LOCUS       sequence-149784-        7908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to subclone in gene of interest into blue motor neuron 
            expression vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7908)
  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 7908)
  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:mTagBFP-NS-MCS
FEATURES             Location/Qualifiers
     source          1..7908
                     /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"
     regulatory      4006..4015
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4012..4713
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     protein_bind    complement(4715..4739)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     4748..4764
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4749..4765
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4805..4825)
                     /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(4833..4849)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    4865..4999
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4889..4908)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4943..4962
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(5438..5625,5626..5637)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5671..5687)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5671..5687)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5724..5744)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5724..5742)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5763..5779)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5776..5798)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5787..5803
                     /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(5811..5817,5818..5835,5836..5841))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5856..5877
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5994..6011)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6165..6753)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6245..6264)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6924..7715,7716..7784))
                     /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     7547..7566
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7785..7889)
                     /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 ttgtgagttg gcaagtttgt acaaaaaagc aggctgccac catggtgagc
     4021 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     4081 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     4141 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     4201 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     4261 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     4321 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     4381 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     4441 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     4501 aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa
     4561 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     4621 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     4681 gacctcccta gcaaactggg gcacaagctt aatgacccag ctttcttgta caaagtggct
     4741 ggatccctcg aggtcgacgg tatcgataag cttgatatcg aattccccgg gtctagaact
     4801 agtgcaactt tattatacat agttgataat tcactggccg tcgttttacg gtaccatcga
     4861 tgatgatcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     4921 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     4981 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     5041 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     5101 tgatcctcta gatcagatct gcgaagatac ggccacgggt gctcttgatc ctgtggctga
     5161 ttttggactg tgctgctcgc agctgctgat gaatcacata cttcctccat tttcttccac
     5221 tgattgactg ttataatttc cctaatttcc aggtcaaggt gctgtgcatt gtggtaatag
     5281 atgtgacatg acgtcacttc caaaggacca atgaacatgt ctgaccaatt tcatataatg
     5341 tgaaaacgat tttcataggc agaataaata acatttaaat taaactgggc atcagcgcaa
     5401 ttcaattggt ttggtaatag caagggaaaa tagaatgaag tgatctccaa aaaataagta
     5461 ctttttgact gtaaataaaa ttgtaaggag taaaaagtac ttttttttct aaaaaaatgt
     5521 aattaagtaa aagtaaaagt attgattttt aattgtactc aagtaaagta aaaatcccca
     5581 aaaataatac ttaagtacag taatcaagta aaattactca agtactttac acctctggtt
     5641 cttgaccccc taccttcagc aagcccagca gatccactag ttctagagcg gccgccaccg
     5701 cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     5761 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5821 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5881 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5941 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6001 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6061 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6121 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6181 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6241 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6301 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6361 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6421 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6481 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6541 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6601 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6661 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6721 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6781 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6841 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6901 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6961 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7021 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7081 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7141 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     7201 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     7261 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     7321 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7381 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7441 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7501 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7561 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7621 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7681 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7741 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7801 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7861 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtg
//
