LOCUS       Cas9m4.        9553 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for expression in mammalian cells of catalytically dead 
            Cas9m4.
ACCESSION   .
VERSION     .
KEYWORDS    Cas9m4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9553)
  AUTHORS   Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang
            L, Church GM.
  TITLE     CAS9 transcriptional activators for target specificity screening and
            paired nickases for cooperative genome engineering.
  JOURNAL   Nat. Biotechnol. 2013;31:833-8.
  PUBMED    23907171
REFERENCE   2  (bases 1 to 9553)
  AUTHORS   Church Lab / Addgene #47316
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9553)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Biotechnol."; date: "2013"; volume: "31"; pages: "833-8"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /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)."
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    complement(258..391)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(570..1361)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     promoter        complement(1428..1757)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      complement(1771..2199)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     polyA_signal    complement(2401..2449)
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(2548..2568)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             complement(2581..6684)
                     /label=Cas9m4
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     promoter        complement(6799..7002)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     enhancer        complement(7003..7382)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7648..7752
                     /label=AmpR promoter
     CDS             7753..8610
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      8784..9372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct
      241 agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac
      301 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
      361 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat
      421 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
      481 catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg
      541 tcatttcgcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
      601 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
      661 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
      721 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
      781 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
      841 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
      901 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
      961 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     1021 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     1081 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     1141 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     1201 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     1261 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     1321 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     1381 gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct
     1441 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg
     1501 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     1561 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     1621 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     1681 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     1741 caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct
     1801 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     1861 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     1921 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     1981 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2041 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2101 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2161 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc
     2221 agctgcgcag atctgctatg gcagggcctg ccgccccgac gttggctgcg agccctgggc
     2281 cttcacccga acttgggggg tggggtgggg aaaaggaaga aacgcgggcg tattggcccc
     2341 aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc ccgcgtttat
     2401 gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg
     2461 gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccgttt aaactcatta
     2521 ctaaccggta gggatcgaac cctttcacac cttcctcttc ttcttggggt cagccctgct
     2581 gtctccaccg agctgagaga ggtcgattct tgtttcatag agccccgtaa ttgactgatg
     2641 aatcagtgtg gcgtccagga cctcctttgt agaggtgtac cgctttctgt ctatggtggt
     2701 gtcgaagtac ttgaaggctg caggcgcgcc caagttggtc agagtaaaca agtggataat
     2761 gttttctgcc tgctccctga tgggcttatc cctgtgctta ttgtaagcag aaagcacctt
     2821 atcgaggtta gcgtcggcga ggatcactct tttggagaat tcgcttattt gctcgatgat
     2881 ctcatcaagg tagtgtttgt gttgttccac gaacagctgc ttctgctcat tatcttcggg
     2941 agaccctttg agcttttcat agtggctggc cagatacaag aaattaacgt atttagaggg
     3001 cagtgccagc tcgttacctt tctgcagctc gcccgcacta gcgagcattc gtttccggcc
     3061 gttttcaagc tcaaagagag agtacttggg aagcttaatg atgaggtctt ttttgacctc
     3121 tttatatcct ttcgcctcga gaaagtcgat ggggtttttt tcgaagcttg atcgctccat
     3181 gattgtgatg cccagcagtt ccttgacgct tttgagtttt ttagacttcc ctttctccac
     3241 tttggccaca accagtacac tgtaagcgac tgtaggagaa tcgaatccgc cgtatttctt
     3301 ggggtcccaa tcttttttgc gtgcgatcag cttgtcgctg ttccttttcg ggaggatact
     3361 ttccttggag aagcctccgg tctgtacttc ggtcttttta acgatgttca cctgcggcat
     3421 ggacaggacc ttccggactg tcgcgaaatc cctacccttg tcccacacga tttctcctgt
     3481 ttctccgttt gtttcgataa gtggtcgctt ccgaatctct ccattggcca gtgtaatctc
     3541 ggtcttgaaa aaattcataa tattgctgta aaagaagtac ttagcggtgg ccttgcctat
     3601 ttcctgctca gactttgcga tcattttcct aacatcgtac actttatagt ctccgtaaac
     3661 aaattcagat tcaagcttgg gatatttttt gataagtgca gtgcctacca ctgcattcag
     3721 gtaggcatca tgcgcatggt ggtaattgtt gatctctctc accttataaa actgaaagtc
     3781 ctttctgaaa tctgagacca gcttagactt cagagtaata actttcacct ctcgaatcag
     3841 tttgtcattt tcatcgtact tggtgttcat gcgtgaatcg agaatttggg ccacgtgctt
     3901 ggtgatctgg cgtgtctcaa caagctgcct tttgatgaag ccggctttat ccaactcaga
     3961 caggccacct cgttcagcct tagtcagatt atcgaacttc cgttgtgtga tcagtttggc
     4021 gttcagcagc tgccgccaat aatttttcat tttcttgaca acttcttctg aggggacgtt
     4081 atcactcttc cctctagctt tatcggatct tgtcaacact ttattatcaa tagaatcatc
     4141 tttgagaaaa gactggggca cgatagcagc cacgtcgtag tcggagagcc gattgatgtc
     4201 cagttcctga tccacgtaca tgtccctgcc gttctgcagg tagtacaggt agagcttctc
     4261 attctgaagc tgggtgtttt caactgggtg ttccttaagg atttgggacc ccagttcttt
     4321 tataccctct tcaatcctct tcatcctttc cctactgttc ttctgtccct tctgggtagt
     4381 ttggttctct cgggccatct cgataacgat attctcgggc ttatgccttc ccattacttt
     4441 gacgagttca tccacgacct taacggtctg cagtattccc tttttgatag ctgggctacc
     4501 tgcaagatta gcgatgtgct cgtgaagact gtccccctgg ccagaaactt gtgctttctg
     4561 gatgtcctcc ttaaaggtga gagagtcatc atggatcaac tgcatgaagt tccggttggc
     4621 aaatccatcg gacttaagaa aatccaggat tgtctttcca ctctgcttgt ctcggatccc
     4681 attgatcagt tttcttgaca gccgccccca tcctgtatat cggcgcctct tgagctgttt
     4741 catgactttg tcgtcgaaga gatgagcgta agttttcaag cgttcttcaa tcatctccct
     4801 atcttcaaac aacgtaaggg tgaggacaat gtcctcaaga atgtcctcgt tctcctcatt
     4861 gtccaggaag tccttgtctt taatgatttt caggagatcg tgatacgttc ccagggatgc
     4921 gttgaagcga tcctccactc cgctgatttc aacagagtcg aaacattcaa tctttttgaa
     4981 atagtcttct ttgagctgtt tcacggtaac tttccggttc gtcttgaaga ggaggtccac
     5041 gatagctttc ttctgctctc cagacaggaa tgctggcttt ctcatccctt ctgtgacgta
     5101 tttgaccttg gtgagctcgt tataaactgt gaagtactcg tacagcagag agtgtttagg
     5161 aagcaccttt tcgttaggca gatttttatc aaagttagtc atcctttcga tgaaggactg
     5221 ggcagaggcc cccttatcca cgacttcctc gaagttccag ggagtgatgg tctcttctga
     5281 tttgcgagtc atccacgcga atctggaatt tccccgggcg agggggccta catagtaggg
     5341 tatccgaaat gtgaggattt tctcaatctt ttccctgtta tctttcaaaa aggggtagaa
     5401 atcctcttgc cgcctgagga tagcgtgcag ttcgcccagg tgaatctggt gggggatgct
     5461 tccattgtcg aaagtgcgct gtttgcgcaa cagatcttct ctgttaagct ttaccagcag
     5521 ctcctcggtg ccgtccattt tttccaagat gggcttaata aatttgtaaa attcctcctg
     5581 gcttgctccg ccgtcaatgt atccggcgta gccattttta gactgatcga agaaaatttc
     5641 cttgtacttc tcaggcagtt gctgtctgac aagggccttc agcaaagtca agtcttggtg
     5701 gtgctcatca tagcgcttga tcatactagc gctcagcgga gctttggtga tctccgtgtt
     5761 cactcgcaga atatcactca gcagaatggc gtctgacagg ttctttgccg ccaaaaaaag
     5821 gtctgcgtac tggtcgccga tctgggccag cagattgtcg agatcatcat cgtaggtgtc
     5881 tttgctcagt tgaagcttgg catcttcggc caggtcgaag ttagatttaa agttgggggt
     5941 cagcccgagt gacagggcga taagattacc aaacaggccg ttcttcttct ccccagggag
     6001 ctgtgcgatg aggttttcga gccgccggga tttggacagc ctagcgctca ggattgcttt
     6061 ggcgtcaact ccggatgcgt tgatcgggtt ctcttcgaaa agctgattgt aagtctgaac
     6121 cagttggata aagagtttgt cgacatcgct gttgtctggg ttcaggtccc cctcgatgag
     6181 gaagtgtccc cgaaatttga tcatatgcgc cagcgcgaga tagatcaacc gcaagtcagc
     6241 cttatcagta ctgtctacaa gcttcttcct cagatgatat atggttgggt acttttcatg
     6301 gtacgccacc tcgtccacga tattgccaaa gattgggtgg cgctcgtgct ttttatcctc
     6361 ctccaccaaa aaggactcct ccagcctatg gaagaaagag tcatccacct tagccatctc
     6421 attactaaag atctcctgca ggtagcagat ccgattcttt ctgcgggtat atctgcgccg
     6481 tgctgttctt ttgagccgcg tggcttcggc cgtctccccg gagtcgaaca ggagggcgcc
     6541 aatgaggttc ttctttatgc tgtggcgatc ggtattgccc agaactttga attttttgct
     6601 cggcaccttg tactcgtccg taatgacggc ccagccgacg ctgtttgtgc cgatagcgag
     6661 cccaatggag tacttcttgt ccatggtggc aagggttcga tcctctagag tccggaggct
     6721 ggatcggtcc cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc tccaggcgat
     6781 ctgacggttc actaaacgag ctctgcttat atagacctcc caccgtacac gcctaccgcc
     6841 catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg
     6901 ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa
     6961 ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac
     7021 taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg
     7081 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
     7141 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
     7201 agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc
     7261 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata
     7321 tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg
     7381 tcaacgcgta tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag
     7441 attcttcatg caattgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca
     7501 ctactcagcg acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc
     7561 agagcagatt gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg
     7621 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     7681 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     7741 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     7801 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     7861 cagttgggtg tacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     7921 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     7981 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     8041 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     8101 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     8161 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     8221 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     8281 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     8341 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     8401 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     8461 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     8521 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     8581 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     8641 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     8701 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     8761 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     8821 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     8881 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     8941 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     9001 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     9061 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     9121 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     9181 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     9241 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     9301 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     9361 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     9421 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     9481 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     9541 gaggaagcgg aag
//
