LOCUS       sequence-264226-        5772 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 5772)
  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 5772)
  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 (Kan)
FEATURES             Location/Qualifiers
     source          1..5772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    40..59
                     /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."
     promoter        complement(join(65..71,72..89,90..95))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(252..271)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             263..1078
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(336..355)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(join(1362..2696,2697..2783))
                     /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"
     promoter        complement(2784..3229)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     rep_origin      3374..3962
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3863..3882
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4206..4223)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4206..4222)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4215..4237)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ctgttcctgt gttatcggat cagcttggta ccgcatgcga attgtgagcg ctcacaattc
       61 cacacattat acgagccgga agcataaagt gtcaagcctg gggtctggat ccatggatat
      121 ctagttctag ccacctgacg tcgggggggg ggaaagccac gttgtgtctc aaaatctctg
      181 atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat
      241 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctcgaggcc
      301 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
      361 cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag ccccatgcgc cagagttgtt
      421 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
      481 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
      541 tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata
      601 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
      661 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
      721 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
      781 gcctgttgaa caagtctgga aagaaatgca taatcttttg ccattctcac cggattcagt
      841 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
      901 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
      961 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     1021 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
     1081 agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgg
     1141 ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc agatcacgca tcttcccgac
     1201 aacgcagacc attccgtggc aaagcaaaag ttcaaaatca ccaactggtc cacctacaac
     1261 aaagctctca ccaaccgtgg ctccctcact ttctctagac gacccaccgc cgagaccgcg
     1321 gccgcaatgc caataggata tcggcatttt cttttgcgtt tttatttgtt aactgttaat
     1381 tgtccttgtt caaggatgct gtctttgaca acagatgttt tcttgccttt gatgttcagc
     1441 aggaagctag gcgcaaacgt tgattgtttg tctgcgtaga atcctctgtt tgtcatatag
     1501 cttgtaatca cgacattgtt tcctttcgct tgaggtacag cgaagtgtga gtaagtaaag
     1561 gttacatcgt taggatcaag atccattttt aacacaaggc cagttttgtt cagcggcttg
     1621 tatgggccag ttaaagaatt agaaacataa ccaagcatgt aaatatcgtt agacgtaatg
     1681 ccgtcaatcg tcatttttga tccgcgggag tcagtgaaca ggtaccattt gccgttcatt
     1741 ttaaagacgt tcgcgcgttc aatttcatct gttactgtgt tagatgcaat cagcggtttc
     1801 atcacttttt tcagtgtgta atcatcgttt agctcaatca taccgagagc gccgtttgct
     1861 aactcagccg tgcgtttttt atcgctttgc agaagttttt gactttcttg acggaagaat
     1921 gatgtgcttt tgccatagta tgctttgtta aataaagatt cttcgccttg gtagccatct
     1981 tcagttccag tgtttgcttc aaatactaag tatttgtggc ctttatcttc tacgtagtga
     2041 ggatctctca gcgtatggtt gtcgcctgag ctgtagttgc cttcatcgat gaactgctgt
     2101 acattttgat acgtttttcc gtcaccgtca aagattgatt tataatcctc tacaccgttg
     2161 atgttcaaag agctgtctga tgctgatacg ttaacttgtg cagttgtcag tgtttgtttg
     2221 ccgtaatgtt taccggagaa atcagtgtag aataaacgga tttttccgtc agatgtaaat
     2281 gtggctgaac ctgaccattc ttgtgtttgg tcttttagga tagaatcatt tgcatcgaat
     2341 ttgtcgctgt ctttaaagac gcggccagcg tttttccagc tgtcaataga agtttcgccg
     2401 actttttgat agaacatgta aatcgatgtg tcatccgcat ttttaggatc tccggctaat
     2461 gcaaagacga tgtggtagcc gtgatagttt gcgacagtgc cgtcagtgtt ttgtaatggc
     2521 cagctgtccc aaacgtccag gccttttgca gaagagatat ttttaattgt ggacgaatcg
     2581 aactcaggaa ctttatattt ttcatttttt tgctgttcag ggatttgcag catatcatgg
     2641 cgtgtaatat gggaaatgcc gtatgtttcc ttatatggct tttggttcgt ttctttcgca
     2701 aacgcttgag ttgcgcctcc tgccagcagt gcggtagtaa aggttaatac tgttgcttgt
     2761 tttgcaaact ttttgatgtt catcgttcat gtctcctttt ttatgtactg tgttagcggt
     2821 ctgcttcttc cagccctcct gtttgaagat ggcaagttag ttacgcacaa taaaaaaaga
     2881 cctaaaatat gtaaggggtg acgccaaagt atacactttg ccctttacac attttaggtc
     2941 ttgcctgctt tatcagtaac aaacccgcgc gatttacttt tcgacctcat tctattagac
     3001 tctcgtttgg attgcaactg gtctattttc ctcttttgtt tgatagaaaa tcataaaagg
     3061 atttgcagac tacgggccta aagaactaaa aaatctatct gtttcttttc attctctgta
     3121 ttttttatag tttctgttgc atgggcataa agttgccttt ttaatcacaa ttcagaaaat
     3181 atcataatat ctcatttcac taaataatag tgaacggcag gtatatgtgg gccggctggg
     3241 cccgatcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3301 cggtatcagc tcactcaggc gcgccttcgt tccactgagc gtcagacccc gtagaaaaga
     3361 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3421 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3481 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     3541 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3601 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3661 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3721 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3781 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3841 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3901 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3961 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcatt
     4021 aggcacccca ggctttaccc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4081 gagcgcccaa tacgcaagga aacagctatg accatgttaa tgcagctggc acgacaggtt
     4141 tcccgactgg aaagcgggca gtgatgctga agagcagatg ctcttcaact tattacgtcg
     4201 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4261 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4321 gaaacgccct tcccaacagt tgcgcagcct gaatggcgaa tgggaggttt ttaccccctc
     4381 tcggctgcat cctctaagtg gaaagaaatt gcaggtcgta gaagcgcgtt gaagcctgag
     4441 agttgcacag gagttgcaac ccggtagcct tgttcacgac gagaggagac ctagttggca
     4501 cgtcgcggat ggggatcgct gaagactcag cgcagcggga ggatccaagc ctcatacgtc
     4561 aacccgcagg acggtgtgag gtactacgcg ctgcaggagt ttcgttcagc actgtcctgc
     4621 tccttgtgat ggtttacaaa cgtaaaaagt ctctttaata cctgtttttg cttcatattg
     4681 ttcagcgaca gcttgctgta cggcaggcac cagttcttcc gggatcagcg cgacgataca
     4741 gccgccaaat ccgccgccgg tcatgcgtac gccacctttg tcgccaatca cagctttgac
     4801 gatttctacc agagtgtcaa tttgcggcac ggtgatttcg aaatcatcgc gcatagaggc
     4861 atgagactcc gccatcaact cgcccatacg tttcaggtcg ccttgctcca gcgcgctggc
     4921 agcttcaacg gtgcgggcgt tttcagtcag tatatgacgc acgcgttttg ccacgatcgg
     4981 gtccagttca tgcgcaacag cgttgaactc ttcaatggtg acatcacgca gggctggctg
     5041 ctggaagaaa cgcgcaccgg tttcgcactg ttcacgacgg gtgttgtatt cgctgccaac
     5101 cagggtacgt ttgaagttac tgttgatgat gacgacagcc acacctttgg gcatggaaac
     5161 tgctttggtc cccagtgagc ggcaatcgat cagcaaggca tgatctttct tgccgagcgc
     5221 ggaaattagc tgatccatga tcccgcagtt acagcctaca aactggtttt ctgcttcctg
     5281 accgttaagc gcgatttgtg cgccgtccag cggcagatga taaagctgct gcaatacggt
     5341 tccgaccgcg acttccagtg aagcggaaga acttaacccg gcaccctgcg gcacattgcc
     5401 gctgatcacc atgtccacgc cgccgaagct gttgttacgc agttgcagat gtttcaccac
     5461 gccacgaacg tagttagccc attgatagtt ttcatgtgcg acaatgggcg catcgaggga
     5521 aaactcgtcg agctgatttt cataatcggc tgccatcacg cgaactttac ggtcatcgcg
     5581 tggtgcacaa ctgatcacgg tttgataatc aatcgcgcag ggcagaacga aaccgtcgtt
     5641 gtagtcggtg tgttcaccaa tcaaattcac gcggccaggc gcctgaatgg tgtgagtggc
     5701 agggagccaa atgcgttggc aaacagagat tgtgtttttt ctttcagact catttcttac
     5761 actccggaag at
//
