LOCUS       Exported               12499 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pSH105, complete sequence.
ACCESSION   MH245114
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12499)
  AUTHORS   Hon S, Holwerda EK, Worthen RS, Maloney MI, Tian L, Cui J, Lin PP, 
            Lynd LR, Olson DG.
  TITLE     Expressing the Thermoanaerobacterium saccharolyticum pforA in 
            engineered Clostridium thermocellum improves ethanol production
  JOURNAL   Biotechnol Biofuels 11, 242 (2018)
  PUBMED    30202437
REFERENCE   2  (bases 1 to 12499)
  AUTHORS   Hon S, Olson DG, Holwerda EK, Worthen RS, Maloney MI, Tian L, Cui J,
            Lin PP, Lynd LR.
  TITLE     Direct Submission
  JOURNAL   Submitted (20-APR-2018) Thayer School of Engineering, Dartmouth 
            College, 14 Engineering Drive, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 12499)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..12499
                     /organism="Cloning vector pSH105"
                     /mol_type="other DNA"
                     /db_xref="taxon:2338062"
     rep_origin      complement(1..828)
                     /direction=LEFT
                     /label=p15A origin of replication
                     /note="p15A origin of replication"
     rep_origin      28..573
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     CDS             complement(840..1418)
                     /codon_start=1
                     /gene="tdk"
                     /product="thymidine kinase"
                     /label=tdk
                     /protein_id="AYA22168.1"
                     /translation="MYGPKDHGYIEVVTGPMFSGKSEELIRRIKRAKIARQKVQVFKPA
                     IDDRYSIDKVVSHNGDNMHAIAIVKASDILAYAEEDTDVFAIDEVQFFDSEIVDIVKEI
                     ADSGKRVICAGLDMDFRGEPFGPTPELMAIAEFVDKLTAICMKCGNPATRTQRLINGKP
                     ANYDDPIIMVGAKESYEARCRKCHEVPRT"
     gene            complement(840..1418)
                     /gene="tdk"
                     /label=tdk
     regulatory      complement(1419..2039)
                     /regulatory_class="promoter"
                     /note="C. thermocellum cbp promoter"
     CDS             complement(2115..3119)
                     /codon_start=1
                     /gene="repB"
                     /product="replication initiator protein"
                     /label=repB
                     /protein_id="AYA22169.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     gene            complement(2115..3119)
                     /gene="repB"
                     /label=repB
     rep_origin      complement(3120..3574)
                     /direction=LEFT
                     /label=C. thermocellum origin of replication
                     /note="C. thermocellum origin of replication"
     CDS             complement(3677..4537)
                     /codon_start=1
                     /gene="bla"
                     /product="ampicillin resistance protein"
                     /label=bla
                     /protein_id="AYA22170.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(3677..4537)
                     /gene="bla"
                     /label=bla
     promoter        complement(4538..4642)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    4732..5266
                     /label=upstream homology recombination flank
                     /note="upstream homology recombination flank"
     regulatory      5267..5444
                     /regulatory_class="promoter"
                     /note="C. thermocellum enolase promoter"
     CDS             5445..8972
                     /codon_start=1
                     /gene="pforA"
                     /product="pyruvate ferredoxin oxidoreductase"
                     /label=pforA
                     /note="derived from Thermoanaerobacterium saccharolyticum"
                     /protein_id="AYA22171.1"
                     /translation="MSKVMKTMDGNTAAAHVAYAFTEVAAIYPITPSSPMAEHVDEWSA
                     HGRKNLFGQEVKVIEMQSEAGAAGAVHGSLAAGALTTTFTASQGLLLMIPNMYKIAGEL
                     LPGVFHVSARALASHALSIFGDHQDVMACRQTGFALLASGSVQEVMDLGSVAHLAAIKG
                     RVPFLHFFDGFRTSHEYQKIEVMDYEDLRKLLDMDAVREFKKRALNPEHPVTRGTAQNP
                     DIYFQEREASNRYYNAVPEIVEEYMKEISKITGREYKLFNYYGAPDAERIVIAMGSVTE
                     TIEETIDYLLKKGEKVGVVKVHLYRPFSFKHFMDAIPKTVKKIAVLDRTKEAGAFGEPL
                     YEDVRAAFYDSEMKPIIVGGRYGLGSKDTTPAQIVAVFDNLKSDTPKNNFTIGIVDDVT
                     YTSLPVGEEIETTAEGTISCKFWGFGSDGTVGANKSAIQIIGDNTDMYAQAYFSYDSKK
                     SGGVTISHLRFGKKPIRSTYLINNADFVACHKQAYVYNYDVLAGLKKGGTFLLNCTWKP
                     EELDEKLPASMKRYIAKNNINFYIINAVDIAKELGLGARINMIMQSAFFKLANIIPIDE
                     AVKHLKDAIVKSYGHKGEKIVNMNYAAVDRGIDALVKVDVPASWANAEDEAKVERNVPD
                     FIKNIADVMNRQEGDKLPVSAFVGMEDGTFPMGTAAYEKRGIAVDVPEWQIDNCIQCNQ
                     CAYVCPHAAIRPFLLNEEEVKNAPEGFTSKKAIGKGLEGLNFRIQVSVLDCTGCGVCAN
                     TCPSKEKSLIMKPLETQLDQAKNWEYAMSLSYKENPLGTDTVKGSQFEKPLLEFSGACA
                     GCGETPYARLVTQLFGDRMLIANATGCSSIWGGSAPSTPYTVNKDGHGPAWANSLFEDN
                     AEFGFGMALAVKQQREKLADIVKEALELDLTQDLKNALKLWLDNFNSSEITKKTANIIV
                     SLIQDYKTDDSKVKELLNEILDRKEYLVKKSQWIFGGDGWAYDIGFGGLDHVLASGEDV
                     NVLVFDTEVYSNTGGQSSKATPVGAIAQFAAAGKGIGKKDLGRIAMSYGYVYVAQIAMG
                     ANQAQTIKALKEAESYPGPSLIIAYAPCINHGIKLGMGCSQIEEKKAVEAGYWHLYRYN
                     PMLKAEGKNPFILDSKAPTASYKEFIMGEVRYSSLAKTFPERAEALFEKAEELAKEKYE
                     TYKKLAEQN"
     gene            5445..8972
                     /gene="pforA"
                     /label=pforA
                     /note="Tsac_0046"
     CDS             8990..9160
                     /codon_start=1
                     /gene="Tsac_2084"
                     /product="ferredoxin"
                     /label=Tsac_2084
                     /note="derived from Thermoanaerobacterium saccharolyticum"
                     /protein_id="AYA22172.1"
                     /translation="MAHIITDECISCGACAAECPVDAIHEGTGKYEVDADTCIDCGACE
                     PVCPTGAIKAE"
     gene            8990..9160
                     /gene="Tsac_2084"
                     /label=Tsac_2084
     misc_feature    9161..9720
                     /label=first downstream homology recombination flank
                     /note="first downstream homology recombination flank"
     regulatory      9724..10298
                     /regulatory_class="promoter"
                     /note="C. thermocellum gapDH promoter"
     CDS             10299..10949
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=cat
                     /protein_id="AYA22173.1"
                     /translation="MNFNKIDLDNWKRKEIFNHYLNQQTTFSITTEIDISVLYRNIKQE
                     GYKFYPAFIFLVTRVINSNTAFRTGYNSDGELGYWDKLEPLYTIFDGVSKTFSGIWTPV
                     KNDFKEFYDLYLSDVEKYNGSGKLFPKTPIPENAFSLSIIPWTSFTGFNLNINNNSNYL
                     LPIITAGKFINKGNSIYLPLSLQVHHSVCDGYHAGLFMNSIQELSDRPNDWLL"
     gene            10299..10949
                     /gene="cat"
                     /label=cat
     CDS             10967..11512
                     /codon_start=1
                     /gene="hpt"
                     /product="hypoxanthine phosphoribosyltransferase"
                     /label=hpt
                     /protein_id="AYA22174.1"
                     /translation="MENLSKDIDEILITEEELKEKIKELGRQITKDYKGKNLMLVGVLK
                     GALMFMADLSRHIDLPLSLDFMAVSSYGSSTHSSGIVKIIKDLDISIEGKDVLIVEDII
                     DSGLTLSYLRETLLGRKPKSLKICTILDKPERREASVKVDYVGFKIPDKFVVGYGLDFD
                     EKYRNLPFIGVLKPEMYS"
     gene            10967..11512
                     /gene="hpt"
                     /label=hpt
     misc_feature    11697..12256
                     /label=second downstream homology recombination flank
                     /note="second downstream homology recombination flank"