LOCUS       sequence-332373-        6202 bp    DNA     circular SYN 15-DEC-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 6202)
  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 6202)
  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"; 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..6202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      545..767
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             815..1765
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     promoter        2799..2901
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2902..3561
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2968..2987)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        3791..4236
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(4237..4323,4324..5658)
                     /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    6066..6122
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
ORIGIN
        1 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
       61 tccccgacag taagacgggt aagcctgttg atgataccgc tgccttactg ggtgcattag
      121 ccagtctgaa tgacctgtca cgggataatc cgaagtggtc agactggaaa atcagagggc
      181 aggaactgct gaacagcaaa aagtcagata gcaccacata gcagacccgc cataaaacgc
      241 cctgagaagc ccgtgacggg cttttcttgt attatgggta gtttccttgc atgaatccat
      301 aaaaggcgcc tgtagtgcca tttaccccca ttcactgcca gagccgtgag cgcagcgaac
      361 tgaatgtcac gaaaaagaca gcgactcagg tgcctgatgg tcggagacaa aaggaatatt
      421 cagcgatttg cccgagcttg cgagggtgct acttaagcct ttagggtttt aaggtctgtt
      481 ttgtagagga gcaaacagcg tttgcgacat ccttttgtaa tactgcggaa ctgactaaag
      541 tagtgagtta tacacagggc tgggatctat tctttttatc tttttttatt ctttctttat
      601 tctataaatt ataaccactt gaatataaac aaaaaaaaca cacaaaggtc tagcggaatt
      661 tacagagggt ctagcagaat ttacaagttt tccagcaaag gtctagcaga atttacagat
      721 acccacaact caaaggaaaa ggactagtaa ttatcattga ctagcccatc tcaattggta
      781 tagtgattaa aatcacctag accaattgag atgtatgtct gaattagttg ttttcaaagc
      841 aaatgaacta gcgattagtc gctatgactt aacggagcat gaaaccaagc taattttatg
      901 ctgtgtggca ctactcaacc ccacgattga aaaccctaca aggaaagaac ggacggtatc
      961 gttcacttat aaccaatacg ttcagatgat gaacatcagt agggaaaatg cttatggtgt
     1021 attagctaaa gcaaccagag agctgatgac gagaactgtg gaaatcagga atcctttggt
     1081 taaaggcttt gagattttcc agtggacaaa ctatgccaag ttctcaagcg aaaaattaga
     1141 attagttttt agtgaagaga tattgcctta tcttttccag ttaaaaaaat tcataaaata
     1201 taatctggaa catgttaagt cttttgaaaa caaatactct atgaggattt atgagtggtt
     1261 attaaaagaa ctaacacaaa agaaaactca caaggcaaat atagagatta gccttgatga
     1321 atttaagttc atgttaatgc ttgaaaataa ctaccatgag tttaaaaggc ttaaccaatg
     1381 ggttttgaaa ccaataagta aagatttaaa cacttacagc aatatgaaat tggtggttga
     1441 taagcgaggc cgcccgactg atacgttgat tttccaagtt gaactagata gacaaatgga
     1501 tctcgtaacc gaacttgaga acaaccagat aaaaatgaat ggtgacaaaa taccaacaac
     1561 cattacatca gattcctacc tacataacgg actaagaaaa acactacacg atgctttaac
     1621 tgcaaaaatt cagctcacca gttttgaggc aaaatttttg agtgacatgc aaagtaagta
     1681 tgatctcaat ggttcgttct catggctcac gcaaaaacaa cgaaccacac tagagaacat
     1741 actggctaaa tacggaagga tctgaggttc ttatggctct tgtatctatc agtgaagcat
     1801 caagactaac aaacaaaagt agaacaactg ttcaccgtta catatcaaag ggaaaactgt
     1861 ccatatgcac agatgaaaac ggtgtaaaaa agatagatac atcagagctt ttacgagttt
     1921 ttggtgcatt taaagctgtt caccatgaac agatcgacaa tgtaacagat gaacagcatg
     1981 taacacctaa tagaacaggt gaaaccagta aaacaaagca actagaacat gaaattgaac
     2041 acctgagaca acttgttaca gctcaacagt cacacataga cagcctgaaa caggcgatgc
     2101 tgcttatcga atcaaagctg ccgacaacac gggagccagt gacgcctccc gtggggaaaa
     2161 aatcatggca attctggaag aaatagcgct ttcagccggc aaaccggctg aagccggatc
     2221 tgcgattctg ataacaaact agcaacacca gaacagcccg tttgcgggca gcaaaacccg
     2281 tacttttgga cgttccggcg gttttttgtg gcgagtggtg ttcgggcggt gcgcgcaaga
     2341 tccattatgt taaacgggcg agtttacatc tcaaaaccgc ccgcttaaca ccatcagaaa
     2401 tcctcagcgc gattttaagc accaaccccc ccccgtaaca cccaaatcca tactgaaagt
     2461 ggctttgttg aataaatcga acttttgctg agttgaagga tcagatcacg catcctcccg
     2521 acaacacaga ccattccgtg gcaaagcaaa agttcagaat caccaactgg tccacctaca
     2581 acaaagctct catcaaccgt ggctccctca ctttctggct ggatgatgag gcgattcagg
     2641 cctggtatga gtcggcaaca ccttcatcac gaggaaggcc gccctttcgt cttcgaataa
     2701 atacctgtga cggaagatca cttcgcagaa taaataaatc ctggtgtccc tgttgatacc
     2761 gggaagccct gggccaactt ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc
     2821 aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt atcgagattt
     2881 tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata
     2941 tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat
     3001 aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac
     3061 aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc
     3121 cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc
     3181 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc
     3241 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
     3301 ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc
     3361 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg
     3421 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat
     3481 gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat
     3541 gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggt
     3601 tgctacgcct gaataagtga taataagcgg atgaatggca gaaattcgaa agcaaattcg
     3661 acccggtcgt cggttcaggg cagggtcgtt aaatagccgc ttatgtctat tgctggttta
     3721 ccggtttatt gactaccgga agcagtgtga ccgtgtgctt ctcaaatgcc tgagatcctt
     3781 tttaacccat cacatatacc tgccgttcac tattatttag tgaaatgaga tattatgata
     3841 ttttctgaat tgtgattaaa aaggcaactt tatgcccatg caacagaaac tataaaaaat
     3901 acagagaatg aaaagaaaca gatagatttt ttagttcttt aggcccgtag tctgcaaatc
     3961 cttttatgat tttctatcaa acaaaagagg aaaatagacc agttgcaatc caaacgagag
     4021 tctaatagaa tgaggtcgaa aagtaaatcg cgcgggtttg ttactgataa agcaggcaag
     4081 acctaaaatg tgtaaagggc aaagtgtata ctttggcgtc accccttaca tattttaggt
     4141 ctttttttat tgtgcgtaac taacttgcca tcttcaaaca ggagggctgg aagaagcaga
     4201 ccgctaacac agtacataaa aaaggagaca tgaacgatga acatcaaaaa gtttgcaaaa
     4261 caagcaacag tattaacctt tactaccgca ctgctggcag gaggcgcaac tcaagcgttt
     4321 gcgaaagaaa cgaaccaaaa gccatataag gaaacatacg gcatttccca tattacacgc
     4381 catgatatgc tgcaaatccc tgaacagcaa aaaaatgaaa aatatcaagt tcctgaattc
     4441 gattcgtcca caattaaaaa tatctcttct gcaaaaggcc tggacgtttg ggacagctgg
     4501 ccattacaaa acgctgacgg cactgtcgca aactatcacg gctaccacat cgtctttgca
     4561 ttagccggag atcctaaaaa tgcggatgac acatcgattt acatgttcta tcaaaaagtc
     4621 ggcgaaactt ctattgacag ctggaaaaac gctggccgcg tctttaaaga cagcgacaaa
     4681 ttcgatgcaa atgattctat cctaaaagac caaacacaag aatggtcagg ttcagccaca
     4741 tttacatctg acggaaaaat ccgtttattc tacactgatt tctccggtaa acattacggc
     4801 aaacaaacac tgacaactgc acaagttaac gtatcagcat cagacagctc tttgaacatc
     4861 aacggtgtag aggattataa atcaatcttt gacggtgacg gaaaaacgta tcaaaatgta
     4921 cagcagttca tcgatgaagg caactacagc tcaggcgaca accatacgct gagagatcct
     4981 cactacgtag aagataaagg ccacaaatac ttagtatttg aagcaaacac tggaactgaa
     5041 gatggctacc aaggcgaaga atctttattt aacaaagcat actatggcaa aagcacatca
     5101 ttcttccgtc aagaaagtca aaaacttctg caaagcgata aaaaacgcac ggctgagtta
     5161 gcaaacggcg ctctcggtat gattgagcta aacgatgatt acacactgaa aaaagtgatg
     5221 aaaccgctga ttgcatctaa cacagtaaca gatgaaattg aacgcgcgaa cgtctttaaa
     5281 atgaacggca aatggtacct gttcactgac tcccgcggat caaaaatgac gattgacggc
     5341 attacgtcta acgatattta catgcttggt tatgtttcta attctttaac tggcccatac
     5401 aagccgctga acaaaactgg ccttgtgtta aaaatggatc ttgatcctaa cgatgtaacc
     5461 tttacttact cacacttcgc tgtacctcaa gcgaaaggaa acaatgtcgt gattacaagc
     5521 tatatgacaa acagaggatt ctacgcagac aaacaatcaa cgtttgcgcc aagcttcctg
     5581 ctgaacatca aaggcaagaa aacatctgtt gtcaaagaca gcatccttga acaaggacaa
     5641 ttaacagtta acaaataaaa acgcaaaaga aaatgccgat tgaggccagt ttgctcaggc
     5701 tctccccgtg gaggtaataa ttgacgatat gatcatttat tctgcctccc agagcctgat
     5761 aaaaacggtt agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     5821 cctgcgatgc agatccggaa cataatggtg cagggcgctt gtttcggcgt gggtatggtg
     5881 gcaggccgcc ccgtggccgg gggactgttg ggcgctgccg gcacctgtcc tacgagttgc
     5941 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     6001 gagctaccgg cagcggtgcg gactgttgta actcagaata agaaatgagg ccgctcatgg
     6061 cgttggaatt cgagctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc
     6121 ttgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tggcaccatt
     6181 ccttgcggcg gcggtgctca ac
//
