LOCUS       40924_2244        7270 bp DNA     circular SYN 13-MAY-2021
DEFINITION  All-in-one AAV plasmid expressing Nme2Cas9 with sgRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7270)
  AUTHORS   Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, 
            Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ
  TITLE     A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo 
            Genome Editing.
  JOURNAL   Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 
            10.1016/j.molcel.2018.12.003.
  PUBMED    30581144
REFERENCE   2  (bases 1 to 7270)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7270)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 
            10.1016/j.molcel.2018.12.003."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..21
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             28..75
                     /codon_start=1
                     /label=nucleoplasmin NLS
                     /note="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3328..3375
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3376..3465
                     /codon_start=1
                     /label=3xHA
                     /note="three tandem HA epitope tags"
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3472..3498
                     /codon_start=1
                     /label=NLS
                     /note="nuclear localization signal (Makkerh et al., 1996)"
                     /translation="PAAKKKKLD"
     primer_bind     complement(3504..3523)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3569..3624
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   3637..3777
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      3852..4307
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4324..4343)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     4443..4465
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4503..4521)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        4589..4693
                     /label=AmpR promoter
     CDS             4694..5551
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5725..6313
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     repeat_region   6375..6504
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_RNA        complement(6532..6624)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(6683..6923)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     regulatory      7259..7268
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 cctaagaaga agagaaaggt ggaagataaa cgcccagcag ctacaaagaa ggcaggtcaa
       61 gccaagaaaa agaaaatggc cgccttcaag cctaacccaa tcaattacat cctgggactg
      121 gacatcggaa tcgcatccgt gggatgggct atggtggaga tcgacgagga ggagaatcct
      181 atccggctga tcgatctggg cgtgagagtg tttgagaggg ccgaggtgcc aaagaccggc
      241 gattctctgg ctatggcccg gagactggca cggagcgtga ggcgcctgac acggagaagg
      301 gcacacaggc tgctgagggc acgccggctg ctgaagagag agggcgtgct gcaggcagca
      361 gacttcgatg agaatggcct gatcaagagc ctgccaaaca ccccctggca gctgagagca
      421 gccgccctgg acaggaagct gacaccactg gagtggtctg ccgtgctgct gcacctgatc
      481 aagcaccgcg gctacctgag ccagcggaag aacgagggag agacagcaga caaggagctg
      541 ggcgccctgc tgaagggagt ggccaacaat gcccacgccc tgcagaccgg cgatttcagg
      601 acacctgccg agctggccct gaataagttt gagaaggagt ccggccacat cagaaaccag
      661 aggggcgact atagccacac cttctcccgc aaggatctgc aggccgagct gatcctgctg
      721 ttcgagaagc agaaggagtt tggcaatcca cacgtgagcg gaggcctgaa ggagggaatc
      781 gagaccctgc tgatgacaca gaggcctgcc ctgtccggcg acgcagtgca gaagatgctg
      841 ggacactgca ccttcgagcc tgcagagcca aaggccgcca agaacaccta cacagccgag
      901 cggtttatct ggctgacaaa gctgaacaat ctgagaatcc tggagcaggg atccgagagg
      961 ccactgaccg acacagagag ggccaccctg atggatgagc cttaccggaa gtctaagctg
     1021 acatatgccc aggccagaaa gctgctgggc ctggaggaca ccgccttctt taagggcctg
     1081 agatacggca aggataatgc cgaggcctcc acactgatgg agatgaaggc ctatcacgcc
     1141 atctctcgcg ccctggagaa ggagggcctg aaggacaaga agtcccccct gaacctgagc
     1201 tccgagctgc aggatgagat cggcaccgcc ttctctctgt ttaagaccga cgaggatatc
     1261 acaggccgcc tgaaggacag ggtgcagcct gagatcctgg aggccctgct gaagcacatc
     1321 tctttcgata agtttgtgca gatcagcctg aaggccctga gaaggatcgt gccactgatg
     1381 gagcagggca agcggtacga cgaggcctgc gccgagatct acggcgatca ctatggcaag
     1441 aagaacacag aggagaagat ctatctgccc cctatccctg ccgacgagat cagaaatcct
     1501 gtggtgctga gggccctgtc ccaggcaaga aaagtgatca acggagtggt gcgccggtac
     1561 ggatctccag cccggatcca catcgagacc gccagagaag tgggcaagag cttcaaggac
     1621 cggaaggaga tcgagaagag acaggaggag aatcgcaagg atcgggagaa ggccgccgcc
     1681 aagtttaggg agtacttccc taactttgtg ggcgagccaa agtctaagga catcctgaag
     1741 ctgcgcctgt acgagcagca gcacggcaag tgtctgtata gcggcaagga gatcaatctg
     1801 gtgcggctga acgagaaggg ctatgtggag atcgatcacg ccctgccttt ctccagaacc
     1861 tgggacgatt cttttaacaa taaggtgctg gtgctgggca gcgagaacca gaataagggc
     1921 aatcagacac catacgagta tttcaatggc aaggacaact ccagggagtg gcaggagttc
     1981 aaggcccgcg tggagacctc tagatttccc aggagcaaga agcagcggat cctgctgcag
     2041 aagttcgacg aggatggctt taaggagtgc aacctgaatg acaccagata cgtgaaccgg
     2101 ttcctgtgcc agtttgtggc cgatcacatc ctgctgaccg gcaagggcaa gagaagggtg
     2161 ttcgcctcta atggccagat cacaaacctg ctgaggggat tttggggact gaggaaggtg
     2221 cgggcagaga atgacagaca ccacgcactg gatgcagtgg tggtggcatg cagcaccgtg
     2281 gcaatgcagc agaagatcac aagattcgtg aggtataagg agatgaacgc ctttgacggc
     2341 aagaccatcg ataaggagac aggcaaggtg ctgcaccaga agacccactt cccccagcct
     2401 tgggagttct ttgcccagga agtgatgatc cgggtgttcg gcaagccaga cggcaagcct
     2461 gagtttgagg aggccgatac cccagagaag ctgaggacac tgctggcaga gaagctgtct
     2521 agcaggccag aggcagtgca cgagtacgtg accccactgt tcgtgtccag ggcacccaat
     2581 cggaagatgt ctggcgccca caaggacaca ctgagaagcg ccaagaggtt tgtgaagcac
     2641 aacgagaaga tctccgtgaa gagagtgtgg ctgaccgaga tcaagctggc cgatctggag
     2701 aacatggtga attacaagaa cggcagggag atcgagctgt atgaggccct gaaggcaagg
     2761 ctggaggcct acggaggaaa tgccaagcag gccttcgacc caaaggataa ccccttttat
     2821 aagaagggag gacagctggt gaaggccgtg cgggtggaga agacccagga gagcggcgtg
     2881 ctgctgaata agaagaacgc ctacacaatc gccgacaatg gcgatatggt gagagtggac
     2941 gtgttctgta aggtggataa gaagggcaag aatcagtact ttatcgtgcc tatctatgcc
     3001 tggcaggtgg ccgagaacat cctgccagac atcgattgca agggctacag aatcgacgat
     3061 agctatacat tctgtttttc cctgcacaag tatgacctga tcgccttcca gaaggatgag
     3121 aagtccaagg tggagtttgc ctactatatc aattgcgact cctctaacgg caggttctac
     3181 ctggcctggc acgataaggg cagcaaggag cagcagtttc gcatctccac ccagaatctg
     3241 gtgctgatcc agaagtatca ggtgaacgag ctgggcaagg agatcaggcc atgtcggctg
     3301 aagaagcgcc cacccgtgcg ggaggataag cggcccgcag caaccaagaa ggcaggacag
     3361 gccaagaaga agaagtaccc ctatgacgtg cctgactacg ccgggtatcc ctacgacgtg
     3421 cctgattacg ccgggtccta tccctacgac gtgccagatt acgctgcagc tccagcagcg
     3481 aagaaaaaga agctggatta agatcttttt ccctctgcca aaaattatgg ggacatcatg
     3541 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     3601 tgttggaatt ttttgtgtct ctcactcggc ggccgcagga acccctagtg atggagttgg
     3661 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     3721 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     3781 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     3841 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3901 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc
     3961 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4021 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     4081 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4141 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc
     4201 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta
     4261 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     4321 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     4381 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4441 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     4501 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     4561 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4621 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     4681 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     4741 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     4801 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     4861 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     4921 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     4981 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5041 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5101 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5161 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5221 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5281 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5341 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5401 tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5461 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5521 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5581 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5641 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5701 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5761 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5821 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     5881 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5941 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6001 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6061 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6121 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6181 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6241 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6301 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6361 ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc
     6421 gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg
     6481 ccaactccat cactaggggt tcctgcggcc tctagagttt aaacaaaaaa ataaacgatg
     6541 ccccttaaag cagaagcttt aaggggcaga gcgttgcggc acatcttttc agacggcctt
     6601 attgtagcaa cggttctcat ttcgtttccg aaatgagaaa gggagctaca actgaagagc
     6661 tggaatgctc ttccggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa
     6721 tactttcaag ttacggtaag catatgatag tccattttaa aacataattt taaaactgca
     6781 aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt
     6841 acagtcaaat taattctaat tatctctcta acagccttgt atcgtatatg caaatatgaa
     6901 ggaatcatgg gaaataggcc ctcttcctgc ccgaccttga cgtcgactct agaatggagg
     6961 cggtactatg tagatgagaa ttcaggagca aactgggaaa agcaactgct tccaaatatt
     7021 tgtgattttt acagtgtagt tttggaaaaa ctcttagcct accaattctt ctaagtgttt
     7081 taaaatgtgg gagccagtac acatgaagtt atagagtgtt ttaatgaggc ttaaatattt
     7141 accgtaacta tgaaatgcta cgcatatcat gctgttcagg ctccgtggcc acgcaactca
     7201 tacttaagca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaacaccgc
     7261 caccatggtg
//
