LOCUS       sequence-442888-        7256 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of truncated NIPP1 with dSaCas9 and empty gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7256)
  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 7256)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pBK1311-AAV-EFSNC-dSaCas9-NIPP1(143-224)
FEATURES             Location/Qualifiers
     source          1..7256
                     /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             513..533
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3714..3761
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4071..4091
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4101..4149
                     /note="synthetic polyadenylation signal"
     promoter        4161..4409
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4161..4181
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4332..4351
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4430..4505
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4519..4659
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4519..4648
                     /label=AAV2 ITR
     rep_origin      4734..5189
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4821..4840)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5031..5052
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5206..5225)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5325..5347
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5385..5403)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5471..5575
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5576..5644,5645..6436)
                     /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(5794..5813)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6607..7195
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7096..7115
                     /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 acatagatta caaggatgac gatgacaagg
      481 gtggatcgcc aaagaaaaag aggaaagtcg ccccaaagaa gaagcggaag gtcggtatcc
      541 acggagtccc agcagccaag cggaactaca tcctgggcct ggccatcggc atcaccagcg
      601 tgggctacgg catcatcgac tacgagacac gggacgtgat cgatgccggc gtgcggctgt
      661 tcaaagaggc caacgtggaa aacaacgagg gcaggcggag caagagaggc gccagaaggc
      721 tgaagcggcg gaggcggcat agaatccaga gagtgaagaa gctgctgttc gactacaacc
      781 tgctgaccga ccacagcgag ctgagcggca tcaaccccta cgaggccaga gtgaagggcc
      841 tgagccagaa gctgagcgag gaagagttct ctgccgccct gctgcacctg gccaagagaa
      901 gaggcgtgca caacgtgaac gaggtggaag aggacaccgg caacgagctg tccaccaaag
      961 agcagatcag ccggaacagc aaggccctgg aagagaaata cgtggccgaa ctgcagctgg
     1021 aacggctgaa gaaagacggc gaagtgcggg gcagcatcaa cagattcaag accagcgact
     1081 acgtgaaaga agccaaacag ctgctgaagg tgcagaaggc ctaccaccag ctggaccaga
     1141 gcttcatcga cacctacatc gacctgctgg aaacccggcg gacctactat gagggacctg
     1201 gcgagggcag ccccttcggc tggaaggaca tcaaagaatg gtacgagatg ctgatgggcc
     1261 actgcaccta cttccccgag gaactgcgga gcgtgaagta cgcctacaac gccgacctgt
     1321 acaacgccct gaacgacctg aacaatctcg tgatcaccag ggacgagaac gagaagctgg
     1381 aatattacga gaagttccag atcatcgaga acgtgttcaa gcagaagaag aagcccaccc
     1441 tgaagcagat cgccaaagaa atcctcgtga acgaagagga tattaagggc tacagagtga
     1501 ccagcaccgg caagcccgag ttcaccaacc tgaaggtgta ccacgacatc aaggacatta
     1561 ccgcccggaa agagattatt gagaacgccg agctgctgga tcagattgcc aagatcctga
     1621 ccatctacca gagcagcgag gacatccagg aagaactgac caatctgaac tccgagctga
     1681 cccaggaaga gatcgagcag atctctaatc tgaagggcta taccggcacc cacaacctga
     1741 gcctgaaggc catcaacctg atcctggacg agctgtggca caccaacgac aaccagatcg
     1801 ctatcttcaa ccggctgaag ctggtgccca agaaggtgga cctgtcccag cagaaagaga
     1861 tccccaccac cctggtggac gacttcatcc tgagccccgt cgtgaagaga agcttcatcc
     1921 agagcatcaa agtgatcaac gccatcatca agaagtacgg cctgcccaac gacatcatta
     1981 tcgagctggc ccgcgagaag aactccaagg acgcccagaa aatgatcaac gagatgcaga
     2041 agcggaaccg gcagaccaac gagcggatcg aggaaatcat ccggaccacc ggcaaagaga
     2101 acgccaagta cctgatcgag aagatcaagc tgcacgacat gcaggaaggc aagtgcctgt
     2161 acagcctgga agccatccct ctggaagatc tgctgaacaa ccccttcaac tatgaggtgg
     2221 accacatcat ccccagaagc gtgtccttcg acaacagctt caacaacaag gtgctcgtga
     2281 agcaggaaga agccagcaag aagggcaacc ggaccccatt ccagtacctg agcagcagcg
     2341 acagcaagat cagctacgaa accttcaaga agcacatcct gaatctggcc aagggcaagg
     2401 gcagaatcag caagaccaag aaagagtatc tgctggaaga acgggacatc aacaggttct
     2461 ccgtgcagaa agacttcatc aaccggaacc tggtggatac cagatacgcc accagaggcc
     2521 tgatgaacct gctgcggagc tacttcagag tgaacaacct ggacgtgaaa gtgaagtcca
     2581 tcaatggcgg cttcaccagc tttctgcggc ggaagtggaa gtttaagaaa gagcggaaca
     2641 aggggtacaa gcaccacgcc gaggacgccc tgatcattgc caacgccgat ttcatcttca
     2701 aagagtggaa gaaactggac aaggccaaaa aagtgatgga aaaccagatg ttcgaggaaa
     2761 agcaggccga gagcatgccc gagatcgaaa ccgagcagga gtacaaagag atcttcatca
     2821 ccccccacca gatcaagcac attaaggact tcaaggacta caagtacagc caccgggtgg
     2881 acaagaagcc taatagagag ctgattaacg acaccctgta ctccacccgg aaggacgaca
     2941 agggcaacac cctgatcgtg aacaatctga acggcctgta cgacaaggac aatgacaagc
     3001 tgaaaaagct gatcaacaag agccccgaaa agctgctgat gtaccaccac gacccccaga
     3061 cctaccagaa actgaagctg attatggaac agtacggcga cgagaagaat cccctgtaca
     3121 agtactacga ggaaaccggg aactacctga ccaagtactc caaaaaggac aacggccccg
     3181 tgatcaagaa gattaagtat tacggcaaca aactgaacgc ccatctggac atcaccgacg
     3241 actaccccaa cagcagaaac aaggtcgtga agctgtccct gaagccctac agattcgacg
     3301 tgtacctgga caatggcgtg tacaagttcg tgaccgtgaa gaatctggat gtgatcaaaa
     3361 aagaaaacta ctacgaagtg aatagcaagt gctatgagga agctaagaag ctgaagaaga
     3421 tcagcaacca ggccgagttt atcgcctcct tctacaacaa cgatctgatc aagatcaacg
     3481 gcgagctgta tagagtgatc ggcgtgaaca acgacctgct gaaccggatc gaagtgaaca
     3541 tgatcgacat cacctaccgc gagtacctgg aaaacatgaa cgacaagagg ccccccagga
     3601 tcattaagac aatcgcctcc aagacccaga gcattaagaa gtacagcaca gacattctgg
     3661 gcaacctgta tgaagtgaaa tctaagaagc accctcagat catcaaaaag ggcaaaaggc
     3721 cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gggatccggt ggaggaagtg
     3781 gcgggtcagg gtcgggtggc actagtatgg gtggagagga tgatgaactc aagggcttac
     3841 tggggcttcc agaggaggaa actgagcttg ataacctgac agagttcaac actgcccaca
     3901 acaagcggat ttctaccctt accattgagg agggaaatct ggacattcaa agaccaaaga
     3961 ggaagaggaa gaactcacgg gtgacattca gtgaggatga tgagatcatc aacccagagg
     4021 atgtggatcc ctcagttggt cgattcagga acgctagcgg aggtggttcg ccaaagaaga
     4081 aacggaaggt gggatcctga aataaaagat ctttgttttc attagatctg tgtgttggtt
     4141 ttttgtgtgc ggccggtacc gagggcctat ttcccatgat tccttcatat ttgcatatac
     4201 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     4261 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     4321 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     4381 tatatatctt gtggaaagga cgaaacaccg gagaccacgg caggtctcag ttttagtact
     4441 ctggaaacag aatctactaa aacaaggcaa aatgccgtgt ttatctcgtc aacttgttgg
     4501 cgagattttt gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     4561 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     4621 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     4681 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     4741 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4801 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4861 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4921 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     4981 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5041 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     5101 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5161 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     5221 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5281 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5341 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     5401 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     5461 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5521 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5581 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5641 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5701 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5761 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5821 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5881 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5941 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6001 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6061 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6121 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6181 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6241 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     6301 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6361 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6421 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6481 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6541 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6601 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6661 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6721 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6781 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6841 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6901 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6961 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7021 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7081 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7141 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7201 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
