LOCUS       sequence-214974-        7073 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 7073)
  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 7073)
  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..7073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     enhancer        165..468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        469..672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     623..643
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     669..693
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      759..768
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             771..791
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3972..4019
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4050..4199
                     /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(4278..4297)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4336..4476
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4336..4465
                     /label=AAV2 ITR
     rep_origin      4551..5006
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4638..4657)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4848..4869
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5023..5042)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5142..5164
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5202..5220)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5288..5392
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5393..5461,5462..6253)
                     /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(5611..5630)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6424..7012
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6913..6932
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtggagt tccgcgttac ataacttacg
      181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      661 ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
      721 gacctccata gaagacaccg gaattcggta ccaccggtgc caccatggcc ccaaagaaga
      781 agcggaaggt cggtatccac ggagtcccag cagccaagcg gaactacatc ctgggcctgg
      841 ccatcggcat caccagcgtg ggctacggca tcatcgacta cgagacacgg gacgtgatcg
      901 atgccggcgt gcggctgttc aaagaggcca acgtggaaaa caacgagggc aggcggagca
      961 agagaggcgc cagaaggctg aagcggcgga ggcggcatag aatccagaga gtgaagaagc
     1021 tgctgttcga ctacaacctg ctgaccgacc acagcgagct gagcggcatc aacccctacg
     1081 aggccagagt gaagggcctg agccagaagc tgagcgagga agagttctct gccgccctgc
     1141 tgcacctggc caagagaaga ggcgtgcaca acgtgaacga ggtggaagag gacaccggca
     1201 acgagctgtc caccaaagag cagatcagcc ggaacagcaa ggccctggaa gagaaatacg
     1261 tggccgaact gcagctggaa cggctgaaga aagacggcga agtgcggggc agcatcaaca
     1321 gattcaagac cagcgactac gtgaaagaag ccaaacagct gctgaaggtg cagaaggcct
     1381 accaccagct ggaccagagc ttcatcgaca cctacatcga cctgctggaa acccggcgga
     1441 cctactatga gggacctggc gagggcagcc ccttcggctg gaaggacatc aaagaatggt
     1501 acgagatgct gatgggccac tgcacctact tccccgagga actgcggagc gtgaagtacg
     1561 cctacaacgc cgacctgtac aacgccctga acgacctgaa caatctcgtg atcaccaggg
     1621 acgagaacga gaagctggaa tattacgaga agttccagat catcgagaac gtgttcaagc
     1681 agaagaagaa gcccaccctg aagcagatcg ccaaagaaat cctcgtgaac gaagaggata
     1741 ttaagggcta cagagtgacc agcaccggca agcccgagtt caccaacctg aaggtgtacc
     1801 acgacatcaa ggacattacc gcccggaaag agattattga gaacgccgag ctgctggatc
     1861 agattgccaa gatcctgacc atctaccaga gcagcgagga catccaggaa gaactgacca
     1921 atctgaactc cgagctgacc caggaagaga tcgagcagat ctctaatctg aagggctata
     1981 ccggcaccca caacctgagc ctgaaggcca tcaacctgat cctggacgag ctgtggcaca
     2041 ccaacgacaa ccagatcgct atcttcaacc ggctgaagct ggtgcccaag aaggtggacc
     2101 tgtcccagca gaaagagatc cccaccaccc tggtggacga cttcatcctg agccccgtcg
     2161 tgaagagaag cttcatccag agcatcaaag tgatcaacgc catcatcaag aagtacggcc
     2221 tgcccaacga catcattatc gagctggccc gcgagaagaa ctccaaggac gcccagaaaa
     2281 tgatcaacga gatgcagaag cggaaccggc agaccaacga gcggatcgag gaaatcatcc
     2341 ggaccaccgg caaagagaac gccaagtacc tgatcgagaa gatcaagctg cacgacatgc
     2401 aggaaggcaa gtgcctgtac agcctggaag ccatccctct ggaagatctg ctgaacaacc
     2461 ccttcaacta tgaggtggac cacatcatcc ccagaagcgt gtccttcgac aacagcttca
     2521 acaacaaggt gctcgtgaag caggaagaag ccagcaagaa gggcaaccgg accccattcc
     2581 agtacctgag cagcagcgac agcaagatca gctacgaaac cttcaagaag cacatcctga
     2641 atctggccaa gggcaagggc agaatcagca agaccaagaa agagtatctg ctggaagaac
     2701 gggacatcaa caggttctcc gtgcagaaag acttcatcaa ccggaacctg gtggatacca
     2761 gatacgccac cagaggcctg atgaacctgc tgcggagcta cttcagagtg aacaacctgg
     2821 acgtgaaagt gaagtccatc aatggcggct tcaccagctt tctgcggcgg aagtggaagt
     2881 ttaagaaaga gcggaacaag gggtacaagc accacgccga ggacgccctg atcattgcca
     2941 acgccgattt catcttcaaa gagtggaaga aactggacaa ggccaaaaaa gtgatggaaa
     3001 accagatgtt cgaggaaaag caggccgaga gcatgcccga gatcgaaacc gagcaggagt
     3061 acaaagagat cttcatcacc ccccaccaga tcaagcacat taaggacttc aaggactaca
     3121 agtacagcca ccgggtggac aagaagccta atagagagct gattaacgac accctgtact
     3181 ccacccggaa ggacgacaag ggcaacaccc tgatcgtgaa caatctgaac ggcctgtacg
     3241 acaaggacaa tgacaagctg aaaaagctga tcaacaagag ccccgaaaag ctgctgatgt
     3301 accaccacga cccccagacc taccagaaac tgaagctgat tatggaacag tacggcgacg
     3361 agaagaatcc cctgtacaag tactacgagg aaaccgggaa ctacctgacc aagtactcca
     3421 aaaaggacaa cggccccgtg atcaagaaga ttaagtatta cggcaacaaa ctgaacgccc
     3481 atctggacat caccgacgac taccccaaca gcagaaacaa ggtcgtgaag ctgtccctga
     3541 agccctacag attcgacgtg tacctggaca atggcgtgta caagttcgtg accgtgaaga
     3601 atctggatgt gatcaaaaaa gaaaactact acgaagtgaa tagcaagtgc tatgaggaag
     3661 ctaagaagct gaagaagatc agcaaccagg ccgagtttat cgcctccttc tacaacaacg
     3721 atctgatcaa gatcaacggc gagctgtata gagtgatcgg cgtgaacaac gacctgctga
     3781 accggatcga agtgaacatg atcgacatca cctaccgcga gtacctggaa aacatgaacg
     3841 acaagaggcc ccccaggatc attaagacaa tcgcctccaa gacccagagc attaagaagt
     3901 acagcacaga cattctgggc aacctgtatg aagtgaaatc taagaagcac cctcagatca
     3961 tcaaaaaggg caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     4021 gatccgaggc cagcggttcc ggacgggctg acgcattgga cgattttgat ctggatatgc
     4081 tgggaagtga cgccctcgat gattttgacc ttgacatgct tggttcggat gcccttgatg
     4141 actttgacct cgacatgctc ggcagtgacg cccttgatga tttcgacctg gacatgctga
     4201 ttaactcaag atgatctaga catatggata tctgactagt ctcgagttta ttgcagctta
     4261 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4321 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4381 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4441 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4501 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4561 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4621 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4681 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4741 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     4801 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4861 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt
     4921 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4981 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     5041 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5101 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5161 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     5221 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5281 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5341 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5401 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5461 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5521 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5581 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5641 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5701 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5761 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5821 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5881 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5941 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6001 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6061 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6121 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6181 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6241 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6301 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6361 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6421 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6481 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6541 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     6601 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6661 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6721 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6781 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6841 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6901 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6961 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7021 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
