LOCUS       40924_10781        7241 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pCK226, complete sequence.
ACCESSION   .
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, King's Road, 
            Newcastle NE1 7RU, United Kingdom
REFERENCE   3  (bases 1 to 7241)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7241)
  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..7241
                     /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..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..5220
                     /label=XylS
                     /note="XylS regulator encoded by the Pseudomonas putida TOL
                     plasmid pWWO"
     promoter        5274..5365
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      5296..5324
                     /label=ampR promoter
                     /note="ampR promoter"
                     /regulatory_class="promoter"
     CDS             5366..6223
                     /label=AmpR
                     /note="beta-lactamase"
     regulatory      6238..6271
                     /label=Ecoli rhaS RBS
                     /note="Ecoli rhaS RBS"
                     /regulatory_class="ribosome_binding_site"
     CDS             6272..7105
                     /label=rhaS
                     /note="positive regulator of the rhaB promoter"
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 tataaatggt ataaacgcag aaaggcccac
     3061 ccgaaggtga gccagtgtga ctctagtaga gagcgttcac cgacaaacaa cagataaaac
     3121 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggct cgagaaaaaa
     3181 agcccggacg actgttcggg cttgtctttt tatatgttgg aaaatcagtg gcaatgcaat
     3241 ggcccaacag aaaagtaggt tcagaatgtc cagattattg cataaagcct aaggggtagg
     3301 ccttactaga gatagccatt ttttgcactc ctgtatccgc ttcttgcaag gctggactta
     3361 tccctatcaa accggacact gcactttatg ctggttatgc tggctgcatg gggcattagt
     3421 tggggagcca gatttgtcat ggagcaggcc gttctgcttt atggatcagg aaaaaactat
     3481 ttgttcttca gtcatggtac tgttctgatg tacctgctgt gtgttttcct ggtataccgc
     3541 cgttggatag ctccgctacc ggtcgttggt cagatcaagc taattcccat gcatggtgaa
     3601 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     3661 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag
     3721 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     3781 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     3841 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac gtaattacgg
     3901 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     3961 tttctttacg gtctttaaaa aggccgtaat atccagcaga tcccctttat ccgccaattc
     4021 gtcgccccat gccccgacag cacgaacttc tggtgttctc gcttcttaaa aagaacgtct
     4081 tcgttctgct tggcgttatt tttgcttgga aaagtggtca ctgattgcaa aaaggatggc
     4141 gcaacgtggc aatgggggta acccgtatac gcatcacgtc gagatgcatt ttcatcgact
     4201 tggcgccttt ctacatcaca ccaagcagcc cacattaaaa taagagaacc gtgaactatg
     4261 gatttttgct tattgaacga gaaaagtcag atcttcgtcc acgccgagcc ctatgcagtc
     4321 tccgattatg ttaaccagta tgtcggtacg cactctattc gcctgcccaa gggcgggcgc
     4381 ccggcaggca ggctgcacca cagaatcttc ggatgcctcg atctgtgtcg aatcagctac
     4441 ggcggtagcg tgagggtaat ctcgcctgga ttagagacct gttatcatct gcaaataata
     4501 ctcaaaggcc attgcctgtg gcgtggccat ggccaggagc actattttgc gccgggcgaa
     4561 ctattgctgc tcaatccgga tgaccaagcc gacctgacct attcagaaga ttgcgagaaa
     4621 tttatcgtta aattgccctc agtggtcctt gatcgggcgt gcagtgacaa caattggcac
     4681 aagccgaggg agggtatccg tttcgccgcg cgacacaatc tccagcaact cgatggcttt
     4741 atcaatctac tcgggttagt ttgtgacgaa gcggaacata caaagtcgat gcctcgggtc
     4801 caagagcact atgcggggat catcgcttcc aagctgctcg aaatgctggg cagcaatgtc
     4861 agccgtgaaa ttttcagcaa aggtaacccg tctttcgagc gagtcgttca attcattgag
     4921 gagaatctca aacggaatat cagccttgag cggttagcgg agctggcgat gatgagtcca
     4981 cgctcgctct acaatttgtt cgagaagcac gccggcacca cgccgaagaa ctacatccgc
     5041 aaccgcaagc tcgaaagcat ccgcgcctgc ttgaacgatc ccagtgccaa tgtgcgtagt
     5101 ataactgaga tagccctaga ctacggcttc ttacatttgg gacgcttcgc tgaaaactat
     5161 aggagcgcgt tcggcgagtt gccttccgac accctgcgtc aatgcaaaaa ggaagtggct
     5221 tgagctcgcg ataatgttca gaattggtta attggttgta acactgaccc ctatttgttt
     5281 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5341 tcaataatat tgaaaaagga agaatatgag tattcaacat ttccgtgtcg cccttattcc
     5401 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5461 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5521 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5581 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5641 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5701 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5761 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5821 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5881 aaacgacgag cgtgacacca cgatgcctgt agcgatggca acaacgttgc gcaaactatt
     5941 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6001 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6061 atccggagcc ggtgagcgtg gttctcgcgg tatcatcgca gcgctggggc cagatggtaa
     6121 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6181 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaagcgg cgcgccatat
     6241 ttcgccgtgt tgacgacatc aggaggccag tatgaccgta ttacatagtg tggatttttt
     6301 tccgtctggt aacgcgtccg tggcgataga accccggctc ccgcaggcgg attttcctga
     6361 acatcatcat gattttcatg aaattgtgat tgtcgaacat ggcacgggta ttcatgtgtt
     6421 taatgggcag ccctatacca tcaccggtgg cacggtctgt ttcgtacgcg atcatgatcg
     6481 gcatctgtat gaacataccg ataatctgtg tctgaccaat gtgctgtatc gctcgccgga
     6541 tcgatttcag tttctcgccg ggctgaatca gttgctgcca caagagctgg atgggcagta
     6601 tccgtctcac tggcgcgtta accacagcgt attgcagcag gtgcgacagc tggttgcaca
     6661 gatggaacag caggaagggg aaaatgattt accctcgacc gccagtcgcg agatcttgtt
     6721 tatgcaatta ctgctcttgc tgcgtaaaag cagtttgcag gagaacctgg aaaacagcgc
     6781 atcacgtctc aacttgcttc tggcctggct ggaggaccat tttgccgatg aggtgaattg
     6841 ggatgccgtg gcggatcaat tttctctttc actgcgtacg ctacatcggc agcttaagca
     6901 gcaaacggga ctgacgcctc agcgatacct gaaccgcctg cgactgatga aagcccgaca
     6961 tctgctacgc cacagcgagg ccagcgttac tgacatcgcc tatcgctgtg gattcagcga
     7021 cagtaaccac ttttcgacgc tttttcgccg agagtttaac tggtcaccgc gtgatattcg
     7081 ccagggacgg gatggctttc tgcaataatc gaatggcgca aaacctttcg cggtatggca
     7141 tgatagcgcc cggaagagag tcaattcagg gtggtgaatc caaaatccct taacgtgagt
     7201 tacgcgcgcg tcgttccact gagcgtcaga ccccgtagaa a
//
