LOCUS       sequence-422556-        3599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Carries the cognate target sites for the homing endonucleases I-OnuI
            and I-GpeMI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3599)
  AUTHORS   Foo GW, Leichthammer CD, Saita IM, Lukas ND, Batko IZ, Heinrichs DE,
            Edgell DR
  TITLE     Intein-based thermoregulated meganucleases for containment of 
            genetic material.
  JOURNAL   Nucleic Acids Res. 2024 Feb 28;52(4):2066-2077. doi: 
            10.1093/nar/gkad1247.
  PUBMED    38180814
REFERENCE   2  (bases 1 to 3599)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2024-02-28- 28"; volume: "52"; issue: 
            "4"; pages: "2066-2077"
COMMENT     Sequence Label: pTarget-Dual
FEATURES             Location/Qualifiers
     source          1..3599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(50..69)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1212..1800
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1701..1720
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1854..1875
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     terminator      1951..2022
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2038..2065
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    2074..2577
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        2578..2764
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             2765..3427
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     primer_bind     complement(3565..3584)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(3576..3599,1..792)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMHNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 tcttgctcga ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
       61 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtacgg gaagcccgat
      121 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
      181 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
      241 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag
      301 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
      361 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
      421 ctcgctcagg cgcaatcacg aatgcataac ggtttggttg atgcgagtga ttttgatgac
      481 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct tttgccattc
      541 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
      601 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
      661 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
      721 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
      781 gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcactcaac caagtcattc
      841 tgatttccgc ttattcaacc ctttagaata gtgtatgcgg cgaccgagtt gctcttgccc
      901 ggcgtcaata cgggatgctt tccacttatt caacctttta tagcctaaac gtcgtatagg
      961 agcatttcta aacgtcgtat aggagcattt ccgcggagac agatcgctga gataggtgcc
     1021 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     1081 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     1141 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1201 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1261 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1321 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     1381 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1441 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1501 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1561 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1621 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1681 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1741 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1801 aacgccagca acgcggcctt tttacggttc ctggcgacgt cgcgcaacgc aattaatgtg
     1861 agttagctca ctcattaggc acccttgagg aggtttctct gtctcactca ttaggcaccc
     1921 ttgaggaggt ttctctgtta atccaggcat caaataaaac gaaaggctca gtcgaaagac
     1981 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc
     2041 tcaccttcgg gtgggccttt ctgcgtttat agcatcacgt gctataaaaa taattataat
     2101 ttaaattttt taatataaat atataaatta aaaatagaaa gtaaaaaaag aaattaaaga
     2161 aaaaatagtt tttgttttcc gaagatgtaa aagactctag ggggatcgcc aacaaatact
     2221 accttttacc ttgctcttcc tgctctcagg tattaatgcc gaattgtttc atcttgtctg
     2281 tgtagaagac cacacacgaa aatcctgtga ttttacattt tacttatcgt taatcgaatg
     2341 tatatctatt taatctgctt ttcttgtcta ataaatatat atgtaaagta cgctttttgt
     2401 tgaaattttt taaacctttg tttatttttt tttcttcatt ccgtaactct tctaccttct
     2461 ttatttactt tctaaaatcc aaatacaaaa cataaaaata aataaacaca gagtaaattc
     2521 ccaaattatt ccatcattaa aagatacgag gcgcgtgtaa gttacaggca agcgatccta
     2581 gtacactcta tattttttta tgcctcggta atgattttca tttttttttt ccacctagcg
     2641 gatgactctt tttttttctt agcgattggc attatcacat aatgaattat acattatata
     2701 aagtaatgtg atttcttcga agaatatact aaaaaatgag caggcaagat aaacgaaggc
     2761 aaagatgaca gagcagaaag ccctagtaaa gcgtattaca aatgaaacca agattcagat
     2821 tgcgatctct ttaaagggtg gtcccctagc gatagagcac tcgatcttcc cagaaaaaga
     2881 ggcagaagca gtagcagaac aggccacaca atcgcaagtg attaacgtcc acacaggtat
     2941 agggtttctg gaccatatga tacatgctct ggccaagcat tccggctggt cgctaatcgt
     3001 tgagtgcatt ggtgacttac acatagacga ccatcacacc actgaagact gcgggattgc
     3061 tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt ggagtaaaaa ggtttggatc
     3121 aggatttgcg cctttggatg aggcactttc cagagcggtg gtagatcttt cgaacaggcc
     3181 gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta ggagatctct cttgcgagat
     3241 gatcccgcat tttcttgaaa gctttgcaga ggctagcaga attaccctcc acgttgattg
     3301 tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg ttcaaggctc ttgcggttgc
     3361 cataagagaa gccacctcgc ccaatggtac caacgatgtt ccctccacca aaggtgttct
     3421 tatgtagttt tacacaggag tctggacttg atcgatttat tcaacaaagc cacgttgtgt
     3481 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac
     3541 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacg
//
