LOCUS       Exported                9103 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pEBM109, complete sequence.
ACCESSION   KC779543
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9103)
  AUTHORS   Mearls EB, Olson DG, Herring CD, Lynd LR.
  TITLE     Development of a regulatable plasmid-based gene expression system 
            for Clostridium thermocellum
  JOURNAL   Appl. Microbiol. Biotechnol. 99 (18), 7589-7599 (2015)
  PUBMED    25994254
REFERENCE   2  (bases 1 to 9103)
  AUTHORS   Argyros A, Mearls EB.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-MAR-2013) Engineering, Dartmouth College, 14 
            Engineering Dr, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 9103)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9103
                     /organism="Expression vector pEBM109"
                     /mol_type="other DNA"
                     /country="USA"
                     /label=derived from Clostridium thermocellum strain BL21
                     /note="derived from Clostridium thermocellum strain BL21"
                     /db_xref="taxon:1381393"
     CDS             22..1059
                     /codon_start=1
                     /gene="glyR3"
                     /product="GlyR3"
                     /label=glyR3
                     /note="repressor"
                     /protein_id="AGT95718.1"
                     /translation="MTSEEIAKLCGVSRATVSRVINNSPNVKEETRQKILAVIKEKNYV
                     PIAPARRLAGIDSNIIGLFVLDIDISESKSRVSESTYFSRLINLIIDQANNFGFQVLVS
                     IITSQKQLSEIRNLFMSRTIFSGIFIGAFNDEIQLDDDIIMQHPTIIIDRQSERMVKKP
                     NRLVVNLDNFEGAYNATQFLIKLGHTRIGHISGDLRKLSGIERYEGYKKALEDAGLGFD
                     KNLVREGNFLDDSGYRLAREILKENVTAIFCANDVMAISAIKAIKETGLSVPDDISVIG
                     FDNTAIGNYIMPALTTVNAPLEHIAEACIESLKYFCEHKHFKQKEIRVKTDLIIRDSTK
                     RALEF"
     gene            22..1059
                     /gene="glyR3"
                     /label=glyR3
     terminator      1114..1361
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      1550..2223
     rep_origin      complement(1609..2197)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2368..3228)
                     /codon_start=1
                     /gene="bla"
                     /product="Bla"
                     /label=bla
                     /note="beta lactamase"
                     /protein_id="AGT95719.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(2368..3228)
                     /gene="bla"
                     /label=bla
     CDS             complement(3324..4127)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4128..4343)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     rep_origin      4366..4884
     misc_feature    4376..4879
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     rep_origin      5033..6567
     CDS             complement(5108..6112)
                     /codon_start=1
                     /gene="repB"
                     /product="RepB"
                     /label=repB
                     /note="replication protein B"
                     /protein_id="AGT95720.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     gene            complement(5108..6112)
                     /gene="repB"
                     /label=repB
     regulatory      6546..7896
                     /regulatory_class="promoter"
                     /gene="gapD"
     gene            6546..7896
                     /gene="gapD"
                     /label=gapD
     CDS             7246..7896
                     /codon_start=1
                     /gene="gapD"
                     /product="Cat"
                     /label=gapD
                     /note="chloramphenicol acetyltransferase"
                     /protein_id="AGT95721.1"
                     /translation="MNFNKIDLDNWKRKEIFNHYLNQQTTFSITTEIDISVLYRNIKQE
                     GYKFYPAFIFLVTRVINSNTAFRTGYNSDGELGYWDKLEPLYTIFDGVSKTFSGIWTPV
                     KNDFKEFYDLYLSDVEKYNGSGKLFPKTPIPENAFSLSIIPWTSFTGFNLNINNNSNYL
                     LPIITAGKFINKGNSIYLPLSLQVHHSVCDGYHAGLFMNSIQELSDRPNDWLL"
     regulatory      8938..8976
                     /regulatory_class="promoter"
                     /gene="celC"
     gene            8938..8976
                     /gene="celC"
                     /label=celC