LOCUS       sequence-442882-        7050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2nd gen. AAV backbone for dSaCas9 (endonuclease dead Cas9 from 
            Staphylococcus aureus) and Sa-gRNA Scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7050)
  AUTHORS   Kantor B, Odonovan B, Rittiner J, Hodgson D, Lindner N, Guerrero S, 
            Dong W, Zhang A, Chiba-Falek O
  TITLE     All-in-one AAV-delivered epigenome-editing platform: 
            proof-of-concept and therapeutic implications for neurodegenerative 
            disorders.
  JOURNAL   bioRxiv [Preprint]. 2024 May 19:2023.04.14.536951. doi: 
            10.1101/2023.04.14.536951.
  PUBMED    38798630
REFERENCE   2  (bases 1 to 7050)
  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: "bioRxiv 
            [Preprint]. 2024 May 19:2023.04.14.536951. doi: 
            10.1101/2023.04.14.536951."
COMMENT     Sequence Label: pBK2043-AAV-2xSp1-2xNFkB-EFSNC-dSaCas9
FEATURES             Location/Qualifiers
     source          1..7050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      167..176
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             176..241
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             251..271
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             275..295
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3476..3523
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3590..3610
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3620..3668
                     /note="synthetic polyadenylation signal"
     promoter        3680..3928
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3680..3700
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3851..3870
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3949..4024
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4038..4178
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4038..4167
                     /label=AAV2 ITR
     rep_origin      4253..4708
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4340..4359)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4550..4571
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4725..4744)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4844..4866
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4904..4922)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4990..5094
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5095..5163,5164..5955)
                     /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(5313..5332)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6126..6714
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6615..6634
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6776..6916
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6787..6916
                     /label=AAV2 ITR
     promoter        join(6998..7050,1..159)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
       61 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
      121 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggtgcca ccatggacta
      181 caaagaccat gacggtgatt ataaagatca tgacatagat tacaaggatg acgatgacaa
      241 gggtggatcg ccaaagaaaa agaggaaagt cgccccaaag aagaagcgga aggtcggtat
      301 ccacggagtc ccagcagcca agcggaacta catcctgggc ctggccatcg gcatcaccag
      361 cgtgggctac ggcatcatcg actacgagac acgggacgtg atcgatgccg gcgtgcggct
      421 gttcaaagag gccaacgtgg aaaacaacga gggcaggcgg agcaagagag gcgccagaag
      481 gctgaagcgg cggaggcggc atagaatcca gagagtgaag aagctgctgt tcgactacaa
      541 cctgctgacc gaccacagcg agctgagcgg catcaacccc tacgaggcca gagtgaaggg
      601 cctgagccag aagctgagcg aggaagagtt ctctgccgcc ctgctgcacc tggccaagag
      661 aagaggcgtg cacaacgtga acgaggtgga agaggacacc ggcaacgagc tgtccaccaa
      721 agagcagatc agccggaaca gcaaggccct ggaagagaaa tacgtggccg aactgcagct
      781 ggaacggctg aagaaagacg gcgaagtgcg gggcagcatc aacagattca agaccagcga
      841 ctacgtgaaa gaagccaaac agctgctgaa ggtgcagaag gcctaccacc agctggacca
      901 gagcttcatc gacacctaca tcgacctgct ggaaacccgg cggacctact atgagggacc
      961 tggcgagggc agccccttcg gctggaagga catcaaagaa tggtacgaga tgctgatggg
     1021 ccactgcacc tacttccccg aggaactgcg gagcgtgaag tacgcctaca acgccgacct
     1081 gtacaacgcc ctgaacgacc tgaacaatct cgtgatcacc agggacgaga acgagaagct
     1141 ggaatattac gagaagttcc agatcatcga gaacgtgttc aagcagaaga agaagcccac
     1201 cctgaagcag atcgccaaag aaatcctcgt gaacgaagag gatattaagg gctacagagt
     1261 gaccagcacc ggcaagcccg agttcaccaa cctgaaggtg taccacgaca tcaaggacat
     1321 taccgcccgg aaagagatta ttgagaacgc cgagctgctg gatcagattg ccaagatcct
     1381 gaccatctac cagagcagcg aggacatcca ggaagaactg accaatctga actccgagct
     1441 gacccaggaa gagatcgagc agatctctaa tctgaagggc tataccggca cccacaacct
     1501 gagcctgaag gccatcaacc tgatcctgga cgagctgtgg cacaccaacg acaaccagat
     1561 cgctatcttc aaccggctga agctggtgcc caagaaggtg gacctgtccc agcagaaaga
     1621 gatccccacc accctggtgg acgacttcat cctgagcccc gtcgtgaaga gaagcttcat
     1681 ccagagcatc aaagtgatca acgccatcat caagaagtac ggcctgccca acgacatcat
     1741 tatcgagctg gcccgcgaga agaactccaa ggacgcccag aaaatgatca acgagatgca
     1801 gaagcggaac cggcagacca acgagcggat cgaggaaatc atccggacca ccggcaaaga
     1861 gaacgccaag tacctgatcg agaagatcaa gctgcacgac atgcaggaag gcaagtgcct
     1921 gtacagcctg gaagccatcc ctctggaaga tctgctgaac aaccccttca actatgaggt
     1981 ggaccacatc atccccagaa gcgtgtcctt cgacaacagc ttcaacaaca aggtgctcgt
     2041 gaagcaggaa gaagccagca agaagggcaa ccggacccca ttccagtacc tgagcagcag
     2101 cgacagcaag atcagctacg aaaccttcaa gaagcacatc ctgaatctgg ccaagggcaa
     2161 gggcagaatc agcaagacca agaaagagta tctgctggaa gaacgggaca tcaacaggtt
     2221 ctccgtgcag aaagacttca tcaaccggaa cctggtggat accagatacg ccaccagagg
     2281 cctgatgaac ctgctgcgga gctacttcag agtgaacaac ctggacgtga aagtgaagtc
     2341 catcaatggc ggcttcacca gctttctgcg gcggaagtgg aagtttaaga aagagcggaa
     2401 caaggggtac aagcaccacg ccgaggacgc cctgatcatt gccaacgccg atttcatctt
     2461 caaagagtgg aagaaactgg acaaggccaa aaaagtgatg gaaaaccaga tgttcgagga
     2521 aaagcaggcc gagagcatgc ccgagatcga aaccgagcag gagtacaaag agatcttcat
     2581 caccccccac cagatcaagc acattaagga cttcaaggac tacaagtaca gccaccgggt
     2641 ggacaagaag cctaatagag agctgattaa cgacaccctg tactccaccc ggaaggacga
     2701 caagggcaac accctgatcg tgaacaatct gaacggcctg tacgacaagg acaatgacaa
     2761 gctgaaaaag ctgatcaaca agagccccga aaagctgctg atgtaccacc acgaccccca
     2821 gacctaccag aaactgaagc tgattatgga acagtacggc gacgagaaga atcccctgta
     2881 caagtactac gaggaaaccg ggaactacct gaccaagtac tccaaaaagg acaacggccc
     2941 cgtgatcaag aagattaagt attacggcaa caaactgaac gcccatctgg acatcaccga
     3001 cgactacccc aacagcagaa acaaggtcgt gaagctgtcc ctgaagccct acagattcga
     3061 cgtgtacctg gacaatggcg tgtacaagtt cgtgaccgtg aagaatctgg atgtgatcaa
     3121 aaaagaaaac tactacgaag tgaatagcaa gtgctatgag gaagctaaga agctgaagaa
     3181 gatcagcaac caggccgagt ttatcgcctc cttctacaac aacgatctga tcaagatcaa
     3241 cggcgagctg tatagagtga tcggcgtgaa caacgacctg ctgaaccgga tcgaagtgaa
     3301 catgatcgac atcacctacc gcgagtacct ggaaaacatg aacgacaaga ggccccccag
     3361 gatcattaag acaatcgcct ccaagaccca gagcattaag aagtacagca cagacattct
     3421 gggcaacctg tatgaagtga aatctaagaa gcaccctcag atcatcaaaa agggcaaaag
     3481 gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagggatccg gtggaggaag
     3541 tggcgggtca gggtcgggtg gcactagtat agctagcgga ggtggttcgc caaagaagaa
     3601 acggaaggtg ggatcctgaa ataaaagatc tttgttttca ttagatctgt gtgttggttt
     3661 tttgtgtgcg gccggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     3721 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     3781 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     3841 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     3901 atatatcttg tggaaaggac gaaacaccgg agaccacggc aggtctcagt tttagtactc
     3961 tggaaacaga atctactaaa acaaggcaaa atgccgtgtt tatctcgtca acttgttggc
     4021 gagatttttg cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4081 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4141 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4201 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4261 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4321 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4381 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4441 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4501 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4561 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     4621 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4681 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     4741 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4801 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4861 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4921 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4981 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5041 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5101 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5161 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5221 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5281 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5341 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5401 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5461 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5521 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5581 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5641 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5701 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5761 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     5821 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5881 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5941 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6001 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6061 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6121 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6181 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6241 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6301 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6361 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6421 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6481 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6541 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6601 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6661 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6721 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtcctgc
     6781 aggcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
     6841 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
     6901 atcactaggg gttcctacgc gtgggcggga cgttaacggg ggcggaacgt taacgggcgt
     6961 aggggacttt ccgttaacgc ggggactttc cgtcgacggg cagagcgcac atcgcccaca
     7021 gtccccgaga agttgggggg aggggtcggc
//
