LOCUS       sequence-442994-        7199 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 7199)
  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 7199)
  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: pBK1306-AAV-EFSNC-dSaCas9-HP1aNH(115-177)
FEATURES             Location/Qualifiers
     source          1..7199
                     /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             4014..4034
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4044..4092
                     /note="synthetic polyadenylation signal"
     promoter        4104..4352
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4104..4124
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4275..4294
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4373..4448
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4462..4602
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4462..4591
                     /label=AAV2 ITR
     rep_origin      4677..5132
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4764..4783)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4974..4995
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5149..5168)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5268..5290
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5328..5346)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5414..5518
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5519..5587,5588..6379)
                     /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(5737..5756)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6550..7138
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7039..7058
                     /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 actagtcggg gctttgagag aggactggaa ccagaaaaga
     3841 tcattggggc aacagattcc tgtggtgatt taatgttcct aatgaaatgg aaagacacag
     3901 atgaagctga cctggttctt gcaaaagaag ctaatgtgaa atgtccacaa attgtgatag
     3961 cattttatga agagagactg acatggcatg catatgctag cggaggtggt tcgccaaaga
     4021 agaaacggaa ggtgggatcc tgaaataaaa gatctttgtt ttcattagat ctgtgtgttg
     4081 gttttttgtg tgcggccggt accgagggcc tatttcccat gattccttca tatttgcata
     4141 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     4201 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     4261 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     4321 ctttatatat cttgtggaaa ggacgaaaca ccggagacca cggcaggtct cagttttagt
     4381 actctggaaa cagaatctac taaaacaagg caaaatgccg tgtttatctc gtcaacttgt
     4441 tggcgagatt tttgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     4501 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4561 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     4621 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     4681 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4741 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4801 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4861 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4921 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4981 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     5041 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5101 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     5161 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     5221 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5281 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     5341 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5401 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5461 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5521 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5581 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5641 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5701 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5761 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5821 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5881 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5941 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6001 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6061 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6121 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6181 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg
     6241 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6301 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6361 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6421 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6481 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6541 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6601 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6661 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6721 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6781 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6841 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6901 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6961 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7021 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7081 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7141 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
