LOCUS       sequence-442892-        7080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of truncated HP1b with dCjCas9 and empty gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7080)
  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 7080)
  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: pBK1297-AAV-EFSNC-dCjCas9-HP1b(79-173)
FEATURES             Location/Qualifiers
     source          1..7080
                     /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             519..539
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3504..3524
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3897..3917
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        3987..4235
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3987..4007
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4158..4177
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4256..4328
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   4343..4483
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4343..4472
                     /label=AAV2 ITR
     rep_origin      4558..5013
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4645..4664)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4855..4876
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5030..5049)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5149..5171
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5209..5227)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5295..5399
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5400..5468,5469..6260)
                     /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(5618..5637)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6431..7019
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6920..6939
                     /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 acatcgatta caaggatgac gatgacaagg
      481 gtggatcgcc aaagaaaaag aggaaagtcg gcagtggacc taaaaagaaa cgaaaggttg
      541 aagcgggatc cgcccgcatc ctcgctttcg ccatcggaat ctctagtatc ggatgggcct
      601 tctctgaaaa cgacgaactg aaagactgcg gcgtgagaat cttcacaaag gttgaaaacc
      661 ctaaaacagg cgagtcttta gctctgccac gtaggttggc ccgctccgcc cgaaaaaggc
      721 tggctcggcg gaaggctcgc ctcaaccact tgaagcattt gatagctaat gagttcaaac
      781 tgaactacga agattaccag tccttcgacg agtcattggc aaaagcctac aaaggcagcc
      841 ttatcagtcc ttatgagttg agatttcgcg cactcaacga actgctttct aagcaagact
      901 ttgctagggt cattctgcac atcgcaaaac ggcgaggtta tgacgatatc aagaactccg
      961 acgataaaga aaagggagcc attctcaagg cgatcaaaca gaatgaggaa aaattggcaa
     1021 actaccagag tgtgggcgag tatctgtata aagagtattt ccagaagttt aaggaaaaca
     1081 gcaaggagtt tacaaacgtc agaaataaaa aggagtctta cgagagatgc atcgcgcagt
     1141 cattcctcaa agatgagctg aagctgatat ttaagaagca acgcgaattt ggtttctcat
     1201 tctctaagaa gttcgaagag gaggttcttt ccgtggcgtt ttacaagagg gcgctcaaag
     1261 acttctccca cctggttggt aactgtagtt tcttcacgga tgagaagcga gctcccaaaa
     1321 attctcccct ggctttcatg tttgttgccc tgactcggat cattaacctg ctgaacaacc
     1381 tgaaaaatac tgaagggatc ttgtatacga aggacgacct aaatgcactc ctgaatgaag
     1441 tgctcaaaaa cggaactcta acctataaac agaccaagaa attactgggg ctctctgacg
     1501 actacgagtt caagggcgag aagggtactt attttatcga attcaaaaag tataaggagt
     1561 tcattaaagc attgggggaa cacaacctca gccaggacga tctcaatgaa attgccaagg
     1621 acatcacgct gattaaagac gagataaaac tgaaaaaggc actggccaag tatgacctca
     1681 accagaacca gatcgactct ctgtccaagc tggagttcaa agaccaccta aacatatcct
     1741 tcaaagccct gaaactggtc acccctctaa tgctcgaagg aaaaaaatac gacgaggcgt
     1801 gtaatgaact gaatcttaag gtggccatca atgaggataa gaaggacttt cttccagcct
     1861 ttaacgagac atattacaaa gacgaggtca caaacccggt tgtgctgagg gccataaaag
     1921 agtatcggaa ggttctgaat gccctcctga agaagtacgg caaagtgcac aaaataaata
     1981 tcgaattggc tagggaggtg gggaagaacc attctcagcg agcaaagatc gagaaagagc
     2041 agaatgagaa ctacaaagcc aagaaagacg ccgaactgga gtgcgaaaag ctggggctta
     2101 aaataaacag taaaaacatc ctgaaattaa gattgttcaa agagcaaaag gagttttgcg
     2161 cctactcagg ggaaaaaatc aaaatatcag acctgcagga cgagaaaatg ctggagatcg
     2221 acgccatcta tccgtatagc aggtcatttg acgattccta catgaacaaa gtgcttgtgt
     2281 ttaccaaaca gaaccaagaa aagctgaacc aaaccccctt tgaggctttc ggaaacgact
     2341 cagccaagtg gcagaaaatc gaagtcctag ccaagaatct gcctacaaaa aaacaaaaga
     2401 ggattcttga taagaactat aaggacaagg aacagaaaaa ctttaaagac aggaacctga
     2461 atgacacgag gtacattgcg cgactggttc taaactatac caaagactac ctggatttcc
     2521 tccctctgag cgacgacgag aatactaaac tgaatgatac ccagaaaggc tcaaaggtcc
     2581 acgttgaggc taagtccggg atgctgacta gcgccctccg ccacacgtgg ggcttcagcg
     2641 ccaaagatcg gaataatcat cttcatcacg ctattgatgc agtaatcata gcctacgcta
     2701 acaacagcat cgtgaaagcc ttctccgatt tcaagaaaga acaggagtct aatagcgccg
     2761 agttgtacgc caagaaaatt tccgaattgg actataaaaa taagagaaaa ttcttcgaac
     2821 ccttctccgg gtttcgccaa aaggtcttag ataagatcga cgagattttc gtttccaagc
     2881 ccgaaagaaa aaagccttca ggggcactgc acgaagagac attccgcaag gaagaggaat
     2941 tttaccaatc ttacggtggt aaagagggag ttctgaaggc tctggagctt gggaagatcc
     3001 gcaaggtaaa cgggaaaatc gtgaaaaacg gggacatgtt cagggtggat atcttcaagc
     3061 acaaaaagac caacaagttc tacgcagtac ccatctacac tatggatttc gctttaaagg
     3121 ttctcccaaa taaggcggtg gctcgatcga agaaaggaga gatcaaggac tggatcttaa
     3181 tggatgaaaa ttacgagttt tgcttctcgc tctacaaaga tagcctgatt ctgatccaga
     3241 caaaagacat gcaggaacca gaatttgttt attataacgc cttcacgagc agtacagtgt
     3301 ccctgattgt gagcaagcat gataacaagt tcgagactct gtctaagaat cagaaaatcc
     3361 ttttcaagaa cgccaacgag aaggaggtca tcgcaaagtc aattggcatc caaaacctga
     3421 aggtgttcga gaaatacata gtgtccgcac tcggtgaagt aactaaagcc gaatttcgac
     3481 agcgcgagga ttttaagaaa agccccaaga agaagagaaa ggtggaggcc agcttagcat
     3541 cgggaggtgg ttcgggtgga ggaagtggcg ggtcagggtc gggtggcact agtaaatcag
     3601 agggaggcaa gcgcaaagct gattctgatt ctgaagataa gggagaggag agcaaaccaa
     3661 agaagaagaa agaagagtca gaaaagccac gaggctttgc tcgaggtttg gagccggagc
     3721 ggattattgg agctacagac tccagtggag agctcatgtt cctgatgaaa tggaaaaact
     3781 ctgatgaggc tgacctggtc cctgccaagg aagccaatgt caagtgccca caggttgtca
     3841 tatccttcta tgaggaaagg ctgacgtggc attcctacgc tagcggaggt ggttcgccaa
     3901 agaagaaacg gaaggtgtga gcatgcaata aaacatcttt gttttcatta gatctgtgtg
     3961 ttggtttttt gtgtgcggcc ggtaccgagg gcctatttcc catgattcct tcatatttgc
     4021 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     4081 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     4141 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     4201 tggctttata tatcttgtgg aaaggacgaa acaccggaga ccacggcagg tctcagtttt
     4261 agtccctgaa aagggactaa aataaagagt ttgcgggact ctgcggggtt acaatcccct
     4321 aaaaccgctt ttttgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     4381 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4441 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     4501 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     4561 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4621 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4681 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4741 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4801 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4861 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     4921 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4981 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     5041 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     5101 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5161 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     5221 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     5281 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5341 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5401 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5461 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5521 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5581 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5641 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5701 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5761 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5821 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5881 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5941 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6001 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6061 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc
     6121 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6181 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6241 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6301 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6361 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6421 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6481 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6541 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6601 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6661 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6721 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6781 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6841 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6901 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6961 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7021 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
