LOCUS       40924_10786        7741 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pCK227, complete sequence.
ACCESSION   .
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, King's Road, 
            Newcastle NE1 7RU, United Kingdom
REFERENCE   3  (bases 1 to 7741)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7741)
  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..7741
                     /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"
     CDS             complement(1349..2311)
                     /label=XylS
                     /note="XylS regulator encoded by the Pseudomonas putida TOL
                     plasmid pWWO"
     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
                     /label=Ecoli rhaS RBS
                     /note="Ecoli rhaS RBS"
                     /regulatory_class="ribosome_binding_site"
     CDS             3326..4159
                     /label=rhaS
                     /note="positive regulator of the rhaB promoter"
     CDS             4160..4870
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Pedelacq et al., 2006)"
     CDS             4889..4906
                     /label=6xHis
                     /note="6xHis affinity tag"
     regulatory      4939..5113
                     /label=rrnB1 B2 T1 term terminator
                     /note="rrnB1 B2 T1 term terminator"
                     /regulatory_class="terminator"
     terminator      complement(5156..5183)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(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"
     promoter        complement(5388..5505)
                     /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        5555..5646
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      5577..5605
                     /label=ampR promoter
                     /note="ampR promoter"
                     /regulatory_class="promoter"
     CDS             5647..6504
                     /label=AmpR
                     /note="beta-lactamase"
     CDS             6590..7669
                     /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 ccctttcaag ccacttcctt tttgcattga cgcagggtgt
     1381 cggaaggcaa ctcgccgaac gcgctcctat agttttcagc gaagcgtccc aaatgtaaga
     1441 agccgtagtc tagggctatc tcagttatac tacgcacatt ggcactggga tcgttcaagc
     1501 aggcgcggat gctttcgagc ttgcggttgc ggatgtagtt cttcggcgtg gtgccggcgt
     1561 gcttctcgaa caaattgtag agcgagcgtg gactcatcat cgccagctcc gctaaccgct
     1621 caaggctgat attccgtttg agattctcct caatgaattg aacgactcgc tcgaaagacg
     1681 ggttaccttt gctgaaaatt tcacggctga cattgctgcc cagcatttcg agcagcttgg
     1741 aagcgatgat ccccgcatag tgctcttgga cccgaggcat cgactttgta tgttccgctt
     1801 cgtcacaaac taacccgagt agattgataa agccatcgag ttgctggaga ttgtgtcgcg
     1861 cggcgaaacg gataccctcc ctcggcttgt gccaattgtt gtcactgcac gcccgatcaa
     1921 ggaccactga gggcaattta acgataaatt tctcgcaatc ttctgaatag gtcaggtcgg
     1981 cttggtcatc cggattgagc agcaatagtt cgcccggcgc aaaatagtgc tcctggccat
     2041 ggccacgcca caggcaatgg cctttgagta ttatttgcag atgataacag gtctctaatc
     2101 caggcgagat taccctcacg ctaccgccgt agctgattcg acacagatcg aggcatccga
     2161 agattctgtg gtgcagcctg cctgccgggc gcccgccctt gggcaggcga atagagtgcg
     2221 taccgacata ctggttaaca taatcggaga ctgcataggg ctcggcgtgg acgaagatct
     2281 gacttttctc gttcaataag caaaaatcca tagttcacgg ttctcttatt ttaatgtggg
     2341 ctgcttggtg tgatgtagaa aggcgccaag tcgatgaaaa tgcatctcga cgtgatgcgt
     2401 atacgggtta cccccattgc cacgttgcgc catccttttt gcaatcagtg accacttttc
     2461 caagcaaaaa taacgccaag cagaacgaag acgttctttt taagaagcga gaacaccaga
     2521 agttcgtgct gtcggggcat ggggcgacga attggcggat aaaggggatc tgctggatat
     2581 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
     2641 cattcttgcc cgcctgatga atgctcatcc gtaattacgt atggcaatga aagacggtga
     2701 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
     2761 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
     2821 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
     2881 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
     2941 caatatggac aacttcttcg cccccgtttt caccatgcat gggaattagc ttgatctgac
     3001 caacgaccgg tagcggagct atccaacggc ggtataccag gaaaacacac agcaggtaca
     3061 tcagaacagt accatgactg aagaacaaat agttttttcc tgatccataa agcagaacgg
     3121 cctgctccat gacaaatctg gctccccaac taatgcccca tgcagccagc ataaccagca
     3181 taaagtgcag tgtccggttt gatagggata agtccagcct tgcaagaagc ggatacagga
     3241 gtgcaaaaaa tggctatctc tagtaaggcc taccccttag gctttatgca atatttcgcc
     3301 gtgttgacga catcaggagg ccagtatgac cgtattacat agtgtggatt tttttccgtc
     3361 tggtaacgcg tccgtggcga tagaaccccg gctcccgcag gcggattttc ctgaacatca
     3421 tcatgatttt catgaaattg tgattgtcga acatggcacg ggtattcatg tgtttaatgg
     3481 gcagccctat accatcaccg gtggcacggt ctgtttcgta cgcgatcatg atcggcatct
     3541 gtatgaacat accgataatc tgtgtctgac caatgtgctg tatcgctcgc cggatcgatt
     3601 tcagtttctc gccgggctga atcagttgct gccacaagag ctggatgggc agtatccgtc
     3661 tcactggcgc gttaaccaca gcgtattgca gcaggtgcga cagctggttg cacagatgga
     3721 acagcaggaa ggggaaaatg atttaccctc gaccgccagt cgcgagatct tgtttatgca
     3781 attactgctc ttgctgcgta aaagcagttt gcaggagaac ctggaaaaca gcgcatcacg
     3841 tctcaacttg cttctggcct ggctggagga ccattttgcc gatgaggtga attgggatgc
     3901 cgtggcggat caattttctc tttcactgcg tacgctacat cggcagctta agcagcaaac
     3961 gggactgacg cctcagcgat acctgaaccg cctgcgactg atgaaagccc gacatctgct
     4021 acgccacagc gaggccagcg ttactgacat cgcctatcgc tgtggattca gcgacagtaa
     4081 ccacttttcg acgctttttc gccgagagtt taactggtca ccgcgtgata ttcgccaggg
     4141 acgggatggc tttctgcaac gtaaaggcga agagctgttc actggtgtcg tccctattct
     4201 ggtggaactg gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg
     4261 tgacgcaact aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt
     4321 accttggccg actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc
     4381 ggaccatatg aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga
     4441 acgcacgatt tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga
     4501 aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag aagacggcaa
     4561 tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga
     4621 taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag
     4681 cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct
     4741 gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg
     4801 cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga
     4861 actgtacaaa ggctccggct ccggctccca ccatcaccat caccattgat aactcgagcc
     4921 ccaagggcga caccccataa ttagcccggg cgaaaggccc agtctttcga ctgagccttt
     4981 cgttttattt gatgcctggc agttccctac tctcgcatgg ggagtcccca cactaccatc
     5041 ggcgctacgg cgtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact
     5101 gccgccaggc aaacaagggg tgttatgagc catattcagg tataaatggt ataaacgcag
     5161 aaaggcccac ccgaaggtga gccagtgtga ctctagtaga gagcgttcac cgacaaacaa
     5221 cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggct
     5281 cgagaaaaaa agcccggacg actgttcggg cttgtctttt tatatgttgg aaaatcagtg
     5341 gcaatgcaat ggcccaacag aaatgttgac gacatcagga ggccagttac gaccagtcta
     5401 aaaagcgcct gaatacgcga ccttctcgtt actgacagga aaatgggcca ttggcaacca
     5461 gggaaagatg aacgtgatga tgttcacaat ttgctgaatt gtgggctcgc gataatgttc
     5521 agaattggtt aattggttgt aacactgacc cctatttgtt tatttttcta aatacattca
     5581 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5641 aagaatatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5701 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5761 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5821 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5881 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5941 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     6001 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6061 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6121 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6181 acgatgcctg tagcgatggc aacaacgttg cgcaaactat taactggcga actacttact
     6241 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6301 ctgcgctcgg cccttccggc tggctggttt attgctgata aatccggagc cggtgagcgt
     6361 ggttctcgcg gtatcatcgc agcgctgggg ccagatggta agccctcccg tatcgtagtt
     6421 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6481 ggtgcctcac tgattaagca ttggtaagcg gcgcgccatc gaatggcgca aaacctttcg
     6541 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaata tgaaaccagt
     6601 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     6661 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     6721 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     6781 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     6841 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     6901 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     6961 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     7021 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     7081 ggacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     7141 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     7201 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     7261 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     7321 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     7381 tggtgcggat atctcggtag tgggatacga cgataccgaa gatagctcat gttatatccc
     7441 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     7501 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgccag tctcactggt
     7561 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     7621 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gactcatgac
     7681 caaaatccct taacgtgagt tacgcgcgcg tcgttccact gagcgtcaga ccccgtagaa
     7741 a
//
