LOCUS       sequence-305685-       10273 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Clostridial vector, encoding a targetron carrying two lox511/71 and 
            loxFAS/66 sites in tandem, in the reverse (antisense) orientation. 
            Retarget intron to insert double-lox site at desired location..
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 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: pQadd1R
FEATURES             Location/Qualifiers
     source          1..10273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    22..38
                     /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     27..49
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     46..62
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     46..62
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    1307..1340
                     /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     1447..1466
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1447..1465
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1628..1650
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(3664..3680)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3841..3857)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(4183..4554)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            4587..4696
                     /note="incP origin of transfer"
     rep_origin      9345..9933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9834..9853
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10087..10104
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10221..10242
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10257..10262,10263..10273,1..7,8..14)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
       61 accatgatta cgccaagctt gcatgcctgc agtttggtca acatttaacc tctaatcttc
      121 tagttcattt atatgtccat gaattgaatt tcatacatag ttttcaattg tatggatgta
      181 gtatgacaga aaatcaaaaa atcatactta ttatatttag gtagttactg ttaattttta
      241 gtatgttaat ttctaatctg agttctttcc gtattataat aagtgtaaag atcacaccat
      301 atttttacag tttttacacg tttcattaat aatttcttat atttctttat ttgtttgtaa
      361 aatttactta aatttcgcca gaaaacaaaa gaaagccttt actaattaat agtttagtga
      421 tactctttta tgtaggtatt ttttaaaata cattaaacct aggtaattga ggaaagttac
      481 aattaccatt atataaggag gatattcaaa ccatatggga tccccagctt cgtcgatcgt
      541 gaagatttaa tcaatgtgcg cccagatagg gtgttaagtc aagtagttta aggtactact
      601 ctgtaagata acacagaaaa cagccaacct aaccgaaaag cgaaagctga tacgggaaca
      661 gagcacggtt ggaaagcgat gagttaccta aagacaatcg ggtacgactg agtcgcaatg
      721 ttaatcagat ataaggtata agttgtgttt actgaacgca agtttctaat ttcggttaaa
      781 tctcgataga ggaaagtgtc tgaaacctct agtacaaaga aaggtaagtt agcattgatg
      841 acttatctgt tatcaccaca tttgtacaat ctgtaggaga acctatggga acgaaacgaa
      901 agcgatgccg agaatctgaa tttaccaaga cttaacacta actggggata ccctaaacaa
      961 gaatgcctaa tagaaaggag gaaaaaggct atagcactag agcttgaaaa tcttgcaagg
     1021 gtacggagta ctcgtagtag tctgagaagg gtaacgccct ttacatggca aaggggtaca
     1081 gttattgtgt actaaaatta aaaattgatt agggaggaaa acctcaaaat gaaaccaaca
     1141 atggcaattt tagaaagaat cagtaaaaat tcacaagaaa atatagacga agtttttaca
     1201 agactttatc gttatctttt acgtccagat atttattacg tggcgacgcg tctctctacc
     1261 gttcgtatag aaaggtatat acgaagttat tctcgtttaa actctcataa cttcgtataa
     1321 tgtatactat acgaacggta ctctcacgcg ttgggaaatg gcaatgatag cgaaacaacg
     1381 taaaactctt gttgtatgct ttcattgtca tcgtcacgtg attcataaac acaagtgaat
     1441 gtcgactaat acgactcact atagggtcga cagtgaattt ttacgaacga acaataacag
     1501 agccgtatac tccgagaggg gtacgtacgg ttcccgaaga gggtggtgca aaccagtcac
     1561 agtaatgtga acaaggcggt acctccctac ttcaccatat cattttctgc agccccctag
     1621 aaataatttt gtttaacttt aagaaggaga tatacatata tggctagatc gtccattccg
     1681 acagcatcgc cagtcactat ggcgtgctgc tagcgctata tgcgttgatg caatttctat
     1741 gcactcgtag tagtctgaga agggtaacgc cctttacatg gcaaaggggt acagttattg
     1801 tgtactaaaa ttaaaaattg attagggagg aaaacctcaa aatgaaacca acaatggcaa
     1861 ttttagaaag aatcagtaaa aattcacaag aaaatataga cgaagttttt acaagacttt
     1921 atcgttatct tttacgtcca gatatttatt acgtggcgta tcaaaattta tattccaata
     1981 aaggagcttc cacaaaagga atattagatg atacagcgga tggctttagt gaagaaaaaa
     2041 taaaaaagat tattcaatct ttaaaagacg gaacttacta tcctcaacct gtacgaagaa
     2101 tgtatattgc aaaaaagaat tctaaaaaga tgagaccttt aggaattcca actttcacag
     2161 ataaattgat ccaagaagct gtgagaataa ttcttgaatc tatctatgaa ccggtattcg
     2221 aagatgtgtc tcacggtttt agacctcaac gaagctgtca cacagctttg aaaacaatca
     2281 aaagagagtt tggcggcgca agatggtttg tggagggaga tataaaaggc tgcttcgata
     2341 atatagacca cgttacactc attggactca tcaatcttaa aatcaaagat atgaaaatga
     2401 gccaattgat ttataaattt ctaaaagcag gttatctgga aaactggcag tatcacaaaa
     2461 cttacagcgg aacacctcaa ggtggaattc tatctcctct tttggccaac atctatcttc
     2521 atgaattgga taagtttgtt ttacaactca aaatgaagtt tgaccgagaa agtccagaaa
     2581 gaataacacc tgaatatcgg gagctccaca atgagataaa aagaatttct caccgtctca
     2641 agaagttgga gggtgaagaa aaagctaaag ttcttttaga atatcaagaa aaacgtaaaa
     2701 gattacccac actcccctgt acctcacaga caaataaagt attgaaatac gtccggtatg
     2761 cggacgactt cattatctct gttaaaggaa gcaaagagga ctgtcaatgg ataaaagaac
     2821 aattaaaact ttttattcat aacaagctaa aaatggaatt gagtgaagaa aaaacactca
     2881 tcacacatag cagtcaaccc gctcgttttc tgggatatga tatacgagta aggagatctg
     2941 gaacgataaa acgatctggt aaagtcaaaa agagaacact caatgggagt gtagaactcc
     3001 ttattcctct tcaagacaaa attcgtcaat ttatttttga caagaaaata gctatccaaa
     3061 agaaagatag ctcatggttt ccagttcaca ggaaatatct tattcgttca acagacttag
     3121 aaatcatcac aatttataat tctgaactcc gcgggatttg taattactac ggtctagcaa
     3181 gtaattttaa ccagctcaat tattttgctt atcttatgga atacagctgt ctaaaaacga
     3241 tagcctccaa acataaggga acactttcaa aaaccatttc catgtttaaa gatggaagtg
     3301 gttcgtgggg gatcccgtat gagataaagc aaggtaagca gcgccgttat tttgcaaatt
     3361 ttagtgaatg taaatcccct tatcaattta cggatgagat aagtcaagct cctgtattgt
     3421 atggctatgc ccggaatact cttgaaaaca ggttaaaagc taaatgttgt gaattatgtg
     3481 ggacgtctga tgaaaatact tcctatgaaa ttcaccatgt caataaggtc aaaaatctta
     3541 aaggcaaaga aaaatgggaa atggcaatga tagcgaaaca acgtaaaact cttgttgtat
     3601 gctttcattg tcatcgtcac gtgattcata aacacaagtg agatgggtac cctcgaggaa
     3661 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3721 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3781 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgggtaccga gctcgaattc
     3841 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     3901 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     3961 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     4021 tacgcatctg tgcggtattt cacaccgcat aggggatcgg tcttgccttg ctcgtcggtg
     4081 atgtacttca ccagctccgc gaagtcgctc ttcttgatgg agcgcatggg gacgtgcttg
     4141 gcaatcacgc gcaccccccg gccgttttag cggctaaaaa agtcatggct ctgccctcgg
     4201 gcggaccacg cccatcatga ccttgccaag ctcgtcctgc ttctcttcga tcttcgccag
     4261 cagggcgagg atcgtggcat caccgaaccg cgccgtgcgc gggtcgtcgg tgagccagag
     4321 tttcagcagg ccgcccaggc ggcccaggtc gccattgatg cgggccagct cgcggacgtg
     4381 ctcatagtcc acgacgcccg tgattttgta gccctggccg acggccagca ggtaggccga
     4441 caggctcatg ccggccgccg ccgccttttc ctcaatcgct cttcgttcgt ctggaaggca
     4501 gtacaccttg ataggtgggc tgcccttcct ggttggcttg gtttcatcag ccatccgctt
     4561 gccctcatct gttacgccgg cggtagccgg ccagcctcgc agagcaggat tcccgttgag
     4621 caccgccagg tgcgaataag ggacagtgaa gaaggaacac ccgctcgcgg gtgggcctac
     4681 ttcacctatc ctgcccggct gacgccgttg gatacaccaa ggaaagtcta cacgaaccct
     4741 ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat accgaaaaaa tcgctataat
     4801 gaccccgaag cagggttatg cagcggaaaa gatcccccga agcaaactta agagtgtgtt
     4861 gatagtgcag tatcttaaaa ttttgtgtat aataggaatt gaagttaaat tagatgctaa
     4921 aaatttgtaa ttaagaagga gggattcgtc atgttggtat tccaaatgcg taatgtagat
     4981 aaaacatcta ctgttttgaa acagactaaa aacagtgatt acgcagataa ataaatacgt
     5041 tagattaatt cctaccagtg actaatctta tgacttttta aacagataac taaaattaca
     5101 aacaaatcgt ttaacttctg tatttattta cagatgtaat cacttcagga gtaattacat
     5161 gaacaaaaat ataaaatatt ctcaaaactt tttaacgagt gaaaaagtac tcaaccaaat
     5221 aataaaacaa ttgaatttaa aagaaaccga taccgtttac gaaattggaa caggtaaagg
     5281 gcatttaacg acgaaactgg ctaaaataag taaacaggta acgtctattg aattagacag
     5341 tcatctattc aacttatcgt cagaaaaatt aaaactgaac attcgtgtca ctttaattca
     5401 ccaagatatt ctacagtttc aattccctaa caaacagagg tataaaattg ttgggagtat
     5461 tccttaccat ttaagcacac aaattattaa aaaagtggtt tttgaaagcc atgcgtctga
     5521 catctatctg attgttgaag aaggattcta caagcgtacc ttggatattc accgaacact
     5581 agggttgctc ttgcacactc aagtctcgat tcagcaattg cttaagctgc cagcggaatg
     5641 ctttcatcct aaaccaaaag taaacagtgt cttaataaaa cttacccgcc ataccacaga
     5701 tgttccagat aaatattgga agctatatac gtactttgtt tcaaaatggg tcaatcgaga
     5761 atatcgtcaa ctgtttacta aaaatcagtt tcatcaagca atgaaacacg ccaaagtaaa
     5821 caatttaagt accattactt atgagcaagt attgtctatt tttaatagtt atctattatt
     5881 taacgggagg aaataattct atgagtcgct tttttaaatt tggaaagtta cacgttacta
     5941 aagggaatgg agataaatta ttagatatac tactgacagc ttccaagaag ctaaagaggt
     6001 ccctagcgcc tacggggaat ttgggcaaat caaaagaaaa attctcccaa gaattagaaa
     6061 tgagtagatc aaattattca cgaatagaat caggaaaatc agatccaacc ataaaaacac
     6121 tagaacaaat tgcaaagtta actaactcaa cgctagtagt ggatttaatc ccaaatgagc
     6181 caacagaacc agaaccagaa acagaatcag aacaagtaac attggattta gaaatggaag
     6241 aagaaaaaag caatgacttc gtgtgaataa tgcacgaaat cgttgcttat tttttttaaa
     6301 agcggtatac tagatataac gaaacaacga actgaataga aacgaaaaaa gagccatgac
     6361 acatttataa aatgtttgac gacattttat aaatgcatag cccgataaga ttgccaaacc
     6421 aacgcttatc agttagtcag atgaactctt ccctcgtaag aagttattta attaactttg
     6481 tttgaagacg gtatataacc gtactatcat tatataggga aatcagagag ttttcaagta
     6541 tctaagctac tgaatttaag aattgttaag caatcaatcg gaaatcgttt gattgctttt
     6601 tttgtattca tttatagaag gtggagtttg tatgaatcat gatgaatgta aaacttatat
     6661 aaaaaatagt ttattggaga taagaaaatt agcaaatatc tatacactag aaacgtttaa
     6721 gaaagagtta gaaaagagaa atatctactt agaaacaaaa tcagataagt atttttcttc
     6781 ggagggggaa gattatatat ataagttaat agaaaataac aaaataattt attcgattag
     6841 tggaaaaaaa ttgacttata aaggaaaaaa atctttttca aaacatgcaa tattgaaaca
     6901 gttgaatgaa aaagcaaacc aagttaatta aacaacctat tttataggat ttataggaaa
     6961 ggagaacagc tgaatgaata tcccttttgt tgtagaaact gtgcttcatg acggcttgtt
     7021 aaagtacaaa tttaaaaata gtaaaattcg ctcaatcact accaagccag gtaaaagcaa
     7081 aggggctatt tttgcgtatc gctcaaaatc aagcatgatt ggcggtcgtg gtgttgttct
     7141 gacttccgag gaagcgattc aagaaaatca agatacattt acacattgga cacccaacgt
     7201 ttatcgttat ggaacgtatg cagacgaaaa ccgttcatac acgaaaggac attctgaaaa
     7261 caatttaaga caaatcaata ccttctttat tgattttgat attcacacgg caaaagaaac
     7321 tatttcagca agcgatattt taacaaccgc tattgattta ggttttatgc ctactatgat
     7381 tatcaaatct gataaaggtt atcaagcata ttttgtttta gaaacgccag tctatgtgac
     7441 ttcaaaatca gaatttaaat ctgtcaaagc agccaaaata atttcgcaaa atatccgaga
     7501 atattttgga aagtctttgc cagttgatct aacgtgtaat cattttggta ttgctcgcat
     7561 accaagaacg gacaatgtag aattttttga tcctaattac cgttattctt tcaaagaatg
     7621 gcaagattgg tctttcaaac aaacagataa taagggcttt actcgttcaa gtctaacggt
     7681 tttaagcggt acagaaggca aaaaacaagt agatgaaccc tggtttaatc tcttattgca
     7741 cgaaacgaaa ttttcaggag aaaagggttt aatagggcgt aataacgtca tgtttaccct
     7801 ctctttagcc tactttagtt caggctattc aatcgaaacg tgcgaatata atatgtttga
     7861 gtttaataat cgattagatc aacccttaga agaaaaagaa gtaatcaaaa ttgttagaag
     7921 tgcctattca gaaaactatc aaggggctaa tagggaatac attaccattc tttgcaaagc
     7981 ttgggtatca agtgatttaa ccagtaaaga tttatttgtc cgtcaagggt ggtttaaatt
     8041 caagaaaaaa agaagcgaac gtcaacgtgt tcatttgtca gaatggaaag aagatttaat
     8101 ggcttatatt agcgaaaaaa gcgatgtata caagccttat ttagtgacga ccaaaaaaga
     8161 gattagagaa gtgctaggca ttcctgaacg gacattagat aaattgctga aggtactgaa
     8221 ggcgaatcag gaaattttct ttaagattaa accaggaaga aatggtggca ttcaacttgc
     8281 tagtgttaaa tcattgttgc tatcgatcat taaagtaaaa aaagaagaaa aagaaagcta
     8341 tataaaggcg ctgacaaatt cttttgactt agagcataca ttcattcaag agactttaaa
     8401 caagctagca gaacgcccta aaacggacac acaactcgat ttgtttagct atgatacagg
     8461 ctgaaaataa aacccgcact atgccattac atttatatct atgatacgtg tttgtttttt
     8521 ctttgctgtt tagcgaatga ttagcagaaa tatacagagt aagattttaa ttaattatta
     8581 gggggagaag gagagagtag cccgaaaact tttagttggc ttggactgaa cgaagtgagg
     8641 gaaaggctac taaaacgtcg aggggcagtg agagcgaagc gaacacttga ttttttaatt
     8701 ttctatcttt tataggtcat tagagtatac ttatttgtcc tataaactat ttagcagcat
     8761 aatagattta ttgaataggt catttaagtt gagcatatta gaggaggaaa atcttggaga
     8821 aatatttgaa gaacccgatt acatggattg gattagttct tgtggttacg tggtttttaa
     8881 ctaaaagtag tgaatttttg atttttggtg tgtgtgtctt gttgttagta tttgctagtc
     8941 aaagtgatta aatagaattg accacttctg cgctcggccc ttccggctgg ctggtttatt
     9001 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     9061 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     9121 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     9181 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     9241 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     9301 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     9361 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     9421 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     9481 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     9541 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     9601 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     9661 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     9721 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     9781 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     9841 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     9901 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     9961 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
    10021 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
    10081 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
    10141 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
    10201 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
    10261 cactttatgc ttc
//
