LOCUS       Exported                7744 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pIJ12739, complete sequence.
ACCESSION   KU523902
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7744)
  AUTHORS   Fernandez-Martinez LT, Bibb MJ.
  TITLE     Use of the meganuclease I-SceI of Saccharomyces cerevisiae to select
            for gene deletions in actinomycetes
  JOURNAL   Sci Rep 4, 7100 (2014)
  PUBMED    25403842
REFERENCE   2  (bases 1 to 7744)
  AUTHORS   Fernandez-Martinez LT, Bibb MJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-JAN-2016) Biology, Edge Hill University, St Helens 
            Road, Ormskirk L39 4QP, UK
REFERENCE   3  (bases 1 to 7744)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7744
                     /organism="Cloning vector pIJ12739"
                     /mol_type="other DNA"
                     /db_xref="taxon:1825502"
     oriT            467..576
                     /note="incP origin of transfer"
     rep_origin      470..718
                     /label=oriT
                     /note="oriT"
     CDS             892..1695
                     /codon_start=1
                     /gene="accIV"
                     /product="apramycin resistance protein"
                     /label=accIV
                     /protein_id="AMV91698.1"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     gene            892..1695
                     /gene="accIV"
                     /label=accIV
     CDS             892..1692
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     rep_origin      1951..2451
     rep_origin      1994..2582
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2870..2891
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2906..2936
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2944..2960
                     /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     2968..2984
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      3163..3197
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     regulatory      3232..3332
                     /regulatory_class="promoter"
                     /gene="tipA"
     gene            3232..3332
                     /gene="tipA"
                     /label=tipA
     CDS             3365..4066
                     /codon_start=1
                     /gene="sceI"
                     /product="SceI meganuclease"
                     /label=sceI
                     /protein_id="AMV91699.1"
                     /translation="MHQKNQVMNLGPNSKLLKEYKSQLIELNIEQFEAGIGLILGDAYI
                     RSRDEGKTYCMQFEWKNKAYMDHVCLLYDQWVLSPPHKKQRVNHLGNLVITWGAQTFKH
                     QAFNKLANLFIVNNKKTIPNNLVENYLTPMSLAYWFMDDGGKWDYNKNSTNKSIVLNTQ
                     SFTFEEVEYLVKGLRNKFQLNCYVKINKNKPIIYIDSMSYLIFYNLIKPYLIPQMMYKL
                     PNTISSETFLK"
     gene            3365..4066
                     /gene="sceI"
                     /label=sceI
                     /note="SceI meganuclease sequence codon optimised for 
                     streptomyces"
     regulatory      4067..4148
                     /regulatory_class="terminator"
                     /note="fd terminator"
     terminator      4070..4118
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     regulatory      4212..4502
                     /regulatory_class="terminator"
                     /note="fd terminator"
     terminator      4342..4390
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             4734..5543
                     /codon_start=1
                     /gene="tsr"
                     /product="thiostrepton resistance protein"
                     /label=tsr
                     /protein_id="AMV91700.1"
                     /translation="MTELDTIANPSDPAVQRIIDVTKPSRSNIKTTLIEDVEPLMHSIA
                     AGVEFIEVYGSDSSPFPSELLDLCGRQNIPVRLIDSSIVNQLFKGERKAKTFGIARVPR
                     PARFGDIASRRGDVVVLDGVKIVGNIGAIVRTSLALGASGIILVDSDITSIADRRLQRA
                     SRGYVFSLPVVLSGREEAIAFIRDSGMQLMTLKADGDISVKELGDNPDRLALLFGSEKG
                     GPSDLFEEASSASVSIPMMSQTESLNVSVSLGIALHERIDRNLAANR"
     gene            4734..5543
                     /gene="tsr"
                     /label=tsr
     CDS             6106..7548
                     /codon_start=1
                     /gene="rep"
                     /product="replication initiator protein from the 
                     Streptomyces ghanaensis plasmid pSG5"
                     /label=pSG5 Rep
                     /translation="MWKPGEATWGNTCRCNNVHTCPWCMSRILAVRGSNVQLAADGLAD
                     AGYGLHLGTNTLRHFERMAFGTVRKGMRHGLVAVLHDGWKGAYGSSGRRWRTMRDDFGI
                     IGYERAFEDTFGWGSGWHLHWHTLWVTREVLGPDAQAAFRDALAGAWAAGVESAGGYTV
                     SETCDRPGCSCEGKGHGTDVRPLNGADAADGDAGKQARYLYKDGDKTKGGVAKIGLELA
                     GQNFKAGRGDDRMGPLDLGDAAAAELQRLRRPGPFVEKYREREFGVFQVRKHYRSQNLN
                     RLIKELGIQQDVRTEEEITDDTEGLVAIAVIPAYIWYRYIARVAGRRLDLIKVAETYGL
                     PGVRRLVESWGLVWGKDVLDPPAPEAPAAPGDLDADQMRFEVMSEEEAAFREARRKANE
                     ARTEELAASLDRVRQPKKEAIRPTISLRKRLKPKPVTVDVKTPPPGAASPVCRRCKGKL
                     APVLQPWGRLPGDCLRVDTAVA"