LOCUS       sequence-442999-        7328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of truncated HP1a with dSaCas9 and empty gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7328)
  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 7328)
  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: pBK1305-AAV-EFSNC-dSaCas9-HP1a(72-177)
FEATURES             Location/Qualifiers
     source          1..7328
                     /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             4143..4163
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4173..4221
                     /note="synthetic polyadenylation signal"
     promoter        4233..4481
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4233..4253
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4404..4423
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4502..4577
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4591..4731
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4591..4720
                     /label=AAV2 ITR
     rep_origin      4806..5261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4893..4912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5103..5124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5278..5297)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5397..5419
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5457..5475)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5543..5647
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5648..5716,5717..6508)
                     /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(5866..5885)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6679..7267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7168..7187
                     /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 actagtaaga tgaaggaggg tgaaaataat aaacccaggg
     3841 agaagtcaga aagtaacaag aggaaatcca atttctcaaa cagtgccgat gacatcaaat
     3901 ctaaaaaaaa gagagagcag agcaatgata tcgctcgggg ctttgagaga ggactggaac
     3961 cagaaaagat cattggggca acagattcct gtggtgattt aatgttccta atgaaatgga
     4021 aagacacaga tgaagctgac ctggttcttg caaaagaagc taatgtgaaa tgtccacaaa
     4081 ttgtgatagc attttatgaa gagagactga catggcatgc atatgctagc ggaggtggtt
     4141 cgccaaagaa gaaacggaag gtgggatcct gaaataaaag atctttgttt tcattagatc
     4201 tgtgtgttgg ttttttgtgt gcggccggta ccgagggcct atttcccatg attccttcat
     4261 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     4321 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     4381 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     4441 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggagaccac ggcaggtctc
     4501 agttttagta ctctggaaac agaatctact aaaacaaggc aaaatgccgt gtttatctcg
     4561 tcaacttgtt ggcgagattt ttgcggccgc aggaacccct agtgatggag ttggccactc
     4621 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4681 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     4741 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     4801 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4861 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     4921 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4981 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     5041 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     5101 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     5161 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     5221 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     5281 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     5341 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5401 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     5461 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5521 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5581 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5641 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5701 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5761 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5821 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5881 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5941 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     6001 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     6061 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     6121 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     6181 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6241 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6301 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6361 tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6421 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6481 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6541 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6601 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6661 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6721 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6781 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6841 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6901 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6961 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     7021 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     7081 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     7141 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     7201 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     7261 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     7321 tcacatgt
//
