LOCUS       sequence-194608-        7627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSa Cas9 fused to optimized combination of truncated 
            activation domains from EFS promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7627)
  AUTHORS   Vora S, Cheng J, Xiao R, VanDusen NJ, Quintino L, Pu WT, 
            Vandenberghe LH, Chavez A, Church G
  TITLE     Rational design of a compact CRISPR-Cas9 activator for AAV-mediated 
            delivery
  JOURNAL   bioRxiv 2018. 298620
REFERENCE   2  (bases 1 to 7627)
  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: "bioRxiv 
            2018. 298620"
COMMENT     Sequence Label: pAAV-EFS-dSa-VPR mini.
FEATURES             Location/Qualifiers
     source          1..7627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..27)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     127..146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(163..618)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(300..321)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     512..531
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   693..833
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   704..833
                     /label=AAV2 ITR
     promoter        891..1102
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1110..1119
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1122..1142
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4323..4370
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4401..4550
                     /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"
     CDS             4575..4595
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4755..5111
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     primer_bind     complement(5367..5384)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5373..5580
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5593..5722
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(5784..6372)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5864..5883)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6543..7334,7335..7403))
                     /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     7166..7185
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7404..7508)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7576..7594
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
       61 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
      121 actatgcggc atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata
      181 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg
      241 aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc
      301 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
      361 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt
      421 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
      481 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
      541 aggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
      601 cgccgctaca gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag
      661 atgcgtaagg agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct
      721 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
      781 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg
      841 cctcgagtag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg
      901 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat ccggtgccta
      961 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     1021 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     1081 cgggtttgcc gccagaacac aggaccggtg ccaccatggc cccaaagaag aagcggaagg
     1141 tcggtatcca cggagtccca gcagccaagc ggaactacat cctgggcctg gccatcggca
     1201 tcaccagcgt gggctacggc atcatcgact acgagacacg ggacgtgatc gatgccggcg
     1261 tgcggctgtt caaagaggcc aacgtggaaa acaacgaggg caggcggagc aagagaggcg
     1321 ccagaaggct gaagcggcgg aggcggcata gaatccagag agtgaagaag ctgctgttcg
     1381 actacaacct gctgaccgac cacagcgagc tgagcggcat caacccctac gaggccagag
     1441 tgaagggcct gagccagaag ctgagcgagg aagagttctc tgccgccctg ctgcacctgg
     1501 ccaagagaag aggcgtgcac aacgtgaacg aggtggaaga ggacaccggc aacgagctgt
     1561 ccaccaaaga gcagatcagc cggaacagca aggccctgga agagaaatac gtggccgaac
     1621 tgcagctgga acggctgaag aaagacggcg aagtgcgggg cagcatcaac agattcaaga
     1681 ccagcgacta cgtgaaagaa gccaaacagc tgctgaaggt gcagaaggcc taccaccagc
     1741 tggaccagag cttcatcgac acctacatcg acctgctgga aacccggcgg acctactatg
     1801 agggacctgg cgagggcagc cccttcggct ggaaggacat caaagaatgg tacgagatgc
     1861 tgatgggcca ctgcacctac ttccccgagg aactgcggag cgtgaagtac gcctacaacg
     1921 ccgacctgta caacgccctg aacgacctga acaatctcgt gatcaccagg gacgagaacg
     1981 agaagctgga atattacgag aagttccaga tcatcgagaa cgtgttcaag cagaagaaga
     2041 agcccaccct gaagcagatc gccaaagaaa tcctcgtgaa cgaagaggat attaagggct
     2101 acagagtgac cagcaccggc aagcccgagt tcaccaacct gaaggtgtac cacgacatca
     2161 aggacattac cgcccggaaa gagattattg agaacgccga gctgctggat cagattgcca
     2221 agatcctgac catctaccag agcagcgagg acatccagga agaactgacc aatctgaact
     2281 ccgagctgac ccaggaagag atcgagcaga tctctaatct gaagggctat accggcaccc
     2341 acaacctgag cctgaaggcc atcaacctga tcctggacga gctgtggcac accaacgaca
     2401 accagatcgc tatcttcaac cggctgaagc tggtgcccaa gaaggtggac ctgtcccagc
     2461 agaaagagat ccccaccacc ctggtggacg acttcatcct gagccccgtc gtgaagagaa
     2521 gcttcatcca gagcatcaaa gtgatcaacg ccatcatcaa gaagtacggc ctgcccaacg
     2581 acatcattat cgagctggcc cgcgagaaga actccaagga cgcccagaaa atgatcaacg
     2641 agatgcagaa gcggaaccgg cagaccaacg agcggatcga ggaaatcatc cggaccaccg
     2701 gcaaagagaa cgccaagtac ctgatcgaga agatcaagct gcacgacatg caggaaggca
     2761 agtgcctgta cagcctggaa gccatccctc tggaagatct gctgaacaac cccttcaact
     2821 atgaggtgga ccacatcatc cccagaagcg tgtccttcga caacagcttc aacaacaagg
     2881 tgctcgtgaa gcaggaagaa gccagcaaga agggcaaccg gaccccattc cagtacctga
     2941 gcagcagcga cagcaagatc agctacgaaa ccttcaagaa gcacatcctg aatctggcca
     3001 agggcaaggg cagaatcagc aagaccaaga aagagtatct gctggaagaa cgggacatca
     3061 acaggttctc cgtgcagaaa gacttcatca accggaacct ggtggatacc agatacgcca
     3121 ccagaggcct gatgaacctg ctgcggagct acttcagagt gaacaacctg gacgtgaaag
     3181 tgaagtccat caatggcggc ttcaccagct ttctgcggcg gaagtggaag tttaagaaag
     3241 agcggaacaa ggggtacaag caccacgccg aggacgccct gatcattgcc aacgccgatt
     3301 tcatcttcaa agagtggaag aaactggaca aggccaaaaa agtgatggaa aaccagatgt
     3361 tcgaggaaaa gcaggccgag agcatgcccg agatcgaaac cgagcaggag tacaaagaga
     3421 tcttcatcac cccccaccag atcaagcaca ttaaggactt caaggactac aagtacagcc
     3481 accgggtgga caagaagcct aatagagagc tgattaacga caccctgtac tccacccgga
     3541 aggacgacaa gggcaacacc ctgatcgtga acaatctgaa cggcctgtac gacaaggaca
     3601 atgacaagct gaaaaagctg atcaacaaga gccccgaaaa gctgctgatg taccaccacg
     3661 acccccagac ctaccagaaa ctgaagctga ttatggaaca gtacggcgac gagaagaatc
     3721 ccctgtacaa gtactacgag gaaaccggga actacctgac caagtactcc aaaaaggaca
     3781 acggccccgt gatcaagaag attaagtatt acggcaacaa actgaacgcc catctggaca
     3841 tcaccgacga ctaccccaac agcagaaaca aggtcgtgaa gctgtccctg aagccctaca
     3901 gattcgacgt gtacctggac aatggcgtgt acaagttcgt gaccgtgaag aatctggatg
     3961 tgatcaaaaa agaaaactac tacgaagtga atagcaagtg ctatgaggaa gctaagaagc
     4021 tgaagaagat cagcaaccag gccgagttta tcgcctcctt ctacaacaac gatctgatca
     4081 agatcaacgg cgagctgtat agagtgatcg gcgtgaacaa cgacctgctg aaccggatcg
     4141 aagtgaacat gatcgacatc acctaccgcg agtacctgga aaacatgaac gacaagaggc
     4201 cccccaggat cattaagaca atcgcctcca agacccagag cattaagaag tacagcacag
     4261 acattctggg caacctgtat gaagtgaaat ctaagaagca ccctcagatc atcaaaaagg
     4321 gcaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag ggatccgagg
     4381 ccagcggttc cggacgggct gacgcattgg acgattttga tctggatatg ctgggaagtg
     4441 acgccctcga tgattttgac cttgacatgc ttggttcgga tgcccttgat gactttgacc
     4501 tcgacatgct cggcagtgac gcccttgatg atttcgacct ggacatgctg attaactcta
     4561 gaagttccgg atctccgaaa aagaaacgca aagttggttc gggaggtggt tcgggtggct
     4621 ctggatcagt gctgcctcag gctcctgctc ctgcaccagc tccagccatg gtgtctgcac
     4681 tggctcaggc accagcaccc gtgcctgtgc tggctcctgg acctccacag gctgtggctc
     4741 caccagcccc taaacctaca caggccggcg agggcacact gtctgaagct ctgctgcagc
     4801 tgcagttcga cgacgaggat ctgggagccc tgctgggaaa cagcaccgat cctgccgtgt
     4861 tcaccgacct ggccagcgtg gacaacagcg agttccagca gctgctgaac cagggcatcc
     4921 ctgtggcccc tcacaccacc gagcccatgc tgatggaata ccccgaggcc atcacccggc
     4981 tcgtgacagg cgctcagagg cctcctgatc cagctcctgc ccctctggga gcaccaggcc
     5041 tgcctaatgg actgctgtct ggcgacgagg acttcagctc tatcgccgat atggatttct
     5101 cagccttgct gtcaggcggt ggtagtggtg ggagcggtag tgacctttcc catccgcccc
     5161 caaggggcca tctggatgag ctgacaacca cacttgagtc catgaccgag gatctgaacc
     5221 tggactcacc cctgaccccg gaattgaacg agattctgga taccttcctg aacgacgagt
     5281 gcctcttgca tgccatgcat atcagcacag gactgtccat cttcgacaca tctctgtttt
     5341 aggaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     5401 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     5461 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     5521 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
     5581 gctagaggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     5641 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggcggcctc agtgagcgag
     5701 cgagcgcgca gctgcctgca ggacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5761 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5821 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5881 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5941 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6001 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6061 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6121 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6181 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6241 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6301 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6361 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6421 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6481 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6541 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6601 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6661 cccagtgctg caatgatacc gcggcttcca cgctcaccgg ctccagattt atcagcaata
     6721 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6781 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6841 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6901 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6961 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7021 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7081 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7141 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7201 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7261 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7321 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7381 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7441 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7501 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     7561 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     7621 gacggtg
//
