LOCUS       sequence-66223-e        4157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E.coli genomic integration at lacZ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4157)
  AUTHORS   Sabri S, Steen JA, Bongers M, Nielsen LK, Vickers CE
  TITLE     Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA
            sequences, including whole metabolic pathways, onto the Escherichia 
            coli chromosome at well-characterised loci.
  JOURNAL   Microb Cell Fact. 2013 Jun 24;12(1):60.
  PUBMED    23799955
REFERENCE   2  (bases 1 to 4157)
  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: "Microb Cell
            Fact."; date: "2013-06-24"; volume: "12(1)"; pages: "60"
COMMENT     Sequence Label: pKIKOlacZKm
FEATURES             Location/Qualifiers
     source          1..4157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      35..391
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(488..1279,1280..1348))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1111..1130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1349..1453)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    complement(2062..2109)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2470..3264
                     /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(2524..2543)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3134..3153
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(3455..3488)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
ORIGIN
        1 ggaggatatt catatggacc atggctaatt cccatgtcag ccgttaagtg ttcctgtgtc
       61 actgaaaatt gctttgagag gctctaaggg cttctcagtg cgttacatcc ctggcttgtt
      121 gtccacaacc gttaaacctt aaaagcttta aaagccttat atattctttt ttttcttata
      181 aaacttaaaa ccttagaggc tatttaagtt gctgatttat attaatttta ttgttcaaac
      241 atgagagctt agtacgtgaa acatgagagc ttagtacgtt agccatgaga gcttagtacg
      301 ttagccatga gggtttagtt cgttaaacat gagagcttag tacgttaaac atgagagctt
      361 agtacgtgaa acatgagagc ttagtacgta ctatcaacag gttgaactgc ggatcttgcg
      421 gccgcaaaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
      481 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
      541 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
      601 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
      661 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
      721 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
      781 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
      841 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
      901 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
      961 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     1021 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     1081 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     1141 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     1201 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     1261 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     1321 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     1381 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     1441 taggggttcc gcgcacattt ccccgaaaag tgccacctgc atccagaaac aagactgtta
     1501 cccatcgcgt gggcgtattc gcaaaggatc agcgggcgca tctctccagg tagcgaaagc
     1561 cattttttga tggaccattt cggcacagcc gggaagggct ggtcttcatc cacgcgcgcg
     1621 tacatcgggc aaataatatc ggtggccgtg gtgtcggctc cgccgccttc atactgcacc
     1681 gggcgggaag gatcgacaga tttgatccag cgatacagcg cgtcgtgatt agcgccgtgg
     1741 cctgattcat tccccagcga ccagatgatc acactcgggt gattacgatc gcgctgcacc
     1801 attcgcgtta cgcgttcgct catcgccggt agccagcgcg gatcatcggt cagacgattc
     1861 attggcacca tgccgtgggt ttcaatattg gcttcatcca ccacatacag gccgtagcgg
     1921 tcgcacagcg tgtaccacag cggatggttc ggataatgcg aacagcgcac ggcgttaaag
     1981 ttgttctgct tcatcagcag gataaacaaa aaaaaccccg cttcggcggg gtttttttta
     2041 atcgtaggct ggagctgctt cgaagttcct atactttcta gagaatagga acttcggaat
     2101 aggaacttca agatcccctc acgctgccgc aagcactcag ggcgcaaggg ctgctaaagg
     2161 aagcggaaca cgtagaaagc cagtccgcag aaacggtgct gaccccggat gaatgtcagc
     2221 tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt
     2281 gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga accggaattg
     2341 ccagctgggg cgccctctgg taaggttggg aagccctgca aagtaaactg gatggctttc
     2401 ttgccgccaa ggatctgatg gcgcagggga tcaagatctg atcaagagac aggatgagga
     2461 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     2521 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     2581 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     2641 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     2701 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     2761 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     2821 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     2881 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     2941 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     3001 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     3061 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     3121 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     3181 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     3241 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     3301 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     3361 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     3421 agttcttcgc ccaccccagc ttcaaaagcg ctctgaagtt cctatacttt ctagagaata
     3481 ggaacttcgg aataggaact aaggaggata ttcatataga ccatgattgc atgcggtacc
     3541 gagctcggat ccactagtag aggataaaac atatggaatt cctgcagtgc gcagtcgacg
     3601 cgcaaaaaac cccgcccctg acagggcggg gttttttcgc tgtttatcct gcaccatcgt
     3661 ctgctcatcc atgacctgac catgcagagg atgatgctcg tgacggttaa cgcctcgaat
     3721 cagcaacggc ttgccgttca gcagcagcag accattttca atccgcacct cgcggaaacc
     3781 gacatcgcag gcttctgctt caatcagcgt gccgtcggcg gtgtgcagtt caaccaccgc
     3841 acgatagaga ttcgggattt cggcgctcca cagcgccggg ttttcgacgt tcagacgtag
     3901 cgtgacgcga tcggcataac caccacgctc atcgataatt tcaccgccga aaggcgcggt
     3961 gccgctggcg acctgcgttt caccctgcca caaagaaacc gtcacccgca gctcatcgcg
     4021 cagctcgccg tacatctgaa cttctgcctc cagtaccgcg cggctgaaat catcattaaa
     4081 gtgagtggca acatggaaat cgctgatttg tgtgctcggt ttgtgcagca acgagacgtc
     4141 acggaaaatg gtttctg
//
