LOCUS       sequence-437475-        5672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2 transposon expresses control non-targeting sgRNA from chick 
            U6.3 promoter expresses GFP reporter from GAGC promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5672)
  AUTHORS   O'Sullivan JDB, Terry S, Scott CA, Bullen A, Jagger DJ, Mann ZF
  TITLE     Mitochondrial dynamics regulate cell size in the developing cochlea
  JOURNAL   bioRxiv 2024.03.04.583298
REFERENCE   2  (bases 1 to 5672)
  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: "bioRxiv 
            2024.03.04.583298"
COMMENT     Sequence Label: Tol2-U6.3-sgRNA-non-targeting-control -GFP
FEATURES             Location/Qualifiers
     source          1..5672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(106..210)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     278..296
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(306..317,318..505)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     protein_bind    577..597
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1011..1035
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        1039..1418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1698..2714
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2638..2660
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2722..2741
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2769..2778
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2775..2777,2778..2780,2781..3494)
                     /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(2775..2777,2778..2780,2781..3491)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2820..2841)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3081..3100)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3428..3449
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3501..3521)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3549..3670
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3560..3579)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3614..3633
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(3796..3958,3959..3970)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3987..4004)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4158..4746)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4238..4257)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4917..5672,1..36,37..105))
                     /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     5540..5559
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
       61 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
      121 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
      181 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
      241 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
      301 tcgtccagag gtgtaaagta cttgagtaat tttacttgat tactgtactt aagtattatt
      361 tttggggatt tttactttac ttgagtacaa ttaaaaatca atacttttac ttttacttaa
      421 ttacattttt ttagaaaaaa aagtactttt tactccttac aattttattt acagtcaaaa
      481 agtacttatt ttttggagat cacttcattc tattttccct tgctattacc aaaccaattg
      541 aattgcgctg atgcccagtt taatttaaat agatctcaac tttgtataga aaagttggcg
      601 ccgccgctcc ttcaggcact gccgacgaca gcccaggcgg aggtcctgag cgccggcgct
      661 aaatttgcat aaagaactac ccaggagccc tcgcgcgcgg aaacgggcaa aaaggggctt
      721 ctaatatgga aatattacgc cgaatcgcgt tacaaatcgg ctaagcgggc ctaagagtta
      781 acaagatgtg ctattaagcg gagccttttg gtgggaagaa atggagtagt cactgtgttc
      841 taaaagaact tgcagaatga gcctttaaat accgcagtct cgatgctctt agtcggatgc
      901 actgctacga tctacaccgt ttaagagcta tgctggaaac agcatagcaa gtttaaataa
      961 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tcaagtttgt
     1021 acaaaaaagc aggctgtcga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1081 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     1141 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     1201 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     1261 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     1321 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     1381 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct
     1441 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
     1501 aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgccr ggsggggsgg
     1561 ggsggggsgr ggggsggggs ggggsgrggs ggagaggkgc ggcggcagcc aatcagagcg
     1621 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     1681 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     1741 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     1801 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
     1861 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
     1921 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc
     1981 ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg
     2041 aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag
     2101 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc
     2161 tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg
     2221 ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg
     2281 gggtgccggg cggggcgggg ccgcctcggg ccggggrggg ctcgggggag gggcgcggcg
     2341 gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     2401 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg
     2461 ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga
     2521 aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc
     2581 agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     2641 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct
     2701 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
     2761 aagaattcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     2821 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     2881 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     2941 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     3001 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     3061 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     3121 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     3181 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     3241 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     3301 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     3361 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     3421 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     3481 agctgtacaa gtaaggatcc caactttatt atacatagtt gatggccggc cgcttcgagc
     3541 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3601 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3661 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     3721 ggaggttttt taaagcaagt aaaacctcta caaatgtggt agatatcaag cttaaacaag
     3781 aatctctagt tttctttctt gcttttactt ttacttcctt aatactcaag tacaatttta
     3841 atggagtact tttttacttt tactcaagta agattctagc cagatacttt tacttttaat
     3901 tgagtaaaat tttccctaag tacttgtact ttcacttgag taaaattttt gagtactttt
     3961 tacacctctg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4021 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4081 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4141 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4201 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4261 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4321 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4381 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4441 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4501 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4561 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4621 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4681 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4741 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4801 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4861 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4921 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4981 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5041 gctgcaatga taccgcgaga tccacgctca ccggctccag atttatcagc aataaaccag
     5101 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5161 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5221 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5281 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5341 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5401 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5461 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5521 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5581 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5641 tcgatgtaac ccactcgtgc acccaactga tc
//
