LOCUS       pCas9D10A_GFP.        9271 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for co-expression in mammalian cells of enhanced GFP (EGFP) 
            and human codon-optimized Cas9 with the D10A nickase mutation.
ACCESSION   .
VERSION     .
KEYWORDS    pCas9D10A_GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9271)
  AUTHORS   Musunuru Lab / Addgene #44720
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 9271)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..665
                     /label=chicken beta-actin promoter
     intron          666..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     CDS             1745..5848
                     /label=Cas9(D10A)
                     /note="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             5861..5881
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             5897..5950
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid
                     protein"
                     /label=2A peptide from Thosea asigna virus capsid
                     protein
                     /note="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5951..6667
                     /label=EGFP
                     /note="enhanced GFP"
     polyA_signal    6713..6846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6883..6899)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(6907..6923)
                     /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(6931..6961)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6976..6997)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(7285..7873)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8047..8904)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8905..9009)
                     /label=AmpR promoter
     promoter        9271
                     /label=CAG
                     /note="CMV early enhancer fused to modified chicken 
                     beta-actin promoter"
ORIGIN
        1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
      541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
      841 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccat ctccagcctc ggggctgtcc
     1561 gcagggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagtgcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgcggccgc
     1741 caccatggac aagaagtact ccattgggct cgctatcggc acaaacagcg tcggctgggc
     1801 cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg gcaataccga
     1861 tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg gggagacggc
     1921 cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa agaatcggat
     1981 ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact ctttcttcca
     2041 taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc acccaatctt
     2101 tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat atcatctgag
     2161 gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc
     2221 gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc cagacaacag
     2281 cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt tcgaagagaa
     2341 cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc
     2401 ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg gcctgtttgg
     2461 taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact tcgacctggc
     2521 cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg acaatctgct
     2581 ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc tgtcagacgc
     2641 cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc cgctgagcgc
     2701 tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga aggcccttgt
     2761 cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta aaaatggcta
     2821 cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta ttaagcccat
     2881 cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag aagatctgtt
     2941 gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc tgggcgaact
     3001 gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata acagggaaaa
     3061 gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg cccggggaaa
     3121 ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct ggaacttcga
     3181 ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga ctaactttga
     3241 taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg agtacttcac
     3301 agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa agccagcatt
     3361 cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga accggaaagt
     3421 taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg actctgttga
     3481 aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg atctcctgaa
     3541 aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc ttgaggacat
     3601 tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct tgaaaactta
     3661 cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat atacaggatg
     3721 ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg gaaagacaat
     3781 cctggatttt cttaagtccg atggatttgc caaccggaac ttcatgcagt tgatccatga
     3841 tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc agggggacag
     3901 tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg gaatactgca
     3961 gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc ccgagaatat
     4021 cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga acagtaggga
     4081 aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc ttaaggaaca
     4141 cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc tgcagaacgg
     4201 cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact acgacgtgga
     4261 tcatatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag tgttgacaag
     4321 atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg tcaagaaaat
     4381 gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga agttcgataa
     4441 tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct tcatcaaaag
     4501 gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg attcacgcat
     4561 gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta ttactctgaa
     4621 gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga gagagatcaa
     4681 caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg cacttatcaa
     4741 aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt acgatgttag
     4801 gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt acttctttta
     4861 cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag agattcggaa
     4921 gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca agggtaggga
     4981 tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta aaaagaccga
     5041 agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct
     5101 gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt ctcctacagt
     5161 cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag
     5221 cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa aaaaccccat
     5281 cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca ttaagcttcc
     5341 caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga
     5401 gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct tgtatctggc
     5461 cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc agctgttcgt
     5521 ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat tctccaaaag
     5581 agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata agcacaggga
     5641 taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga ccaacttggg
     5701 cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt acacctctac
     5761 aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct atgaaacaag
     5821 aatcgacctc tctcagctcg gtggagacag cagggctgac cccaagaaga agaggaaggt
     5881 gaggtccggc ggcggagagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa
     5941 tcccggccca atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     6001 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     6061 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     6121 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     6181 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     6241 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     6301 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     6361 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     6421 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     6481 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     6541 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     6601 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     6661 gtacaagtaa ctgcagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     6721 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     6781 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     6841 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     6901 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     6961 agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     7021 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     7081 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     7141 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     7201 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     7261 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     7321 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     7381 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     7441 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     7501 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     7561 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     7621 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     7681 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     7741 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     7801 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     7861 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     7921 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     7981 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     8041 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     8101 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     8161 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     8221 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     8281 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     8341 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     8401 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     8461 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     8521 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     8581 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     8641 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     8701 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     8761 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     8821 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     8881 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     8941 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     9001 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc
     9061 tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca
     9121 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     9181 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     9241 tgcgctgctt cgcgatgtac gggccagata t
//
