LOCUS       40924_10776        5939 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pCK222, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5939)
  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 5939)
  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 5939)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5939)
  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..5939
                     /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"
     regulatory      1346..1489
                     /label=proD promoter
                     /note="proD promoter"
                     /regulatory_class="promoter"
     regulatory      1490..1523
                     /label=Ecoli rhaS RBS
                     /note="Ecoli rhaS RBS"
                     /regulatory_class="ribosome_binding_site"
     CDS             1524..2357
                     /label=rhaS
                     /note="positive regulator of the rhaB promoter"
     CDS             2358..3068
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Pedelacq et al., 2006)"
     CDS             3087..3104
                     /label=6xHis
                     /note="6xHis affinity tag"
     regulatory      3137..3311
                     /label=rrnB1 B2 T1 term terminator
                     /note="rrnB1 B2 T1 term terminator"
                     /regulatory_class="terminator"
     terminator      complement(3354..3381)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(3397..3468)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     misc_feature    complement(3483..3561)
                     /label=micC Hfq binding scaffold
                     /note="micC Hfq binding scaffold"
     misc_feature    3562..3585
                     /label=sRNA rhaS from Start
                     /note="sRNA rhaS from Start"
     promoter        complement(3586..3703)
                     /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        3753..3844
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      3775..3803
                     /label=ampR promoter
                     /note="ampR promoter"
                     /regulatory_class="promoter"
     CDS             3845..4702
                     /label=AmpR
                     /note="beta-lactamase"
     CDS             4788..5867
                     /label=lacI
                     /note="lac repressor"
ORIGIN
        1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
       61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      181 agttagccca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      601 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      661 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      721 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      781 cggaaggcga gagtagggaa ctgccaggca tcaaactaag cagaaggccc ctgacggatg
      841 gcctttttgc gtttctacaa actctttctg tgttgtaaaa cgacggccag tcttaagctc
      901 gggccccctg ggcggttctg ataacgagta atcgttaatc cgcaaataac gtaaaaaccc
      961 gcttcggcgg gtttttttat ggggggagtt tagggaaaga gcatttgtca gaatatttaa
     1021 gggcgcctgt cactttgctt gatatatgag aattatttaa ccttataaat gagaaaaaag
     1081 caacgcactt taaataagat acgttgcttt ttcgattgat gaacacctat aattaaacta
     1141 ttcatctatt atttatgatt ttttgtatat acaatatttc tagtttgtta aagagaatta
     1201 agaaaataaa tctcgaaaat aataaaggga aaatcagttt ttgatatcaa aattatacat
     1261 gtcaacgata atacaaaata taatacaaac tataagatgt tatcagtatt tattatgcat
     1321 ttagaataaa ttttgtgtcg cccttcacag ctaacaccac gtcgtcccta tctgctgccc
     1381 taggtctatg agtggttgct ggataacttt acgggcatgc ataaggctcg tataatatat
     1441 tcagggagac cacaacggtt tccctctaca aataattttg tttaactttt atttcgccgt
     1501 gttgacgaca tcaggaggcc agtatgaccg tattacatag tgtggatttt tttccgtctg
     1561 gtaacgcgtc cgtggcgata gaaccccggc tcccgcaggc ggattttcct gaacatcatc
     1621 atgattttca tgaaattgtg attgtcgaac atggcacggg tattcatgtg tttaatgggc
     1681 agccctatac catcaccggt ggcacggtct gtttcgtacg cgatcatgat cggcatctgt
     1741 atgaacatac cgataatctg tgtctgacca atgtgctgta tcgctcgccg gatcgatttc
     1801 agtttctcgc cgggctgaat cagttgctgc cacaagagct ggatgggcag tatccgtctc
     1861 actggcgcgt taaccacagc gtattgcagc aggtgcgaca gctggttgca cagatggaac
     1921 agcaggaagg ggaaaatgat ttaccctcga ccgccagtcg cgagatcttg tttatgcaat
     1981 tactgctctt gctgcgtaaa agcagtttgc aggagaacct ggaaaacagc gcatcacgtc
     2041 tcaacttgct tctggcctgg ctggaggacc attttgccga tgaggtgaat tgggatgccg
     2101 tggcggatca attttctctt tcactgcgta cgctacatcg gcagcttaag cagcaaacgg
     2161 gactgacgcc tcagcgatac ctgaaccgcc tgcgactgat gaaagcccga catctgctac
     2221 gccacagcga ggccagcgtt actgacatcg cctatcgctg tggattcagc gacagtaacc
     2281 acttttcgac gctttttcgc cgagagttta actggtcacc gcgtgatatt cgccagggac
     2341 gggatggctt tctgcaacgt aaaggcgaag agctgttcac tggtgtcgtc cctattctgg
     2401 tggaactgga tggtgatgtc aacggtcata agttttccgt gcgtggcgag ggtgaaggtg
     2461 acgcaactaa tggtaaactg acgctgaagt tcatctgtac tactggtaaa ctgccggtac
     2521 cttggccgac tctggtaacg acgctgactt atggtgttca gtgctttgct cgttatccgg
     2581 accatatgaa gcagcatgac ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac
     2641 gcacgatttc ctttaaggat gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag
     2701 gcgataccct ggtaaaccgc attgagctga aaggcattga ctttaaagaa gacggcaata
     2761 tcctgggcca taagctggaa tacaatttta acagccacaa tgtttacatc accgccgata
     2821 aacaaaaaaa tggcattaaa gcgaatttta aaattcgcca caacgtggag gatggcagcg
     2881 tgcagctggc tgatcactac cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc
     2941 cagacaatca ctatctgagc acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg
     3001 atcatatggt tctgctggag ttcgtaaccg cagcgggcat cacgcatggt atggatgaac
     3061 tgtacaaagg ctccggctcc ggctcccacc atcaccatca ccattgataa ctcgagcccc
     3121 aagggcgaca ccccataatt agcccgggcg aaaggcccag tctttcgact gagcctttcg
     3181 ttttatttga tgcctggcag ttccctactc tcgcatgggg agtccccaca ctaccatcgg
     3241 cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc gcgctactgc
     3301 cgccaggcaa acaaggggtg ttatgagcca tattcaggta taaatggtat aaacgcagaa
     3361 aggcccaccc gaaggtgagc cagtgtgact ctagtagaga gcgttcaccg acaaacaaca
     3421 gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctggctcg
     3481 agaaaaaaag cccggacgac tgttcgggct tgtcttttta tatgttggaa aatcagtggc
     3541 aatgcaatgg cccaacagaa atgttgacga catcaggagg ccagttacga ccagtctaaa
     3601 aagcgcctga atacgcgacc ttctcgttac tgacaggaaa atgggccatt ggcaaccagg
     3661 gaaagatgaa cgtgatgatg ttcacaattt gctgaattgt gggctcgcga taatgttcag
     3721 aattggttaa ttggttgtaa cactgacccc tatttgttta tttttctaaa tacattcaaa
     3781 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3841 gaatatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3901 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3961 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4021 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4081 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4141 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4201 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4261 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4321 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4381 gatgcctgta gcgatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4441 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4501 gcgctcggcc cttccggctg gctggtttat tgctgataaa tccggagccg gtgagcgtgg
     4561 ttctcgcggt atcatcgcag cgctggggcc agatggtaag ccctcccgta tcgtagttat
     4621 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4681 tgcctcactg attaagcatt ggtaagcggc gcgccatcga atggcgcaaa acctttcgcg
     4741 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatatg aaaccagtaa
     4801 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     4861 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     4921 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     4981 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     5041 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     5101 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     5161 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     5221 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgagg
     5281 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     5341 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     5401 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     5461 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     5521 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     5581 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga tagctcatgt tatatcccgc
     5641 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     5701 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgccagtc tcactggtga
     5761 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     5821 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga ctcatgacca
     5881 aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaa
//
