LOCUS       sequence-311217-        4268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plant mitoTALEN vector assembly, Entry clone 1 with attR4 R3 (last 
            HD).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4268)
  AUTHORS   Kazama T, Okuno M, Watari Y, Yanase S, Koizuka C, Tsuruta Y, Sugaya 
            H, Toyoda A, Itoh T, Tsutsumi N, Toriyama K, Koizuka N, Arimura SI
  TITLE     Curing cytoplasmic male sterility via TALEN-mediated mitochondrial 
            genome editing.
  JOURNAL   Nat Plants. 2019 Jul;5(7):722-730. doi: 10.1038/s41477-019-0459-z. 
            Epub 2019 Jul 8.
  PUBMED    31285556
REFERENCE   2  (bases 1 to 4268)
  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"; doi: 
            "10.1038/s41477-019-0459-z"; journalName: "Nat Plants"; date: 
            "2019-07"; volume: "5"; issue: "7"; pages: "722-730"
COMMENT     Sequence Label: pENTR E1 pF5A L1-L4
FEATURES             Location/Qualifiers
     source          1..4268
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             142..729
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     protein_bind    complement(767..862)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(888..906)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(911..927)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1029..1048)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1040..1849
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1107..1126)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1996..2584
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2485..2504
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2738..2755
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2914..2941)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3033..3119)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3183..3199
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3215..3314
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3317..3343
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3650..4060)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRASRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLLER
                     GTRQCIAPCMHCLHCGHAATFCNLEKLACVQL"
     primer_bind     3925..3941
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3951..3969
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4017..4037)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4017..4035)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4056..4072)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4069..4091)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4080..4096
                     /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(4104..4110,4111..4128,4129..4134))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4149..4170
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcaattgttg cccagttatc tcgccctgat ccggcgttgg ccgcgttgac caacgaccac
       61 ctcgtcgcct tggcctgcct cggcgggcgt cctgcgctgg atgcagtgaa aaagggattg
      121 ggggatccta tcagccgttc ccagctggtg aagtccgagc tggaggagaa gaaatccgag
      181 ttgaggcaca agctgaagta cgtgccccac gagtacatcg agctgatcga gatcgcccgg
      241 aacagcaccc aggaccgtat cctggagatg aaggtgatgg agttcttcat gaaggtgtac
      301 ggctacaggg gcaagcacct gggcggctcc aggaagcccg acggcgccat ctacaccgtg
      361 ggctccccca tcgactacgg cgtgatcgtg gacaccaagg cctactccgg cggctacaac
      421 ctgcccatcg gccaggccga cgaaatgcag aggtacgtgg aggagaacca gaccaggaac
      481 aagcacatca accccaacga gtggtggaag gtgtacccct ccagcgtgac cgagttcaag
      541 ttcctgttcg tgtccggcca cttcaagggc aactacaagg cccagctgac caggctgaac
      601 cacatcacca actgcaacgg cgccgtgctg tccgtggagg agctcctgat cggcggcgag
      661 atgatcaagg ccggcaccct gaccctggag gaggtgagga ggaagttcaa caacggcgag
      721 atcaacttcg cggccgactg ataactcgag cgatcctcta gacacccaac ttttctatac
      781 aaagttggca ttataagaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc
      841 agtcaaaata aaatcattat ttgccatcca gctgcagggc ggccgcccct atagtgagtc
      901 gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg
      961 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     1021 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa
     1081 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     1141 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     1201 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     1261 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     1321 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc
     1381 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     1441 ttgtaattgt ccttttaaca gcgatcgcgt atttcgcctt gctcaggcgc aatcacgaat
     1501 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     1561 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     1621 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     1681 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     1741 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     1801 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     1861 taattggttg taacactggc agagcattac gctgacttga cgggacggcg caagctcatg
     1921 accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa
     1981 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2041 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2101 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     2161 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2221 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2281 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2341 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2401 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcatgg tcggaacagg
     2461 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2521 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2581 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2641 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2701 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2761 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     2821 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg
     2881 taccgctagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag gatggccttc
     2941 tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt
     3001 tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc
     3061 gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg ttttatttga
     3121 tgcctggcag ttccctactc tcgcgttaac gctagcatgg atgttttccc agtcacgacg
     3181 ttgtaaaacg acggccagtc ttaagctcgg gccccaaata atgattttat tttgactgat
     3241 agtgacctgt tcgttgcaac aaattgatga gcaatgcttt tttataatgc caactttgta
     3301 caaaaaagca ggcttttacc catacgatgt tccagattac gctgatatcg ccgatctacg
     3361 cacgctcggc tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt
     3421 ggcgcagcac cacgaggcac tggtcggcca cgggtttaca cacgcgcaca tcgttgcgtt
     3481 aagccaacac ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc
     3541 gttgccagag gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg
     3601 cgctctggag gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga
     3661 cacaggccaa cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca
     3721 tgcatggcgc aatgcactga cgggtgcccc gctcaaggag acgggcgccg ctacagggcg
     3781 cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     3841 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     3901 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact
     3961 cactataggg cgaattgggt accgggcccc ccctcgaggc cctccagctt ttgttccctt
     4021 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4081 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4141 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccac
     4201 cggtcgtctc cacccctgag caggtagtgg ctattgcatc ccacgacggg ggcagacccg
     4261 cactggag
//
