LOCUS       sequence-452438-        4737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Conjugation knockout plasmid (pK18msB) to delete the C. necator 
            addiction system toxin PemK, to enable deletion of the pHG1 
            megaplasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4737)
  AUTHORS   Calvey CH, Sanchez I Nogue V, White AM, Kneucker CM, Woodworth SP, 
            Alt HM, Eckert CA, Johnson CW
  TITLE     Improving growth of Cupriavidus necator H16 on formate using 
            adaptive laboratory evolution-informed engineering.
  JOURNAL   Metab Eng. 2023 Jan;75:78-90. doi: 10.1016/j.ymben.2022.10.016. Epub
            2022 Nov 9.
  PUBMED    36368470
REFERENCE   2  (bases 1 to 4737)
  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: "Metab 
            Eng."; date: "2023-01"; volume: "75:78-90. doi"; pages: " 
            10.1016/j.ymben.2022.10.016. Epub 2022 Nov 9"
COMMENT     Sequence Label: pCHC027 (∆pemK)
FEATURES             Location/Qualifiers
     source          1..4737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     601..617
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     601..617
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2141..2158)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2141..2157)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2239..3033
                     /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(2293..2312)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2903..2922
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(3040..3126,3127..4461)
                     /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            4478..4586
                     /note="incP origin of transfer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgcctcaca
      601 caggaaacag ctatgacatg attacgaatt cagctgctac ctcgaggctg cacaagagat
      661 tcgagccgat tgccataacc cactgcgaca ggtcacctgg taggggctca gcgtcgcgcc
      721 aagtgtggca gccgtggcgt cggccagcga agaaagcgcc ccgcggcggt gcagatttcc
      781 ggcgggttgc cgagaaagga ggctcaaatg tcctcacagg aaaacagcgg ccatgtcgaa
      841 caaggcaatg gcaaccgtgt cgaagctggc gctagcgggg ctgcggcatg gcgccggtgc
      901 ggtagccgga gcccggcgcg gcgccaagca gatacgcagg cttctgccgg cagactcctt
      961 cgcgcgttcg gctgcgcagc gccgctgcgc cgtcagtcgt cacgcgggca aatttcattt
     1021 gttggcagcg atggcgagca acgctcgagg gagcgatgta gatacgcgtt gaaacatgga
     1081 tctcttatgt ttatacttgt atcaacattg tttggaggca tctattatgc gaaaaagcgc
     1141 aaccctgacg attcaaaagt ggggcaacag cttggcggtt cgaatcccca ctgcggtggc
     1201 tcgttctgca catttcgccg agggccagga agtggaggta tccgtcgatg agattggcgt
     1261 aactgttcga ccagttggtc gtcgtgccct cactctcgcg gaaaagcttg ctctgtttga
     1321 ccccatcaag cacggcggcg aagctatggc cacgcagcgt gtcggcgcgg aggccatgta
     1381 acctccatgc ttgaccgcat ggcgcgttgt cttggcgaac acgccgagcc gtcgcaagct
     1441 ggtcactgcc agcgcacaca tgcggctcga ctggatgaag ccagccacgg tagacgtaga
     1501 cgtgatcagc acatcaacct gaccaccgag cagatcattg atggccgacc cggcgccctt
     1561 gtacggcacg tgctgcagcg gcacgctgct gttcttgtcg agcacaacgc ctatcaggtg
     1621 caacagcgtg ccgatgccgg gcgtggcgta ggtgatcttc tgcagttgtg ccttggcctg
     1681 tgtggccagc gccgggcagt gggcgggact cgtcgcgccg cgcggtccgc gtgccgaagt
     1741 cactgcgccg cccaaggttc gcgctgcgga actgggcgcc ggcctgcgtg agcgtcaatg
     1801 ctggcggcgg cccccccggc agcatggcgc gccgccaacc ccatgttgag gttgtgtccc
     1861 ggcgctactg gtcaacgcgc acaatcaacc tcacctggcg cacaacctac ctcaccgatc
     1921 cctgccggct gtttccttcc agcctttgtc cagcttggga gataagacat atgcacaggt
     1981 cacgcacaat acatctcacc ttaggggcat caacacaaca aacctcacct tcttggggcg
     2041 gcttcggatg cggtgccgtt catcaggcat cgtgtccgcc gtaacgggga tgtcgatgta
     2101 caaacagtag tcggcaaaca ctatgcaaat aaagcttggc actggccgtc gttttacaac
     2161 gtcgtgaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaaca
     2221 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     2281 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     2341 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     2401 ggtgccctga atgaactcca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     2461 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     2521 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     2581 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     2641 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     2701 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     2761 aaggcgcgga tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     2821 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     2881 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     2941 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     3001 gccttctatc gccttcttga cgagttcttc tgagcgacga tgaacatcaa aaagtttgca
     3061 aaacaagcaa cagtattaac ctttactacc gcactgctgg caggaggcgc aactcaagcg
     3121 tttgcgaaag aaacgaacca aaagccatat aaggaaacat acggcatttc ccatattaca
     3181 cgccatgata tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca agtttctgaa
     3241 tttgattcgt ccacaattaa aaatatctct tctgcaaaag gcctggacgt ttgggacagc
     3301 tggccattac aaaacgctga cggcactgtc gcaaactatc acggctacca catcgtcttt
     3361 gcattagccg gagatcctaa aaatgcggat gacacatcga tttacatgtt ctatcaaaaa
     3421 gtcggcgaaa cttctattga cagctggaaa aacgctggcc gcgtctttaa agacagcgac
     3481 aaattcgatg caaatgattc tatcctaaaa gaccaaacac aagaatggtc aggttcagcc
     3541 acatttacat ctgacggaaa aatccgttta ttctacactg atttctccgg taaacattac
     3601 ggcaaacaaa cactgacaac tgcacaagtt aacgtatcag catcagacag ctctttgaac
     3661 atcaacggtg tagaggatta taaatcaatc tttgacggtg acggaaaaac gtatcaaaat
     3721 gtacagcagt tcatcgatga aggcaactac agctcaggcg acaaccatac gctgagagat
     3781 cctcactacg tagaagataa aggccacaaa tacttagtat ttgaagcaaa cactggaact
     3841 gaagatggct accaaggcga agaatcttta tttaacaaag catactatgg caaaagcaca
     3901 tcattcttcc gtcaagaaag tcaaaaactt ctgcaaagcg ataaaaaacg cacggctgag
     3961 ttagcaaacg gcgctctcgg tatgattgag ctaaacgatg attacacact gaaaaaagtg
     4021 atgaaaccgc tgattgcatc taacacagta acagatgaaa ttgaacgcgc gaacgtcttt
     4081 aaaatgaacg gcaaatggta cctgttcact gactcccgcg gatcaaaaat gacgattgac
     4141 ggcattacgt ctaacgatat ttacatgctt ggttatgttt ctaattcttt aactggccca
     4201 tacaagccgc tgaacaaaac tggccttgtg ttaaaaatgg atcttgatcc taacgatgta
     4261 acctttactt actcacactt cgctgtacct caagcgaaag gaaacaatgt cgtgattaca
     4321 agctatatga caaacagagg attctacgca gacaaacaat caacgtttgc gccgagcttc
     4381 ctgctgaaca tcaaaggcaa gaaaacatct gttgtcaaag acagcatcct tgaacaagga
     4441 caattaacag ttaacaaata atcagacccc gtagaaacgg ccagcctcgc agagcaggat
     4501 tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac ccgctcgcgg
     4561 gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa ggaaagtcta
     4621 cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat accgaaaaaa
     4681 tcgctataat gaccccgaag cagggttatg cagcggaaaa gagatcaaag gatcttc
//
