LOCUS       Exported                7270 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  All-in-one AAV plasmid expressing Nme2Cas9 with sgRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    Nme2Cas9_AAV
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)
  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."
FEATURES             Location/Qualifiers
     source          1..7270
                     /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"
     CDS             3328..3375
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3376..3465
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3472..3498
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /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"
     repeat_region   3637..3766
                     /label=AAV2 ITR
     rep_origin      3852..4307
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3939..3958)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4149..4170
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     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
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4694..5554
                     /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(4912..4931)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5725..6313
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6214..6233
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     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"
     primer_bind     complement(6733..6752)
                     /label=LKO.1 5
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(6903..6923)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     regulatory      7259..7268
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"