LOCUS       Exported               11112 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pHCT2, complete sequence.
ACCESSION   KT725395
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11112)
  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 11112)
  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 11112)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..11112
                     /organism="Cloning vector pHCT2"
                     /mol_type="other DNA"
                     /db_xref="taxon:1758559"
     CDS             complement(28..1035)
                     /codon_start=1
                     /gene="rtTA"
                     /product="tetracycline-responsive transcription activator"
                     /label=rtTA
                     /protein_id="ALP32061.1"
                     /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"
     CDS             2510..4960
                     /codon_start=1
                     /gene="HO"
                     /product="homothallic switching endonuclease"
                     /label=HO
                     /protein_id="ALP32064.1"
                     /translation="MLSENTTILMANGEIKDIANVTANSYVMCADGSAARVINVTQGYQ
                     KIYNIQQKTKHRAFEGEPGRLDPRRRTVYQRLALQCTAGHKLSVRVPTKPLLEKSGRNA
                     TKYKVRWRNLQQCQTLDGRIIIIPKNHHKTFPMTVEGEFAAKRFIEEMERSKGEYFNFD
                     IEVRDLDYLDAQLRISSCIRFGPVLAGNGVLSKFLTGRSDLVTPAVKSMAWMLGLWLGD
                     STTKEPEISVDSLDPKLMESLRENAKIWGLYLTVCDDHVPLRAKHVRLHYGDGPDENRK
                     TRNLRKNNPFWKAVTILKFKRDLDGEKQIPEFMYGEHIEVREAFLAGLIDSDGYVVKKG
                     EGPESYKIAIQTVYSSIMDGIVHISRSLGMSATVTTRSAREEIIEGRKVQCQFTYDCNV
                     AGGTTSQNVLSYCRSGHKTREVPPIIKREPVYFSFTDDFQGESTVYGLTIEGHKNFLLG
                     NKIEVKSCRGCCVGEQLKISQKKNLKHCVACPRKGIKYFYKDWSGKNRVCARCYGRYKF
                     SGHHCINCKYVPEAREVKKAKDKGEKLGITPEGLPVKGPECIKCGGILQFDAVRGPHKS
                     CGNNAGARICRIPGLINGGGGGGGGGEQKLISEEDLNGEQKLISEEDLNGLDGEQKLIS
                     EEDLNGEQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISEEDLNGEQ
                     KLISEEDLNGLDGEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLDGEQKLISE
                     EDLNGEQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISEEDLNGEQK
                     LINH"
     gene            2510..4960
                     /gene="HO"
                     /label=HO
     CDS             3659..3670
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    4292..4315
                     /gene="HO"
                     /label=6x glycine linker
                     /note="6x glycine linker"
     misc_feature    4316..4927
                     /gene="HO"
                     /label=16x myc epitope tag
                     /note="16x myc epitope tag"
     CDS             4316..4813
                     /codon_start=1
                     /product="13 tandem Myc epitope tags"
                     /label=13xMyc
                     /translation="EQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLIS
                     EEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLD
                     GEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISE
                     EDL"
     CDS             4433..4930
                     /codon_start=1
                     /product="13 tandem Myc epitope tags"
                     /label=13xMyc
                     /translation="EQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLIS
                     EEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLD
                     GEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGLDGEQKLISEEDLNGEQKLISE
                     EDL"
     terminator      5006..5253
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5276..5292)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5300..5316
                     /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(5324..5354)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5369..5390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      5676..6276
                     /label=colE1 ori
                     /note="colE1 ori"
     rep_origin      complement(5678..6266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6437..7297)
                     /codon_start=1
                     /gene="ampR"
                     /product="beta-lactamase"
                     /label=ampR
                     /note="ampicillin resistance"
                     /protein_id="ALP32062.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(6437..7297)
                     /gene="ampR"
                     /label=ampR
     promoter        complement(7298..7402)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      7570..7949
                     /regulatory_class="promoter"
     gene            7610..8729
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        7610..7953
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             7954..8526
                     /codon_start=1
                     /gene="natR"
                     /product="nourseothricin-resistance"
                     /label=natR
                     /protein_id="ALP32063.1"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     gene            7954..8526
                     /gene="natR"
                     /label=natR
     regulatory      8527..8793
                     /regulatory_class="terminator"
     terminator      8532..8729
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      8794..11112
                     /label=CEN4/ARS416 yeast rep ori/centromere
                     /note="CEN4/ARS416 yeast rep ori/centromere"
     rep_origin      9072..9909
                     /label=ARS1
                     /note="S. cerevisiae autonomously replicating sequence 
                     ARS1/ARS416"
     misc_feature    10045..10337
                     /label=CEN4
                     /note="S. cerevisiae CEN4 centromere"
     primer_bind     11096..11112
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"