LOCUS       sequence-401179-        8187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2 based genomic integration of zebrafish 4kb Wnt3 promoter 
            driving the expression of PMTmApple for zebrafish transgenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8187)
  AUTHORS   Veerapathiran S, Teh C, Zhu S, Kartigayen I, Korzh V, Matsudaira PT,
            Wohland T
  TITLE     Wnt3 distribution in the zebrafish brain is determined by 
            expression, diffusion and multiple molecular interactions.
  JOURNAL   Elife. 2020 Nov 25;9:e59489. doi: 10.7554/eLife.59489.
  PUBMED    33236989
REFERENCE   2  (bases 1 to 8187)
  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: "Elife."; 
            date: "2020-11-25"; volume: "9:e59489. doi"; pages: " 
            10.7554/eLife.59489"
COMMENT     Sequence Label: pminiTol2-4kb wnt3 promoter-PMTmApple
FEATURES             Location/Qualifiers
     source          1..8187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..41)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(24..40)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(33..55)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      181..636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     478..499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        714..818
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(819..887,888..1679)
                     /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(1037..1056)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1850..2438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2339..2358
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2592..2609
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2726..2747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2762..2767,2768..2785,2786..2792)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2800..2816
                     /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     2805..2827
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2824..2840
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2824..2840
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2858..2876
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2858..2875
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    join(2927..2938,2939..3101)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     CDS             7179..7889
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNF
                     EDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALK
                     SEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     7592..7611
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    join(7974..8161,8162..8173)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(join(8185..8187,1..17))
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(join(8186..8187,1..17))
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
       61 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
      121 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
      181 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      241 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      301 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      361 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      421 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      481 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      541 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      601 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
      661 gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa
      721 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      781 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
      841 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
      901 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
      961 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1021 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1081 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1141 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1201 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1261 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1321 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     1381 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1441 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1501 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     1561 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1621 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     1681 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1741 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1801 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1861 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1921 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1981 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2041 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2101 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2161 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2221 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2281 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2341 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2401 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2461 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2521 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2581 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     2641 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2701 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     2761 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     2821 acacaggaaa cagctatgac catgattacg ccaagctatt taggtgacac tatagaatac
     2881 tcaagctatg catccaacgc gttgggagct ctcccatatg gtcgagcaga ggtgtaaaaa
     2941 gtactcaaaa attttactca agtgaaagta caagtactta gggaaaattt tactcaatta
     3001 aaagtaaaag tatctggcta gaatcttact tgagtaaaag taaaaaagta ctccattaaa
     3061 attgtacttg agtattaagg aagtaaaagt aaaagcaaga aagaaaacta gagattcttg
     3121 tttaagcttc aataaaagaa gtgtctgttg tggggccgag ggtcctgggg ggctgcggag
     3181 ctttaggcat tgtcctggag ggtgattgag agcgagagga ggggagccga gaagcattgt
     3241 gcgaggacgg agcctttcta aaggtggtta ttaggagaga aatggtgtgg gggtggctgt
     3301 tgactcttca tcttgtgtga acaaagtatc tggcgtatag ctttctctct ctctctctcg
     3361 ctctcttgcg gctttcgctt tacgacggcc atgaatactc tcggcgggct cgcccagacc
     3421 tgcgcgaagg cttattgtcc ggcttaagtg cggtttaatt aagcgagctc tgtgtcctga
     3481 gagaagagag gagctttaga gcgctcgggt cctgcggaga tgcagaggat gatgcgtaat
     3541 atcagggcta ttgaagggga aagtgtttgc ggctctaatg aaaaaggttt gtttgggggg
     3601 gatgagtggg gaggggattg aggagtgtct ggcctttgtc tccagcttac aaagtaggga
     3661 caaaagcagt gatattatac aaatgcaccc atttccagga catgatgcga ggacgggggc
     3721 ctggagatgg gtcggactgc tccatctcag ccccgttggc accttacaaa atgatttttt
     3781 ccccccacca caaagatagg cattggggat aaaccccgag agctggggct tctgggtacc
     3841 gcaaatttgg gggttgtgga cttaagcgga ttttctctgc aactgtaaga acaagctaat
     3901 tcctcagacc tcctttatct ctaacatggt aataacaaac aatgaaattc tgaagagaaa
     3961 ggggcaaagg aagaggaggg aggagagacg cagacacaaa agaaggagag atggagggaa
     4021 tgaaagagag gagagacgtg tgtgaggaga gggtgggctt gggcgtgtta agaaactttc
     4081 tcgctctagt gttatgtgtg aacactctgc taatccaggt tttattgctt gcctgtgtcc
     4141 ggccacactg tccggattga gtgtgtgtca gggatagtgg gggtctacac atgtccctct
     4201 ttgaggaaag cctgagaaag agccggacct ggttacggcc gttttggggt cgcctaagaa
     4261 acctgcacca caggggtcct ctgtgtatgt ctctgaattc aacgtgcatg ttgctagcaa
     4321 acacatacgg attaatgcta atgatgaagg tgtggctata ttaggtacaa aaaaaaactt
     4381 tgacaatagt tgtataaact cctgtcaaca gtccatcaat gtatgaagca gaactcgctt
     4441 tgaaaccaaa cattgttgtg tgggtccatt tggaccagtt taaggaaatc ttttgcccta
     4501 taaattccag cctgcatgaa tttttatgat tattgctcat aaacatactt ttaaaaatgt
     4561 gcattttaac taactatcaa cttgtgcccg tccactcaaa ggaagccaaa agggtgtctc
     4621 tcccagatca cagggactac cttgtgggtg tgtagggtga tgtattagct aggcgctaat
     4681 aaaatgcagg agtgtccaca aacatgactt aatgagggag gatgagagac tcctgaccca
     4741 gaggtcaaag ctccgggcga ctctctttat tcccattgat ctgctggtgc acttcatttc
     4801 catgctctag ggcccacttc agtaaattca ttaagtgcga ttgatacgag aatcaagact
     4861 tcctccaaaa cacaatggct atgctaggag ggaatggtct tgtttgtgct gcagctatgt
     4921 caagcatctc tggcttttgt cctggtttcg caccattgcg gtgcatgctg gggtttgagt
     4981 cctcgagatt ctaatcgtaa gctcatttcc agctggttcg ttgtgttgcg cccctatggc
     5041 cttgacctgg gaaacgtggt ttcagtgttg ttgagatgcc gaccgcagac cgttccttgc
     5101 ctccactcgt aaattacgct ccgatcaggt tttcccagcc aatcctggcg agcagatctg
     5161 catctgtcac tctgcaagaa tgctgaatca aaccgctgat ctaggcacaa tacagctgat
     5221 ggaaagcaga tctttattcc aacatcagga acgtagcacg agccgagtgc ttctggaagc
     5281 tgcaatttaa ccaacaggtt catatctgat gagatcaagg atgcatcatc ctggaagaaa
     5341 cactgtgatg aacatttaag atgtcagaat gacccgtcat gcctctgagc tgagtccccg
     5401 cacagcgctt attcttttga agagggagaa taaaagcaaa aaaacagagg cgatggaaat
     5461 atgacatcca gaccgcggtc agaactgtga gtgtctctcc tgggctccta tggttgtggt
     5521 gcacagtcca tgtccctgct atgagttaga tagacagatg cactggcaga tagacagaca
     5581 gacaggcttt actacttggt cgctggattg caggaaagag ctttatccaa ccagagtcaa
     5641 gcaagcaaca tgggacaata acataattaa atattctggg gaaaagtggc acttatgcaa
     5701 atgttttttg taatgggttg gagaaaattg gccggaacct acataatttt taatgacagg
     5761 atgattcatt tgtgaaattg taattcaaag aaattcagta aactaactaa caatctaaaa
     5821 aattctttga tttgcagttt aattctactt cctaacattg atttgtttca actttattca
     5881 aatggcaaaa ctaaattagt actcaaagct ccaaatgata tgattcctag actgtttaaa
     5941 gtgctaaaag tgccaaaact tacagctacc cacatgaact tatatttcat tctgcctcca
     6001 tttgcaattc ctccagtgtt tgtacctgaa actgttgtca gttctcacct ctgtcctgcc
     6061 gttcattctg acacagagac tctatatttg aatgtattgt caacttctaa tgccgtgtct
     6121 actactctgt gctgtctgct ctcatcccat ccgaatgaca atagtgttta tgtaaaatgt
     6181 gctgcctttg ggccgctggg gcacagaaca ctgcatgcct ggccctgcca caccaccttt
     6241 tacggctttt tattgtgttg cctttgtgtc acaaggggag gacgcggcca tcaaaattct
     6301 ccaagacaaa attcttgaat gtatgtgcac gcacacgcat ccggaacaat tagttcagga
     6361 gcacgggtgc ttaaaaaacg gagacgacac ttctcgccac cacctcctcg ccgagcagca
     6421 ggcccgagtc tggcacagct gggtttcctt gatgctaacg cagacaatat agatcttaat
     6481 aagacaaatc tacctctccg aaactcagtc ctgacaccct ttcattctta ttccctccca
     6541 gcctcctctc tctctctctc tctctttctt tctttctttg ctacattacc agggggtggt
     6601 gcatgattga cagatgggca ggaataataa tttttttttt ttttttgcgt aacctaaggc
     6661 catgctctgt gattggcttg ccagctgaca tctccaaacc tctcttgtcg tttttttccc
     6721 ctctctctct ctcgctttct ctctctcact ctctctctct aatattggcc ctgatgatcc
     6781 taaggggaaa aaagtgggga gagaaaaaga tatgtacctt agggaagaac ctcagtaaga
     6841 gggagggaat cagaggacga gtaggagcgt gtgtgcgagt gtgtgaaaga gacacttact
     6901 ctcctaacta ctactggaag acacacactt ttctcaccca actcaaagca gcagcaaacc
     6961 ttggagaatt gttttatttt tgttttttcg cgaacccccc taaaaaaatt cctaattatt
     7021 ctctcaagca tccatctttg ccttgccggg atttaatttt caccccctct ctaggaatcc
     7081 tatgtactga tcaatagacc ctttccatcc acacaagaca tacctcgatg ggctgcttct
     7141 tcagcaagcg gcggaaggcc gacaaggaga gcactagtat ggtgagcaag ggcgaggaga
     7201 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     7261 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag gcctttcaga
     7321 ccgctaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     7381 ctcagttcat gtacggctcc aaggtctaca ttaagcaccc agccgacatc cccgactact
     7441 tcaagctgtc cttccccgag ggcttcaggt gggagcgcgt gatgaacttc gaggacggcg
     7501 gcattattca cgttaaccag gactcctccc tgcaggacgg cgtgttcatc tacaaggtga
     7561 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
     7621 gggaggcctc cgaggagcgg atgtaccccg aggacggcgc cctgaagagc gagatcaaga
     7681 agaggctgaa gctgaaggac ggcggccact acgccgccga ggtcaagacc acctacaagg
     7741 ccaagaagcc cgtgcagctg cccggcgcct acatcgtcga catcaagttg gacatcgtgt
     7801 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     7861 ccggcggcat ggacgagctg tacaagtaag ctctagatgg ccagatctat ttaaattaaa
     7921 ctgggcatca gcgcaattca attggtttgg taatagcaag ggaaaataga atgaagtgat
     7981 ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt
     8041 ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt
     8101 aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta
     8161 ctttacacct ctgggcccaa ttcgccc
//
