LOCUS       hCas9_D10A.        9553 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian vector with a G418 resistance marker for expressing human 
            codon-optimized Cas9 with the D10A nickase mutation.
ACCESSION   .
VERSION     .
KEYWORDS    hCas9_D10A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9553)
  AUTHORS   Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE,
            Church GM.
  TITLE     RNA-guided human genome engineering via Cas9.
  JOURNAL   Science 2013;339:823-6.
  PUBMED    23287722
REFERENCE   2  (bases 1 to 9553)
  AUTHORS   Church Lab / Addgene #41816
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9553)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science"; 
            date: "2013"; volume: "339"; pages: "823-6"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             748..4851
                     /label=Cas9(D10A)
                     /note="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             4864..4884
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     polyA_signal    4983..5031
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5233..5661
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5675..6004
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             6071..6862
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    7041..7174
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7211..7227)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7235..7251)
                     /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)."
     promoter        complement(7259..7289)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7304..7325)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(7613..8201)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8375..9232)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(9233..9337)
                     /label=AmpR promoter
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagagg atcgaaccct tgccaccatg gacaagaagt actccattgg gctcgctatc
      781 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
      841 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
      901 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
      961 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
     1021 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
     1081 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
     1141 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
     1201 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
     1261 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact
     1321 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
     1381 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
     1441 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
     1501 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
     1561 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
     1621 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
     1681 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
     1741 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
     1801 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
     1861 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
     1921 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
     1981 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
     2041 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
     2101 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
     2161 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
     2221 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
     2281 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
     2341 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
     2401 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
     2461 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
     2521 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
     2581 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
     2641 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
     2701 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
     2761 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaacagg
     2821 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
     2881 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
     2941 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
     3001 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
     3061 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
     3121 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
     3181 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
     3241 cggctctccg actacgacgt ggatcatatc gtgccccagt cttttctcaa agatgattct
     3301 attgataata aagtgttgac aagatccgat aaaaatagag ggaagagtga taacgtcccc
     3361 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
     3421 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
     3481 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
     3541 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
     3601 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
     3661 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
     3721 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
     3781 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
     3841 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
     3901 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
     3961 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
     4021 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
     4081 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
     4141 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
     4201 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
     4261 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
     4321 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
     4381 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
     4441 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
     4501 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
     4561 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
     4621 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
     4681 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
     4741 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
     4801 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
     4861 gaccccaaga agaagaggaa ggtgtgaaag ggttcgatcc ctaccggtta gtaatgagtt
     4921 taaacggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     4981 gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg
     5041 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     5101 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     5161 tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc tggggctcta
     5221 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     5281 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     5341 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5401 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     5461 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5521 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     5581 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5641 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     5701 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     5761 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     5821 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     5881 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     5941 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     6001 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     6061 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     6121 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     6181 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     6241 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     6301 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     6361 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     6421 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     6481 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     6541 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     6601 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     6661 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     6721 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     6781 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     6841 tcgccttctt gacgagttct tctgagcggg actctggggt tcgcgaaatg accgaccaag
     6901 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     6961 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     7021 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     7081 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     7141 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     7201 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     7261 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     7321 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     7381 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     7441 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     7501 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     7561 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     7621 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     7681 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     7741 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     7801 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     7861 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     7921 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     7981 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     8041 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     8101 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     8161 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     8221 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     8281 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     8341 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     8401 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     8461 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     8521 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     8581 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     8641 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     8701 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     8761 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     8821 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     8881 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     8941 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     9001 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     9061 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     9121 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     9181 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     9241 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     9301 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     9361 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     9421 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     9481 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     9541 gaatctgctt agg
//
