LOCUS       sequence-325874-        6201 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Suicide vector with the sacB gene (as a contraselective marker), 
            CmlR gene, and temperature sensitive ori, used to perform allelic 
            exchange mutagenesis..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6201)
  AUTHORS   Morona R, van den Bosch L, Manning PA
  TITLE     Molecular, genetic, and topological characterization of O-antigen 
            chain length regulation in Shigella flexneri.
  JOURNAL   J Bacteriol. 1995 Feb;177(4):1059-68. doi: 
            10.1128/jb.177.4.1059-1068.1995.
  PUBMED    7532168
REFERENCE   2  (bases 1 to 6201)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1128/jb.177.4.1059-1068"; journalName: "J Bacteriol"; date: 
            "1995-02"; volume: "177"; issue: "4"; pages: "1059-68"
COMMENT     Sequence Label: pCACTUS
FEATURES             Location/Qualifiers
     source          1..6201
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        126..228
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             229..888
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(295..314)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        1116..1561
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1562..1648,1649..2983)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     misc_feature    3392..3448
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     rep_origin      4069..4291
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             4339..5289
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 gccctttcgt cttcaagaag cgaataaata cctgtgacgg aagatcactt cgcagaataa
       61 ataaatcctg gtgtccctgt tgataccggg aagccctggg ccaacttttg gcgaaaatga
      121 gacgttgatc ggcacgtaag aggttccaac tttcaccata atgaaataag atcactaccg
      181 ggcgtatttt ttgagttatc gagattttca ggagctaagg aagctaaaat ggagaaaaaa
      241 atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca
      301 tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt
      361 ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc
      421 cgcctgatga atgctcatcc ggaattccgt atggcaatga aagacggtga gctggtgata
      481 tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg
      541 ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg
      601 gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc
      661 gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac
      721 aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg
      781 atgccgctgg cgattcaggt tcatcatgcc gtctgtgatg gcttccatgt cggcagaatg
      841 cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc
      901 agttattggt gcccttaaac gcctggtgct acgcctgaat aagtgataat aagcggatga
      961 atggcagaaa ttcgaaagca aattcgaccc ggtcgtcggt tcagggcagg gtcgttaaat
     1021 agccgcttat gtctattgct ggtttaccgg tttattgact accggaagca gtgtgaccgt
     1081 gtgcttctca aatgcctgaa tcctttttaa cccatcacat atacctgccg ttcactatta
     1141 tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc aactttatgc
     1201 ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag attttttagt
     1261 tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa agaggaaaat
     1321 agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta aatcgcgcgg
     1381 gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt gtatactttg
     1441 gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact tgccatcttc
     1501 aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg agacatgaac
     1561 gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta ccgcactgct
     1621 ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat ataaggaaac
     1681 atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac agcaaaaaaa
     1741 tgaaaaatat caagttcctg aattcgattc gtccacaatt aaaaatatct cttctgcaaa
     1801 aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg tcgcaaacta
     1861 tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg atgacacatc
     1921 gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga aaaacgctgg
     1981 ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa aagaccaaac
     2041 acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt tattctacac
     2101 tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag ttaacgtatc
     2161 agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa tctttgacgg
     2221 tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact acagctcagg
     2281 cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca aatacttagt
     2341 atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt tatttaacaa
     2401 agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac ttctgcaaag
     2461 cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg agctaaacga
     2521 tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag taacagatga
     2581 aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca ctgactcccg
     2641 cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc ttggttatgt
     2701 ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg tgttaaaaat
     2761 ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac ctcaagcgaa
     2821 aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg cagacaaaca
     2881 atcaacgttt gcgccaagct tcctgctgaa catcaaaggc aagaaaacat ctgttgtcaa
     2941 agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca aaagaaaatg
     3001 ccgattgagg ccagtttgct caggctctcc ccgtggaggt aataattgac gatatgatca
     3061 tttattctgc ctcccagagc ctgataaaaa cggttagcgc ttcgttaata cagatgtagg
     3121 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     3181 cgcttgtttc ggcgtgggta tggtggcagg ccgccccgtg gccgggggac tgttgggcgc
     3241 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     3301 agtcatgccc cgcgcccacc ggaaggagct accggacagc ggtgcggact gttgtaactc
     3361 agaataagaa atgaggccgc tcatggcgtt tgaattcgag ctcggtaccc ggggatcctc
     3421 tagagtcgac ctgcaggcat gcaagcttac tgcattaatg aatcggccaa cgcgcgggga
     3481 gaggcggttt gcgtattggc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     3541 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtccccg acagtaagac
     3601 gggtaagcct gttgatgata ccgctgcctt actgggtgca ttagccagtc tgaatgacct
     3661 gtcacgggat aatccgaagt ggtcagactg gaaaatcaga gggcaggaac tgctgaacag
     3721 caaaaagtca gatagcacca catagcagac ccgccataaa acgccctgag aagcccgtga
     3781 cgggcttttc ttgtattatg ggtagtttcc ttgcatgaat ccataaaagg cgcctgtagt
     3841 gccatttacc cccattcact gccagagccg tgagcgcagc gaactgaatg tcacgaaaaa
     3901 gacagcgact caggtgcctg atggtcggag acaaaaggaa tattcagcga tttgcccgag
     3961 cttgcgaggg tgctacttaa gcctttaggg ttttaaggtc tgttttgtag aggagcaaac
     4021 agcgtttgcg acatcctttt gtaatactgc ggaactgact aaagtagtga gttatacaca
     4081 gggctgggat ctattctttt tatctttttt tattctttct ttattctata aattataacc
     4141 acttgaatat aaacaaaaaa aacacacaaa ggtctagcgg aatttacaga gggtctagca
     4201 gaatttacaa gttttccagc aaaggtctag cagaatttac agatacccac aactcaaagg
     4261 aaaaggacta gtaattatca ttgactagcc catctcaatt ggtatagtga ttaaaatcac
     4321 ctagaccaat tgagatgtat gtctgaatta gttgttttca aagcaaatga actagcgatt
     4381 agtcgctatg acttaacgga gcatgaaacc aagctaattt tatgctgtgt ggcactactc
     4441 aaccccacga ttgaaaaccc tacaaggaaa gaacggacgg tatcgttcac ttataaccaa
     4501 tacgctcaga tgatgaacat cagtagggaa aatgcttatg gtgtattagc taaagcaacc
     4561 agagagctga tgacgagaac tgtggaaatc aggaatcctt tggttaaagg ctttgagatt
     4621 ttccagtgga caaactatgc caagttctca agcgaaaaat tagaattagt ttttagtgaa
     4681 gagatattgc cttatctttt ccagttaaaa aaattcataa aatataatct ggaacatgtt
     4741 aagtcttttg aaaacaaata ctctatgagg atttatgagt ggttattaaa agaactaaca
     4801 caaaagaaaa ctcacaaggc aaatatagag attagccttg atgaatttaa gttcatgtta
     4861 atgcttgaaa ataactacca tgagtttaaa aggcttaacc aatgggtttt gaaaccaata
     4921 agtaaagatt taaacactta cagcaatatg aaattggtgg ttgataagcg aggccgcccg
     4981 actgatacgt tgattttcca agttgaacta gatagacaaa tggatctcgt aaccgaactt
     5041 gagaacaacc agataaaaat gaatggtgac aaaataccaa caaccattac atcagattcc
     5101 tacctacgta acggactaag aaaaacacta cacgatgctt taactgcaaa aattcagctc
     5161 accagttttg aggcaaaatt tttgagtgac atgcaaagta agcatgatct caatggttcg
     5221 ttctcatggc tcacgcaaaa acaacgaacc acactagaga acatactggc taaatacgga
     5281 aggatctgag gttcttatgg ctcttgtatc tatcagtgaa gcatcaagac taacaaacaa
     5341 aagtagaaca actgttcacc gttagatatc aaagggaaaa ctgtccatat gcacagatga
     5401 aaacggtgta aaaaagatag atacatcaga gcttttacga gtttttggtg catttaaagc
     5461 tgttcaccat gaacagatcg acaatgtaac agatgaacag catgtaacac ctaatagaac
     5521 aggtgaaacc agtaaaacaa agcaactaga acatgaaatt gaacacctga gacaacttgt
     5581 tacagctcaa cagtcacaca tagacagcct gaaacaggcg atgctgctta tcgaatcaaa
     5641 gctgccgaca acacgggagc cagtgacgcc tcccgtgggg aaaaaatcat ggcaattctg
     5701 gaagaaatag cgctttcagc cggcaaacct gaagccggat ctgcgattct gataacaaac
     5761 tagcaacacc agaacagccc gtttgcgggc agcaaaaccc gtacttttgg acgttccggc
     5821 ggttttttgt ggcgagtggt gttcgggcgg tgcgcgcaag atccattatg ttaaacgggc
     5881 gagtttacat ctcaaaaccg cccgcttaac accatcagaa atcctcagcg cgattttaag
     5941 caccaacccc cccccgtaac acccaaatcc atactgaaag tggctttgtt gaataaatcg
     6001 aacttttgct gagttgaagg atcagatcac gcatcctccc gacaacacag accattccgt
     6061 ggcaaagcaa aagttcagaa tcaccaactg gtccacctac aacaaagctc tcatcaaccg
     6121 tggctccctc actttctggc tggatgatga ggcgattcag gcctggtatg agtcggcaac
     6181 accttcatca cgaggaaggc c
//
