LOCUS       Exported                8009 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pWCD2503, complete sequence.
ACCESSION   KR232282
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8009)
  AUTHORS   DeLoache WC, Russ ZN, Narcross L, Gonzales AM, Martin VJ, Dueber JE.
  TITLE     An enzyme-coupled biosensor enables (S)-reticuline production in 
            yeast from glucose
  JOURNAL   Nat. Chem. Biol. 11 (7), 465-471 (2015)
  PUBMED    25984720
REFERENCE   2  (bases 1 to 8009)
  AUTHORS   DeLoache WC, Russ ZN, Narcross L, Gonzales AM, Martin VJJ., Dueber 
            JE.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-APR-2015) Department of Bioengineering, University of 
            California, Berkeley, 2151 Berkeley Way Room 542, Berkeley, CA 
            94704, USA
REFERENCE   3  (bases 1 to 8009)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8009
                     /organism="Expression vector pWCD2503"
                     /mol_type="other DNA"
                     /db_xref="taxon:1655414"
     misc_feature    12..17
                     /label=BsmBI recognition site
                     /note="BsmBI recognition site"
     misc_feature    19..22
                     /label=BsmBI recognition site
                     /note="BsmBI recognition site"
     regulatory      189..888
                     /regulatory_class="promoter"
     CDS             895..3087
                     /codon_start=1
                     /product="CYP76AD1/P450 tyrosinase/mKate2 red fluorescent 
                     protein fusion protein"
                     /label=CYP76AD1/P450 tyrosinase/mKate2 red fluorescent
                     /note="CYP76AD1 derived from Beta vulgaris - P450 
                     tyrosinase with W13L/F309L mutations plus mKate2 red 
                     fluorescent protein"
                     /protein_id="AKH61429.1"
                     /translation="MDHATLAMILAILFISFHFIKLLFSQQTTKLLPPGPKPLPIIGNI
                     LEVGKKPHRSFANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSNRTIPNS
                     VTAGDHHKLTMSWLPVSPKWRNFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECA
                     QKGQAVDIGKAAFTTSLNLLSKLFFSVELAHHKSHTSQEFKELIWNIMEDIGKPNYADY
                     FPILGCVDPSGIRRRLACSFDKLIAVFQGIICERLAPDSSTTTTTTTDDVLDVLLQLFK
                     QNELTMGEINHLLVDIFDAGTDTTSSTLEWVMTELIRNPEMMEKAQEEIKQVLGKDKQI
                     QESDIINLPYLQAIIKETLRLHPPTVFLLPRKADTDVELYGYIVPKDAQILVNLWAIGR
                     DPNAWQNADIFSPERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQ
                     FFNWKLEGDISPKDLDMDEKFGIALQKTKPLKLIPIPRYGSVSELIKENMHMKLYMEGT
                     VNNHHFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILATSFMYGSKTFINHTQGIP
                     DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKK
                     TLGWEASTETLYPADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYV
                     DRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHR"
     misc_feature    898..2385
                     /note="CYP76AD1 derived from Beta vulgaris - P450 
                     tyrosinase with W13L/F309L mutations"
     misc_feature    903
                     /label=9T>C relative to WT CYP76AD1
                     /note="9T>C relative to WT CYP76AD1"
     misc_feature    931..933
                     /label=W13L mutation relative to wild type CYP76AD1
                     /note="W13L mutation relative to wild type CYP76AD1"
     misc_feature    1819..1821
                     /label=F309L mutation relative to wildtype CYP76AD1
                     /note="F309L mutation relative to wildtype CYP76AD1"
     CDS             2392..3087
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     misc_feature    2392..3084
                     /label=mKate2 red fluorescent protein
                     /note="mKate2 red fluorescent protein"
     regulatory      3094..3318
                     /regulatory_class="terminator"
     terminator      3094..3281
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     misc_feature    complement(3488..3491)
                     /label=BsmBI recognition site
                     /note="BsmBI recognition site"
     misc_feature    complement(3493..3498)
                     /label=BsmBI recognition site
                     /note="BsmBI recognition site"
     misc_feature    3510..4013
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     rep_origin      3510..3626
     rep_origin      3627..4017
     regulatory      complement(4019..4495)
                     /regulatory_class="terminator"
     CDS             complement(4496..5590)
                     /codon_start=1
                     /product="LEU2p auxotrophy marker"
                     /label=LEU2p auxotrophy marker
                     /protein_id="AKH61430.1"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     regulatory      complement(5591..6244)
                     /regulatory_class="promoter"
     promoter        complement(5591..5998)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      6255..7018
     rep_origin      complement(6354..6942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     regulatory      7019..7166
                     /regulatory_class="promoter"
     CDS             7167..7982
                     /codon_start=1
                     /product="kanamycin resistance protein"
                     /label=kanamycin resistance protein
                     /protein_id="AKH61431.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     regulatory      7983..8009
                     /regulatory_class="terminator"