LOCUS       Exported                9002 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTOPO-1705, complete sequence.
ACCESSION   KX272606
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9002)
  AUTHORS   Tian L, Lo J, Shao X, Zheng T, Olson DG, Lynd LR.
  TITLE     Identification of a ferredoxin:NAD+ oxidoreductase enzyme in 
            Thermoanaerobacterium saccharolyticum and its role in ethanol 
            formation
  JOURNAL   Appl. Environ. Microbiol. (2016) In press
  PUBMED    27694237
REFERENCE   2  (bases 1 to 9002)
  AUTHORS   Tian L.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-MAY-2016) Thayer School of Engineering, Dartmouth 
            College, 14 Engineering Dr, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 9002)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9002
                     /organism="Cloning vector pTOPO-1705"
                     /mol_type="other DNA"
                     /db_xref="taxon:1906604"
     misc_recomb     1..1500
     CDS             1501..2262
                     /codon_start=1
                     /product="Histag-putative ferredoxin:NAD+ oxidoreductase"
                     /label=Histag-putative ferredoxin:NAD+ oxidoreductase
                     /note="Histag-Tsac_1705"
                     /protein_id="AOU74536.1"
                     /translation="MSGSHHHHHHGRYVVRENREISNGIYRLRLEFDGIPTPGQFIMIN
                     CGGNTLLKRPFSICDHDADEITIVYRIKGKGTYNLSKVKALDTLEVTGPHGHGFDMFDK
                     FNNILIVGGGIGIPPLLFLSKKLKSKNLKIALGFKSDVFLVDEFKKYGDVVIATEDGNT
                     GIKGYVTDAMKDEIKDIDMIYGCGPMQMLKSVKDMAEKYDVPCQISIEEHMGCGIGACM
                     VCACKTKTDSGFQYKKVCKDGPVFWSREVVF"
     CDS             1513..1530
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_recomb     3609..5046
     promoter        complement(5081..5099)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5106..5122)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5260..5562
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             5911..6705
                     /codon_start=1
                     /product="kanamycin resistance protein"
                     /label=kanamycin resistance protein
                     /protein_id="AOU74537.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(6955..7815)
                     /codon_start=1
                     /product="ampicillin resistance protein"
                     /label=ampicillin resistance protein
                     /protein_id="AOU74538.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7923..8542
     rep_origin      7939..8527
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    8815..8836
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8851..8881
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8889..8905
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     8913..8929
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8950..8968
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"