LOCUS       sequence-237140-        7043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An EFS driven inverted de-catalyzed SaCas9 fused to the 
            transcription repressor KRAB. dSaCas9-KRAB is floxed to render the 
            system cre-dependent..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7043)
  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 7043)
  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: pJEP309-pAAV-EFS-dSaCas9-KRAB-Dio-pA
FEATURES             Location/Qualifiers
     source          1..7043
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     192..211
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   373..513
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   384..513
                     /label=AAV2 ITR
     primer_bind     552..571
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(624..657)
                     /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(708..741)
                     /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)."
     regulatory      763..772
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             775..795
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3976..4023
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4075..4260
                     /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"
     protein_bind    4286..4319
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4370..4403
                     /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(4438..4649)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     repeat_region   4689..4818
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4880..5468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4960..4979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5639..6430,6431..6499))
                     /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     6262..6281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6500..6604)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6672..6690
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6728..6750)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6850..6869
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(join(6886..7043,1..298))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(join(7023..7043,1))
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
       61 ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta
      121 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
      181 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta aggcgctggc aagtgtagcg
      241 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtac
      301 tatggttgct ttgacgtatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
      361 gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa
      421 agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
      481 agggagtggc caactccatc actaggggtt cctgcggccg ctcggtccag tgaaaaaaat
      541 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
      601 aactcgagac tagtatcgaa ttcataactt cgtatagcat acattatacg aagttattct
      661 ttgcaccatt ctaaagaata acagtgataa tttctgggtt aaggcaaata acttcgtata
      721 ggatacttta tacgaagtta tgctagcgcc accatgaccg gtgccaccat ggccccaaag
      781 aagaagcgga aggtcggtat ccacggagtc ccagcagcca agcggaacta catcctgggc
      841 ctggccatcg gcatcaccag cgtgggctac ggcatcatcg actacgagac acgggacgtg
      901 atcgatgccg gcgtgcggct gttcaaagag gccaacgtgg aaaacaacga gggcaggcgg
      961 agcaagagag gcgccagaag gctgaagcgg cggaggcggc atagaatcca gagagtgaag
     1021 aagctgctgt tcgactacaa cctgctgacc gaccacagcg agctgagcgg catcaacccc
     1081 tacgaggcca gagtgaaggg cctgagccag aagctgagcg aggaagagtt ctctgccgcc
     1141 ctgctgcacc tggccaagag aagaggcgtg cacaacgtga acgaggtgga agaggacacc
     1201 ggcaacgagc tgtccaccaa agagcagatc agccggaaca gcaaggccct ggaagagaaa
     1261 tacgtggccg aactgcagct ggaacggctg aagaaagacg gcgaagtgcg gggcagcatc
     1321 aacagattca agaccagcga ctacgtgaaa gaagccaaac agctgctgaa ggtgcagaag
     1381 gcctaccacc agctggacca gagcttcatc gacacctaca tcgacctgct ggaaacccgg
     1441 cggacctact atgagggacc tggcgagggc agccccttcg gctggaagga catcaaagaa
     1501 tggtacgaga tgctgatggg ccactgcacc tacttccccg aggaactgcg gagcgtgaag
     1561 tacgcctaca acgccgacct gtacaacgcc ctgaacgacc tgaacaatct cgtgatcacc
     1621 agggacgaga acgagaagct ggaatattac gagaagttcc agatcatcga gaacgtgttc
     1681 aagcagaaga agaagcccac cctgaagcag atcgccaaag aaatcctcgt gaacgaagag
     1741 gatattaagg gctacagagt gaccagcacc ggcaagcccg agttcaccaa cctgaaggtg
     1801 taccacgaca tcaaggacat taccgcccgg aaagagatta ttgagaacgc cgagctgctg
     1861 gatcagattg ccaagatcct gaccatctac cagagcagcg aggacatcca ggaagaactg
     1921 accaatctga actccgagct gacccaggaa gagatcgagc agatctctaa tctgaagggc
     1981 tataccggca cccacaacct gagcctgaag gccatcaacc tgatcctgga cgagctgtgg
     2041 cacaccaacg acaaccagat cgctatcttc aaccggctga agctggtgcc caagaaggtg
     2101 gacctgtccc agcagaaaga gatccccacc accctggtgg acgacttcat cctgagcccc
     2161 gtcgtgaaga gaagcttcat ccagagcatc aaagtgatca acgccatcat caagaagtac
     2221 ggcctgccca acgacatcat tatcgagctg gcccgcgaga agaactccaa ggacgcccag
     2281 aaaatgatca acgagatgca gaagcggaac cggcagacca acgagcggat cgaggaaatc
     2341 atccggacca ccggcaaaga gaacgccaag tacctgatcg agaagatcaa gctgcacgac
     2401 atgcaggaag gcaagtgcct gtacagcctg gaagccatcc ctctggaaga tctgctgaac
     2461 aaccccttca actatgaggt ggaccacatc atccccagaa gcgtgtcctt cgacaacagc
     2521 ttcaacaaca aggtgctcgt gaagcaggaa gaagccagca agaagggcaa ccggacccca
     2581 ttccagtacc tgagcagcag cgacagcaag atcagctacg aaaccttcaa gaagcacatc
     2641 ctgaatctgg ccaagggcaa gggcagaatc agcaagacca agaaagagta tctgctggaa
     2701 gaacgggaca tcaacaggtt ctccgtgcag aaagacttca tcaaccggaa cctggtggat
     2761 accagatacg ccaccagagg cctgatgaac ctgctgcgga gctacttcag agtgaacaac
     2821 ctggacgtga aagtgaagtc catcaatggc ggcttcacca gctttctgcg gcggaagtgg
     2881 aagtttaaga aagagcggaa caaggggtac aagcaccacg ccgaggacgc cctgatcatt
     2941 gccaacgccg atttcatctt caaagagtgg aagaaactgg acaaggccaa aaaagtgatg
     3001 gaaaaccaga tgttcgagga aaagcaggcc gagagcatgc ccgagatcga aaccgagcag
     3061 gagtacaaag agatcttcat caccccccac cagatcaagc acattaagga cttcaaggac
     3121 tacaagtaca gccaccgggt ggacaagaag cctaatagag agctgattaa cgacaccctg
     3181 tactccaccc ggaaggacga caagggcaac accctgatcg tgaacaatct gaacggcctg
     3241 tacgacaagg acaatgacaa gctgaaaaag ctgatcaaca agagccccga aaagctgctg
     3301 atgtaccacc acgaccccca gacctaccag aaactgaagc tgattatgga acagtacggc
     3361 gacgagaaga atcccctgta caagtactac gaggaaaccg ggaactacct gaccaagtac
     3421 tccaaaaagg acaacggccc cgtgatcaag aagattaagt attacggcaa caaactgaac
     3481 gcccatctgg acatcaccga cgactacccc aacagcagaa acaaggtcgt gaagctgtcc
     3541 ctgaagccct acagattcga cgtgtacctg gacaatggcg tgtacaagtt cgtgaccgtg
     3601 aagaatctgg atgtgatcaa aaaagaaaac tactacgaag tgaatagcaa gtgctatgag
     3661 gaagctaaga agctgaagaa gatcagcaac caggccgagt ttatcgcctc cttctacaac
     3721 aacgatctga tcaagatcaa cggcgagctg tatagagtga tcggcgtgaa caacgacctg
     3781 ctgaaccgga tcgaagtgaa catgatcgac atcacctacc gcgagtacct ggaaaacatg
     3841 aacgacaaga ggccccccag gatcattaag acaatcgcct ccaagaccca gagcattaag
     3901 aagtacagca cagacattct gggcaacctg tatgaagtga aatctaagaa gcaccctcag
     3961 atcatcaaaa agggcaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa
     4021 aagggatccg gcggtggcgg agggatggat gctaagtcac taactgcctg gtcccggaca
     4081 ctggtgacct tcaaggatgt atttgtggac ttcaccaggg aggagtggaa gctgctggac
     4141 actgctcagc agatcgtgta cagaaatgtg atgctggaga actataagaa cctggtttcc
     4201 ttgggttatc agcttactaa gccagatgtg atcctccggt tggagaaggg agaagagccc
     4261 tagtctagaa caagtaaggc gcgccataac ttcgtataat gtatgctata cgaagttatt
     4321 aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgca taacttcgta
     4381 taaagtatcc tatacgaagt tattccggag tcgactcgaa ttcccgcgtc acgacacctg
     4441 tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata
     4501 cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt
     4561 taccccgcgc caccttctct aggcacccgt tcaattgccg acccctcccc ccaacttctc
     4621 ggggactgtg ggcgatgtgc gctctgccca ctgacgggca ccggagccac tcgaacgcgt
     4681 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4741 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc ggcctcagtg agcgagcgag
     4801 cgcgcagctg cctgcaggac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4861 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4921 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4981 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5041 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5101 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5161 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5221 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5281 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5341 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5401 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5461 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5521 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5581 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5641 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5701 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5761 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5821 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5881 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5941 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6001 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6061 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6121 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6181 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6241 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6301 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6361 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6421 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6481 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6541 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6601 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6661 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     6721 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     6781 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     6841 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccataaaatt gtaaacgtta
     6901 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaataga
     6961 ccgaaatcgg caaaatccct tataaatcaa aagaatagcc cgagatagag ttgagtgttg
     7021 ttccagtttg gaacaagagt cca
//
