LOCUS       40924_10766        6147 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pCK210, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6147)
  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 6147)
  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 6147)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6147)
  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..6147
                     /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"
     promoter        3090..3181
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      3112..3140
                     /label=ampR promoter
                     /note="ampR promoter"
                     /regulatory_class="promoter"
     CDS             3182..4039
                     /label=AmpR
                     /note="beta-lactamase"
     regulatory      4054..4087
                     /label=Ecoli rhaS RBS
                     /note="Ecoli rhaS RBS"
                     /regulatory_class="ribosome_binding_site"
     CDS             4088..4921
                     /label=rhaS
                     /note="positive regulator of the rhaB promoter"
     CDS             4996..6075
                     /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 ccctttccct atcagtgata gagattgaca tccctatcag
     1381 tgatagagat actgagcaca tcgataaaca cagagaagta ggttcagaat gtccagatta
     1441 gataaaagta aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt
     1501 ttaacaaccc gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat
     1561 gtaaaaaata agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat
     1621 actcactttt gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa
     1681 agttttagat gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg
     1741 cctacagaaa aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt
     1801 ttttcactag agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc
     1861 gtattggaag atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat
     1921 agtatgccgc cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca
     1981 gccttcttat tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa
     2041 agtgggtccc gtaaaggcga agagctgttc actggtgtcg tccctattct ggtggaactg
     2101 gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg tgacgcaact
     2161 aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt accttggccg
     2221 actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg
     2281 aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga acgcacgatt
     2341 tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc
     2401 ctggtaaacc gcattgagct gaaaggcatt gactttaaag aagacggcaa tatcctgggc
     2461 cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga taaacaaaaa
     2521 aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag cgtgcagctg
     2581 gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct gccagacaat
     2641 cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg cgatcatatg
     2701 gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga actgtacaaa
     2761 ggctccggct ccggctccca ccatcaccat caccattgat aactcgagcc ccaagggcga
     2821 caccccataa ttagcccggg cgaaaggccc agtctttcga ctgagccttt cgttttattt
     2881 gatgcctggc agttccctac tctcgcatgg ggagtcccca cactaccatc ggcgctacgg
     2941 cgtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc
     3001 aaacaagggg tgttatgagc catattcagg tataaatggg ctcgcgataa tgttcagaat
     3061 tggttaattg gttgtaacac tgacccctat ttgtttattt ttctaaatac attcaaatat
     3121 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagaa
     3181 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3241 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3301 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3361 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3421 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3481 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3541 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3601 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3661 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3721 gcctgtagcg atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3781 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3841 ctcggccctt ccggctggct ggtttattgc tgataaatcc ggagccggtg agcgtggttc
     3901 tcgcggtatc atcgcagcgc tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3961 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4021 ctcactgatt aagcattggt aagcggcgcg ccatatttcg ccgtgttgac gacatcagga
     4081 ggccagtatg accgtattac atagtgtgga tttttttccg tctggtaacg cgtccgtggc
     4141 gatagaaccc cggctcccgc aggcggattt tcctgaacat catcatgatt ttcatgaaat
     4201 tgtgattgtc gaacatggca cgggtattca tgtgtttaat gggcagccct ataccatcac
     4261 cggtggcacg gtctgtttcg tacgcgatca tgatcggcat ctgtatgaac ataccgataa
     4321 tctgtgtctg accaatgtgc tgtatcgctc gccggatcga tttcagtttc tcgccgggct
     4381 gaatcagttg ctgccacaag agctggatgg gcagtatccg tctcactggc gcgttaacca
     4441 cagcgtattg cagcaggtgc gacagctggt tgcacagatg gaacagcagg aaggggaaaa
     4501 tgatttaccc tcgaccgcca gtcgcgagat cttgtttatg caattactgc tcttgctgcg
     4561 taaaagcagt ttgcaggaga acctggaaaa cagcgcatca cgtctcaact tgcttctggc
     4621 ctggctggag gaccattttg ccgatgaggt gaattgggat gccgtggcgg atcaattttc
     4681 tctttcactg cgtacgctac atcggcagct taagcagcaa acgggactga cgcctcagcg
     4741 atacctgaac cgcctgcgac tgatgaaagc ccgacatctg ctacgccaca gcgaggccag
     4801 cgttactgac atcgcctatc gctgtggatt cagcgacagt aaccactttt cgacgctttt
     4861 tcgccgagag tttaactggt caccgcgtga tattcgccag ggacgggatg gctttctgca
     4921 ataatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     4981 ttcagggtgg tgaatatgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     5041 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     5101 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     5161 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     5221 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     5281 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     5341 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     5401 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     5461 atcaacagta ttattttctc ccatgaggac ggtacgcgac tgggcgtgga gcatctggtc
     5521 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     5581 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     5641 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     5701 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     5761 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
     5821 accgaagata gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     5881 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     5941 aatcagctgt tgccagtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     6001 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     6061 ctggaaagcg ggcagtgact catgaccaaa atcccttaac gtgagttacg cgcgcgtcgt
     6121 tccactgagc gtcagacccc gtagaaa
//
