LOCUS       sequence-274040-        7566 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intracellular control plasmid for HiBiTopt experiments.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7566)
  AUTHORS   Oliveira Paiva AM, de Jong L, Friggen AH, Smits WK, Corver J
  TITLE     The C-terminal domain of Clostridioides difficile TcdC is exposed on
            the bacterial cell surface.
  JOURNAL   J Bacteriol. 2020 Aug 31. pii: JB.00771-19. doi: 
            10.1128/JB.00771-19.
  PUBMED    32868401
REFERENCE   2  (bases 1 to 7566)
  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: "J 
            Bacteriol. 2020 Aug 31. pii: JB.00771-19. doi: 10.1128/JB.00771-19."
COMMENT     Sequence Label: pAF302
FEATURES             Location/Qualifiers
     source          1..7566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      244..832
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     733..752
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     986..1003
                     /label=L4440
                     /note="L4440 vector, forward primer"
     oriT            2194..2303
                     /note="incP origin of transfer"
     CDS             2336..2707
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     protein_bind    3483..3501
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             3608..4234
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(3684..3703)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 tatctaaact tatctatatc gctttttccg tcgtttggtt ctctagttac gatacagggg
       61 atatgcttat attgagttat agtactaatc agtgcttaat atagttaata aaattatagt
      121 taccatagtt tagtaactat gatgtatgtt agttagaaac ttgcatttca tgaccaaaat
      181 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
      241 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
      301 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
      361 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
      421 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
      481 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
      541 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
      601 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
      661 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      721 ggagcttcaa gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      781 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      841 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      901 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      961 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgctc
     1021 ggccggggat cgatccccgg gtacgtaccc ggcagttttt ctttttcggc aagtgttcaa
     1081 gaagttatta agtcgggagt gcagtcgaag tgggcaagtt gaaaaattca caaaaatgtg
     1141 gtataatatc tttgttcatt agagcgataa acttgaattt gagagggaac ttagatggta
     1201 tttgaaaaaa ttgataaaaa tagttggaac agaaaagagt attttgacca ctactttgca
     1261 agtgtacctt gtacctacag catgaccgtt aaagtggata tcacacaaat aaaggaaaag
     1321 ggaatgaaac tatatcctgc aatgctttat tatattgcaa tgattgtaaa ccgccattca
     1381 gagtttagga cggcaatcaa tcaagatggt gaattgggga tatatgatga gatgatacca
     1441 agctatacaa tatttcacaa tgatactgaa acattttcca gcctttggac tgagtgtaag
     1501 tctgacttta aatcattttt agcagattat gaaagtgata cgcaacggta tggaaacaat
     1561 catagaatgg aaggaaagcc aaatgctccg gaaaacattt ttaatgtatc tatgataccg
     1621 tggtcaacct tcgatggctt taatctgaat ttgcagaaag gatatgatta tttgattcct
     1681 atttttacta tggggaaata ttataaagaa gataacaaaa ttatacttcc tttggcaatt
     1741 caagttcatc acgcagtatg tgacggattt cacatttgcc gttttgtaaa cgaattgcag
     1801 gaattgataa atagttaact tcaggtttgt ctgtaactaa aaacaagtat ttaagcaaaa
     1861 acatcgtaga aatacggtgt tttttgttac cctaaaatct acaattttat acataaccac
     1921 aggttagtac aaagaccttg tgtttctttt tgaaaggctt aaaacaagga tttttccttg
     1981 atttaagccc cgaaaagcaa cacaaccaag gttttagtat caatctgtgg tttttatatt
     2041 ttcagagacc ggtcagatct gcagaattcg cccttcctgc ttcggggtca ttatagcgat
     2101 tttttcggta tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt
     2161 agactttcct tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc
     2221 cgcgagcggg tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa
     2281 tcctgctctg cgaggctggc cggctaccgc cggcgtaaca gatgagggca agcggatggc
     2341 tgatgaaacc aagccaacca ggaagggcag cccacctatc aaggtgtact gccttccaga
     2401 cgaacgaaga gcgattgagg aaaaggcggc ggcggccggc atgagcctgt cggcctacct
     2461 gctggccgtc ggccagggct acaaaatcac gggcgtcgtg gactatgagc acgtccgcga
     2521 gctggcccgc atcaatggcg acctgggccg cctgggcggc ctgctgaaac tctggctcac
     2581 cgacgacccg cgcacggcgc ggttcggtga tgccacgatc ctcgccctgc tggcgaagat
     2641 cgaagagaag caggacgagc ttggcaaggt catgatgggc gtggtccgcc cgagggcaga
     2701 gccatgactt ttttagccgc taaaacggcc ggggggtgcg cgtgattgcc aagcacgtcc
     2761 ccatgcgctc catcaagaag agcgacttcg cggagctggt gaagtacatc accgacgagc
     2821 aaggcaagac cgatccccta ctactgacag cttccaagga gctaaagagg tccctagcgc
     2881 agcctaaggg cgaattccag cacactggca tctttttatt tagggatttc tcacataaaa
     2941 tagaggctat ttatagcctc tattttattt tatatttatt ttaaataaaa aagacttctc
     3001 atgagagaag cctttttcta tttaaagttt tattaaaact tataggatcc ctaagaaatt
     3061 ttcttaaaaa gtctccaacc actaacacca ctagaaccac caccaccaga accaccacca
     3121 ccactagaac cccctcgaga tccatttatt atatccttta atccttttcc agctttgaaa
     3181 actggtgctt tagaagctgg tatatctata acttgctctg gatctcttgg atttcttcct
     3241 tgtctagcag ctctttctct tgtctcaaat gttccaaatc caactaattg aactttttca
     3301 ttatttacta gtgcatcttg aacagaactc ataaatgcgt ttaacgcagc ttctgcttcc
     3361 ttctttgtta atccactttt ttctgccatc tttgatacta attcagcttt attcacaaaa
     3421 ttttctcctt tactgcagga gctcagatct gttaacgcta cgatcaagct tattttaatt
     3481 atactctatc aatgatagag tgtcaatatt ttttttagtt tttcatgaag tccagggcat
     3541 cgaaataaaa aactagtttg acaaataact ctatcaatga tataatgtca acaaaaagga
     3601 ggaattaatg atgtctagat tagataaaag taaagtgatt aacagcgcat tagagctgct
     3661 taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga
     3721 gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat
     3781 tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga
     3841 ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc
     3901 aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt
     3961 agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt
     4021 ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga
     4081 aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt
     4141 tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt
     4201 agaaaaacaa cttaaatgtg aaagtgggtc ttaaggtacc ataaaaataa gaagcctgca
     4261 aatgcaggct tcttattttt atgctagctt gatgcagaat tcgcccttaa gtctaaaaat
     4321 taggggagat gtaaggattt gggaaaaata gaagatgtta taatcataaa tatggtattc
     4381 gtaggcttaa agtcaaaaag gaggtgaaat ataaatagat ttttagctaa attaagtaag
     4441 aaataggagg agatttattg aacaaaaaat tagaaaaacc atttgtatat aagagagagt
     4501 acgatttgac tggatatgat gttgaaattt tacaaaaata tgagttagaa caagcaatat
     4561 atgtttatgt tgggagtagt tgtgcatata acatgagagc tagaagtagt aaatggagat
     4621 accatataag aacaaataat aagtctatat gttgtaacat taaaaatttt atacataact
     4681 tggaattgtt ttataaaatg gaattaaagt tgtcagataa tattattaat gataagctat
     4741 actatagcaa tatagcagag tttgaagaat ttgaaacact agaaaaagct agagaggtag
     4801 aaagtactat tataagtcaa tatcaatttt tagattctat aaatcatatg ttaaaacaaa
     4861 aaataatttt attgagtaat aaggatagtg tgttaaacat aactaaaaat ggaaatacaa
     4921 attatttgaa agtaaaaaat aaatacatag aaaaacataa gaacaagcca ataatgagat
     4981 accatatcaa ctgtcaattc aatacagatg gaagtgtcaa aagtattaca caggagtttg
     5041 aaccaatatt ggaattaaac aaaaaaaata ccctaagccg accaagcaga gtatttttaa
     5101 aataatattt taagataaca acaaaatgag ataatactac tagacaatga caactcaact
     5161 accaattgag tttatggagc taccaactcc aatatcggtc taactgatta agtatctgta
     5221 gttatataat aatattgcta tcaattttag catcttaaca atattattat acatactaag
     5281 ctaaaattat tcaatagttg taaaagttga ttagtcaata agtatatatt taatgtagtg
     5341 ttatctctta aaaaaactag ataaggagat aataaatata tggaacaatt agattcaaaa
     5401 tataagttga aaaaatttct aatggcagta tttagagatg gtataggaca aggaaataat
     5461 cttattgata atgaatatgt tagagtattt caaaataata aaagtaatag taaacaatta
     5521 gaactcggag aagaatttaa agaatatagt aaaacaactt tttttaaaaa tatagatgat
     5581 atagtagaat ttaccttcgc aaaaaatatt tattatgaaa atacattttt taacctatgt
     5641 acaactgatg gaaaagcagg aaccaatgaa aacttaataa atagatatgc attaggattt
     5701 gattttgaca aaaaagaatt aggacaaggt tttaattata aagatataat taatttattt
     5761 actaagatag gattacatta tcatatccta gttgatagtg gaaatggatt ccatgtttat
     5821 gtgctaatta ataaaactaa taacattaag ttagtatcag aagttacaaa tacattaata
     5881 aataaattgg gtgcagataa acaagcaaat ttatctactc aagtattaag agtaccttat
     5941 acatataata ttaaaaatac tactaaacaa gtaaaaataa tacaccaaga caaaaatata
     6001 tatagatatg acatagaaaa gttagctaaa aaatattgca aagatgtaaa aacagtaggt
     6061 aatactaata caaaatatat attagatagt aagctaccaa attgtatagt agatatttta
     6121 aaaaatggta gtaaagatgg acataaaaac ctagatttgc aaaaaatagt tgtgacttta
     6181 agattgagga ataaaagttt aagtcaagta atatccgttg ctagagaatg gaactatata
     6241 tcacaaaata gtctttcaaa tagtgagcta gaatatcaag tcaagtatat gtatgagaag
     6301 cttaaaacgg ttaattttgg ttgtacaggt tgtgagttta atagtgattg ttggaataaa
     6361 atagaatcag attttatata tagtgatgaa gatactttgt tcaatatgcc acataagcac
     6421 tcaaaggatt tgaaatataa gaataggaaa ggggttaaaa taatgactgg taatcaattg
     6481 tttatctata atgtgttact taacaataaa gatagagaat taaacataga cgatataatg
     6541 gagctgataa cctataaacg taagaagaaa gttaaaaaca ttgttatgag tgaaaagaca
     6601 ttaagagaaa cattaaaaga acttcaacat aatgattata ttacaaaaac aaaaggtgtt
     6661 acaaagctag gaataaaaga tacatacaat gtaaaagaag ttagatgtaa tatagataaa
     6721 caatatacta ttagttactt tgttaccatg gcagtaattt ggggaataat ttcaactgaa
     6781 gaattaagat tatatactca tatgagatat aagcaagatt tattggtcaa agatgataaa
     6841 ataaaaggaa atatattaag aattaatcaa gaggaattag caaaagattt aggagtaaca
     6901 cagcaaagaa tttcaaatat gatagaatct ttattagata ctaaaatttt agatgtatgg
     6961 gaaactaaaa taaatgatag aggatttatg tactatacat atagattaaa caagtagatt
     7021 tttgatagga ttagaattga ttttctagtc ctatttttat gcaaaaaaac taattaataa
     7081 aaatttcttt tggtaaaata attgtacgag aattgcaaaa aaaaaatggc atcaaagtat
     7141 tgaaattaag ccgttttaaa aatttctttt ggtaaaataa ttgtacatat atatgtagta
     7201 tatatatata tgttttttag agaatgtata actagaatat agagctagaa tatagagaat
     7261 gtataactag aatatagagc tagaatatag agaatgtata actagaatat agagctagaa
     7321 tatagagaat gtataactag aatatagagc tagaatatag agaatgtata actagaatat
     7381 agagctagaa tatagagaat gtataactag aatatagagc tagaatatag agaatgtata
     7441 actagaatat agagctagaa tcctaattag taggtgcttt tttaaaacaa gttaaaaatc
     7501 aaaaatagta ttagtaagca ttggaaatgc tagattctaa aatagaaaag taaaaaattg
     7561 gtgcac
//
