LOCUS       sequence-442897-        7116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of truncated MECP2 with dCjCas9 and empty gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7116)
  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 7116)
  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: pBK1303-AAV-EFSNC-dCjCas9-MECP2(204-310)
FEATURES             Location/Qualifiers
     source          1..7116
                     /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             3933..3953
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        4023..4271
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4023..4043
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4194..4213
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4292..4364
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   4379..4519
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4379..4508
                     /label=AAV2 ITR
     rep_origin      4594..5049
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4681..4700)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4891..4912
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5066..5085)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5185..5207
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5245..5263)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5331..5435
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5436..5504,5505..6296)
                     /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(5654..5673)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6467..7055
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6956..6975
                     /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 agttcggagg
     3601 gtgtgcaggt gaaaagggtc ctggagaaaa gtcctgggaa gctccttgtc aagatgcctt
     3661 ttcaaacttc gccagggggc aaggctgagg ggggtggggc caccacatcc acccaggtca
     3721 tggtgatcaa acgccccggc aggaagcgaa aagctgaggc cgaccctcag gccattccca
     3781 agaaacgggg ccgaaagccg gggagtgtgg tggcagccgc tgccgccgag gccaaaaaga
     3841 aagccgtgaa ggagtcttct atccgatctg tgcaggagac agtactcccc atcaagaagc
     3901 gcaagacccg ggaggctagc ggaggtggtt cgccaaagaa gaaacggaag gtgtgagcat
     3961 gcaataaaac atctttgttt tcattagatc tgtgtgttgg ttttttgtgt gcggccggta
     4021 ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     4081 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     4141 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     4201 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     4261 gacgaaacac cggagaccac ggcaggtctc agttttagtc cctgaaaagg gactaaaata
     4321 aagagtttgc gggactctgc ggggttacaa tcccctaaaa ccgctttttt gcggccgcag
     4381 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     4441 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4501 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4561 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     4621 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     4681 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4741 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4801 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     4861 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     4921 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     4981 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     5041 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     5101 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5161 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5221 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     5281 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5341 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5401 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5461 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5521 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5581 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5641 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5701 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5761 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5821 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5881 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5941 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6001 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6061 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6121 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     6181 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6241 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6301 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6361 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6421 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6481 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6541 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6601 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6661 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6721 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6781 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6841 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6901 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6961 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7021 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7081 ggttcctggc cttttgctgg ccttttgctc acatgt
//
