LOCUS       Exported                5887 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pECJ3, complete sequence.
ACCESSION   KM521210
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5887)
  AUTHORS   Cameron DE, Collins JJ.
  TITLE     Tunable protein degradation in bacteria
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5887)
  AUTHORS   Cameron DE, Collins JJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (12-SEP-2014) Biomedical Engineering, Boston University, 
            44 Cummington St., Boston, MA 02215, USA
REFERENCE   3  (bases 1 to 5887)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5887
                     /organism="Cloning vector pECJ3"
                     /mol_type="other DNA"
                     /db_xref="taxon:1565074"
     misc_feature    49..54
                     /label=PvuII
                     /note="PvuII"
     CDS             complement(55..771)
                     /codon_start=1
                     /product="gfp"
                     /label=gfp
                     /note="mut3b"
                     /protein_id="AIU94523.1"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     misc_feature    772..777
                     /label=ApaLI
                     /note="ApaLI"
     misc_feature    817..822
                     /label=SphI
                     /note="SphI"
     regulatory      complement(823..896)
                     /regulatory_class="promoter"
                     /note="PLtetO promoter"
     promoter        complement(823..896)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     misc_feature    842
                     /label=transcription start
                     /note="transcription start"
     protein_bind    853..871
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    878..896
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    897..902
                     /label=AclI
                     /note="AclI"
     misc_feature    933..938
                     /label=SacII
                     /note="SacII"
     misc_feature    complement(939..1022)
                     /label=pdt#3
                     /note="pdt#3"
     CDS             complement(1023..2111)
                     /codon_start=1
                     /product="LacI"
                     /label=LacI
                     /protein_id="AIU94524.1"
                     /translation="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     misc_feature    2112..2117
                     /label=BsrGI
                     /note="BsrGI"
     misc_feature    2142..2147
                     /label=BamHI
                     /note="BamHI"
     regulatory      complement(2148..2221)
                     /regulatory_class="promoter"
                     /note="PLtetO promoter"
     promoter        complement(2148..2221)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     misc_feature    2167
                     /label=transcription start
                     /note="transcription start"
     protein_bind    2178..2196
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2203..2221
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    2222..2227
                     /label=EagI
                     /note="EagI"
     misc_feature    2234..2239
                     /label=NcoI
                     /note="NcoI"
     regulatory      2240..2314
                     /regulatory_class="promoter"
                     /note="Ptrc-2 promoter"
     promoter        2253..2282
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     misc_feature    2288
                     /label=transcription start
                     /note="transcription start"
     protein_bind    2290..2306
                     /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    2315..2320
                     /label=SalI
                     /note="SalI"
     regulatory      2337..2340
                     /regulatory_class="ribosome_binding_site"
                     /note="RBS8"
     misc_feature    2347..2352
                     /label=NheI
                     /note="NheI"
     CDS             2353..2976
                     /codon_start=1
                     /product="tetR"
                     /label=tetR
                     /protein_id="AIU94525.1"
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     misc_feature    2977..2982
                     /label=SacI
                     /note="SacI"
     misc_feature    3001..3006
                     /label=NdeI
                     /note="NdeI"
     misc_feature    3013..3018
                     /label=XmaI
                     /note="XmaI"
     regulatory      3019..3093
                     /regulatory_class="promoter"
                     /note="Ptrc-2 promoter"
     promoter        3032..3061
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3069..3085
                     /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    3069
                     /label=transcription start
                     /note="transcription start"
     misc_feature    3094..3099
                     /label=MfeI
                     /note="MfeI"
     misc_feature    3129..3134
                     /label=SfoI
                     /note="SfoI"
     CDS             3136..3846
                     /codon_start=1
                     /product="mCherry"
                     /label=mCherry
                     /protein_id="AIU94526.1"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     misc_feature    3847..3852
                     /label=EcoRV
                     /note="EcoRV"
     misc_feature    3859..3864
                     /label=AvrII
                     /note="AvrII"
     misc_feature    3871..3876
                     /label=XhoI
                     /note="XhoI"
     regulatory      3901..4005
                     /regulatory_class="terminator"
                     /note="T1 terminator"
     terminator      3906..3992
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      4012..4819
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      complement(4156..4744)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     regulatory      4826..4931
                     /regulatory_class="terminator"
                     /note="T0 terminator"
     terminator      4832..4926
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4957..5751)
                     /codon_start=1
                     /product="KanR"
                     /label=KanR
                     /note="kanamycin resistance"
                     /protein_id="AIU94527.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"