LOCUS       sequence-442926-        7326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of KRAB and truncated MECP2 with dCjCas9 and empty gRNA 
            scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7326)
  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 7326)
  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: pBK1304-AAV-EFSNC-dCjCas9-KRAB-MECP2
FEATURES             Location/Qualifiers
     source          1..7326
                     /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             3594..3779
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             4143..4163
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     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..4574
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   4589..4729
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4589..4718
                     /label=AAV2 ITR
     rep_origin      4804..5259
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4891..4910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5101..5122
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5276..5295)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5395..5417
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5455..5473)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5541..5645
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5646..5714,5715..6506)
                     /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(5864..5883)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6677..7265
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7166..7185
                     /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 agtcggacac
     3601 tggtgacctt caaggatgta tttgtggact tcaccaggga ggagtggaag ctgctggaca
     3661 ctgctcagca gatcgtgtac agaaatgtga tgctggagaa ctataagaac ctggtttcct
     3721 tgggttatca gcttactaag ccagatgtga tcctccggtt ggagaaggga gaagagccct
     3781 cgggaggtgg ttcgggaggt ggttcggagg gtgtgcaggt gaaaagggtc ctggagaaaa
     3841 gtcctgggaa gctccttgtc aagatgcctt ttcaaacttc gccagggggc aaggctgagg
     3901 ggggtggggc caccacatcc acccaggtca tggtgatcaa acgccccggc aggaagcgaa
     3961 aagctgaggc cgaccctcag gccattccca agaaacgggg ccgaaagccg gggagtgtgg
     4021 tggcagccgc tgccgccgag gccaaaaaga aagccgtgaa ggagtcttct atccgatctg
     4081 tgcaggagac agtactcccc atcaagaagc gcaagacccg ggaggctagc ggaggtggtt
     4141 cgccaaagaa gaaacggaag gtgtgagcat gcaataaaac 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 agttttagtc cctgaaaagg gactaaaata aagagtttgc gggactctgc ggggttacaa
     4561 tcccctaaaa ccgctttttt gcggccgcag gaacccctag tgatggagtt ggccactccc
     4621 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4681 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     4741 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     4801 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4861 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4921 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4981 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     5041 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     5101 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     5161 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     5221 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     5281 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     5341 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     5401 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     5461 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     5521 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5581 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5641 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5701 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5761 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5821 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5881 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5941 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6001 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6061 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6121 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6181 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6241 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6301 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6361 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6421 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6481 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6541 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6601 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6661 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6721 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6781 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6841 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6901 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6961 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     7021 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7081 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7141 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7201 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7261 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7321 acatgt
//
