LOCUS       sequence-229793-        4437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kanamycin resistance cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4437)
  AUTHORS   Diebold-Durand ML, Burmann F, Gruber S
  TITLE     High-Throughput Allelic Replacement Screening in Bacillus subtilis.
  JOURNAL   Methods Mol Biol. 2019;2004:49-61. doi: 10.1007/978-1-4939-9520-2_5.
  PUBMED    31147909
REFERENCE   2  (bases 1 to 4437)
  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: "Methods Mol
            Biol."; date: "2019"; volume: "2004:49-61. doi"; pages: " 
            10.1007/978-1-4939-9520-2_5"
COMMENT     Sequence Label: pJet1.2 kanR cassette
FEATURES             Location/Qualifiers
     source          1..4437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     305..324
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        305..323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             920..1714
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        complement(join(2269..2275,2276..2293,2294..2299))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    2314..2335
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2455..2472)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2626..3214)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2706..2725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3385..4176,4177..4245))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4008..4027
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4246..4350)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4418..4436
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc
       61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc
      121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact
      181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt
      241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc
      301 ggcgtaatac gactcactat agggagagcg gccgccagat cttccggatg gctcgagttt
      361 ttcagcaaga tgttacaggt ctcgaataaa acccagcgaa ccatttgagg tgataggtaa
      421 gattataccg aggtatgaaa acgagaattg gacctttaca gaattactct atgaagcgcc
      481 atatttaaaa agctaccaag acgaagagga tgaagaggat gaggaggcag attgccttga
      541 atatattgac aatactgata agataatata tcttttatat agaagatatc gccgtatgta
      601 aggatttcag ggggcaaggc ataggcagcg cgcttatcaa tatatctata gaatgggcaa
      661 agcataaaaa cttgcatgga ctaatgcttg aaacccagga caataacctt atagcttgta
      721 aattctatca taattgtggt ttcaaaatcg gctccgtcga tactatgtta tacgccaact
      781 ttgaaaacaa ctttgaaaaa gctgttttct ggtatttaag gttttagaat gcaaggaaca
      841 gtgaattgga gttcgtcttg ttataattag cttcttgggg tatctttaaa tactgtagaa
      901 aagaggaagg aaataataaa tggctaaaat gagaatatca ccggaattga aaaaactgat
      961 cgaaaaatac cgctgcgtaa aagatacgga aggaatgtct cctgctaagg tatataagct
     1021 ggtgggagaa aatgaaaacc tatatttaaa aatgacggac agccggtata aagggaccac
     1081 ctatgatgtg gaacgggaaa aggacatgat gctatggctg gaaggaaagc tgcctgttcc
     1141 aaaggtcctg cactttgaac ggcatgatgg ctggagcaat ctgctcatga gtgaggccga
     1201 tggcgtcctt tgctcggaag agtatgaaga tgaacaaagc cctgaaaaga ttatcgagct
     1261 gtatgcggag tgcatcaggc tctttcactc catcgacata tcggattgtc cctatacgaa
     1321 tagcttagac agccgcttag ccgaattgga ttacttactg aataacgatc tggccgatgt
     1381 ggattgcgaa aactgggaag aagacactcc atttaaagat ccgcgcgagc tgtatgattt
     1441 tttaaagacg gaaaagcccg aagaggaact tgtcttttcc cacggcgacc tgggagacag
     1501 caacatcttt gtgaaagatg gcaaagtaag tggctttatt gatcttggga gaagcggcag
     1561 ggcggacaag tggtatgaca ttgccttctg cgtccggtcg atcagggagg atatcgggga
     1621 agaacagtat gtcgagctat tttttgactt actggggatc aagcctgatt gggagaaaat
     1681 aaaatattat attttactgg atgaattgtt ttagcattat tcaaaggtga aagcactcgt
     1741 taagtatatg aaaaattcta tagaaacttc tctcaattaa gaaaaaggcc ccaacatctt
     1801 ggggcctttt tcttttttat ctacgagacc catgatcttt ctagaagatc tcctacaata
     1861 ttctcagctg ccatggaaaa tcgatgttct tcttttattc tctcaagatt ttcaggctgt
     1921 atattaaaac ttatattaag aactatgcta accacctcat caggaaccgt tgtaggtggc
     1981 gtgggttttc ttggcaatcg actctcatga aaactacgag ctaaatattc aatatgttcc
     2041 tcttgaccaa ctttattctg catttttttt gaacgaggtt tagagcaagc ttcaggaaac
     2101 tgagacagga attttattaa aaatttaaat tttgaagaaa gttcagggtt aatagcatcc
     2161 attttttgct ttgcaagttc ctcagcattc ttaacaaaag acgtctcttt tgacatgttt
     2221 aaagtttaaa cctcctgtgt gaaattatta tccgctcata attccacaca ttatacgagc
     2281 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     2341 gttgcgctca ctgccaattg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2401 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2461 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2521 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2581 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2641 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2701 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2761 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2821 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2881 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2941 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3001 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3061 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3121 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3181 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3241 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3301 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3361 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3421 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3481 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3541 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3601 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3661 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3721 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3781 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     3841 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3901 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3961 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4021 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4081 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4141 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4201 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4261 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4321 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4381 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggcc
//
