LOCUS       Exported                7827 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTrc-At4CL1m-AtCHI-PhCHS, complete sequence.
ACCESSION   MH488946
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7827)
  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 7827)
  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 7827)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7827
                     /organism="Cloning vector pTrc-At4CL1m-AtCHI-PhCHS"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /db_xref="taxon:2301172"
     regulatory      193..262
                     /regulatory_class="promoter"
                     /note="Trc promoter"
     promoter        193..222
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    230..246
                     /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             267..1952
                     /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="AXN70059.1"
                     /translation="MAPQEQAVSQVMEKQSNNNNSDVIFRSKLPDIYIPNHLSLHDYIF
                     QNISEFATKPCLINGPTGHVYTYSDVHVISRQIAANFHKLGVNQNDVVMLLLPNCPEFV
                     LSFLAASFRGATATAANPFFTPAEIAKQAKASNTKLIITEARYVDKIKPLQNDDGVVIV
                     CIDDNESVPIPEGCLRFTELTQSTTEASEVIDSVEISPDDVVALPYSSGTTGLPKGVML
                     THKGLVTSVAQQVDGENPNLYFHSDDVLLCVLPMFHIYALNSIMLCGLRVGAAILIMPK
                     FEINLLLELIQRCKVTVAPMVPPIVLAIAKSSETEKYDLSSIRVVKSGAAPLGKELEDA
                     VNAKFPNAKLGQGYGMTEAGPVLAMSLGFAKEPFPVKSGACGTVVRNAEMKIVDPDTGD
                     SLSRKQPGEICIRGHQIMKGYLNNPAATAETIDKDGWLHTGDIGLIDDDDELFIVDRLK
                     ELVKYKGFQVAPAELEALLIGHPDITDVAVVAMKEEAAGEVPVAFVVKSKDSELSEDDV
                     KQFVSKQVVFYKRINKVFFTESIPKAPSGKILRKDLRAKLANGL"
     gene            267..1952
                     /gene="4CL1m"
                     /label=4CL1m
     CDS             1978..2718
                     /codon_start=1
                     /gene="CHI"
                     /product="chalcone isomerase"
                     /label=CHI
                     /note="derived from Arabidopsis thaliana"
                     /protein_id="AXN70060.1"
                     /translation="MSSSNACASPSPFPAVTKLHVDSVTFVPSVKSPASSNPLFLGGAG
                     VRGLDIQGKFVIFTVIGVYLEGNAVPSLSVKWKGKTTEELTESIPFFREIVTGAFEKFI
                     KVTMKLPLTGQQYSEKVTENCVAIWKQLGLYTDCEAKAVEKFLEIFKEETFPPGSSILF
                     ALSPTGSLTVAFSKDDSIPETGIAVIENKLLAEAVLESIIGKNGVSPGTRLSVAERLSQ
                     LMMKNKDEKEVSDHSVEEKLAKEN"
     gene            1978..2718
                     /gene="CHI"
                     /label=CHI
     CDS             2778..3947
                     /codon_start=1
                     /gene="CHS"
                     /product="chalcone synthase"
                     /label=CHS
                     /note="derived from Petunia x hybrida"
                     /protein_id="AXN70061.1"
                     /translation="MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTYPDYYFRITN
                     SEHKTDLKEKFKRMCEKSMIKKRYMHLTEEILKENPSMCEYMAPSLDARQDIVVVEVPK
                     LGKEAAQKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLTKLLGLRPSVKRLMMYQQG
                     CFAGGTVLRLAKDLAENNKGARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGAGAI
                     IIGSDPIPGVERPLFELVSAAQTLLPDSHGAIDGHLREVGLTFHLLKDVPGLISKNIEK
                     SLEEAFRPLSISDWNSLFWIAHPGGPAILDQVEIKLGLKPEKLKATRNVLSNYGNMSSA
                     CVLFILDEMRKASAKEGLGTTGEGLEWGVLFGFGPGLTVETVVLHSVAT"
     gene            2778..3947
                     /gene="CHS"
                     /label=CHS
     regulatory      4034..4397
                     /regulatory_class="terminator"
                     /note="rrnBT1T2"
     terminator      4180..4266
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4358..4385
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4405..4496
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4497..5357
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /protein_id="AXN70062.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPTAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            4497..5357
                     /gene="bla"
                     /label=bla
     rep_origin      5512..6131
                     /label=pBR322
                     /note="pBR322"
     rep_origin      5528..6116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    6302..6442
                     /label=bom
                     /note="basis of mobility region from pBR322"
     promoter        6628..6705
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6697..7788
                     /codon_start=1
                     /gene="lacIq"
                     /product="lac repressor protein"
                     /label=lacIq
                     /protein_id="AXN70063.1"
                     /translation="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     gene            6697..7788
                     /gene="lacIq"
                     /label=lacIq
     CDS             6706..7788
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"