LOCUS       sequence-233640-        6773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A EFS driven de-catalyzed SaCas9 fused to KRAB domain for inhibition
            of transcription in targeted region..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6773)
  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 6773)
  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: pJEP305-pAAV-EFS-dSaCas9-KRAB-pA
FEATURES             Location/Qualifiers
     source          1..6773
                     /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             3726..3911
                     /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"
     primer_bind     complement(3978..3997)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4036..4176
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4036..4165
                     /label=AAV2 ITR
     rep_origin      4251..4706
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4338..4357)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4548..4569
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4723..4742)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4842..4864
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4902..4920)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4988..5092
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5093..5161,5162..5953)
                     /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(5311..5330)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6124..6712
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6613..6632
                     /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 ggcggtggcg gagggatgga tgctaagtca ctaactgcct
     3721 ggtcccggac actggtgacc ttcaaggatg tatttgtgga cttcaccagg gaggagtgga
     3781 agctgctgga cactgctcag cagatcgtgt acagaaatgt gatgctggag aactataaga
     3841 acctggtttc cttgggttat cagcttacta agccagatgt gatcctccgg ttggagaagg
     3901 gagaagagcc ctagtctaga catatggata tctgactagt ctcgagttta ttgcagctta
     3961 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4021 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4081 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4141 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4201 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4261 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4321 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4381 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4441 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     4501 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4561 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     4621 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4681 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     4741 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4801 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4861 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     4921 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     4981 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5041 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5101 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5161 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5221 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5281 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5341 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5401 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5461 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5521 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5581 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5641 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5701 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     5761 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     5821 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     5881 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     5941 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6001 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6061 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6121 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6181 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6241 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6301 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6361 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6421 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6481 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6541 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6601 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6661 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6721 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
