LOCUS       Exported               14612 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pSH074, complete sequence.
ACCESSION   KY852357
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14612)
  AUTHORS   Hon S, Olson DG, Holwerda EK, Lanahan AA, Murphy SJL., Maloney MI, 
            Zheng T, Papanek B, Guss AM, Lynd LR.
  TITLE     The ethanol pathway from Thermoanaerobacterium saccharolyticum 
            improves ethanol production in Clostridium thermocellum
  JOURNAL   Metab. Eng. 42, 175-184 (2017)
  PUBMED    28663138
REFERENCE   2  (bases 1 to 14612)
  AUTHORS   Hon S, Olson DG, Holwerda EK, Lanahan AA, Murphy SJL., Maloney MI, 
            Zheng T, Papanek BA, Guss AM, Lynd LR.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-MAR-2017) Thayer School of Engineering, Dartmouth 
            College, 14 Engineering Drive, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 14612)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..14612
                     /organism="Cloning vector pSH074"
                     /mol_type="other DNA"
                     /label=derived from Clostridium thermocellum
                     /note="derived from Clostridium thermocellum"
                     /db_xref="taxon:2018469"
     CDS             complement(1..579)
                     /codon_start=1
                     /gene="tdk"
                     /product="Tdk"
                     /label=tdk
                     /note="thymidine kinase"
                     /protein_id="ASK05943.1"
                     /translation="MYGPKDHGYIEVVTGPMFSGKSEELIRRIKRAKIARQKVQVFKPA
                     IDDRYSIDKVVSHNGDNMHAIAIVKASDILAYAEEDTDVFAIDEVQFFDSEIVDIVKEI
                     ADSGKRVICAGLDMDFRGEPFGPTPELMAIAEFVDKLTAICMKCGNPATRTQRLINGKP
                     ANYDDPIIMVGAKESYEARCRKCHEVPRT"
     gene            complement(1..579)
                     /gene="tdk"
                     /label=tdk
     regulatory      complement(580..1200)
                     /regulatory_class="promoter"
                     /note="C therm cbp promoter"
     CDS             complement(1276..2280)
                     /codon_start=1
                     /gene="repB"
                     /product="RepB"
                     /label=repB
                     /note="replication initiator protein"
                     /protein_id="ASK05944.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     gene            complement(1276..2280)
                     /gene="repB"
                     /label=repB
     rep_origin      complement(2281..2735)
                     /direction=LEFT
                     /label=C thermocellum replication origin
                     /note="C thermocellum replication origin"
     CDS             complement(2838..3698)
                     /codon_start=1
                     /gene="ampR"
                     /product="AmpR"
                     /label=ampR
                     /note="ampicillin resistance"
                     /protein_id="ASK05945.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(2838..3698)
                     /gene="ampR"
                     /label=ampR
     promoter        complement(3699..3803)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    3893..4392
                     /label=upstream homology region
                     /note="upstream homology region"
     regulatory      4396..4970
                     /regulatory_class="promoter"
                     /note="C thermocellum gapDH promoter"
     CDS             4971..5621
                     /codon_start=1
                     /gene="cat"
                     /product="Cat"
                     /label=cat
                     /note="chloramphenicol acetyltransferase"
                     /protein_id="ASK05946.1"
                     /translation="MNFNKIDLDNWKRKEIFNHYLNQQTTFSITTEIDISVLYRNIKQE
                     GYKFYPAFIFLVTRVINSNTAFRTGYNSDGELGYWDKLEPLYTIFDGVSKTFSGIWTPV
                     KNDFKEFYDLYLSDVEKYNGSGKLFPKTPIPENAFSLSIIPWTSFTGFNLNINNNSNYL
                     LPIITAGKFINKGNSIYLPLSLQVHHSVCDGYHAGLFMNSIQELSDRPNDWLL"
     gene            4971..5621
                     /gene="cat"
                     /label=cat
     CDS             5639..6184
                     /codon_start=1
                     /gene="hpt"
                     /product="Hpt"
                     /label=hpt
                     /note="hypoxanthine phosphoribosyltransferase"
                     /protein_id="ASK05947.1"
                     /translation="MENLSKDIDEILITEEELKEKIKELGRQITKDYKGKNLMLVGVLK
                     GALMFMADLSRHIDLPLSLDFMAVSSYGSSTHSSGIVKIIKDLDISIEGKDVLIVEDII
                     DSGLTLSYLRETLLGRKPKSLKICTILDKPERREASVKVDYVGFKIPDKFVVGYGLDFD
                     EKYRNLPFIGVLKPEMYS"
     gene            5639..6184
                     /gene="hpt"
                     /label=hpt
     misc_feature    6369..6868
                     /label=upstream homology region
                     /note="upstream homology region"
     CDS             6869..8068
                     /codon_start=1
                     /gene="adhA"
                     /product="AdhA"
                     /label=adhA
                     /note="NADPH-dependent alcohol dehydrogenase"
                     /protein_id="ASK05948.1"
                     /translation="MWETKVNPSKIFELRCKNTTYFGVGSIHKIKDILENLKINGINNV
                     IFITGKGSYKTSGAWDVVRPVLEELDLKYSLYDKVGPNPTVDMIDEAAKIGRESGAKAV
                     IGIGGGSPIDTAKSVAVLLKYTDKNARELYKQKFIPDDAVPIIAINLTHGTGTEVDRFA
                     VATIPEKNYKPAIAYDCLYPMFAIDDPSLMTKLDKKQTIAVTVDALNHITEAATTLVAS
                     PYSILTAKETVRLIVRYLPAAVNDPLNIVARYYLLYASALAGISFDNGLLHLTHALEHP
                     LSAVKPEIAHGLGLGAILPAVIKAIYPATAEVLADVYSPIVPGLKGLPVEAEYVAEKVQ
                     EWLFSVGCIQKLSDFGFTKDDIPNLVKLAKTTPSLDGLLSIAPVEATESVIEKIYLKSL
                     "
     gene            6869..8068
                     /gene="adhA"
                     /label=adhA
     CDS             8086..8940
                     /codon_start=1
                     /gene="nfnA"
                     /product="NfnA"
                     /label=nfnA
                     /note="NADH-dependent reduced ferredoxin:NADP+ 
                     oxidoreductase"
                     /protein_id="ASK05949.1"
                     /translation="MNEILEKKQLNPTVKMMVINAPLMAKKAKPGQFVIVRVDEKGERI
                     PLTIADYDRNKGTITIIFQEVGMSTKKLGTLNVGDRLHDFVGPLGKPVEFSKDTKRVLA
                     IGGGVGVAPLYPKVKMLNEMKVPVDSIIGGRSAEYVILEDEMKKVSENLYITTDDGTKG
                     RKGFVTDVLKELIEKDNKYDEVIAIGPLIMMKMVCNITKEYNIPTMVSMNPIMIDGTGM
                     CGGCRVTVGGETKFACVDGPAFDGLKVDFDEAMRRQNMYKDMERKVLENYEHECKLGGI
                     LNG"
     gene            8086..8940
                     /gene="nfnA"
                     /label=nfnA
     CDS             8933..10324
                     /codon_start=1
                     /gene="nfnB"
                     /product="NfnB"
                     /label=nfnB
                     /note="NADH-dependent reduced ferredoxin:NADP+ 
                     oxidoreductase"
                     /protein_id="ASK05950.1"
                     /translation="MANMSLKKVPMPEQEPDQRNKNFKEVALGYEENMAVEEAERCIQC
                     KNQPCVEGCPVHVKIPEFIKLIANRDFEGAYQKIKETNNLPAICGRVCPQESQCESVCT
                     RGKKGEPVAIGRLERFTADWHMKNNEDKIEKPETNGRKVAVIGSGPAGLSCAGDLAKMG
                     YDTTIFEAFHTPGGVLMYGIPEFRLPKEIVQKEIDSLKKLGVKIETNMVIGKILTIDDL
                     FDMGYEAVFIGTGAGLPKFMNIPGENLNGVYSANEFLTRINLMKAYDFPNSPTPVKVGK
                     KVAVVGGGNVAMDAARSAKRMGAEEVYIVYRRSEEEMPARLEEIHHAKEEGIIFKLLTN
                     PVRIIGDESGSVKGIECVNMVLGDVDESGRRRPVEEKGSEHVIDVDTVIIAIGQSPNPL
                     ITSTTEGLEKQRWGGIIVNEETLETSRRGVFAGGDAVTGAATVILAMGAGKKAAASIHK
                     YLSEK"
     gene            8933..10324
                     /gene="nfnB"
                     /label=nfnB
     CDS             10342..12921
                     /codon_start=1
                     /gene="adhE"
                     /product="AdhE"
                     /label=adhE
                     /note="bifunctional alcohol and aldehyde dehydrogenase; 
                     bifunctional alcohol and aldehyde dehydrognase"
                     /protein_id="ASK05951.1"
                     /translation="MATTKTELDVQKQIDLLVSRAQEAQKKFMSYTQEQIDAIVKAMAL
                     AGVDKHVELAKMAYEETKMGVYEDKITKNLFATEYVYHDIKNEKTVGIINENIEENYME
                     VAEPIGVIAGVTPVTNPTSTTMFKCLISIKTRNPIIFSFHPKAIKCSIAAAKVMYEAAL
                     KAGAPEGCIGWIETPSIEATQLLMTHPGVSLILATGGAGMVKAAYSSGKPALGVGPGNV
                     PCYIEKSANIKRAVSDLILSKTFDNGVICASEQAVIIDEEIADEVKKLMKEYGCYFLNK
                     DEIKKLEKFAIDEQSCAMSPAVVGQPAAKIAEMAGFKVPEGTKILVAEYEGVGPKYPLS
                     REKLSPILACYTVKDYNEGIKKCEEMTEFGGLGHSAVIHSENQNVINEFARRVRTGRLI
                     VNSPSSQGAIGDIYNTNTPSLTLGCGSMGRNSTTDNVSVKNLLNIKRVVIRKDRMKWFK
                     IPPKIYFESGSLQYLCKVKRKKAFIVTDPFMVKLGFVDKVTYQLDKANIEYEIFSEVEP
                     DPSVDTVMNGVKIMNSYNPDLIIAVGDGSAIDAAKGMWLFYEYPDTEFETLRLKFADIR
                     KRAFKFPELGKKALFIAIPTTSGTGSEVTAFAVITDKKRNIKYPLADYELTPDIAIIDP
                     DLTKTVPPSVTADTGMDVLTHAIEAYVSVMASDYTDALAEKAIKIVFEYLPRAYKNGND
                     EEAREKMHNASCMAGMAFTNAFLGINHSMAHILGGKFHIPHGRANAILLPYVIRYNAEK
                     PTKFVAFPQYEYPKAAERYAEIAKFLGLPASTVEEGVESLIEAIKNLMKELNIPLTLKD
                     AGINKEQFEKEIEEMSDIAFNDQCTGTNPRMPLTKEIAEIYRKAYGA"
     gene            10342..12921
                     /gene="adhE"
                     /label=adhE
     misc_feature    12939..13457
                     /label=downstream homology region
                     /note="downstream homology region"
     rep_origin      complement(13938..14526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"