pCK221 vector (V008505)

LOCUS       40924_10771        5412 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pCK221, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5412)
  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 5412)
  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, King's Road, 
            Newcastle NE1 7RU, United Kingdom
REFERENCE   3  (bases 1 to 5412)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5412)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. (2018) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (03-SEP-2018) School of Natural and Environmental Sciences, 
            Newcastle University, Agriculture Building, King's Road, Newcastle 
            NE1 7RU, United Kingdom"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5412
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..605
                     /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)
                     /label=rpn txn term terminator
                     /note="rpn txn term terminator"
                     /regulatory_class="terminator"
     regulatory      complement(1028..1328)
                     /label=bla txn term terminator
                     /note="bla txn term terminator"
                     /regulatory_class="terminator"
     protein_bind    1346..1364
                     /label=tet operator
                     /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
                     /label=Synthetic RBS
                     /note="Synthetic RBS"
                     /regulatory_class="ribosome_binding_site"
     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
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Pedelacq et al., 2006)"
     CDS             2779..2796
                     /label=6xHis
                     /note="6xHis affinity tag"
     regulatory      2829..3003
                     /label=rrnB1 B2 T1 term terminator
                     /note="rrnB1 B2 T1 term terminator"
                     /regulatory_class="terminator"
     terminator      complement(3046..3073)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(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"
     promoter        complement(3278..3395)
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     promoter        3445..3536
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      3467..3495
                     /label=ampR promoter
                     /note="ampR promoter"
                     /regulatory_class="promoter"
     CDS             3537..4394
                     /label=AmpR
                     /note="beta-lactamase"
     regulatory      4409..4442
                     /label=Ecoli rhaS RBS
                     /note="Ecoli rhaS RBS"
                     /regulatory_class="ribosome_binding_site"
     CDS             4443..5276
                     /label=rhaS
                     /note="positive regulator of the rhaB promoter"