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"
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 cagattatac gaccagtcta aaaagcgcct
     3301 gaatacgcga ccttctcgtt actgacagga aaatgggcca ttggcaacca gggaaagatg
     3361 aacgtgatga tgttcacaat ttgctgaatt gtgggctcgc gataatgttc agaattggtt
     3421 aattggttgt aacactgacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3481 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagaatatga
     3541 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3601 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3661 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3721 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     3781 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3841 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3901 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3961 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4021 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4081 tagcgatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4141 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4201 cccttccggc tggctggttt attgctgata aatccggagc cggtgagcgt ggttctcgcg
     4261 gtatcatcgc agcgctgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4321 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4381 tgattaagca ttggtaagcg gcgcgccata tttcgccgtg ttgacgacat caggaggcca
     4441 gtatgaccgt attacatagt gtggattttt ttccgtctgg taacgcgtcc gtggcgatag
     4501 aaccccggct cccgcaggcg gattttcctg aacatcatca tgattttcat gaaattgtga
     4561 ttgtcgaaca tggcacgggt attcatgtgt ttaatgggca gccctatacc atcaccggtg
     4621 gcacggtctg tttcgtacgc gatcatgatc ggcatctgta tgaacatacc gataatctgt
     4681 gtctgaccaa tgtgctgtat cgctcgccgg atcgatttca gtttctcgcc gggctgaatc
     4741 agttgctgcc acaagagctg gatgggcagt atccgtctca ctggcgcgtt aaccacagcg
     4801 tattgcagca ggtgcgacag ctggttgcac agatggaaca gcaggaaggg gaaaatgatt
     4861 taccctcgac cgccagtcgc gagatcttgt ttatgcaatt actgctcttg ctgcgtaaaa
     4921 gcagtttgca ggagaacctg gaaaacagcg catcacgtct caacttgctt ctggcctggc
     4981 tggaggacca ttttgccgat gaggtgaatt gggatgccgt ggcggatcaa ttttctcttt
     5041 cactgcgtac gctacatcgg cagcttaagc agcaaacggg actgacgcct cagcgatacc
     5101 tgaaccgcct gcgactgatg aaagcccgac atctgctacg ccacagcgag gccagcgtta
     5161 ctgacatcgc ctatcgctgt ggattcagcg acagtaacca cttttcgacg ctttttcgcc
     5221 gagagtttaa ctggtcaccg cgtgatattc gccagggacg ggatggcttt ctgcaataat
     5281 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     5341 ggtggtgaat ccaaaatccc ttaacgtgag ttacgcgcgc gtcgttccac tgagcgtcag
     5401 accccgtaga aa
//
