LOCUS       Exported               13274 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pWM91-L6-PT3-T4, complete sequence.
ACCESSION   KX264180
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13274)
  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 13274)
  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 13274)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..13274
                     /organism="Cloning vector pWM91-L6-PT3-T4"
                     /mol_type="other DNA"
                     /db_xref="taxon:1883155"
     rep_origin      complement(60..515)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     657..673
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        683..701
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     727..743
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    776..1194
                     /label=similar to lacZ (upstream)
                     /note="similar to lacZ (upstream)"
     regulatory      1231..1336
                     /regulatory_class="terminator"
                     /note="T0 transcriptional terminator from phage lambda"
     terminator      1237..1331
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1391..2107)
                     /codon_start=1
                     /product="green fluorescent protein mut3b"
                     /label=green fluorescent protein mut3b
                     /note="gfp(mut3b)"
                     /protein_id="ANW61922.1"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     regulatory      complement(2108..2131)
                     /regulatory_class="ribosome_binding_site"
                     /note="G5"
     RBS             2113..2121
                     /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(2149..2222)
                     /regulatory_class="promoter"
                     /note="PLtetO"
     promoter        complement(2149..2222)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2204..2222
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             complement(2265..3428)
                     /codon_start=1
                     /product="LacI"
                     /label=LacI
                     /protein_id="ANW61921.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQAANKNEENTNEVPTFMLNAGQANYLSQ"
     misc_feature    complement(2265..2348)
                     /label=mfLon-specific tag pdt2
                     /note="mfLon-specific tag pdt2"
     regulatory      complement(3429..3547)
                     /regulatory_class="ribosome_binding_site"
                     /note="L6 (promoter+RBS)"
     RBS             3434..3442
                     /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(3474..3547)
                     /regulatory_class="promoter"
                     /note="PLtetO"
     promoter        complement(3474..3547)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     prim_transcript 3493
                     /label=transcription start
                     /note="transcription start"
     protein_bind    3529..3547
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      3566..3683
                     /regulatory_class="promoter"
                     /note="PT3"
     protein_bind    3566..3585
                     /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    3602..3621
                     /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        3622..3651
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3658..3677
                     /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      3690..3721
                     /regulatory_class="ribosome_binding_site"
                     /note="T4"
     RBS             3708..3716
                     /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             3722..4345
                     /codon_start=1
                     /product="TetR"
                     /label=TetR
                     /protein_id="ANW61920.1"
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     regulatory      4388..4462
                     /regulatory_class="promoter"
                     /note="Ptrc-2"
     promoter        4401..4430
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4438..4454
                     /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 4438
                     /label=transcription start
                     /note="transcription start"
     regulatory      4470..4496
                     /regulatory_class="ribosome_binding_site"
                     /note="M2"
     RBS             4483..4491
                     /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             4497..5207
                     /codon_start=1
                     /product="mCherry"
                     /label=mCherry
                     /protein_id="ANW61919.1"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     regulatory      5262..5366
                     /regulatory_class="terminator"
                     /note="T1 transcriptional terminator from rrnB operon"
     terminator      5267..5353
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    5422..5821
                     /label=similar to lacZ (downstream)
                     /note="similar to lacZ (downstream)"
     promoter        complement(5840..5858)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5879..5895)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5903..5919
                     /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)."
     promoter        complement(5927..5957)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5972..5993
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(6694..7065)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            7098..7207
                     /note="incP origin of transfer"
     rep_origin      7099..7342
                     /label=RP4 OriT origin
                     /note="RP4 OriT origin"
     promoter        8374..8819
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             8820..10241
                     /codon_start=1
                     /product="SacB"
                     /label=SacB
                     /protein_id="ANW61917.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             complement(11808..12668)
                     /codon_start=1
                     /product="ampicillin resistance protein"
                     /label=ampicillin resistance protein
                     /note="beta-lactamase; Bla"
                     /protein_id="ANW61918.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(12669..12773)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      12880..13274
                     /label=Ori R6K
                     /note="Ori R6K"
     rep_origin      12883..13271
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"