LOCUS       Exported                4504 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pKIKOrbsARKm, complete sequence.
ACCESSION   KC503970
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. 12 (1), 60 (2013)
  PUBMED    23799955
REFERENCE   2  (bases 1 to 4504)
  AUTHORS   Steen JA, Sabri S, Bongers M, Nielsen LK, Vickers CE.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-JAN-2013) Australian Institute for Bioengineering and 
            Nanotechnology, University of Queensland, Crn College and Cooper 
            Rds, St Lucia, QLD 4072, Australia
REFERENCE   3  (bases 1 to 4504)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4504
                     /organism="Cloning vector pKIKOrbsARKm"
                     /mol_type="other DNA"
                     /db_xref="taxon:1352340"
     rep_origin      32..418
                     /label=R6K origin
                     /note="R6K origin"
     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(488..1348)
                     /codon_start=1
                     /product="ampicillin resistance protein"
                     /label=ampicillin resistance protein
                     /protein_id="AGO96865.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1349..1453)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    complement(1491..2035)
                     /label=partial rbsR fragment
                     /note="partial rbsR fragment"
     regulatory      2235..2266
                     /regulatory_class="terminator"
                     /note="hairpin loop"
     misc_feature    2288..2336
                     /label=FRT site for Flp/Frt recombination
                     /note="FRT site for Flp/Frt recombination"
     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="aphA"
                     /product="Tn5 neomycin phosphotransferase"
                     /label=aphA
                     /note="kanamycin resistance protein"
                     /protein_id="AGO96864.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            2697..3491
                     /gene="aphA"
                     /label=aphA
     misc_feature    3681..3729
                     /label=FRT site for Flp/Frt recombination
                     /note="FRT site for Flp/Frt recombination"
     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)"
     regulatory      3828..3867
                     /regulatory_class="terminator"
                     /note="hairpin loop"
     misc_feature    complement(3968..4497)
                     /label=partial rbsA fragment
                     /note="partial rbsA fragment"