LOCUS       Exported                7835 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pWCD1955, complete sequence.
ACCESSION   KR232327
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7835)
  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 7835)
  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 7835)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7835
                     /organism="Expression vector pWCD1955"
                     /mol_type="other DNA"
                     /db_xref="taxon:1655369"
     misc_feature    6..11
                     /label=BsmBI recognition site
                     /note="BsmBI recognition site"
     misc_feature    13..16
                     /label=BsmBI recognition site
                     /note="BsmBI recognition site"
     misc_feature    17..516
                     /label=similar to LEU2 5
                     /note="similar to LEU2 5"
     regulatory      523..1202
                     /regulatory_class="promoter"
     promoter        536..1202
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
     CDS             1209..3401
                     /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 various mutations (mutant 6) fused to 
                     mKate2 red fluorescent protein"
                     /protein_id="AKH61592.1"
                     /translation="MDHATLAMILAIWFISFHFIKLLFSQQTTKLLPPGPKPLPIIGNI
                     LEVGKKPHRSFANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSNRTIPNS
                     VTAGDHHKLTMSWLPVSPKWRNFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECA
                     QKGQAVDIGKAAFTTSLNLLSKLFFSVELAHHKSHTSQEFKELIWNIMEDIGKPNYADY
                     FPILGCVDPSGIRRRLACSFDKLIAVFQGIICERLAPDSSTTTTTTTDDVLDVLLQLFK
                     QNELTMGEINHLLVDIFDAGTDTTSSTLEWVMTELIRNPEMMEKAQEEIKQVLGKDKQI
                     QESDIINLPYLQAIIKETLRLHPPTVFLLPRKADTDVELYGYIVPKDAQILVNLWAIGR
                     DPNAWQNADIFSPERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQ
                     FFNWKLEGDISPKDLDMDEKFGIALQKTKPLKLIPIPRYGSVSELIKENMHMKLYMEGT
                     VNNHHFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILATSFMYGSKTFINHTQGIP
                     DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKK
                     TLGWEASTETLYPADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYV
                     DRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHR"
     misc_feature    1212..2699
                     /note="CYP76AD1 derived from Beta vulgaris - P450 
                     tyrosinase with various mutations"
     CDS             2706..3401
                     /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    2706..3398
                     /label=mKate2 red fluorescent protein
                     /note="mKate2 red fluorescent protein"
     regulatory      3408..3632
                     /regulatory_class="terminator"
     terminator      3408..3595
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     regulatory      3640..4293
                     /regulatory_class="promoter"
     promoter        3886..4293
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             4294..5388
                     /codon_start=1
                     /product="LEU2p auxotrophy marker"
                     /label=LEU2p auxotrophy marker
                     /protein_id="AKH61593.1"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     regulatory      5389..5865
                     /regulatory_class="terminator"
     misc_feature    5451..5940
                     /label=similar to LEU2 3
                     /note="similar to LEU2 3"
     misc_feature    complement(5942..5945)
                     /label=BsmBI recognition site
                     /note="BsmBI recognition site"
     misc_feature    complement(5947..5952)
                     /label=BsmBI recognition site
                     /note="BsmBI recognition site"
     rep_origin      5972..6735
     rep_origin      complement(6071..6659)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     regulatory      6736..6832
                     /regulatory_class="terminator"
     CDS             complement(6830..7690)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /protein_id="AKH61594.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     regulatory      complement(7691..7821)
                     /regulatory_class="promoter"
     promoter        complement(7691..7795)
                     /gene="bla"
                     /label=AmpR promoter