LOCUS       sequence-437471-        5672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty vector to clone custom sgRNA into BsaI sites to express 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-empty-GFP
FEATURES             Location/Qualifiers
     source          1..5672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..35
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(254..358)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     426..444
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(454..465,466..653)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     protein_bind    725..745
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1160..1184
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        1188..1567
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1569..1845
                     /label=chicken beta-actin promoter
     intron          1846..2862
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2786..2808
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2870..2889
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2917..2926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2923..2925,2926..2928,2929..3642)
                     /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(2923..2925,2926..2928,2929..3639)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2968..2989)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3229..3248)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3576..3597
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3649..3669)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3697..3818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3708..3727)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3762..3781
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(3944..4106,4107..4118)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4135..4152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4306..4894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4386..4405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5065..5672,1..184,185..253))
                     /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 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
       61 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
      121 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
      181 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
      241 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
      301 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
      361 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
      421 ataaaaatag gcgtatcacg aggccctttc gtccagaggt gtaaagtact tgagtaattt
      481 tacttgatta ctgtacttaa gtattatttt tggggatttt tactttactt gagtacaatt
      541 aaaaatcaat acttttactt ttacttaatt acattttttt agaaaaaaaa gtacttttta
      601 ctccttacaa ttttatttac agtcaaaaag tacttatttt ttggagatca cttcattcta
      661 ttttcccttg ctattaccaa accaattgaa ttgcgctgat gcccagttta atttaaatag
      721 atctcaactt tgtatagaaa agttggcgcc gccgctcctt caggcactgc cgacgacagc
      781 ccaggcggag gtcctgagcg ccggcgctaa atttgcataa agaactaccc aggagccctc
      841 gcgcgcggaa acgggcaaaa aggggcttct aatatggaaa tattacgccg aatcgcgtta
      901 caaatcggct aagcgggcct aagagttaac aagatgtgct attaagcgga gccttttggt
      961 gggaagaaat ggagtagtca ctgtgttcta aaagaacttg cagaatgagc ctttaaatac
     1021 cgcagtctcg atgctcttag tcggatagag accgagagag ggtctcagtt taagagctat
     1081 gctggaaaca gcatagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc
     1141 accgagtcgg tgcttttttt caagtttgta caaaaaagca ggctgtcgac attgattatt
     1201 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     1261 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     1321 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     1381 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     1441 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     1501 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     1561 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1621 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1681 gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg
     1741 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
     1801 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
     1861 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1921 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1981 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
     2041 cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg
     2101 gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
     2161 gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg
     2221 tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga
     2281 gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt
     2341 gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc
     2401 gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc
     2461 ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
     2521 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
     2581 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
     2641 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
     2701 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
     2761 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga
     2821 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
     2881 gttattgtgc tgtctcatca ttttggcaaa gaattcgcca ccatggtgag caagggcgag
     2941 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     3001 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     3061 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     3121 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     3181 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     3241 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     3301 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     3361 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     3421 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     3481 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     3541 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     3601 gccgccggga tcactctcgg catggacgag ctgtacaagt aaggatccca actttattat
     3661 acatagttga tggccggccg cttcgagcag acatgataag atacattgat gagtttggac
     3721 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     3781 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     3841 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     3901 aatgtggtag atatcaagct taaacaagaa tctctagttt tctttcttgc ttttactttt
     3961 acttccttaa tactcaagta caattttaat ggagtacttt tttactttta ctcaagtaag
     4021 attctagcca gatactttta cttttaattg agtaaaattt tccctaagta cttgtacttt
     4081 cacttgagta aaatttttga gtacttttta cacctctggg cgctcttccg cttcctcgct
     4141 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4201 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4261 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4321 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4381 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4441 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4501 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4561 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4621 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4681 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4741 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4801 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4861 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4921 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4981 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5041 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5101 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5161 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagatc cacgctcacc
     5221 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5281 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5341 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5401 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5461 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5521 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5581 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5641 atagtgtatg cggcgaccga gttgctcttg cc
//
