LOCUS       sequence-251117-        6808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes YhaV toxin and amilCP chromoprotein. CAM-R.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6808)
  AUTHORS   Lazarus JE, Warr AR, Kuehl CJ, Giorgio RT, Davis BM, Waldor MK
  TITLE     A New Suite of Allelic Exchange Vectors for the Scarless 
            Modification of Proteobacterial Genomes.
  JOURNAL   Appl Environ Microbiol. 2019 Jun 14. pii: AEM.00990-19. doi: 
            10.1128/AEM.00990-19.
  PUBMED    31201277
REFERENCE   2  (bases 1 to 6808)
  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: "Appl 
            Environ Microbiol. 2019 Jun 14. pii: AEM.00990-19. doi: 
            10.1128/AEM.00990-19."
COMMENT     Sequence Label: pTOX4
FEATURES             Location/Qualifiers
     source          1..6808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        276..378
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             379..1038
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(445..464)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1139..1225
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1317..1344
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     regulatory      1398..1407
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        1547..1665
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     terminator      2213..2459
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     protein_bind    2495..2511
                     /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)."
     rep_origin      2546..2934
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(3552..3923)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            3956..4065
                     /note="incP origin of transfer"
     primer_bind     complement(4746..4765)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(4904..4926)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     RBS             5090..5098
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        join(5976..5981,5982..5997,5998..6004)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 ccccgcttcg gcggggtttt ttcgctgatc acgtacgata tcttcgaacc ggtgcacatg
       61 gtgtacaggg ccctaggata ggacgtctta aggtttaaac caggttaatt aatttaaatg
      121 catcccggga cgtctcgagc tcgatcggac cgcggccgct agcacgtata ccaagtgtcc
      181 tgttttcttg ccgccaagga tctgatggcg caggggatca agatctctgt tgataccggg
      241 aagccctggg ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag aggttccaac
      301 tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca
      361 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc
      421 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac
      481 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
      541 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattacgt
      601 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
      661 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
      721 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
      781 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
      841 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc
      901 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
      961 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     1021 tggcagggcg gggcgtaatt tttttaaggc agttattgtc ttcaaattcc cgttgcatcg
     1081 atggcccccg atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca
     1141 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     1201 gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg
     1261 gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa
     1321 ggccatcctg acggatggcc tttttgcgtg gccagtgcca agcttgcatg cctgcagctg
     1381 tccggaacta gtcgaggcca tggtggcctc ctgatgtcgt caacacggcg aaatagtaat
     1441 cacgaggtca ggttcttacc ttaaattttc gacggaaaac cacgtaaaaa acgtcgattt
     1501 ttcaatatac agcgtgaatt ttcaggaaat gcggtgagca tcacatcacc acaattcagc
     1561 aaattgtgaa catcatcacg ttcatctttc cctggttgcc aatggcccat tttcctgtca
     1621 gtaacgagaa ggtcgcgaat tcaggcgctt tttagactgg tcgtaatgaa attcagcagg
     1681 atcaccatat ggattttcca caaagggtta atggttgggc gctatatgct catccctgtt
     1741 ttcaggaaac ctacgacgct ttagttgccg aagtcgagac attaaaggga aaagatcctg
     1801 aaaattatca gagaaaagcc gccacaaagt tattggcggt agtccataaa gtgattgagg
     1861 agcatatcac ggtcaatcca tcatcaccgg cattccgtca tggcaagtcg ttaggctctg
     1921 ggaaaaataa agactggtca cgggtaaaat ttggtgctgg tcgttatcgt ctcttctttc
     1981 gttatagtga aaaagagaaa gtcatcattc tgggatggat gaacgatgaa aacactctgc
     2041 gcacctacgg taaaaaaaca gatgcctata ccgtattcag caaaatgtta aaaagaggac
     2101 atcctcctgc cgactgggaa accctcaccc gagaaacaga agaaacccat tgaggatccg
     2161 cgaattctca tgtttgacag cttatcactg atcagtgaat taatggcgat gacgcatcct
     2221 cacgataata tccgggtagg cgcaatcact ttcgtctcta ctccgttaca aagcgaggct
     2281 gggtatttcc cggcctttct gttatccgaa atccactgaa agcacagcgg ctggctgagg
     2341 agataaataa taaacgaggg gctgtatgca caaagcatct tctgttgagt taagaacgag
     2401 tatcgagatg gcacatagcc ttgctcaaat tggaatcagg tttgtgccaa taccagtaga
     2461 aacagacgaa gaagtcgtgg gggaattcca caaattgtta tccgctcaca attccacatg
     2521 tggaattcca catgtggaat tcccatgtca gccgttaagt gttcctgtgt cactcaaaat
     2581 tgctttgaga ggctctaagg gcttctcagt gcgttacatc cctggcttgt tgtccacaac
     2641 cgttaaacct taaaagcttt aaaagcctta tatattcttt tttttcttat aaaacttaaa
     2701 accttagagg ctatttaagt tgctgattta tattaatttt attgttcaaa catgagagct
     2761 tagtacgtga aacatgagag cttagtacgt tagccatgag agcttagtac gttagccatg
     2821 agggtttagt tcgttaaaca tgagagctta gtacgttaaa catgagagct tagtacgtga
     2881 aacatgagag cttagtacgt actatcaaca ggttgaactg ctgatcttca gatcctctac
     2941 gccggacgca tcgtggccgg atccagccga ccaggctttc cacgcccgcg tgccgctcca
     3001 tgtcgttcgc gcggttctcg gaaacgcgct gccgcgtttc gtgattgtca cgctcaagcc
     3061 cgtagtcccg ttcgagcgtc gcgcagaggt cagcgagggc gcggtaggcc cgatacggct
     3121 catggatggt gtttcgggtc gggtgaatct tgttgatggc gatatggatg tgcaggttgt
     3181 cggtgtcgtg atgcacggca ctgacgcgct gatgctcggc gaagccaagc ccagcgcaga
     3241 tgcggtcctc aatcgcgcgc aacgtctccg cgtcgggctt ctctcccgcg cggaagctaa
     3301 ccagcaggtg ataggtcttg tcggcctcgg aacgggtgtt gccgtgctgg gtcgccatca
     3361 cctcggccat gacagcgggc agggtgtttg cctcgcagtt cgtgacgcgc acgtgaccca
     3421 ggcgctcggt cttgccttgc tcgtcggtga tgtacttcac cagctccgcg aagtcgctct
     3481 tcttgatgga gcgcatgggg acgtgcttgg caatcacgcg caccccccgg ccgttttagc
     3541 ggctaaaaaa gtcatggctc tgccctcggg cggaccacgc ccatcatgac cttgccaagc
     3601 tcgtcctgct tctcttcgat cttcgccagc agggcgagga tcgtggcatc accgaaccgc
     3661 gccgtgcgcg ggtcgtcggt gagccagagt ttcagcaggc cgcccaggcg gcccaggtcg
     3721 ccattgatgc gggccagctc gcggacgtgc tcatagtcca cgacgcccgt gattttgtag
     3781 ccctggccga cggccagcag gtaggccgac aggctcatgc cggccgccgc cgccttttcc
     3841 tcaatcgctc ttcgttcgtc tggaaggcag tacaccttga taggtgggct gcccttcctg
     3901 gttggcttgg tttcatcagc catccgcttg ccctcatctg ttacgccggc ggtagccggc
     3961 cagcctcgca gagcaggatt cccgttgagc accgccaggt gcgaataagg gacagtgaag
     4021 aaggaacacc cgctcgcggg tgggcctact tcacctatcc tgcccggctg acgccgttgg
     4081 atacaccaag gaaagtctac acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag
     4141 gatggatata ccgaaaaaat cgctataatg accccgaagc agggttatgc agcggaaaag
     4201 cgctgcttcc ctgctgtttt gtggaatatc taccgactgg aaacaggcaa atgcaggaaa
     4261 ttactgaact gaggggacag gcgagagacg atgccaaaga gctacaccga cgagctggcc
     4321 gagtgggttg aatcccgcgc ggccaagaag cgccggcgtg atgaggctgc ggttgcgttc
     4381 ctggcggtga gggcggatgt cgaggcggcg ttagcgtccg gctatgcgct cgtcaccatt
     4441 tgggagcaca tgcgggaaac ggggaaggtc aagttctcct acgagacgtt ccgctcgcac
     4501 gccaggcggc acatcaaggc caagcccgcc gatgtgcccg caccgcaggc caaggctgcg
     4561 gaacccgcgc cggcacccaa gacgccggag ccacggcggc cgaagcaggg gggcaaggct
     4621 gaaaagccgg cccccgctgc ggccccgacc ggcttcacct tcaacccaac accggacaaa
     4681 aaggatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc
     4741 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat
     4801 gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc
     4861 catctccttg ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     4921 tcccggagac ggtcacagct tgtctgtaag cggatgccgt aagtaaggta aaggcctcgc
     4981 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5041 taaccctgat aaatgcttca ataatattga aaaaggagct tatcaccgat aaggaggttt
     5101 tttaatgacg gtgctgcact cggttgactt cttccctagc ggcaatgcca gcgttgccat
     5161 tgagccgcgc ctgcctcaag ccgacttccc ggagcaccac cacgacttcc acgagatcgt
     5221 tatcgtggag cacggtaccg gcatccacgt tttcaacggc caaccgtaca cgattacggg
     5281 cggtaccgtg tgcttcgttc gtgatcacga ccgccactta tacgagcaca cggacaactt
     5341 atgcttaacc aacgttttat accgtagccc tgaccgcttc caattcctgg cgggcttaaa
     5401 ccaactgtta ccgcaggaat tagacggcca ataccctagc cattggcgtg tgaatcattc
     5461 ggtgctgcaa caagttcgcc aattagtggc gcaaatggag caacaagagg gcgagaacga
     5521 cctgccgagc acggcgagcc gtgaaattct gttcatgcag ctgttactgc tgttacgcaa
     5581 gtcgagcctg caagaaaatt tagagaattc ggcgagccgc ctgaatctgc tgttagcgtg
     5641 gttagaagat cacttcgcgg acgaagttaa ctgggatgcg gttgccgacc agttcagcct
     5701 gagcttacgc accctgcacc gccaactgaa acaacagacc ggcttaaccc cgcaacgcta
     5761 tttaaaccgt ttacgcttaa tgaaggcgcg ccacttactg cgccattcgg aagcgtcggt
     5821 gaccgatatt gcgtaccact gtggcttttc ggatagcaat catttcagca ccctgttccg
     5881 tcgcgaattc aattggagcc ctcgcgacat ccgccaaggc cgcgacggtt tcttacagtg
     5941 aaggccttct agagtcgacc tgcatcgacc tgcagttgac aattaatcat cggctcgtat
     6001 aatgtgtgga aaggcggttc accgccgttt tcacacagga aacagaattc tttaagaagg
     6061 agatatacat atgtcagtga tagcaaagca gatgacatac aaagtatata tgagcggtac
     6121 tgtaaatggt cactacttcg aagtagaagg tgatggcaaa gggaagccgt atgagggtga
     6181 acaaacagtg aagcttacag ttacgaaggg tggccctttg ccgttcgcgt gggacatttt
     6241 gtcgccacag tgccagtacg ggagcatacc attcaccaag tatccagagg acataccaga
     6301 ctacgtgaaa cagtcctttc ccgaaggcta tacctgggag cgcataatga actttgaaga
     6361 cggtgcggtt tgtacggtat cgaacgattc atcaatccaa ggaaattgct ttatttatca
     6421 tgttaaattt tcggggctta actttccgcc aaatggcccc gtgatgcaga agaaaactca
     6481 gggctgggaa ccaaataccg agcgcctttt cgctcgggac ggaatgctgt tgggaaacaa
     6541 ttttatggcg ttgaagctgg aagggggtgg ccactatctc tgtgaattca agacaacata
     6601 caaagccaag aagcctgtca agatgcccgg ttatcactat gtggaccgga agctcgatgt
     6661 cactaatcac aacaaggatt atacttcagt tgaacagtgc gaaatttcga tcgctcgcaa
     6721 acctgtcgta gcataagggg catgcaagct tggctgtttt ggcggatgag agggtaccgc
     6781 atgagcttcg agctaatcgc gcaaaaaa
//
