LOCUS       Exported               10452 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pBAC-L6-PT3-T4Ci, complete sequence.
ACCESSION   KX264182
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10452)
  AUTHORS   Lee JW, Gyorgy A, Cameron DE, Pyenson N, Choi KR, Way JC, Silver PA,
            Del Vecchio D, Collins JJ.
  TITLE     Creating Single-Copy Genetic Circuits
  JOURNAL   Mol. Cell 63 (2), 329-336 (2016)
  PUBMED    27425413
REFERENCE   2  (bases 1 to 10452)
  AUTHORS   Lee JW, Gyorgy A, Cameron DE, Pyenson N, Choi KR, Way JC, Silver PA,
            Del Vecchio D, Collins JJ.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-MAY-2016) Biological Engineering, MIT, 45 Carleton St,
            MIT Building E25-Rm302, Cambridge, MA 02142, USA
REFERENCE   3  (bases 1 to 10452)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..10452
                     /organism="Cloning vector pBAC-L6-PT3-T4Ci"
                     /mol_type="other DNA"
                     /db_xref="taxon:1883150"
     CDS             complement(102..1073)
                     /codon_start=1
                     /gene="sopB"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopB
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     CDS             complement(1073..2248)
                     /codon_start=1
                     /gene="sopA"
                     /product="partitioning protein for the bacterial F plasmid"
                     /label=sopA
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     misc_feature    2574..2824
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     CDS             complement(2827..3582)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein for the bacterial 
                     F plasmid"
                     /label=repE
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     rep_origin      3673..3892
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     rep_origin      3674..3891
                     /label=OriS origin
                     /note="OriS origin"
     rep_origin      3962..4586
                     /label=OriV origin
                     /note="OriV origin"
     rep_origin      3968..4582
                     /label=oriV
                     /note="origin of replication for the bacterial F plasmid"
     misc_feature    5425..5472
                     /label=FRT site
                     /note="FRT site"
     protein_bind    complement(5425..5472)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        5496..5598
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             5599..6258
                     /codon_start=1
                     /product="CamR"
                     /label=CamR
                     /protein_id="ANW61929.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     misc_feature    complement(6384..6418)
                     /label=FRT site
                     /note="FRT site"
     protein_bind    complement(6384..6417)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    6437..6663
                     /gene="attP"
                     /label=HK022 attP
                     /bound_moiety="HK022 integrase"
                     /note="attachment site of phage HK022"
     misc_feature    complement(6508..6532)
                     /label=HK022 attachment site
                     /note="HK022 attachment site"
     regulatory      6711..6816
                     /regulatory_class="terminator"
                     /note="T0 transcriptional terminator from phage lambda"
     terminator      6717..6811
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6859..7164)
                     /codon_start=1
                     /product="CcdB"
                     /label=CcdB
                     /protein_id="ANW61928.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     regulatory      complement(7171..7185)
                     /regulatory_class="ribosome_binding_site"
                     /note="RBS"
     regulatory      complement(7192..7265)
                     /regulatory_class="promoter"
                     /note="PLtetO"
     promoter        complement(7192..7265)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     prim_transcript 7211
                     /label=transcription start
                     /note="transcription start"
     protein_bind    7247..7265
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             complement(7308..8471)
                     /codon_start=1
                     /product="LacI"
                     /label=LacI
                     /protein_id="ANW61932.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQAANKNEENTNEVPTFMLNAGQANYLSQ"
     misc_feature    complement(7308..7391)
                     /label=mfLon-specific tag pdt2
                     /note="mfLon-specific tag pdt2"
     regulatory      complement(8472..8590)
                     /regulatory_class="ribosome_binding_site"
                     /note="L6 (promoter+RBS)"
     RBS             8477..8485
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3 end of the 16S rRNA (Chen et al., 1994)"
     regulatory      complement(8517..8590)
                     /regulatory_class="promoter"
                     /note="PLtetO"
     promoter        complement(8517..8590)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     prim_transcript 8536
                     /label=transcription start
                     /note="transcription start"
     protein_bind    8572..8590
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      8609..8726
                     /regulatory_class="promoter"
                     /note="PT3"
     protein_bind    8609..8628
                     /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."
     protein_bind    8645..8664
                     /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."
     promoter        8665..8694
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    8701..8720
                     /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      8733..8764
                     /regulatory_class="ribosome_binding_site"
                     /note="T4"
     RBS             8751..8759
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3 end of the 16S rRNA (Chen et al., 1994)"
     CDS             8765..9388
                     /codon_start=1
                     /product="TetR"
                     /label=TetR
                     /protein_id="ANW61931.1"
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     regulatory      9431..9505
                     /regulatory_class="promoter"
                     /note="Ptrc-2"
     promoter        9444..9473
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    9481..9497
                     /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)."
     prim_transcript 9481
                     /label=transcription start
                     /note="transcription start"
     regulatory      9513..9539
                     /regulatory_class="ribosome_binding_site"
                     /note="M2"
     RBS             9526..9534
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3 end of the 16S rRNA (Chen et al., 1994)"
     CDS             9540..10250
                     /codon_start=1
                     /product="mCherry"
                     /label=mCherry
                     /protein_id="ANW61930.1"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     regulatory      10305..10409
                     /regulatory_class="terminator"
                     /note="T1 transcriptional terminator from rrnB operon"
     terminator      10310..10396
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"