LOCUS       Exported                6685 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTac-At4CL1m-opr-VvSTS, complete sequence.
ACCESSION   MH488943
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6685)
  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 6685)
  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 6685)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6685
                     /organism="Cloning vector pTac-At4CL1m-opr-VvSTS"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /db_xref="taxon:2301154"
     CDS             8..1693
                     /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="AXN70050.1"
                     /translation="MAPQEQAVSQVMEKQSNNNNSDVIFRSKLPDIYIPNHLSLHDYIF
                     QNISEFATKPCLINGPTGHVYTYSDVHVISRQIAANFHKLGVNQNDVVMLLLPNCPEFV
                     LSFLAASFRGATATAANPFFTPAEIAKQAKASNTKLIITEARYVDKIKPLQNDDGVVIV
                     CIDDNESVPIPEGCLRFTELTQSTTEASEVIDSVEISPDDVVALPYSSGTTGLPKGVML
                     THKGLVTSVAQQVDGENPNLYFHSDDVLLCVLPMFHIYALNSIMLCGLRVGAAILIMPK
                     FEINLLLELIQRCKVTVAPMVPPIVLAIAKSSETEKYDLSSIRVVKSGAAPLGKELEDA
                     VNAKFPNAKLGQGYGMTEAGPVLAMSLGFAKEPFPVKSGACGTVVRNAEMKIVDPDTGD
                     SLSRKQPGEICIRGHQIMKGYLNNPAATAETIDKDGWLHTGDIGLIDDDDELFIVDRLK
                     ELVKYKGFQVAPAELEALLIGHPDITDVAVVAMKEEAAGEVPVAFVVKSKDSELSEDDV
                     KQFVSKQVVFYKRINKVFFTESIPKAPSGKILRKDLRAKLANGL"
     gene            8..1693
                     /gene="4CL1m"
                     /label=4CL1m
     CDS             1757..2935
                     /codon_start=1
                     /gene="STS"
                     /product="stilbene synthase"
                     /label=STS
                     /note="Vitis vinifera"
                     /protein_id="AXN70051.1"
                     /translation="MASVEEFRNAQRAKGPATILAIGTATPDHCVYQSDYADFYFRVTK
                     SEHMTALKKKFNRICDKSMIKKRYIHLTEEMLEEHPNIGAYMAPSLNIRQEIITAEVPK
                     LGKEAALKALKEWGQPKSKITHLVFCTTSGVEMPGADYKLANLLGLEPSVRRVMLYHQG
                     CYAGGTVLRTAKDLAENNAGARVLVVCSEITVVTFRGPSEDALDSLVGQALFGDGSAAV
                     IVGSDPDISIERPLFQLVSAAQTFIPNSAGAIAGNLREVGLTFHLWPNVPTLISENIEK
                     CLTQAFDPLGISDWNSLFWIAHPGGPAILDAVEAKLNLDKKKLEATRHVLSEYGNMSSA
                     CVLFILDEMRKKSLKGERATTGEGLDWGVLFGFGPGLTIETVVLHSIPMVTN"
     gene            1757..2935
                     /gene="STS"
                     /label=STS
     regulatory      3000..3317
                     /regulatory_class="terminator"
                     /note="rrnBT1T2"
     terminator      3205..3291
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3383..3410
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3430..3521
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3834..3850)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4093..4908
                     /codon_start=1
                     /product="kanamycin resistance protein"
                     /label=kanamycin resistance protein
                     /protein_id="AXN70052.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      5437..5982
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     rep_origin      5490..5940
                     /label=p15A
                     /note="p15A"
     regulatory      6544..6572
                     /regulatory_class="promoter"
                     /note="Tac promoter"
     regulatory      6619..6647
                     /regulatory_class="promoter"
                     /note="Tac promoter"
     promoter        6619..6647
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    6655..6671
                     /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)."