LOCUS       Exported                7359 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTrcCDF-At4CL1m-AtCHI-PhCHS, complete sequence.
ACCESSION   MH488947
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7359)
  AUTHORS   Kim B, Binkley R, Kim HU, Lee SY.
  TITLE     Metabolic engineering of Escherichia coli for the enhanced 
            production of l-tyrosine
  JOURNAL   Biotechnol. Bioeng. (2018) In press
  PUBMED    30019750
REFERENCE   2  (bases 1 to 7359)
  AUTHORS   Yang D, Kim WJ, Yoo SM, Choi JH, Ha SH, Lee MH, Lee SY.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-JUN-2018) Dept. Chemical and Biomolecular Engineering,
            Korea Advanced Institute of Science and Technology, Daehak-ro 291, 
            Daejeon 34141, South Korea
REFERENCE   3  (bases 1 to 7359)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7359
                     /organism="Cloning vector pTrcCDF-At4CL1m-AtCHI-PhCHS"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /db_xref="taxon:2301185"
     regulatory      175..204
                     /regulatory_class="promoter"
                     /note="Trc promoter"
     promoter        175..204
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    212..228
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             249..1934
                     /codon_start=1
                     /gene="4CL1m"
                     /product="4-coumarate:CoA ligase 1 (4AT mutant)"
                     /label=4CL1m
                     /note="I250L/N404K/I461V; derived from Arabidopsis 
                     thaliana"
                     /protein_id="AXN70064.1"
                     /translation="MAPQEQAVSQVMEKQSNNNNSDVIFRSKLPDIYIPNHLSLHDYIF
                     QNISEFATKPCLINGPTGHVYTYSDVHVISRQIAANFHKLGVNQNDVVMLLLPNCPEFV
                     LSFLAASFRGATATAANPFFTPAEIAKQAKASNTKLIITEARYVDKIKPLQNDDGVVIV
                     CIDDNESVPIPEGCLRFTELTQSTTEASEVIDSVEISPDDVVALPYSSGTTGLPKGVML
                     THKGLVTSVAQQVDGENPNLYFHSDDVLLCVLPMFHIYALNSIMLCGLRVGAAILIMPK
                     FEINLLLELIQRCKVTVAPMVPPIVLAIAKSSETEKYDLSSIRVVKSGAAPLGKELEDA
                     VNAKFPNAKLGQGYGMTEAGPVLAMSLGFAKEPFPVKSGACGTVVRNAEMKIVDPDTGD
                     SLSRKQPGEICIRGHQIMKGYLNNPAATAETIDKDGWLHTGDIGLIDDDDELFIVDRLK
                     ELVKYKGFQVAPAELEALLIGHPDITDVAVVAMKEEAAGEVPVAFVVKSKDSELSEDDV
                     KQFVSKQVVFYKRINKVFFTESIPKAPSGKILRKDLRAKLANGL"
     gene            249..1934
                     /gene="4CL1m"
                     /label=4CL1m
     CDS             1960..2700
                     /codon_start=1
                     /gene="CHI"
                     /product="chalcone isomerase"
                     /label=CHI
                     /note="derived from Arabidopsis thaliana"
                     /protein_id="AXN70065.1"
                     /translation="MSSSNACASPSPFPAVTKLHVDSVTFVPSVKSPASSNPLFLGGAG
                     VRGLDIQGKFVIFTVIGVYLEGNAVPSLSVKWKGKTTEELTESIPFFREIVTGAFEKFI
                     KVTMKLPLTGQQYSEKVTENCVAIWKQLGLYTDCEAKAVEKFLEIFKEETFPPGSSILF
                     ALSPTGSLTVAFSKDDSIPETGIAVIENKLLAEAVLESIIGKNGVSPGTRLSVAERLSQ
                     LMMKNKDEKEVSDHSVEEKLAKEN"
     gene            1960..2700
                     /gene="CHI"
                     /label=CHI
     CDS             2760..3929
                     /codon_start=1
                     /gene="CHS"
                     /product="chalcone synthase"
                     /label=CHS
                     /note="derived from Petunia x hybrida"
                     /protein_id="AXN70066.1"
                     /translation="MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTYPDYYFRITN
                     SEHKTDLKEKFKRMCEKSMIKKRYMHLTEEILKENPSMCEYMAPSLDARQDIVVVEVPK
                     LGKEAAQKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLTKLLGLRPSVKRLMMYQQG
                     CFAGGTVLRLAKDLAENNKGARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGAGAI
                     IIGSDPIPGVERPLFELVSAAQTLLPDSHGAIDGHLREVGLTFHLLKDVPGLISKNIEK
                     SLEEAFRPLSISDWNSLFWIAHPGGPAILDQVEIKLGLKPEKLKATRNVLSNYGNMSSA
                     CVLFILDEMRKASAKEGLGTTGEGLEWGVLFGFGPGLTVETVVLHSVAT"
     gene            2760..3929
                     /gene="CHS"
                     /label=CHS
     regulatory      3957..4104
                     /regulatory_class="terminator"
                     /note="rrnBT1T2"
     CDS             complement(4275..5066)
                     /codon_start=1
                     /product="spectinomycin resistance protein"
                     /label=spectinomycin resistance protein
                     /protein_id="AXN70067.1"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(5067..5158)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5206..5944)
                     /direction=LEFT
                     /label=CDF
                     /note="CDF"
     CDS             complement(6154..7236)
                     /codon_start=1
                     /gene="lacIq"
                     /product="lac repressor protein"
                     /label=lacIq
                     /protein_id="AXN70068.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(6154..7236)
                     /gene="lacIq"
                     /label=lacIq
     promoter        complement(7237..7314)
                     /gene="lacI"
                     /label=lacI promoter