LOCUS       Exported                8084 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle vector pPxylA+-cel8A, complete sequence.
ACCESSION   MH328012
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8084)
  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  JOURNAL   Unpublished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 8084)
  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 8084)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8084
                     /organism="Shuttle vector pPxylA+-cel8A"
                     /mol_type="other DNA"
                     /label=derived from Escherichia coli TOP10
                     /note="derived from Escherichia coli TOP10"
                     /db_xref="taxon:2282839"
     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="AXF54488.1"
                     /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="AXF54489.1"
                     /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="AXF54490.1"
                     /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(5063..6229)
                     /codon_start=1
                     /gene="xylR"
                     /product="XylR"
                     /label=xylR
                     /note="XylR from B. megaterium xyl-operon"
                     /protein_id="AXF54491.1"
                     /translation="MVIIQIADQALVKKMNQKLILDEILKNSPVSRATLSEITGLNKST
                     VSSQVNTLLEKDFIFEIGAGQSRGGRRPVMLVFNKNAGYSIGIDIGVDYLNGILTDLEG
                     NIILEKTSDLSSSSASEVKEILFALIHGFVTHMPESPYGLVGIGICVPGLVDRHQQIIF
                     MPNLNWNIKDLQFLIESEFNVPVFVENEANAGAYGEKVFGMTKNYENIVYISINIGIGT
                     GLVINNELYKGVQGFSGEMGHMTIDFNGPKCSCGNRGCWELYASEKALLASLSKEEKNI
                     SRKEIVERANKNDVEMLNALQNFGFYIGIGLTNILNTFDIEAVILRNHIIESHPIVLNT
                     IKNEVSSRVHSHLDNKCELLPSSLGKNAPALGAVSIVIDSFLSVTPIS"
     gene            complement(5063..6229)
                     /gene="xylR"
                     /label=xylR
     regulatory      6333..6367
                     /regulatory_class="promoter"
                     /note="PxylA, xylose inducible promoter, derived from 
                     pHIS1525"
     misc_feature    6369..6397
                     /label=XylR binding site
                     /note="XylR binding site"
     regulatory      6438..6447
                     /regulatory_class="ribosome_binding_site"
                     /note="rbs+, ribosome binding site optimized for B. 
                     megaterium"
     gene            6461..7924
                     /gene="SPLipB-Cel8A-His6"
                     /label=SPLipB-Cel8A-His6
     CDS             6461..6544
                     /codon_start=1
                     /gene="SPLipB-Cel8A-His6"
                     /product="LipB signal peptide"
                     /label=SPLipB-Cel8A-His6
                     /protein_id="AXF54492.1"
                     /translation="MKKVLMAFIICLSLILSVLAAPPSGAKA"
     CDS             6572..7924
                     /codon_start=1
                     /gene="SPLipB-Cel8A-His6"
                     /product="Cel8A with C-terminal hexahistidine-tag"
                     /label=SPLipB-Cel8A-His6
                     /protein_id="AXF54493.1"
                     /translation="PFNTKYPYGPTSIADNQSEVTAMLKAEWEDWKSKRITSNGAGGYK
                     RVQRDASTNYDTVSEGMGYGLLLAVCFNEQALFDDLYRYVKSHFNGNGLMHWHIDANNN
                     VTSHDGGDGAATDADEDIALALIFADKLWGSSGAINYGQEARTLINNLYNHCVEHGSYV
                     LKPGDRWGGSSVTNPSYFAPAWYKVYAQYTGDTRWNQVADKCYQIVEEVKKYNNGTGLV
                     PDWCTASGTPASGQSYDYKYDATRYGWRTAVDYSWFGDQRAKANCDMLTKFFARDGAKG
                     IVDGYTIQGSKISNNHNASFIGPVAAASMTGYDLNFAKELYRETVAVKDSEYYGYYGNS
                     LRLLTLLYITGNFPNPLSDLSGQPTPPSNPTPSLPPQVVYGDVNGDGNVNSTDLTMLKR
                     YLLKSVTNINREAADVNRDGAINSSDMTILKRYLIKSIPHLPYSRHHHHHH"
     CDS             7904..7921
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"