LOCUS       sequence-442870-        7579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2nd gen. vector for expression of KRAB and truncated MECP2 with 
            dSaCas9 and gRNA2 targeting human/mouse APOE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7579)
  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 7579)
  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: 
            pBK2047-AAV-2xSp1-2xNFkB-EFSNC-dSaCas9-KRAB-MECP2(APOE-gRNA2)
FEATURES             Location/Qualifiers
     source          1..7579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      167..176
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             176..241
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             251..271
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             275..295
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3476..3523
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3569..3754
                     /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             4118..4138
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4148..4196
                     /note="synthetic polyadenylation signal"
     promoter        4208..4456
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4208..4228
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4379..4398
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4478..4553
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4567..4707
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4567..4696
                     /label=AAV2 ITR
     rep_origin      4782..5237
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4869..4888)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5079..5100
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5254..5273)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5373..5395
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5433..5451)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5519..5623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5624..5692,5693..6484)
                     /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(5842..5861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6655..7243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7144..7163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7305..7445
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7316..7445
                     /label=AAV2 ITR
     promoter        join(7527..7579,1..159)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
       61 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
      121 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggtgcca ccatggacta
      181 caaagaccat gacggtgatt ataaagatca tgacatagat tacaaggatg acgatgacaa
      241 gggtggatcg ccaaagaaaa agaggaaagt cgccccaaag aagaagcgga aggtcggtat
      301 ccacggagtc ccagcagcca agcggaacta catcctgggc ctggccatcg gcatcaccag
      361 cgtgggctac ggcatcatcg actacgagac acgggacgtg atcgatgccg gcgtgcggct
      421 gttcaaagag gccaacgtgg aaaacaacga gggcaggcgg agcaagagag gcgccagaag
      481 gctgaagcgg cggaggcggc atagaatcca gagagtgaag aagctgctgt tcgactacaa
      541 cctgctgacc gaccacagcg agctgagcgg catcaacccc tacgaggcca gagtgaaggg
      601 cctgagccag aagctgagcg aggaagagtt ctctgccgcc ctgctgcacc tggccaagag
      661 aagaggcgtg cacaacgtga acgaggtgga agaggacacc ggcaacgagc tgtccaccaa
      721 agagcagatc agccggaaca gcaaggccct ggaagagaaa tacgtggccg aactgcagct
      781 ggaacggctg aagaaagacg gcgaagtgcg gggcagcatc aacagattca agaccagcga
      841 ctacgtgaaa gaagccaaac agctgctgaa ggtgcagaag gcctaccacc agctggacca
      901 gagcttcatc gacacctaca tcgacctgct ggaaacccgg cggacctact atgagggacc
      961 tggcgagggc agccccttcg gctggaagga catcaaagaa tggtacgaga tgctgatggg
     1021 ccactgcacc tacttccccg aggaactgcg gagcgtgaag tacgcctaca acgccgacct
     1081 gtacaacgcc ctgaacgacc tgaacaatct cgtgatcacc agggacgaga acgagaagct
     1141 ggaatattac gagaagttcc agatcatcga gaacgtgttc aagcagaaga agaagcccac
     1201 cctgaagcag atcgccaaag aaatcctcgt gaacgaagag gatattaagg gctacagagt
     1261 gaccagcacc ggcaagcccg agttcaccaa cctgaaggtg taccacgaca tcaaggacat
     1321 taccgcccgg aaagagatta ttgagaacgc cgagctgctg gatcagattg ccaagatcct
     1381 gaccatctac cagagcagcg aggacatcca ggaagaactg accaatctga actccgagct
     1441 gacccaggaa gagatcgagc agatctctaa tctgaagggc tataccggca cccacaacct
     1501 gagcctgaag gccatcaacc tgatcctgga cgagctgtgg cacaccaacg acaaccagat
     1561 cgctatcttc aaccggctga agctggtgcc caagaaggtg gacctgtccc agcagaaaga
     1621 gatccccacc accctggtgg acgacttcat cctgagcccc gtcgtgaaga gaagcttcat
     1681 ccagagcatc aaagtgatca acgccatcat caagaagtac ggcctgccca acgacatcat
     1741 tatcgagctg gcccgcgaga agaactccaa ggacgcccag aaaatgatca acgagatgca
     1801 gaagcggaac cggcagacca acgagcggat cgaggaaatc atccggacca ccggcaaaga
     1861 gaacgccaag tacctgatcg agaagatcaa gctgcacgac atgcaggaag gcaagtgcct
     1921 gtacagcctg gaagccatcc ctctggaaga tctgctgaac aaccccttca actatgaggt
     1981 ggaccacatc atccccagaa gcgtgtcctt cgacaacagc ttcaacaaca aggtgctcgt
     2041 gaagcaggaa gaagccagca agaagggcaa ccggacccca ttccagtacc tgagcagcag
     2101 cgacagcaag atcagctacg aaaccttcaa gaagcacatc ctgaatctgg ccaagggcaa
     2161 gggcagaatc agcaagacca agaaagagta tctgctggaa gaacgggaca tcaacaggtt
     2221 ctccgtgcag aaagacttca tcaaccggaa cctggtggat accagatacg ccaccagagg
     2281 cctgatgaac ctgctgcgga gctacttcag agtgaacaac ctggacgtga aagtgaagtc
     2341 catcaatggc ggcttcacca gctttctgcg gcggaagtgg aagtttaaga aagagcggaa
     2401 caaggggtac aagcaccacg ccgaggacgc cctgatcatt gccaacgccg atttcatctt
     2461 caaagagtgg aagaaactgg acaaggccaa aaaagtgatg gaaaaccaga tgttcgagga
     2521 aaagcaggcc gagagcatgc ccgagatcga aaccgagcag gagtacaaag agatcttcat
     2581 caccccccac cagatcaagc acattaagga cttcaaggac tacaagtaca gccaccgggt
     2641 ggacaagaag cctaatagag agctgattaa cgacaccctg tactccaccc ggaaggacga
     2701 caagggcaac accctgatcg tgaacaatct gaacggcctg tacgacaagg acaatgacaa
     2761 gctgaaaaag ctgatcaaca agagccccga aaagctgctg atgtaccacc acgaccccca
     2821 gacctaccag aaactgaagc tgattatgga acagtacggc gacgagaaga atcccctgta
     2881 caagtactac gaggaaaccg ggaactacct gaccaagtac tccaaaaagg acaacggccc
     2941 cgtgatcaag aagattaagt attacggcaa caaactgaac gcccatctgg acatcaccga
     3001 cgactacccc aacagcagaa acaaggtcgt gaagctgtcc ctgaagccct acagattcga
     3061 cgtgtacctg gacaatggcg tgtacaagtt cgtgaccgtg aagaatctgg atgtgatcaa
     3121 aaaagaaaac tactacgaag tgaatagcaa gtgctatgag gaagctaaga agctgaagaa
     3181 gatcagcaac caggccgagt ttatcgcctc cttctacaac aacgatctga tcaagatcaa
     3241 cggcgagctg tatagagtga tcggcgtgaa caacgacctg ctgaaccgga tcgaagtgaa
     3301 catgatcgac atcacctacc gcgagtacct ggaaaacatg aacgacaaga ggccccccag
     3361 gatcattaag acaatcgcct ccaagaccca gagcattaag aagtacagca cagacattct
     3421 gggcaacctg tatgaagtga aatctaagaa gcaccctcag atcatcaaaa agggcaaaag
     3481 gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagggatccg gtggaggaag
     3541 tggcgggtca gggtcgggtg gcactagtcg gacactggtg accttcaagg atgtatttgt
     3601 ggacttcacc agggaggagt ggaagctgct ggacactgct cagcagatcg tgtacagaaa
     3661 tgtgatgctg gagaactata agaacctggt ttccttgggt tatcagctta ctaagccaga
     3721 tgtgatcctc cggttggaga agggagaaga gccctcggga ggtggttcgg gaggtggttc
     3781 ggagggtgtg caggtgaaaa gggtcctgga gaaaagtcct gggaagctcc ttgtcaagat
     3841 gccttttcaa acttcgccag ggggcaaggc tgaggggggt ggggccacca catccaccca
     3901 ggtcatggtg atcaaacgcc ccggcaggaa gcgaaaagct gaggccgacc ctcaggccat
     3961 tcccaagaaa cggggccgaa agccggggag tgtggtggca gccgctgccg ccgaggccaa
     4021 aaagaaagcc gtgaaggagt cttctatccg atctgtgcag gagacagtac tccccatcaa
     4081 gaagcgcaag acccgggagg ctagcggagg tggttcgcca aagaagaaac ggaaggtggg
     4141 atcctgaaat aaaagatctt tgttttcatt agatctgtgt gttggttttt tgtgtgcggc
     4201 cggtaccgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     4261 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     4321 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     4381 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     4441 gaaaggacga aacaccgtag ctcttccctc ccaaggtgtt ttagtactct ggaaacagaa
     4501 tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg agatttttgc
     4561 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4621 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4681 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4741 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4801 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     4861 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4921 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4981 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5041 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5101 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     5161 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5221 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5281 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5341 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5401 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5461 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5521 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5581 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5641 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5701 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5761 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5821 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5881 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5941 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6001 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6061 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6121 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6181 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6241 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6301 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     6361 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6421 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6481 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6541 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6601 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6661 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6721 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6781 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6841 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6901 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6961 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7021 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7081 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7141 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7201 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7261 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg
     7321 cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg
     7381 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg
     7441 ttcctacgcg tgggcgggac gttaacgggg gcggaacgtt aacgggcgta ggggactttc
     7501 cgttaacgcg gggactttcc gtcgacgggc agagcgcaca tcgcccacag tccccgagaa
     7561 gttgggggga ggggtcggc
//
