LOCUS       Exported               10360 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Gene deletion vector pJL2, complete sequence.
ACCESSION   HQ157352
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10360)
  AUTHORS   Olson DG, Tripathi SA, Giannone RJ, Lo J, Caiazza NC, Hogsett DA, 
            Hettich RL, Guss AM, Dubrovsky G, Lynd LR.
  TITLE     Deletion of the Cel48S cellulase from Clostridium thermocellum
  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 107 (41), 17727-17732 (2010)
  PUBMED    20837514
REFERENCE   2  (bases 1 to 10360)
  AUTHORS   Olson DG.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-AUG-2010) Thayer School of Engineering, Dartmouth 
            College, 8000 Cummings, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 10360)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10360
                     /organism="Gene deletion vector pJL2"
                     /lab_host="Clostridium thermocellum"
                     /mol_type="other DNA"
                     /db_xref="taxon:981226"
     terminator      27..274
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      463..1136
                     /label=ColE1
                     /note="ColE1"
     rep_origin      complement(522..1110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     regulatory      1182..1526
                     /regulatory_class="promoter"
                     /note="pfk; derived from Clostridium thermocellum strain 
                     DSM 1313"
     RBS             1512..1520
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3 end of the 16S rRNA (Chen et al., 1994)"
     CDS             1527..2471
                     /codon_start=1
                     /product="pyrF"
                     /label=pyrF
                     /note="5FOA negative-selection in Clostridium thermocellum"
                     /protein_id="ADW95859.1"
                     /translation="MFIDTLIEKIREKDNPSVVGLDPKIEYVPSFIKEDMYKKYGKNLK
                     AVAEAILLFNKYIIDAVYDIVPAVKPQLAYYEMYGLEGMRVFYETCKYAKEKGLLVIAD
                     GKRNDIGSTAQCYSAAYLGKTDIDEGISEAVFDVDALTVNPYLGVDGIKPFIDDCVKYN
                     KGIFVLVKTSNKSSGEIQDILTQEGRSIYEIVAEYVESWGENKKGKYGYSCVGAVVGAT
                     YPNLAKILRKILKNSYILVPGYGAQGGTARDVAHCFNYDGLGAIVNASRSIMCAYKSEQ
                     WKNVYSEEKFYEASRAEAIRMRDDINSALRDRK"
     CDS             complement(2571..3431)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /note="bla; ampicillin resistance in E. coli"
                     /protein_id="ADW95860.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     CDS             complement(3527..4330)
                     /codon_start=1
                     /product="URA3"
                     /label=URA3
                     /note="selectable marker for yeast mediated ligation"
                     /protein_id="ADW95861.1"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4331..4546)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     rep_origin      4569..5087
                     /label=CEN6/ARSH4
                     /note="CEN6/ARSH4"
     misc_feature    4579..5082
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(5311..6315)
                     /codon_start=1
                     /product="repB"
                     /label=repB
                     /note="replication protein for plasmid replication in 
                     Clostridium thermocellum"
                     /protein_id="ADW95862.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     misc_feature    complement(6784..7783)
                     /note="cel48Y 3 flanking region; derived from Clostridium 
                     thermocellum strain DSM 1313"
     regulatory      7784..8431
                     /regulatory_class="promoter"
                     /note="gapDH; derived from Clostridium thermocellum strain 
                     DSM 1313"
     CDS             8432..9226
                     /codon_start=1
                     /product="kan"
                     /label=kan
                     /note="kanamycin resistance gene derived from plasmid 
                     pIKM1; provides neomycin resistance"
                     /protein_id="ADW95863.1"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    complement(9227..10281)
                     /note="cel48Y 5 flanking region; derived from Clostridium 
                     thermocellum strain DSM 1313"