LOCUS       40924_17294        7267 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Delivery of human-codon-optimized Cas9 from Neisseria meningitidis 
            (NmeCas9) and its single-guide RNA in a single AAV vector for in 
            vivo genome editing..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7267)
  AUTHORS   Ibraheim R, Song CQ, Mir A, Amrani N, Xue W, Sontheimer EJ
  TITLE     All-in-one adeno-associated virus delivery and genome editing by 
            Neisseria meningitidis Cas9 in vivo.
  JOURNAL   Genome Biol. 2018 Sep 19;19(1):137. doi: 10.1186/s13059-018-1515-0.
  PUBMED    30231914
REFERENCE   2  (bases 1 to 7267)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7267)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Genome
            Biol."; date: "2018-09-19"; pages: "
            10.1186/s13059-018-1515-0"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_RNA        complement(158..250)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(309..549)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     regulatory      885..894
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             897..917
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             924..971
                     /codon_start=1
                     /label=nucleoplasmin NLS
                     /note="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             972..4214
                     /codon_start=1
                     /label=NmCas9
                     /note="Cas9 endonuclease from the Neisseria meningitidis
                     Type II CRISPR/Cas system (Esvelt et al., 2013)"
                     /translation="AAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVF
                     ERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKS
                     LPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAG
                     NAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFG
                     NPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTK
                     LNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDN
                     AEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
                     DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE
                     EKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKE
                     IEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGR
                     LNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFK
                     ARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRV
                     FASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFD
                     GKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAEK
                     LSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDL
                     EKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKT
                     GVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQL
                     IDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEG
                     IGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     CDS             4221..4268
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4269..4358
                     /codon_start=1
                     /label=3xHA
                     /note="three tandem HA epitope tags"
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             4365..4391
                     /codon_start=1
                     /label=NLS
                     /note="nuclear localization signal (Makkerh et al., 1996)"
                     /translation="PAAKKKKLD"
     primer_bind     complement(4397..4416)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4462..4517
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   4530..4670
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4745..5200
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5217..5236)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     5336..5358
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5396..5414)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        5482..5586
                     /label=AmpR promoter
     CDS             5587..6444
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6618..7206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gagtttaaac aaaaaaataa acgatgcccc ttaaagcaga
      181 agctttaagg ggcagagcgt tgcggcacat cttttcagac ggccttattg tagcaacggt
      241 tctcatttcg tttccgaaat gagaaaggga gctacaactg aagagctgga atgctcttcc
      301 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
      361 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
      421 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
      481 tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
      541 taggccctct tcctgcccga ccttgacgtc gactctagaa tggaggcggt actatgtaga
      601 tgagaattca ggagcaaact gggaaaagca actgcttcca aatatttgtg atttttacag
      661 tgtagttttg gaaaaactct tagcctacca attcttctaa gtgttttaaa atgtgggagc
      721 cagtacacat gaagttatag agtgttttaa tgaggcttaa atatttaccg taactatgaa
      781 atgctacgca tatcatgctg ttcaggctcc gtggccacgc aactcatact taagcagaca
      841 gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa caccgccacc atggtgccta
      901 agaagaagag aaaggtggaa gataaacgcc cagcagctac aaagaaggca ggtcaagcca
      961 agaaaaagaa agcagcattc aagccaaact caatcaatta catcctggga ctggacatcg
     1021 gcatcgcatc cgtcgggtgg gctatggtcg aaatcgacga ggaggagaac cccatccgcc
     1081 tgatcgatct gggcgtgcgc gtgtttgaga gggcagaggt gcctaagacc ggcgacagcc
     1141 tggccatggc acggagactg gcacgctccg tgaggcgcct gacccggaga agggcccaca
     1201 gactgctgag gacacgccgg ctgctgaaga gggagggcgt gctgcaggcc gccaacttcg
     1261 atgagaatgg cctgatcaag tccctgccca ataccccttg gcagctgagg gcagccgccc
     1321 tggaccgcaa gctgacacct ctggagtggt ccgccgtgct gctgcacctg atcaagcacc
     1381 ggggctacct gtctcagaga aagaacgagg gcgagacagc cgataaggag ctgggcgccc
     1441 tgctgaaggg agtggcagga aatgcacacg ccctgcagac cggcgacttt cgcacaccag
     1501 ccgagctggc cctgaacaag ttcgagaagg agagcggcca catccgcaat cagcggtctg
     1561 actatagcca caccttctcc cggaaggatc tgcaggccga gctgatcctg ctgtttgaga
     1621 agcagaagga gttcggcaac ccacacgtgt ctggcggcct gaaggagggc atcgagacac
     1681 tgctgatgac acagcggccc gccctgagcg gcgacgcagt gcagaagatg ctgggacact
     1741 gcacctttga gccagccgag cccaaggccg ccaagaatac ctacacagcc gagcggttca
     1801 tctggctgac aaagctgaac aatctgagga tcctggagca gggaagcgag cgcccactga
     1861 ccgacacaga gagggccacc ctgatggatg agccctaccg caagtccaag ctgacatatg
     1921 cacaggcaag gaagctgctg ggcctggagg acaccgcctt ctttaagggc ctgagatacg
     1981 gcaaggataa cgccgaggcc tctacactga tggagatgaa ggcctatcac gccatcagca
     2041 gggccctgga gaaggagggc ctgaaggaca agaagtcccc actgaatctg tctcccgagc
     2101 tgcaggatga gatcggcacc gcctttagcc tgttcaagac cgacgaggat atcacaggca
     2161 gactgaagga caggatccag ccagagatcc tggaggccct gctgaagcac atcagctttg
     2221 ataagttcgt gcagatcagc ctgaaggccc tgcggaggat cgtgccactg atggagcagg
     2281 gcaagaggta cgacgaggcc tgcgccgaaa tctacggcga tcactatggc aagaagaaca
     2341 cagaggagaa aatctacctg ccccctatcc ccgccgatga gatcaggaac cctgtggtgc
     2401 tgcgcgccct gtctcaggca agaaaagtga tcaacggagt ggtgcgccgg tacggcagcc
     2461 ccgccagaat ccacatcgag acagccaggg aagtgggcaa gtcctttaag gacagaaagg
     2521 agatcgagaa gaggcaggag gagaacagaa aggataggga gaaggccgcc gccaagttca
     2581 gagagtactt tcctaatttc gtgggcgagc caaagtccaa ggatatcctg aagctgaggc
     2641 tgtacgagca gcagcacggc aagtgtctgt attctggcaa ggagatcaac ctgggccgcc
     2701 tgaatgagaa gggctatgtg gagatcgacc acgccctgcc tttttctcgg acctgggacg
     2761 atagcttcaa caataaggtg ctggtgctgg gctctgagaa ccagaataag ggcaaccaga
     2821 caccctacga gtatttcaac ggcaaggaca atagccgcga gtggcaggag tttaaggcaa
     2881 gggtggagac aagcaggttc cctcggtcca agaagcagag aatcctgctg cagaagtttg
     2941 acgaggatgg cttcaaggag aggaacctga atgacacccg ctacgtgaat cggtttctgt
     3001 gccagttcgt ggccgataga atgaggctga ccggcaaggg caagaagaga gtgtttgcct
     3061 ccaacggcca gatcacaaat ctgctgaggg gcttctgggg cctgagaaag gtgagggcag
     3121 agaacgacag gcaccacgca ctggatgcag tggtggtggc atgttctacc gtggccatgc
     3181 agcagaagat cacacgcttt gtgcggtata aggagatgaa tgccttcgac ggcaagacca
     3241 tcgataagga gacaggcgag gtgctgcacc agaagacaca ctttcctcag ccatgggagt
     3301 tctttgccca ggaagtgatg atccgggtgt ttggcaagcc tgacggcaag ccagagttcg
     3361 aggaggccga taccctggag aagctgagaa cactgctggc agagaagctg agctccaggc
     3421 ccgaggcagt gcacgagtac gtgaccccac tgttcgtgtc tagagccccc aacaggaaga
     3481 tgagcggcca gggccacatg gagacagtga agtccgccaa gagactggac gagggcgtgt
     3541 ctgtgctgag ggtgcctctg acacagctga agctgaagga tctggagaag atggtgaatc
     3601 gcgagcggga gccaaagctg tatgaggccc tgaaggcaag gctggaggca cacaaggacg
     3661 atcctgccaa ggcctttgcc gagccattct acaagtatga taaggccggc aacagaaccc
     3721 agcaggtgaa ggccgtgagg gtggagcagg tgcagaagac aggcgtgtgg gtgcgcaacc
     3781 acaatggcat cgccgacaat gctaccatgg tgcgggtgga cgtgtttgag aagggcgata
     3841 agtactatct ggtgcccatc tacagctggc aggtggccaa gggcatcctg cctgatagag
     3901 ccgtggtgca gggcaaggac gaggaggatt ggcagctgat cgacgattcc ttcaacttta
     3961 agttctctct gcaccccaat gacctggtgg aagtgatcac caagaaggcc aggatgtttg
     4021 gctacttcgc ctcctgccac cgcggcacag gcaacatcaa tatccggatc cacgacctgg
     4081 atcacaagat cggcaagaac ggcatcctgg agggcatcgg cgtgaagaca gccctgagct
     4141 tccagaagta tcagatcgac gagctgggca aggagatcag accttgtagg ctgaagaagc
     4201 gcccacccgt gcgggaggat aagcggcccg cagcaaccaa gaaggcagga caggccaaga
     4261 agaagaagta cccctatgac gtgcctgact acgccgggta tccctacgac gtgcctgatt
     4321 acgccgggtc ctatccctac gacgtgccag attacgctgc agctccagca gcgaagaaaa
     4381 agaagctgga ttaagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc
     4441 ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga
     4501 attttttgtg tctctcactc ggcggccgca ggaaccccta gtgatggagt tggccactcc
     4561 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4621 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     4681 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     4741 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4801 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     4861 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4921 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     4981 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5041 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     5101 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     5161 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     5221 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5281 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5341 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5401 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5461 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5521 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5581 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5641 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5701 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5761 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5821 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5881 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5941 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6001 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6061 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6121 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6181 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6241 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6301 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6361 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6421 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6481 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6541 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6601 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6661 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6721 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6781 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6841 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6901 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6961 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7021 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7081 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7141 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7201 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7261 cacatgt
//
