LOCUS       sequence-275772-       10213 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Clostridial vector, encoding a targetron carrying a lox66 site in 
            the forward (sense) orientation. Retarget intron to insert lox66 
            onto genome at desired site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10213)
  AUTHORS   Cerisy T, Rostain W, Chhun A, Boutard M, Salanoubat M, Tolonen AC
  TITLE     A Targetron-Recombinase System for Large-Scale Genome Engineering of
            Clostridia.
  JOURNAL   mSphere. 2019 Dec 11;4(6). pii: 4/6/e00710-19. doi: 
            10.1128/mSphere.00710-19.
  PUBMED    31826971
REFERENCE   2  (bases 1 to 10213)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "mSphere."; 
            date: "2019-12-11"; volume: "4(6). pii: 4/6/e00710-19. doi"; pages: 
            " 10.1128/mSphere.00710-19"
COMMENT     Sequence Label: pQlox66F
FEATURES             Location/Qualifiers
     source          1..10213
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(194..565)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            598..707
                     /note="incP origin of transfer"
     rep_origin      5356..5944
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5845..5864
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6098..6115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6232..6253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6268..6273,6274..6291,6292..6298)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6306..6322
                     /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)."
     primer_bind     6311..6333
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6330..6346
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6330..6346
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(7536..7569)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     7671..7690
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7671..7689
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             7852..7874
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(9888..9904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10065..10081)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
       61 taggggatcg gtcttgcctt gctcgtcggt gatgtacttc accagctccg cgaagtcgct
      121 cttcttgatg gagcgcatgg ggacgtgctt ggcaatcacg cgcacccccc ggccgtttta
      181 gcggctaaaa aagtcatggc tctgccctcg ggcggaccac gcccatcatg accttgccaa
      241 gctcgtcctg cttctcttcg atcttcgcca gcagggcgag gatcgtggca tcaccgaacc
      301 gcgccgtgcg cgggtcgtcg gtgagccaga gtttcagcag gccgcccagg cggcccaggt
      361 cgccattgat gcgggccagc tcgcggacgt gctcatagtc cacgacgccc gtgattttgt
      421 agccctggcc gacggccagc aggtaggccg acaggctcat gccggccgcc gccgcctttt
      481 cctcaatcgc tcttcgttcg tctggaaggc agtacacctt gataggtggg ctgcccttcc
      541 tggttggctt ggtttcatca gccatccgct tgccctcatc tgttacgccg gcggtagccg
      601 gccagcctcg cagagcagga ttcccgttga gcaccgccag gtgcgaataa gggacagtga
      661 agaaggaaca cccgctcgcg ggtgggccta cttcacctat cctgcccggc tgacgccgtt
      721 ggatacacca aggaaagtct acacgaaccc tttggcaaaa tcctgtatat cgtgcgaaaa
      781 aggatggata taccgaaaaa atcgctataa tgaccccgaa gcagggttat gcagcggaaa
      841 agatcccccg aagcaaactt aagagtgtgt tgatagtgca gtatcttaaa attttgtgta
      901 taataggaat tgaagttaaa ttagatgcta aaaatttgta attaagaagg agggattcgt
      961 catgttggta ttccaaatgc gtaatgtaga taaaacatct actgttttga aacagactaa
     1021 aaacagtgat tacgcagata aataaatacg ttagattaat tcctaccagt gactaatctt
     1081 atgacttttt aaacagataa ctaaaattac aaacaaatcg tttaacttct gtatttattt
     1141 acagatgtaa tcacttcagg agtaattaca tgaacaaaaa tataaaatat tctcaaaact
     1201 ttttaacgag tgaaaaagta ctcaaccaaa taataaaaca attgaattta aaagaaaccg
     1261 ataccgttta cgaaattgga acaggtaaag ggcatttaac gacgaaactg gctaaaataa
     1321 gtaaacaggt aacgtctatt gaattagaca gtcatctatt caacttatcg tcagaaaaat
     1381 taaaactgaa cattcgtgtc actttaattc accaagatat tctacagttt caattcccta
     1441 acaaacagag gtataaaatt gttgggagta ttccttacca tttaagcaca caaattatta
     1501 aaaaagtggt ttttgaaagc catgcgtctg acatctatct gattgttgaa gaaggattct
     1561 acaagcgtac cttggatatt caccgaacac tagggttgct cttgcacact caagtctcga
     1621 ttcagcaatt gcttaagctg ccagcggaat gctttcatcc taaaccaaaa gtaaacagtg
     1681 tcttaataaa acttacccgc cataccacag atgttccaga taaatattgg aagctatata
     1741 cgtactttgt ttcaaaatgg gtcaatcgag aatatcgtca actgtttact aaaaatcagt
     1801 ttcatcaagc aatgaaacac gccaaagtaa acaatttaag taccattact tatgagcaag
     1861 tattgtctat ttttaatagt tatctattat ttaacgggag gaaataattc tatgagtcgc
     1921 ttttttaaat ttggaaagtt acacgttact aaagggaatg gagataaatt attagatata
     1981 ctactgacag cttccaagaa gctaaagagg tccctagcgc ctacggggaa tttgggcaaa
     2041 tcaaaagaaa aattctccca agaattagaa atgagtagat caaattattc acgaatagaa
     2101 tcaggaaaat cagatccaac cataaaaaca ctagaacaaa ttgcaaagtt aactaactca
     2161 acgctagtag tggatttaat cccaaatgag ccaacagaac cagaaccaga aacagaatca
     2221 gaacaagtaa cattggattt agaaatggaa gaagaaaaaa gcaatgactt cgtgtgaata
     2281 atgcacgaaa tcgttgctta ttttttttaa aagcggtata ctagatataa cgaaacaacg
     2341 aactgaatag aaacgaaaaa agagccatga cacatttata aaatgtttga cgacatttta
     2401 taaatgcata gcccgataag attgccaaac caacgcttat cagttagtca gatgaactct
     2461 tccctcgtaa gaagttattt aattaacttt gtttgaagac ggtatataac cgtactatca
     2521 ttatataggg aaatcagaga gttttcaagt atctaagcta ctgaatttaa gaattgttaa
     2581 gcaatcaatc ggaaatcgtt tgattgcttt ttttgtattc atttatagaa ggtggagttt
     2641 gtatgaatca tgatgaatgt aaaacttata taaaaaatag tttattggag ataagaaaat
     2701 tagcaaatat ctatacacta gaaacgttta agaaagagtt agaaaagaga aatatctact
     2761 tagaaacaaa atcagataag tatttttctt cggaggggga agattatata tataagttaa
     2821 tagaaaataa caaaataatt tattcgatta gtggaaaaaa attgacttat aaaggaaaaa
     2881 aatctttttc aaaacatgca atattgaaac agttgaatga aaaagcaaac caagttaatt
     2941 aaacaaccta ttttatagga tttataggaa aggagaacag ctgaatgaat atcccttttg
     3001 ttgtagaaac tgtgcttcat gacggcttgt taaagtacaa atttaaaaat agtaaaattc
     3061 gctcaatcac taccaagcca ggtaaaagca aaggggctat ttttgcgtat cgctcaaaat
     3121 caagcatgat tggcggtcgt ggtgttgttc tgacttccga ggaagcgatt caagaaaatc
     3181 aagatacatt tacacattgg acacccaacg tttatcgtta tggaacgtat gcagacgaaa
     3241 accgttcata cacgaaagga cattctgaaa acaatttaag acaaatcaat accttcttta
     3301 ttgattttga tattcacacg gcaaaagaaa ctatttcagc aagcgatatt ttaacaaccg
     3361 ctattgattt aggttttatg cctactatga ttatcaaatc tgataaaggt tatcaagcat
     3421 attttgtttt agaaacgcca gtctatgtga cttcaaaatc agaatttaaa tctgtcaaag
     3481 cagccaaaat aatttcgcaa aatatccgag aatattttgg aaagtctttg ccagttgatc
     3541 taacgtgtaa tcattttggt attgctcgca taccaagaac ggacaatgta gaattttttg
     3601 atcctaatta ccgttattct ttcaaagaat ggcaagattg gtctttcaaa caaacagata
     3661 ataagggctt tactcgttca agtctaacgg ttttaagcgg tacagaaggc aaaaaacaag
     3721 tagatgaacc ctggtttaat ctcttattgc acgaaacgaa attttcagga gaaaagggtt
     3781 taatagggcg taataacgtc atgtttaccc tctctttagc ctactttagt tcaggctatt
     3841 caatcgaaac gtgcgaatat aatatgtttg agtttaataa tcgattagat caacccttag
     3901 aagaaaaaga agtaatcaaa attgttagaa gtgcctattc agaaaactat caaggggcta
     3961 atagggaata cattaccatt ctttgcaaag cttgggtatc aagtgattta accagtaaag
     4021 atttatttgt ccgtcaaggg tggtttaaat tcaagaaaaa aagaagcgaa cgtcaacgtg
     4081 ttcatttgtc agaatggaaa gaagatttaa tggcttatat tagcgaaaaa agcgatgtat
     4141 acaagcctta tttagtgacg accaaaaaag agattagaga agtgctaggc attcctgaac
     4201 ggacattaga taaattgctg aaggtactga aggcgaatca ggaaattttc tttaagatta
     4261 aaccaggaag aaatggtggc attcaacttg ctagtgttaa atcattgttg ctatcgatca
     4321 ttaaagtaaa aaaagaagaa aaagaaagct atataaaggc gctgacaaat tcttttgact
     4381 tagagcatac attcattcaa gagactttaa acaagctagc agaacgccct aaaacggaca
     4441 cacaactcga tttgtttagc tatgatacag gctgaaaata aaacccgcac tatgccatta
     4501 catttatatc tatgatacgt gtttgttttt tctttgctgt ttagcgaatg attagcagaa
     4561 atatacagag taagatttta attaattatt agggggagaa ggagagagta gcccgaaaac
     4621 ttttagttgg cttggactga acgaagtgag ggaaaggcta ctaaaacgtc gaggggcagt
     4681 gagagcgaag cgaacacttg attttttaat tttctatctt ttataggtca ttagagtata
     4741 cttatttgtc ctataaacta tttagcagca taatagattt attgaatagg tcatttaagt
     4801 tgagcatatt agaggaggaa aatcttggag aaatatttga agaacccgat tacatggatt
     4861 ggattagttc ttgtggttac gtggttttta actaaaagta gtgaattttt gatttttggt
     4921 gtgtgtgtct tgttgttagt atttgctagt caaagtgatt aaatagaatt gaccacttct
     4981 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5041 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     5101 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     5161 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     5221 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5281 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5341 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5401 aaaaccaccg ctaccagcgg tggttttttt gccggatcaa gagctaccaa ctctttttcc
     5461 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5521 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5581 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5641 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5701 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5761 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5821 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5881 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5941 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6001 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     6061 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     6121 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     6181 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     6241 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     6301 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     6361 gcttgcatgc ctgcagtttg gtcaacattt aacctctaat cttctagttc atttatatgt
     6421 ccatgaattg aatttcatac atagttttca attgtatgga tgtagtatga cagaaaatca
     6481 aaaaatcata cttattatat ttaggtagtt actgttaatt tttagtatgt taatttctaa
     6541 tctgagttct ttccgtatta taataagtgt aaagatcaca ccatattttt acagttttta
     6601 cacgtttcat taataatttc ttatatttct ttatttgttt gtaaaattta cttaaatttc
     6661 gccagaaaac aaaagaaagc ctttactaat taatagttta gtgatactct tttatgtagg
     6721 tattttttaa aatacattaa acctaggtaa ttgaggaaag ttacaattac cattatataa
     6781 ggaggatatt caaaccatat gggatcccca gcttcgtcga tcgtgaagat ttaatcaatg
     6841 tgcgcccaga tagggtgtta agtcaagtag tttaaggtac tactctgtaa gataacacag
     6901 aaaacagcca acctaaccga aaagcgaaag ctgatacggg aacagagcac ggttggaaag
     6961 cgatgagtta cctaaagaca atcgggtacg actgagtcgc aatgttaatc agatataagg
     7021 tataagttgt gtttactgaa cgcaagtttc taatttcggt taaatctcga tagaggaaag
     7081 tgtctgaaac ctctagtaca aagaaaggta agttagcatt gatgacttat ctgttatcac
     7141 cacatttgta caatctgtag gagaacctat gggaacgaaa cgaaagcgat gccgagaatc
     7201 tgaatttacc aagacttaac actaactggg gataccctaa acaagaatgc ctaatagaaa
     7261 ggaggaaaaa ggctatagca ctagagcttg aaaatcttgc aagggtacgg agtactcgta
     7321 gtagtctgag aagggtaacg ccctttacat ggcaaagggg tacagttatt gtgtactaaa
     7381 attaaaaatt gattagggag gaaaacctca aaatgaaacc aacaatggca attttagaaa
     7441 gaatcagtaa aaattcacaa gaaaatatag acgaagtttt tacaagactt tatcgttatc
     7501 ttttacgtcc agatatttat tacgtggcga cgcgtataac ttcgtatagc atacattata
     7561 cgaacggtaa cgcgttggga aatggcaatg atagcgaaac aacgtaaaac tcttgttgta
     7621 tgctttcatt gtcatcgtca cgtgattcat aaacacaagt gaatgtcgac taatacgact
     7681 cactataggg tcgacagtga atttttacga acgaacaata acagagccgt atactccgag
     7741 aggggtacgt acggttcccg aagagggtgg tgcaaaccag tcacagtaat gtgaacaagg
     7801 cggtacctcc ctacttcacc atatcatttt ctgcagcccc ctagaaataa ttttgtttaa
     7861 ctttaagaag gagatataca tatatggcta gatcgtccat tccgacagca tcgccagtca
     7921 ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt ctatgcactc gtagtagtct
     7981 gagaagggta acgcccttta catggcaaag gggtacagtt attgtgtact aaaattaaaa
     8041 attgattagg gaggaaaacc tcaaaatgaa accaacaatg gcaattttag aaagaatcag
     8101 taaaaattca caagaaaata tagacgaagt ttttacaaga ctttatcgtt atcttttacg
     8161 tccagatatt tattacgtgg cgtatcaaaa tttatattcc aataaaggag cttccacaaa
     8221 aggaatatta gatgatacag cggatggctt tagtgaagaa aaaataaaaa agattattca
     8281 atctttaaaa gacggaactt actatcctca acctgtacga agaatgtata ttgcaaaaaa
     8341 gaattctaaa aagatgagac ctttaggaat tccaactttc acagataaat tgatccaaga
     8401 agctgtgaga ataattcttg aatctatcta tgaaccggta ttcgaagatg tgtctcacgg
     8461 ttttagacct caacgaagct gtcacacagc tttgaaaaca atcaaaagag agtttggcgg
     8521 cgcaagatgg tttgtggagg gagatataaa aggctgcttc gataatatag accacgttac
     8581 actcattgga ctcatcaatc ttaaaatcaa agatatgaaa atgagccaat tgatttataa
     8641 atttctaaaa gcaggttatc tggaaaactg gcagtatcac aaaacttaca gcggaacacc
     8701 tcaaggtgga attctatctc ctcttttggc caacatctat cttcatgaat tggataagtt
     8761 tgttttacaa ctcaaaatga agtttgaccg agaaagtcca gaaagaataa cacctgaata
     8821 tcgggagctc cacaatgaga taaaaagaat ttctcaccgt ctcaagaagt tggagggtga
     8881 agaaaaagct aaagttcttt tagaatatca agaaaaacgt aaaagattac ccacactccc
     8941 ctgtacctca cagacaaata aagtattgaa atacgtccgg tatgcggacg acttcattat
     9001 ctctgttaaa ggaagcaaag aggactgtca atggataaaa gaacaattaa aactttttat
     9061 tcataacaag ctaaaaatgg aattgagtga agaaaaaaca ctcatcacac atagcagtca
     9121 acccgctcgt tttctgggat atgatatacg agtaaggaga tctggaacga taaaacgatc
     9181 tggtaaagtc aaaaagagaa cactcaatgg gagtgtagaa ctccttattc ctcttcaaga
     9241 caaaattcgt caatttattt ttgacaagaa aatagctatc caaaagaaag atagctcatg
     9301 gtttccagtt cacaggaaat atcttattcg ttcaacagac ttagaaatca tcacaattta
     9361 taattctgaa ctccgcggga tttgtaatta ctacggtcta gcaagtaatt ttaaccagct
     9421 caattatttt gcttatctta tggaatacag ctgtctaaaa acgatagcct ccaaacataa
     9481 gggaacactt tcaaaaacca tttccatgtt taaagatgga agtggttcgt gggggatccc
     9541 gtatgagata aagcaaggta agcagcgccg ttattttgca aattttagtg aatgtaaatc
     9601 cccttatcaa tttacggatg agataagtca agctcctgta ttgtatggct atgcccggaa
     9661 tactcttgaa aacaggttaa aagctaaatg ttgtgaatta tgtgggacgt ctgatgaaaa
     9721 tacttcctat gaaattcacc atgtcaataa ggtcaaaaat cttaaaggca aagaaaaatg
     9781 ggaaatggca atgatagcga aacaacgtaa aactcttgtt gtatgctttc attgtcatcg
     9841 tcacgtgatt cataaacaca agtgagatgg gtaccctcga ggaattcact ggccgtcgtt
     9901 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     9961 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
    10021 ttgcgcagcc tgaatggcga atggcgggta ccgagctcga attcactggc cgtcgtttta
    10081 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
    10141 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
    10201 cgcagcctga atg
//
