LOCUS       sequence-442865-        7578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2nd gen. vector for expression of KRAB and truncated MECP2 with 
            dSaCas9 and empty gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7578)
  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 7578)
  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: pBK2044-AAV-2xSp1-2xNFkB-EFSNC-dSaCas9-KRAB-MECP2
FEATURES             Location/Qualifiers
     source          1..7578
                     /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        4477..4552
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4566..4706
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4566..4695
                     /label=AAV2 ITR
     rep_origin      4781..5236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4868..4887)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5078..5099
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5253..5272)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5372..5394
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5432..5450)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5518..5622
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5623..5691,5692..6483)
                     /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(5841..5860)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6654..7242
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7143..7162
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7304..7444
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7315..7444
                     /label=AAV2 ITR
     promoter        join(7526..7578,1..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 aacaccggag accacggcag gtctcagttt tagtactctg gaaacagaat
     4501 ctactaaaac aaggcaaaat gccgtgttta tctcgtcaac ttgttggcga gatttttgcg
     4561 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4621 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4681 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     4741 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     4801 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     4861 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4921 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4981 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5041 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5101 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     5161 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5221 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5281 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5341 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5401 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5461 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5521 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5581 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5641 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5701 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5761 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5821 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5881 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5941 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6001 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6061 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6121 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6181 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6241 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6301 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     6361 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6421 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6481 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6541 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6601 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6661 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6721 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6781 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6841 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6901 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6961 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7021 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7081 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7141 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7201 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7261 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc
     7321 gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc
     7381 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt
     7441 tcctacgcgt gggcgggacg ttaacggggg cggaacgtta acgggcgtag gggactttcc
     7501 gttaacgcgg ggactttccg tcgacgggca gagcgcacat cgcccacagt ccccgagaag
     7561 ttggggggag gggtcggc
//
