LOCUS       sequence-437474-        5670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tol2 transposon expressing sgRNA targeting chick DNM1L 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 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-DNM1L -GFP
FEATURES             Location/Qualifiers
     source          1..5670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..36
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(255..359)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     427..445
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(455..466,467..654)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     protein_bind    726..746
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1161..1185
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        1189..1568
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1570..1844
                     /label=chicken beta-actin promoter
     intron          1845..2861
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2785..2807
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2869..2888
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2916..2925
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2922..2924,2925..2927,2928..3641)
                     /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(2922..2924,2925..2927,2928..3638)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2967..2988)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3228..3247)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3575..3596
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3648..3668)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3696..3817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3707..3726)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3761..3780
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(3943..4105,4106..4117)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4134..4151)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4305..4893)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4385..4404)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5064..5670,1..185,186..254))
                     /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"
ORIGIN
        1 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
       61 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
      121 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
      181 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
      241 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
      301 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
      361 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
      421 tataaaaata ggcgtatcac gaggcccttt cgtccagagg tgtaaagtac ttgagtaatt
      481 ttacttgatt actgtactta agtattattt ttggggattt ttactttact tgagtacaat
      541 taaaaatcaa tacttttact tttacttaat tacatttttt tagaaaaaaa agtacttttt
      601 actccttaca attttattta cagtcaaaaa gtacttattt tttggagatc acttcattct
      661 attttccctt gctattacca aaccaattga attgcgctga tgcccagttt aatttaaata
      721 gatctcaact ttgtatagaa aagttggcgc cgccgctcct tcaggcactg ccgacgacag
      781 cccaggcgga ggtcctgagc gccggcgcta aatttgcata aagaactacc caggagccct
      841 cgcgcgcgga aacgggcaaa aaggggcttc taatatggaa atattacgcc gaatcgcgtt
      901 acaaatcggc taagcgggcc taagagttaa caagatgtgc tattaagcgg agccttttgg
      961 tgggaagaaa tggagtagtc actgtgttct aaaagaactt gcagaatgag cctttaaata
     1021 ccgcagtctc gatgctctta gtcggatgtg ttttccgacc atcctctggt ttaagagcta
     1081 tgctggaaac agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg
     1141 caccgagtcg gtgctttttt tcaagtttgt acaaaaaagc aggctgtcga cattgattat
     1201 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     1261 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     1321 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     1381 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     1441 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     1501 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     1561 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
     1621 cccccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
     1681 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
     1741 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
     1801 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1861 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1921 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1981 gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc
     2041 gggagggccc tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg
     2101 ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg
     2161 ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt
     2221 gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag
     2281 cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct gcacccccct ccccgagttg
     2341 ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg
     2401 tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg
     2461 gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg
     2521 cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt
     2581 cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg
     2641 gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc
     2701 cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc
     2761 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     2821 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     2881 ttattgtgct gtctcatcat tttggcaaag aattcgccac catggtgagc aagggcgagg
     2941 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3001 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3061 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3121 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3181 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3241 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3301 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     3361 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     3421 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     3481 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     3541 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     3601 ccgccgggat cactctcggc atggacgagc tgtacaagta aggatcccaa ctttattata
     3661 catagttgat ggccggccgc ttcgagcaga catgataaga tacattgatg agtttggaca
     3721 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     3781 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     3841 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     3901 atgtggtaga tatcaagctt aaacaagaat ctctagtttt ctttcttgct tttactttta
     3961 cttccttaat actcaagtac aattttaatg gagtactttt ttacttttac tcaagtaaga
     4021 ttctagccag atacttttac ttttaattga gtaaaatttt ccctaagtac ttgtactttc
     4081 acttgagtaa aatttttgag tactttttac acctctgggc gctcttccgc ttcctcgctc
     4141 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4201 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4261 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4321 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4381 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4441 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4501 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4561 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4621 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4681 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4741 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4801 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4861 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4921 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4981 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5041 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5101 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5161 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagatcc acgctcaccg
     5221 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5281 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5341 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5401 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5461 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5521 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5581 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5641 tagtgtatgc ggcgaccgag ttgctcttgc
//
