LOCUS       sequence-422555-        7785 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses thermosensitive variants of the homing endonucleases 
            I-OnuI and I-GpeMI, each in a different open reading frame. Both 
            were made thermosensitive by a fusion with the L212P VMA1 intein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 3623 to 3657)
  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 7785)
  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: pDual
FEATURES             Location/Qualifiers
     source          1..7785
                     /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"
     promoter        3623..3657
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     primer_bind     complement(6405..6423)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6491..6595
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6596..6664,6665..7456)
                     /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(6814..6833)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7621..7785,1..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 agttgacggc tagctcagtc ctaggtacag tgctagcgca
     3661 tccggcagtt caacagatcg ggaagggaag gagatatacc catgggccct accagaaacg
     3721 agagtataaa tccttgggtg ctgactggat tcgccgatgc agaagggagt ttcatcctgc
     3781 gtattcggaa taacaataaa agttccgccg gatacagtac ggaacttggg tttcaaataa
     3841 cgctgcacaa aaaagacata tctattttgg agaatataca atccacgtgg aaggttggtg
     3901 tgatcgccaa tagcggtgac aatgctgtca gcttaaaagt caccagattc gaggacttac
     3961 gcgttgtgct taatcatttc gagaaatacc ccctgattac tcagaaactt ggtgactatc
     4021 ttttgtttaa gcaagcgttc agcgtgatgg aaaataagga gcacctgaaa atagaaggaa
     4081 tcaagagact ggtcgggatt aaggccaacc tgaattgggg acttactgac gagttgaagg
     4141 aagcgtttgt tgcctccggc ggcgagaata ttttcgttgc aagtgggggg gaacgtagcc
     4201 ttattaacaa gaacatacct aatagcggtt ggttagcagg atttacgagt ggggaggggt
     4261 gtttcttcgt tagtttaatt aaaagcaagt ccaagcttgg agtacaggta caacttgtgt
     4321 tttcaattac tcaacacgct cgggacagag ccctgatgga taacttggtt acgtatttag
     4381 gatgctttgc aaagggtaca aatgttttga tggcggacgg gtctatcgaa tgtatcgaaa
     4441 acatcgaggt tggcaataag gtgatgggta aggatggccg tcctcgtgaa gtaattaagt
     4501 tgccccgtgg aagagagact atgtatagcg tcgttcagaa aagccagcac cgtgcccaca
     4561 agagtgacag cagtcgtgag gtgccagagt tactcaaatt tacgtgcaat gcgacacatg
     4621 agctggttgt tcgtacacct cgcagtgtcc gtcgtttgag ccgtaccatc aagggtgtgg
     4681 aatatttcga agttatcact tttgaaatgg gccagaagaa agcacccgac ggcagaattg
     4741 tggagcttgt gaaggaagtg tcaaagagtt acccaatttc tgagggtcct gagcgtgcca
     4801 acgagttagt agagtcctat cgtaaggcta gcaataaagc ctattttgaa tggactatcg
     4861 aggcccgtga tcttagtctg ttgggctccc atgtgcgtaa agccacctac caaacttacg
     4921 ccccaattct gtatgagaac gaccacttct tcgactatat gcaaaagagt aagttccatc
     4981 tcacaattga aggcccaaaa gtgcttgctt acttacctgg cttatggatc ggtgatggct
     5041 tgtctgatag ggcaaccttt tcggtggatt cccgtgatac tagcttgatg gagcgtgtta
     5101 ccgaatatgc cgaaaagctg aatttgtgtg ccgagtacaa ggaccgtaaa gaaccgcaag
     5161 ttgcaaaaac tgtgaatttg tattctaaag tggtcagagg caatggtatc cgcaataacc
     5221 ttaacaccga gaatccgtta tgggatgcta ttgtgggctt aggcttcttg aaagacggtg
     5281 tgaaaaatat cccttctttt ttgtctaccg acaatattgg tactcgcgaa acattccttg
     5341 ctggcctaat tgactctgat ggttatgtta ccgatgagca cggtatcaag gcaacaatta
     5401 agacaatcca tactagcgtc cgtgacggtt tggtgtccct tgcccgttct ctgggcttag
     5461 tggtctcggt gaacgcagag cctgctaaag ttgacatgaa cggcacaaaa cataagatta
     5521 gttacgctat ttacatgtct ggcggagatg tgttgcttaa tgttctttca aagtgtgcag
     5581 gctctaagaa attccgtcct gctccggccg ctgcctttgt acgcgagtgc cgtggatttt
     5641 acttcgagct gcaagaatta aaggaagatg attattacgg gattacctta tctgacgatt
     5701 ctgaccatca gttcttgctt gcaaaccagg tggtcgtcca caattgcggg tatatcaagg
     5761 agaaaaagaa aagcgagttt tcttggctgg aatttgtggt cacaaagttc tctgacataa
     5821 aagacaagat tataccggtc tttcaagtga ataatattat aggtgtcaaa cttgaggact
     5881 tcgaagactg gtgcaaggtg gccaagctga ttgaggagaa gaagcattta actgaaagcg
     5941 gtttggaaga aatccgcaat atcaaattga acatgaacaa agggagagta ttataactgg
     6001 atttgctgag gatgaaggtg gaggaaggtg atgtcattct ggtgaagaag ctcgaccgtc
     6061 ttggccgcga caccgccgac atgatccaac tgataaaaga gtttgatgct cagggtgtag
     6121 cggttcggtt tattgacgac gggatcagta ccgacggtga tatggggcaa atggtggtca
     6181 ccatcctgtc ggctgtggca caggctgaac gccggaggat cctagagcgc acgaatgagg
     6241 gccgacagga agcaaagctg aaaggaatca aatttggccg caggcgtacc gtggacagga
     6301 acgtcgtgct gacgcttcat cagaagggca ctggtgcaac ggaaattgct catcagctca
     6361 gtattgcccg ctccacggtt tataaaattc ttgaagacga aagggcctcg tgatacgcct
     6421 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     6481 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     6541 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6601 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6661 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6721 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6781 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     6841 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6901 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6961 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     7021 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     7081 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     7141 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     7201 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     7261 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     7321 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     7381 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     7441 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     7501 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     7561 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccttaataa gatgatcttc
     7621 ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca
     7681 gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta actggcttgg
     7741 aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taacc
//
