LOCUS       Exported                5150 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTHSSe_41, complete sequence.
ACCESSION   MH029758
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5150)
  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 5150)
  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 5150)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5150
                     /organism="Cloning vector pTHSSe_41"
                     /mol_type="other DNA"
                     /label=PTac driving ColE2 RepA
                     /note="PTac driving ColE2 RepA"
                     /db_xref="taxon:2137922"
     regulatory      1..56
                     /regulatory_class="promoter"
                     /note="PTac"
     promoter        3..31
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..56
                     /label=LacI binding site
                     /bound_moiety="LacI"
                     /note="symmetric LacI operator"
     ncRNA           72..146
                     /product="RiboJ"
                     /label=ribozyme
                     /note="insulator"
                     /ncRNA_class="ribozyme"
     misc_RNA        74..123
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      147..165
                     /regulatory_class="ribosome_binding_site"
                     /note="RBS32A; derived from BBa_B0032"
     CDS             166..1059
                     /codon_start=1
                     /product="RepA"
                     /label=RepA
                     /note="ColE2 replication protein"
                     /protein_id="AVR55117.1"
                     /translation="MSAVLQRFREKLPHKPYCTNDFAYGVRILPKNIAILARFIQQNQP
                     HALYWLPFDVDRTGASIDWSDRNCPAPNITVKNPRNGHAHLLYALALPVRTAPDASASA
                     LRYAAAIERALCEKLGADVNYSGLICKNPCHPEWQEVEWREEPYTLDELADYLDLSASA
                     RRSVDKNYGLGRNYHLFEKVRKWAYRAIRQGWPVFSQWLDAVIQRVEMYNASLPVPLSP
                     AECRAIGKSIAKYTHRKFSPEGFSAVQAARGRKGGTKSKRAAVPTSARSLKPWEALGIS
                     RATYYRKLKCDPDLAK"
     regulatory      1065..1193
                     /regulatory_class="terminator"
                     /note="B0015; BBa_B0015"
     terminator      1073..1144
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1160..1187
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1194..1214
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1215..1272
                     /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."
     CDS             complement(1430..2221)
                     /codon_start=1
                     /product="streptomycin 3-adenylyltransferase"
                     /label=streptomycin 3-adenylyltransferase
                     /note="streptomycin/spectinomycin resistance protein"
                     /protein_id="AVR55118.1"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPVESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRTDQLEEFVHYVKGEITKVVGK"
     rep_origin      complement(2553..3377)
                     /direction=LEFT
                     /label=p15A origin
                     /note="p15A origin"
     rep_origin      complement(2816..3360)
                     /direction=LEFT
                     /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."
     terminator      3850..3893
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(3936..5018)
                     /codon_start=1
                     /product="LacI"
                     /label=LacI
                     /note="Lac repressor"
                     /protein_id="AVR55119.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     regulatory      complement(5019..5100)
                     /regulatory_class="promoter"
                     /note="LacI promoter"