LOCUS       Exported                7389 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pWCD2469, complete sequence.
ACCESSION   KR232288
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7389)
  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 7389)
  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 7389)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7389
                     /organism="Expression vector pWCD2469"
                     /mol_type="other DNA"
                     /db_xref="taxon:1655408"
     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..3598
                     /codon_start=1
                     /product="CYP76AD1/P450 tyrosinase/Venus yellow fluorescent
                     protein fusion protein"
                     /label=CYP76AD1/P450 tyrosinase/Venus yellow fluoresce
                     /note="CYP76AD1 derived from Beta vulgaris - P450 
                     tyrosinase with F309L mutation plus Venus yellow 
                     fluorescent protein"
                     /protein_id="AKH61447.1"
                     /translation="MDHATLAMILAIWFISFHFIKLLFSQQTTKLLPPGPKPLPIIGNI
                     LEVGKKPHRSFANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSNRTIPNS
                     VTAGDHHKLTMSWLPVSPKWRNFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECA
                     QKGQAVDIGKAAFTTSLNLLSKLFFSVELAHHKSHTSQEFKELIWNIMEDIGKPNYADY
                     FPILGCVDPSGIRRRLACSFDKLIAVFQGIICERLAPDSSTTTTTTTDDVLDVLLQLFK
                     QNELTMGEINHLLVDIFDAGTDTTSSTLEWVMTELIRNPEMMEKAQEEIKQVLGKDKQI
                     QESDIINLPYLQAIIKETLRLHPPTVFLLPRKADTDVELYGYIVPKDAQILVNLWAIGR
                     DPNAWQNADIFSPERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQ
                     FFNWKLEGDISPKDLDMDEKFGIALQKTKPLKLIPIPRYGSSKGEELFTGVVPILVELD
                     GDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHM
                     KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEGGNIL
                     GHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLP
                     DNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK"
     misc_feature    1391..2878
                     /note="CYP76AD1 derived from Beta vulgaris - P450 
                     tyrosinase with F309L mutation"
     misc_feature    2312..2314
                     /label=F309L mutation relative to wildtype CYP76AD1
                     /note="F309L mutation relative to wildtype CYP76AD1"
     CDS             2885..3598
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
                     ICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEGGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     misc_feature    2885..3595
                     /label=Venus yellow fluorescent protein
                     /note="Venus yellow fluorescent protein"
     regulatory      3609..3832
                     /regulatory_class="terminator"
     regulatory      4031..4207
                     /regulatory_class="promoter"
     CDS             4208..5011
                     /codon_start=1
                     /product="URA3p uracil auxotrophy marker"
                     /label=URA3p uracil auxotrophy marker
                     /protein_id="AKH61449.1"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     regulatory      5012..5089
                     /regulatory_class="terminator"
     misc_feature    5094..5613
                     /label=similar to URA3 3
                     /note="similar to URA3 3"
     regulatory      complement(5626..5652)
                     /regulatory_class="terminator"
     CDS             complement(5653..6468)
                     /codon_start=1
                     /product="kanamycin resistance protein"
                     /label=kanamycin resistance protein
                     /protein_id="AKH61448.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     regulatory      complement(6469..6616)
                     /regulatory_class="promoter"
     rep_origin      complement(6617..7380)
                     /direction=LEFT
     rep_origin      6693..7281
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"