LOCUS Exported 6571 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Single pathway reporter; hedgehog promoter-driven expression of firefly luciferase; MultiSite Gateway entry vector.. ACCESSION . VERSION . KEYWORDS pMuLE_ENTR_12GLI-FLuc_R4-R3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6571) 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 6571) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6571 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 296..358 /label=Mini-TK promoter /note="minimal herpes simplex virus (HSV) thymidine kinase promoter" CDS 414..2066 /codon_start=1 /gene="luc" /product="firefly luciferase" /label=luciferase /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL" primer_bind complement(480..498) /label=LucNrev /note="Firefly luciferase, reverse primer" intron 2197..2262 /label=small t intron /note="SV40 (simian virus 40) small t antigen intron" CDS 2392..2412 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 2837..2971 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" protein_bind 3523..3646 /gene="mutant version of attR" /label=attR3 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(3701..3720) /label=T7 /note="T7 promoter, forward primer" promoter complement(3702..3720) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3725..3741) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3725..3741) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3843..3862) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 3854..4663 /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(3921..3940) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 4810..5398 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5299..5318 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5552..5569 /label=L4440 /note="L4440 vector, forward primer" terminator 5728..5755 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 5847..5933 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 5982..6004 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 5996..6013 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 5997..6013 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind complement(6063..6187) /gene="mutant version of attR" /label=attR4 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind 6339..6361 /label=pGEX 3' /note="pGEX vectors, reverse primer" polyA_signal 6492..55 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" ORIGIN 1 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccaagc 61 tcagatccaa gcttatcgat accgtcgagt gggtggtctg ggtggtctgg gtggtctggg 121 tggtcgagtg ggtggtctgg gtggtctggg tggtctgggt ggtcgagtgg gtggtctggg 181 tggtctgggt ggtctgggtg gtcgacctcg agatccggca aaccccgccc agcgtcttgt 241 cattggcgaa ttcgaacacg cagatgcagt cggggcggcg cggtccgagg tccacttcgc 301 atattaaggt gacgcgtgtg gcctcgaaca ccgagcgacc ctgcagcgac ccgcttaaca 361 gcgtcaacag cgtgccgcag atctgagctt ggcattccgg tactgttggt aaaatggaag 421 acgccaaaaa cataaagaaa ggcccggcgc cattctatcc tctagaggat ggaaccgctg 481 gagagcaact gcataaggct atgaagagat acgccctggt tcctggaaca attgctttta 541 cagatgcaca tatcgaggtg aacatcacgt acgcggaata cttcgaaatg tccgttcggt 601 tggcagaagc tatgaaacga tatgggctga atacaaatca cagaatcgtc gtatgcagtg 661 aaaactctct tcaattcttt atgccggtgt tgggcgcgtt atttatcgga gttgcagttg 721 cgcccgcgaa cgacatttat aatgaacgtg aattgctcaa cagtatgaac atttcgcagc 781 ctaccgtagt gtttgtttcc aaaaaggggt tgcaaaaaat tttgaacgtg caaaaaaaat 841 taccaataat ccagaaaatt attatcatgg attctaaaac ggattaccag ggatttcagt 901 cgatgtacac gttcgtcaca tctcatctac ctcccggttt taatgaatac gattttgtac 961 cagagtcctt tgatcgtgac aaaacaattg cactgataat gaattcctct ggatctactg 1021 ggttacctaa gggtgtggcc cttccgcata gaactgcctg cgtcagattc tcgcatgcca 1081 gagatcctat ttttggcaat caaatcattc cggatactgc gattttaagt gttgttccat 1141 tccatcacgg ttttggaatg tttactacac tcggatattt gatatgtgga tttcgagtcg 1201 tcttaatgta tagatttgaa gaagagctgt ttttacgatc ccttcaggat tacaaaattc 1261 aaagtgcgtt gctagtacca accctatttt cattcttcgc caaaagcact ctgattgaca 1321 aatacgattt atctaattta cacgaaattg cttctggggg cgcacctctt tcgaaagaag 1381 tcggggaagc ggttgcaaaa cgcttccatc ttccagggat acgacaagga tatgggctca 1441 ctgagactac atcagctatt ctgattacac ccgaggggga tgataaaccg ggcgcggtcg 1501 gtaaagttgt tccatttttt gaagcgaagg ttgtggatct ggataccggg aaaacgctgg 1561 gcgttaatca gagaggcgaa ttatgtgtca gaggacctat gattatgtcc ggttatgtaa 1621 acaatccgga agcgaccaac gccttgattg acaaggatgg atggctacat tctggagaca 1681 tagcttactg ggacgaagac gaacacttct tcatagttga ccgcttgaag tctttaatta 1741 aatacaaagg atatcaggtg gcccccgctg aattggaatc gatattgtta caacacccca 1801 acatcttcga cgcgggcgtg gcaggtcttc ccgacgatga cgccggtgaa cttcccgccg 1861 ccgttgttgt tttggagcac ggaaagacga tgacggaaaa agagatcgtg gattacgtcg 1921 ccagtcaagt aacaaccgcg aaaaagttgc gcggaggagt tgtgtttgtg gacgaagtac 1981 cgaaaggtct taccggaaaa ctcgacgcaa gaaaaatcag agagatcctc ataaaggcca 2041 agaagggcgg aaagtccaaa ttgtaaaatg taactgtatt cagcgatgac gaaattctta 2101 gctattgtaa tactctagag gatctttgtg aaggaacctt acttctgtgg tgtgacataa 2161 ttggacaaac tacctacaga gatttaaagc tctaaggtaa atataaaatt tttaagtgta 2221 taatgtgtta aactactgat tctaattgtt tgtgtatttt agattccaac ctatggaact 2281 gatgaatggg agcagtggtg gaatgccttt aatgaggaaa acctgttttg ctcagaagaa 2341 atgccatcta gtgatgatga ggctactgct gactctcaac attctactcc tccaaaaaag 2401 aagagaaagg tagaagaccc caaggacttt ccttcagaat tgctaagttt tttgagtcat 2461 gctgtgttta gtaatagaac tcttgcttgc tttgctattt acaccacaaa ggaaaaagct 2521 gcactgctat acaagaaaat tatggaaaaa tattctgtaa cctttataag taggcataac 2581 agttataatc ataacatact gttttttctt actccacaca ggcatagagt gtctgctatt 2641 aataactatg ctcaaaaatt gtgtaccttt agctttttaa tttgtaaagg ggttaataag 2701 gaatatttga tgtatagtgc cttgactaga gatcataatc agccatacca catttgtaga 2761 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa 2821 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag 2881 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 2941 actcatcaat gtatcttatc atgtctggat cgatccccag gaagctcctc tgtgtcctca 3001 taaaccctaa cctcctctac ttgagaggac attccaatca taggctgccc atccaccctc 3061 tgtgtcctcc tgttaattag gtcacttaac aaaaaggaaa ttgggtaggg gtttttcaca 3121 gaccgctttc taagggtaat tttaaaatat ctgggaagtc ccttccactg ctgtgttcca 3181 gaagtgttgg taaacagccc acaaatgtca acagcagaaa catacaagct gtcagctttg 3241 cacaagggcc caacaccctg ctcatcaaga agcactgtgg ttgctgtgtt agtaatgtgc 3301 aaaacaggag gcacattttc cccacctgtg taggttccaa aatatctagt gttttcattt 3361 ttacttggat caggaaccca gcactccact ggataagcat tatccttatc caaaacagcc 3421 ttgtggtcag tgttcatctg ctgactagta acggccgcca gtgtgctgga attctgcaga 3481 tatccatcac actggcggcc gctcgagcat gcatctagag gacaactttg tataataaag 3541 ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa 3601 aacagactac ataatactgt aaaacacaac atatccagtc actatgaatc aactacttag 3661 atggtattag tgacctgtac tgcagggcgg ccgcgatatc ccctatagtg agtcgtatta 3721 catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct gatgttacat 3781 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa 3841 tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa 3901 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc 3961 gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa 4021 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt 4081 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac 4141 cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga 4201 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa 4261 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa 4321 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt 4381 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga 4441 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg 4501 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga 4561 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat 4621 gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg 4681 gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa 4741 atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa 4801 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 4861 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 4921 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg 4981 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 5041 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 5101 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 5161 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 5221 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 5281 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 5341 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 5401 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 5461 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 5521 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 5581 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 5641 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 5701 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 5761 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 5821 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 5881 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 5941 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 6001 aacgacggcc agtcttaagc tcgggcccct acaggtcact aataccatct aagtagttga 6061 ttcatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa 6121 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcaact tttctataca 6181 aagttgggag acccaagctt ggtaccgagc tctatacact ccgctatcgc tacgtgactg 6241 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 6301 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 6361 gttttcaccg tcatcaccga aacgcgcgag gcaggatcag ccatatcaca tttgtagagg 6421 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg 6481 caattgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 6541 caaatttcac aaataaagca tttttttcac t //