LOCUS       sequence-442868-        7577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2nd gen. vector for expression of KRAB and truncated MECP2 with 
            dSaCas9 and gRNA1 targeting human/mouse APOE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7577)
  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 7577)
  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: 
            pBK2046-AAV-2xSp1-2xNFkB-EFSNC-dSaCas9-KRAB-MECP2(APOE-gRNA1)
FEATURES             Location/Qualifiers
     source          1..7577
                     /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        4476..4551
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4565..4705
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4565..4694
                     /label=AAV2 ITR
     rep_origin      4780..5235
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4867..4886)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5077..5098
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5252..5271)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5371..5393
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5431..5449)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5517..5621
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5622..5690,5691..6482)
                     /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(5840..5859)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6653..7241
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7142..7161
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7303..7443
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7314..7443
                     /label=AAV2 ITR
     promoter        join(7525..7577,1..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 aacaccgagg agggggcggg acagggtttt agtactctgg aaacagaatc
     4501 tactaaaaca aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag atttttgcgg
     4561 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     4621 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     4681 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     4741 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     4801 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
     4861 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4921 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4981 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     5041 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     5101 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     5161 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5221 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5281 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5341 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5401 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     5461 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     5521 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5581 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5641 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5701 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5761 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5821 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5881 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5941 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     6001 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     6061 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6121 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6181 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6241 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6301 ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac
     6361 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6421 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6481 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6541 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6601 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6661 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6721 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6781 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6841 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6901 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6961 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     7021 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7081 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7141 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7201 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7261 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg
     7321 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc
     7381 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     7441 cctacgcgtg ggcgggacgt taacgggggc ggaacgttaa cgggcgtagg ggactttccg
     7501 ttaacgcggg gactttccgt cgacgggcag agcgcacatc gcccacagtc cccgagaagt
     7561 tggggggagg ggtcggc
//
