LOCUS       sequence-316821-        6203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Suicide vector for generation of gene deletions in mycobacteria. 
            Contains the two genes sacB and galK for negative selection. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6203)
  AUTHORS   Arnold FM, Hohl M, Remm S, Koliwer-Brandl H, Adenau S, Chusri S, 
            Sander P, Hilbi H, Seeger MA
  TITLE     A uniform cloning platform for mycobacterial genetics and protein 
            production.
  JOURNAL   Sci Rep. 2018 Jun 22;8(1):9539. doi: 10.1038/s41598-018-27687-5.
  PUBMED    29934571
REFERENCE   2  (bases 1 to 6203)
  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: "Sci Rep."; 
            date: "2018-06-22"; volume: "8(1):9539. doi"; pages: " 
            10.1038/s41598-018-27687-5"
COMMENT     Sequence Label: pKO (Hyg)
FEATURES             Location/Qualifiers
     source          1..6203
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(21..26,27..44,45..51)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    57..76
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     primer_bind     1652..1674
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1666..1683
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1667..1683
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(1927..2515)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2007..2026)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2660..3105
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(3106..3192,3193..4527)
                     /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
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
ORIGIN
        1 catggatcca gaccccaggc ttgacacttt atgcttccgg ctcgtataat gtgtggaatt
       61 gtgagcgctc acaattcgca tgcggtacca agctgatccg ataacacagg aacagatctt
      121 ccggagtgta agaaatgagt ctgaaagaaa aaacacaatc tctgtttgcc aacgcatttg
      181 gctaccctgc cactcacacc attcaggcgc ctggccgcgt gaatttgatt ggtgaacaca
      241 ccgactacaa cgacggtttc gttctgccct gcgcgattga ttatcaaacc gtgatcagtt
      301 gtgcaccacg cgatgaccgt aaagttcgcg tgatggcagc cgattatgaa aatcagctcg
      361 acgagttttc cctcgatgcg cccattgtcg cacatgaaaa ctatcaatgg gctaactacg
      421 ttcgtggcgt ggtgaaacat ctgcaactgc gtaacaacag cttcggcggc gtggacatgg
      481 tgatcagcgg caatgtgccg cagggtgccg ggttaagttc ttccgcttca ctggaagtcg
      541 cggtcggaac cgtattgcag cagctttatc atctgccgct ggacggcgca caaatcgcgc
      601 ttaacggtca ggaagcagaa aaccagtttg taggctgtaa ctgcgggatc atggatcagc
      661 taatttccgc gctcggcaag aaagatcatg ccttgctgat cgattgccgc tcactgggga
      721 ccaaagcagt ttccatgccc aaaggtgtgg ctgtcgtcat catcaacagt aacttcaaac
      781 gtaccctggt tggcagcgaa tacaacaccc gtcgtgaaca gtgcgaaacc ggtgcgcgtt
      841 tcttccagca gccagccctg cgtgatgtca ccattgaaga gttcaacgct gttgcgcatg
      901 aactggaccc gatcgtggca aaacgcgtgc gtcatatact gactgaaaac gcccgcaccg
      961 ttgaagctgc cagcgcgctg gagcaaggcg acctgaaacg tatgggcgag ttgatggcgg
     1021 agtctcatgc ctctatgcgc gatgatttcg aaatcaccgt gccgcaaatt gacactctgg
     1081 tagaaatcgt caaagctgtg attggcgaca aaggtggcgt acgcatgacc ggcggcggat
     1141 ttggcggctg tatcgtcgcg ctgatcccgg aagaactggt gcctgccgta cagcaagctg
     1201 tcgctgaaca atatgaagca aaaacaggta ttaaagagac tttttacgtt tgtaaaccat
     1261 cacaaggagc aggacagtgc tgaacgaaac tcctgcagcg cgtagtacct cacaccgtcc
     1321 tgcgggttga cgtatgaggc ttggatcctc ccgctgcgct gagtcttcag cgatccccat
     1381 ccgcgacgtg ccaactaggt ctcctctcgt cgtgaacaag gctaccgggt tgcaactcct
     1441 gtgcaactct caggcttcaa cgcgcttcta cgacctgcaa tttctttcca cttagaggat
     1501 gcagccgaga gggggtaaaa acctcccatt cgccattcag gctgcgcaac tgttgggaag
     1561 ggcgtttcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     1621 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     1681 agtgagcgcg acgtaataag ttgaagagca tctgctcttc agcatcactg cccgctttcc
     1741 agtcgggaaa cctgtcgtgc cagctgcatt aacatggtca tagctgtttc cttgcgtatt
     1801 gggcgctctc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg gtaaagcctg
     1861 gggtgcctaa tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     1921 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     1981 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2041 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2101 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2161 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2221 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2281 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2341 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2401 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2461 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2521 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaggcgcgc ctgagtgagc
     2581 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     2641 agatcgggcc cagccggccc acatatacct gccgttcact attatttagt gaaatgagat
     2701 attatgatat tttctgaatt gtgattaaaa aggcaacttt atgcccatgc aacagaaact
     2761 ataaaaaata cagagaatga aaagaaacag atagattttt tagttcttta ggcccgtagt
     2821 ctgcaaatcc ttttatgatt ttctatcaaa caaaagagga aaatagacca gttgcaatcc
     2881 aaacgagagt ctaatagaat gaggtcgaaa agtaaatcgc gcgggtttgt tactgataaa
     2941 gcaggcaaga cctaaaatgt gtaaagggca aagtgtatac tttggcgtca ccccttacat
     3001 attttaggtc tttttttatt gtgcgtaact aacttgccat cttcaaacag gagggctgga
     3061 agaagcagac cgctaacaca gtacataaaa aaggagacat gaacgatgaa catcaaaaag
     3121 tttgcaaaac aagcaacagt attaaccttt actaccgcac tgctggcagg aggcgcaact
     3181 caagcgtttg cgaaagaaac gaaccaaaag ccatataagg aaacatacgg catttcccat
     3241 attacacgcc atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa atataaagtt
     3301 cctgagttcg attcgtccac aattaaaaat atctcttctg caaaaggcct ggacgtttgg
     3361 gacagctggc cattacaaaa cactgacggc actgtcgcaa actatcacgg ctaccacatc
     3421 gtctttgcat tagccggaga tcctaaaaat gcggatgaca catcgattta catgttctat
     3481 caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt ctttaaagac
     3541 agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga atggtcaggt
     3601 tcagccacat ttacatctga cggaaaaatc cgtttattct acactgattt ctccggtaaa
     3661 cattacggca aacaaacact gacaactgca caagttaacg tatcagcatc agacagctct
     3721 ttgaacatca acggtgtaga ggattataaa tcaatctttg acggtgacgg aaaaacgtat
     3781 caaaatgtac agcagttcat cgatgaaggc aactacagct caggcgacaa ccatacgctg
     3841 agagatcctc actacgtaga agataaaggc cacaaatact tagtatttga agcaaacact
     3901 ggaactgaag atggctacca aggcgaagaa tctttattta acaaagcata ctatggcaaa
     3961 agcacatcat tcttccgtca agaaagtcaa aaacttctgc aaagcgataa aaaacgcacg
     4021 gctgagttag caaacggcgc tctcggtatg attgagctaa acgatgatta cacactgaaa
     4081 aaagtgatga aaccgctgat tgcatctaac acagtaacag atgaaattga acgcgcgaac
     4141 gtctttaaaa tgaacggcaa atggtacctg ttcactgact cccgcggatc aaaaatgacg
     4201 attgacggca ttacgtctaa cgatatttac atgcttggtt atgtttctaa ttctttaact
     4261 ggcccataca agccgctgaa caaaactggc cttgtgttaa aaatggatct tgatcctaac
     4321 gatgtaacct ttacttactc acacttcgct gtacctcaag cgaaaggaaa caatgtcgtg
     4381 attacaagct atatgacaaa cagaggattc tacgcagaca aacaatcaac gtttgcgcct
     4441 agcttcctgc tgaacatcaa aggcaagaaa acatctgttg tcaaagacag catccttgaa
     4501 caaggacaat taacagttaa caaataaaaa cgcaaaagaa aatgccgata tcctattggc
     4561 attgcggccg cggtctcggc ggtgggtcgt ctagaggtct gacgctcagt ggaacgaaaa
     4621 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     4681 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4741 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4801 agttgcctga ctccccgacg tggccgacca gcccgtcatc gtcaacgcct gatccgcggt
     4861 gcggacaggc cgtgtcgcga ccggccgtgc ggaattaagc cggcccgtac cctgtgaata
     4921 gaggtccgct gtgacacaag aatccctgtt acttctcgac cgtattgatt cggatgattc
     4981 ctacgcgagc ctgcggaacg accaggaatt ctgggagccg ctggcccgcc gagccctgga
     5041 ggagctcggg ctgccggtgc cgccggtgct gcgggtgccc ggcgagagca ccaaccccgt
     5101 actggtcggc gagcccggcc cggtgatcaa gctgttcggc gagcactggt gcggtccgga
     5161 gagcctcgcg tcggagtcgg aggcgtacgc ggtcctggcg gacgccccgg tgccggtgcc
     5221 ccgcctcctc ggccgcggcg agctgcggcc cggcaccgga gcctggccgt ggccctacct
     5281 ggtgatgagc cggatgaccg gcaccacctg gcggtccgcg atggacggca cgaccgaccg
     5341 gaacgcgctg ctcgccctgg cccgcgaact cggccgggtg ctcggccggc tgcacagggt
     5401 gccgctgacc gggaacaccg tgctcacccc ccattccgag gtcttcccgg aactgctgcg
     5461 ggaacgccgc gcggcgaccg tcgaggacca ccgcgggtgg ggctacctct cgccccggct
     5521 gctggaccgc ctggaggact ggctgccgga cgtggacacg ctgctggccg gccgcgaacc
     5581 ccggttcgtc cacggcgacc tgcacgggac caacatcttc gtggacctgg ccgcgaccga
     5641 ggtcaccggg atcgtcgact tcaccgacgt ctatgcggga gactcccgct acagcctggt
     5701 gcaactgcat ctcaacgcct tccggggcga ccgcgagatc ctggccgcgc tgctcgacgg
     5761 ggcgcagtgg aagcggaccg aggacttcgc ccgcgaactg ctcgccttca ccttcctgca
     5821 cgacttcgag gtgttcgagg agaccccgct ggatctctcc ggcttcaccg atccggagga
     5881 actggcgcag ttcctctggg ggccgccgga caccgccccc ggcgcctgac gccccgggcc
     5941 cgcgccgaag cccgctgctg cgagcccgga gcgggccggc cgacggcggt gcgggcccgg
     6001 cggcggacgc tcagcagcgg cgggcgtgaa aggccctggc atcctcgatc atctcctcca
     6061 gggtgggtcg gccgggcttc catcccagct cggcaaggat caacgaggac agtcgcacga
     6121 cgaagttctt ctggatcgcg cccgtgctgg aagcactcaa cctcgaagcg tgtggttgcg
     6181 gagccatcta gcaaccacac gac
//
