LOCUS       sequence-305687-       10273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Clostridial vector, encoding a targetron carrying two lox511/71 and 
            loxFAS/66 sites in tandem in the forward (sense) orientation. 
            Retarget intron to insert double-lox site onto genome at desired 
            site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10273)
  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 10273)
  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"; journalName: "mSphere."; 
            date: "2019-12-11"; volume: "4(6). pii: 4/6/e00710-19. doi"; pages: 
            " 10.1128/mSphere.00710-19"
COMMENT     Sequence Label: pQadd1F
FEATURES             Location/Qualifiers
     source          1..10273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2032..2048)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2209..2225)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2551..2922)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            2955..3064
                     /note="incP origin of transfer"
     rep_origin      7713..8301
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8202..8221
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8455..8472
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8589..8610
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8625..8630,8631..8648,8649..8655)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8663..8679
                     /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     8668..8690
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8687..8703
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8687..8703
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(9898..9931)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     10088..10107
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        10088..10106
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             join(10269..10273,1..18)
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ttaactttaa gaaggagata tacatatatg gctagatcgt ccattccgac agcatcgcca
       61 gtcactatgg cgtgctgcta gcgctatatg cgttgatgca atttctatgc actcgtagta
      121 gtctgagaag ggtaacgccc tttacatggc aaaggggtac agttattgtg tactaaaatt
      181 aaaaattgat tagggaggaa aacctcaaaa tgaaaccaac aatggcaatt ttagaaagaa
      241 tcagtaaaaa ttcacaagaa aatatagacg aagtttttac aagactttat cgttatcttt
      301 tacgtccaga tatttattac gtggcgtatc aaaatttata ttccaataaa ggagcttcca
      361 caaaaggaat attagatgat acagcggatg gctttagtga agaaaaaata aaaaagatta
      421 ttcaatcttt aaaagacgga acttactatc ctcaacctgt acgaagaatg tatattgcaa
      481 aaaagaattc taaaaagatg agacctttag gaattccaac tttcacagat aaattgatcc
      541 aagaagctgt gagaataatt cttgaatcta tctatgaacc ggtattcgaa gatgtgtctc
      601 acggttttag acctcaacga agctgtcaca cagctttgaa aacaatcaaa agagagtttg
      661 gcggcgcaag atggtttgtg gagggagata taaaaggctg cttcgataat atagaccacg
      721 ttacactcat tggactcatc aatcttaaaa tcaaagatat gaaaatgagc caattgattt
      781 ataaatttct aaaagcaggt tatctggaaa actggcagta tcacaaaact tacagcggaa
      841 cacctcaagg tggaattcta tctcctcttt tggccaacat ctatcttcat gaattggata
      901 agtttgtttt acaactcaaa atgaagtttg accgagaaag tccagaaaga ataacacctg
      961 aatatcggga gctccacaat gagataaaaa gaatttctca ccgtctcaag aagttggagg
     1021 gtgaagaaaa agctaaagtt cttttagaat atcaagaaaa acgtaaaaga ttacccacac
     1081 tcccctgtac ctcacagaca aataaagtat tgaaatacgt ccggtatgcg gacgacttca
     1141 ttatctctgt taaaggaagc aaagaggact gtcaatggat aaaagaacaa ttaaaacttt
     1201 ttattcataa caagctaaaa atggaattga gtgaagaaaa aacactcatc acacatagca
     1261 gtcaacccgc tcgttttctg ggatatgata tacgagtaag gagatctgga acgataaaac
     1321 gatctggtaa agtcaaaaag agaacactca atgggagtgt agaactcctt attcctcttc
     1381 aagacaaaat tcgtcaattt atttttgaca agaaaatagc tatccaaaag aaagatagct
     1441 catggtttcc agttcacagg aaatatctta ttcgttcaac agacttagaa atcatcacaa
     1501 tttataattc tgaactccgc gggatttgta attactacgg tctagcaagt aattttaacc
     1561 agctcaatta ttttgcttat cttatggaat acagctgtct aaaaacgata gcctccaaac
     1621 ataagggaac actttcaaaa accatttcca tgtttaaaga tggaagtggt tcgtggggga
     1681 tcccgtatga gataaagcaa ggtaagcagc gccgttattt tgcaaatttt agtgaatgta
     1741 aatcccctta tcaatttacg gatgagataa gtcaagctcc tgtattgtat ggctatgccc
     1801 ggaatactct tgaaaacagg ttaaaagcta aatgttgtga attatgtggg acgtctgatg
     1861 aaaatacttc ctatgaaatt caccatgtca ataaggtcaa aaatcttaaa ggcaaagaaa
     1921 aatgggaaat ggcaatgata gcgaaacaac gtaaaactct tgttgtatgc tttcattgtc
     1981 atcgtcacgt gattcataaa cacaagtgag atgggtaccc tcgaggaatt cactggccgt
     2041 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     2101 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     2161 acagttgcgc agcctgaatg gcgaatggcg ggtaccgagc tcgaattcac tggccgtcgt
     2221 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     2281 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     2341 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     2401 cggtatttca caccgcatag gggatcggtc ttgccttgct cgtcggtgat gtacttcacc
     2461 agctccgcga agtcgctctt cttgatggag cgcatgggga cgtgcttggc aatcacgcgc
     2521 accccccggc cgttttagcg gctaaaaaag tcatggctct gccctcgggc ggaccacgcc
     2581 catcatgacc ttgccaagct cgtcctgctt ctcttcgatc ttcgccagca gggcgaggat
     2641 cgtggcatca ccgaaccgcg ccgtgcgcgg gtcgtcggtg agccagagtt tcagcaggcc
     2701 gcccaggcgg cccaggtcgc cattgatgcg ggccagctcg cggacgtgct catagtccac
     2761 gacgcccgtg attttgtagc cctggccgac ggccagcagg taggccgaca ggctcatgcc
     2821 ggccgccgcc gccttttcct caatcgctct tcgttcgtct ggaaggcagt acaccttgat
     2881 aggtgggctg cccttcctgg ttggcttggt ttcatcagcc atccgcttgc cctcatctgt
     2941 tacgccggcg gtagccggcc agcctcgcag agcaggattc ccgttgagca ccgccaggtg
     3001 cgaataaggg acagtgaaga aggaacaccc gctcgcgggt gggcctactt cacctatcct
     3061 gcccggctga cgccgttgga tacaccaagg aaagtctaca cgaacccttt ggcaaaatcc
     3121 tgtatatcgt gcgaaaaagg atggatatac cgaaaaaatc gctataatga ccccgaagca
     3181 gggttatgca gcggaaaaga tcccccgaag caaacttaag agtgtgttga tagtgcagta
     3241 tcttaaaatt ttgtgtataa taggaattga agttaaatta gatgctaaaa atttgtaatt
     3301 aagaaggagg gattcgtcat gttggtattc caaatgcgta atgtagataa aacatctact
     3361 gttttgaaac agactaaaaa cagtgattac gcagataaat aaatacgtta gattaattcc
     3421 taccagtgac taatcttatg actttttaaa cagataacta aaattacaaa caaatcgttt
     3481 aacttctgta tttatttaca gatgtaatca cttcaggagt aattacatga acaaaaatat
     3541 aaaatattct caaaactttt taacgagtga aaaagtactc aaccaaataa taaaacaatt
     3601 gaatttaaaa gaaaccgata ccgtttacga aattggaaca ggtaaagggc atttaacgac
     3661 gaaactggct aaaataagta aacaggtaac gtctattgaa ttagacagtc atctattcaa
     3721 cttatcgtca gaaaaattaa aactgaacat tcgtgtcact ttaattcacc aagatattct
     3781 acagtttcaa ttccctaaca aacagaggta taaaattgtt gggagtattc cttaccattt
     3841 aagcacacaa attattaaaa aagtggtttt tgaaagccat gcgtctgaca tctatctgat
     3901 tgttgaagaa ggattctaca agcgtacctt ggatattcac cgaacactag ggttgctctt
     3961 gcacactcaa gtctcgattc agcaattgct taagctgcca gcggaatgct ttcatcctaa
     4021 accaaaagta aacagtgtct taataaaact tacccgccat accacagatg ttccagataa
     4081 atattggaag ctatatacgt actttgtttc aaaatgggtc aatcgagaat atcgtcaact
     4141 gtttactaaa aatcagtttc atcaagcaat gaaacacgcc aaagtaaaca atttaagtac
     4201 cattacttat gagcaagtat tgtctatttt taatagttat ctattattta acgggaggaa
     4261 ataattctat gagtcgcttt tttaaatttg gaaagttaca cgttactaaa gggaatggag
     4321 ataaattatt agatatacta ctgacagctt ccaagaagct aaagaggtcc ctagcgccta
     4381 cggggaattt gggcaaatca aaagaaaaat tctcccaaga attagaaatg agtagatcaa
     4441 attattcacg aatagaatca ggaaaatcag atccaaccat aaaaacacta gaacaaattg
     4501 caaagttaac taactcaacg ctagtagtgg atttaatccc aaatgagcca acagaaccag
     4561 aaccagaaac agaatcagaa caagtaacat tggatttaga aatggaagaa gaaaaaagca
     4621 atgacttcgt gtgaataatg cacgaaatcg ttgcttattt tttttaaaag cggtatacta
     4681 gatataacga aacaacgaac tgaatagaaa cgaaaaaaga gccatgacac atttataaaa
     4741 tgtttgacga cattttataa atgcatagcc cgataagatt gccaaaccaa cgcttatcag
     4801 ttagtcagat gaactcttcc ctcgtaagaa gttatttaat taactttgtt tgaagacggt
     4861 atataaccgt actatcatta tatagggaaa tcagagagtt ttcaagtatc taagctactg
     4921 aatttaagaa ttgttaagca atcaatcgga aatcgtttga ttgctttttt tgtattcatt
     4981 tatagaaggt ggagtttgta tgaatcatga tgaatgtaaa acttatataa aaaatagttt
     5041 attggagata agaaaattag caaatatcta tacactagaa acgtttaaga aagagttaga
     5101 aaagagaaat atctacttag aaacaaaatc agataagtat ttttcttcgg agggggaaga
     5161 ttatatatat aagttaatag aaaataacaa aataatttat tcgattagtg gaaaaaaatt
     5221 gacttataaa ggaaaaaaat ctttttcaaa acatgcaata ttgaaacagt tgaatgaaaa
     5281 agcaaaccaa gttaattaaa caacctattt tataggattt ataggaaagg agaacagctg
     5341 aatgaatatc ccttttgttg tagaaactgt gcttcatgac ggcttgttaa agtacaaatt
     5401 taaaaatagt aaaattcgct caatcactac caagccaggt aaaagcaaag gggctatttt
     5461 tgcgtatcgc tcaaaatcaa gcatgattgg cggtcgtggt gttgttctga cttccgagga
     5521 agcgattcaa gaaaatcaag atacatttac acattggaca cccaacgttt atcgttatgg
     5581 aacgtatgca gacgaaaacc gttcatacac gaaaggacat tctgaaaaca atttaagaca
     5641 aatcaatacc ttctttattg attttgatat tcacacggca aaagaaacta tttcagcaag
     5701 cgatatttta acaaccgcta ttgatttagg ttttatgcct actatgatta tcaaatctga
     5761 taaaggttat caagcatatt ttgttttaga aacgccagtc tatgtgactt caaaatcaga
     5821 atttaaatct gtcaaagcag ccaaaataat ttcgcaaaat atccgagaat attttggaaa
     5881 gtctttgcca gttgatctaa cgtgtaatca ttttggtatt gctcgcatac caagaacgga
     5941 caatgtagaa ttttttgatc ctaattaccg ttattctttc aaagaatggc aagattggtc
     6001 tttcaaacaa acagataata agggctttac tcgttcaagt ctaacggttt taagcggtac
     6061 agaaggcaaa aaacaagtag atgaaccctg gtttaatctc ttattgcacg aaacgaaatt
     6121 ttcaggagaa aagggtttaa tagggcgtaa taacgtcatg tttaccctct ctttagccta
     6181 ctttagttca ggctattcaa tcgaaacgtg cgaatataat atgtttgagt ttaataatcg
     6241 attagatcaa cccttagaag aaaaagaagt aatcaaaatt gttagaagtg cctattcaga
     6301 aaactatcaa ggggctaata gggaatacat taccattctt tgcaaagctt gggtatcaag
     6361 tgatttaacc agtaaagatt tatttgtccg tcaagggtgg tttaaattca agaaaaaaag
     6421 aagcgaacgt caacgtgttc atttgtcaga atggaaagaa gatttaatgg cttatattag
     6481 cgaaaaaagc gatgtataca agccttattt agtgacgacc aaaaaagaga ttagagaagt
     6541 gctaggcatt cctgaacgga cattagataa attgctgaag gtactgaagg cgaatcagga
     6601 aattttcttt aagattaaac caggaagaaa tggtggcatt caacttgcta gtgttaaatc
     6661 attgttgcta tcgatcatta aagtaaaaaa agaagaaaaa gaaagctata taaaggcgct
     6721 gacaaattct tttgacttag agcatacatt cattcaagag actttaaaca agctagcaga
     6781 acgccctaaa acggacacac aactcgattt gtttagctat gatacaggct gaaaataaaa
     6841 cccgcactat gccattacat ttatatctat gatacgtgtt tgttttttct ttgctgttta
     6901 gcgaatgatt agcagaaata tacagagtaa gattttaatt aattattagg gggagaagga
     6961 gagagtagcc cgaaaacttt tagttggctt ggactgaacg aagtgaggga aaggctacta
     7021 aaacgtcgag gggcagtgag agcgaagcga acacttgatt ttttaatttt ctatctttta
     7081 taggtcatta gagtatactt atttgtccta taaactattt agcagcataa tagatttatt
     7141 gaataggtca tttaagttga gcatattaga ggaggaaaat cttggagaaa tatttgaaga
     7201 acccgattac atggattgga ttagttcttg tggttacgtg gtttttaact aaaagtagtg
     7261 aatttttgat ttttggtgtg tgtgtcttgt tgttagtatt tgctagtcaa agtgattaaa
     7321 tagaattgac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     7381 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     7441 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     7501 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     7561 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     7621 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7681 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7741 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7801 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     7861 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7921 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     7981 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     8041 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     8101 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     8161 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     8221 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     8281 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     8341 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     8401 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     8461 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     8521 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     8581 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     8641 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     8701 gaccatgatt acgccaagct tgcatgcctg cagtttggtc aacatttaac ctctaatctt
     8761 ctagttcatt tatatgtcca tgaattgaat ttcatacata gttttcaatt gtatggatgt
     8821 agtatgacag aaaatcaaaa aatcatactt attatattta ggtagttact gttaattttt
     8881 agtatgttaa tttctaatct gagttctttc cgtattataa taagtgtaaa gatcacacca
     8941 tatttttaca gtttttacac gtttcattaa taatttctta tatttcttta tttgtttgta
     9001 aaatttactt aaatttcgcc agaaaacaaa agaaagcctt tactaattaa tagtttagtg
     9061 atactctttt atgtaggtat tttttaaaat acattaaacc taggtaattg aggaaagtta
     9121 caattaccat tatataagga ggatattcaa accatatggg atccccagct tcgtcgatcg
     9181 tgaagattta atcaatgtgc gcccagatag ggtgttaagt caagtagttt aaggtactac
     9241 tctgtaagat aacacagaaa acagccaacc taaccgaaaa gcgaaagctg atacgggaac
     9301 agagcacggt tggaaagcga tgagttacct aaagacaatc gggtacgact gagtcgcaat
     9361 gttaatcaga tataaggtat aagttgtgtt tactgaacgc aagtttctaa tttcggttaa
     9421 atctcgatag aggaaagtgt ctgaaacctc tagtacaaag aaaggtaagt tagcattgat
     9481 gacttatctg ttatcaccac atttgtacaa tctgtaggag aacctatggg aacgaaacga
     9541 aagcgatgcc gagaatctga atttaccaag acttaacact aactggggat accctaaaca
     9601 agaatgccta atagaaagga ggaaaaaggc tatagcacta gagcttgaaa atcttgcaag
     9661 ggtacggagt actcgtagta gtctgagaag ggtaacgccc tttacatggc aaaggggtac
     9721 agttattgtg tactaaaatt aaaaattgat tagggaggaa aacctcaaaa tgaaaccaac
     9781 aatggcaatt ttagaaagaa tcagtaaaaa ttcacaagaa aatatagacg aagtttttac
     9841 aagactttat cgttatcttt tacgtccaga tatttattac gtggcgacgc gtgagagtac
     9901 cgttcgtata gtatacatta tacgaagtta tgagagttta aacgagaata acttcgtata
     9961 tacctttcta tacgaacggt agagagacgc gttgggaaat ggcaatgata gcgaaacaac
    10021 gtaaaactct tgttgtatgc tttcattgtc atcgtcacgt gattcataaa cacaagtgaa
    10081 tgtcgactaa tacgactcac tatagggtcg acagtgaatt tttacgaacg aacaataaca
    10141 gagccgtata ctccgagagg ggtacgtacg gttcccgaag agggtggtgc aaaccagtca
    10201 cagtaatgtg aacaaggcgg tacctcccta cttcaccata tcattttctg cagcccccta
    10261 gaaataattt tgt
//
