LOCUS       40924_46658        7692 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pwtcas9, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7692)
  AUTHORS   Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J, 
            Turczyk BM, Tung M, Collins JJ, Church GM.
  TITLE     Precise Cas9 targeting enables genomic mutation prevention
  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. (2018) In press
  PUBMED    29555762
REFERENCE   2  (bases 1 to 7692)
  AUTHORS   Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J, 
            Turczyk BM, Tung M, Collins JJ, Church GM.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-MAR-2018) Synthetic Biology, Wyss Institute, 3 
            Blackfan Circle, Floor 5, Boston, MA 02115, USA
REFERENCE   3  (bases 1 to 7692)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7692)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc. Natl.
            Acad. Sci. U.S.A. (2018) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-MAR-2018) Synthetic Biology, Wyss Institute, 3 Blackfan Circle, 
            Floor 5, Boston, MA 02115, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(157..780)
                     /label=TetR
                     /note="tetracycline repressor TetR"
     promoter        796..851
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     RBS             869..880
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             887..4990
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     misc_feature    4991..5020
                     /label=SGS linker
                     /note="SGS linker"
     CDS             5021..5053
                     /label=ssrA tag
                     /note="C-terminal peptide that mediates degradation in
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
     misc_feature    5054..5059
                     /label=double stop
                     /note="double stop"
     terminator      5083..5154
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      5170..5197
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        5437..5515
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     regulatory      complement(5553..5594)
                     /note="derived from Streptococcus pyogenes; tracrRNA
                     terminator"
                     /regulatory_class="terminator"
     regulatory      5651..5715
                     /label=J23100 promoter
                     /note="J23100 promoter"
                     /regulatory_class="promoter"
     repeat_region   5731..5766
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes
                     CRISPR/Cas system"
     rep_origin      5948..6686
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     promoter        6734..6825
                     /label=AmpR promoter
     CDS             6826..7614
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
ORIGIN
        1 accaggcacg cctaaccgtc agtgagattg gatgagtgaa cgatattgat cgagaagagc
       61 cctgcgcagc cgctgccgtg cctgcaggaa gcaacggccc ggagggtggc gggcaggacg
      121 cccgccataa actgccaggc atcaaattaa gccttaagac ccactttcac atttaagttg
      181 tttttctaat ccgcatatga tcaattcaag gccgaataag aaggctggct ctgcaccttg
      241 gtgatcaaat aattcgatag cttgtcgtaa taatggcggc atactatcag tagtaggtgt
      301 ttccctttct tctttagcga cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa
      361 atgccccaca gcgctgagtg catataatgc attctctagt gaaaaacctt gttggcataa
      421 aaaggctaat tgattttcga gagtttcata ctgtttttct gtaggccgtg tacctaaatg
      481 tacttttgct ccatcgcgat gacttagtaa agcacatcta aaacttttag cgttattacg
      541 taaaaaatct tgccagcttt ccccttctaa agggcaaaag tgagtatggt gcctatctaa
      601 catctcaatg gctaaggcgt cgagcaaagc ccgcttattt tttacatgcc aatacaatgt
      661 aggctgctct acacctagct tctgggcgag tttacgggtt gttaaacctt cgattccgac
      721 ctcattaagc agctctaatg cgctgttaat cactttactt ttatctaatc tagacatcat
      781 taattcctaa tttttgttga cactctatcg ttgatagagt tattttacca ctccctatca
      841 gtgatagaga aaagaattca aaagatctaa agaggagaaa ggatctctgg ataagaaata
      901 ctcaataggc ttagatatcg gcacaaatag cgtcggatgg gcggtgatca ctgatgaata
      961 taaggttccg tctaaaaagt tcaaggttct gggaaataca gaccgccaca gtatcaaaaa
     1021 aaatcttata ggggctcttt tatttgacag tggagagaca gcggaagcga ctcgtctcaa
     1081 acggacagct cgtagaaggt atacacgtcg gaagaatcgt atttgttatc tacaggagat
     1141 tttttcaaat gagatggcga aagtagatga tagtttcttt catcgacttg aagagtcttt
     1201 tttggtggaa gaagacaaga agcatgaacg tcatcctatt tttggaaata tagtagatga
     1261 agttgcttat catgagaaat atccaactat ctatcatctg cgaaaaaaat tggtagattc
     1321 tactgataaa gcggatttgc gcttaatcta tttggcctta gcgcatatga ttaagtttcg
     1381 tggtcatttt ttgattgagg gagatttaaa tcctgataat agtgatgtgg acaaactatt
     1441 tatccagttg gtacaaacct acaatcaatt atttgaagaa aaccctatta acgcaagtgg
     1501 agtagatgct aaagcgattc tttctgcacg attgagtaaa tcaagacgat tagaaaatct
     1561 cattgctcag ctccccggtg agaagaaaaa tggcttattt gggaatctca ttgctttgtc
     1621 attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg ctaaattaca
     1681 gctttcaaaa gatacttacg atgatgattt agataattta ttggcgcaaa ttggagatca
     1741 atatgctgat ttgtttttgg cagctaagaa tttatcagat gctattttac tttcagatat
     1801 cctaagagta aatactgaaa taactaaggc tcccctatca gcttcaatga ttaaacgcta
     1861 cgatgaacat catcaagact tgactctttt aaaagcttta gttcgacaac aacttccaga
     1921 aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt atattgatgg
     1981 gggagctagc caagaagaat tttataaatt tatcaaacca attttagaaa aaatggatgg
     2041 tactgaggaa ttattggtga aactaaatcg tgaagatttg ctgcgcaagc aacggacctt
     2101 tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta ttttgagaag
     2161 acaagaagac ttttatccat ttttaaaaga caatcgtgag aagattgaaa aaatcttgac
     2221 ttttcgaatt ccttattatg ttggtccatt ggcgcgtggc aatagtcgtt ttgcatggat
     2281 gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg tcgataaagg
     2341 tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc ttccaaatga
     2401 aaaagtacta ccaaaacata gtttgcttta tgagtatttt acggtttata acgaattgac
     2461 aaaggtcaaa tatgttactg aaggaatgcg aaaaccagca tttctttcag gtgaacagaa
     2521 gaaagccatt gttgatttac tcttcaaaac aaatcgaaaa gtaaccgtta agcaattaaa
     2581 agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag gagttgaaga
     2641 tagatttaat gcttcattag gtacctacca tgatttgcta aaaattatta aagataaaga
     2701 ttttttggat aatgaagaaa atgaagatat cttagaggat attgttttaa cattgacctt
     2761 atttgaagat agggagatga ttgaggaaag acttaaaaca tatgctcacc tctttgatga
     2821 taaggtgatg aaacagctta aacgtcgccg ttatactggt tggggacgtt tgtctcgaaa
     2881 attgattaat ggtattaggg ataagcaatc tggcaaaaca atattagatt ttttgaaatc
     2941 agatggtttt gccaatcgca attttatgca gctgatccat gatgatagtt tgacatttaa
     3001 agaagacatt caaaaagcac aagtgtctgg acaaggcgat agtttacatg aacatattgc
     3061 aaatttagct ggtagccctg ctattaaaaa aggtatttta cagactgtaa aagttgttga
     3121 tgaattggtc aaagtaatgg ggcggcataa gccagaaaat atcgttattg aaatggcacg
     3181 tgaaaatcag acaactcaaa agggccagaa aaattcgcga gagcgtatga aacgaatcga
     3241 agaaggtatc aaagaattag gaagtcagat tcttaaagag catcctgttg aaaatactca
     3301 attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagaca tgtatgtgga
     3361 ccaagaatta gatattaatc gtttaagtga ttatgatgtc gatcacattg ttccacaaag
     3421 tttccttaaa gacgattcaa tagacaataa ggtcttaacg cgttctgata aaaatcgtgg
     3481 taaatcggat aacgttccaa gtgaagaagt agtcaaaaag atgaaaaact attggagaca
     3541 acttctaaac gccaagttaa tcactcaacg taagtttgat aatttaacga aagctgaacg
     3601 tggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg ttgaaactcg
     3661 ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta aatacgatga
     3721 aaatgataaa cttattcgag aggttaaagt gattacctta aaatctaaat tagtttctga
     3781 cttccgaaaa gatttccaat tctataaagt acgtgagatt aacaattacc atcatgccca
     3841 tgatgcgtat ctaaatgccg tcgttggaac tgctttgatt aagaaatatc caaaacttga
     3901 atcggagttt gtctatggtg attataaagt ttatgatgtt cgtaaaatga ttgctaagtc
     3961 tgagcaagaa ataggcaaag caaccgcaaa atatttcttt tactctaata tcatgaactt
     4021 cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc taatcgaaac
     4081 taatggggaa actggagaaa ttgtctggga taaagggcga gattttgcca cagtgcgcaa
     4141 agtattgtcc atgccccaag tcaatattgt caagaaaaca gaagtacaga caggcggatt
     4201 ctccaaggag tcaattttac caaaaagaaa ttcggacaag cttattgctc gtaaaaaaga
     4261 ctgggatcca aaaaaatatg gtggttttga tagtccaacg gtagcttatt cagtcctagt
     4321 ggttgctaag gtggaaaaag ggaaatcgaa gaagttaaaa tccgttaaag agttactagg
     4381 gatcacaatt atggaaagaa gttcctttga aaaaaatccg attgactttt tagaagctaa
     4441 aggatataag gaagttaaaa aagacttaat cattaaacta cctaaatata gtctttttga
     4501 gttagaaaac ggtcgtaaac ggatgctggc tagtgccgga gaattacaaa aaggaaatga
     4561 gctggctctg ccaagcaaat atgtgaattt tttatattta gctagtcatt atgaaaagtt
     4621 gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc ataagcatta
     4681 tttagatgag attattgagc aaatcagtga attttctaag cgtgttattt tagcagatgc
     4741 caatttagat aaagttctta gtgcatataa caaacataga gacaaaccaa tacgtgaaca
     4801 agcagaaaat attattcatt tatttacgtt gacgaatctt ggagctcccg ctgcttttaa
     4861 atattttgat acaacaattg atcgtaaacg atatacgtct acaaaagaag ttttagatgc
     4921 cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt tgagtcagct
     4981 aggaggtgac tccggcggcg gttcgggtag tggaggcagc gcagctaacg atgaaaatta
     5041 cgcgctggct gcttaatagc tcgagtaagg atctccaggc atcaaataaa acgaaaggct
     5101 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag
     5161 agtcacactg gctcaccttc gggtgggcct ttctgcgttt atacctaggg agacgtcgag
     5221 ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat acgtctacaa
     5281 aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat gaaacacgca
     5341 ttgatttgag tcagctagga ggtgactgac cggctgataa atttctttga atttctcctt
     5401 gattatttgt tataaatgtt ataaaataat cttgttggaa ccattcaaaa cagcatagca
     5461 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     5521 ttgatacttc tattctactc tgactgcaaa ccaaaaaaac aagcgctttc aaaacgcttg
     5581 ttttatcatt tttagggaaa ttaatctctt aatcctttta tcattctaca tttaggcgct
     5641 gccatcttgg gacaatgaaa acgttagtca tggcgcgcct tgacggctag ctcagtccta
     5701 ggtacagtgc tagcttaatt agtctacgag gttttagagc tatgctgttt tgaatggtcc
     5761 caaaacctgc agacaagccc ggccggccta aggcgatgcc ccctcgacct cgatcaggga
     5821 ggcgttcagg acgactcaca aagaaagccg ggcaatgccc ggctttttcc acgcctcctg
     5881 ggctgacttc aggtgctaca tttgaagaga taaattgcac tgaaatctag agcggttcag
     5941 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc ttttgccctg
     6001 taaacgaaaa aaccacctgg ggaggtggtt tgatcgaagg ttaagtcagt tggggaactg
     6061 cttaaccgtg gtaactggct ttcgcagagc acagcaacca aatctgtcct tccagtgtag
     6121 ccggactttg gcgcacactt caagagcaac cgcgtgttta gctaaacaaa tcctctgcga
     6181 actcccagtt accaatggct gctgccagtg gcgttttacc gtgcttttcc gggttggact
     6241 caagtgaaca gttaccggat aaggcgcagc agtcgggctg aacggggagt tcttgcttac
     6301 agcccagctt ggagcgaacg acctacaccg agccgagata ccagtgtgtg agctatgaga
     6361 aagcgccaca cttcccgtaa gggagaaagg cggaacaggt atccggtaaa cggcagggtc
     6421 ggaacaggag agcgcaagag ggagcgaccc gccggaaacg gtggggatct ttaagtcctg
     6481 tcgggtttcg cccgtactgt cagattcatg gttgagcctc acggctccca cagatgcacc
     6541 ggaaaagcgt ctgtttatgt gaactctggc aggagggcgg agcctatgga aaaacgccac
     6601 cggcgcggcc ctgctgtttt gcctcacatg ttagtcccct gcttatccac ggaatctgtg
     6661 ggtaactttg tatgtgtccg cagcgcccgc cgcagtctca cgcccggagc gtagcgaccg
     6721 agtgagctag ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     6781 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag ggaagcggtg
     6841 atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa
     6901 ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac
     6961 agtgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct
     7021 ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct
     7081 gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc
     7141 gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc
     7201 acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg
     7261 gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg
     7321 ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat
     7381 gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag
     7441 gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt
     7501 gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag
     7561 ttggaagaat ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg caaataatgt
     7621 ctaacaattc gttcaagccg aggggccgca agatccggcc acgatgaccc ggtcgtcggt
     7681 tcagggcagg gt
//
