LOCUS       sequence-66224-e        4504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E.coli genomic integration rbsAR locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4504)
  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 4504)
  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: pKIKOrbsARKm
FEATURES             Location/Qualifiers
     source          1..4504
                     /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(2289..2336)
                     /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             2697..3491
                     /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(2751..2770)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3361..3380
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(3682..3715)
                     /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 cccagttcat
     1501 ctttcggttg gtggatagtg gttaatggtg gcgtcataaa gcttgccagt tcgatatcgt
     1561 catagccaat caccgcgata tcctgcggaa cctgtaactc tgcctgatat aacgcctggt
     1621 aaacgccaac agccatagcg tcatttccgg taaagacggc ctgaggacgc agcggatgtg
     1681 ataacagttg gcgcatggcg tcaaacccgc cgttaaattc aaaatcacca gtgacttcat
     1741 agccatcagg aatgttgaga cccgcacgtt tcatcgccgc ccgataacct tccaaccgca
     1801 ggcgcgccgg agttttatcc agcggaccgg taatacaggc gatacgggta tgacctttat
     1861 cgatcagata ttgcgttgct aagtctccgc ccagcaacga gttatcctga ataagatcgc
     1921 tgtcgccatc gaacggagcc cagtccatca tcacagtagg cactgtcgga taacgttgca
     1981 tgatttcacg cgaaggttga tgcgtttcgg tgcacagtaa cagccaccag cgcattgact
     2041 tcaatgccga cgatagatgc agcaactgcg ccggttagcg ccaacagaga acctacagac
     2101 aagtcgatgc ccgacgtcag gatcaccagc gtcatcccga ccgccataat ggcgttcacc
     2161 gaggtttgct ggagaatatt gaataagtta ttgatggtga aaaagttcgg gcttaacgtc
     2221 gagacaatcg aaacaaaaaa aaccccgctt cggcggggtt ttttttaatc gtaggctgga
     2281 gctgcttcga agttcctata ctttctagag aataggaact tcggaatagg aacttcaaga
     2341 tcccctcacg ctgccgcaag cactcagggc gcaagggctg ctaaaggaag cggaacacgt
     2401 agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct
     2461 ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc
     2521 gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc
     2581 cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga
     2641 tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga
     2701 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     2761 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     2821 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     2881 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     2941 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3001 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3061 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3121 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3181 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     3241 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3301 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3361 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3421 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3481 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     3541 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     3601 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     3661 ccccagcttc aaaagcgctc tgaagttcct atactttcta gagaatagga acttcggaat
     3721 aggaactaag gaggatattc atatagacca tgattgcatg cggtaccgag ctcggatcca
     3781 ctagtagagg ataaaacata tggaattcct gcagtgcgca gtcgacgcgc aaaaaacccc
     3841 gcccctgaca gggcggggtt ttttcgctgt ttcgatcagc accagcagag cgataagcga
     3901 tttctgctcc atcagccacg ctttcgtgaa ataacggcga ccagagacag tctgggttgt
     3961 cattttttta ctcctgattc acgcgattaa gcttgcccac agcggcagcc attaacactt
     4021 cctgggtggc ctgctcacga gtaaattccc cgctgagatg cccttcatgc atgacgatga
     4081 tgcgatcgct catgcctaat acttctggca tctccgatga caccagaatg atgctcaagc
     4141 catcggcttt gaactggtta atcagttgat agatctcttt tttcgcgccg acatctacgc
     4201 cacgggtagg ttcatcaagg atcaacactt tggggcgtgt catcagaccg cgggcaatcg
     4261 ccactttttg ctgattgcca ccggaaagca gaccaattgc ctgttccatc gacggtgttt
     4321 tcacattaaa cagacgaatg aaatcactca cagcctgctg ttcatcggca tgcttcaaac
     4381 tgccgccagc gcggctgaag tagcgcagcg ctgtcagcga catgttctct tttactgaca
     4441 tgcccaacac taaaccgtca cgtttacggt cttcggagat atacacaatg ccgcttggtt
     4501 tctg
//
