LOCUS       Exported                7741 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pCK227, complete sequence.
ACCESSION   MH822884
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7741)
  AUTHORS   Kelly CL, Harris AW, Steel H, Hancock EJ, Heap JT, Papachristodoulou
            A.
  TITLE     Synthetic negative feedback circuits using engineered small RNAs
  JOURNAL   Nucleic Acids Res. (2018) In press
  PUBMED    30212900
REFERENCE   2  (bases 1 to 7741)
  AUTHORS   Kelly CL, Harris AWK., Steel H, Hancock EJ, Heap JT, 
            Papachristodoulou A.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-SEP-2018) School of Natural and Environmental 
            Sciences, Newcastle University, Agriculture Building, Kings Road, 
            Newcastle NE1 7RU, United Kingdom
REFERENCE   3  (bases 1 to 7741)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7741
                     /organism="Expression vector pCK227"
                     /mol_type="other DNA"
                     /db_xref="taxon:2339205"
     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      complement(908..1021)
                     /regulatory_class="terminator"
                     /note="rpn txn term terminator"
     regulatory      complement(1028..1328)
                     /regulatory_class="terminator"
                     /note="bla txn term terminator"
     CDS             complement(1346..2311)
                     /codon_start=1
                     /product="XylS"
                     /label=XylS
                     /protein_id="AYC07587.1"
                     /translation="MDFCLLNEKSQIFVHAEPYAVSDYVNQYVGTHSIRLPKGGRPAGR
                     LHHRIFGCLDLCRISYGGSVRVISPGLETCYHLQIILKGHCLWRGHGQEHYFAPGELLL
                     LNPDDQADLTYSEDCEKFIVKLPSVVLDRACSDNNWHKPREGIRFAARHNLQQLDGFIN
                     LLGLVCDEAEHTKSMPRVQEHYAGIIASKLLEMLGSNVSREIFSKGNPSFERVVQFIEE
                     NLKRNISLERLAELAMMSPRSLYNLFEKHAGTTPKNYIRNRKLESIRACLNDPSANVRS
                     ITEIALDYGFLHLGRFAENYRSAFGELPSDTLRQCKKEVA"
     regulatory      3211..3291
                     /regulatory_class="promoter"
                     /note="Pm promoter"
     promoter        3211..3291
                     /label=Pm promoter
                     /note="The bacterial Pm promoter is activated by XylS in 
                     the presence of benzoate or m-toluate (Marques et al., 
                     1999)."
     regulatory      3292..3325
                     /regulatory_class="ribosome_binding_site"
                     /note="Ecoli rhaS RBS"
     CDS             3326..4909
                     /codon_start=1
                     /product="RhaS/superfolderGFP fusion protein"
                     /label=RhaS/superfolderGFP fusion protein
                     /protein_id="AYC07588.1"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQRKG
                     EELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTT
                     LTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNR
                     IELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADH
                     YQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGS
                     GSGSHHHHHH"
     CDS             3326..4159
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter"
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     CDS             4160..4870
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="RKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             4889..4906
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      4939..5113
                     /regulatory_class="terminator"
                     /note="rrnB1 B2 T1 term terminator"
     regulatory      5150..5278
                     /regulatory_class="terminator"
                     /note="T1/TE"
     terminator      5156..5183
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      5199..5270
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    complement(5285..5363)
                     /label=micC Hfq binding scaffold
                     /note="micC Hfq binding scaffold"
     misc_feature    5364..5387
                     /label=sRNA rhaS from Start
                     /note="sRNA rhaS from Start"
     regulatory      complement(5388..5504)
                     /regulatory_class="promoter"
                     /note="rhaBAD promoter"
     promoter        5555..5646
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      5577..5605
                     /regulatory_class="promoter"
                     /note="ampR promoter"
     CDS             5647..6507
                     /codon_start=1
                     /product="AmpR"
                     /label=AmpR
                     /protein_id="AYC07589.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     CDS             6590..7672
                     /codon_start=1
                     /product="LacI"
                     /label=LacI
                     /protein_id="AYC07590.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"