LOCUS       sequence-66222-e        4232 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 4232)
  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 4232)
  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: pKIKOarsBKm
FEATURES             Location/Qualifiers
     source          1..4232
                     /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             2525..3319
                     /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(2579..2598)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3189..3208
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(3510..3543)
                     /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 cttcaagatc ccctcacgct gccgcaagca ctcagggcgc aagggctgct aaaggaagcg
     2221 gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg
     2281 ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct
     2341 tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc
     2401 tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc
     2461 gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt
     2521 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     2581 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     2641 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     2701 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     2761 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     2821 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     2881 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     2941 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3001 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     3061 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     3121 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3181 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     3241 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     3301 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     3361 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     3421 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     3481 ttcgcccacc ccagcttcaa aagcgctctg aagttcctat actttctaga gaataggaac
     3541 ttcggaatag gaactaagga ggatattcat atagaccatg attgcatgcg gtaccgagct
     3601 cggatccact agtagaggat aaaacatatg gaattcctgc agtgcgcagt cgacgcgcaa
     3661 aaaaccccgc ccctgacagg gcggggtttt ttcgctgttt atccaccggc accatcaccg
     3721 aggcgtattc gcgaaagccg aggccaaaga aatcagcgga aacgatattc accaggttgg
     3781 agacaataag cggcaggctg gcggtatcgg caatgaatcc ggccgccatc acgaacgcca
     3841 gcgtagtgcc tttactgaac cctaaagcca gcagcatggc gatgacaatc ggtgtcaaaa
     3901 taagcgccgc gccatcattg gcaaacaggg cggcaacggc agcaccgagc aggacaatcc
     3961 aggtaaacag caagcgacca cgaccattac cccagcgtga gacgtgcagc gccgcccatt
     4021 caaaaaagcc ggactcatcc agcagcaggc tgatgataat gacggcgata aacgcagccg
     4081 tcgcgttcca gacgatattc cacaccaccg gaatatcacc cggatggacc acgcccgtaa
     4141 ctaacgccag tactgcgccg agcgttgcac tccagccgat gcctaaacct ttcggctgcc
     4201 agataaccaa tacgatggtc aggacgtttc tg
//
