LOCUS       sequence-401150-        8170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2 based genomic integration of zebrafish 4kb Wnt3 promoter 
            driving the expression of EGFP for zebrafish transgenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8170)
  AUTHORS   Teh C, Sun G, Shen H, Korzh V, Wohland T
  TITLE     Modulating the expression level of secreted Wnt3 influences 
            cerebellum development in zebrafish transgenics.
  JOURNAL   Development. 2015 Nov 1;142(21):3721-33. doi: 10.1242/dev.127589. 
            Epub 2015 Sep 22.
  PUBMED    26395493
REFERENCE   2  (bases 1 to 8170)
  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: 
            "10.1242/dev.127589"; journalName: "Development"; date: "2015-11-1- 
            1"; volume: "142"; issue: "21"; pages: "3721-33"
COMMENT     Sequence Label: pminiTol2-4kb Wnt3 promoter-EGFP
FEATURES             Location/Qualifiers
     source          1..8170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..42
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     24..41
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    join(93..104,105..267)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      4306..4315
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4312..4314,4315..4317,4318..5031)
                     /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(4312..4314,4315..4317,4318..5028)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4357..4378)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4618..4637)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4965..4986
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(5123..5310,5311..5322)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5334..5353)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5335..5353)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5360..5377)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5360..5376)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5369..5391)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5517..5972
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5604..5623)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5814..5835
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6050..6154
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6155..6223,6224..7015)
                     /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(6373..6392)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7186..7774
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7675..7694
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7928..7945
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8062..8083
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8098..8103,8104..8121,8122..8128)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8136..8152
                     /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     8141..8163
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(8160..8170,1..6)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(8160..8170,1..6)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tatgaccatg attacgccaa gctatttagg tgacactata gaatactcaa gctatgcatc
       61 caacgcgttg ggagctctcc catatggtcg agcagaggtg taaaaagtac tcaaaaattt
      121 tactcaagtg aaagtacaag tacttaggga aaattttact caattaaaag taaaagtatc
      181 tggctagaat cttacttgag taaaagtaaa aaagtactcc attaaaattg tacttgagta
      241 ttaaggaagt aaaagtaaaa gcaagaaaga aaactagaga ttcttgttta agcttcaata
      301 aaagaagtgt ctgttgtggg gccgagggtc ctggggggct gcggagcttt aggcattgtc
      361 ctggagggtg attgagagcg agaggagggg agccgagaag cattgtgcga ggacggagcc
      421 tttctaaagg tggttattag gagagaaatg gtgtgggggt ggctgttgac tcttcatctt
      481 gtgtgaacaa agtatctggc gtatagcttt ctctctctct ctctcgctct cttgcggctt
      541 tcgctttacg acggccatga atactctcgg cgggctcgcc cagacctgcg cgaaggctta
      601 ttgtccggct taagtgcggt ttaattaagc gagctctgtg tcctgagaga agagaggagc
      661 tttagagcgc tcgggtcctg cggagatgca gaggatgatg cgtaatatca gggctattga
      721 aggggaaagt gtttgcggct ctaatgaaaa aggtttgttt gggggggatg agtggggagg
      781 ggattgagga gtgtctggcc tttgtctcca gcttacaaag tagggacaaa agcagtgata
      841 ttatacaaat gcacccattt ccaggacatg atgcgaggac gggggcctgg agatgggtcg
      901 gactgctcca tctcagcccc gttggcacct tacaaaatga ttttttcccc ccaccacaaa
      961 gataggcatt ggggataaac cccgagagct ggggcttctg ggtaccgcaa atttgggggt
     1021 tgtggactta agcggatttt ctctgcaact gtaagaacaa gctaattcct cagacctcct
     1081 ttatctctaa catggtaata acaaacaatg aaattctgaa gagaaagggg caaaggaaga
     1141 ggagggagga gagacgcaga cacaaaagaa ggagagatgg agggaatgaa agagaggaga
     1201 gacgtgtgtg aggagagggt gggcttgggc gtgttaagaa actttctcgc tctagtgtta
     1261 tgtgtgaaca ctctgctaat ccaggtttta ttgcttgcct gtgtccggcc acactgtccg
     1321 gattgagtgt gtgtcaggga tagtgggggt ctacacatgt ccctctttga ggaaagcctg
     1381 agaaagagcc ggacctggtt acggccgttt tggggtcgcc taagaaacct gcaccacagg
     1441 ggtcctctgt gtatgtctct gaattcaacg tgcatgttgc tagcaaacac atacggatta
     1501 atgctaatga tgaaggtgtg gctatattag gtacaaaaaa aaactttgac aatagttgta
     1561 taaactcctg tcaacagtcc atcaatgtat gaagcagaac tcgctttgaa accaaacatt
     1621 gttgtgtggg tccatttgga ccagtttaag gaaatctttt gccctataaa ttccagcctg
     1681 catgaatttt tatgattatt gctcataaac atacttttaa aaatgtgcat tttaactaac
     1741 tatcaacttg tgcccgtcca ctcaaaggaa gccaaaaggg tgtctctccc agatcacagg
     1801 gactaccttg tgggtgtgta gggtgatgta ttagctaggc gctaataaaa tgcaggagtg
     1861 tccacaaaca tgacttaatg agggaggatg agagactcct gacccagagg tcaaagctcc
     1921 gggcgactct ctttattccc attgatctgc tggtgcactt catttccatg ctctagggcc
     1981 cacttcagta aattcattaa gtgcgattga tacgagaatc aagacttcct ccaaaacaca
     2041 atggctatgc taggagggaa tggtcttgtt tgtgctgcag ctatgtcaag catctctggc
     2101 ttttgtcctg gtttcgcacc attgcggtgc atgctggggt ttgagtcctc gagattctaa
     2161 tcgtaagctc atttccagct ggttcgttgt gttgcgcccc tatggccttg acctgggaaa
     2221 cgtggtttca gtgttgttga gatgccgacc gcagaccgtt ccttgcctcc actcgtaaat
     2281 tacgctccga tcaggttttc ccagccaatc ctggcgagca gatctgcatc tgtcactctg
     2341 caagaatgct gaatcaaacc gctgatctag gcacaataca gctgatggaa agcagatctt
     2401 tattccaaca tcaggaacgt agcacgagcc gagtgcttct ggaagctgca atttaaccaa
     2461 caggttcata tctgatgaga tcaaggatgc atcatcctgg aagaaacact gtgatgaaca
     2521 tttaagatgt cagaatgacc cgtcatgcct ctgagctgag tccccgcaca gcgcttattc
     2581 ttttgaagag ggagaataaa agcaaaaaaa cagaggcgat ggaaatatga catccagacc
     2641 gcggtcagaa ctgtgagtgt ctctcctggg ctcctatggt tgtggtgcac agtccatgtc
     2701 cctgctatga gttagataga cagatgcact ggcagataga cagacagaca ggctttacta
     2761 cttggtcgct ggattgcagg aaagagcttt atccaaccag agtcaagcaa gcaacatggg
     2821 acaataacat aattaaatat tctggggaaa agtggcactt atgcaaatgt tttttgtaat
     2881 gggttggaga aaattggccg gaacctacat aatttttaat gacaggatga ttcatttgtg
     2941 aaattgtaat tcaaagaaat tcagtaaact aactaacaat ctaaaaaatt ctttgatttg
     3001 cagtttaatt ctacttccta acattgattt gtttcaactt tattcaaatg gcaaaactaa
     3061 attagtactc aaagctccaa atgatatgat tcctagactg tttaaagtgc taaaagtgcc
     3121 aaaacttaca gctacccaca tgaacttata tttcattctg cctccatttg caattcctcc
     3181 agtgtttgta cctgaaactg ttgtcagttc tcacctctgt cctgccgttc attctgacac
     3241 agagactcta tatttgaatg tattgtcaac ttctaatgcc gtgtctacta ctctgtgctg
     3301 tctgctctca tcccatccga atgacaatag tgtttatgta aaatgtgctg cctttgggcc
     3361 gctggggcac agaacactgc atgcctggcc ctgccacacc accttttacg gctttttatt
     3421 gtgttgcctt tgtgtcacaa ggggaggacg cggccatcaa aattctccaa gacaaaattc
     3481 ttgaatgtat gtgcacgcac acgcatccgg aacaattagt tcaggagcac gggtgcttaa
     3541 aaaacggaga cgacacttct cgccaccacc tcctcgccga gcagcaggcc cgagtctggc
     3601 acagctgggt ttccttgatg ctaacgcaga caatatagat cttaataaga caaatctacc
     3661 tctccgaaac tcagtcctga caccctttca ttcttattcc ctcccagcct cctctctctc
     3721 tctctctctc tttctttctt tctttgctac attaccaggg ggtggtgcat gattgacaga
     3781 tgggcaggaa taataatttt tttttttttt ttgcgtaacc taaggccatg ctctgtgatt
     3841 ggcttgccag ctgacatctc caaacctctc ttgtcgtttt tttcccctct ctctctctcg
     3901 ctttctctct ctcactctct ctctctaata ttggccctga tgatcctaag gggaaaaaag
     3961 tggggagaga aaaagatatg taccttaggg aagaacctca gtaagaggga gggaatcaga
     4021 ggacgagtag gagcgtgtgt gcgagtgtgt gaaagagaca cttactctcc taactactac
     4081 tggaagacac acacttttct cacccaactc aaagcagcag caaaccttgg agaattgttt
     4141 tatttttgtt ttttcgcgaa cccccctaaa aaaattccta attattctct caagcatcca
     4201 tctttgcctt gccgggattt aattttcacc ccctctctag gaatcctatg tactgatcaa
     4261 tagacccttt ccatccacac aagacatacc tcggatccac cggtcgccac catggtgagc
     4321 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     4381 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     4441 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     4501 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     4561 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     4621 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     4681 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     4741 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     4801 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     4861 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     4921 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     4981 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc
     5041 tctagatggc cagatctatt taaattaaac tgggcatcag cgcaattcaa ttggtttggt
     5101 aatagcaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt tgactgtaaa
     5161 taaaattgta aggagtaaaa agtacttttt tttctaaaaa aatgtaatta agtaaaagta
     5221 aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat aatacttaag
     5281 tacagtaatc aagtaaaatt actcaagtac tttacacctc tgggcccaat tcgccctata
     5341 gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     5401 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     5461 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggacgc
     5521 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5581 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5641 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5701 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     5761 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5821 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     5881 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5941 gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat
     6001 ctgtgcggta tttcacaccg catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6061 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     6121 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     6181 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     6241 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     6301 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     6361 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     6421 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     6481 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6541 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6601 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6661 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6721 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6781 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6841 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6901 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6961 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7021 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     7081 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     7141 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     7201 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     7261 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     7321 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     7381 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     7441 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     7501 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     7561 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     7621 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7681 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7741 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7801 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     7861 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     7921 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     7981 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     8041 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     8101 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     8161 aggaaacagc
//
