LOCUS       sequence-229549-        6728 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 6728)
  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 6728)
  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..6728
                     /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        170..381
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      414..423
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             426..446
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3627..3674
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3705..3854
                     /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(3933..3952)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3991..4131
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3991..4120
                     /label=AAV2 ITR
     rep_origin      4206..4661
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4293..4312)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4503..4524
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4678..4697)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4797..4819
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4857..4875)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4943..5047
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5048..5116,5117..5908)
                     /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(5266..5285)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6079..6667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6568..6587
                     /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 gcggccgcac gcgttcgagt ggctccggtg cccgtcagtg ggcagagcgc
      181 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag
      241 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
      301 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
      361 gggtttgccg ccagaacaca ggtgtcgtga cgcgggaatt cggtaccacc ggtgccacca
      421 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc aagcggaact
      481 acatcctggg cctggccatc ggcatcacca gcgtgggcta cggcatcatc gactacgaga
      541 cacgggacgt gatcgatgcc ggcgtgcggc tgttcaaaga ggccaacgtg gaaaacaacg
      601 agggcaggcg gagcaagaga ggcgccagaa ggctgaagcg gcggaggcgg catagaatcc
      661 agagagtgaa gaagctgctg ttcgactaca acctgctgac cgaccacagc gagctgagcg
      721 gcatcaaccc ctacgaggcc agagtgaagg gcctgagcca gaagctgagc gaggaagagt
      781 tctctgccgc cctgctgcac ctggccaaga gaagaggcgt gcacaacgtg aacgaggtgg
      841 aagaggacac cggcaacgag ctgtccacca aagagcagat cagccggaac agcaaggccc
      901 tggaagagaa atacgtggcc gaactgcagc tggaacggct gaagaaagac ggcgaagtgc
      961 ggggcagcat caacagattc aagaccagcg actacgtgaa agaagccaaa cagctgctga
     1021 aggtgcagaa ggcctaccac cagctggacc agagcttcat cgacacctac atcgacctgc
     1081 tggaaacccg gcggacctac tatgagggac ctggcgaggg cagccccttc ggctggaagg
     1141 acatcaaaga atggtacgag atgctgatgg gccactgcac ctacttcccc gaggaactgc
     1201 ggagcgtgaa gtacgcctac aacgccgacc tgtacaacgc cctgaacgac ctgaacaatc
     1261 tcgtgatcac cagggacgag aacgagaagc tggaatatta cgagaagttc cagatcatcg
     1321 agaacgtgtt caagcagaag aagaagccca ccctgaagca gatcgccaaa gaaatcctcg
     1381 tgaacgaaga ggatattaag ggctacagag tgaccagcac cggcaagccc gagttcacca
     1441 acctgaaggt gtaccacgac atcaaggaca ttaccgcccg gaaagagatt attgagaacg
     1501 ccgagctgct ggatcagatt gccaagatcc tgaccatcta ccagagcagc gaggacatcc
     1561 aggaagaact gaccaatctg aactccgagc tgacccagga agagatcgag cagatctcta
     1621 atctgaaggg ctataccggc acccacaacc tgagcctgaa ggccatcaac ctgatcctgg
     1681 acgagctgtg gcacaccaac gacaaccaga tcgctatctt caaccggctg aagctggtgc
     1741 ccaagaaggt ggacctgtcc cagcagaaag agatccccac caccctggtg gacgacttca
     1801 tcctgagccc cgtcgtgaag agaagcttca tccagagcat caaagtgatc aacgccatca
     1861 tcaagaagta cggcctgccc aacgacatca ttatcgagct ggcccgcgag aagaactcca
     1921 aggacgccca gaaaatgatc aacgagatgc agaagcggaa ccggcagacc aacgagcgga
     1981 tcgaggaaat catccggacc accggcaaag agaacgccaa gtacctgatc gagaagatca
     2041 agctgcacga catgcaggaa ggcaagtgcc tgtacagcct ggaagccatc cctctggaag
     2101 atctgctgaa caaccccttc aactatgagg tggaccacat catccccaga agcgtgtcct
     2161 tcgacaacag cttcaacaac aaggtgctcg tgaagcagga agaagccagc aagaagggca
     2221 accggacccc attccagtac ctgagcagca gcgacagcaa gatcagctac gaaaccttca
     2281 agaagcacat cctgaatctg gccaagggca agggcagaat cagcaagacc aagaaagagt
     2341 atctgctgga agaacgggac atcaacaggt tctccgtgca gaaagacttc atcaaccgga
     2401 acctggtgga taccagatac gccaccagag gcctgatgaa cctgctgcgg agctacttca
     2461 gagtgaacaa cctggacgtg aaagtgaagt ccatcaatgg cggcttcacc agctttctgc
     2521 ggcggaagtg gaagtttaag aaagagcgga acaaggggta caagcaccac gccgaggacg
     2581 ccctgatcat tgccaacgcc gatttcatct tcaaagagtg gaagaaactg gacaaggcca
     2641 aaaaagtgat ggaaaaccag atgttcgagg aaaagcaggc cgagagcatg cccgagatcg
     2701 aaaccgagca ggagtacaaa gagatcttca tcacccccca ccagatcaag cacattaagg
     2761 acttcaagga ctacaagtac agccaccggg tggacaagaa gcctaataga gagctgatta
     2821 acgacaccct gtactccacc cggaaggacg acaagggcaa caccctgatc gtgaacaatc
     2881 tgaacggcct gtacgacaag gacaatgaca agctgaaaaa gctgatcaac aagagccccg
     2941 aaaagctgct gatgtaccac cacgaccccc agacctacca gaaactgaag ctgattatgg
     3001 aacagtacgg cgacgagaag aatcccctgt acaagtacta cgaggaaacc gggaactacc
     3061 tgaccaagta ctccaaaaag gacaacggcc ccgtgatcaa gaagattaag tattacggca
     3121 acaaactgaa cgcccatctg gacatcaccg acgactaccc caacagcaga aacaaggtcg
     3181 tgaagctgtc cctgaagccc tacagattcg acgtgtacct ggacaatggc gtgtacaagt
     3241 tcgtgaccgt gaagaatctg gatgtgatca aaaaagaaaa ctactacgaa gtgaatagca
     3301 agtgctatga ggaagctaag aagctgaaga agatcagcaa ccaggccgag tttatcgcct
     3361 ccttctacaa caacgatctg atcaagatca acggcgagct gtatagagtg atcggcgtga
     3421 acaacgacct gctgaaccgg atcgaagtga acatgatcga catcacctac cgcgagtacc
     3481 tggaaaacat gaacgacaag aggcccccca ggatcattaa gacaatcgcc tccaagaccc
     3541 agagcattaa gaagtacagc acagacattc tgggcaacct gtatgaagtg aaatctaaga
     3601 agcaccctca gatcatcaaa aagggcaaaa ggccggcggc cacgaaaaag gccggccagg
     3661 caaaaaagaa aaagggatcc gaggccagcg gttccggacg ggctgacgca ttggacgatt
     3721 ttgatctgga tatgctggga agtgacgccc tcgatgattt tgaccttgac atgcttggtt
     3781 cggatgccct tgatgacttt gacctcgaca tgctcggcag tgacgccctt gatgatttcg
     3841 acctggacat gctgattaac tcaagatgat ctagacatat ggatatctga ctagtctcga
     3901 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3961 agcatttttt tcactggacc gagcggccgc aggaacccct agtgatggag ttggccactc
     4021 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4081 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     4141 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     4201 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4261 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     4321 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4381 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     4441 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4501 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     4561 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     4621 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     4681 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     4741 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4801 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     4861 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     4921 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4981 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5041 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5101 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5161 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5221 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5281 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5341 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5401 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5461 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5521 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5581 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5641 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5701 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5761 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5821 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5881 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5941 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6001 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6061 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6121 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6181 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6241 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6301 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6361 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6421 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6481 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6541 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6601 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6661 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6721 tcacatgt
//
