LOCUS       sequence-311221-        4291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plant mitoTALEN vector assembly, Entry clone 3 with attL3 L2 (last 
            HD).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4291)
  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 4291)
  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 E3 pR5A L3-L2
FEATURES             Location/Qualifiers
     source          1..4291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             139..726
                     /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(760..859)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(890..908)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(913..929)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1031..1050)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1042..1851
                     /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(1109..1128)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1998..2586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2487..2506
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2740..2757
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2916..2943)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3035..3121)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3185..3201
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3218..3313
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3316..3360
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(3670..4080)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRASRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLLER
                     GTRQCIAPCMHCLHCGHAATFCNLEKLACVQL"
     primer_bind     3945..3961
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3971..3989
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4037..4057)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4037..4055)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4076..4092)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4089..4111)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4100..4116
                     /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(4124..4130,4131..4148,4149..4154))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4169..4190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc
       61 gtcgccttgg cctgcctcgg cgggcgtcct gcgctggatg cagtgaaaaa gggattgggg
      121 gatcctatca gccgttccca gctggtgaag tccgagctgg aggagaagaa atccgagttg
      181 aggcacaagc tgaagtacgt gccccacgag tacatcgagc tgatcgagat cgcccggaac
      241 agcacccagg accgtatcct ggagatgaag gtgatggagt tcttcatgaa ggtgtacggc
      301 tacaggggca agcacctggg cggctccagg aagcccgacg gcgccatcta caccgtgggc
      361 tcccccatcg actacggcgt gatcgtggac accaaggcct actccggcgg ctacaacctg
      421 cccatcggcc aggccgacga aatgcagagg tacgtggagg agaaccagac caggaacaag
      481 cacatcaacc ccaacgagtg gtggaaggtg tacccctcca gcgtgaccga gttcaagttc
      541 ctgttcgtgt ccggccactt caagggcaac tacaaggccc agctgaccag gctgaaccac
      601 atcaccaact gcaacggcgc cgtgctgtcc gtggaggagc tcctgatcgg cggcgagatg
      661 atcaaggccg gcaccctgac cctggaggag gtgaggagga agttcaacaa cggcgagatc
      721 aacttcgcgg ccgactgata actcgagcga tcctctagaa cccagctttc ttgtacaaag
      781 ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc
      841 aaaataaaat cattatttgc catccagctg cagggcggcc gcgatatccc ctatagtgag
      901 tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
      961 tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata
     1021 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta
     1081 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     1141 tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa
     1201 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     1261 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     1321 ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat
     1381 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     1441 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgcc ttgctcaggc gcaatcacga
     1501 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     1561 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     1621 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     1681 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     1741 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     1801 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg
     1861 gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca
     1921 tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa
     1981 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2041 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2101 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     2161 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2221 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2281 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2341 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2401 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2461 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2521 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2581 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2641 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2701 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2761 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     2821 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatacg
     2881 cgtaccgcta gccaggaaga gtttgtagaa acgcaaaaag gccatccgtc aggatggcct
     2941 tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc
     3001 gttgcttcac aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca
     3061 ccgacaaaca acagataaaa cgaaaggccc agtcttccga ctgagccttt cgttttattt
     3121 gatgcctggc agttccctac tctcgcgtta acgctagcat ggatgttttc ccagtcacga
     3181 cgttgtaaaa cgacggccag tcttaagctc gggccccaaa taatgatttt attttgactg
     3241 atagtgacct gttcgttgca acaaattgat gagcaatgct tttttataat gccaactttg
     3301 tataataaag ttgataagga gaccgccgct gccaagttcg agagacagca catggacagc
     3361 atcgatatcg ccgatctacg cacgctcggc tacagccagc agcaacagga gaagatcaaa
     3421 ccgaaggttc gttcgacagt ggcgcagcac cacgaggcac tggtcggcca cgggtttaca
     3481 cacgcgcaca tcgttgcgtt aagccaacac ccggcagcgt tagggaccgt cgctgtcaag
     3541 tatcaggaca tgatcgcagc gttgccagag gcgacacacg aagcgatcgt tggcgtcggc
     3601 aaacagtggt ccggcgcacg cgctctggag gccttgctca cggtggcggg agagttgaga
     3661 ggtccaccgt tacagttgga cacaggccaa cttctcaaga ttgcaaaacg tggcggcgtg
     3721 accgcagtgg aggcagtgca tgcatggcgc aatgcactga cgggtgcccc gctcaaggag
     3781 acgggcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
     3841 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     3901 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     3961 tgagcgcgcg taatacgact cactataggg cgaattgggt accgggcccc ccctcgaggc
     4021 cctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     4081 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4141 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4201 gctcactgcc cgctttccac cggtcgtctc cacccctgag caggtagtgg ctattgcatc
     4261 ccacgacggg ggcagacccg cactggagtc a
//
