LOCUS       sequence-363053-        3038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for gene replacement using kanamycin/sucrose 
            selection/counterselection. Derived from pK18mobsacB but reduced in 
            size and lacking oriT required for conjugation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3038)
  AUTHORS   Jayakody LN, Johnson CW, Whitham JM, Giannone RJ, Black BA, 
            Cleveland NS, Klingeman DM, Michener WE, Olstad JL, Vardon DR, Brown
            RC, Brown SD, Hettich RL, Guss AM, Beckham GT
  TITLE     Thermochemical wastewater valorization via enhanced microbial 
            toxicity tolerance
  JOURNAL   Energy Environ. Sci., 2018,11, 1625-1638
REFERENCE   2  (bases 1 to 3038)
  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: "Energy 
            Environ. Sci., 2018,11, 1625-1638"
COMMENT     Sequence Label: pK18sB
FEATURES             Location/Qualifiers
     source          1..3038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      106..694
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     595..614
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     706..722
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     706..722
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    731..787
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(791..808)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(791..807)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             889..1683
                     /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(943..962)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1553..1572
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(1690..1776,1777..3038,1..73)
                     /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"
ORIGIN
        1 catcaaaggc aagaaaacat ctgttgtcaa agacagcatc cttgaacaag gacaattaac
       61 agttaacaaa taatcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
      121 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
      181 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
      241 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
      301 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      361 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      421 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      481 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      541 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      601 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      661 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc tcacacagga aacagctatg
      721 acatgattac gaattcgagc tcggtacccg gggatcctct agagtcgacc tgcaggcatg
      781 caagcttggc actggccgtc gttttacaac gtcgtgaccg gaattgccag ctggggcgcc
      841 ctctggtaag gttgggaagc cctgcaaaca ggatgaggat cgtttcgcat gattgaacaa
      901 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
      961 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     1021 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactcca agacgaggca
     1081 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     1141 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     1201 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     1261 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     1321 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     1381 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgga tgcccgacgg cgaggatctc
     1441 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     1501 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     1561 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     1621 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     1681 tgagcgacga tgaacatcaa aaagtttgca aaacaagcaa cagtattaac ctttactacc
     1741 gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca aaagccatat
     1801 aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat ccctgaacag
     1861 caaaaaaatg aaaaatatca agtttctgaa tttgattcgt ccacaattaa aaatatctct
     1921 tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacgctga cggcactgtc
     1981 gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa aaatgcggat
     2041 gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa
     2101 aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc tatcctaaaa
     2161 gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa aatccgttta
     2221 ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac tgcacaagtt
     2281 aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta taaatcaatc
     2341 tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga aggcaactac
     2401 agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa aggccacaaa
     2461 tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga agaatcttta
     2521 tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt
     2581 ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg tatgattgag
     2641 ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc taacacagta
     2701 acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta cctgttcact
     2761 gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat ttacatgctt
     2821 ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac tggccttgtg
     2881 ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt cgctgtacct
     2941 caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg attctacgca
     3001 gacaaacaat caacgtttgc gccgagcttc ctgctgaa
//
