LOCUS       Exported                6771 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pWCD2480, complete sequence.
ACCESSION   KR232283
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6771)
  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 6771)
  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 6771)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6771
                     /organism="Expression vector pWCD2480"
                     /mol_type="other DNA"
                     /db_xref="taxon:1655413"
     misc_feature    5..524
                     /label=similar to URA3 5
                     /note="similar to URA3 5"
     regulatory      702..1381
                     /regulatory_class="promoter"
     promoter        715..1381
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
     CDS             1388..2980
                     /codon_start=1
                     /product="CYP76AD1/P450 tyrosinase/FLAG/hexahistidine tag 
                     fusion protein"
                     /label=CYP76AD1/P450 tyrosinase/FLAG/hexahistidine tag
                     /note="CYP76AD1 derived from Beta vulgaris - P450 
                     tyrosinase with W13L/F309L mutations fused to triple FLAG 
                     tag and hexahistidine tag"
                     /protein_id="AKH61432.1"
                     /translation="MDHATLAMILAILFISFHFIKLLFSQQTTKLLPPGPKPLPIIGNI
                     LEVGKKPHRSFANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSNRTIPNS
                     VTAGDHHKLTMSWLPVSPKWRNFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECA
                     QKGQAVDIGKAAFTTSLNLLSKLFFSVELAHHKSHTSQEFKELIWNIMEDIGKPNYADY
                     FPILGCVDPSGIRRRLACSFDKLIAVFQGIICERLAPDSSTTTTTTTDDVLDVLLQLFK
                     QNELTMGEINHLLVDIFDAGTDTTSSTLEWVMTELIRNPEMMEKAQEEIKQVLGKDKQI
                     QESDIINLPYLQAIIKETLRLHPPTVFLLPRKADTDVELYGYIVPKDAQILVNLWAIGR
                     DPNAWQNADIFSPERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQ
                     FFNWKLEGDISPKDLDMDEKFGIALQKTKPLKLIPIPRYGSDYKDDDDKDYKDDDDKDY
                     KDDDDKGHHHHHH"
     misc_feature    1396
                     /label=9T>C relative to WT CYP76AD1
                     /note="9T>C relative to WT CYP76AD1"
     misc_feature    1424..1426
                     /label=W13L mutation relative to wild type CYP76AD1
                     /note="W13L mutation relative to wild type CYP76AD1"
     misc_feature    2312..2314
                     /label=F309L mutation relative to wildtype CYP76AD1
                     /note="F309L mutation relative to wildtype CYP76AD1"
     misc_feature    2885..2956
                     /label=triplicate FLAG tag
                     /note="triplicate FLAG tag"
     CDS             2885..2956
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     misc_feature    2960..2977
                     /label=hexahistidine tag
                     /note="hexahistidine tag"
     CDS             2960..2977
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2991..3214
                     /regulatory_class="terminator"
     regulatory      3413..3589
                     /regulatory_class="promoter"
     CDS             3590..4393
                     /codon_start=1
                     /product="URA3p uracil auxotrophy marker"
                     /label=URA3p uracil auxotrophy marker
                     /protein_id="AKH61434.1"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     regulatory      4394..4471
                     /regulatory_class="terminator"
     misc_feature    4476..4995
                     /label=similar to URA3 3
                     /note="similar to URA3 3"
     regulatory      complement(5008..5034)
                     /regulatory_class="terminator"
     CDS             complement(5035..5850)
                     /codon_start=1
                     /product="kanamycin resistance protein"
                     /label=kanamycin resistance protein
                     /protein_id="AKH61433.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     regulatory      complement(5851..5998)
                     /regulatory_class="promoter"
     rep_origin      complement(5999..6762)
                     /direction=LEFT
     rep_origin      6075..6663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"