LOCUS       sequence-472828-        5366 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for marker free integration of E. coli appA at the phac1 
            locus of Cupriavidus necator..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5366)
  AUTHORS   Arhar S, Pirchner J, Stolterfoht-Stock H, Reicher K, Kourist R, 
            Emmerstorfer-Augustin A
  TITLE     CnRed: Efficient, Marker-free Genome Engineering of Cupriavidus 
            necator H16 by Adapted Lambda Red Recombineering.
  JOURNAL   ACS Synth Biol. 2025 Feb 24. doi: 10.1021/acssynbio.4c00757.
  PUBMED    39989320
REFERENCE   2  (bases 1 to 5366)
  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: "ACS Synth 
            Biol. 2025 Feb 24. doi: 10.1021/acssynbio.4c00757."
COMMENT     Sequence Label: pHInt-AppA-KanR-loxP
FEATURES             Location/Qualifiers
     source          1..5366
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             829..851
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2158..2175
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2217..2234)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2387..2473
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2565..2592
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    2599..2632
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2737..2756)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2748..3563
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2748..3563
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2821..2840)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     protein_bind    3652..3685
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(4647..5235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4727..4746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggtcccgcc agtggcaggg cactgaaggc aacggccacg cggccgcgtc cggcattccg
       61 ggcctggatg cgctggcagg cgtcaagatc gcgccggcgc agctgggtga tatccagcag
      121 cgctacatga aggacttctc agcgctgtgg caggccatgg ccgagggcaa ggccgaggcc
      181 accggtccgc tgcacgaccg gcgcttcgcc ggcgacgcat ggcgcaccaa cctcccatat
      241 cgcttcgctg ccgcgttcta cctgctcaat gcgcgcgcct tgaccgagct ggccgatgcc
      301 gtcgaggccg atgccaagac ccgccagcgc atccgcttcg cgatctcgca atgggtcgat
      361 gcgatgtcgc ccgccaactt ccttgccacc aatcccgagg cgcagcgcct gctgatcgag
      421 tcgggcggcg aatcgctgcg tgccggcgtg cgcaacatga tggaagacct gacacgcggc
      481 aagatctcgc agaccgacga gagcgcgttt gaggtcggcc gcaatgtcgc ggtgaccgaa
      541 ggcgccgtgg tcttcgagaa cgagtacttc cagctgttgc agtacaagcc gctgaccgac
      601 aaggtgcacg cgcgcccgct gctgatggtg ccgccgtgca tcaacaagta ctacatcctg
      661 gacctgcagc cggagagctc gctggtgcgc catgtggtgg agcagggaca tacggtgttt
      721 ctggtgtcgt ggcgcaatcc ggacgccagc atggccggca gcacctggga cgactgcatg
      781 caaaaaccgt tattgacaca ggtggaaatt tagaatatac tgaagctttt tgtttaactt
      841 taagaaggag atataggatc catgaaagcg atcttaatcc catttttatc tcttctgatt
      901 ccgttaaccc cgcaatctgc attcgctcag agtgagccgg agctgaagct ggaaagtgtg
      961 gtgattgtca gtcgtcatgg tgtgcgtgct ccaaccaagg ccacgcaact gatgcaggat
     1021 gtcaccccag acgcatggcc aacctggccg gtaaaactgg gttggctgac accgcgcggt
     1081 ggtgagctaa tcgcctatct cggacattac caacgccagc gtctggtagc cgacggattg
     1141 ctggcgaaaa agggctgccc gcagtctggt caggtcgcga ttattgctga tgtcgacgag
     1201 cgtacccgta aaacaggcga agccttcgcc gccgggctgg cacctgactg tgcaataacc
     1261 gtacataccc aggcagatac gtccagtccc gatccgttat ttaatcctct aaaaactggc
     1321 gtttgccaac tggataacgc gaacgtgact gacgcgatcc tcagcagggc aggagggtca
     1381 attgctgact ttaccgggca tcggcaaacg gcgtttcgcg aactggaacg ggtgcttaat
     1441 tttccgcaat caaacttgtg ccttaaacgt gagaaacagg acgaaagctg ttcattaacg
     1501 caggcattac catcggaact caaggtgagc gccgacaatg tctcattaac cggtgcggta
     1561 agcctcgcat caatgctgac ggagatattt ctcctgcaac aagcacaggg aatgccggag
     1621 ccggggtggg gaaggatcac cgattcacac cagtggaaca ccttgctaag tttgcataac
     1681 gcgcaatttt atttgctaca acgcacgcca gaggttgccc gcagccgcgc caccccgtta
     1741 ttagatttga tcaagacagc gttgacgccc catccaccgc aaaaacaggc gtatggtgtg
     1801 acattaccca cttcagtgct gtttatcgcc ggacacgata ctaatctggc aaatctcggc
     1861 ggcgcactgg agctcaactg gacgcttccc ggtcagccgg ataacacgcc gccaggtggt
     1921 gaactggtgt ttgaacgctg gcgtcggcta agcgataaca gccagtggat tcaggtttcg
     1981 ctggtcttcc agactttaca gcagatgcgt gataaaacgc cgctgtcatt aaatacgccg
     2041 cccggagagg tgaaactgac cctggcagga tgtgaagagc gaaatgcgca gggcatgtgt
     2101 tcgttggcag gttttacgca aatcgtgaat gaagcacgca taccggcgtg cagtttgcat
     2161 caccatcacc atcactaaga attcggctgt tttggcggat gagagaagat tttcagcctg
     2221 atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt
     2281 agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat
     2341 ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa
     2401 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     2461 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     2521 gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac
     2581 ggatggcctt ttaattaaat aacttcgtat aatgtatgct atacgaagtt atcatatgtt
     2641 tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaatatgt atccgctcat
     2701 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     2761 aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg
     2821 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
     2881 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     2941 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     3001 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa
     3061 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     3121 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     3181 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     3241 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     3301 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     3361 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     3421 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     3481 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     3541 tgatgctcga tgagtttttc taagaattaa ttcatgacca aaatccctta acgtgagttt
     3601 tcgttccact gagcgtcaga caggcctcct aggacggatg gccttactag tataacttcg
     3661 tataatgtat gctatacgaa gttatacatc gagcacgcgg ccatccgcgc catcgaagtc
     3721 gcgcgcgaca tcagcggcca ggacaagatc aacgtgctcg gcttctgcgt gggcggcacc
     3781 attgtctcga ccgcgctggc ggtgctggcc gcgcgcggcg agcacccggc cgccagcgtc
     3841 acgctgctga ccacgctgct ggactttgcc gacacgggca tcctcgacgt ctttgtcgac
     3901 gagggccatg tgcagttgcg cgaggccacg ctgggcggcg gcgccggcgc gccgtgcgcg
     3961 ctgctgcgcg gccttgagct ggccaatacc ttctcgttct tgcgcccgaa cgacctggtg
     4021 tggaactacg tggtcgacaa ctacctgaag ggcaacacgc cggtgccgtt cgacctgctg
     4081 ttctggaacg gcgacgccac caacctgccg gggccgtggt actgctggta cctgcgccac
     4141 acctacctgc agaacgagct caaggtaccg ggcaagctga ccgtgtgcgg cgtgccggtg
     4201 gacctggcca gcatcgacgt gccgacctat atctacggct cgcgcgaaga ccatatcgtg
     4261 ccgtggaccg cggcctatgc ctcgaccgcg ctgctggcga acaagctgcg cttcgtgctg
     4321 ggtgcgtcgg gccatatcgc cggtgtgatc aacccgccgg ccaagaacaa gcgcagccac
     4381 tggactaacg atgcgctgcc ggagtcgccg cagcaatggc tggccggcgc catcgagcat
     4441 cacggcagct ggtggccgga ctggaccgca tggctggccg ggcaggccgg cgcgaaacgc
     4501 gccgcgcccg ccaactatgg caatgcgcgc tatcgcgcaa tcgaacccgc gcctgggcga
     4561 tacgtcaaag ccaaggcatg actcgagatg tgagcaaaag gccagcaaaa ggccaggaac
     4621 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4681 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4741 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4801 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4861 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4921 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4981 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5041 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     5101 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5161 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5221 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg gtctagaatg gcgaccggca
     5281 aaggcgcggc agcttccacg caggaaggca agtcccaacc attcaaggtc acgccggggc
     5341 cattcgatcc agccacatgg ctggaa
//
