LOCUS       Exported                8042 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle vector pPgrac-cel8A, complete sequence.
ACCESSION   MH328010
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8042)
  AUTHORS   Heinze S, Zimmermann K, Ludwig C, Heinzlmeir S, Schwarz WH, Zverlov 
            VV, Liebl W, Kornberger P.
  TITLE     Efficient secretory heterologous enzyme production in the novel host
            Paenibacillus polymyxa by usage of its promoter sequences
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8042)
  AUTHORS   Heinze S, Zimmermann K, Ludwig C, Heinzlmeir S, Schwarz WH, Zverlov 
            VV, Liebl W, Kornberger P.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-MAY-2018) Department for Microbiology, Technical 
            University of Munich, Emil-Ramann-Str. 4, Freising, Bavaria 85354, 
            Germany
REFERENCE   3  (bases 1 to 8042)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8042
                     /organism="Shuttle vector pPgrac-cel8A"
                     /mol_type="other DNA"
                     /label=derived from Escherichia coli TOP10
                     /note="derived from Escherichia coli TOP10"
                     /db_xref="taxon:2282837"
     regulatory      complement(77..676)
                     /regulatory_class="replication_regulatory_region"
                     /note="ColE1 origin of replication, E. coli"
     rep_origin      complement(79..667)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(838..1698)
                     /codon_start=1
                     /gene="ampR"
                     /product="AmpR"
                     /label=ampR
                     /note="Ampicillin Resistance"
                     /protein_id="AXF54476.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(838..1698)
                     /gene="ampR"
                     /label=ampR
     promoter        complement(1699..1803)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      complement(1832..2353)
                     /regulatory_class="other"
                     /note="origin of transfer for conjugation, contains traJ 
                     and oriT"
     CDS             complement(1832..2116)
                     /codon_start=1
                     /gene="traJ"
                     /product="TraJ"
                     /label=traJ
                     /protein_id="AXF54477.1"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGQQKAHYVNHHPNQVFWGRGAVKH"
     gene            complement(1832..2116)
                     /gene="traJ"
                     /label=traJ
     regulatory      complement(2117..2353)
                     /regulatory_class="other"
                     /note="oriT (origin of transfer)"
     oriT            2149..2258
                     /note="incP origin of transfer"
     CDS             complement(2515..3276)
                     /codon_start=1
                     /gene="kanR"
                     /product="KanR"
                     /label=kanR
                     /note="Kanamycin Resistance"
                     /protein_id="AXF54478.1"
                     /translation="MNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGRQTD
                     GPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFDSEEILLDYASQVESDWPLTHGQFF
                     SILPIYDSGGYLEKVYQTAKSVEAQTFHDAICALIVEELFEYAGKWRNIRVQGPTTFLP
                     SLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQLSDSEK
                     LLESLENFWNGIQEWTERHGYIVDVSKRIPF"
     gene            complement(2515..3276)
                     /gene="kanR"
                     /label=kanR
     regulatory      complement(3445..4612)
                     /regulatory_class="replication_regulatory_region"
                     /note="pUB origin of replication, Bacillus"
     CDS             complement(3445..4449)
                     /codon_start=1
                     /gene="repB"
                     /product="RepB replication protein"
                     /label=repB
                     /note="from Enterococcus faecalis plasmid pAM-alpha-1"
                     /db_xref="GI:22652809"
                     /protein_id="AAN03827.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     CDS             complement(4902..5984)
                     /codon_start=1
                     /gene="lacI"
                     /product="LacI"
                     /label=lacI
                     /note="LacI from E. coli lac-operon"
                     /protein_id="AXF54479.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(4902..5984)
                     /gene="lacI"
                     /label=lacI
     regulatory      6228..6405
                     /regulatory_class="promoter"
                     /note="Pgrac, IPTG inducible promoter, derived from pHT43"
     protein_bind    6368..6392
                     /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)."
     gene            6419..7882
                     /gene="SPLipB-Cel8A-His6"
                     /label=SPLipB-Cel8A-His6
     CDS             6419..6502
                     /codon_start=1
                     /gene="SPLipB-Cel8A-His6"
                     /product="LipB signal peptide"
                     /label=SPLipB-Cel8A-His6
                     /protein_id="AXF54480.1"
                     /translation="MKKVLMAFIICLSLILSVLAAPPSGAKA"
     CDS             6530..7882
                     /codon_start=1
                     /gene="SPLipB-Cel8A-His6"
                     /product="Cel8A with C-terminal hexahistidine-tag"
                     /label=SPLipB-Cel8A-His6
                     /protein_id="AXF54481.1"
                     /translation="PFNTKYPYGPTSIADNQSEVTAMLKAEWEDWKSKRITSNGAGGYK
                     RVQRDASTNYDTVSEGMGYGLLLAVCFNEQALFDDLYRYVKSHFNGNGLMHWHIDANNN
                     VTSHDGGDGAATDADEDIALALIFADKLWGSSGAINYGQEARTLINNLYNHCVEHGSYV
                     LKPGDRWGGSSVTNPSYFAPAWYKVYAQYTGDTRWNQVADKCYQIVEEVKKYNNGTGLV
                     PDWCTASGTPASGQSYDYKYDATRYGWRTAVDYSWFGDQRAKANCDMLTKFFARDGAKG
                     IVDGYTIQGSKISNNHNASFIGPVAAASMTGYDLNFAKELYRETVAVKDSEYYGYYGNS
                     LRLLTLLYITGNFPNPLSDLSGQPTPPSNPTPSLPPQVVYGDVNGDGNVNSTDLTMLKR
                     YLLKSVTNINREAADVNRDGAINSSDMTILKRYLIKSIPHLPYSRHHHHHH"
     CDS             7862..7879
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"