LOCUS       Exported                7241 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pCK226, complete sequence.
ACCESSION   MH822876
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7241)
  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 7241)
  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 7241)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7241
                     /organism="Expression vector pCK226"
                     /mol_type="other DNA"
                     /db_xref="taxon:2339204"
     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"
     regulatory      1346..1400
                     /regulatory_class="promoter"
                     /note="tet promoter"
     misc_binding    1346..1364
                     /label=tetracycline repressor TetR binding site
                     /bound_moiety="tetracycline repressor TetR"
                     /note="operator 1"
     protein_bind    1346..1364
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_binding    1371..1389
                     /label=tetracycline repressor TetR binding site
                     /bound_moiety="tetracycline repressor TetR"
                     /note="operator 2"
     protein_bind    1371..1389
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      1401..1428
                     /regulatory_class="ribosome_binding_site"
                     /note="Synthetic RBS"
     CDS             1429..2799
                     /codon_start=1
                     /product="RNA-dependent RNA polymerase/superfolderGFP 
                     fusion protein"
                     /label=RNA-dependent RNA polymerase/superfolderGFP fus
                     /protein_id="AYC07572.1"
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSRKGEELFTGVVPILV
                     ELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYP
                     DHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDG
                     NILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPV
                     LLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSGSGSHHHHHH"
     CDS             1429..2046
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     CDS             2050..2760
                     /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             2779..2796
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2829..3003
                     /regulatory_class="terminator"
                     /note="rrnB1 B2 T1 term terminator"
     regulatory      3040..3168
                     /regulatory_class="terminator"
                     /note="T1/TE"
     terminator      3046..3073
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      3089..3160
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    complement(3175..3253)
                     /label=micC Hfq binding scaffold
                     /note="micC Hfq binding scaffold"
     misc_feature    complement(3254..3277)
                     /label=sRNA tetR from SD
                     /note="sRNA tetR from SD"
     regulatory      complement(3278..3358)
                     /regulatory_class="promoter"
                     /note="Pm promoter"
     promoter        complement(3278..3358)
                     /label=Pm promoter
                     /note="The bacterial Pm promoter is activated by XylS in 
                     the presence of benzoate or m-toluate (Marques et al., 
                     1999)."
     CDS             4258..5223
                     /codon_start=1
                     /product="XylS"
                     /label=XylS
                     /protein_id="AYC07573.1"
                     /translation="MDFCLLNEKSQIFVHAEPYAVSDYVNQYVGTHSIRLPKGGRPAGR
                     LHHRIFGCLDLCRISYGGSVRVISPGLETCYHLQIILKGHCLWRGHGQEHYFAPGELLL
                     LNPDDQADLTYSEDCEKFIVKLPSVVLDRACSDNNWHKPREGIRFAARHNLQQLDGFIN
                     LLGLVCDEAEHTKSMPRVQEHYAGIIASKLLEMLGSNVSREIFSKGNPSFERVVQFIEE
                     NLKRNISLERLAELAMMSPRSLYNLFEKHAGTTPKNYIRNRKLESIRACLNDPSANVRS
                     ITEIALDYGFLHLGRFAENYRSAFGELPSDTLRQCKKEVA"
     promoter        5274..5365
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      5296..5324
                     /regulatory_class="promoter"
                     /note="ampR promoter"
     CDS             5366..6226
                     /codon_start=1
                     /product="AmpR"
                     /label=AmpR
                     /protein_id="AYC07574.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     regulatory      6238..6271
                     /regulatory_class="ribosome_binding_site"
                     /note="Ecoli rhaS RBS"
     CDS             6272..7108
                     /codon_start=1
                     /product="RhaS"
                     /label=RhaS
                     /protein_id="AYC07575.1"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"