LOCUS       sequence-161970-        5714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2-based hedgehog signal reporter plasmid for zebrafish 
            transgenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5714)
  AUTHORS   Mich JK, Payumo AY, Rack PG, Chen JK
  TITLE     In vivo imaging of Hedgehog pathway activation with a nuclear 
            fluorescent reporter.
  JOURNAL   PLoS One. 2014 Jul 28;9(7):e103661. doi: 
            10.1371/journal.pone.0103661. eCollection 2014.
  PUBMED    25068273
REFERENCE   2  (bases 1 to 5714)
  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: "2014-07-28"; volume: "9(7):e103661. doi"; pages: " 
            10.1371/journal.pone.0103661. eCollection 2014"
COMMENT     Sequence Label: 8xGliBS-IVS2-mCherry-NLS-polyA-Tol2
FEATURES             Location/Qualifiers
     source          1..5714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        20..124
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(125..193,194..985)
                     /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     complement(343..362)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1156..1744
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1645..1664
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1898..1915
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2032..2053
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2068..2073,2074..2091,2092..2098)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2106..2122
                     /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)."
     primer_bind     2111..2133
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2130..2146
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2130..2146
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2165..2185
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2167..2185
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2222..2238
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2222..2238
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(2272..2283,2284..2471)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    2492..2626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     intron          3109..3681
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             3742..4449
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3887..3905)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4155..4174
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4456..4476
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4480..4500
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4504..4524
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4631..4765
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(4887..5024,5025..5036)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5066..5085)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5067..5085)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5095..5112)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5095..5111)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5104..5126)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5253..5708
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5340..5359)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5550..5571
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
       61 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      121 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
      181 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
      241 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
      301 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
      361 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
      421 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
      481 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
      541 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
      601 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
      661 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
      721 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
      781 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
      841 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
      901 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
      961 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     1021 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1081 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1141 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1201 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1261 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     1321 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1381 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1441 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1501 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1561 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1621 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1681 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1741 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1801 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     1861 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     1921 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     1981 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     2041 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     2101 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     2161 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg
     2221 ctctagaact agtggatctg ctgggcttgc tgaaggtagg gggtcaagaa ccagaggtgt
     2281 aaagtacttg agtaatttta cttgattact gtacttaagt attatttttg gggattttta
     2341 ctttacttga gtacaattaa aaatcaatac ttttactttt acttaattac atttttttag
     2401 aaaaaaaagt actttttact ccttacaatt ttatttacag tcaaaaagta cttatttttt
     2461 ggagatcact tgggcccggc tcgagcgagc taacttgttt attgcagctt ataatggtta
     2521 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2581 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccccg ggaacagatt
     2641 cgcgatcgac ctcgacaagc agggaacacc caagtagaag ttccgacaag cagggaacac
     2701 ccaagtagaa gctctcgacc tcgacaagca gggaacaccc aagtagaagc tccgacaagc
     2761 agggaacacc caagtagaag ctctcgacct cgacaagcag ggaacaccca agtagaagct
     2821 ccgacaagca gggaacaccc aagtagaagc tctcgacctc gacaagcagg gaacacccaa
     2881 gtagaagctc cgacaagcag ggaacaccca agtagaagct ctcgatcgcg aatctgttcc
     2941 cggggatcct ctagagtcga cctgcagggc cagcccaggc tccggggcac gtaaaagcgg
     3001 ggctgtgaga ccggagagca cggagcgacc agccagggct gagctgcgga gacggtgagc
     3061 agggctgtgc gggcagccaa gctagcgacc gatcctgaga acttcagggt gagtttgggg
     3121 acccttgatt gttctttctt tttcgctatt gtaaaattca tgttatatgg agggggcaaa
     3181 gttttcaggg tgttgtttag aatgggaaga tgtcccttgt atcaccatgg accctcatga
     3241 taattttgtt tctttcactt tctactctgt tgacaaccat tgtctcctct tattttcttt
     3301 tcattttctg taactttttc gttaaacttt agcttgcatt tgtaacgaat ttttaaattc
     3361 acttttgttt atttgtcaga ttgtaagtac tttctctaat cacttttttt tcaaggcaat
     3421 cagggtatat tatattgtac ttcagcacag ttttagagaa caattgttat aattaaatga
     3481 taaggtagaa tatttctgca tataaattct ggctggcgtg gaaatattct tattggtaga
     3541 aacaactaca ccctggtcat catcctgcct ttctctttat ggttacaatg atatacactg
     3601 tttgagatga ggataaaata ctctgagtcc aaaccgggcc cctctgctaa ccatgttcat
     3661 gccttcttct ctttcctaca gctcctgggc aacgtgctgg ttgttgtgct gtctcatcat
     3721 tttggcaaag agatctccac catggtgagc aagggcgagg aggataacat ggccatcatc
     3781 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     3841 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     3901 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     3961 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     4021 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     4081 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     4141 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
     4201 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
     4261 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
     4321 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
     4381 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     4441 ctgtacaagt ccgatccaaa aaagaagaga aaggtagatc caaaaaagaa gagaaaggta
     4501 gatccaaaaa agaagagaaa ggtagggtac aagaagtgat aggaattcct gcagaagctt
     4561 gatatcgatg gatcctgtac aagtaaagcg gccgccaccg cggtggagct cgaattaatt
     4621 catcgatgat gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag
     4681 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     4741 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     4801 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc
     4861 tgattatgat cctctagatc agatctaata ctcaagtaca attttaatgg agtacttttt
     4921 tacttttact caagtaagat tctagccaga tacttttact tttaattgag taaaattttc
     4981 cctaagtact tgtactttca cttgagtaaa atttttgagt actttttaca cctctgtcaa
     5041 gaaccatatg ccggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     5101 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     5161 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     5221 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     5281 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     5341 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5401 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5461 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5521 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5581 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     5641 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     5701 tacaatttag gtgg
//
