LOCUS       sequence-224737-        4544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gli protein-dependent EGFP reporter for analysis of Hedgehog 
            signaling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4544)
  AUTHORS   Hyman JM, Firestone AJ, Heine VM, Zhao Y, Ocasio CA, Han K, Sun M, 
            Rack PG, Sinha S, Wu JJ, Solow-Cordero DE, Jiang J, Rowitch DH, Chen
            JK
  TITLE     Small-molecule inhibitors reveal multiple strategies for Hedgehog 
            pathway blockade.
  JOURNAL   Proc Natl Acad Sci U S A. 2009 Aug 18;106(33):14132-7. doi: 
            10.1073/pnas.0907134106. Epub 2009 Aug 5.
  PUBMED    19666565
REFERENCE   2  (bases 1 to 4544)
  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"; doi: 
            "10.1073/pnas.0907134106"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2009-08-18- 18"; volume: "106"; issue: "33"; pages: "14132-7"
COMMENT     Sequence Label: pGL3b-8XGliBS:EGFP
FEATURES             Location/Qualifiers
     source          1..4544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    154..202
                     /note="synthetic polyadenylation signal"
     misc_feature    216..307
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     256..275
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    322..456
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      972..981
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(978..980,981..983,984..1697)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(978..980,981..983,984..1694)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1023..1044)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1284..1303)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1631..1652
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1821..1942
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2190..2207)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2361..2949)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2441..2460)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3120..3911,3912..3980))
                     /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     3743..3762
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3981..4085)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4112..4540
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4199..4218)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4409..4430
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 caaaatatta acgcttacaa tttgccattc gccattcagg ctgcgcaact gttgggaagg
       61 gcgatcggtg cgggcctctt cgctattacg ccagcccaag ctaccatgat aagtaagtaa
      121 tattaaggta cgggaggtac ttggagcggc cgcaataaaa tatctttatt ttcattacat
      181 ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca tcaaaacaaa
      241 acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg tgccagaaca
      301 tttctctatc gataggtacc gaacttgttt attgcagctt ataatggtta caaataaagc
      361 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
      421 tccaaactca tcaatgtatc ttatcatgtc tggatccccg ggaacagatt cgcgatcgac
      481 ctcgacaagc agggaacacc caagtagaag ttccgacaag cagggaacac ccaagtagaa
      541 gctctcgacc tcgacaagca gggaacaccc aagtagaagc tccgacaagc agggaacacc
      601 caagtagaag ctctcgacct cgacaagcag ggaacaccca agtagaagct ccgacaagca
      661 gggaacaccc aagtagaagc tctcgacctc gacaagcagg gaacacccaa gtagaagctc
      721 cgacaagcag ggaacaccca agtagaagct ctcgatcgcg aatctgttcc cggggatcct
      781 ctagagtcga cctgcagggc cagcccaggc tccggggcac gtaaaagcgg ggctgtgaga
      841 ccggagagca cggagcgacc agccagggct gagctgcgga gacggtgagc agggctgtgc
      901 gggcagccaa gctagcccgg gctcgagatc tgcgatctaa gtaagcttgg cattccggta
      961 ctgttggtaa agccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1021 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1081 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1141 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1201 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1261 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1321 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1381 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1441 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1501 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1561 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1621 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1681 acgagctgta caagtaaagc ggccgcgact ctagatcata atcagccata ccacatttgt
     1741 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     1801 gaatgcaatt gttgttgttt taagatacat tgatgagttt ggacaaacca caactagaat
     1861 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     1921 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     1981 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga
     2041 taaggatccg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     2101 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     2161 gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2221 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2281 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2341 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2401 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2461 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2521 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2581 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2641 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2701 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2761 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     2821 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2881 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2941 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3001 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3061 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3121 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3181 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3241 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3301 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3361 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3421 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3481 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3541 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3601 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3661 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3721 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3781 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3841 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3901 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3961 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4021 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4081 ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg
     4141 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4201 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4261 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4321 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4381 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4441 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     4501 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaa
//
