg gtcttcacac tcgaagattt cgttggggac
      181 tggcgacaga cagccggcta caacctggac caagtccttg aacagggagg tgtgtccagt
      241 ttgtttcaga atctcggggt gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa
      301 aatgggctga agatcgacat ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa
      361 atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg tggatgatca tcactttaag
      421 gtgatcctgc actatggcac actggtaatc gacggggtta cgccgaacat gatcgactat
      481 ttcggacggc cgtatgaagg catcgccgtg ttcgacggca aaaagatcac tgtaacaggg
      541 accctgtgga acggcaacaa aattatcgac gagcgcctga tcaaccccga cggctccctg
      601 ctgttccgag taaccatcaa cggagtgacc ggctggcggc tgtgcgaacg cattctggcg
      661 taaggccgcg actctagagt cggggcggcc ggccgcttcg agcagacatg ataagataca
      721 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
      781 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
      841 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
      901 agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc actagtaacg gccgccagtg
      961 tgctggaatt ctgcagatat ccatcacact ggcggccgct cgagcatgca tctagaggca
     1021 cccaactttt ctatacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa
     1081 cgaacaggtc actatcagtc aaaataaaat cattatttgc catccagctg cagggcggcc
     1141 gcgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc
     1201 ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     1261 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     1321 aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     1381 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg
     1441 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     1501 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     1561 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg
     1621 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     1681 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     1741 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     1801 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     1861 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     1921 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     1981 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     2041 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     2101 agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt
     2161 gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg tcgttccact
     2221 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2281 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2341 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2401 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2461 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2521 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2581 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2641 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2701 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2761 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2821 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2881 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2941 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3001 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     3061 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     3121 agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa acgcaaaaag
     3181 gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc
     3241 tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg gcggatttgt
     3301 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtcttccga
     3361 ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta acgctagcat
     3421 ggatgttttc ccagtcacga cgttgtaaaa cgacggccag tcttaagctc gggccccaaa
     3481 taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat gagcaatgct
     3541 tttttataat gccaactttg tatacaaaag ttgggagacc caagcttggt accgagctct
     3601 tacgcgagat caaagggggt aagatcaaag ggggtaagat caaaggggcg cgagatcaaa
     3661 gggggtaaga tcaaaggggg taagatcaaa ggg
//
