LOCUS       sequence-66221-e        4043 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 4043)
  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 4043)
  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: pKIKOrbsARCm
FEATURES             Location/Qualifiers
     source          1..4043
                     /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             complement(2353..3012)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2927..2946
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3013..3115)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(3221..3254)
                     /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 aacttcattt
     2341 aaatggcgcg ccttacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag
     2401 cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat
     2461 cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt
     2521 gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac
     2581 gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc
     2641 cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag
     2701 cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca
     2761 tatcaccagc tcaccgtctt tcattgccat acgtaattcc ggatgagcat tcatcaggcg
     2821 ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa
     2881 aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa
     2941 tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat
     3001 ttttttctcc attttagctt ccttagctcc tgaaaatctc gacaactcaa aaaatacgcc
     3061 cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc
     3121 tcattttcgc caaaagttgg cccagggctt cccggtatca acagggacac caggatttat
     3181 ttattctgcg aagtgatctt ccgtcacagg taggcgcgcc gaagttccta tactttctag
     3241 agaataggaa cttcggaata ggaactaagg aggatattca tatagaccat gattgcatgc
     3301 ggtaccgagc tcggatccac tagtagagga taaaacatat ggaattcctg cagtgcgcag
     3361 tcgacgcgca aaaaaccccg cccctgacag ggcggggttt tttcgctgtt tcgatcagca
     3421 ccagcagagc gataagcgat ttctgctcca tcagccacgc tttcgtgaaa taacggcgac
     3481 cagagacagt ctgggttgtc atttttttac tcctgattca cgcgattaag cttgcccaca
     3541 gcggcagcca ttaacacttc ctgggtggcc tgctcacgag taaattcccc gctgagatgc
     3601 ccttcatgca tgacgatgat gcgatcgctc atgcctaata cttctggcat ctccgatgac
     3661 accagaatga tgctcaagcc atcggctttg aactggttaa tcagttgata gatctctttt
     3721 ttcgcgccga catctacgcc acgggtaggt tcatcaagga tcaacacttt ggggcgtgtc
     3781 atcagaccgc gggcaatcgc cactttttgc tgattgccac cggaaagcag accaattgcc
     3841 tgttccatcg acggtgtttt cacattaaac agacgaatga aatcactcac agcctgctgt
     3901 tcatcggcat gcttcaaact gccgccagcg cggctgaagt agcgcagcgc tgtcagcgac
     3961 atgttctctt ttactgacat gcccaacact aaaccgtcac gtttacggtc ttcggagata
     4021 tacacaatgc cgcttggttt ctg
//
