LOCUS       sequence-229519-        7062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A CMV 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 7062)
  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 7062)
  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: pJEP303-pAAV-CMV-dSaCas9-VP64-pA
FEATURES             Location/Qualifiers
     source          1..7062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..457
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        458..661
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     612..632
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     658..682
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      748..757
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             760..780
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3961..4008
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4039..4188
                     /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(4267..4286)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4325..4465
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4325..4454
                     /label=AAV2 ITR
     rep_origin      4540..4995
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4627..4646)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4837..4858
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5012..5031)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5131..5153
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5191..5209)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5277..5381
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5382..5450,5451..6242)
                     /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(5600..5619)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6413..7001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6902..6921
                     /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 gcgtggagtt ccgcgttaca taacttacgg taaatggccc
      181 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      241 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      301 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      361 cagtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      421 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      481 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      541 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      601 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      661 tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag
      721 aagacaccgg aattcggtac caccggtgcc accatggccc caaagaagaa gcggaaggtc
      781 ggtatccacg gagtcccagc agccaagcgg aactacatcc tgggcctggc catcggcatc
      841 accagcgtgg gctacggcat catcgactac gagacacggg acgtgatcga tgccggcgtg
      901 cggctgttca aagaggccaa cgtggaaaac aacgagggca ggcggagcaa gagaggcgcc
      961 agaaggctga agcggcggag gcggcataga atccagagag tgaagaagct gctgttcgac
     1021 tacaacctgc tgaccgacca cagcgagctg agcggcatca acccctacga ggccagagtg
     1081 aagggcctga gccagaagct gagcgaggaa gagttctctg ccgccctgct gcacctggcc
     1141 aagagaagag gcgtgcacaa cgtgaacgag gtggaagagg acaccggcaa cgagctgtcc
     1201 accaaagagc agatcagccg gaacagcaag gccctggaag agaaatacgt ggccgaactg
     1261 cagctggaac ggctgaagaa agacggcgaa gtgcggggca gcatcaacag attcaagacc
     1321 agcgactacg tgaaagaagc caaacagctg ctgaaggtgc agaaggccta ccaccagctg
     1381 gaccagagct tcatcgacac ctacatcgac ctgctggaaa cccggcggac ctactatgag
     1441 ggacctggcg agggcagccc cttcggctgg aaggacatca aagaatggta cgagatgctg
     1501 atgggccact gcacctactt ccccgaggaa ctgcggagcg tgaagtacgc ctacaacgcc
     1561 gacctgtaca acgccctgaa cgacctgaac aatctcgtga tcaccaggga cgagaacgag
     1621 aagctggaat attacgagaa gttccagatc atcgagaacg tgttcaagca gaagaagaag
     1681 cccaccctga agcagatcgc caaagaaatc ctcgtgaacg aagaggatat taagggctac
     1741 agagtgacca gcaccggcaa gcccgagttc accaacctga aggtgtacca cgacatcaag
     1801 gacattaccg cccggaaaga gattattgag aacgccgagc tgctggatca gattgccaag
     1861 atcctgacca tctaccagag cagcgaggac atccaggaag aactgaccaa tctgaactcc
     1921 gagctgaccc aggaagagat cgagcagatc tctaatctga agggctatac cggcacccac
     1981 aacctgagcc tgaaggccat caacctgatc ctggacgagc tgtggcacac caacgacaac
     2041 cagatcgcta tcttcaaccg gctgaagctg gtgcccaaga aggtggacct gtcccagcag
     2101 aaagagatcc ccaccaccct ggtggacgac ttcatcctga gccccgtcgt gaagagaagc
     2161 ttcatccaga gcatcaaagt gatcaacgcc atcatcaaga agtacggcct gcccaacgac
     2221 atcattatcg agctggcccg cgagaagaac tccaaggacg cccagaaaat gatcaacgag
     2281 atgcagaagc ggaaccggca gaccaacgag cggatcgagg aaatcatccg gaccaccggc
     2341 aaagagaacg ccaagtacct gatcgagaag atcaagctgc acgacatgca ggaaggcaag
     2401 tgcctgtaca gcctggaagc catccctctg gaagatctgc tgaacaaccc cttcaactat
     2461 gaggtggacc acatcatccc cagaagcgtg tccttcgaca acagcttcaa caacaaggtg
     2521 ctcgtgaagc aggaagaagc cagcaagaag ggcaaccgga ccccattcca gtacctgagc
     2581 agcagcgaca gcaagatcag ctacgaaacc ttcaagaagc acatcctgaa tctggccaag
     2641 ggcaagggca gaatcagcaa gaccaagaaa gagtatctgc tggaagaacg ggacatcaac
     2701 aggttctccg tgcagaaaga cttcatcaac cggaacctgg tggataccag atacgccacc
     2761 agaggcctga tgaacctgct gcggagctac ttcagagtga acaacctgga cgtgaaagtg
     2821 aagtccatca atggcggctt caccagcttt ctgcggcgga agtggaagtt taagaaagag
     2881 cggaacaagg ggtacaagca ccacgccgag gacgccctga tcattgccaa cgccgatttc
     2941 atcttcaaag agtggaagaa actggacaag gccaaaaaag tgatggaaaa ccagatgttc
     3001 gaggaaaagc aggccgagag catgcccgag atcgaaaccg agcaggagta caaagagatc
     3061 ttcatcaccc cccaccagat caagcacatt aaggacttca aggactacaa gtacagccac
     3121 cgggtggaca agaagcctaa tagagagctg attaacgaca ccctgtactc cacccggaag
     3181 gacgacaagg gcaacaccct gatcgtgaac aatctgaacg gcctgtacga caaggacaat
     3241 gacaagctga aaaagctgat caacaagagc cccgaaaagc tgctgatgta ccaccacgac
     3301 ccccagacct accagaaact gaagctgatt atggaacagt acggcgacga gaagaatccc
     3361 ctgtacaagt actacgagga aaccgggaac tacctgacca agtactccaa aaaggacaac
     3421 ggccccgtga tcaagaagat taagtattac ggcaacaaac tgaacgccca tctggacatc
     3481 accgacgact accccaacag cagaaacaag gtcgtgaagc tgtccctgaa gccctacaga
     3541 ttcgacgtgt acctggacaa tggcgtgtac aagttcgtga ccgtgaagaa tctggatgtg
     3601 atcaaaaaag aaaactacta cgaagtgaat agcaagtgct atgaggaagc taagaagctg
     3661 aagaagatca gcaaccaggc cgagtttatc gcctccttct acaacaacga tctgatcaag
     3721 atcaacggcg agctgtatag agtgatcggc gtgaacaacg acctgctgaa ccggatcgaa
     3781 gtgaacatga tcgacatcac ctaccgcgag tacctggaaa acatgaacga caagaggccc
     3841 cccaggatca ttaagacaat cgcctccaag acccagagca ttaagaagta cagcacagac
     3901 attctgggca acctgtatga agtgaaatct aagaagcacc ctcagatcat caaaaagggc
     3961 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg atccgaggcc
     4021 agcggttccg gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac
     4081 gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc
     4141 gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taactcaaga
     4201 tgatctagac atatggatat ctgactagtc tcgagtttat tgcagcttat aatggttaca
     4261 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg gaccgagcgg
     4321 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     4381 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     4441 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     4501 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     4561 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
     4621 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4681 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4741 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4801 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4861 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     4921 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     4981 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5041 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5101 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5161 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     5221 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     5281 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5341 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5401 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5461 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5521 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5581 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5641 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5701 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5761 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5821 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5881 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5941 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6001 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6061 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6121 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6181 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6241 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6301 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6361 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6421 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6481 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6541 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6601 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6661 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6721 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6781 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6841 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6901 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6961 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7021 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
