LOCUS       sequence-161964-        5838 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 5838)
  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 5838)
  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-Odc1-polyA-Tol2
FEATURES             Location/Qualifiers
     source          1..5838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(34..462)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     356..375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     589..611
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     603..620
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     604..620
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(679..690,691..828)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    950..1084
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1314..1334)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1338..1358)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1362..1382)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1389..2096)
                     /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(1664..1683)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1933..1951
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     intron          2157..2730
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_signal    3213..3347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(3368..3555,3556..3567)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3601..3617)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3601..3617)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3654..3674)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3654..3672)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3693..3709)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3693..3709)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3706..3728)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3717..3733
                     /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)."
     promoter        complement(join(3741..3747,3748..3765,3766..3771))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3786..3807
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3924..3941)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4095..4683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4175..4194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4854..5645,5646..5714))
                     /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     5477..5496
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5715..5819)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
      421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 ccggcatatg gttcttgaca gaggtgtaaa aagtactcaa aaattttact caagtgaaag
      721 tacaagtact tagggaaaat tttactcaat taaaagtaaa agtatctggc tagaatctta
      781 cttgagtaaa agtaaaaaag tactccatta aaattgtact tgagtattag atctgatcta
      841 gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
      901 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
      961 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     1021 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     1081 gatcatcatc gatgaattaa ttcgagctcc accgcggtgg cggccgcttt acttgtacag
     1141 gatccatcga tatcaagctt ctgcaggaat tcctatcaca cattgatcct agcagaagca
     1201 caggctgcag ggtgacggtc catcccgctc tcctgggcac aagacatggg cagcgtgcca
     1261 tcatcctgcg ccgccaccgc cggcgggaag ccatggctaa gcttcttgta ccctaccttt
     1321 ctcttctttt ttggatctac ctttctcttc ttttttggat ctacctttct cttctttttt
     1381 ggatcggact tgtacagctc gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg
     1441 tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg
     1501 ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg
     1561 tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg
     1621 tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt ctgcattacg
     1681 gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc
     1741 tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc
     1801 tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc
     1861 ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc
     1921 agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc
     1981 tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg
     2041 aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct caccatggtg
     2101 gagatctctt tgccaaaatg atgagacagc acaacaacca gcacgttgcc caggagctgt
     2161 aggaaagaga agaaggcatg aacatggtta gcagaggggc ccggtttgga ctcagagtat
     2221 tttatcctca tctcaaacag tgtatatcat tgtaaccata aagagaaagg caggatgatg
     2281 accagggtgt agttgtttct accaataaga atatttccac gccagccaga atttatatgc
     2341 agaaatattc taccttatca tttaattata acaattgttc tctaaaactg tgctgaagta
     2401 caatataata taccctgatt gccttgaaaa aaaagtgatt agagaaagta cttacaatct
     2461 gacaaataaa caaaagtgaa tttaaaaatt cgttacaaat gcaagctaaa gtttaacgaa
     2521 aaagttacag aaaatgaaaa gaaaataaga ggagacaatg gttgtcaacc agagtagaaa
     2581 gtgaaagaaa caaaattatc atgagggtcc atggtgatac aagggacatc ttcccattct
     2641 aaacaacacc ctgaaaactt tgccccctcc atataacatg aattttacaa tagcgaaaaa
     2701 gaaagaacaa tcaagggtcc ccaaactcac cctgaagttc tcaggatcgg tcgctagctt
     2761 ggctgcccgc acagccctgc tcaccgtctc cgcagctcag ccctggctgg tcgctccgtg
     2821 ctctccggtc tcacagcccc gcttttacgt gccccggagc ctgggctggc cctgcaggtc
     2881 gactctagag gatccccggg aacagattcg cgatcgagag cttctacttg ggtgttccct
     2941 gcttgtcgga gcttctactt gggtgttccc tgcttgtcga ggtcgagagc ttctacttgg
     3001 gtgttccctg cttgtcggag cttctacttg ggtgttccct gcttgtcgag gtcgagagct
     3061 tctacttggg tgttccctgc ttgtcggagc ttctacttgg gtgttccctg cttgtcgagg
     3121 tcgagagctt ctacttgggt gttccctgct tgtcggaact tctacttggg tgttccctgc
     3181 ttgtcgaggt cgatcgcgaa tctgttcccg gggatccaga catgataaga tacattgatg
     3241 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     3301 atgctattgc tttatttgta accattataa gctgcaataa acaagttagc tcgctcgagc
     3361 cgggcccaag tgatctccaa aaaataagta ctttttgact gtaaataaaa ttgtaaggag
     3421 taaaaagtac ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt attgattttt
     3481 aattgtactc aagtaaagta aaaatcccca aaaataatac ttaagtacag taatcaagta
     3541 aaattactca agtactttac acctctggtt cttgaccccc taccttcagc aagcccagca
     3601 gatccactag ttctagagcg gccgccaccg cggtggagct ccagcttttg ttccctttag
     3661 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3721 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3781 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3841 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3901 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3961 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4021 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4081 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4141 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4201 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4261 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4321 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4381 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4441 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4501 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     4561 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4621 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4681 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4741 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4801 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4861 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4921 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4981 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5041 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5101 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5161 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5221 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5281 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5341 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5401 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5461 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5521 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5581 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5641 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5701 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5761 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5821 acatttcccc gaaaagtg
//
