LOCUS       Exported                7715 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRMTn-Km DNA, complete sequence.
ACCESSION   AB777651
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7715)
  AUTHORS   Miyazaki R, van der Meer JR.
  TITLE     A New Large-DNA-Fragment Delivery System Based on Integrase Activity
            from an Integrative and Conjugative Element
  JOURNAL   Appl. Environ. Microbiol. 79 (14), 4440-4447 (2013)
  PUBMED    23686268
REFERENCE   2  (bases 1 to 7715)
  AUTHORS   Miyazaki R, van der Meer JR.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-JAN-2013) Contact:Ryo Miyazaki University of Lausanne,
            Department of Fundamental Microbiology; Batiment Biophore, Quartier 
            UNIL-Sorge, Lausanne 1015, Switzerland
REFERENCE   3  (bases 1 to 7715)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7715
                     /organism="Cloning vector pRMTn-Km"
                     /mol_type="other DNA"
                     /db_xref="taxon:1284605"
     misc_feature    1..19
                     /label=Tn5 inverted repeat
                     /note="Tn5 inverted repeat"
     misc_feature    38..85
                     /label=FRT
                     /note="FRT"
     protein_bind    complement(38..85)
                     /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)."
     misc_feature    90..343
                     /label=oriT
                     /note="oriT"
     oriT            225..333
                     /note="incP origin of transfer"
     CDS             612..1406
                     /codon_start=1
                     /gene="npt"
                     /product="kanamycin phosphotransferase"
                     /label=npt
                     /protein_id="BAN28923.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            612..1406
                     /gene="npt"
                     /label=npt
     misc_feature    1581..1628
                     /label=FRT
                     /note="FRT"
     protein_bind    complement(1581..1628)
                     /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(1986..3959)
                     /codon_start=1
                     /gene="intB13"
                     /product="P4-type integrase"
                     /label=intB13
                     /protein_id="BAN28924.1"
                     /translation="MTSIGPKSLLRSLEMALSDLIVRQAKTTGKRYTLYDNDCLSLMVS
                     AAGGKSWMFRYSWLGKQKRMALGGYPALSLREARAERDKAQALIARGIDPQIERDQRRH
                     AAKLAGEYTFKNVFDAWVEHRRKELKEGRQSTLSQILRIFNKDVLPTLGKMSIYDIRRP
                     QLLGVLAAIEKRKAFTTAEKVRTWFNQMFRYALVIAEGLEVNPAADLDVVAEPKPPVAH
                     NPYLHLPELPEFLQKLRRYNPRGWQTQLGVRLLFLTGVRTGELRLAEPEQFDLDRGFWI
                     IPPEVVKQLQDEMRKAGKRPQDVPPYIVPLSLQAIEIVRYLLGVMRPAQKYLLSHRSEL
                     KKRISENTLNKAVQLMGYEGRLTGHGIRGTISTALNEIGYPKIWVDAQLSHSDPNKVSS
                     AYNHAKYVEPRRRMMQDWADRLDLLEQGEVQAASAHLTIRIDGVPAMAEVEEAVDVAPT
                     VAEPGVSGSPPVAATPIVVTPNSGGITFQRLSQVPPPPAHAPESEVSAIQREREEMLAM
                     YESPNNLPVALFGKLAGKSKDQINRELKAGKLLSISLGNRGQRVPDWQLVPLKRRLAQA
                     LMNQCPHVDSWALYRLLTKPHSNLGNRAAIDVVTPTNVGKVLQAVTPYKEFERSSTDET
                     PQFSEFVRQLQRHVNALEEAPC"
     gene            complement(1986..3959)
                     /gene="intB13"
                     /label=intB13
     misc_feature    4053..4071
                     /label=lacO
                     /note="lacO"
     protein_bind    4055..4071
                     /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)."
     misc_feature    4163..4180
                     /label=attP
                     /note="attP"
     regulatory      4249..4277
                     /regulatory_class="promoter"
                     /note="Pcirc"
     regulatory      4334..4622
                     /regulatory_class="terminator"
                     /note="bacteriophage lambda T0, Escherichia coli rrnB T1"
     terminator      4339..4425
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4528..4622
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     misc_feature    4623..4667
                     /label=multiple cloning site
                     /note="multiple cloning site"
     misc_feature    4705..4723
                     /label=Tn5 inverted repeat
                     /note="Tn5 inverted repeat"
     rep_origin      4735..5126
                     /label=oriR6K
                     /note="oriR6K"
     rep_origin      4738..5126
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      5143..5182
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             complement(5186..6046)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /protein_id="BAN28925.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(5186..6046)
                     /gene="bla"
                     /label=bla
     promoter        complement(6047..6138)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(6192..7634)
                     /codon_start=1
                     /gene="tnpA"
                     /product="Tn5 hyperactive transposase"
                     /label=tnpA
                     /note="contains substitutions E54K and L372P"
                     /protein_id="BAN28926.1"
                     /translation="MVTFMITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKY
                     SGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTS
                     LSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADE
                     KESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDV
                     ESGLYLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNI
                     TLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGA
                     GAERQRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAET
                     VLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWE
                     ALQSKLDGFLAAKDLMAQGIKI"
     gene            complement(6192..7634)
                     /gene="tnpA"
                     /label=tnpA
     CDS             complement(6195..7622)
                     /codon_start=1
                     /product="transposase from the bacterial Tn5 transposon 
                     (Reznikoff, 1993)"
                     /label=Tn5 transposase
                     /translation="MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKS
                     ITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYR
                     HQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESG
                     KWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGL
                     YLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNA
                     VLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAER
                     QRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTP
                     DECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQS
                     KLDGFLAAKDLMAQGIKI"