LOCUS       Exported                4343 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Transposon donor vector pRZ101, complete sequence.
ACCESSION   KJ939358
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4343)
  AUTHORS   Zordan RE, Beliveau BJ, Trow JA, Craig NL, Cormack BP.
  TITLE     Transposon-based method for internal epitope tagging
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4343)
  AUTHORS   Zordan RE, Cormack BP.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-JUN-2014) Molecular Biology and Genetics, Johns 
            Hopkins School of Medicine, 725 N Wolfe St., Hunterian 609, 
            Baltimore, MD 21205, USA
REFERENCE   3  (bases 1 to 4343)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4343
                     /organism="Transposon donor vector pRZ101"
                     /mol_type="other DNA"
                     /db_xref="taxon:1552217"
     CDS             361..1155
                     /codon_start=1
                     /gene="kanR"
                     /product="aminoglycoside-3-O-phosphotransferase"
                     /label=kanR
                     /note="kanamycin resistance protein"
                     /protein_id="AIS76353.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            361..1155
                     /gene="kanR"
                     /label=kanR
     protein_bind    1430..1451
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1466..1496
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1504..1520
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     1528..1544
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1597..1795
                     /label=Tn7 left end
                     /note="Tn7 left end"
     misc_feature    1820..1849
                     /label=linker 1
                     /note="linker 1"
     misc_feature    1850..1921
                     /label=3x FLAG
                     /note="3x FLAG"
     CDS             1850..1921
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     misc_feature    1922..1951
                     /label=linker 2
                     /note="linker 2"
     misc_feature    1967..2747
                     /label=dhfR gene cassette
                     /note="dhfR gene cassette"
     CDS             2130..2603
                     /codon_start=1
                     /gene="dhfR"
                     /product="dihydrofolate reductase"
                     /label=dhfR
                     /protein_id="AIS76352.1"
                     /translation="MKLSLMVAISKNGVIGNGPDIPWSAKGEQLLFKAITYNQWLLVGR
                     KTFGSMGALPNRKYAVVTRSSFTSDNENVLIFPSIKDALTNLKKITDHVIVSGGGEIYK
                     SLIDQVDTLHISTIDIEPEGDVYFPEIPSNFRPVFTQDFASNINYSYQIWQKG"
     gene            2130..2603
                     /gene="dhfR"
                     /label=dhfR
     repeat_region   2754..2824
                     /label=Tn7 right end
                     /note="Tn7 right end"
     primer_bind     complement(2831..2847)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3153..3926
                     /label=mob RP4
                     /note="mob RP4"
     CDS             complement(3279..3650)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            3683..3792
                     /note="incP origin of transfer"
     rep_origin      3952..4343
                     /label=R6K gamma
                     /note="R6K gamma"
     rep_origin      3952..4340
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"