LOCUS       sequence-437473-        5670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2 transposon expressing sgRNA targeting chick MFN1 from chick 
            U6.3 promoter expresses GFP reporter from GAGC promote.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5670)
  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 5670)
  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-MFN1-GFP
FEATURES             Location/Qualifiers
     source          1..5670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          64..1080
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1004..1026
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1088..1107
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1135..1144
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1141..1143,1144..1146,1147..1860)
                     /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(1141..1143,1144..1146,1147..1857)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1186..1207)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1447..1466)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1794..1815
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1867..1887)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    1915..2036
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1926..1945)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1980..1999
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(2162..2324,2325..2336)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(2353..2370)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2524..3112)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2604..2623)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3283..4074,4075..4143))
                     /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     3906..3925
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4144..4248)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4316..4334
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(4344..4355,4356..4543)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     protein_bind    4615..4635
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    5049..5073
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        5077..5456
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(5458..5670,1..63)
                     /label=chicken beta-actin promoter
ORIGIN
        1 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
       61 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      121 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      181 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      241 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
      301 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
      361 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
      421 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
      481 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
      541 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
      601 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
      661 gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc
      721 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
      781 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
      841 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
      901 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
      961 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1021 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1081 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcgccacc
     1141 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1201 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1261 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1321 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1381 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1441 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1501 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1561 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1621 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1681 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1741 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1801 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     1861 ggatcccaac tttattatac atagttgatg gccggccgct tcgagcagac atgataagat
     1921 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     1981 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     2041 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     2101 gcaagtaaaa cctctacaaa tgtggtagat atcaagctta aacaagaatc tctagttttc
     2161 tttcttgctt ttacttttac ttccttaata ctcaagtaca attttaatgg agtacttttt
     2221 tacttttact caagtaagat tctagccaga tacttttact tttaattgag taaaattttc
     2281 cctaagtact tgtactttca cttgagtaaa atttttgagt actttttaca cctctgggcg
     2341 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2401 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2461 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2521 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2581 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2641 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2701 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2761 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2821 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2881 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2941 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3001 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3061 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3121 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3181 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3241 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3301 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3361 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3421 gcgagatcca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3481 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3541 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3601 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3661 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3721 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3781 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3841 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3901 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     3961 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4021 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4081 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4141 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4201 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4261 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     4321 gcgtatcacg aggccctttc gtccagaggt gtaaagtact tgagtaattt tacttgatta
     4381 ctgtacttaa gtattatttt tggggatttt tactttactt gagtacaatt aaaaatcaat
     4441 acttttactt ttacttaatt acattttttt agaaaaaaaa gtacttttta ctccttacaa
     4501 ttttatttac agtcaaaaag tacttatttt ttggagatca cttcattcta ttttcccttg
     4561 ctattaccaa accaattgaa ttgcgctgat gcccagttta atttaaatag atctcaactt
     4621 tgtatagaaa agttggcgcc gccgctcctt caggcactgc cgacgacagc ccaggcggag
     4681 gtcctgagcg ccggcgctaa atttgcataa agaactaccc aggagccctc gcgcgcggaa
     4741 acgggcaaaa aggggcttct aatatggaaa tattacgccg aatcgcgtta caaatcggct
     4801 aagcgggcct aagagttaac aagatgtgct attaagcgga gccttttggt gggaagaaat
     4861 ggagtagtca ctgtgttcta aaagaacttg cagaatgagc ctttaaatac cgcagtctcg
     4921 atgctcttag tcggatgaga agaagagcgt caaggtgttt aagagctatg ctggaaacag
     4981 catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     5041 gctttttttc aagtttgtac aaaaaagcag gctgtcgaca ttgattattg actagttatt
     5101 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     5161 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     5221 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     5281 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     5341 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     5401 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     5461 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     5521 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg ggggggggcg
     5581 cgccaggggg gggggggggg gggggggggg cggggcgggg cgaggcggag aggtgcggcg
     5641 gcagccaatc agagcggcgc gctccgaaag
//
