LOCUS       Exported                7225 bp ds-DNA     circular SYN 04-JAN-2021
DEFINITION  All-in-one AAV plasmid expressing Nme2Cas9 with sgRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    15-Nme2Cas9_AAV
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7225)
  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 7225)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Sep 16, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7225
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1..21
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             28..75
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(51..75)
                     /label=nme2Cas9-R
     CDS             76..3282
                     /codon_start=1
                     /label=15-nme2
                     /translation="SHFRLKQLNYILGLDLGIASVGWAVVEIDTEENPTRLIDVGVRTF
                     ERAEVPKTGDSLAAARRQARSARRLIRRRTHRLLRARRLLKREGILHADDLNAEGLVKE
                     LPNTPWQLRVDGLDRKLMPKEWAAVLLHLVKHRGYLSQRKNESEGADKELGRLLAGVHE
                     NHQALQSSEYRTPAELAVKKFQAQSGHIRNQRGDYTHTFDRKDLQQELHQLFAAQQKYG
                     NPFVSDGLKQGIETLLMQQRPALSGDAVQKMLGRCTFETEEFKAAKHTYSAERFIWLTK
                     LNNLRIQENGNERCLTAQERGQIIDLPYKKAKLTYAQVRKELSLPENAFFKGLRYGKEN
                     AETAALMEMKAYHAIRKALEKAGLKTEWQGLACKPELLDAIGTVFSLYKTDEDISTRLQ
                     GQLSEDVINALLAGLNFDKFIQLSLKALSKILPLMEQGQRYDEAHAALYVRSTPQPQRL
                     LPQITADEICNPVVLRTLSQARKVINAIVRRYGSPARIHIETGREVGKSFKDRQEIQKR
                     QEDNHKERKQAAEKFLEYFPNFVGQPKAKDILKLRLYQHQHGQCLYSGKTLDICRLNEK
                     GYAEIDHALPFSRTWDDSFNNKVLVLAGENQNKGNQTPYEWLGGASNSPRWQEFVARVG
                     SSRFHHSKKQRILLRQLDEEGFKERNLNDTRYVARFLCKFIEENMLLLGRGKRRVFASN
                     GQITSFLRSRWGLRKVREENDRHHALDAVVVACSTVAIQQRITYFVRQREMNIFDGELI
                     DKETSEVSRPYFPQPWDFFRQEVMMRVFGKPDGDPSFPEADTKETLCTLLETRLSSRPQ
                     AVHEYVTPLFVSRAPTRKMSGQGHKETVKSAKLLHEGKSVLRVPLTQLKPNLLEKMVNR
                     DREPQLYEALKERLAAYGNDPAKAFATEFRKKGGQLVKAVRVEDTQKSGVLLHKGTGVA
                     DNATMVRVDVFEKSGKYYLVPIYSWQVAQGILPNKAVIQGKDEKNWDEMDETYTFKFSM
                     YPNDLVEINTGKQRLFGYYIGLDRATGAISIREHDLDNFKGKNGIYRVGVKTAISFKKY
                     QTDELGKVVVPCRPKKRLPVR"
     misc_feature    2878..3282
                     /label=15PI
     CDS             3283..3330
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     3283..3300
                     /label=nme2Cas9-F
     primer_bind     3283..3294
                     /label=Nme2PI-F
     CDS             3331..3420
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3427..3453
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     primer_bind     complement(3459..3478)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_site      3524..3579
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     repeat_region   3592..3732
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3592..3721
                     /label=AAV2 ITR
     primer_bind     3736..3754
                     /label=ORI-F
     rep_origin      3807..4262
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3894..3913)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4104..4125
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4279..4298)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4398..4420
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4458..4476)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4544..4648
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4649..5509
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4867..4886)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5680..6268
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6169..6188
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6304..6321)
                     /label=ORI-R
     repeat_region   6330..6459
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_RNA        complement(6487..6579)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(6638..6878)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(6688..6707)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(6858..6878)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     regulatory      7214..7223
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
ORIGIN
        1 cctaagaaga agagaaaggt ggaagataaa cgcccagcag ctacaaagaa ggcaggtcaa
       61 gccaagaaaa agaaatccca cttcagactg aaacagctga actacatcct cggacttgac
      121 ctgggcatcg ccagcgtggg atgggctgta gtggaaatcg acaccgaaga gaaccccacc
      181 agactgatcg atgtgggcgt tcggaccttc gaacgggccg aggttccaaa aaccggcgac
      241 tctctggctg ctgcgagacg gcaggccaga agcgccagac ggctgatcag gcggcgcacc
      301 cacaggctgc tgagagcccg gcgcctactt aagagggaag gcatcttgca tgcggatgac
      361 ctgaacgccg agggcctggt gaaggagctg cccaataccc cttggcagct tcgggtcgac
      421 ggactcgata gaaagctgat gcctaaggaa tgggccgccg tgctgctgca tctggttaaa
      481 cacagaggat acctgtcaca gaggaaaaac gagagcgaag gtgccgacaa ggaactgggc
      541 cggctgctgg ctggcgtgca cgagaaccac caggccctgc agagctctga gtacagaacc
      601 cctgctgaac tggccgtgaa aaagtttcag gctcagagcg gccacatccg gaaccagaga
      661 ggcgactaca cccacacttt tgacagaaag gacctgcagc aggagctgca ccagcttttc
      721 gccgcccagc agaagtacgg taatcccttc gtgtccgatg gcctgaagca gggcattgag
      781 acactgctga tgcagcaaag acccgccctg tccggcgacg ccgtgcagaa gatgctgggc
      841 agatgcacct tcgagacaga agagtttaag gcagccaagc acacctacag cgccgagaga
      901 ttcatctggc tgaccaagct gaacaatctg cggatccaag agaacggcaa cgagagatgt
      961 ctgaccgccc aggaaagagg ccagatcatt gacctgccat ataagaaggc caaactgaca
     1021 tacgcccagg tgagaaagga gctgtccctg cctgagaatg cttttttcaa gggcctgcgg
     1081 tacggcaaag agaatgccga gaccgccgcc ctcatggaaa tgaaagccta ccacgccatc
     1141 cggaaggccc tggaaaaagc cggcctgaaa accgagtggc aaggcctggc ttgcaagcct
     1201 gaactgctgg atgccatcgg caccgtgttc agcctgtaca agacagacga agatatctcc
     1261 acaagactgc aggggcagct gagcgaggac gtgatcaacg ccctgctggc tggactaaac
     1321 ttcgacaagt tcatccagct gagcctgaaa gctttgtcca agatcttgcc tctgatggaa
     1381 cagggccaac gctacgacga agcccacgcc gccctgtacg tgagaagcac acctcagcct
     1441 cagcggctgc tgccacagat cacagccgac gagatctgca accccgtggt gctgagaacc
     1501 ctgagccagg ccaggaaggt gatcaacgct atcgtgcgga gatacggctc tccagccaga
     1561 atccacatcg agacaggacg ggaagtcggc aagagcttca aggacagaca ggagattcag
     1621 aagagacagg aggataacca caaggaacgg aagcaggccg ccgaaaagtt cctggaatac
     1681 ttccccaact tcgtgggcca gccaaaggcc aaggacatcc tgaagctgcg gctgtaccag
     1741 caccagcacg gccagtgcct ctacagcggc aagacccttg acatctgtag actgaatgag
     1801 aaaggctacg ccgagattga ccacgccctg cctttcagcc ggacctggga cgacagcttt
     1861 aacaacaagg tgctggtcct ggctggagag aaccagaaca aaggcaatca gacaccttac
     1921 gagtggctgg gaggagcctc taacagccct agatggcagg aattcgtggc cagagtggga
     1981 tctagccggt ttcaccacag caagaagcag cggatcctgc tcagacagct agacgaggag
     2041 ggctttaagg aaagaaacct gaacgacacc agatacgtgg ccagattcct ctgtaaattc
     2101 atcgaggaaa acatgctgct gctgggacgc ggaaagagaa gggtgttcgc tagcaacggc
     2161 cagatcacca gcttcctgag aagcagatgg ggcctgagaa aggtgagaga ggaaaatgac
     2221 cggcaccacg ccctggatgc cgtggtggtg gcctgcagca ccgtggccat tcagcaaaga
     2281 atcacatatt tcgtcagaca gcgcgaaatg aacatcttcg atggcgagct gatcgataag
     2341 gagaccagcg aagtctcaag accttacttt cctcagcctt gggatttctt cagacaggaa
     2401 gtgatgatgc gggtgttcgg caagcccgac ggcgacccta gcttccctga ggccgacaca
     2461 aaggaaacgc tgtgcaccct gctggagacc cggctgagct ctagacctca agctgtgcac
     2521 gagtatgtga ctcccctgtt cgtgtctaga gcccctaccc ggaagatgag cggccaggga
     2581 cacaaggaaa ccgtgaagtc tgccaagctg ctgcatgagg gcaagtccgt gctgagagtg
     2641 cctctgaccc agctgaaacc aaacctgctg gaaaaaatgg tgaacagaga tagagagcct
     2701 cagctgtacg aggccctgaa ggagagactg gccgcttatg gcaatgatcc cgccaaggcc
     2761 ttcgccaccg agttcagaaa gaagggcgga caactggtga aggctgtccg cgttgaagat
     2821 acacagaagt ctggcgtgct gctgcacaag ggcaccggcg tggccgataa cgccacaatg
     2881 gtgcgggtgg acgtgttcga gaagtccgga aagtactacc tggtgcctat ctacagttgg
     2941 caagtggccc agggaatcct gcctaacaag gccgtgatcc agggcaagga cgagaagaac
     3001 tgggatgaga tggacgagac ctacacattc aagtttagca tgtaccctaa tgacctggtg
     3061 gaaatcaaca caggcaaaca acgactgttt ggctattaca tcggcctgga ccgggccaca
     3121 ggcgccatca gcatcagaga gcacgacctg gacaatttca aggggaagaa cggcatctac
     3181 cgggtgggcg tgaaaaccgc aatcagcttt aaaaagtacc agaccgatga gctgggcaag
     3241 gtggtggtgc cctgcagacc taagaaaaga ctgcccgtga ggaagcggcc cgcagcaacc
     3301 aagaaggcag gacaggccaa gaagaagaag tacccctatg acgtgcctga ctacgccggg
     3361 tatccctacg acgtgcctga ttacgccggg tcctatccct acgacgtgcc agattacgct
     3421 gcagctccag cagcgaagaa aaagaagctg gattaagatc tttttccctc tgccaaaaat
     3481 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     3541 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggcggccg caggaacccc
     3601 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     3661 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     3721 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3781 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     3841 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     3901 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3961 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4021 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4081 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     4141 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     4201 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     4261 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     4321 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4381 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4441 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4501 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4561 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4621 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4681 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4741 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4801 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4861 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4921 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4981 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5041 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5101 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5161 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5221 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5281 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5341 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     5401 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5461 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5521 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5581 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5641 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5701 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5761 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5821 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5881 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5941 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6001 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6061 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6121 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6181 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6241 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6301 ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac
     6361 tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
     6421 gcagagaggg agtggccaac tccatcacta ggggttcctg cggcctctag agtttaaaca
     6481 aaaaaataaa cgatgcccct taaagcagaa gctttaaggg gcagagcgtt gcggcacatc
     6541 ttttcagacg gccttattgt agcaacggtt ctcatttcgt ttccgaaatg agaaagggag
     6601 ctacaactga agagctggaa tgctcttccg gtgtttcgtc ctttccacaa gatatataaa
     6661 gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat tttaaaacat
     6721 aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt attttgtact
     6781 aatatctttg tgtttacagt caaattaatt ctaattatct ctctaacagc cttgtatcgt
     6841 atatgcaaat atgaaggaat catgggaaat aggccctctt cctgcccgac cttgacgtcg
     6901 actctagaat ggaggcggta ctatgtagat gagaattcag gagcaaactg ggaaaagcaa
     6961 ctgcttccaa atatttgtga tttttacagt gtagttttgg aaaaactctt agcctaccaa
     7021 ttcttctaag tgttttaaaa tgtgggagcc agtacacatg aagttataga gtgttttaat
     7081 gaggcttaaa tatttaccgt aactatgaaa tgctacgcat atcatgctgt tcaggctccg
     7141 tggccacgca actcatactt aagcagacag tggttcaaag tttttttctt ccatttcagg
     7201 tgtcgtgaac accgccacca tggtg
//
