LOCUS       Exported               10875 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pWCD2249, complete sequence.
ACCESSION   KR232306
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10875)
  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 10875)
  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 10875)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10875
                     /organism="Expression vector pWCD2249"
                     /mol_type="other DNA"
                     /db_xref="taxon:1655390"
     misc_feature    5..524
                     /label=similar to URA3 5
                     /note="similar to URA3 5"
     regulatory      573..1252
                     /regulatory_class="promoter"
     promoter        586..1252
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
     CDS             1259..2758
                     /codon_start=1
                     /product="CYP76AD1/P450 tyrosinase fusion protein"
                     /label=CYP76AD1/P450 tyrosinase fusion protein
                     /note="CYP76AD1 derived from Beta vulgaris - P450 
                     tyrosinase with W13L/F309L mutations"
                     /protein_id="AKH61510.1"
                     /translation="MDHATLAMILAILFISFHFIKLLFSQQTTKLLPPGPKPLPIIGNI
                     LEVGKKPHRSFANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSNRTIPNS
                     VTAGDHHKLTMSWLPVSPKWRNFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECA
                     QKGQAVDIGKAAFTTSLNLLSKLFFSVELAHHKSHTSQEFKELIWNIMEDIGKPNYADY
                     FPILGCVDPSGIRRRLACSFDKLIAVFQGIICERLAPDSSTTTTTTTDDVLDVLLQLFK
                     QNELTMGEINHLLVDIFDAGTDTTSSTLEWVMTELIRNPEMMEKAQEEIKQVLGKDKQI
                     QESDIINLPYLQAIIKETLRLHPPTVFLLPRKADTDVELYGYIVPKDAQILVNLWAIGR
                     DPNAWQNADIFSPERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQ
                     FFNWKLEGDISPKDLDMDEKFGIALQKTKPLKLIPIPRYGS"
     misc_feature    1267
                     /label=9T>C relative to WT CYP76AD1
                     /note="9T>C relative to WT CYP76AD1"
     misc_feature    1295..1297
                     /label=W13L mutation relative to wild type CYP76AD1
                     /note="W13L mutation relative to wild type CYP76AD1"
     misc_feature    2183..2185
                     /label=F309L mutation relative to wildtype CYP76AD1
                     /note="F309L mutation relative to wildtype CYP76AD1"
     regulatory      2765..2988
                     /regulatory_class="terminator"
     regulatory      3022..3721
                     /regulatory_class="promoter"
     CDS             3728..5146
                     /codon_start=1
                     /product="DOPA decarboxylase"
                     /label=DOPA decarboxylase
                     /note="PpDODC (DOPA decarboxylase) derived from Pseudomonas
                     putida"
                     /protein_id="AKH61511.1"
                     /translation="MTPEQFRQYGHQLIDLIADYRQTVGERPVMAQVEPGYLKAALPAT
                     APQQGEPFAAILDDVNNLVMPGLSHWQHPDFYGYFPSNGTLSSVLGDFLSTGLGVLGLS
                     WQSSPALSELEETTLDWLRQLLGLSGQWSGVIQDTASTSTLVALISARERATDYALVRG
                     GLQAEPKPLIVYVSAHAHSSVDKAALLAGFGRDNIRLIPTDERYALRPEALQAAIEQDL
                     AAGNQPCAVVATTGTTTTTALDPLRPVGEIAQANGLWLHVDSAMAGSAMILPECRWMWD
                     GIELADSVVVNAHKWLGVAFDCSIYYVRDPQHLIRVMSTNPSYLQSAVDGEVKNLRDWG
                     IPLGRRFRALKLWFMLRSEGVDALQARLRRDLDNAQWLAGQVEAAAEWEVLAPVQLQTL
                     CIRHRPAGLEGEALDAHTKGWAERLNASGAAYVTPATLDGRWMVRVSIGALPTERGDVQ
                     RLWARLQDVIKGGS"
     regulatory      5153..5377
                     /regulatory_class="terminator"
     terminator      5153..5340
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     regulatory      5411..6110
                     /regulatory_class="promoter"
     CDS             6117..7229
                     /codon_start=1
                     /product="ARO4 protein"
                     /label=ARO4 protein
                     /note="ARO4 derived from Saccharomyces cerevisiae with 
                     K229L feedback resistant mutation"
                     /protein_id="AKH61512.1"
                     /translation="MSESPMFAANGMPKVNQGAEEDVRILGYDPLASPALLQVQIPATP
                     TSLETAKRGRREAIDIITGKDDRVLVIVGPCSIHDLEAAQEYALRLKKLSDELKGDLSI
                     IMRAYLEKPRTTVGWKGLINDPDVNNTFNINKGLQSARQLFVNLTNIGLPIGSEMLDTI
                     SPQYLADLVSFGAIGARTTESQLHRELASGLSFPVGFKNGTDGTLNVAVDACQAAAHSH
                     HFMGVTLHGVAAITTTKGNEHCFVILRGGKKGTNYDAKSVAEAKAQLPAGSNGLMIDYS
                     HGNSNKDFRNQPKVNDVVCEQIANGENAITGVMIESNINEGNQGIPAEGKAGLKYGVSI
                     TDACIGWETTEDVLRKLAAAVRQRREVNKK"
     regulatory      7245..7469
                     /regulatory_class="terminator"
     regulatory      7518..7694
                     /regulatory_class="promoter"
     CDS             7695..8498
                     /codon_start=1
                     /product="URA3p uracil auxotrophy marker"
                     /label=URA3p uracil auxotrophy marker
                     /protein_id="AKH61514.1"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     regulatory      8499..8576
                     /regulatory_class="terminator"
     misc_feature    8581..9100
                     /label=similar to URA3 3
                     /note="similar to URA3 3"
     regulatory      complement(9113..9139)
                     /regulatory_class="terminator"
     CDS             complement(9140..9955)
                     /codon_start=1
                     /product="kanamycin resistance protein"
                     /label=kanamycin resistance protein
                     /protein_id="AKH61513.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     regulatory      complement(9956..10103)
                     /regulatory_class="promoter"
     rep_origin      complement(10104..10867)
                     /direction=LEFT
     rep_origin      10180..10768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"