LOCUS       sequence-412663-        5441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a fusion of the homing endonuclease I-OnuI with the 
            thermosensitive L212P VMA1 intein. Endonuclease activity is 
            restricted to lower temperatures..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5441)
  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 5441)
  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: pEndo-I-OnuI-TSM
FEATURES             Location/Qualifiers
     source          1..5441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     282..301
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     498..515
                     /label=L4440
                     /note="L4440 vector, forward primer"
     RBS             974..996
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1670..2488
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="5' region of the intein from the yeast Vma1 
                     subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 5' region
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLPGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
     CDS             2944..2955
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4061..4079)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4147..4251
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4252..4320,4321..5112)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4470..4489)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5277..5441,1..381)
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
ORIGIN
        1 ggcgcatgac ttcaagacta actcctctaa atcaattacc agtggctgct gccagtggtg
       61 cttttgcatg tctttccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
      121 cggactgaac ggggggttcg tgcatacagt ccagcttgga gcgaactgcc tacccggaac
      181 tgagtgtcag gcgtggaatg agacaaacgc ggccataaca gcggaatgac accggtaaac
      241 cgaaaggcag gaacaggaga gcgcacgagg gagccgccag ggggaaacgc ctggtatctt
      301 tatagtcctg tcgggtttcg ccaccactga tttgagcgtc agatttcgtg atgcttgtca
      361 ggggggcgga gcctatggaa aaacggcttt gccgcggccc tctcacttcc ctgttaagta
      421 tcttcctggc atcttccagg aaatctccgc cccgttcgta agccatttcc gctcgccgca
      481 gtcgaacgac cgagcgtagc gagtcagtga gcgaggaagc ggaatatatc ctgtatcaca
      541 tattctgctg acgcaccggt gcagcctttt ttctcctgcc acatgaagca cttcactgac
      601 accctcatca gtgccaacat agtaagccag tatacactcc gctagcgctg aggtctgcct
      661 cgtgaagaag gtgttgctga ctcataccag gcctgaatcg ccccatcatc cagccagaaa
      721 gtgagggagc cacggttgat gagagctttg ttgtaggtgg accagttggt gattttgaac
      781 ttttgctttg ccacggaacg gtctgcgttg tcggcatgcg cataatgtgc ctgtcaaatg
      841 gacgaagcag ggattctgca aaccctatgc tactccgtca agccgtcaat tgtctgattc
      901 gttaccaact gacgctagct cagtcctagg tataatgcta gctccatacc cgtttttttg
      961 ggctagaaat aattttgttt aactttaaga aggagatata cccatgggat ccgcctacat
     1021 gtcccgtcgc gagtccatta acccgtggat tctcaccggt ttcgccgacg cggaaggctc
     1081 ctttttgctg cgcatccgca acaacaacaa gtccagcgtc ggctactcca ctgagctcgg
     1141 cttccaaatt acacttcata acaaggacaa gagcattctt gagaacatcc agtcaacatg
     1201 gaaggtgggc gtgatcgcca acagcggtga caacgccgtg tcgctgaagg tcacgcgttt
     1261 tgaggacctg aaggtcatta tcgaccattt tgaaaaatac ccactgatta cgcagaagct
     1321 cggtgactac atgctgttta agcaggcgtt ttgcgtcatg gagaacaagg agcatttgaa
     1381 gattaatggt atcaaggagc tggtgcgcat taaggcaaag ctcaattggg gtctgacgga
     1441 tgagctgaag aaggcctttc cggagatcat ctcgaaggag cgctccctca tcaacaagaa
     1501 catccctaat ttcaagtggc tggcgggttt tacctcgggc gagggttgct tctttgttaa
     1561 cctgatcaag tcaaagtcga agctaggtgt ccaggtgcag ctggtgttca gcattaccca
     1621 acacatcaag gataagaacc tcatgaactc tctgattacc tacttgggct gctttgccaa
     1681 gggtaccaat gttttaatgg cggatgggtc tattgaatgt attgaaaaca ttgaggttgg
     1741 taataaggtc atgggtaaag atggcagacc tcgtgaggta attaaattgc ccagaggaag
     1801 agaaactatg tacagcgtcg tgcagaaaag tcagcacaga gcccacaaaa gtgactcaag
     1861 tcgtgaagtg ccagaattac tcaagtttac gtgtaatgcg acccatgagt tggttgttag
     1921 aacacctcgt agtgtccgcc gtttgtctcg taccattaag ggtgtcgaat attttgaagt
     1981 tattactttt gagatgggcc aaaagaaagc ccccgacggt agaattgttg agcttgtcaa
     2041 ggaagtttca aagagctacc caatatctga ggggcctgag agagccaacg aattagtaga
     2101 atcctataga aaggcttcaa ataaagctta ttttgagtgg actattgagg ccagagatct
     2161 ttctctgttg ggttcccatg ttcgtaaagc tacctaccag acttacgctc caattcttta
     2221 tgagaatgac cactttttcg actacatgca aaaaagtaag tttcatctca ccattgaagg
     2281 tccaaaagta cttgcttatt tacctggttt atggattggt gatggattgt ctgacagggc
     2341 aactttttcg gttgattcca gagatacttc tttgatggaa cgtgttactg aatatgctga
     2401 aaagttgaat ttgtgcgccg agtataagga cagaaaagaa ccacaagttg ccaaaactgt
     2461 taatttgtac tctaaagttg tcagaggtaa tggtattcgc aataatctta atactgagaa
     2521 tccattatgg gacgctattg ttggcttagg attcttgaag gacggtgtca aaaatattcc
     2581 ttctttcttg tctacggaca atatcggtac tcgtgaaaca tttcttgctg gtctaattga
     2641 ttctgatggc tatgttactg atgagcatgg tattaaagca acaataaaga caattcatac
     2701 ttctgtcaga gatggtttgg tttcccttgc tcgttcttta ggcttagtag tctcggttaa
     2761 cgcagaacct gctaaggttg acatgaatgg caccaaacat aaaattagtt atgctattta
     2821 tatgtctggt ggagatgttt tgcttaacgt tctttcgaag tgtgccggct ctaaaaaatt
     2881 caggcctgct cccgccgctg cttttgcacg tgagtgccgc ggattttatt tcgagttaca
     2941 agaattgaag gaagacgatt attatgggat tactttatct gatgattctg atcatcagtt
     3001 tttgcttgcc aaccaggttg tcgtccataa ttgcggctac attaaggaga aaaacaagag
     3061 tgagttctcc tggcttgact tcgtcgtcac gaaattctcc gacatcaacg acaagatcat
     3121 tccggtcttt caggaaaaca cgctcatcgg cgtgaagctc gaggacttcg aggattggtg
     3181 taaggtcgct aagctgatcg aggagaaaaa gcacctgaca gaaagtggcc tggacgagat
     3241 caagaagatt aagctgaaca tgaacaaggg cagagtattc taagcggccg ctcagaattg
     3301 gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg cggctttgtt
     3361 gaataaatcg aacttttgct gagttgaagg atcagatcac gcatcttccc gacaacgcag
     3421 accgttccgt ggcaaagcaa aagttcaaaa tcaccaactg gtccacctac aacaaagctc
     3481 tcatcaaccg tggctccctc actttctggc tggatgatgg ggcgattcag gcctggtatg
     3541 agtcagcaac accttcttca cgaggcagac ctcagcgctc aaagatgcag gggtaaaagc
     3601 taaccgcatc tttaccgaca aggcatccgg cagttcaaca gatcgggaag ggctggattt
     3661 gctgaggatg aaggtggagg aaggtgatgt cattctggtg aagaagctcg accgtcttgg
     3721 ccgcgacacc gccgacatga tccaactgat aaaagagttt gatgctcagg gtgtagcggt
     3781 tcggtttatt gacgacggga tcagtaccga cggtgatatg gggcaaatgg tggtcaccat
     3841 cctgtcggct gtggcacagg ctgaacgccg gaggatccta gagcgcacga atgagggccg
     3901 acaggaagca aagctgaaag gaatcaaatt tggccgcagg cgtaccgtgg acaggaacgt
     3961 cgtgctgacg cttcatcaga agggcactgg tgcaacggaa attgctcatc agctcagtat
     4021 tgcccgctcc acggtttata aaattcttga agacgaaagg gcctcgtgat acgcctattt
     4081 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4141 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4201 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4261 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4321 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4381 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4441 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     4501 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4561 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4621 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4681 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4741 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     4801 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4861 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4921 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4981 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5041 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5101 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5161 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5221 ccttaacgtg agttttcgtt ccactgagcg tcagacccct taataagatg atcttcttga
     5281 gatcgttttg gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc cttgcagggc
     5341 ggtttttcga aggttctctg agctaccaac tctttgaacc gaggtaactg gcttggagga
     5401 gcgcagtcac caaaacttgt cctttcagtt tagccttaac c
//
