LOCUS       sequence-214976-        6784 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A EFS driven de-catalyzed SaCas9 fused to KRAB domain for inhibition
            of transcription in targeted region..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6784)
  AUTHORS   Kumar N, Stanford W, de Solis C, Aradhana, Abraham ND, Dao TJ, 
            Thaseen S, Sairavi A, Gonzalez CU, Ploski JE
  TITLE     The Development of an AAV-Based CRISPR SaCas9 Genome Editing System 
            That Can Be Delivered to Neurons in vivo and Regulated via 
            Doxycycline and Cre-Recombinase.
  JOURNAL   Front Mol Neurosci. 2018 Nov 13;11:413. doi: 
            10.3389/fnmol.2018.00413. eCollection 2018.
  PUBMED    30483052
REFERENCE   2  (bases 1 to 6784)
  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: "Front Mol 
            Neurosci."; date: "2018-11-13"; volume: "11:413. doi"; pages: " 
            10.3389/fnmol.2018.00413. eCollection 2018"
COMMENT     Sequence Label: pJEP305-pAAV-EFS-dSaCas9-KRAB-pA
FEATURES             Location/Qualifiers
     source          1..6784
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        181..392
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      425..434
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             437..457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3638..3685
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3737..3922
                     /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"
     primer_bind     complement(3989..4008)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4047..4187
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4047..4176
                     /label=AAV2 ITR
     rep_origin      4262..4717
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4349..4368)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4559..4580
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4734..4753)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4853..4875
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4913..4931)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4999..5103
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5104..5172,5173..5964)
                     /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(5322..5341)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6135..6723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6624..6643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgttcgag tggctccggt gcccgtcagt
      181 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
      241 cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      301 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      361 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggaat tcggtaccac
      421 cggtgccacc atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
      481 caagcggaac tacatcctgg gcctggccat cggcatcacc agcgtgggct acggcatcat
      541 cgactacgag acacgggacg tgatcgatgc cggcgtgcgg ctgttcaaag aggccaacgt
      601 ggaaaacaac gagggcaggc ggagcaagag aggcgccaga aggctgaagc ggcggaggcg
      661 gcatagaatc cagagagtga agaagctgct gttcgactac aacctgctga ccgaccacag
      721 cgagctgagc ggcatcaacc cctacgaggc cagagtgaag ggcctgagcc agaagctgag
      781 cgaggaagag ttctctgccg ccctgctgca cctggccaag agaagaggcg tgcacaacgt
      841 gaacgaggtg gaagaggaca ccggcaacga gctgtccacc aaagagcaga tcagccggaa
      901 cagcaaggcc ctggaagaga aatacgtggc cgaactgcag ctggaacggc tgaagaaaga
      961 cggcgaagtg cggggcagca tcaacagatt caagaccagc gactacgtga aagaagccaa
     1021 acagctgctg aaggtgcaga aggcctacca ccagctggac cagagcttca tcgacaccta
     1081 catcgacctg ctggaaaccc ggcggaccta ctatgaggga cctggcgagg gcagcccctt
     1141 cggctggaag gacatcaaag aatggtacga gatgctgatg ggccactgca cctacttccc
     1201 cgaggaactg cggagcgtga agtacgccta caacgccgac ctgtacaacg ccctgaacga
     1261 cctgaacaat ctcgtgatca ccagggacga gaacgagaag ctggaatatt acgagaagtt
     1321 ccagatcatc gagaacgtgt tcaagcagaa gaagaagccc accctgaagc agatcgccaa
     1381 agaaatcctc gtgaacgaag aggatattaa gggctacaga gtgaccagca ccggcaagcc
     1441 cgagttcacc aacctgaagg tgtaccacga catcaaggac attaccgccc ggaaagagat
     1501 tattgagaac gccgagctgc tggatcagat tgccaagatc ctgaccatct accagagcag
     1561 cgaggacatc caggaagaac tgaccaatct gaactccgag ctgacccagg aagagatcga
     1621 gcagatctct aatctgaagg gctataccgg cacccacaac ctgagcctga aggccatcaa
     1681 cctgatcctg gacgagctgt ggcacaccaa cgacaaccag atcgctatct tcaaccggct
     1741 gaagctggtg cccaagaagg tggacctgtc ccagcagaaa gagatcccca ccaccctggt
     1801 ggacgacttc atcctgagcc ccgtcgtgaa gagaagcttc atccagagca tcaaagtgat
     1861 caacgccatc atcaagaagt acggcctgcc caacgacatc attatcgagc tggcccgcga
     1921 gaagaactcc aaggacgccc agaaaatgat caacgagatg cagaagcgga accggcagac
     1981 caacgagcgg atcgaggaaa tcatccggac caccggcaaa gagaacgcca agtacctgat
     2041 cgagaagatc aagctgcacg acatgcagga aggcaagtgc ctgtacagcc tggaagccat
     2101 ccctctggaa gatctgctga acaacccctt caactatgag gtggaccaca tcatccccag
     2161 aagcgtgtcc ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg aagaagccag
     2221 caagaagggc aaccggaccc cattccagta cctgagcagc agcgacagca agatcagcta
     2281 cgaaaccttc aagaagcaca tcctgaatct ggccaagggc aagggcagaa tcagcaagac
     2341 caagaaagag tatctgctgg aagaacggga catcaacagg ttctccgtgc agaaagactt
     2401 catcaaccgg aacctggtgg ataccagata cgccaccaga ggcctgatga acctgctgcg
     2461 gagctacttc agagtgaaca acctggacgt gaaagtgaag tccatcaatg gcggcttcac
     2521 cagctttctg cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt acaagcacca
     2581 cgccgaggac gccctgatca ttgccaacgc cgatttcatc ttcaaagagt ggaagaaact
     2641 ggacaaggcc aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg ccgagagcat
     2701 gcccgagatc gaaaccgagc aggagtacaa agagatcttc atcacccccc accagatcaa
     2761 gcacattaag gacttcaagg actacaagta cagccaccgg gtggacaaga agcctaatag
     2821 agagctgatt aacgacaccc tgtactccac ccggaaggac gacaagggca acaccctgat
     2881 cgtgaacaat ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa agctgatcaa
     2941 caagagcccc gaaaagctgc tgatgtacca ccacgacccc cagacctacc agaaactgaa
     3001 gctgattatg gaacagtacg gcgacgagaa gaatcccctg tacaagtact acgaggaaac
     3061 cgggaactac ctgaccaagt actccaaaaa ggacaacggc cccgtgatca agaagattaa
     3121 gtattacggc aacaaactga acgcccatct ggacatcacc gacgactacc ccaacagcag
     3181 aaacaaggtc gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc tggacaatgg
     3241 cgtgtacaag ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa actactacga
     3301 agtgaatagc aagtgctatg aggaagctaa gaagctgaag aagatcagca accaggccga
     3361 gtttatcgcc tccttctaca acaacgatct gatcaagatc aacggcgagc tgtatagagt
     3421 gatcggcgtg aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg acatcaccta
     3481 ccgcgagtac ctggaaaaca tgaacgacaa gaggcccccc aggatcatta agacaatcgc
     3541 ctccaagacc cagagcatta agaagtacag cacagacatt ctgggcaacc tgtatgaagt
     3601 gaaatctaag aagcaccctc agatcatcaa aaagggcaaa aggccggcgg ccacgaaaaa
     3661 ggccggccag gcaaaaaaga aaaagggatc cggcggtggc ggagggatgg atgctaagtc
     3721 actaactgcc tggtcccgga cactggtgac cttcaaggat gtatttgtgg acttcaccag
     3781 ggaggagtgg aagctgctgg acactgctca gcagatcgtg tacagaaatg tgatgctgga
     3841 gaactataag aacctggttt ccttgggtta tcagcttact aagccagatg tgatcctccg
     3901 gttggagaag ggagaagagc cctagtctag acatatggat atctgactag tctcgagttt
     3961 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4021 tttttttcac tggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     4081 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     4141 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     4201 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     4261 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4321 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4381 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4441 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     4501 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4561 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta
     4621 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4681 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     4741 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     4801 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4861 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     4921 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4981 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5041 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5101 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5161 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5221 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5281 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5341 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5401 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5461 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5521 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5581 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5641 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     5701 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5761 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     5821 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     5881 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5941 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6001 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6061 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6121 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6181 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6241 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     6301 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6361 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6421 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6481 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6541 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6601 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6661 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6721 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     6781 atgt
//
