LOCUS       V003400                10906 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V003400
VERSION     V003400
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 10906)
  AUTHORS   Zhao C, Sun B.
  TITLE     Construction of a gene knockdown system based on dCas9 in
            Staphylococcus aureus
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 10906)
  AUTHORS   Zhao C, Sun B.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-AUG-2016) School of Life Sciences, University of
            Science and Technology of China, No.443, Huangshan Road, Hefei,
            Anhui 230027, China
REFERENCE   3  (bases 1 to 10906)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 10906)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName:
            "Unpublished"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (10-AUG-2016) School of Life Sciences, University of Science and
            Technology of China, No.443, Huangshan Road, Hefei, Anhui 230027,
            China"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     379..395
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     misc_RNA        complement(408..483)
                     /label="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     misc_feature    complement(481..510)
                     /label="SapI box"
                     /note="SapI box"
     regulatory      complement(508..692)
                     /note="sgRNA; constitutive expression"
                     /regulatory_class="promoter"
     terminator      complement(705..732)
                     /label="T7Te terminator"
                     /note="phage T7 early transcription terminator"
     terminator      complement(748..819)
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(838..4941)
                     /label="dCas9"
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     regulatory      complement(4949..4953)
                     /label="dcas9"
                     /note="dcas9"
                     /regulatory_class="ribosome_binding_site"
     protein_bind    complement(4993..5011)
                     /label="tet operator"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             5118..5741
                     /label="TetR"
                     /note="tetracycline repressor TetR"
     CDS             complement(5985..5999)
                     /label="enterokinase site"
                     /note="enterokinase recognition and cleavage site"
     CDS             7492..8139
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     primer_bind     complement(8685..8701)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    8709..8725
                     /label="lac operator"
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(8733..8763)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(8778..8799)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(9087..9675)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(9849..10706)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(10707..10811)
                     /label="AmpR promoter"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt caaaaaagca ccgactcggt
      421 gccacttttt caagttgata acggactagc cttattttaa cttgctattt ctagctctaa
      481 aacagaagag cctgacatgg ctcttcatag tcattgtagc atgtttgttg tgattttttt
      541 cacataaatt atgtacaatt ttcatcaaat gtatgtcaaa acatagaaac tgtactaatt
      601 tgtgtaacag taatatgcta attgcattct gttacacaac agttataatg ttcactattt
      661 tcattgcttt aaaagttaat ttaggagtat atcccgggta taaacgcaga aaggcccacc
      721 cgaaggtgag ccagtgtgac tctagtagag agcgttcacc gacaaacaac agataaaacg
      781 aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctggctc tagttcagtc
      841 acctcctagc tgactcaaat caatgcgtgt ttcataaaga ccagtgatgg attgatggat
      901 aagagtggca tctaaaactt cttttgtaga cgtatatcgt ttacgatcaa ttgttgtatc
      961 aaaatattta aaagcagcgg gagctccaag attcgtcaac gtaaataaat gaataatatt
     1021 ttctgcttgt tcacgtattg gtttgtctct atgtttgtta tatgcactaa gaactttatc
     1081 taaattggca tctgctaaaa taacacgctt agaaaattca ctgatttgct caataatctc
     1141 atctaaataa tgcttatgct gctccacaaa caattgtttt tgttcgttat cttctggact
     1201 acccttcaac ttttcataat gactagctaa atataaaaaa ttcacatatt tgcttggcag
     1261 agccagctca tttccttttt gtaattctcc ggcactagcc agcatccgtt tacgaccgtt
     1321 ttctaactca aaaagactat atttaggtag tttaatgatt aagtcttttt taacttcctt
     1381 atatccttta gcttctaaaa agtcaatcgg atttttttca aaggaacttc tttccataat
     1441 tgtgatccct agtaactctt taacggattt taacttcttc gatttccctt tttccacctt
     1501 agcaaccact aggactgaat aagctaccgt tggactatca aaaccaccat atttttttgg
     1561 atcccagtct tttttacgag caataagctt gtccgaattt ctttttggta aaattgactc
     1621 cttggagaat ccgcctgtct gtacttctgt tttcttgaca atattgactt ggggcatgga
     1681 caatactttg cgcactgtgg caaaatctcg ccctttatcc cagacaattt ctccagtttc
     1741 cccattagtt tcgattagag ggcgtttgcg aatctctcca tttgcaagtg taatttctgt
     1801 tttgaagaag ttcatgatat tagagtaaaa gaaatatttt gcggttgctt tgcctatttc
     1861 ttgctcagac ttagcaatca ttttacgaac atcataaact ttataatcac catagacaaa
     1921 ctccgattca agttttggat atttcttaat caaagcagtt ccaacgacgg catttagata
     1981 cgcatcatgg gcatgatggt aattgttaat ctcacgtact ttatagaatt ggaaatcttt
     2041 tcggaagtca gaaactaatt tagattttaa ggtaatcact ttaacctctc gaataagttt
     2101 atcattttca tcgtatttag tattcatgcg actatccaaa atttgtgcca catgcttagt
     2161 gatttggcga gtttcaacca attggcgttt gataaaacca gctttatcaa gttcactcaa
     2221 acctccacgt tcagctttcg ttaaattatc aaacttacgt tgagtgatta acttggcgtt
     2281 tagaagttgt ctccaatagt ttttcatctt tttgactact tcttcacttg gaacgttatc
     2341 cgatttacca cgatttttat cagaacgcgt taagacctta ttgtctattg aatcgtcttt
     2401 aaggaaactt tgtggaacaa tggcatcgac atcataatca cttaaacgat taatatctaa
     2461 ttcttggtcc acatacatgt ctcttccatt ttggagataa tagagataga gcttttcatt
     2521 ttgcaattga gtattttcaa caggatgctc tttaagaatc tgacttccta attctttgat
     2581 accttcttcg attcgtttca tacgctctcg cgaatttttc tggccctttt gagttgtctg
     2641 attttcacgt gccatttcaa taacgatatt ttctggctta tgccgcccca ttactttgac
     2701 caattcatca acaactttta cagtctgtaa aatacctttt ttaatagcag ggctaccagc
     2761 taaatttgca atatgttcat gtaaactatc gccttgtcca gacacttgtg ctttttgaat
     2821 gtcttcttta aatgtcaaac tatcatcatg gatcagctgc ataaaattgc gattggcaaa
     2881 accatctgat ttcaaaaaat ctaatattgt tttgccagat tgcttatccc taataccatt
     2941 aatcaatttt cgagacaaac gtccccaacc agtataacgg cgacgtttaa gctgtttcat
     3001 caccttatca tcaaagaggt gagcatatgt tttaagtctt tcctcaatca tctccctatc
     3061 ttcaaataag gtcaatgtta aaacaatatc ctctaagata tcttcatttt cttcattatc
     3121 caaaaaatct ttatctttaa taatttttag caaatcatgg taggtaccta atgaagcatt
     3181 aaatctatct tcaactcctg aaatttcaac actatcaaaa cattctattt ttttgaaata
     3241 atcttctttt aattgcttaa cggttacttt tcgatttgtt ttgaagagta aatcaacaat
     3301 ggctttcttc tgttcacctg aaagaaatgc tggttttcgc attccttcag taacatattt
     3361 gacctttgtc aattcgttat aaaccgtaaa atactcataa agcaaactat gttttggtag
     3421 tactttttca tttggaagat ttttatcaaa gtttgtcatg cgttcaataa atgattgagc
     3481 tgaagcacct ttatcgacaa cttcttcaaa attccatggg gtaattgttt cttcagactt
     3541 ccgagtcatc catgcaaaac gactattgcc acgcgccaat ggaccaacat aataagggat
     3601 tcgaaaagtc aagatttttt caatcttctc acgattgtct tttaaaaatg gataaaagtc
     3661 ttcttgtctt ctcaaaatag catgcagctc acccaagtga atttgatggg gaatagagcc
     3721 gttgtcaaag gtccgttgct tgcgcagcaa atcttcacga tttagtttca ccaataattc
     3781 ctcagtacca tccatttttt ctaaaattgg tttgataaat ttataaaatt cttcttggct
     3841 agctccccca tcaatataac ctgcatatcc gttttttgat tgatcaaaaa agatttcttt
     3901 atacttttct ggaagttgtt gtcgaactaa agcttttaaa agagtcaagt cttgatgatg
     3961 ttcatcgtag cgtttaatca ttgaagctga taggggagcc ttagttattt cagtatttac
     4021 tcttaggata tctgaaagta aaatagcatc tgataaattc ttagctgcca aaaacaaatc
     4081 agcatattga tctccaattt gcgccaataa attatctaaa tcatcatcgt aagtatcttt
     4141 tgaaagctgt aatttagcat cttctgccaa atcaaaattt gatttaaaat taggggtcaa
     4201 acccaatgac aaagcaatga gattcccaaa taagccattt ttcttctcac cggggagctg
     4261 agcaatgaga ttttctaatc gtcttgattt actcaatcgt gcagaaagaa tcgctttagc
     4321 atctactcca cttgcgttaa tagggttttc ttcaaataat tgattgtagg tttgtaccaa
     4381 ctggataaat agtttgtcca catcactatt atcaggattt aaatctccct caatcaaaaa
     4441 atgaccacga aacttaatca tatgcgctaa ggccaaatag attaagcgca aatccgcttt
     4501 atcagtagaa tctaccaatt tttttcgcag atgatagata gttggatatt tctcatgata
     4561 agcaacttca tctactatat ttccaaaaat aggatgacgt tcatgcttct tgtcttcttc
     4621 caccaaaaaa gactcttcaa gtcgatgaaa gaaactatca tctactttcg ccatctcatt
     4681 tgaaaaaatc tcctgtagat aacaaatacg attcttccga cgtgtatacc ttctacgagc
     4741 tgtccgtttg agacgagtcg cttccgctgt ctctccactg tcaaataaaa gagcccctat
     4801 aagatttttt ttgatactgt ggcggtctgt atttcccaga accttgaact ttttagacgg
     4861 aaccttatat tcatcagtga tcaccgccca tccgacgcta tttgtgccga tagctaagcc
     4921 tattgagtat ttcttatcca tgaaacggcc tcccagatct gttaacggta ccatcaagct
     4981 tattttaatt atactctatc aatgatagag tgtcaatatt ttttttagtt tttcatgaac
     5041 tcgaggggat ccaaataaaa aactagtttg acaaataact ctatcaatga tataatgtca
     5101 acaaaaagga ggaattaatg atgtctagat tagataaaag taaagtgatt aacagcgcat
     5161 tagagctgct taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc
     5221 taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg
     5281 ccttagccat tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa
     5341 gctggcaaga ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc
     5401 gcgatggagc aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg
     5461 aaaatcaatt agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac
     5521 tcagcgctgt ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg
     5581 ctaaagaaga aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta
     5641 tcgaattatt tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca
     5701 tatgcggatt agaaaaacaa cttaaatgtg aaagtgggtc ttaactgcag gcatgcaagc
     5761 ttttaaaaag caaatatgag ccaaataaat atattctaat tctacaaaca aaaatttgag
     5821 caaattcagt gtcgattttt taagacactg cccagttaca tgcaaattaa aattttcatg
     5881 attttttata gttcctaaca gggttaaaat ttgtataacg aaagtataat gtttatataa
     5941 cgttagtata ataaagcatt ttaacattat acttttgata atcgtttatc gtcgtcatca
     6001 caataacttt taaaatactc gtgcataatt cacgctgacc tcccaataac tacatggtgt
     6061 tatcgggagg tcagctgtta gcacttatat tttgttattg ttcttcctcg atttcgtcta
     6121 tcattttgtg attaatttct cttttttctt gttctgttaa gtcataaagt tcactagcta
     6181 aatactcttt ttgtttccaa atataaaaaa tttgatagat atattacggt tggatcaatt
     6241 tcttttaagt aatctaaatc cccatttttt aatttctttt tagcctcttt aaataatcct
     6301 gaataaacta atacctgttt acctttaagt gatttataaa atgcatcaaa gactttttga
     6361 tttatttatt aaataatcac tatctttacc agaatactta gccatttcat ataattcttt
     6421 attattattt tgtcttattt tttgaacttg aacttgtgtt atttctgaaa tgcccgttac
     6481 atcacgccat aaatctaacc attcttgttg gctaatataa tatcttttat ctgtgaaata
     6541 cgatttattt actgcaatta acacatgaaa atgaggatta taatcatctc tttttttatt
     6601 atatgtaatc tctaacttac gaacatatcc ctttataaca ctacctactt tttttctctt
     6661 tataagtttt ctaaaagaat tattataacg ttttatttca ttttctaatt catcactcat
     6721 tacattaggt gtagtcaaag ttaaaaagat aaactccttt ttctcttgct gcttaatata
     6781 ttgcatcatc aaagataaac ccaatgcatc ttttctagct tttctccaag cacagacagg
     6841 acaaaatcga tttttacaag aattagcttt atataatttc tgtttttcta aagttttatc
     6901 agctacaaaa gacagaaatg tattgcaatc ttcaactaaa tccatttgat tctctccaat
     6961 atgacgttta ataaatttct gaaatacttg atttctttgt tttttctcag tatacttttc
     7021 catgttataa cacataaaaa caacttagtt ttcacaaact atgacaataa aaaaagttgc
     7081 tttttcccct ttctatgtat gttttttact agtcatttaa aacgatacat taataggtac
     7141 gaaaaagcaa ctttttttgc gcttaaaacc agtcatacca ataacttaag ggtaactagc
     7201 ctcgccggca atagttaccc ttattatcaa gataagaaag aaaaggattt ttcgctacgc
     7261 tcaaatcctt taaaaaaaca caaaagacca cattttttaa tgtggtcttt attcttcaac
     7321 taaagcaccc attagttcaa caaacgaaaa ttggataaag tgggatattt ttaaaatata
     7381 tatttatgtt acagtaatat tgacttttaa aaaaggattg attctaatga agaaagcaga
     7441 caagtaagcc tcctaaattc actttagata aaaatttagg aggcatatca aatgaacttt
     7501 aataaaattg atttagacaa ttggaagaga aaagagatat ttaatcatta tttgaaccaa
     7561 caaacgactt ttagtataac cacagaaatt gatattagtg ttttataccg aaacataaaa
     7621 caagaaggat ataaatttta ccctgcattt attttcttag tgacaagggt gataaactca
     7681 aatacagctt ttagaactgg ttacaatagc gacggagagt taggttattg ggataagtta
     7741 gagccacttt atacaatttt tgatggtgta tctaaaacat tctctggtat ttggactcct
     7801 gtaaagaatg acttcaaaga gttttatgat ttataccttt ctgatgtaga gaaatataat
     7861 ggttcgggga aattgtttcc caaaacacct atacctgaaa atgctttttc tctttctatt
     7921 attccatgga cttcatttac tgggtttaac ttaaatatca ataataatag taattacctt
     7981 ctacccatta ttacagcagg aaaattcatt aataaaggta attcaatata tttaccgcta
     8041 tctttacagg tacatcattc tgtttgtgat ggttatcatg caggattgtt tatgaactct
     8101 attcaggaat tgtcagatag gcctaatgac tggcttttat aatatgagat aatgccgact
     8161 gtacttttta cagtcggttt tctaatgtca ctaacctgcc ccgttagttg aagaaggttt
     8221 ttatattaca gctccagatc catatccttc tttttctgaa ccgacttctc ctttttcgct
     8281 tctttattcc aattgcttta ttgacgttga gcctcggaac ccttaacaat cccaaaactt
     8341 gtcgaatggt cggcttaata gctcacgcta tgccgacatt cgtctgcaag tttagttaag
     8401 ggttcttctc aacgcacaat aaattttctc ggcataaatg cgtggtctaa tttttatttt
     8461 taataacctt gatagcaaaa aatgccattc caatacaaaa ccacatacct ataatcgata
     8521 accacataac agtcataaaa ccactccttt ttaacaaact ttatcacaag aaatatttaa
     8581 attttaaatg cctttatttt gaattttaag gggcatttta aagatttagg ggtaaatcat
     8641 atagttttat gcctaaaaac ctacagaagc ttggcgtaat catggtcata gctgtttcct
     8701 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     8761 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     8821 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     8881 agaggcggtt tgcgtattgg gcgctctacc gcttcctcgc tcactgactc gctgcgctcg
     8941 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     9001 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     9061 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     9121 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     9181 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     9241 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     9301 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     9361 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     9421 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     9481 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     9541 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     9601 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     9661 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     9721 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     9781 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     9841 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     9901 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     9961 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
    10021 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
    10081 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
    10141 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
    10201 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
    10261 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
    10321 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
    10381 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
    10441 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
    10501 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
    10561 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
    10621 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
    10681 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
    10741 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
    10801 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
    10861 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
