LOCUS       Exported                7391 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_70, complete sequence.
ACCESSION   MH029787
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7391)
  AUTHORS   Segall-Shapiro TH, Sontag ED, Voigt CA.
  TITLE     Engineered promoters enable constant gene expression at any copy 
            number in bacteria
  JOURNAL   Nat. Biotechnol. (2018) In press
  PUBMED    29553576
REFERENCE   2  (bases 1 to 7391)
  AUTHORS   Segall-Shapiro TH, Sontag ED, Voigt CA.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-MAR-2018) Synthetic Biology Center, Department of 
            Biological Engineering, Massachusetts Institute of Technology, 77 
            Massachusetts Avenue, Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 7391)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7391
                     /organism="Cloning vector pTHSSe_70"
                     /mol_type="other DNA"
                     /note="insulated constitutive promoter driving VioE VioA 
                     BioB in ColE1 KanR"
                     /db_xref="taxon:2137951"
     regulatory      complement(13..102)
                     /regulatory_class="terminator"
                     /note="ECK120029600"
     regulatory      107..175
                     /regulatory_class="promoter"
                     /note="PT7A1w1"
     ncRNA           176..250
                     /product="RiboJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     misc_RNA        178..227
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      251..273
                     /regulatory_class="ribosome_binding_site"
                     /note="RBSVioEc"
     CDS             274..849
                     /codon_start=1
                     /product="VioE"
                     /label=VioE
                     /protein_id="AVR55197.1"
                     /translation="MENREPPLLPARWSSAYVSYWSPMLPDDQLTSGYCWFDYERDICR
                     IDGLFNPWSERDTGYRLWMSEVGNAASGRTWKQKVAYGRERTALGEQLCERPLDDETGP
                     FAELFLPRDVLRRLGARHIGRRVVLGREADGWRYQRPGKGPSTLYLDAASGTPLRMVTG
                     DEASRASLRDFPNVSEAEIPDAVFAAKR"
     regulatory      850..873
                     /regulatory_class="ribosome_binding_site"
                     /note="RBSVioAc"
     CDS             874..2130
                     /codon_start=1
                     /product="VioA"
                     /label=VioA
                     /protein_id="AVR55195.1"
                     /translation="MKHSSDICIVGAGISGLTCASHLLDSPACRGLSLRIFDMQQEAGG
                     RIRSKMLDGKASIELGAGRYSPQLHPHFQSAMQHYSQKSEVYPFTQLKFKSHVQQKLKR
                     AMNELSPRLKEHGKESFLQFVSRYQGHDSAVGMIRSMGYDALFLPDISAEMAYDIVGKH
                     PEIQSVTDNDANQWFAAETGFAGLIQGIKAKVKAAGARFSLGYRLLSVRTDGDGYLLQL
                     AGDDGWKLEHRTRHLILAIPPSAMAGLNVDFPEAWSGARYGSLPLFKGFLTYGEPWWLD
                     YKLDDQVLIVDNPLRKIYFKGDKYLFFYTDSEMANYWRGCVAEGEDGYLEQIRTHLASA
                     LGIVRERIPQPLAHVHKYWAHGVEFCRDSDIDHPSALSHRDSGIIACSDAYTEHCGWME
                     GGLLSAREASRLLLQRIAA"
     regulatory      2131..2154
                     /regulatory_class="ribosome_binding_site"
                     /note="RBSVioBc"
     CDS             2155..5151
                     /codon_start=1
                     /product="VioB"
                     /label=VioB
                     /protein_id="AVR55196.1"
                     /translation="MSILDFPRIHFRGWARVNAPTANRDPHGHIDMASNTVAMAGEPFD
                     LARHPTEFHRHLRSLGPRFGLDGRADPEGPFSLAEGYNAAGNNHFSWESATVSHVQWDG
                     GEADRGDGLVGARLALWGHYNDYLRTTFNRARWVDSDPTRRDAAQIYAGQFTISPAGAG
                     PGTPWLFTADIDDSHGARWTRGGHIAERGGHFLDEEFGLARLFQFSVPKDHPHFLFHPG
                     PFDSEAWRRLQLALEDDDVLGLTVQYALFNMSTPPQPNSPVFHDMVGVVGLWRRGELAS
                     YPAGRLLRPRQPGLGDLTLRVNGGRVALNLACAIPFSTRAAQPSAPDRLTPDLGAKLPL
                     GDLLLRDEDGALLARVPQALYQDYWTNHGIVDLPLLREPRGSLTLSSELAEWREQDWVT
                     QSDASNLYLEAPDRRHGRFFPESIALRSYFRGEARARPDIPHRIEGMGLVGVESRQDGD
                     AAEWRLTGLRPGPARIVLDDGAEAIPLRVLPDDWALDDATVEEVDYAFLYRHVMAYYEL
                     VYPFMSDKVFSLADRCKCETYARLMWQMCDPQNRNKSYYMPSTRELSAPKARLFLKYLA
                     HVEGQARLQAPPPAGPARIESKAQLAAELRKAVDLELSVMLQYLYAAYSIPNYAQGQQR
                     VRDGAWTAEQLQLACGSGDRRRDGGIRAALLEIAHEEMIHYLVVNNLLMALGEPFYAGV
                     PLMGEAARQAFGLDTEFALEPFSESTLARFVRLEWPHFIPAPGKSIADCYAAIRQAFLD
                     LPDLFGGEAGKRGGEHHLFLNELTNRAHPGYQLEVFDRDSALFGIAFVTDQGEGGALDS
                     PHYEHSHFQRLREMSARIMAQSAPFEPALPALRNPVLDESPGCQRVADGRARALMALYQ
                     GVYELMFAMMAQHFAVKPLGSLRRSRLMNAAIDLMTGLLRPLSCALMNLPSGIAGRTAG
                     PPLPGPVDTRSYDDYALGCRMLARRCERLLEQASMLEPGWLPDAQMELLDFYRRQMLDL
                     ACGKLSREA"
     regulatory      5158..5286
                     /regulatory_class="terminator"
                     /note="B0015; BBa_B0015"
     terminator      5166..5237
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5253..5280
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    5299..5319
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      5320..5377
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     rep_origin      5473..6236
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      5549..6137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6335..7129)
                     /codon_start=1
                     /product="aminoglycoside O-phosphotransferase"
                     /label=aminoglycoside O-phosphotransferase
                     /note="kanamycin resistance protein"
                     /protein_id="AVR55194.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     terminator      7324..7367
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    7370..7391
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""