LOCUS       Exported                9405 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pBM5155, complete sequence.
ACCESSION   KT725394
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9405)
  AUTHORS   Alexander WG, Peris D, Pfannenstiel BT, Opulente DA, Kuang M, 
            Hittinger CT.
  TITLE     Efficient engineering of marker-free synthetic allotetraploids of 
            Saccharomyces
  JOURNAL   Fungal Genet. Biol. (2015) In press
  PUBMED    26555931
REFERENCE   2  (bases 1 to 9405)
  AUTHORS   Alexander WG, Peris D, Pfannenstiel B, Opulente DA, Kuang M, 
            Hittinger CT.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-SEP-2015) Laboratory of Genetics, University of 
            Wisconsin-Madison, 425-G Henry Mall, 4350 Genetics Biotechnology 
            Center, Madison, WI 53706, USA
REFERENCE   3  (bases 1 to 9405)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9405
                     /organism="Cloning vector pBM5155"
                     /mol_type="other DNA"
                     /db_xref="taxon:1758558"
     CDS             complement(28..1035)
                     /codon_start=1
                     /gene="rtTA"
                     /product="tetracycline-responsive transcription activator"
                     /label=rtTA
                     /protein_id="ALP32058.1"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     gene            complement(28..1035)
                     /gene="rtTA"
                     /label=rtTA
     regulatory      complement(1140..1343)
                     /regulatory_class="promoter"
     promoter        complement(1140..1343)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      complement(1344..1723)
                     /regulatory_class="enhancer"
     enhancer        1344..1723
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     terminator      1835..2022
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     regulatory      2044..2491
                     /regulatory_class="promoter"
     protein_bind    2052..2322
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     misc_feature    2540..2547
                     /label=NotI site for gap-repair cloning
                     /note="NotI site for gap-repair cloning"
     misc_feature    2585..2608
                     /label=6x glycine linker
                     /note="6x glycine linker"
     misc_feature    2609..3220
                     /label=16x myc epitope tag
                     /note="16x myc epitope tag"
     CDS             2609..3106
                     /codon_start=1
                     /product="13 tandem Myc epitope tags"
                     /label=13xMyc
                     /translation="EQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLIS
                     EEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLD
                     GEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISE
                     EDL"
     CDS             2726..3223
                     /codon_start=1
                     /product="13 tandem Myc epitope tags"
                     /label=13xMyc
                     /translation="EQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLIS
                     EEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLD
                     GEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISE
                     EDL"
     misc_feature    3251..3253
                     /label=in-frame amber stop codon
                     /note="in-frame amber stop codon"
     terminator      3299..3546
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3569..3585)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3593..3609
                     /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)."
     promoter        complement(3617..3647)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3662..3683
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      3969..4569
                     /label=colE1 ori
                     /note="colE1 ori"
     rep_origin      complement(3971..4559)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4730..5590)
                     /codon_start=1
                     /gene="ampR"
                     /product="beta-lactamase"
                     /label=ampR
                     /note="ampicillin resistance"
                     /protein_id="ALP32059.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(4730..5590)
                     /gene="ampR"
                     /label=ampR
     promoter        complement(5591..5695)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      5863..6242
                     /regulatory_class="promoter"
     gene            5903..7022
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        5903..6246
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             6247..6819
                     /codon_start=1
                     /gene="natR"
                     /product="nourseothricin-resistance"
                     /label=natR
                     /protein_id="ALP32060.1"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     gene            6247..6819
                     /gene="natR"
                     /label=natR
     regulatory      6820..7086
                     /regulatory_class="terminator"
     terminator      6825..7022
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      7087..9405
                     /label=CEN4/ARS416 yeast rep ori/centromere
                     /note="CEN4/ARS416 yeast rep ori/centromere"
     rep_origin      7365..8202
                     /label=ARS1
                     /note="S. cerevisiae autonomously replicating sequence 
                     ARS1/ARS416"
     misc_feature    8338..8630
                     /label=CEN4
                     /note="S. cerevisiae CEN4 centromere"
     primer_bind     9389..9405
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"