LOCUS       sequence-311220-        4268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plant mitoTALEN vector assembly, Entry clone 1 with attR4 R3 (last 
            NN).
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 pF8A L1-L4
FEATURES             Location/Qualifiers
     source          1..4268
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             199..786
                     /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(824..919)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(945..963)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(968..984)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1086..1105)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1097..1906
                     /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(1164..1183)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2053..2641
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2542..2561
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2795..2812
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2971..2998)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3090..3176)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3240..3256
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3272..3371
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3374..3400
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3707..4117)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLLER
                     GTRQCIAPCMHCLHCGHAATFCNLEKLACVQL"
     primer_bind     3982..3998
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4008..4026
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4074..4094)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4074..4092)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4113..4129)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4126..4148)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4137..4153
                     /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(4161..4167,4168..4185,4186..4191))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4206..4227
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 acccctgagc aggtagtggc tattgcatcc aacaacgggg gcagacccgc actggagtca
       61 attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc
      121 gtcgccttgg cctgcctcgg cgggcgtcct gcgctggatg cagtgaaaaa gggattgggg
      181 gatcctatca gccgttccca gctggtgaag tccgagctgg aggagaagaa atccgagttg
      241 aggcacaagc tgaagtacgt gccccacgag tacatcgagc tgatcgagat cgcccggaac
      301 agcacccagg accgtatcct ggagatgaag gtgatggagt tcttcatgaa ggtgtacggc
      361 tacaggggca agcacctggg cggctccagg aagcccgacg gcgccatcta caccgtgggc
      421 tcccccatcg actacggcgt gatcgtggac accaaggcct actccggcgg ctacaacctg
      481 cccatcggcc aggccgacga aatgcagagg tacgtggagg agaaccagac caggaacaag
      541 cacatcaacc ccaacgagtg gtggaaggtg tacccctcca gcgtgaccga gttcaagttc
      601 ctgttcgtgt ccggccactt caagggcaac tacaaggccc agctgaccag gctgaaccac
      661 atcaccaact gcaacggcgc cgtgctgtcc gtggaggagc tcctgatcgg cggcgagatg
      721 atcaaggccg gcaccctgac cctggaggag gtgaggagga agttcaacaa cggcgagatc
      781 aacttcgcgg ccgactgata actcgagcga tcctctagac acccaacttt tctatacaaa
      841 gttggcatta taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt
      901 caaaataaaa tcattatttg ccatccagct gcagggcggc cgcccctata gtgagtcgta
      961 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     1021 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     1081 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     1141 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     1201 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     1261 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     1321 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     1381 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     1441 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     1501 taattgtcct tttaacagcg atcgcgtatt tcgccttgct caggcgcaat cacgaatgaa
     1561 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     1621 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     1681 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     1741 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     1801 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     1861 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     1921 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc
     1981 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     2041 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2101 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2161 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2221 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2281 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2341 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2401 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2461 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcatggtcg gaacaggaga
     2521 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2581 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     2641 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     2701 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     2761 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     2821 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     2881 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac
     2941 cgctagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc
     3001 ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc
     3061 ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac
     3121 aaacaacaga taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc
     3181 ctggcagttc cctactctcg cgttaacgct agcatggatg ttttcccagt cacgacgttg
     3241 taaaacgacg gccagtctta agctcgggcc ccaaataatg attttatttt gactgatagt
     3301 gacctgttcg ttgcaacaaa ttgatgagca atgctttttt ataatgccaa ctttgtacaa
     3361 aaaagcaggc ttttacccat acgatgttcc agattacgct gatatcgccg atctacgcac
     3421 gctcggctac agccagcagc aacaggagaa gatcaaaccg aaggttcgtt cgacagtggc
     3481 gcagcaccac gaggcactgg tcggccacgg gtttacacac gcgcacatcg ttgcgttaag
     3541 ccaacacccg gcagcgttag ggaccgtcgc tgtcaagtat caggacatga tcgcagcgtt
     3601 gccagaggcg acacacgaag cgatcgttgg cgtcggcaaa cagtggtccg gcgcacgcgc
     3661 tctggaggcc ttgctcacgg tggcgggaga gttgagaggt ccaccgttac agttggacac
     3721 aggccaactt ctcaagattg caaaacgtgg cggcgtgacc gcagtggagg cagtgcatgc
     3781 atggcgcaat gcactgacgg gtgccccgct caaggagacg ggcgccgcta cagggcgcgt
     3841 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     3901 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     3961 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac
     4021 tatagggcga attgggtacc gggccccccc tcgaggtcct ccagcttttg ttccctttag
     4081 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4141 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4201 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccaccgg
     4261 tcgtctcc
//
