LOCUS       sequence-349446-        3282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for gene replacement using kanamycin/sucrose 
            selection/counterselection. Derived from pK18mobsacB but reduced in 
            size..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3282)
  AUTHORS   Ling C, Peabody GL, Salvachua D, Kim YM, Kneucker CM, Calvey CH, 
            Monninger MA, Munoz NM, Poirier BC, Ramirez KJ, St John PC, 
            Woodworth SP, Magnuson JK, Burnum-Johnson KE, Guss AM, Johnson CW, 
            Beckham GT
  TITLE     Muconic acid production from glucose and xylose in Pseudomonas 
            putida via evolution and metabolic engineering.
  JOURNAL   Nat Commun. 2022 Aug 22;13(1):4925. doi: 10.1038/s41467-022-32296-y.
  PUBMED    35995792
REFERENCE   2  (bases 1 to 3282)
  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: "Nat 
            Commun."; date: "2022-08-22"; volume: "13(1):4925. doi"; pages: " 
            10.1038/s41467-022-32296-y"
COMMENT     Sequence Label: pK18msB
FEATURES             Location/Qualifiers
     source          1..3282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     292..311
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(429..515,516..1850)
                     /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
                     ITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     oriT            1867..1975
                     /note="incP origin of transfer"
     rep_origin      2127..2715
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2616..2635
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2727..2743
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2727..2743
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    2752..2808
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2812..2829)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2812..2828)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(2910..3282,1..422)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2964..2983)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
       61 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
      121 tcgcgccagc cgaactgttc gccaggctca aggcgcggat gcccgacggc gaggatctcg
      181 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
      241 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
      301 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
      361 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
      421 gagcgacgat gaacatcaaa aagtttgcaa aacaagcaac agtattaacc tttactaccg
      481 cactgctggc aggaggcgca actcaagcgt ttgcgaaaga aacgaaccaa aagccatata
      541 aggaaacata cggcatttcc catattacac gccatgatat gctgcaaatc cctgaacagc
      601 aaaaaaatga aaaatatcaa gtttctgaat ttgattcgtc cacaattaaa aatatctctt
      661 ctgcaaaagg cctggacgtt tgggacagct ggccattaca aaacgctgac ggcactgtcg
      721 caaactatca cggctaccac atcgtctttg cattagccgg agatcctaaa aatgcggatg
      781 acacatcgat ttacatgttc tatcaaaaag tcggcgaaac ttctattgac agctggaaaa
      841 acgctggccg cgtctttaaa gacagcgaca aattcgatgc aaatgattct atcctaaaag
      901 accaaacaca agaatggtca ggttcagcca catttacatc tgacggaaaa atccgtttat
      961 tctacactga tttctccggt aaacattacg gcaaacaaac actgacaact gcacaagtta
     1021 acgtatcagc atcagacagc tctttgaaca tcaacggtgt agaggattat aaatcaatct
     1081 ttgacggtga cggaaaaacg tatcaaaatg tacagcagtt catcgatgaa ggcaactaca
     1141 gctcaggcga caaccatacg ctgagagatc ctcactacgt agaagataaa ggccacaaat
     1201 acttagtatt tgaagcaaac actggaactg aagatggcta ccaaggcgaa gaatctttat
     1261 ttaacaaagc atactatggc aaaagcacat cattcttccg tcaagaaagt caaaaacttc
     1321 tgcaaagcga taaaaaacgc acggctgagt tagcaaacgg cgctctcggt atgattgagc
     1381 taaacgatga ttacacactg aaaaaagtga tgaaaccgct gattgcatct aacacagtaa
     1441 cagatgaaat tgaacgcgcg aacgtcttta aaatgaacgg caaatggtac ctgttcactg
     1501 actcccgcgg atcaaaaatg acgattgacg gcattacgtc taacgatatt tacatgcttg
     1561 gttatgtttc taattcttta actggcccat acaagccgct gaacaaaact ggccttgtgt
     1621 taaaaatgga tcttgatcct aacgatgtaa cctttactta ctcacacttc gctgtacctc
     1681 aagcgaaagg aaacaatgtc gtgattacaa gctatatgac aaacagagga ttctacgcag
     1741 acaaacaatc aacgtttgcg ccgagcttcc tgctgaacat caaaggcaag aaaacatctg
     1801 ttgtcaaaga cagcatcctt gaacaaggac aattaacagt taacaaataa tcagaccccg
     1861 tagaaacggc cagcctcgca gagcaggatt cccgttgagc accgccaggt gcgaataagg
     1921 gacagtgaag aaggaacacc cgctcgcggg tgggcctact tcacctatcc tgcccggctg
     1981 acgccgttgg atacaccaag gaaagtctac acgaaccctt tggcaaaatc ctgtatatcg
     2041 tgcgaaaaag gatggatata ccgaaaaaat cgctataatg accccgaagc agggttatgc
     2101 agcggaaaag agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2161 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2221 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     2281 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2341 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2401 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2461 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2521 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     2581 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     2641 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     2701 cggagcctat ggaaaaacgc ctcacacagg aaacagctat gacatgatta cgaattcgag
     2761 ctcggtaccc ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cactggccgt
     2821 cgttttacaa cgtcgtgacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
     2881 ccctgcaaac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     2941 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3001 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3061 ccgacctgtc cggtgccctg aatgaactcc aagacgaggc agcgcggcta tcgtggctgg
     3121 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3181 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3241 agaaagtatc catcatggct gatgcaatgc ggcggctgca ta
//
