LOCUS       sequence-66218-e        3771 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E.coli genomic integration at arsB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3771)
  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 3771)
  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: pKIKOarsBCm
FEATURES             Location/Qualifiers
     source          1..3771
                     /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(2117..2164)
                     /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(2181..2840)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2755..2774
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2841..2943)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(3049..3082)
                     /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 gtcccaaatc
     1501 gcagccaatc acattggcat aaaccatcgc ttctttgata acgccagatg ccgtgctgcc
     1561 atcaatggac aacgcgccaa ccagtaccgt cggcatattg ttcataatag aagagaggaa
     1621 ggcggtgagg aatccggtgc cgagcgtcgc ggcccacagg ccgttatccg ccagcacgtt
     1681 gagtacgcca gaaagatatt ccgttaatcc ggcattgcgc aggccataaa ccaccagata
     1741 catgccgagc gagaagatga caatctgcca gggggcaccg cgcaggactt tacccgtatt
     1801 aatcgcatga ccgcgtttag cgacgacaaa taatatcagc gcgcccacag ctgcaatggc
     1861 gctcaccgga ctgccgagcg gttccaggac gaaaaatccc accagcagaa gcagtaaaac
     1921 aacccagcca gttttgaacg tagcaggatc tttgatcgct tctgcgggag atttcagcag
     1981 cgccatatcg tagttctgcg gaatatcttt gcgaaaatag agatgtaaca tcaccagcgt
     2041 ggcaacaatc gcggcgataa acaaaaaaaa ccccgcttcg gcggggtttt ttttaatcgt
     2101 aggctggagc tgcttcgaag ttcctatact ttctagagaa taggaacttc ggaataggaa
     2161 cttcatttaa atggcgcgcc ttacgccccg ccctgccact catcgcagta ctgttgtaat
     2221 tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc
     2281 agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg
     2341 aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg
     2401 gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg
     2461 taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca
     2521 ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca
     2581 ctatcccata tcaccagctc accgtctttc attgccatac gtaattccgg atgagcattc
     2641 atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg
     2701 gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact
     2761 gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat
     2821 ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga caactcaaaa
     2881 aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga
     2941 tcaacgtctc attttcgcca aaagttggcc cagggcttcc cggtatcaac agggacacca
     3001 ggatttattt attctgcgaa gtgatcttcc gtcacaggta ggcgcgccga agttcctata
     3061 ctttctagag aataggaact tcggaatagg aactaaggag gatattcata tagaccatga
     3121 ttgcatgcgg taccgagctc ggatccacta gtagaggata aaacatatgg aattcctgca
     3181 gtgcgcagtc gacgcgcaaa aaaccccgcc cctgacaggg cggggttttt tcgctgttta
     3241 tccaccggca ccatcaccga ggcgtattcg cgaaagccga ggccaaagaa atcagcggaa
     3301 acgatattca ccaggttgga gacaataagc ggcaggctgg cggtatcggc aatgaatccg
     3361 gccgccatca cgaacgccag cgtagtgcct ttactgaacc ctaaagccag cagcatggcg
     3421 atgacaatcg gtgtcaaaat aagcgccgcg ccatcattgg caaacagggc ggcaacggca
     3481 gcaccgagca ggacaatcca ggtaaacagc aagcgaccac gaccattacc ccagcgtgag
     3541 acgtgcagcg ccgcccattc aaaaaagccg gactcatcca gcagcaggct gatgataatg
     3601 acggcgataa acgcagccgt cgcgttccag acgatattcc acaccaccgg aatatcaccc
     3661 ggatggacca cgcccgtaac taacgccagt actgcgccga gcgttgcact ccagccgatg
     3721 cctaaacctt tcggctgcca gataaccaat acgatggtca ggacgtttct g
//
