LOCUS       Exported                7090 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  A catalytically inactive SaCas9 (dSaCas9) with the D10A and N580A 
            mutations. .
ACCESSION   .
VERSION     .
KEYWORDS    pX603-AAV-CMV::NLS-dSaCas9(D10A,N580A)-NLS-3xHA-bGHpA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7090)
  AUTHORS   Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B,
            Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F
  TITLE     In vivo genome editing using Staphylococcus aureus Cas9.
  JOURNAL   Nature. 2015 Apr 1. doi: 10.1038/nature14299.
  PUBMED    25830891
REFERENCE   2  (bases 1 to 7090)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2015 Apr 1. doi: 10.1038/nature14299."
FEATURES             Location/Qualifiers
     source          1..7090
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   42..182
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   53..182
                     /label=AAV2 ITR
     enhancer        202..581
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        582..785
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     736..756
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      804..813
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             816..836
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4017..4064
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4071..4151
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4179..4196)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4185..4392
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4405..4534
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4596..5184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4676..4695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5355..6215)
                     /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     5978..5997
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6216..6320)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6388..6406
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6444..6466)
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     6566..6585
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6602..7057)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6739..6760)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6951..6970
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"