LOCUS       sequence-442890-        7295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of truncated HP1b with dSaCas9 and empty gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7295)
  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 7295)
  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: pBK1307-AAV-EFSNC-dSaCas9-HP1b(79-173)
FEATURES             Location/Qualifiers
     source          1..7295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        186..397
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      405..414
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             414..479
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             489..509
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             513..533
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3714..3761
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4110..4130
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4140..4188
                     /note="synthetic polyadenylation signal"
     promoter        4200..4448
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4200..4220
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4371..4390
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4469..4544
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4558..4698
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4558..4687
                     /label=AAV2 ITR
     rep_origin      4773..5228
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4860..4879)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5070..5091
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5245..5264)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5364..5386
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5424..5442)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5510..5614
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5615..5683,5684..6475)
                     /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(5833..5852)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6646..7234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7135..7154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gctaggtctt gaaaggagtg ggaattggct ccggtgcccg
      181 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
      241 ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
      301 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
      361 cgttcttttt cgcaacgggt ttgccgccag aacacaggac cggtgccacc atggactaca
      421 aagaccatga cggtgattat aaagatcatg acatagatta caaggatgac gatgacaagg
      481 gtggatcgcc aaagaaaaag aggaaagtcg ccccaaagaa gaagcggaag gtcggtatcc
      541 acggagtccc agcagccaag cggaactaca tcctgggcct ggccatcggc atcaccagcg
      601 tgggctacgg catcatcgac tacgagacac gggacgtgat cgatgccggc gtgcggctgt
      661 tcaaagaggc caacgtggaa aacaacgagg gcaggcggag caagagaggc gccagaaggc
      721 tgaagcggcg gaggcggcat agaatccaga gagtgaagaa gctgctgttc gactacaacc
      781 tgctgaccga ccacagcgag ctgagcggca tcaaccccta cgaggccaga gtgaagggcc
      841 tgagccagaa gctgagcgag gaagagttct ctgccgccct gctgcacctg gccaagagaa
      901 gaggcgtgca caacgtgaac gaggtggaag aggacaccgg caacgagctg tccaccaaag
      961 agcagatcag ccggaacagc aaggccctgg aagagaaata cgtggccgaa ctgcagctgg
     1021 aacggctgaa gaaagacggc gaagtgcggg gcagcatcaa cagattcaag accagcgact
     1081 acgtgaaaga agccaaacag ctgctgaagg tgcagaaggc ctaccaccag ctggaccaga
     1141 gcttcatcga cacctacatc gacctgctgg aaacccggcg gacctactat gagggacctg
     1201 gcgagggcag ccccttcggc tggaaggaca tcaaagaatg gtacgagatg ctgatgggcc
     1261 actgcaccta cttccccgag gaactgcgga gcgtgaagta cgcctacaac gccgacctgt
     1321 acaacgccct gaacgacctg aacaatctcg tgatcaccag ggacgagaac gagaagctgg
     1381 aatattacga gaagttccag atcatcgaga acgtgttcaa gcagaagaag aagcccaccc
     1441 tgaagcagat cgccaaagaa atcctcgtga acgaagagga tattaagggc tacagagtga
     1501 ccagcaccgg caagcccgag ttcaccaacc tgaaggtgta ccacgacatc aaggacatta
     1561 ccgcccggaa agagattatt gagaacgccg agctgctgga tcagattgcc aagatcctga
     1621 ccatctacca gagcagcgag gacatccagg aagaactgac caatctgaac tccgagctga
     1681 cccaggaaga gatcgagcag atctctaatc tgaagggcta taccggcacc cacaacctga
     1741 gcctgaaggc catcaacctg atcctggacg agctgtggca caccaacgac aaccagatcg
     1801 ctatcttcaa ccggctgaag ctggtgccca agaaggtgga cctgtcccag cagaaagaga
     1861 tccccaccac cctggtggac gacttcatcc tgagccccgt cgtgaagaga agcttcatcc
     1921 agagcatcaa agtgatcaac gccatcatca agaagtacgg cctgcccaac gacatcatta
     1981 tcgagctggc ccgcgagaag aactccaagg acgcccagaa aatgatcaac gagatgcaga
     2041 agcggaaccg gcagaccaac gagcggatcg aggaaatcat ccggaccacc ggcaaagaga
     2101 acgccaagta cctgatcgag aagatcaagc tgcacgacat gcaggaaggc aagtgcctgt
     2161 acagcctgga agccatccct ctggaagatc tgctgaacaa ccccttcaac tatgaggtgg
     2221 accacatcat ccccagaagc gtgtccttcg acaacagctt caacaacaag gtgctcgtga
     2281 agcaggaaga agccagcaag aagggcaacc ggaccccatt ccagtacctg agcagcagcg
     2341 acagcaagat cagctacgaa accttcaaga agcacatcct gaatctggcc aagggcaagg
     2401 gcagaatcag caagaccaag aaagagtatc tgctggaaga acgggacatc aacaggttct
     2461 ccgtgcagaa agacttcatc aaccggaacc tggtggatac cagatacgcc accagaggcc
     2521 tgatgaacct gctgcggagc tacttcagag tgaacaacct ggacgtgaaa gtgaagtcca
     2581 tcaatggcgg cttcaccagc tttctgcggc ggaagtggaa gtttaagaaa gagcggaaca
     2641 aggggtacaa gcaccacgcc gaggacgccc tgatcattgc caacgccgat ttcatcttca
     2701 aagagtggaa gaaactggac aaggccaaaa aagtgatgga aaaccagatg ttcgaggaaa
     2761 agcaggccga gagcatgccc gagatcgaaa ccgagcagga gtacaaagag atcttcatca
     2821 ccccccacca gatcaagcac attaaggact tcaaggacta caagtacagc caccgggtgg
     2881 acaagaagcc taatagagag ctgattaacg acaccctgta ctccacccgg aaggacgaca
     2941 agggcaacac cctgatcgtg aacaatctga acggcctgta cgacaaggac aatgacaagc
     3001 tgaaaaagct gatcaacaag agccccgaaa agctgctgat gtaccaccac gacccccaga
     3061 cctaccagaa actgaagctg attatggaac agtacggcga cgagaagaat cccctgtaca
     3121 agtactacga ggaaaccggg aactacctga ccaagtactc caaaaaggac aacggccccg
     3181 tgatcaagaa gattaagtat tacggcaaca aactgaacgc ccatctggac atcaccgacg
     3241 actaccccaa cagcagaaac aaggtcgtga agctgtccct gaagccctac agattcgacg
     3301 tgtacctgga caatggcgtg tacaagttcg tgaccgtgaa gaatctggat gtgatcaaaa
     3361 aagaaaacta ctacgaagtg aatagcaagt gctatgagga agctaagaag ctgaagaaga
     3421 tcagcaacca ggccgagttt atcgcctcct tctacaacaa cgatctgatc aagatcaacg
     3481 gcgagctgta tagagtgatc ggcgtgaaca acgacctgct gaaccggatc gaagtgaaca
     3541 tgatcgacat cacctaccgc gagtacctgg aaaacatgaa cgacaagagg ccccccagga
     3601 tcattaagac aatcgcctcc aagacccaga gcattaagaa gtacagcaca gacattctgg
     3661 gcaacctgta tgaagtgaaa tctaagaagc accctcagat catcaaaaag ggcaaaaggc
     3721 cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gggatccggt ggaggaagtg
     3781 gcgggtcagg gtcgggtggc actagtaaat cagagggagg caagcgcaaa gctgattctg
     3841 attctgaaga taagggagag gagagcaaac caaagaagaa gaaagaagag tcagaaaagc
     3901 cacgaggctt tgctcgaggt ttggagccgg agcggattat tggagctaca gactccagtg
     3961 gagagctcat gttcctgatg aaatggaaaa actctgatga ggctgacctg gtccctgcca
     4021 aggaagccaa tgtcaagtgc ccacaggttg tcatatcctt ctatgaggaa aggctgacgt
     4081 ggcattccta cgctagcgga ggtggttcgc caaagaagaa acggaaggtg ggatcctgaa
     4141 ataaaagatc tttgttttca ttagatctgt gtgttggttt tttgtgtgcg gccggtaccg
     4201 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     4261 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     4321 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     4381 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     4441 gaaacaccgg agaccacggc aggtctcagt tttagtactc tggaaacaga atctactaaa
     4501 acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatttttg cggccgcagg
     4561 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     4621 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     4681 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4741 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     4801 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     4861 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4921 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4981 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5041 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5101 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     5161 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     5221 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5281 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5341 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5401 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5461 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5521 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5581 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5641 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5701 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5761 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5821 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5881 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5941 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6001 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6061 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6121 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6181 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6241 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6301 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     6361 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6421 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6481 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6541 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6601 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6661 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6721 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6781 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6841 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6901 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6961 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     7021 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     7081 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7141 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7201 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7261 gttcctggcc ttttgctggc cttttgctca catgt
//
