LOCUS       Exported                5618 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Promoter vector pCK302, complete sequence.
ACCESSION   KU555410
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5618)
  AUTHORS   Kelly CL, Liu Z, Yoshihara A, Jenkinson SF, Wormald MR, Otero JM, 
            Estevez AM, Kato A, Marqvorsen MH, Fleet GW, Estevez RJ, Izumori K, 
            Heap JT.
  TITLE     Synthetic chemical inducers and genetic decoupling enable orthogonal
            control of the rhaBAD promoter
  JOURNAL   ACS Synth Biol (2016) In press
  PUBMED    27247275
REFERENCE   2  (bases 1 to 5618)
  AUTHORS   Kelly CL, Liu Z, Yoshihara A, Jenkinson SF, Wormald MR, Otero J, 
            Estevez A, Kato A, Marqvorsen MHS., Fleet GWJ., Estevez RJ, Izumori 
            K, Heap JT.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-JAN-2016) Centre for Synthetic Biology and Innovation,
            Department of Life Sciences, Imperial College London, South 
            Kensington Campus, London SW7 2AZ, United Kingdom
REFERENCE   3  (bases 1 to 5618)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5618
                     /organism="Promoter vector pCK302"
                     /mol_type="other DNA"
                     /label=rhaS-expressing rhaBAD promoter
                     /note="rhaS-expressing rhaBAD promoter"
                     /db_xref="taxon:1857249"
     rep_origin      1..620
                     /label=pBR322 origin
                     /note="pBR322 origin"
     rep_origin      17..605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     875..891
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      908..1021
                     /regulatory_class="terminator"
                     /note="rpn txn term terminator"
     regulatory      1028..1328
                     /regulatory_class="terminator"
                     /note="bla txn term terminator"
     regulatory      1346..1467
                     /regulatory_class="promoter"
                     /note="rhaBAD promoter"
     CDS             1518..2270
                     /codon_start=1
                     /gene="sfGFP"
                     /product="sfGFP"
                     /label=sfGFP
                     /protein_id="ANJ04197.1"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYKGSGSGSHHHHHH"
     gene            1518..2270
                     /gene="sfGFP"
                     /label=sfGFP
     CDS             1518..2231
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2250..2267
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2300..2474
                     /regulatory_class="terminator"
                     /note="rrnB1 B2 T1 term terminator"
     promoter        2561..2652
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      2583..2611
                     /regulatory_class="promoter"
                     /note="Pamp promoter"
     CDS             2653..3513
                     /codon_start=1
                     /gene="ampR"
                     /product="ampR"
                     /label=ampR
                     /protein_id="ANJ04198.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            2653..3513
                     /gene="ampR"
                     /label=ampR
     regulatory      3525..3558
                     /regulatory_class="ribosome_binding_site"
                     /note="Translation initiation rate of 20000 using Salis RBS
                     calculator; MG1655 rhaS RBS"
     CDS             3559..4395
                     /codon_start=1
                     /gene="rhaS"
                     /product="rhaS"
                     /label=rhaS
                     /protein_id="ANJ04199.1"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     gene            3559..4395
                     /gene="rhaS"
                     /label=rhaS
     CDS             4467..5549
                     /codon_start=1
                     /gene="lacI"
                     /product="lacI"
                     /label=lacI
                     /protein_id="ANJ04200.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            4467..5549
                     /gene="lacI"
                     /label=lacI