LOCUS       sequence-453517-        4735 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Conjugation knockout plasmid (pK18msB) to delete the C. necator 
            phaCAB operon, to prevent accumulation of PHB..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4735)
  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 4735)
  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: pCHC042 (∆phaCAB)
FEATURES             Location/Qualifiers
     source          1..4735
                     /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(2139..2156)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2139..2155)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2237..3031
                     /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(2291..2310)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2901..2920
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(3038..3124,3125..4459)
                     /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            4476..4584
                     /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 ccgccggtcg cttctactcc tatcggcgcg
      661 atggcgtgac cggccgcatg gccagcctgg tctggctggc ggactgagcc cgccgctgcc
      721 tcactcgtcc ttgcccctgg ccgcctgcgc gcgctcggct tcagccttgc gtcggcggcg
      781 gccgggcgtg cccatgatgt agagcaccag cgccaccggc gccatgccat acatcaggaa
      841 ggtggcaacg cctgccacca cgttgtgctc ggtgatcgcc atcatcagcg ccacgtagag
      901 ccagccaatg gccacgatgt acatcaaaaa ttcatccttc tcgcctatgc tctggggcct
      961 cggcagatgc gagcgctgca taccgtccgg taggtcggga agcgtgcagt gccgaggcgg
     1021 attcccgcat tgacagcgcg tgcgttgcaa ggcaacaatg gactcaaatg tctcggaatc
     1081 gctgacgatt cccaggtttc tccggcaagc atagcgcatg gcgtctccat gcgagaatgt
     1141 cgcgcttgcc ggataaaagg ggagccgcta tcggaatgga cgcaagccac ggccgcagca
     1201 ggtgcggtcg agggcttcca gccagttcca gggcagatgt gccggcagac cctcccgctt
     1261 tgggggaggc gcaagccggg tccattcgga tagcatctcc ccatgcaaag tgccggccag
     1321 ggcaatgccc ggagccggtt cgaatagtga cggcagagag acaatcaaat cgtttaaacc
     1381 ctgccggcct ggttcaacca gtcggcagcc ggcgctggcg cccgcgtatt gcggtgcagc
     1441 cagcgcggcg cacaaggcgg cgggcgtttc gtttcgccgc ccgtttcgcg ggccgtcaag
     1501 gcccgcgaat cgtttctgcc cgcgcggcat tcctcgcttt ttgcgccaat tcaccgggtt
     1561 ttccttaagc cccgtcgctt ttcttagtgc cttgttgggc atagaatcag ggcagcggcg
     1621 cagccagcac catgttcgtg cagcgcggcc ctcgcggggg cgaggctgca ggccgccacg
     1681 cgcagccatg cgcgaacggg ccaccagatg gccggcacga caacaagcag atggcgcggg
     1741 cgataccgat ttgcgcactg caccccatgc ggtgcagcag cgcgcaaaca gcgatgacac
     1801 aaggacagag caccgatggc cacgaccaaa aaaggcgcag agcgactgat caaaaagtat
     1861 ccgaaccgta ggctctacga cacccagacc agcacctaca tcaccctggc cgacgtcaag
     1921 cagctggtca tggattcaga agaattcaag gtcgtcgacg ccaagtctgg tgacgaactg
     1981 acccgcagca tcttgctgca gatcatcctg gaagaagaaa cgggcggcgt gccgatgttc
     2041 tccagcgcga tgctgtcgca gatcatccgc ttctacggcc atgccatgca gggcatgatg
     2101 ggcacctacc tggaaaagaa catccaggca agcttggcac tggccgtcgt tttacaacgt
     2161 cgtgaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaacagg
     2221 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     2281 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     2341 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     2401 tgccctgaat gaactccaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     2461 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     2521 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     2581 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     2641 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     2701 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     2761 ggcgcggatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     2821 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     2881 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     2941 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     3001 cttctatcgc cttcttgacg agttcttctg agcgacgatg aacatcaaaa agtttgcaaa
     3061 acaagcaaca gtattaacct ttactaccgc actgctggca ggaggcgcaa ctcaagcgtt
     3121 tgcgaaagaa acgaaccaaa agccatataa ggaaacatac ggcatttccc atattacacg
     3181 ccatgatatg ctgcaaatcc ctgaacagca aaaaaatgaa aaatatcaag tttctgaatt
     3241 tgattcgtcc acaattaaaa atatctcttc tgcaaaaggc ctggacgttt gggacagctg
     3301 gccattacaa aacgctgacg gcactgtcgc aaactatcac ggctaccaca tcgtctttgc
     3361 attagccgga gatcctaaaa atgcggatga cacatcgatt tacatgttct atcaaaaagt
     3421 cggcgaaact tctattgaca gctggaaaaa cgctggccgc gtctttaaag acagcgacaa
     3481 attcgatgca aatgattcta tcctaaaaga ccaaacacaa gaatggtcag gttcagccac
     3541 atttacatct gacggaaaaa tccgtttatt ctacactgat ttctccggta aacattacgg
     3601 caaacaaaca ctgacaactg cacaagttaa cgtatcagca tcagacagct ctttgaacat
     3661 caacggtgta gaggattata aatcaatctt tgacggtgac ggaaaaacgt atcaaaatgt
     3721 acagcagttc atcgatgaag gcaactacag ctcaggcgac aaccatacgc tgagagatcc
     3781 tcactacgta gaagataaag gccacaaata cttagtattt gaagcaaaca ctggaactga
     3841 agatggctac caaggcgaag aatctttatt taacaaagca tactatggca aaagcacatc
     3901 attcttccgt caagaaagtc aaaaacttct gcaaagcgat aaaaaacgca cggctgagtt
     3961 agcaaacggc gctctcggta tgattgagct aaacgatgat tacacactga aaaaagtgat
     4021 gaaaccgctg attgcatcta acacagtaac agatgaaatt gaacgcgcga acgtctttaa
     4081 aatgaacggc aaatggtacc tgttcactga ctcccgcgga tcaaaaatga cgattgacgg
     4141 cattacgtct aacgatattt acatgcttgg ttatgtttct aattctttaa ctggcccata
     4201 caagccgctg aacaaaactg gccttgtgtt aaaaatggat cttgatccta acgatgtaac
     4261 ctttacttac tcacacttcg ctgtacctca agcgaaagga aacaatgtcg tgattacaag
     4321 ctatatgaca aacagaggat tctacgcaga caaacaatca acgtttgcgc cgagcttcct
     4381 gctgaacatc aaaggcaaga aaacatctgt tgtcaaagac agcatccttg aacaaggaca
     4441 attaacagtt aacaaataat cagaccccgt agaaacggcc agcctcgcag agcaggattc
     4501 ccgttgagca ccgccaggtg cgaataaggg acagtgaaga aggaacaccc gctcgcgggt
     4561 gggcctactt cacctatcct gcccggctga cgccgttgga tacaccaagg aaagtctaca
     4621 cgaacccttt ggcaaaatcc tgtatatcgt gcgaaaaagg atggatatac cgaaaaaatc
     4681 gctataatga ccccgaagca gggttatgca gcggaaaaga gatcaaagga tcttc
//
