LOCUS       sequence-442869-        7580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2nd gen. vector for expression of KRAB and truncated MECP2 with 
            dSaCas9 and gRNA1 targeting CMV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7580)
  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 7580)
  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: 
            pBK2045-AAV-2xSp1-2xNFkB-EFSNC-dSaCas9-KRAB-MECP2(CMV-gRNA1)
FEATURES             Location/Qualifiers
     source          1..7580
                     /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        4479..4554
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4568..4708
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4568..4697
                     /label=AAV2 ITR
     rep_origin      4783..5238
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4870..4889)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5080..5101
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5255..5274)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5374..5396
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5434..5452)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5520..5624
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5625..5693,5694..6485)
                     /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(5843..5862)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6656..7244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7145..7164
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7306..7446
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7317..7446
                     /label=AAV2 ITR
     promoter        join(7528..7580,1..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 aacaccggtg gatagcggtt tgactcacgt tttagtactc tggaaacaga
     4501 atctactaaa acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatttttg
     4561 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4621 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     4681 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     4741 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     4801 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     4861 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4921 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4981 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5041 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5101 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     5161 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5221 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5281 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     5341 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     5401 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     5461 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     5521 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5581 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5641 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5701 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5761 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5821 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5881 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5941 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6001 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6061 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6121 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6181 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6241 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6301 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag
     6361 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6421 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6481 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6541 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6601 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6661 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6721 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6781 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6841 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6901 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6961 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7021 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7081 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7141 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7201 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7261 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtcctgc aggcagctgc
     7321 gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg acctttggtc
     7381 gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
     7441 gttcctacgc gtgggcggga cgttaacggg ggcggaacgt taacgggcgt aggggacttt
     7501 ccgttaacgc ggggactttc cgtcgacggg cagagcgcac atcgcccaca gtccccgaga
     7561 agttgggggg aggggtcggc
//
