LOCUS       sequence-358582-        8602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector containing nuclease null SaCas9 fused to VPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8602)
  AUTHORS   Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian
            J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJ, Ebrahimkhani MR,
            Collins JJ, Weiss R, Church G
  TITLE     Cas9 gRNA engineering for genome editing, activation and repression.
  JOURNAL   Nat Methods. 2015 Sep 7. doi: 10.1038/nmeth.3580.
  PUBMED    26344044
REFERENCE   2  (bases 1 to 8602)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2015 Sep 7. doi: 10.1038/nmeth.3580."
COMMENT     Sequence Label: AAV_NLS-dSaCas9-NLS-VPR
FEATURES             Location/Qualifiers
     source          1..8602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     enhancer        161..540
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        541..744
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     695..715
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      763..772
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             775..795
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3976..4023
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4054..4203
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             4228..4248
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4678..5034
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             5053..5622
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     primer_bind     complement(5650..5667)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5656..5863
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5876..6005
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(6067..6655)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6147..6166)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6826..7617,7618..7686))
                     /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     7449..7468
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7687..7791)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7859..7877
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7915..7937)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8037..8056
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(8073..8528)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8210..8231)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8422..8441
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgcc tcgaggcgtt gacattgatt attgactagt
      181 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      241 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      301 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      361 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      421 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      481 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      541 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      601 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      661 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      721 tgggaggtct atataagcag agctctctgg ctaactaccg gtgccaccat ggccccaaag
      781 aagaagcgga aggtcggtat ccacggagtc ccagcagcca agcggaacta catcctgggc
      841 ctggccatcg gcatcaccag cgtgggctac ggcatcatcg actacgagac acgggacgtg
      901 atcgatgccg gcgtgcggct gttcaaagag gccaacgtgg aaaacaacga gggcaggcgg
      961 agcaagagag gcgccagaag gctgaagcgg cggaggcggc atagaatcca gagagtgaag
     1021 aagctgctgt tcgactacaa cctgctgacc gaccacagcg agctgagcgg catcaacccc
     1081 tacgaggcca gagtgaaggg cctgagccag aagctgagcg aggaagagtt ctctgccgcc
     1141 ctgctgcacc tggccaagag aagaggcgtg cacaacgtga acgaggtgga agaggacacc
     1201 ggcaacgagc tgtccaccaa agagcagatc agccggaaca gcaaggccct ggaagagaaa
     1261 tacgtggccg aactgcagct ggaacggctg aagaaagacg gcgaagtgcg gggcagcatc
     1321 aacagattca agaccagcga ctacgtgaaa gaagccaaac agctgctgaa ggtgcagaag
     1381 gcctaccacc agctggacca gagcttcatc gacacctaca tcgacctgct ggaaacccgg
     1441 cggacctact atgagggacc tggcgagggc agccccttcg gctggaagga catcaaagaa
     1501 tggtacgaga tgctgatggg ccactgcacc tacttccccg aggaactgcg gagcgtgaag
     1561 tacgcctaca acgccgacct gtacaacgcc ctgaacgacc tgaacaatct cgtgatcacc
     1621 agggacgaga acgagaagct ggaatattac gagaagttcc agatcatcga gaacgtgttc
     1681 aagcagaaga agaagcccac cctgaagcag atcgccaaag aaatcctcgt gaacgaagag
     1741 gatattaagg gctacagagt gaccagcacc ggcaagcccg agttcaccaa cctgaaggtg
     1801 taccacgaca tcaaggacat taccgcccgg aaagagatta ttgagaacgc cgagctgctg
     1861 gatcagattg ccaagatcct gaccatctac cagagcagcg aggacatcca ggaagaactg
     1921 accaatctga actccgagct gacccaggaa gagatcgagc agatctctaa tctgaagggc
     1981 tataccggca cccacaacct gagcctgaag gccatcaacc tgatcctgga cgagctgtgg
     2041 cacaccaacg acaaccagat cgctatcttc aaccggctga agctggtgcc caagaaggtg
     2101 gacctgtccc agcagaaaga gatccccacc accctggtgg acgacttcat cctgagcccc
     2161 gtcgtgaaga gaagcttcat ccagagcatc aaagtgatca acgccatcat caagaagtac
     2221 ggcctgccca acgacatcat tatcgagctg gcccgcgaga agaactccaa ggacgcccag
     2281 aaaatgatca acgagatgca gaagcggaac cggcagacca acgagcggat cgaggaaatc
     2341 atccggacca ccggcaaaga gaacgccaag tacctgatcg agaagatcaa gctgcacgac
     2401 atgcaggaag gcaagtgcct gtacagcctg gaagccatcc ctctggaaga tctgctgaac
     2461 aaccccttca actatgaggt ggaccacatc atccccagaa gcgtgtcctt cgacaacagc
     2521 ttcaacaaca aggtgctcgt gaagcaggaa gaagccagca agaagggcaa ccggacccca
     2581 ttccagtacc tgagcagcag cgacagcaag atcagctacg aaaccttcaa gaagcacatc
     2641 ctgaatctgg ccaagggcaa gggcagaatc agcaagacca agaaagagta tctgctggaa
     2701 gaacgggaca tcaacaggtt ctccgtgcag aaagacttca tcaaccggaa cctggtggat
     2761 accagatacg ccaccagagg cctgatgaac ctgctgcgga gctacttcag agtgaacaac
     2821 ctggacgtga aagtgaagtc catcaatggc ggcttcacca gctttctgcg gcggaagtgg
     2881 aagtttaaga aagagcggaa caaggggtac aagcaccacg ccgaggacgc cctgatcatt
     2941 gccaacgccg atttcatctt caaagagtgg aagaaactgg acaaggccaa aaaagtgatg
     3001 gaaaaccaga tgttcgagga aaagcaggcc gagagcatgc ccgagatcga aaccgagcag
     3061 gagtacaaag agatcttcat caccccccac cagatcaagc acattaagga cttcaaggac
     3121 tacaagtaca gccaccgggt ggacaagaag cctaatagag agctgattaa cgacaccctg
     3181 tactccaccc ggaaggacga caagggcaac accctgatcg tgaacaatct gaacggcctg
     3241 tacgacaagg acaatgacaa gctgaaaaag ctgatcaaca agagccccga aaagctgctg
     3301 atgtaccacc acgaccccca gacctaccag aaactgaagc tgattatgga acagtacggc
     3361 gacgagaaga atcccctgta caagtactac gaggaaaccg ggaactacct gaccaagtac
     3421 tccaaaaagg acaacggccc cgtgatcaag aagattaagt attacggcaa caaactgaac
     3481 gcccatctgg acatcaccga cgactacccc aacagcagaa acaaggtcgt gaagctgtcc
     3541 ctgaagccct acagattcga cgtgtacctg gacaatggcg tgtacaagtt cgtgaccgtg
     3601 aagaatctgg atgtgatcaa aaaagaaaac tactacgaag tgaatagcaa gtgctatgag
     3661 gaagctaaga agctgaagaa gatcagcaac caggccgagt ttatcgcctc cttctacaac
     3721 aacgatctga tcaagatcaa cggcgagctg tatagagtga tcggcgtgaa caacgacctg
     3781 ctgaaccgga tcgaagtgaa catgatcgac atcacctacc gcgagtacct ggaaaacatg
     3841 aacgacaaga ggccccccag gatcattaag acaatcgcct ccaagaccca gagcattaag
     3901 aagtacagca cagacattct gggcaacctg tatgaagtga aatctaagaa gcaccctcag
     3961 atcatcaaaa agggcaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa
     4021 aagggatccg aggccagcgg ttccggacgg gctgacgcat tggacgattt tgatctggat
     4081 atgctgggaa gtgacgccct cgatgatttt gaccttgaca tgcttggttc ggatgccctt
     4141 gatgactttg acctcgacat gctcggcagt gacgcccttg atgatttcga cctggacatg
     4201 ctgattaact ctagaagttc cggatctccg aaaaagaaac gcaaagttgg tagccagtac
     4261 ctgcccgaca ccgacgaccg gcaccggatc gaggaaaagc ggaagcggac ctacgagaca
     4321 ttcaagagca tcatgaagaa gtcccccttc agcggcccca ccgaccctag acctccacct
     4381 agaagaatcg ccgtgcccag cagatccagc gccagcgtgc caaaacctgc cccccagcct
     4441 taccccttca ccagcagcct gagcaccatc aactacgacg agttccctac catggtgttc
     4501 cccagcggcc agatctctca ggcctctgct ctggctccag cccctcctca ggtgctgcct
     4561 caggctcctg ctcctgcacc agctccagcc atggtgtctg cactggctca ggcaccagca
     4621 cccgtgcctg tgctggctcc tggacctcca caggctgtgg ctccaccagc ccctaaacct
     4681 acacaggccg gcgagggcac actgtctgaa gctctgctgc agctgcagtt cgacgacgag
     4741 gatctgggag ccctgctggg aaacagcacc gatcctgccg tgttcaccga cctggccagc
     4801 gtggacaaca gcgagttcca gcagctgctg aaccagggca tccctgtggc ccctcacacc
     4861 accgagccca tgctgatgga ataccccgag gccatcaccc ggctcgtgac aggcgctcag
     4921 aggcctcctg atccagctcc tgcccctctg ggagcaccag gcctgcctaa tggactgctg
     4981 tctggcgacg aggacttcag ctctatcgcc gatatggatt tctcagcctt gctgggctct
     5041 ggcagcggca gccgggattc cagggaaggg atgtttttgc cgaagcctga ggccggctcc
     5101 gctattagtg acgtgtttga gggccgcgag gtgtgccagc caaaacgaat ccggccattt
     5161 catcctccag gaagtccatg ggccaaccgc ccactccccg ccagcctcgc accaacacca
     5221 accggtccag tacatgagcc agtcgggtca ctgaccccgg caccagtccc tcagccactg
     5281 gatccagcgc ccgcagtgac tcccgaggcc agtcacctgt tggaggatcc cgatgaagag
     5341 acgagccagg ctgtcaaagc ccttcgggag atggccgata ctgtgattcc ccagaaggaa
     5401 gaggctgcaa tctgtggcca aatggacctt tcccatccgc ccccaagggg ccatctggat
     5461 gagctgacaa ccacacttga gtccatgacc gaggatctga acctggactc acccctgacc
     5521 ccggaattga acgagattct ggataccttc ctgaacgacg agtgcctctt gcatgccatg
     5581 catatcagca caggactgtc catcttcgac acatctctgt tttgagaatt cctagagctc
     5641 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     5701 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     5761 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     5821 gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagctagag gccgcaggaa
     5881 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     5941 cgaccaaagg tcgcccgacg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     6001 gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6061 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6121 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6181 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6241 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6301 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6361 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6421 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6481 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     6541 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6601 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6661 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6721 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6781 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6841 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6901 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6961 accgcggctt ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7021 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7081 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7141 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7201 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7261 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7321 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7381 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7441 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7501 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7561 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7621 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7681 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     7741 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7801 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     7861 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     7921 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     7981 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     8041 atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata ttttgttaaa
     8101 attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg aaatcggcaa
     8161 aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc cagtttggaa
     8221 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     8281 gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg
     8341 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     8401 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta aggcgctggc
     8461 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     8521 gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag atgcgtaagg
     8581 agaaaatacc gcatcaggcg cc
//
