LOCUS       Cas9m4-VP64.        9771 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for expression in mammalian cells of catalytically dead 
            Cas9m4 fused to the VP64 transcriptional activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    Cas9m4-VP64
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9771)
  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 9771)
  AUTHORS   Church Lab / Addgene #47319
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9771)
  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..9771
                     /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(2559..2708)
                     /label=VP64
                     /note="tetrameric repeat of the minimal activation domain
                     of herpes simplex virus VP16 (Beerli et al., 1998)"
     CDS             complement(2733..2753)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             complement(2766..6869)
                     /label=Cas9m4
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             complement(6879..6899)
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             complement(6900..6902)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     promoter        complement(7017..7220)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     enhancer        complement(7221..7600)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7866..7970
                     /label=AmpR promoter
     CDS             7971..8828
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      9002..9590
                     /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 ccttcatcta gagttaatca gcatgtccag gtcgaaatca
     2581 tcaagggcgt cactgccgag catgtcgagg tcaaagtcat caagggcatc cgaaccaagc
     2641 atgtcaaggt caaaatcatc gagggcgtca cttcccagca tatccagatc aaaatcgtcc
     2701 aatgcgtcag cccgtccgga accgctggcc tccaccttcc tcttcttctt ggggtcagcc
     2761 ctgctgtctc caccgagctg agagaggtcg attcttgttt catagagccc cgtaattgac
     2821 tgatgaatca gtgtggcgtc caggacctcc tttgtagagg tgtaccgctt tctgtctatg
     2881 gtggtgtcga agtacttgaa ggctgcaggc gcgcccaagt tggtcagagt aaacaagtgg
     2941 ataatgtttt ctgcctgctc cctgatgggc ttatccctgt gcttattgta agcagaaagc
     3001 accttatcga ggttagcgtc ggcgaggatc actcttttgg agaattcgct tatttgctcg
     3061 atgatctcat caaggtagtg tttgtgttgt tccacgaaca gctgcttctg ctcattatct
     3121 tcgggagacc ctttgagctt ttcatagtgg ctggccagat acaagaaatt aacgtattta
     3181 gagggcagtg ccagctcgtt acctttctgc agctcgcccg cactagcgag cattcgtttc
     3241 cggccgtttt caagctcaaa gagagagtac ttgggaagct taatgatgag gtcttttttg
     3301 acctctttat atcctttcgc ctcgagaaag tcgatggggt ttttttcgaa gcttgatcgc
     3361 tccatgattg tgatgcccag cagttccttg acgcttttga gttttttaga cttccctttc
     3421 tccactttgg ccacaaccag tacactgtaa gcgactgtag gagaatcgaa tccgccgtat
     3481 ttcttggggt cccaatcttt tttgcgtgcg atcagcttgt cgctgttcct tttcgggagg
     3541 atactttcct tggagaagcc tccggtctgt acttcggtct ttttaacgat gttcacctgc
     3601 ggcatggaca ggaccttccg gactgtcgcg aaatccctac ccttgtccca cacgatttct
     3661 cctgtttctc cgtttgtttc gataagtggt cgcttccgaa tctctccatt ggccagtgta
     3721 atctcggtct tgaaaaaatt cataatattg ctgtaaaaga agtacttagc ggtggccttg
     3781 cctatttcct gctcagactt tgcgatcatt ttcctaacat cgtacacttt atagtctccg
     3841 taaacaaatt cagattcaag cttgggatat tttttgataa gtgcagtgcc taccactgca
     3901 ttcaggtagg catcatgcgc atggtggtaa ttgttgatct ctctcacctt ataaaactga
     3961 aagtcctttc tgaaatctga gaccagctta gacttcagag taataacttt cacctctcga
     4021 atcagtttgt cattttcatc gtacttggtg ttcatgcgtg aatcgagaat ttgggccacg
     4081 tgcttggtga tctggcgtgt ctcaacaagc tgccttttga tgaagccggc tttatccaac
     4141 tcagacaggc cacctcgttc agccttagtc agattatcga acttccgttg tgtgatcagt
     4201 ttggcgttca gcagctgccg ccaataattt ttcattttct tgacaacttc ttctgagggg
     4261 acgttatcac tcttccctct agctttatcg gatcttgtca acactttatt atcaatagaa
     4321 tcatctttga gaaaagactg gggcacgata gcagccacgt cgtagtcgga gagccgattg
     4381 atgtccagtt cctgatccac gtacatgtcc ctgccgttct gcaggtagta caggtagagc
     4441 ttctcattct gaagctgggt gttttcaact gggtgttcct taaggatttg ggaccccagt
     4501 tcttttatac cctcttcaat cctcttcatc ctttccctac tgttcttctg tcccttctgg
     4561 gtagtttggt tctctcgggc catctcgata acgatattct cgggcttatg ccttcccatt
     4621 actttgacga gttcatccac gaccttaacg gtctgcagta ttcccttttt gatagctggg
     4681 ctacctgcaa gattagcgat gtgctcgtga agactgtccc cctggccaga aacttgtgct
     4741 ttctggatgt cctccttaaa ggtgagagag tcatcatgga tcaactgcat gaagttccgg
     4801 ttggcaaatc catcggactt aagaaaatcc aggattgtct ttccactctg cttgtctcgg
     4861 atcccattga tcagttttct tgacagccgc ccccatcctg tatatcggcg cctcttgagc
     4921 tgtttcatga ctttgtcgtc gaagagatga gcgtaagttt tcaagcgttc ttcaatcatc
     4981 tccctatctt caaacaacgt aagggtgagg acaatgtcct caagaatgtc ctcgttctcc
     5041 tcattgtcca ggaagtcctt gtctttaatg attttcagga gatcgtgata cgttcccagg
     5101 gatgcgttga agcgatcctc cactccgctg atttcaacag agtcgaaaca ttcaatcttt
     5161 ttgaaatagt cttctttgag ctgtttcacg gtaactttcc ggttcgtctt gaagaggagg
     5221 tccacgatag ctttcttctg ctctccagac aggaatgctg gctttctcat cccttctgtg
     5281 acgtatttga ccttggtgag ctcgttataa actgtgaagt actcgtacag cagagagtgt
     5341 ttaggaagca ccttttcgtt aggcagattt ttatcaaagt tagtcatcct ttcgatgaag
     5401 gactgggcag aggccccctt atccacgact tcctcgaagt tccagggagt gatggtctct
     5461 tctgatttgc gagtcatcca cgcgaatctg gaatttcccc gggcgagggg gcctacatag
     5521 tagggtatcc gaaatgtgag gattttctca atcttttccc tgttatcttt caaaaagggg
     5581 tagaaatcct cttgccgcct gaggatagcg tgcagttcgc ccaggtgaat ctggtggggg
     5641 atgcttccat tgtcgaaagt gcgctgtttg cgcaacagat cttctctgtt aagctttacc
     5701 agcagctcct cggtgccgtc cattttttcc aagatgggct taataaattt gtaaaattcc
     5761 tcctggcttg ctccgccgtc aatgtatccg gcgtagccat ttttagactg atcgaagaaa
     5821 atttccttgt acttctcagg cagttgctgt ctgacaaggg ccttcagcaa agtcaagtct
     5881 tggtggtgct catcatagcg cttgatcata ctagcgctca gcggagcttt ggtgatctcc
     5941 gtgttcactc gcagaatatc actcagcaga atggcgtctg acaggttctt tgccgccaaa
     6001 aaaaggtctg cgtactggtc gccgatctgg gccagcagat tgtcgagatc atcatcgtag
     6061 gtgtctttgc tcagttgaag cttggcatct tcggccaggt cgaagttaga tttaaagttg
     6121 ggggtcagcc cgagtgacag ggcgataaga ttaccaaaca ggccgttctt cttctcccca
     6181 gggagctgtg cgatgaggtt ttcgagccgc cgggatttgg acagcctagc gctcaggatt
     6241 gctttggcgt caactccgga tgcattgatc gggttctctt cgaaaagctg attgtaagtc
     6301 tgaaccagtt ggataaagag tttgtcgaca tcgctgttgt ctgggttcag gtccccctcg
     6361 atgaggaagt gtccccgaaa tttgatcata tgcgccagcg cgagatagat caaccgcaag
     6421 tcagccttat cagtactgtc tacaagcttc ttcctcagat gatatatggt tgggtacttt
     6481 tcatggtacg ccacctcgtc cacgatattg ccaaagattg ggtggcgctc gtgcttttta
     6541 tcctcctcca ccaaaaagga ctcctccagc ctatggaaga aagagtcatc caccttagcc
     6601 atctcattac taaagatctc ctgcaggtag cagatccgat tctttctgcg ggtatatctg
     6661 cgccgtgctg ttcttttgag ccgcgtggct tcggccgtct ccccggagtc gaacaggagg
     6721 gcgccaatga ggttcttctt tatgctgtgg cgatcggtat tgcccagaac tttgaatttt
     6781 ttgctcggca ccttgtactc gtccgtaatg acggcccagc cgacgctgtt tgtgccgata
     6841 gcgagcccaa tggagtactt cttgtccatc ccccttccca ccttcctctt cttcttgggc
     6901 atggtggcaa gggttcgatc ctctagagtc cggaggctgg atcggtcccg gtgtcttcta
     6961 tggaggtcaa aacagcgtgg atggcgtctc caggcgatct gacggttcac taaacgagct
     7021 ctgcttatat agacctccca ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag
     7081 ttgttacgac attttggaaa gtcccgttga ttttggtgcc aaaacaaact cccattgacg
     7141 tcaatggggt ggagacttgg aaatccccgt gagtcaaacc gctatccacg cccattgatg
     7201 tactgccaaa accgcatcac catggtaata gcgatgacta atacgtagat gtactgccaa
     7261 gtaggaaagt cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca
     7321 ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc caagtgggca
     7381 gtttaccgta aatactccac ccattgacgt caatggaaag tccctattgg cgttactatg
     7441 ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg
     7501 ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac taatgacccc
     7561 gtaattgatt actattaata actagtcaat aatcaatgtc aacgcgtata tctggcccgt
     7621 acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca attgtcggtc
     7681 aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact actcagcgac ctccaacaca
     7741 caagcaggga gcagatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     7801 caccataggg gatcgggaga tctcccgatc cgtcgacgtc aggtggcact tttcggggaa
     7861 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     7921 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     7981 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     8041 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     8101 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     8161 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     8221 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     8281 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     8341 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     8401 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     8461 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     8521 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     8581 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     8641 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     8701 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     8761 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     8821 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     8881 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     8941 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     9001 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     9061 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     9121 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     9181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     9241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     9301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     9361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     9421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     9481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     9541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     9601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     9661 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     9721 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa g
//
