LOCUS       sequence-149769-        8922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to subclone in tagged gene of interest into inducible 
            motor neuron expression vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8922)
  AUTHORS   Don EK, Formella I, Badrock AP, Hall TE, Morsch M, Hortle E, Hogan 
            A, Chow S, Gwee SS, Stoddart JJ, Nicholson G, Chung R, Cole NJ
  TITLE     A Tol2 Gateway-Compatible Toolbox for the Study of the Nervous 
            System and Neurodegenerative Disease.
  JOURNAL   Zebrafish. 2016 Sep 15.
  PUBMED    27631880
REFERENCE   2  (bases 1 to 8922)
  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: "Zebrafish. 
            2016 Sep 15."
COMMENT     Sequence Label: 
            pTol2p2A--3mnx1:loxP-mTagBFP2-stop-loxP-mCherry-NS-MCS
FEATURES             Location/Qualifiers
     source          1..8922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(514..525,526..688)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     1061..1077
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1097..1117
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    3981..4005
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4008..4041)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4048..4749
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4787..4921
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(4975..5008)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(5012..5036)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      5038..5047
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     5762..5778
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     5763..5779
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5819..5839)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(5847..5863)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    5879..6013
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(6452..6639,6640..6651)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6685..6701)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6685..6701)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(6738..6758)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6738..6756)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6777..6793)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6790..6812)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6801..6817
                     /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(6825..6831,6832..6849,6850..6855))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6870..6891
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7008..7025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7179..7767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7259..7278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7938..8729,8730..8798))
                     /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     8561..8580
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8799..8903)
                     /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 tggcccacgc tggctaagaa
      241 ctcatcagcc tccccggtcc atctacccac gtaccaatgc accaattggc cacaatgacg
      301 gctactacat ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca
      361 aggctcttgg atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa
      421 tgccctctga tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc
      481 acacaggtca gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt
      541 ttactcaagt gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat
      601 ctggctagaa tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt
      661 attaaggaag taaaagtaaa agcaagaaag aaaactagag attcttgttt aagcttttaa
      721 tctcaaaaaa cattaaatga aatgcataca aggttttatc ctgctttaga actgtttgta
      781 tttaattatc aaactataag acagacaatc taatgccagt acacgctact caaagttgta
      841 aaacctcaga tttaacttca gtagaagctg attctcaaaa ttgttagtgt caagcctagc
      901 tcttttgggg ctgaaaagca atcctgcagt gctgaaaagc ctctcacagg cagccgatgc
      961 gggaagaggt gtattagtct tgatagagag gctgcaaata gcaggaaacg tgagcagaga
     1021 ctccctggtg tctgaaacac aggccagatg ggccctcgag caggaaacag ctatgaccat
     1081 gattacgcca agctatcaac tttgtataga aaagttgctg aggaaaacgc tttcgactac
     1141 agaaggactt tgtattatgc aatgttttca aaaaaatatc ttcatattca gtgtgggaga
     1201 taataagaaa ggaaaaagca tgaagcactg aatcggtgtc ctttaatgac ttttatagtt
     1261 gggaacaatt agccgttctc cacaaattaa attgataaaa agattacgag atccaaatgc
     1321 gtttaaaagc tgtggtacgc acggcccatt tgtgcaatta tgatcgaaaa tatttattgc
     1381 aataaaagag acagtgcagg tcccgttaaa ttggcttttc ataatgaaca catttaacat
     1441 ggataattaa tatataaact atttcgattt gtcacgatga tatgtgttgc acttaattgt
     1501 gaaagtaatt accgagcgaa tgaacatctt tgaggagagt ccctggtttc aggccctcgt
     1561 atcatcgggt gcaataataa ttttactctc tcctgatgtg atgaaaaaga gccataaatc
     1621 tctcagatcg ctgcaggtcc acggcttcat atttcaccgc tgctttaata gaaatattca
     1681 tgcgatccct taatgaaatt aaaactgaga agcggacaaa gcggccctag gctatgtgca
     1741 acgtgtaaag acacaagagc atttgattgg gaaaatgttg catccttcaa atatgaggat
     1801 gttgttgcac tgtaaaccac gtaaaacaat ttttttctta cagtccagta ttggccacac
     1861 ttgataattg ttacatttgt tatcaattac acgcgaggct cattgaatgt catttcagaa
     1921 ttggtcagtg catgcgtcat tacggcacgc ttttattctc atcctttcgg ccggtgactt
     1981 gttgatttcc ctcgagcaaa taaacgcatg cgaccagtct ctcgagagtg gttagctgat
     2041 gaattgagga aaactaatcc gctttattgg tagacaggtt aagggcaaca gagtgtcaat
     2101 aattctctcc ctgacctctt tctccattag cttaaagagg cttataagag gaacagaccc
     2161 cacaacatga caccgatcct ccacatcacc atctcacatg tagccaacct aggcttggaa
     2221 aatcatttcg atttataaag ctcaagatcc aacatttgcc aaaaattcat gaaaacaggg
     2281 cacaaatagg gcatcatttt ctcacaaagt attatatagg cctaattaga tagatagata
     2341 gatagataga tagatagata gatagataga tagatagata gatagataga tagatagata
     2401 gatagataga tagatagata gatagataga tagatagata gatagataga tagatagata
     2461 gatagataga tagatagata gatagacaga tagatagata gaaaaaaaac aagattttgc
     2521 ttgtgattca ttatgcataa taatttgcct atgtttatta ataaacatcg tgttaggatt
     2581 ataaatgtgt tgtgatgaat gtgattaaat gtttaaatat ttactgacag ttgttagcct
     2641 atatttacag atgtaaaaaa tctacgtcta attagtatgt gtttcttaaa tctataataa
     2701 ggaaaggctt attaagttta tctgtacaat ttagaacaat tttacgaatt tatcctttag
     2761 agaaaaaaaa tgtaataaca ccatatttaa atatcctgtt gagaaattat taaatcaaat
     2821 ttttacattc taagagcaag gttaaatatt gagtgaaagg ttgaattgtt tttaagacag
     2881 ttgttttatt tttttaagca tattgtggtg ggtgagtttt caaagattta cacatttcaa
     2941 acaatttatt aaaacaatat ttgttgcata aaacactgat ttggtcagtt ttaccactga
     3001 ctttaactta tttatttaat taattatatt tctattgttc attgttatta aattttgcct
     3061 acctaatgct ctctaacaaa taaggaataa cttgaatatt tcctctgcgc aaaatgttca
     3121 tcgtaatgta aaagatccgt ttgacattta aaatggttct cttctcataa agatagtaat
     3181 tcttttaaga actgtcctct aaaaggttct ttggggaaac aaaaaaatat acgtgtacaa
     3241 catcaatgtg aaaatcaaat agtttttttt attatcgcag gctgttcaga taaaaaaata
     3301 caattgaata aaaaagtatg gacattaaaa tggtgtcata gcaaaatgac aaatgcgtgt
     3361 atagactatg gttttttgtt aattaatttg atgaaataaa aaaaaattat acaaatgaaa
     3421 aaaatgtcat gcctctacat ctatgcgaat aaaatgcatg gctgtttttt tttttttttt
     3481 ttttgttcaa acaaactatt tcgccaaaat aatattttcg agttaacgtg ctttatcaat
     3541 cttcgtttat attaagatta taggtcggtg tttaaataat gaaatcttga taactttata
     3601 aatgtttcat gtttcaaagt tgcatgttaa taattgccct ttagactcta caaatctgga
     3661 attattcata aattaagtag atgtaaaatg tatgtgtgca tgttgtgttt cccacttaaa
     3721 aagctttcat tttatgggag aacatactta tgacaactct tgaacttcta caagcacacc
     3781 gtgaacactg gaatcactgt ccaatcggaa atgcacaaga atgacgtgaa tgaccaatcc
     3841 actccctctc gggcagcagt tgacaagtgg acttctggct tgcacacctt ggccaagatc
     3901 ataactttac gaagaggagc ggtgacactc ctgacagcct tttaaagtta cgaacgacgc
     3961 gttaatctgt ttgtgaggtg gcaagtttgt acaaaaaagc aggcttaata acttcgtata
     4021 gcatacatta tacgaagtta tgccaccatg agcgagctga ttaaggagaa catgcacatg
     4081 aagctgtaca tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa
     4141 ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc
     4201 cccttcgcct tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac
     4261 cacacccagg gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag
     4321 agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag
     4381 gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct
     4441 gtgatgcaga agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac
     4501 ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc
     4561 gcaaacgcca agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc
     4621 gtctactatg tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc
     4681 gagcagcacg aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag
     4741 cttaattaag gcgcgcctct agaactatag tgagtcgtat tacgtagatc cagacatgat
     4801 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     4861 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     4921 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaataact
     4981 tcgtatagca tacattatac gaagttatac tacccagctt tcttgtacaa agtggctgcc
     5041 accatggtgt ccaagggcga agaggacaac atggccatca tcaaagagtt catgcggttc
     5101 aaggtgcaca tggaaggcag cgtgaacggc cacgagttcg agattgaggg cgagggcgaa
     5161 ggcagaccct acgagggaac acagaccgcc aagctgaaag tgaccaaggg cggacccctg
     5221 cccttcgcct gggatattct gagcccccag tttatgtacg gcagcaaggc ctacgtgaag
     5281 caccccgccg acatccccga ctacctgaag ctgagcttcc ccgagggctt caagtgggag
     5341 agagtgatga acttcgagga cggcggcgtc gtgaccgtga cccaggatag ctctctgcag
     5401 gacggcgagt tcatctacaa agtgaagctg cggggcacca acttccccag cgacggaccc
     5461 gtgatgcaga aaaagaccat gggctgggag gccagcagcg agagaatgta ccctgaggat
     5521 ggcgccctga agggcgagat caagcagcgg ctgaaactga aggatggcgg ccactacgac
     5581 gccgaagtga aaaccaccta caaggccaag aaacccgtgc agctgcctgg cgcctacaac
     5641 gtgaacatca agctggacat caccagccac aacgaggact acaccatcgt ggaacagtac
     5701 gagcgggccg agggcagaca ctctacaggc ggaatggacg agctgtacaa gcacggatcc
     5761 ctcgaggtcg acggtatcga taagcttgat atcgaattcc ccgggtctag aactagtgca
     5821 actttattat acatagttga taattcactg gccgtcgttt tacggtacca tcgatgatga
     5881 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     5941 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     6001 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     6061 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatcc
     6121 tctagatcag atctgcgaag atacggccac gggtgctctt gatcctgtgg ctgattttgg
     6181 actgtgctgc tcgcagctgc tgatgaatca catacttcct ccattttctt ccactgattg
     6241 actgttataa tttccctaat ttccaggtca aggtgctgtg cattgtggta atagatgtga
     6301 catgacgtca cttccaaagg accaatgaac atgtctgacc aatttcatat aatgtgaaaa
     6361 cgattttcat aggcagaata aataacattt aaattaaact gggcatcagc gcaattcaat
     6421 tggtttggta atagcaaggg aaaatagaat gaagtgatct ccaaaaaata agtacttttt
     6481 gactgtaaat aaaattgtaa ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa
     6541 gtaaaagtaa aagtattgat ttttaattgt actcaagtaa agtaaaaatc cccaaaaata
     6601 atacttaagt acagtaatca agtaaaatta ctcaagtact ttacacctct ggttcttgac
     6661 cccctacctt cagcaagccc agcagatcca ctagttctag agcggccgcc accgcggtgg
     6721 agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     6781 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     6841 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     6901 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6961 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     7021 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     7081 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     7141 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     7201 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     7261 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     7321 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     7381 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     7441 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     7501 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     7561 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     7621 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     7681 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     7741 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     7801 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     7861 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     7921 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     7981 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     8041 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     8101 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     8161 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     8221 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     8281 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     8341 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     8401 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     8461 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     8521 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     8581 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     8641 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     8701 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     8761 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     8821 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8881 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tg
//
