LOCUS       sequence-476941-        3733 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 3733)
  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 3733)
  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: pKIKOlacZCm
FEATURES             Location/Qualifiers
     source          1..3733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      35..423
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(525..1316,1317..1385))
                     /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     1148..1167
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1386..1490)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    complement(2099..2146)
                     /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             complement(2163..2822)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2737..2756
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2823..2925)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(3031..3064)
                     /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 caaatggacc atggctaatt cccatgtcag ccgttaagtg ttcctgtgtc
       61 actcaaaatt 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 tgatcttcag
      421 atcctctacg ccggacgcat cgtggccgga tcttgcggcc gcaaaaatta aaaatgaagt
      481 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
      541 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
      601 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
      661 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
      721 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
      781 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
      841 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
      901 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
      961 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1021 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     1081 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     1141 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     1201 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     1261 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     1321 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     1381 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     1441 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     1501 cgaaaagtgc cacctgcatc cagaaacaag actgttaccc atcgcgtggg cgtattcgca
     1561 aaggatcagc gggcgcatct ctccaggtag cgaaagccat tttttgatgg accatttcgg
     1621 cacagccggg aagggctggt cttcatccac gcgcgcgtac atcgggcaaa taatatcggt
     1681 ggccgtggtg tcggctccgc cgccttcata ctgcaccggg cgggaaggat cgacagattt
     1741 gatccagcga tacagcgcgt cgtgattagc gccgtggcct gattcattcc ccagcgacca
     1801 gatgatcaca ctcgggtgat tacgatcgcg ctgcaccatt cgcgttacgc gttcgctcat
     1861 cgccggtagc cagcgcggat catcggtcag acgattcatt ggcaccatgc cgtgggtttc
     1921 aatattggct tcatccacca catacaggcc gtagcggtcg cacagcgtgt accacagcgg
     1981 atggttcgga taatgcgaac agcgcacggc gttaaagttg ttctgcttca tcagcaggat
     2041 aaacaaaaaa aaccccgctt cggcggggtt ttttttaatc gtaggctgga gctgcttcga
     2101 agttcctata ctttctagag aataggaact tcggaatagg aacttcattt aaatggcgcg
     2161 ccttacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
     2221 acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg
     2281 tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg
     2341 gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata
     2401 ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc
     2461 gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac
     2521 gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc
     2581 tcaccgtctt tcattgccat acgtaattcc ggatgagcat tcatcaggcg ggcaagaatg
     2641 tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta
     2701 atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa
     2761 tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc
     2821 attttagctt ccttagctcc tgaaaatctc gacaactcaa aaaatacgcc cggtagtgat
     2881 cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc
     2941 caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg
     3001 aagtgatctt ccgtcacagg taggcgcgcc gaagttccta tactttctag agaataggaa
     3061 cttcggaata ggaactaagg aggatattca tatagaccat gattgcatgc ggtaccgagc
     3121 tcggatccac tagtagagga taaaacatat ggaattcctg cagtgcgcag tcgacgcgca
     3181 aaaaaccccg cccctgacag ggcggggttt tttcgctgtt tatcctgcac catcgtctgc
     3241 tcatccatga cctgaccatg cagaggatga tgctcgtgac ggttaacgcc tcgaatcagc
     3301 aacggcttgc cgttcagcag cagcagacca ttttcaatcc gcacctcgcg gaaaccgaca
     3361 tcgcaggctt ctgcttcaat cagcgtgccg tcggcggtgt gcagttcaac caccgcacga
     3421 tagagattcg ggatttcggc gctccacagc gccgggtttt cgacgttcag acgtagcgtg
     3481 acgcgatcgg cataaccacc acgctcatcg ataatttcac cgccgaaagg cgcggtgccg
     3541 ctggcgacct gcgtttcacc ctgccacaaa gaaaccgtca cccgcagctc atcgcgcagc
     3601 tcgccgtaca tctgaacttc tgcctccagt accgcgcggc tgaaatcatc attaaagtga
     3661 gtggcaacat ggaaatcgct gatttgtgtg ctcggtttgt gcagcaacga gacgtcacgg
     3721 aaaatggttt ctg
//
