LOCUS       Exported                9414 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Yeast truncation assay backbone vector pLSK870, complete sequence.
ACCESSION   AY297714
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9414)
  AUTHORS   Zan Y, Haag JD, Chen KS, Shepel LA, Wigington D, Wang YR, Hu R, 
            Lopez-Guajardo CC, Brose HL, Porter KI, Leonard RA, Hitt AA, 
            Schommer SL, Elegbede AF, Gould MN.
  TITLE     Production of knockout rats using ENU mutagenesis and a yeast-based 
            screening assay
  JOURNAL   Nat. Biotechnol. 21 (6), 645-651 (2003)
  PUBMED    12754522
REFERENCE   2  (bases 1 to 9414)
  AUTHORS   Chen K-S., Gould MN.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-MAY-2003) Oncology, University of Wisconsin, 1400 
            University Avenue, Madison, WI 53706, USA
REFERENCE   3  (bases 1 to 9414)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9414
                     /organism="Yeast truncation assay backbone vector pLSK870"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:232236"
     misc_feature    70..573
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        610..714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             715..1575
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1746..2334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2622..2643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2658..2688
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2696..2712
                     /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     2720..2736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      2807..4265
                     /regulatory_class="promoter"
                     /note="yeast ADH1 promoter"
     promoter        3559..4263
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     misc_feature    4266..4271
                     /label=HindIII site
                     /note="HindIII site"
     CDS             4273..6027
                     /codon_start=1
                     /product="chimeric p53-yADE2"
                     /label=chimeric p53-yADE2
                     /note="yeast phosphoribosylamino-imidazole-carboxylase with
                     rat p53 leader sequence; reporter gene"
                     /protein_id="AAP51419.1"
                     /translation="MEDSQSDMSIERPLDSRTVGILGGGQLGRMIVEAANRLNIKTVIL
                     DAENSPAKQISNSNDHVNGSFSNPLDIEKLAEKCDVLTIEIEHVDVPTLKNLQVKHPKL
                     KIYPSPETIRLIQDKYIQKEHLIKNGIAVTQSVPVEQASETSLLNVGRDLGFPFVLKSR
                     TLAYDGRGNFVVKNKEMIPEALEVLKDRPLYAEKWAPFTKELAVMIVRSVNGLVFSYPI
                     VETIHKDNICDLCYAPARVPDSVQLKAKLLAENAIKSFPGCGIFGVEMFYLETGELLIN
                     EIAPRPHNSGHYTIDACVTSQFEAHLRSILDLPMPKNFTSFSTITTNAIMLNVLGDKHT
                     KDKELETCERALATPGSSVYLYGKESRPNRKVGHINIIASSMAECEQRLNYITGRTDIP
                     IKISVAQKLDLEAMVKPLVGIIMGSDSDLPVMSAACAVLKDFGVPFEVTIVSAHRTPHR
                     MSAYAISASKRGIKTIIAGAGGAAHLPGMVAAMTPLPVIGVPVKGSCLDGVDSLHSIVQ
                     MPRGVPVATVAINNSTNAALLAVRLLGAYDSSYTTKMEQFLLKQEEEVLVKAQKLETVG
                     YEAYLENK"
     misc_feature    4273..4305
                     /label=encodes rat p53 leader sequence
                     /note="encodes rat p53 leader sequence"
     misc_feature    4305..4312
                     /label=NotI cloning site
                     /note="NotI cloning site"
     misc_feature    4315..6027
                     /label=encodes yeast ADE2 reporter gene
                     /note="encodes yeast ADE2 reporter gene"
     CDS             4315..6027
                     /codon_start=1
                     /gene="S. cerevisiae ADE2"
                     /product="phosphoribosylaminoimidazole carboxylase, 
                     required for adenine biosynthesis"
                     /label=ADE2
                     /note="yeast auxotrophic marker"
                     /translation="DSRTVGILGGGQLGRMIVEAANRLNIKTVILDAENSPAKQISNSN
                     DHVNGSFSNPLDIEKLAEKCDVLTIEIEHVDVPTLKNLQVKHPKLKIYPSPETIRLIQD
                     KYIQKEHLIKNGIAVTQSVPVEQASETSLLNVGRDLGFPFVLKSRTLAYDGRGNFVVKN
                     KEMIPEALEVLKDRPLYAEKWAPFTKELAVMIVRSVNGLVFSYPIVETIHKDNICDLCY
                     APARVPDSVQLKAKLLAENAIKSFPGCGIFGVEMFYLETGELLINEIAPRPHNSGHYTI
                     DACVTSQFEAHLRSILDLPMPKNFTSFSTITTNAIMLNVLGDKHTKDKELETCERALAT
                     PGSSVYLYGKESRPNRKVGHINIIASSMAECEQRLNYITGRTDIPIKISVAQKLDLEAM
                     VKPLVGIIMGSDSDLPVMSAACAVLKDFGVPFEVTIVSAHRTPHRMSAYAISASKRGIK
                     TIIAGAGGAAHLPGMVAAMTPLPVIGVPVKGSCLDGVDSLHSIVQMPRGVPVATVAINN
                     STNAALLAVRLLGAYDSSYTTKMEQFLLKQEEEVLVKAQKLETVGYEAYLENK"
     terminator      6040..6287
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     promoter        complement(6311..6329)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6336..6352)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      6493..6948
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        7249..7656
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             7657..8751
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"