LOCUS       sequence-442990-        6984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of truncated HP1a with dCjCas9 and empty gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6984)
  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 6984)
  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: pBK1296-AAV-EFSNC-dCjCas9-HP1aNH(115-177)
FEATURES             Location/Qualifiers
     source          1..6984
                     /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             3801..3821
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        3891..4139
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3891..3911
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4062..4081
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4160..4232
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   4247..4387
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4247..4376
                     /label=AAV2 ITR
     rep_origin      4462..4917
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4549..4568)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4759..4780
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4934..4953)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5053..5075
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5113..5131)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5199..5303
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5304..5372,5373..6164)
                     /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(5522..5541)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6335..6923
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6824..6843
                     /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 agtcggggct
     3601 ttgagagagg actggaacca gaaaagatca ttggggcaac agattcctgt ggtgatttaa
     3661 tgttcctaat gaaatggaaa gacacagatg aagctgacct ggttcttgca aaagaagcta
     3721 atgtgaaatg tccacaaatt gtgatagcat tttatgaaga gagactgaca tggcatgcat
     3781 atgctagcgg aggtggttcg ccaaagaaga aacggaaggt gtgagcatgc aataaaacat
     3841 ctttgttttc attagatctg tgtgttggtt ttttgtgtgc ggccggtacc gagggcctat
     3901 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     3961 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     4021 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     4081 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     4141 gagaccacgg caggtctcag ttttagtccc tgaaaaggga ctaaaataaa gagtttgcgg
     4201 gactctgcgg ggttacaatc ccctaaaacc gcttttttgc ggccgcagga acccctagtg
     4261 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4321 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4381 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4441 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4501 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     4561 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4621 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     4681 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4741 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     4801 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     4861 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     4921 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     4981 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5041 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5101 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     5161 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5221 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5281 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5341 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5401 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5461 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5521 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5581 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5641 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5701 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5761 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5821 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     5881 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     5941 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6001 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     6061 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6121 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6181 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6241 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6301 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6361 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6421 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6481 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6541 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6601 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6661 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6721 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6781 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6841 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6901 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6961 tttgctggcc ttttgctcac atgt
//
