LOCUS       sequence-442993-        6981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of KRAB with dCjCas9 and empty gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6981)
  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 6981)
  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: pBK1302-AAV-EFSNC-dCjCas9-KRAB
FEATURES             Location/Qualifiers
     source          1..6981
                     /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             3798..3818
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        3888..4136
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3888..3908
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4059..4078
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4157..4229
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   4244..4384
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4244..4373
                     /label=AAV2 ITR
     rep_origin      4459..4914
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4546..4565)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4756..4777
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4931..4950)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5050..5072
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5110..5128)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5196..5300
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5301..5369,5370..6161)
                     /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(5519..5538)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6332..6920
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6821..6840
                     /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 gaagagcccg
     3781 ctagcggagg tggttcgcca aagaagaaac ggaaggtgtg agcatgcaat aaaacatctt
     3841 tgttttcatt agatctgtgt gttggttttt tgtgtgcggc cggtaccgag ggcctatttc
     3901 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     3961 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     4021 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     4081 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggag
     4141 accacggcag gtctcagttt tagtccctga aaagggacta aaataaagag tttgcgggac
     4201 tctgcggggt tacaatcccc taaaaccgct tttttgcggc cgcaggaacc cctagtgatg
     4261 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4321 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     4381 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4441 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4501 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     4561 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4621 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     4681 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4741 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     4801 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     4861 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     4921 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     4981 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5041 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5101 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5161 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5221 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5281 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5341 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5401 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5461 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5521 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5581 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5641 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5701 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5761 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5821 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5881 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5941 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6001 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6061 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6121 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6181 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6241 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6301 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6361 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6421 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6481 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6541 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6601 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6661 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6721 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6781 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6841 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6901 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6961 gctggccttt tgctcacatg t
//
