LOCUS       sequence-264191-        4346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Analogous to pKO but without the negative selection gene galK..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4346)
  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 4346)
  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: pKOdeltagalK (Apr)
FEATURES             Location/Qualifiers
     source          1..4346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(206..794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(286..305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        939..1384
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1385..1471,1472..2806)
                     /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"
     CDS             2991..3791
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     primer_bind     4277..4299
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4291..4308
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4292..4308
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gcatcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta acatggtcat
       61 agctgtttcc ttgcgtattg ggcgctctcc gcttcctcgc tcactgactc gctgcgctcg
      121 gtcgttcggg taaagcctgg ggtgcctaat gagcaaaagg ccagcaaaag gccaggaacc
      181 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      241 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      301 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      361 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      421 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      481 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      541 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      601 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      661 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      721 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      781 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      841 aaggcgcgcc tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      901 cagtgagcga ggaagcggaa gatcgggccc agccggccca catatacctg ccgttcacta
      961 ttatttagtg aaatgagata ttatgatatt ttctgaattg tgattaaaaa ggcaacttta
     1021 tgcccatgca acagaaacta taaaaaatac agagaatgaa aagaaacaga tagatttttt
     1081 agttctttag gcccgtagtc tgcaaatcct tttatgattt tctatcaaac aaaagaggaa
     1141 aatagaccag ttgcaatcca aacgagagtc taatagaatg aggtcgaaaa gtaaatcgcg
     1201 cgggtttgtt actgataaag caggcaagac ctaaaatgtg taaagggcaa agtgtatact
     1261 ttggcgtcac cccttacata ttttaggtct ttttttattg tgcgtaacta acttgccatc
     1321 ttcaaacagg agggctggaa gaagcagacc gctaacacag tacataaaaa aggagacatg
     1381 aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta ttaaccttta ctaccgcact
     1441 gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg aaccaaaagc catataagga
     1501 aacatacggc atttcccata ttacacgcca tgatatgctg caaatccctg aacagcaaaa
     1561 aaatgaaaaa tataaagttc ctgagttcga ttcgtccaca attaaaaata tctcttctgc
     1621 aaaaggcctg gacgtttggg acagctggcc attacaaaac actgacggca ctgtcgcaaa
     1681 ctatcacggc taccacatcg tctttgcatt agccggagat cctaaaaatg cggatgacac
     1741 atcgatttac atgttctatc aaaaagtcgg cgaaacttct attgacagct ggaaaaacgc
     1801 tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat gattctatcc taaaagacca
     1861 aacacaagaa tggtcaggtt cagccacatt tacatctgac ggaaaaatcc gtttattcta
     1921 cactgatttc tccggtaaac attacggcaa acaaacactg acaactgcac aagttaacgt
     1981 atcagcatca gacagctctt tgaacatcaa cggtgtagag gattataaat caatctttga
     2041 cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc gatgaaggca actacagctc
     2101 aggcgacaac catacgctga gagatcctca ctacgtagaa gataaaggcc acaaatactt
     2161 agtatttgaa gcaaacactg gaactgaaga tggctaccaa ggcgaagaat ctttatttaa
     2221 caaagcatac tatggcaaaa gcacatcatt cttccgtcaa gaaagtcaaa aacttctgca
     2281 aagcgataaa aaacgcacgg ctgagttagc aaacggcgct ctcggtatga ttgagctaaa
     2341 cgatgattac acactgaaaa aagtgatgaa accgctgatt gcatctaaca cagtaacaga
     2401 tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa tggtacctgt tcactgactc
     2461 ccgcggatca aaaatgacga ttgacggcat tacgtctaac gatatttaca tgcttggtta
     2521 tgtttctaat tctttaactg gcccatacaa gccgctgaac aaaactggcc ttgtgttaaa
     2581 aatggatctt gatcctaacg atgtaacctt tacttactca cacttcgctg tacctcaagc
     2641 gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac agaggattct acgcagacaa
     2701 acaatcaacg tttgcgccta gcttcctgct gaacatcaaa ggcaagaaaa catctgttgt
     2761 caaagacagc atccttgaac aaggacaatt aacagttaac aaataaaaac gcaaaagaaa
     2821 atgccgatat cctattggca ttgcggccgc ggtctcggcg gtgggtcgtc tagaaggatc
     2881 ttcacctaga tccttttggt tcatgtgcag ctccatcagc aaaaggggat gataagttta
     2941 tcaccaccga ctatttgcaa cagtgccgtt gatcgtgcta tgatcgactg atgtcatcag
     3001 cggtggagtg caatgtcgtg caatacgaat ggcgaaaagc cgagctcatc ggtcagcttc
     3061 tcaaccttgg ggttaccccc ggcggtgtgc tgctggtcca cagctccttc cgtagcgtcc
     3121 ggcccctcga agatgggcca cttggactga tcgaggccct gcgtgctgcg ctgggtccgg
     3181 gagggacgct cgtcatgccc tcgtggtcag gtctggacga cgagccgttc gatcctgcca
     3241 cgtcgcccgt tacaccggac cttggagttg tctctgacac attctggcgc ctgccaaatg
     3301 taaagcgcag cgcccatcca tttgcctttg cggcagcggg gccacaggca gagcagatca
     3361 tctctgatcc attgcccctg ccacctcact cgcctgcaag cccggtcgcc cgtgtccatg
     3421 aactcgatgg gcaggtactt ctcctcggcg tgggacacga tgccaacacg acgctgcatc
     3481 ttgccgagtt gatggcaaag gttccctatg gggtgccgag acactgcacc attcttcagg
     3541 atggcaagtt ggtacgcgtc gattatctcg agaatgacca ctgctgtgag cgctttgcct
     3601 tggcggacag gtggctcaag gaaaagagcc ttcagaagga aggtccagtc ggtcatgcct
     3661 ttgctcggtt gatccgctcc cgcgacattg tggcgacagc cctgggtcaa ctgggccgag
     3721 atccgttgat cttcctgcat ccgccagagg cgggatgcga agaatgcgat gccgctcgcc
     3781 agtcgattgg ctgagctcat gagcggagaa cgagatgacg ttggaggggc aaggtcgcgc
     3841 tgattgctgg ggcaacacgt ggagcggatc ggggattgtc tttcttcagc tcgctgatga
     3901 tatgctgacg ctcaatgccg tttggcctcc gactaacgaa aatcccgcat ttggacggct
     3961 gatccatgga tcctcccgct gcgctgagtc ttcagcgatc cccatccgcg acgtgccaac
     4021 taggtctcct ctcgtcgtga acaaggctac cgggttgcaa ctcctgtgca actctcaggc
     4081 ttcaacgcgc ttctacgacc tgcaatttct ttccacttag aggatgcagc cgagaggggg
     4141 taaaaacctc ccattcgcca ttcaggctgc gcaactgttg ggaagggcgt ttcggtgcgg
     4201 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     4261 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgacgta
     4321 ataagttgaa gagcatctgc tcttca
//
