LOCUS       Exported                4304 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pAJM.336, complete sequence.
ACCESSION   MH101731
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4304)
  AUTHORS   Meyer AJ, Segall-Shapiro TH, Voigt CA.
  TITLE     Marionette: E. coli containing 12 highly-optimized small molecule 
            sensors
  JOURNAL   bioRxivorg 285866, 1-26 (2018)
REFERENCE   2  (bases 1 to 4304)
  AUTHORS   Meyer AJ, Segall-Shapiro TH, Voigt CA.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-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 4304)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4304
                     /organism="Cloning vector pAJM.336"
                     /mol_type="other DNA"
                     /db_xref="taxon:2461527"
     regulatory      1..53
                     /regulatory_class="terminator"
                     /note="L3S3P21"
     regulatory      73..128
                     /regulatory_class="promoter"
                     /note="PTac"
     promoter        75..103
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     regulatory      75..80
                     /regulatory_class="minus_35_signal"
     regulatory      97..102
                     /regulatory_class="minus_10_signal"
     misc_feature    109..128
                     /label=LacO operator
                     /note="LacO operator"
     protein_bind    109..128
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     regulatory      110
                     /regulatory_class="other"
                     /note="+1"
     misc_feature    129..203
                     /label=RiboJ ribozyme insulator
                     /note="RiboJ ribozyme insulator"
     misc_RNA        131..180
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     regulatory      212..223
                     /regulatory_class="ribosome_binding_site"
                     /note="B0064"
     CDS             230..949
                     /codon_start=1
                     /product="YFP"
                     /label=YFP
                     /protein_id="AYJ72238.1"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     regulatory      950..1010
                     /regulatory_class="terminator"
                     /note="L3S2P21"
     regulatory      1015..1068
                     /regulatory_class="promoter"
                     /note="PLacI"
     regulatory      1033..1038
                     /regulatory_class="minus_35_signal"
     regulatory      1056..1061
                     /regulatory_class="minus_10_signal"
     regulatory      1069..1096
                     /regulatory_class="ribosome_binding_site"
                     /note="lac1"
     CDS             1097..2179
                     /codon_start=1
                     /product="LacI-AM"
                     /label=LacI-AM
                     /protein_id="AYJ72239.1"
                     /translation="MKPVTLYDVAEYAGVSYMTVSRVVNQASHVSAKTREKVEAAMVEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIYSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     regulatory      2189..2443
                     /regulatory_class="terminator"
                     /note="induction operon terminator region"
     rep_origin      2481..3317
                     /label=p15A origin
                     /note="p15A origin"
     rep_origin      complement(2755..3300)
                     /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."
     CDS             complement(3389..4204)
                     /codon_start=1
                     /product="kanamycin phosphotransferase"
                     /label=kanamycin phosphotransferase
                     /protein_id="AYJ72240.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"