LOCUS       pCAG-T3-hCasD10A        9272 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for mammalian cell expression or in vitro transcription of 
            human codon-optimized Cas9 with the D10A nickase mutation.
ACCESSION   .
VERSION     .
KEYWORDS    pCAG-T3-hCasD10A-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9272)
  AUTHORS   Fujii W, Onuma A, Sugiura K, Naito K.
  TITLE     Efficient generation of genome-modified mice via offset-nicking by 
            CRISPR/Cas system.
  JOURNAL   Biochem. Biophys. Res. Commun. 2014;445:791-4.
  PUBMED    24491566
REFERENCE   2  (bases 1 to 9272)
  AUTHORS   
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9272)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biochem. 
            Biophys. Res. Commun."; date: "2014"; volume: "445"; pages: "791-4"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9272
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    complement(6..140)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(146..342)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     protein_bind    400..421
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        436..466
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    474..490
                     /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     498..514
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    complement(875..930)
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     misc_feature    1103..1197
                     /label=poly(A)
                     /note="poly(A)"
                     /note="enables microinjection of poly(A)-tailed mRNA after
                     in vitro transcription"
     3'UTR           1198..1267
                     /label=portion of the 3' UTR from the human TBPL1 gene
                     /note="portion of the 3' UTR from the human TBPL1 gene"
     CDS             complement(1283..1303)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             complement(1316..5416)
                     /label=Cas9(D10A)
                     /note="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             complement(5441..5461)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             complement(5471..5494)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /label=FLAG
                     /note="FLAG"
                     /translation="DYKDDDDK"
     CDS             complement(5495..5497)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     promoter        complement(5555..5573)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     intron          complement(5630..6646)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     promoter        complement(6647..6903)
                     /note="chicken beta-actin promoter"
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
     enhancer        6905..7284
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7315..7419
                     /label=AmpR promoter
     CDS             7420..8277
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      8451..9039
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cagcggatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
       61 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
      121 taagctgcaa taaacaagtt agctttttgc aaaagcctag gcctccaaaa aagcctcctc
      181 actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa
      241 ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg
      301 gagttagggg cgggactatg gttgctgact aattgagatg cggatccgct ggcacgacag
      361 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      421 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      481 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgggctgca
      541 ggtcgaggga tctccataag agaagaggga cagctatgac tgggagtagt caggagagga
      601 ggaaaaatct ggctagtaaa acatgtaagg aaaattttag ggatgttaaa gaaaaaaata
      661 acacaaaaca aaatataaaa aaaatctaac ctcaagtcaa ggcttttcta tggaataagg
      721 aatggacagc agggggctgt ttcatatact gatgacctct ttatagccaa cctttgttca
      781 tggcagccag catatgggca tatgttgcca aactctaaac caaatactca ttctgatgtt
      841 ttaaatgatt tgccctccca tatgtccttc cgagtgagag acacaaaaaa ttccaacaca
      901 ctattgcaat gaaaataaat ttcctttatt agccagaagt cagatgctca aggggcttca
      961 tgatgtcccc ataatttttg gcagagggaa aaagatctca gtggtatttg tgagccaggg
     1021 cattggccac accagccacc accttctgat aggcagcctg cacctgagga gtgaattccg
     1081 tacacttgca tgctacctcg agtttttttt tttttttttt tttttttttt tttttttttt
     1141 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttctt
     1201 ttccaaactt ttatttgtgc acattcaatt taaaaagata acgtttacag gttctttggt
     1261 gcccataggt accatcgatt cacaccttcc tcttcttctt ggggtcagcc ctgctgtctc
     1321 caccgagctg agagaggtcg attcttgttt catagagccc cgtaattgac tgatgaatca
     1381 gtgtggcgtc caggacctcc tttgtagagg tgtaccgctt tctgtctatg gtggtgtcga
     1441 agtacttgaa ggctgcaggc gcgcccaagt tggtcagagt aaacaagtgg ataatgtttt
     1501 ctgcctgctc cctgatgggc ttatccctgt gcttattgta agcagaaagc accttatcga
     1561 ggttagcgtc ggcgaggatc actcttttgg agaattcgct tatttgctcg atgatctcat
     1621 caaggtagtg tttgtgttgt tccacgaaca gctgcttctg ctcattatct tcgggagacc
     1681 ctttgagctt ttcatagtgg ctggccagat acaagaaatt aacgtattta gagggcagtg
     1741 ccagctcgtt acctttctgc agctcgcccg cactagcgag cattcgtttc cggccgtttt
     1801 caagctcaaa gagagagtac ttgggaagct taatgatgag gtcttttttg acctctttat
     1861 atcctttcgc ctcgagaaag tcgatggggt ttttttcgaa gcttgatcgc tccatgattg
     1921 tgatgcccag cagttccttg acgcttttga gttttttaga cttccctttc tccactttgg
     1981 ccacaaccag tacactgtaa gcgactgtag gagaatcgaa tccgccgtat ttcttggggt
     2041 cccaatcttt tttgcgtgcg atcagcttgt cgctgttcct tttcgggagg atactttcct
     2101 tggagaagcc tccggtctgt acttcggtct ttttaacgat gttcacctgc ggcatggaca
     2161 ggaccttccg gactgtcgcg aaatccctac ccttgtccca cacgatttct cctgtttctc
     2221 cgtttgtttc gataagtggt cgcttccgaa tctctccatt ggccagtgta atctcggtct
     2281 tgaaaaaatt cataatattg ctgtaaaaga agtacttagc ggtggccttg cctatttcct
     2341 gctcagactt tgcgatcatt ttcctaacat cgtacacttt atagtctccg taaacaaatt
     2401 cagattcaag cttgggatat tttttgataa gtgcagtgcc taccactgca ttcaggtagg
     2461 catcatgcgc atggtggtaa ttgttgatct ctctcacctt ataaaactga aagtcctttc
     2521 tgaaatctga gaccagctta gacttcagag taataacttt cacctctcga atcagtttgt
     2581 cattttcatc gtacttggtg ttcatgcgtg aatcgagaat ttgggccacg tgcttggtga
     2641 tctggcgtgt ctcaacaagc tgccttttga tgaagccggc tttatccaac tcagacaggc
     2701 cacctcgttc agccttagtc agattatcga acttccgttg tgtgatcagt ttggcgttca
     2761 gcagctgccg ccaataattt ttcattttct tgacaacttc ttctgagggg acgttatcac
     2821 tcttccctct atttttatcg gatcttgtca acactttatt atcaatagaa tcatctttga
     2881 gaaaagactg gggcacgata tgatccacgt cgtagtcgga gagccgattg atgtccagtt
     2941 cctgatccac gtacatgtcc ctgccgttct gcaggtagta caggtagagc ttctcattct
     3001 gaagctgggt gttttcaact gggtgttcct taaggatttg ggaccccagt tcttttatac
     3061 cctcttcaat cctcttcatc ctttccctac tgttcttctg tcccttctgg gtagtttggt
     3121 tctctcgggc catctcgata acgatattct cgggcttatg ccttcccatt actttgacga
     3181 gttcatccac gaccttaacg gtctgcagta ttcccttttt gatagctggg ctacctgcaa
     3241 gattagcgat gtgctcgtga agactgtccc cctggccaga aacttgtgct ttctggatgt
     3301 cctccttaaa ggtgagagag tcatcatgga tcaactgcat gaagttccgg ttggcaaatc
     3361 catcggactt aagaaaatcc aggattgtct ttccactctg cttgtctcgg atcccattga
     3421 tcagttttct tgacagccgc ccccatcctg tatatcggcg cctcttgagc tgtttcatga
     3481 ctttgtcgtc gaagagatga gcgtaagttt tcaagcgttc ttcaatcatc tccctatctt
     3541 caaacaacgt aagggtgagg acaatgtcct caagaatgtc ctcgttctcc tcattgtcca
     3601 ggaagtcctt gtctttaatg attttcagga gatcgtgata cgttcccagg gatgcgttga
     3661 agcgatcctc cactccgctg atttcaacag agtcgaaaca ttcaatcttt ttgaaatagt
     3721 cttctttgag ctgtttcacg gtaactttcc ggttcgtctt gaagaggagg tccacgatag
     3781 ctttcttctg ctctccagac aggaatgctg gctttctcat cccttctgtg acgtatttga
     3841 ccttggtgag ctcgttataa actgtgaagt actcgtacag cagagagtgt ttaggaagca
     3901 ccttttcgtt aggcagattt ttatcaaagt tagtcatcct ttcgatgaag gactgggcag
     3961 aggccccctt atccacgact tcctcgaagt tccagggagt gatggtctct tctgatttgc
     4021 gagtcatcca cgcgaatctg gaatttcccc gggcgagggg gcctacatag tagggtatcc
     4081 gaaatgtgag gattttctca atcttttccc tgttatcttt caaaaagggg tagaaatcct
     4141 cttgccgcct gaggatagcg tgcagttcgc ccaggtgaat ctggtggggg atgcttccat
     4201 tgtcgaaagt gcgctgtttg cgcaacagat cttctctgtt aagctttacc agcagctcct
     4261 cggtgccgtc cattttttcc aagatgggct taataaattt gtaaaattcc tcctggcttg
     4321 ctccgccgtc aatgtatccg gcgtagccat ttttagactg atcgaagaaa atttccttgt
     4381 acttctcagg cagttgctgt ctgacaaggg ccttcagcaa agtcaagtct tggtggtgct
     4441 catcatagcg cttgatcata ctagcgctca gcggagcttt ggtgatctcc gtgttcactc
     4501 gcagaatatc actcagcaga atggcgtctg acaggttctt tgccgccaaa aaaaggtctg
     4561 cgtactggtc gccgatctgg gccagcagat tgtcgagatc atcatcgtag gtgtctttgc
     4621 tcagttgaag cttggcatct tcggccaggt cgaagttaga tttaaagttg ggggtcagcc
     4681 cgagtgacag ggcgataaga ttaccaaaca ggccgttctt cttctcccca gggagctgtg
     4741 cgatgaggtt ttcgagccgc cgggatttgg acagcctagc gctcaggatt gctttggcgt
     4801 caactccgga tgcgttgatc gggttctctt cgaaaagctg attgtaagtc tgaaccagtt
     4861 ggataaagag tttgtcgaca tcgctgttgt ctgggttcag gtccccctcg atgaggaagt
     4921 gtccccgaaa tttgatcata tgcgccagcg cgagatagat caaccgcaag tcagccttat
     4981 cagtactgtc tacaagcttc ttcctcagat gatatatggt tgggtacttt tcatggtacg
     5041 ccacctcgtc cacgatattg ccaaagattg ggtggcgctc gtgcttttta tcctcctcca
     5101 ccaaaaagga ctcctccagc ctatggaaga aagagtcatc caccttagcc atctcattac
     5161 taaagatctc ctgcaggtag cagatccgat tctttctgcg ggtatatctg cgccgtgctg
     5221 ttcttttgag ccgcgtggct tcggccgtct ccccggagtc gaacaggagg gcgccaatga
     5281 ggttcttctt tatgctgtgg cgatcggtat tgcccagaac tttgaatttt ttgctcggca
     5341 ccttgtactc gtccgtaatg acggcccagc cgacgctgtt tgtgccgatg gcgagcccaa
     5401 tggagtactt cttgtcggct gctgggactc cgtggatacc gaccttccgc ttcttctttg
     5461 gggccatgat cttatcgtcg tcatccttgt aatccatggt ggatccgccc gggctagagc
     5521 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta atttcgaatt
     5581 ctttgccaaa atgatgagac agcacaataa ccagcacgtt gcccaggagc tgtaggaaaa
     5641 agaagaaggc atgaacatgg ttagcagagg ctctagagcc gccggtcaca cgccagaagc
     5701 cgaaccccgc cctgccccgt cccccccgaa ggcagccgtc cccccgcgga cagccccgag
     5761 gctggagagg gagaagggga cggcggcgcg gcgacgcacg aaggccctcc ccgcccattt
     5821 ccttcctgcc ggcgccgcac cgcttcgccc cgcgcccgct agagggggtg cggcggcgcc
     5881 tcccagattt cggctccgca cagatttggg acaaaggaag tccctgcgcc ctctcgcacg
     5941 attaccataa aaggcaatgg ctgcggctcg ccgcgcctcg acagccgccg gcgctccggg
     6001 ggccgccgcg cccctccccc gagccctccc cggcccgagg cggccccgcc ccgcccggca
     6061 cccccacctg ccgccacccc ccgcccggca cggcgagccc cgcgccacgc cccgtacgga
     6121 gccccgcacc cgaagccggg ccgtgctcag caactcgggg aggggggtgc agggggggtt
     6181 gcagcccgac cgacgcgccc acaccccctg ctcacccccc cacgcacaca ccccgcacgc
     6241 agcctttgtt cccctcgcag ccccccccgc accgcggggc accgcccccg gccgcgctcc
     6301 cctcgcgcac actgcggagc gcacaaagcc ccgcgccgcg cccgcagcgc tcacagccgc
     6361 cgggcagcgc ggagccgcac gcggcgctcc ccacgcacac acacacgcac gcaccccccg
     6421 agccgctccc cccgcacaaa gggccctccc ggagcccctc aaggctttca cgcagccaca
     6481 gaaaagaaac aagccgtcat taaaccaagc gctaattaca gcccggagga gaagggccgt
     6541 cccgcccgct cacctgtggg agtaacgcgg tcagtcagag ccggggcggg cggcgcgagg
     6601 cggcggcgga gcggggcacg gggcgaaggc agcgcgcagc gactcccgcc cgccgcgcgc
     6661 ttcgcttttt atagggccgc cgccgccgcc gcctcgccat aaaaggaaac tttcggagcg
     6721 cgccgctctg attggctgcc gccgcacctc tccgcctcgc cccgccccgc ccctcgcccc
     6781 gccccccccc cccccccgcc cccatcgctg cacaaaataa ttaaaaaata aataaataca
     6841 aaattggggg tggggagggg ggggagatgg ggagagtgaa gcagaacgtg gggctcacct
     6901 cgaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata
     6961 aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg
     7021 gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact
     7081 ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta
     7141 ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt
     7201 atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt
     7261 aataactagt caataatcaa tgtcgaccca ggtggcactt ttcggggaaa tgtgcgcgga
     7321 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     7381 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     7441 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     7501 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     7561 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     7621 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     7681 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     7741 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     7801 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     7861 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     7921 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     7981 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     8041 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     8101 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     8161 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     8221 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     8281 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     8341 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     8401 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     8461 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     8521 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     8581 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     8641 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     8701 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     8761 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     8821 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     8881 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     8941 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     9001 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     9061 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     9121 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     9181 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     9241 cctctccccg cgcgttggcc gattcattaa tg
//
