LOCUS       Exported                5596 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pKD227, complete sequence.
ACCESSION   MG323874
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5596)
  AUTHORS   Landry BP, Palanki R, Dyulgyarov N, Hartsough LA, Tabor JJ.
  TITLE     Phosphatase activity tunes two-component system sensor detection 
            threshold
  JOURNAL   Nat Commun 9 (1), 1433 (2018)
  PUBMED    29650958
REFERENCE   2  (bases 1 to 5596)
  AUTHORS   Landry BP.
  TITLE     Direct Submission
  JOURNAL   Submitted (29-OCT-2017) Bioengineering, Rice University, 6100 Main 
            St, Houston, TX 77005, USA
REFERENCE   3  (bases 1 to 5596)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5596
                     /organism="Cloning vector pKD227"
                     /mol_type="other DNA"
                     /db_xref="taxon:2219085"
     regulatory      1..148
                     /regulatory_class="promoter"
                     /note="pKanR from E. coli tn5"
     CDS             149..940
                     /codon_start=1
                     /product="SpecR"
                     /label=SpecR
                     /note="from P. Mirabilis R plasmid NR1"
                     /protein_id="AWU50965.1"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     regulatory      948..1053
                     /regulatory_class="terminator"
                     /note="T0"
     terminator      952..1046
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     misc_feature    1048..1049
                     /label=GC>CA
                     /note="GC>CA"
     misc_feature    1059..1855
                     /label=pProLar p15a Fragment
                     /note="pProLar p15a Fragment"
     misc_feature    1159..1197
                     /label=RNAII Promoter
                     /note="RNAII Promoter"
     regulatory      1159..1165
                     /regulatory_class="minus_35_signal"
     rep_origin      1160..1705
                     /direction=RIGHT
                     /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."
     rep_origin      complement(1168..1855)
                     /direction=LEFT
                     /label=p15A
                     /note="p15A"
     regulatory      1183..1188
                     /regulatory_class="minus_10_signal"
     misc_feature    1197..1705
                     /label=Putative RNAII
                     /note="Putative RNAII"
     misc_feature    1197
                     /label=+1
                     /note="+1"
     misc_feature    complement(1199..1303)
                     /label=Putative RNAI
                     /note="Putative RNAI"
     misc_feature    complement(1303..1339)
                     /label=RNAI Promoter
                     /note="RNAI Promoter"
     misc_feature    1303
                     /label=+1
                     /note="+1"
     regulatory      complement(1310..1315)
                     /regulatory_class="minus_10_signal"
     regulatory      complement(1334..1339)
                     /regulatory_class="minus_35_signal"
     regulatory      1856..1955
                     /regulatory_class="promoter"
                     /note="LacI Upstream Region"
     promoter        1878..1955
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             1956..3038
                     /codon_start=1
                     /product="LacI"
                     /label=LacI
                     /note="lacI"
                     /protein_id="AWU50964.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     regulatory      3039..3143
                     /regulatory_class="terminator"
                     /note="T1"
     terminator      3044..3130
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    complement(3144..3149)
                     /label=AvrII Scar
                     /note="AvrII Scar"
     misc_feature    complement(3149..3196)
                     /label=ColE1 Fragment
                     /note="ColE1 Fragment"
     misc_feature    3197..3318
                     /label=Ptrp Fragment
                     /note="Ptrp Fragment"
     misc_feature    complement(3238..3318)
                     /label=PyciVOQ
                     /note="PyciVOQ"
     misc_feature    3258
                     /label=+1
                     /note="+1"
     regulatory      3302..3363
                     /regulatory_class="promoter"
                     /note="PtacI"
     promoter        3302..3330
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     regulatory      3302..3307
                     /regulatory_class="minus_35_signal"
     regulatory      3324..3330
                     /regulatory_class="minus_10_signal"
     misc_binding    3336..3356
                     /label=LacO binding site
                     /bound_moiety="LacO"
     misc_feature    3336
                     /label=+1
                     /note="+1"
     protein_bind    3338..3354
                     /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)."
     misc_feature    3364..3397
                     /label=sRBS (1096.98)
                     /note="sRBS (1096.98)"
     CDS             3398..5413
                     /codon_start=1
                     /product="Sbal195_3859 (TtrS)"
                     /label=Sbal195_3859
                     /protein_id="AWU50966.1"
                     /translation="MFSSGMSLKWPLSIQGSIEPKMTPRIIFRMALRTKVFITLLACLF
                     ASANVFAVEPQNQSAPSDVMDAEVVAPVATEEPGYQVVDVGVLAIRGYKATINRWQPLM
                     GWLETQIPNSYFRLHPLSLDELAKGVETQGLDFVITNPGQSVLLARQYSLTWLATLRSP
                     LNNGAAMQVGSALVVRADSPYQTLTDLKGRRLGIVSKNAFGGYLTLVYEAQLKGIDLPR
                     FVGEIIPLGFPLDNLLYQLDDQKSGNDAAKDNRLDATVVPVCQLEQMQAEGLINIGHYR
                     VLDNQTPVGFHCQVSTRLYPNWSMAKTNRASQSLAKSVTQALLALPEDHLAAKSSDSAG
                     WTTAVSQLAIDQLLKDLDMHPLQTPWWQRAWQWVKLHQQWAWFILGILVLLNAYHFWLE
                     YRFSRRGRELINTQRQLNENRALLEHAQRIAIAGELGASLSHELNQPLAAIGHYCHGAE
                     VRLQRGTSPEELQSVLTLIQQEVTRADSIISRLRNLLKKRPVSKQALYLHELVNETVPL
                     LAYEFEQHQINLAVNVSGEPYLQSLDEVGMQQLLLNLLKNAFDACVQRLELESSGTEQS
                     GMRKPYVPTIDIDLRYQERTLLLTVTDNGTGLTEETSLLMQAFYSTKSEGLGLGLVICR
                     DIAESHGGTFSLESAMGGGCQAQVAIPRKPEPNGVL"
     misc_feature    3503..3557
                     /label=TM
                     /note="TM"
     regulatory      4490..4499
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_feature    4532..4588
                     /label=TM
                     /note="TM"
     misc_feature    4718..4720
                     /label=Catalytic His
                     /note="Catalytic His"
     regulatory      5423..5551
                     /regulatory_class="terminator"
                     /note="B0015"
     regulatory      5423..5502
                     /regulatory_class="terminator"
                     /note="B0010"
     terminator      5431..5502
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    5503..5510
                     /label=BB Scar
                     /note="BB Scar"
     regulatory      5511..5551
                     /regulatory_class="terminator"
                     /note="B0012"
     terminator      5518..5545
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    5552..5596
                     /label=pUC19 MCS
                     /note="pUC19 MCS"