LOCUS       sequence-453519-        7671 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Conjugation knockout plasmid (pK18mobsacB) to delete H16_0006 (Type 
            I restriction endonuclease subunit)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7671)
  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 7671)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pCHC005 (∆RE)
FEATURES             Location/Qualifiers
     source          1..7671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             266..1060
                     /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(320..339)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     930..949
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        1106..1551
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1552..1638,1639..2973)
                     /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            3327..3436
                     /note="incP origin of transfer"
     promoter        complement(3671..3761)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        complement(3955..4045)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      4405..4993
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4894..4913
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5147..5164
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5281..5301
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     5300..5316
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5300..5316
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7416..7433)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7416..7432)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7425..7447)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa
       61 gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc
      121 aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt
      181 aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gatctgatca
      241 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
      301 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
      361 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
      421 cctgtccggt gccctgaatg aactccaaga cgaggcagcg cggctatcgt ggctggccac
      481 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
      541 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
      601 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
      661 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
      721 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
      781 caggctcaag gcgcggatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
      841 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
      901 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
      961 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     1021 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgct
     1081 agaggatcga tcctttttaa cccatcacat atacctgccg ttcactatta tttagtgaaa
     1141 tgagatatta tgatattttc tgaattgtga ttaaaaaggc aactttatgc ccatgcaaca
     1201 gaaactataa aaaatacaga gaatgaaaag aaacagatag attttttagt tctttaggcc
     1261 cgtagtctgc aaatcctttt atgattttct atcaaacaaa agaggaaaat agaccagttg
     1321 caatccaaac gagagtctaa tagaatgagg tcgaaaagta aatcgcgcgg gtttgttact
     1381 gataaagcag gcaagaccta aaatgtgtaa agggcaaagt gtatactttg gcgtcacccc
     1441 ttacatattt taggtctttt tttattgtgc gtaactaact tgccatcttc aaacaggagg
     1501 gctggaagaa gcagaccgct aacacagtac ataaaaaagg agacatgaac gatgaacatc
     1561 aaaaagtttg caaaacaagc aacagtatta acctttacta ccgcactgct ggcaggaggc
     1621 gcaactcaag cgtttgcgaa agaaacgaac caaaagccat ataaggaaac atacggcatt
     1681 tcccatatta cacgccatga tatgctgcaa atccctgaac agcaaaaaaa tgaaaaatat
     1741 caagtttctg aatttgattc gtccacaatt aaaaatatct cttctgcaaa aggcctggac
     1801 gtttgggaca gctggccatt acaaaacgct gacggcactg tcgcaaacta tcacggctac
     1861 cacatcgtct ttgcattagc cggagatcct aaaaatgcgg atgacacatc gatttacatg
     1921 ttctatcaaa aagtcggcga aacttctatt gacagctgga aaaacgctgg ccgcgtcttt
     1981 aaagacagcg acaaattcga tgcaaatgat tctatcctaa aagaccaaac acaagaatgg
     2041 tcaggttcag ccacatttac atctgacgga aaaatccgtt tattctacac tgatttctcc
     2101 ggtaaacatt acggcaaaca aacactgaca actgcacaag ttaacgtatc agcatcagac
     2161 agctctttga acatcaacgg tgtagaggat tataaatcaa tctttgacgg tgacggaaaa
     2221 acgtatcaaa atgtacagca gttcatcgat gaaggcaact acagctcagg cgacaaccat
     2281 acgctgagag atcctcacta cgtagaagat aaaggccaca aatacttagt atttgaagca
     2341 aacactggaa ctgaagatgg ctaccaaggc gaagaatctt tatttaacaa agcatactat
     2401 ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac ttctgcaaag cgataaaaaa
     2461 cgcacggctg agttagcaaa cggcgctctc ggtatgattg agctaaacga tgattacaca
     2521 ctgaaaaaag tgatgaaacc gctgattgca tctaacacag taacagatga aattgaacgc
     2581 gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca ctgactcccg cggatcaaaa
     2641 atgacgattg acggcattac gtctaacgat atttacatgc ttggttatgt ttctaattct
     2701 ttaactggcc catacaagcc gctgaacaaa actggccttg tgttaaaaat ggatcttgat
     2761 cctaacgatg taacctttac ttactcacac ttcgctgtac ctcaagcgaa aggaaacaat
     2821 gtcgtgatta caagctatat gacaaacaga ggattctacg cagacaaaca atcaacgttt
     2881 gcgccgagct tcctgctgaa catcaaaggc aagaaaacat ctgttgtcaa agacagcatc
     2941 cttgaacaag gacaattaac agttaacaaa taaaaacgca aaagaaaatg ccgatgggta
     3001 ccgagcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     3061 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccct cgcggacgtg
     3121 ctcatagtcc acgacgcccg tgattttgta gccctggccg acggccagca ggtaggccga
     3181 caggctcatg ccggccgccg ccgccttttc ctcaatcgct cttcgttcgt ctggaaggca
     3241 gtacaccttg ataggtgggc tgcccttcct ggttggcttg gtttcatcag ccatccgctt
     3301 gccctcatct gttacgccgg cggtagccgg ccagcctcgc agagcaggat tcccgttgag
     3361 caccgccagg tgcgaataag ggacagtgaa gaaggaacac ccgctcgcgg gtgggcctac
     3421 ttcacctatc ctgcccggct gacgccgttg gatacaccaa ggaaagtcta cacgaaccct
     3481 ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat accgaaaaaa tcgctataat
     3541 gaccccgaag cagggttatg cagcggaaaa gcgctgcttc cctgctgttt tgtggaatat
     3601 ctaccgactg gaaacaggca aatgcaggaa attactgaac tgaggggaca ggcgagagac
     3661 gatgccaaag agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta
     3721 tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaaa
     3781 agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat tctgcgaagt
     3841 gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct gccaacttac
     3901 tgatttagtg tatgatggtg tttttgaggt gctccagtgg cttctgtttc tatcagctcc
     3961 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa
     4021 gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt
     4081 cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg
     4141 tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac ttactgattt agtgtatgat
     4201 ggtgtttttg aggtgctcca gtggcttctg tttctatcag ggctggatga tcctccagcg
     4261 cggggatctc atgctggagt tcttcgccca ccccaaaagg atctaggtga agatcctttt
     4321 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4381 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4441 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4501 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4561 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4621 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4681 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4741 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4801 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4861 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4921 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4981 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5041 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5101 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5161 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     5221 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     5281 taatgtgagt tagctcactc aggaaacagc tatgacatga ttacgaattc gacgatgacg
     5341 aagatttctc cgagcagtgt ttttcgtccg gcatactttt ttcatgtcgc caagcaagct
     5401 gagcgattcc tgaaggctca aaccaatggc actggcattc cacacgttga ccgagagctt
     5461 ctcgagggga taaaggtctt ttgtcctggc tctacggagc agcaattact tgcggaaatc
     5521 ctcgacactc tcgacaccgc catctacgaa actgaagcga tcatcgccaa gctcaaggcg
     5581 gtcaagcaag gcctgctgca tgacctcttg acgcgcggca tcgacgccaa cggcgaattg
     5641 cgcccacctc aggccgaggc accgcatctc tacgagtcgt caccgttggg ttggattccg
     5701 aatgagtggg gtcttgctcc tacagcaact cgctgccatc tgataaccaa aggcactacc
     5761 cctgcggcta atgagatgtg gcagggtggc gcgggaatta ggtttctgcg agtcgataat
     5821 ctttctttcg atggacaact ggatctagat gcaagcacgt ttcgagttag ccttgccacg
     5881 cacaaaggtt ttctggctcg ttcaagatgc cttgaaggtg atgtgctgac gaacatcgtt
     5941 ggcccacctc tagggaaact ggggcttgtt accaaagaaa ttggtgaggt caatattaat
     6001 caagcaattg cgttatttcg accaaccgaa caactactgc caaagttcct attaatctgg
     6061 cttagtagct caatctcgca gtcttggctg aggaaccgag ccaagcagac gtcgggacaa
     6121 gtgaatctga ccctcgctct atgccaggag cttcctctac ctcggatgac gatcaatgag
     6181 caacaggcaa tcgttgaccg agttgatgcc gcgcaggaac aaatctggtg tgaggaggaa
     6241 ctgatccgaa agatgcgact tgagaaatct ggccttatgg atgacctcct caccggccgc
     6301 gtccgcgtca agccgcagct ggcggaaacc aaacaagcag ggagcgcctg agtttaaact
     6361 gccttcgccg gtgaaattgc caagccttca gatcggtgac ctccggttca cgctccagcg
     6421 gagcgcgcgc cgcagaacta tgcagatcac cgtggagcgc agtggcgact tgatgctctg
     6481 cgcaccgccg gaggtggacg aggccgcgct gcgagcattc gtgctggaga agcgcttctg
     6541 gatctacacc aagctggccg agaaggaccg cttgcagcgc caggttccgc gcaaggaatt
     6601 cgtcggaggc gagggattct tgtatctcgg ccgcagccat cggctgaagg tggtcgatga
     6661 acagaatgtg ccactgaagt tgaatggagg ccgcttttgt ctgcgccgtg acgccctacc
     6721 cgccgcgcgc gagcatttca tccgctggta cggcgagcgt gccaaggcct ggctttcggg
     6781 gcgtgtagct gactaccagt cgcgaatgga ggtgacgcct gccggcgtca aggtgcagga
     6841 ccttggatat cgctggggtt cgtgtggcaa gggcgactgg ctgtacttcc actggaaggc
     6901 aatcctgctg ccggcgcgca tcgctgagta tgtcgtggtg catgagattg cccatctgca
     6961 tgagccgcac cacacgcctg cgttctggct tcgagtggag cgtgccatgc cggactatgc
     7021 gcaacgcaag gcctggctgg ccgagcatgg aatcgatgtt gaaggaatct aaagaacgat
     7081 ggctgactat ttcaccagtg actacttcaa gctgctgaac aagtggaagg ggcagaagcg
     7141 tgacgagtcc aaccccgagc agaaccgcgc ttatgaagat ctgaagaagg cctacgaggt
     7201 gacggaggcg tgggcggaca aggttaaggc cgagttgttc cctgtcgggc gcgtcgagat
     7261 tcgtaagcgc ccgaccaacc agggcaacaa ctttgccagc tacaactggg ccaaaatcta
     7321 cccttcatct gaggcgccga aagagttggc ttacacagtt ggcatcggcg ccgatgacgg
     7381 cttcgtagtc aagatcgata ccgttgaagc ttggcactgg ccgtcgtttt acaacgtcgt
     7441 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     7501 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     7561 aatggcgaat ggcgataagc tagcttcacg ctgccgcaag cactcagggc gcaagggctg
     7621 ctaaaggaag cggaacacgt agaaagccag tccgcagaaa cggtgctgac c
//
