LOCUS       sequence-214975-        6739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A EFS driven de-catalyzed SaCas9 fused to VP64 domain for increased 
            transcription in targeted region.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6739)
  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 6739)
  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: pJEP304-pAAV-EFS-dSaCas9-VP64-pA
FEATURES             Location/Qualifiers
     source          1..6739
                     /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             3716..3865
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     primer_bind     complement(3944..3963)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4002..4142
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4002..4131
                     /label=AAV2 ITR
     rep_origin      4217..4672
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4304..4323)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4514..4535
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4689..4708)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4808..4830
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4868..4886)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4954..5058
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5059..5127,5128..5919)
                     /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(5277..5296)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6090..6678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6579..6598
                     /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 cgaggccagc ggttccggac gggctgacgc
     3721 attggacgat tttgatctgg atatgctggg aagtgacgcc ctcgatgatt ttgaccttga
     3781 catgcttggt tcggatgccc ttgatgactt tgacctcgac atgctcggca gtgacgccct
     3841 tgatgatttc gacctggaca tgctgattaa ctcaagatga tctagacata tggatatctg
     3901 actagtctcg agtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3961 ttcacaaata aagcattttt ttcactggac cgagcggccg caggaacccc tagtgatgga
     4021 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     4081 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     4141 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4201 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4261 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4321 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4381 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4441 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4501 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc
     4561 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4621 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     4681 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4741 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4801 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     4861 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4921 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4981 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5041 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5101 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5161 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5221 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5281 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5341 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5401 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5461 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5521 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5581 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5641 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5701 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5761 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5821 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5881 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5941 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6001 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6061 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6121 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6181 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     6241 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6301 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6361 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6421 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6481 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6541 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6601 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6661 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6721 tggccttttg ctcacatgt
//
