LOCUS       Exported                6485 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pET-AaOKS, complete sequence.
ACCESSION   MH651714
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6485)
  AUTHORS   Yang D, Kim WJ, Yoo SM, Choi JH, Ha SH, Lee MH, Lee SY.
  TITLE     Repurposing type III polyketide synthase as a malonyl-CoA biosensor 
            for metabolic engineering in bacteria
  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. (2018) In press
  PUBMED    30232266
REFERENCE   2  (bases 1 to 6485)
  AUTHORS   Yang D, Kim WJ, Yoo SM, Choi JH, Ha SH, Lee MH, Lee SY.
  TITLE     Novel Enzyme-coupled Malonyl-CoA Biosensor based on Type III 
            Polyketide Synthase and Use of the same (KR 10-2018-0066323)
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 6485)
  AUTHORS   Yang D, Kim WJ, Yoo SM, Choi JH, Ha SH, Lee MH, Lee SY.
  TITLE     Direct Submission
  JOURNAL   Submitted (19-JUL-2018) Dept. Chemical and Biomolecular Engineering,
            Korea Advanced Institute of Science and Technology, Daehak-ro 291, 
            Daejeon 34141, South Korea
REFERENCE   4  (bases 1 to 6485)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6485
                     /organism="Cloning vector pET-AaOKS"
                     /mol_type="other DNA"
                     /db_xref="taxon:2364892"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(560..1375)
                     /codon_start=1
                     /gene="aphIII"
                     /product="kanamycin resistance protein"
                     /label=aphIII
                     /protein_id="AYD60169.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(560..1375)
                     /gene="aphIII"
                     /label=aphIII
     rep_origin      1497..2085
                     /label=pBR322
                     /note="pBR322"
     misc_feature    2271..2413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2515..2706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3515..4597)
                     /codon_start=1
                     /gene="lacIq"
                     /product="lac repressor protein"
                     /label=lacIq
                     /protein_id="AYD60170.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(3515..4597)
                     /gene="lacIq"
                     /label=lacIq
     promoter        complement(4598..4675)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4988..5006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4988..5004
                     /regulatory_class="promoter"
                     /note="T7 promoter"
     protein_bind    5007..5031
                     /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)."
     RBS             5046..5068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5076..6287
                     /codon_start=1
                     /gene="OKS"
                     /product="octaketide synthase"
                     /label=OKS
                     /note="Aloe arborescens"
                     /protein_id="AYD60171.1"
                     /translation="MSSLSNASHLMEDVQGIRKAQRADGTATVMAIGTAHPPHIFPQDT
                     YADFYFRATNSEHKVELKKKFDRICKKTMIGKRYFNYDEEFLKKYPNITSFDEPSLNDR
                     QDICVPGVPALGAEAAVKAIAEWGRPKSEITHLVFCTSCGVDMPSADFQCAKLLGLRTN
                     VNKYCVYMQGCYAGGTVMRYAKDLAENNRGARVLVVCAELTIIGLRGPNESHLDNAIGN
                     SLFGDGAAALIVGSDPIIGVEKPMFEIVCAKQTVIPNSEDVIHLHMREAGLMFYMSKDS
                     PETISNNVEACLVDVFKSVGMTPPEDWNSLFWIPHPGGRAILDQVEAKLKLRPEKFRAT
                     RTVLWDCGNMVSACVLYILDEMRRKSADEGLETYGEGLEWGVLLGFGPGMTVETILLHS
                     LPLM"
     gene            5076..6287
                     /gene="OKS"
                     /label=OKS
     CDS             6329..6346
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      6413..6460
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"