LOCUS       Exported               10275 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_71, complete sequence.
ACCESSION   MH029788
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10275)
  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 10275)
  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 10275)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10275
                     /organism="Cloning vector pTHSSe_71"
                     /mol_type="other DNA"
                     /note="TALEsp2 stabilized promoter driving VioE VioA BioB 
                     in ColE1 KanR"
                     /db_xref="taxon:2137952"
     misc_feature    9..12
                     /label=GGAG seam
                     /note="GGAG seam"
     regulatory      13..73
                     /regulatory_class="terminator"
                     /note="L3S2P21"
     regulatory      78..146
                     /regulatory_class="promoter"
                     /note="PT7A1w3"
     ncRNA           147..225
                     /product="SarJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     regulatory      226..245
                     /regulatory_class="ribosome_binding_site"
                     /note="RBSsp2"
     CDS             246..2885
                     /codon_start=1
                     /product="TALEsp2"
                     /label=TALEsp2
                     /protein_id="AVR55202.1"
                     /translation="MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVAL
                     SQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQL
                     DTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLL
                     PVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQ
                     ALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVA
                     IASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH
                     GLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQR
                     LLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGG
                     KQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQV
                     VAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQ
                     DHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETV
                     QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNG
                     GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNR
                     RIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTEL
                     EARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMH
                     EGDQTRAS"
     regulatory      complement(2891..2980)
                     /regulatory_class="terminator"
                     /note="ECK120029600"
     regulatory      2985..3059
                     /regulatory_class="promoter"
                     /note="PUPsp2"
     misc_feature    3010..3016
                     /label=bfpT7A1
                     /note="bfpT7A1"
     protein_bind    complement(3018..3035)
                     /label=TALEsp2 binding site
                     /bound_moiety="TALEsp2"
     ncRNA           3060..3134
                     /product="RiboJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     misc_RNA        3062..3111
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      3135..3157
                     /regulatory_class="ribosome_binding_site"
                     /note="RBSVioEs"
     CDS             3158..3733
                     /codon_start=1
                     /product="VioE"
                     /label=VioE
                     /protein_id="AVR55201.1"
                     /translation="MENREPPLLPARWSSAYVSYWSPMLPDDQLTSGYCWFDYERDICR
                     IDGLFNPWSERDTGYRLWMSEVGNAASGRTWKQKVAYGRERTALGEQLCERPLDDETGP
                     FAELFLPRDVLRRLGARHIGRRVVLGREADGWRYQRPGKGPSTLYLDAASGTPLRMVTG
                     DEASRASLRDFPNVSEAEIPDAVFAAKR"
     regulatory      3734..3757
                     /regulatory_class="ribosome_binding_site"
                     /note="RBSVioAs"
     CDS             3758..5014
                     /codon_start=1
                     /product="VioA"
                     /label=VioA
                     /protein_id="AVR55198.1"
                     /translation="MKHSSDICIVGAGISGLTCASHLLDSPACRGLSLRIFDMQQEAGG
                     RIRSKMLDGKASIELGAGRYSPQLHPHFQSAMQHYSQKSEVYPFTQLKFKSHVQQKLKR
                     AMNELSPRLKEHGKESFLQFVSRYQGHDSAVGMIRSMGYDALFLPDISAEMAYDIVGKH
                     PEIQSVTDNDANQWFAAETGFAGLIQGIKAKVKAAGARFSLGYRLLSVRTDGDGYLLQL
                     AGDDGWKLEHRTRHLILAIPPSAMAGLNVDFPEAWSGARYGSLPLFKGFLTYGEPWWLD
                     YKLDDQVLIVDNPLRKIYFKGDKYLFFYTDSEMANYWRGCVAEGEDGYLEQIRTHLASA
                     LGIVRERIPQPLAHVHKYWAHGVEFCRDSDIDHPSALSHRDSGIIACSDAYTEHCGWME
                     GGLLSAREASRLLLQRIAA"
     regulatory      5015..5038
                     /regulatory_class="ribosome_binding_site"
                     /note="RBSVioBs"
     CDS             5039..8035
                     /codon_start=1
                     /product="VioB"
                     /label=VioB
                     /protein_id="AVR55200.1"
                     /translation="MSILDFPRIHFRGWARVNAPTANRDPHGHIDMASNTVAMAGEPFD
                     LARHPTEFHRHLRSLGPRFGLDGRADPEGPFSLAEGYNAAGNNHFSWESATVSHVQWDG
                     GEADRGDGLVGARLALWGHYNDYLRTTFNRARWVDSDPTRRDAAQIYAGQFTISPAGAG
                     PGTPWLFTADIDDSHGARWTRGGHIAERGGHFLDEEFGLARLFQFSVPKDHPHFLFHPG
                     PFDSEAWRRLQLALEDDDVLGLTVQYALFNMSTPPQPNSPVFHDMVGVVGLWRRGELAS
                     YPAGRLLRPRQPGLGDLTLRVNGGRVALNLACAIPFSTRAAQPSAPDRLTPDLGAKLPL
                     GDLLLRDEDGALLARVPQALYQDYWTNHGIVDLPLLREPRGSLTLSSELAEWREQDWVT
                     QSDASNLYLEAPDRRHGRFFPESIALRSYFRGEARARPDIPHRIEGMGLVGVESRQDGD
                     AAEWRLTGLRPGPARIVLDDGAEAIPLRVLPDDWALDDATVEEVDYAFLYRHVMAYYEL
                     VYPFMSDKVFSLADRCKCETYARLMWQMCDPQNRNKSYYMPSTRELSAPKARLFLKYLA
                     HVEGQARLQAPPPAGPARIESKAQLAAELRKAVDLELSVMLQYLYAAYSIPNYAQGQQR
                     VRDGAWTAEQLQLACGSGDRRRDGGIRAALLEIAHEEMIHYLVVNNLLMALGEPFYAGV
                     PLMGEAARQAFGLDTEFALEPFSESTLARFVRLEWPHFIPAPGKSIADCYAAIRQAFLD
                     LPDLFGGEAGKRGGEHHLFLNELTNRAHPGYQLEVFDRDSALFGIAFVTDQGEGGALDS
                     PHYEHSHFQRLREMSARIMAQSAPFEPALPALRNPVLDESPGCQRVADGRARALMALYQ
                     GVYELMFAMMAQHFAVKPLGSLRRSRLMNAAIDLMTGLLRPLSCALMNLPSGIAGRTAG
                     PPLPGPVDTRSYDDYALGCRMLARRCERLLEQASMLEPGWLPDAQMELLDFYRRQMLDL
                     ACGKLSREA"
     regulatory      8042..8170
                     /regulatory_class="terminator"
                     /note="B0015; BBa_B0015"
     terminator      8050..8121
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      8137..8164
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    8183..8203
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      8204..8261
                     /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      8357..9120
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      8433..9021
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9219..10013)
                     /codon_start=1
                     /product="aminoglycoside O-phosphotransferase"
                     /label=aminoglycoside O-phosphotransferase
                     /note="kanamycin resistance protein"
                     /protein_id="AVR55199.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     terminator      10208..10251
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    10254..10275
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""