LOCUS       sequence-232027-        6998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An EFS driven inverted de-catalyzed SaCas9 fused to the 
            transcription activator VP64. dSaCas9-VP64 is floxed to render the 
            system cre-dependent..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6998)
  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 6998)
  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: pJEP308-pAAV-EFS-dSaCas9-VP64-Dio-pA
FEATURES             Location/Qualifiers
     source          1..6998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      10..19
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             22..42
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3223..3270
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3301..3450
                     /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"
     protein_bind    3488..3521
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3572..3605
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     promoter        complement(3640..3851)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     repeat_region   3891..4020
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4082..4670)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4162..4181)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4841..5632,5633..5701))
                     /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     5464..5483
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5702..5806)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5874..5892
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5930..5952)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6052..6071
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6088..6543)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6225..6246)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6437..6456
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6618..6758
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6629..6758
                     /label=AAV2 ITR
     primer_bind     6797..6816
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(6869..6902)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(6953..6986)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
ORIGIN
        1 atgaccggtg ccaccatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca
       61 gcagccaagc ggaactacat cctgggcctg gccatcggca tcaccagcgt gggctacggc
      121 atcatcgact acgagacacg ggacgtgatc gatgccggcg tgcggctgtt caaagaggcc
      181 aacgtggaaa acaacgaggg caggcggagc aagagaggcg ccagaaggct gaagcggcgg
      241 aggcggcata gaatccagag agtgaagaag ctgctgttcg actacaacct gctgaccgac
      301 cacagcgagc tgagcggcat caacccctac gaggccagag tgaagggcct gagccagaag
      361 ctgagcgagg aagagttctc tgccgccctg ctgcacctgg ccaagagaag aggcgtgcac
      421 aacgtgaacg aggtggaaga ggacaccggc aacgagctgt ccaccaaaga gcagatcagc
      481 cggaacagca aggccctgga agagaaatac gtggccgaac tgcagctgga acggctgaag
      541 aaagacggcg aagtgcgggg cagcatcaac agattcaaga ccagcgacta cgtgaaagaa
      601 gccaaacagc tgctgaaggt gcagaaggcc taccaccagc tggaccagag cttcatcgac
      661 acctacatcg acctgctgga aacccggcgg acctactatg agggacctgg cgagggcagc
      721 cccttcggct ggaaggacat caaagaatgg tacgagatgc tgatgggcca ctgcacctac
      781 ttccccgagg aactgcggag cgtgaagtac gcctacaacg ccgacctgta caacgccctg
      841 aacgacctga acaatctcgt gatcaccagg gacgagaacg agaagctgga atattacgag
      901 aagttccaga tcatcgagaa cgtgttcaag cagaagaaga agcccaccct gaagcagatc
      961 gccaaagaaa tcctcgtgaa cgaagaggat attaagggct acagagtgac cagcaccggc
     1021 aagcccgagt tcaccaacct gaaggtgtac cacgacatca aggacattac cgcccggaaa
     1081 gagattattg agaacgccga gctgctggat cagattgcca agatcctgac catctaccag
     1141 agcagcgagg acatccagga agaactgacc aatctgaact ccgagctgac ccaggaagag
     1201 atcgagcaga tctctaatct gaagggctat accggcaccc acaacctgag cctgaaggcc
     1261 atcaacctga tcctggacga gctgtggcac accaacgaca accagatcgc tatcttcaac
     1321 cggctgaagc tggtgcccaa gaaggtggac ctgtcccagc agaaagagat ccccaccacc
     1381 ctggtggacg acttcatcct gagccccgtc gtgaagagaa gcttcatcca gagcatcaaa
     1441 gtgatcaacg ccatcatcaa gaagtacggc ctgcccaacg acatcattat cgagctggcc
     1501 cgcgagaaga actccaagga cgcccagaaa atgatcaacg agatgcagaa gcggaaccgg
     1561 cagaccaacg agcggatcga ggaaatcatc cggaccaccg gcaaagagaa cgccaagtac
     1621 ctgatcgaga agatcaagct gcacgacatg caggaaggca agtgcctgta cagcctggaa
     1681 gccatccctc tggaagatct gctgaacaac cccttcaact atgaggtgga ccacatcatc
     1741 cccagaagcg tgtccttcga caacagcttc aacaacaagg tgctcgtgaa gcaggaagaa
     1801 gccagcaaga agggcaaccg gaccccattc cagtacctga gcagcagcga cagcaagatc
     1861 agctacgaaa ccttcaagaa gcacatcctg aatctggcca agggcaaggg cagaatcagc
     1921 aagaccaaga aagagtatct gctggaagaa cgggacatca acaggttctc cgtgcagaaa
     1981 gacttcatca accggaacct ggtggatacc agatacgcca ccagaggcct gatgaacctg
     2041 ctgcggagct acttcagagt gaacaacctg gacgtgaaag tgaagtccat caatggcggc
     2101 ttcaccagct ttctgcggcg gaagtggaag tttaagaaag agcggaacaa ggggtacaag
     2161 caccacgccg aggacgccct gatcattgcc aacgccgatt tcatcttcaa agagtggaag
     2221 aaactggaca aggccaaaaa agtgatggaa aaccagatgt tcgaggaaaa gcaggccgag
     2281 agcatgcccg agatcgaaac cgagcaggag tacaaagaga tcttcatcac cccccaccag
     2341 atcaagcaca ttaaggactt caaggactac aagtacagcc accgggtgga caagaagcct
     2401 aatagagagc tgattaacga caccctgtac tccacccgga aggacgacaa gggcaacacc
     2461 ctgatcgtga acaatctgaa cggcctgtac gacaaggaca atgacaagct gaaaaagctg
     2521 atcaacaaga gccccgaaaa gctgctgatg taccaccacg acccccagac ctaccagaaa
     2581 ctgaagctga ttatggaaca gtacggcgac gagaagaatc ccctgtacaa gtactacgag
     2641 gaaaccggga actacctgac caagtactcc aaaaaggaca acggccccgt gatcaagaag
     2701 attaagtatt acggcaacaa actgaacgcc catctggaca tcaccgacga ctaccccaac
     2761 agcagaaaca aggtcgtgaa gctgtccctg aagccctaca gattcgacgt gtacctggac
     2821 aatggcgtgt acaagttcgt gaccgtgaag aatctggatg tgatcaaaaa agaaaactac
     2881 tacgaagtga atagcaagtg ctatgaggaa gctaagaagc tgaagaagat cagcaaccag
     2941 gccgagttta tcgcctcctt ctacaacaac gatctgatca agatcaacgg cgagctgtat
     3001 agagtgatcg gcgtgaacaa cgacctgctg aaccggatcg aagtgaacat gatcgacatc
     3061 acctaccgcg agtacctgga aaacatgaac gacaagaggc cccccaggat cattaagaca
     3121 atcgcctcca agacccagag cattaagaag tacagcacag acattctggg caacctgtat
     3181 gaagtgaaat ctaagaagca ccctcagatc atcaaaaagg gcaaaaggcc ggcggccacg
     3241 aaaaaggccg gccaggcaaa aaagaaaaag ggatccgagg ccagcggttc cggacgggct
     3301 gacgcattgg acgattttga tctggatatg ctgggaagtg acgccctcga tgattttgac
     3361 cttgacatgc ttggttcgga tgcccttgat gactttgacc tcgacatgct cggcagtgac
     3421 gcccttgatg atttcgacct ggacatgctg attaactcaa gatgatctag aacaagtaag
     3481 gcgcgccata acttcgtata atgtatgcta tacgaagtta ttaagaggtt tcatattgct
     3541 aatagcagct acaatccagc taccattctg cataacttcg tataaagtat cctatacgaa
     3601 gttattccgg agtcgactcg aattcccgcg tcacgacacc tgtgttctgg cggcaaaccc
     3661 gttgcgaaaa agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc
     3721 gggaaaaagg cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct
     3781 ctaggcaccc gttcaattgc cgacccctcc ccccaacttc tcggggactg tgggcgatgt
     3841 gcgctctgcc cactgacggg caccggagcc actcgaacgc gtgcggccgc aggaacccct
     3901 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     3961 aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     4021 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4081 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4141 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4201 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4261 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4321 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4381 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4441 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4501 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4561 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4621 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4681 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4741 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4801 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4861 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4921 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4981 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5041 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5101 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5161 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5221 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5281 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5341 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5401 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5461 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5521 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5581 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5641 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5701 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5761 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5821 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5881 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     5941 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     6001 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     6061 agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg
     6121 cgttaaattt ttgttaaatc agctcatttt ttaaccaata gaccgaaatc ggcaaaatcc
     6181 cttataaatc aaaagaatag cccgagatag agttgagtgt tgttccagtt tggaacaaga
     6241 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     6301 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag
     6361 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     6421 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctaaggcg ctggcaagtg
     6481 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     6541 cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa
     6601 ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg
     6661 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
     6721 cagagaggga gtggccaact ccatcactag gggttcctgc ggccgctcgg tccagtgaaa
     6781 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     6841 caataaactc gagactagta tcgaattcat aacttcgtat agcatacatt atacgaagtt
     6901 attctttgca ccattctaaa gaataacagt gataatttct gggttaaggc aaataacttc
     6961 gtataggata ctttatacga agttatgcta gcgccacc
//
