LOCUS       sequence-194506-        7800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSa Cas9 fused to optimized combination of truncated 
            activation domains with syn pA (poly adenylation) signal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7800)
  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 7800)
  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-CMV-dSa-VPR mini.-syn pA
FEATURES             Location/Qualifiers
     source          1..7800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..38
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(55..510)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(192..213)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     404..423
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   585..725
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   596..725
                     /label=AAV2 ITR
     enhancer        745..1124
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1125..1328
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1279..1299
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1347..1356
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1359..1379
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4560..4607
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4638..4787
                     /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             4812..4832
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4992..5348
                     /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"
     polyA_signal    5586..5634
                     /note="synthetic polyadenylation signal"
     repeat_region   5647..5787
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5647..5776
                     /label=AAV2 ITR
     rep_origin      complement(5849..6437)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5929..5948)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6608..7399,7400..7468))
                     /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     7231..7250
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7469..7573)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7641..7659
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7697..7719)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac cataaaattg
       61 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
      121 accaatagac cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagagt
      181 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
      241 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca cccaaatcaa
      301 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
      361 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
      421 gagcgggcgc taaggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
      481 ccgcgcttaa tgcgccgcta cagggcgcgt actatggttg ctttgacgta tgcggtgtga
      541 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgcccctgca ggcagctgcg
      601 cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg
      661 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg
      721 ttcctgcggc cgcctcgagg cgttgacatt gattattgac tagttattaa tagtaatcaa
      781 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      841 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      901 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      961 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1021 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1081 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     1141 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     1201 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     1261 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     1321 gcagagctct ctggctaact accggtgcca ccatggcccc aaagaagaag cggaaggtcg
     1381 gtatccacgg agtcccagca gccaagcgga actacatcct gggcctggcc atcggcatca
     1441 ccagcgtggg ctacggcatc atcgactacg agacacggga cgtgatcgat gccggcgtgc
     1501 ggctgttcaa agaggccaac gtggaaaaca acgagggcag gcggagcaag agaggcgcca
     1561 gaaggctgaa gcggcggagg cggcatagaa tccagagagt gaagaagctg ctgttcgact
     1621 acaacctgct gaccgaccac agcgagctga gcggcatcaa cccctacgag gccagagtga
     1681 agggcctgag ccagaagctg agcgaggaag agttctctgc cgccctgctg cacctggcca
     1741 agagaagagg cgtgcacaac gtgaacgagg tggaagagga caccggcaac gagctgtcca
     1801 ccaaagagca gatcagccgg aacagcaagg ccctggaaga gaaatacgtg gccgaactgc
     1861 agctggaacg gctgaagaaa gacggcgaag tgcggggcag catcaacaga ttcaagacca
     1921 gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca gaaggcctac caccagctgg
     1981 accagagctt catcgacacc tacatcgacc tgctggaaac ccggcggacc tactatgagg
     2041 gacctggcga gggcagcccc ttcggctgga aggacatcaa agaatggtac gagatgctga
     2101 tgggccactg cacctacttc cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg
     2161 acctgtacaa cgccctgaac gacctgaaca atctcgtgat caccagggac gagaacgaga
     2221 agctggaata ttacgagaag ttccagatca tcgagaacgt gttcaagcag aagaagaagc
     2281 ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga agaggatatt aagggctaca
     2341 gagtgaccag caccggcaag cccgagttca ccaacctgaa ggtgtaccac gacatcaagg
     2401 acattaccgc ccggaaagag attattgaga acgccgagct gctggatcag attgccaaga
     2461 tcctgaccat ctaccagagc agcgaggaca tccaggaaga actgaccaat ctgaactccg
     2521 agctgaccca ggaagagatc gagcagatct ctaatctgaa gggctatacc ggcacccaca
     2581 acctgagcct gaaggccatc aacctgatcc tggacgagct gtggcacacc aacgacaacc
     2641 agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga
     2701 aagagatccc caccaccctg gtggacgact tcatcctgag ccccgtcgtg aagagaagct
     2761 tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaacgaca
     2821 tcattatcga gctggcccgc gagaagaact ccaaggacgc ccagaaaatg atcaacgaga
     2881 tgcagaagcg gaaccggcag accaacgagc ggatcgagga aatcatccgg accaccggca
     2941 aagagaacgc caagtacctg atcgagaaga tcaagctgca cgacatgcag gaaggcaagt
     3001 gcctgtacag cctggaagcc atccctctgg aagatctgct gaacaacccc ttcaactatg
     3061 aggtggacca catcatcccc agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc
     3121 tcgtgaagca ggaagaagcc agcaagaagg gcaaccggac cccattccag tacctgagca
     3181 gcagcgacag caagatcagc tacgaaacct tcaagaagca catcctgaat ctggccaagg
     3241 gcaagggcag aatcagcaag accaagaaag agtatctgct ggaagaacgg gacatcaaca
     3301 ggttctccgt gcagaaagac ttcatcaacc ggaacctggt ggataccaga tacgccacca
     3361 gaggcctgat gaacctgctg cggagctact tcagagtgaa caacctggac gtgaaagtga
     3421 agtccatcaa tggcggcttc accagctttc tgcggcggaa gtggaagttt aagaaagagc
     3481 ggaacaaggg gtacaagcac cacgccgagg acgccctgat cattgccaac gccgatttca
     3541 tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg
     3601 aggaaaagca ggccgagagc atgcccgaga tcgaaaccga gcaggagtac aaagagatct
     3661 tcatcacccc ccaccagatc aagcacatta aggacttcaa ggactacaag tacagccacc
     3721 gggtggacaa gaagcctaat agagagctga ttaacgacac cctgtactcc acccggaagg
     3781 acgacaaggg caacaccctg atcgtgaaca atctgaacgg cctgtacgac aaggacaatg
     3841 acaagctgaa aaagctgatc aacaagagcc ccgaaaagct gctgatgtac caccacgacc
     3901 cccagaccta ccagaaactg aagctgatta tggaacagta cggcgacgag aagaatcccc
     3961 tgtacaagta ctacgaggaa accgggaact acctgaccaa gtactccaaa aaggacaacg
     4021 gccccgtgat caagaagatt aagtattacg gcaacaaact gaacgcccat ctggacatca
     4081 ccgacgacta ccccaacagc agaaacaagg tcgtgaagct gtccctgaag ccctacagat
     4141 tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac cgtgaagaat ctggatgtga
     4201 tcaaaaaaga aaactactac gaagtgaata gcaagtgcta tgaggaagct aagaagctga
     4261 agaagatcag caaccaggcc gagtttatcg cctccttcta caacaacgat ctgatcaaga
     4321 tcaacggcga gctgtataga gtgatcggcg tgaacaacga cctgctgaac cggatcgaag
     4381 tgaacatgat cgacatcacc taccgcgagt acctggaaaa catgaacgac aagaggcccc
     4441 ccaggatcat taagacaatc gcctccaaga cccagagcat taagaagtac agcacagaca
     4501 ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc tcagatcatc aaaaagggca
     4561 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggga tccgaggcca
     4621 gcggttccgg acgggctgac gcattggacg attttgatct ggatatgctg ggaagtgacg
     4681 ccctcgatga ttttgacctt gacatgcttg gttcggatgc ccttgatgac tttgacctcg
     4741 acatgctcgg cagtgacgcc cttgatgatt tcgacctgga catgctgatt aactctagaa
     4801 gttccggatc tccgaaaaag aaacgcaaag ttggttcggg aggtggttcg ggtggctctg
     4861 gatcagtgct gcctcaggct cctgctcctg caccagctcc agccatggtg tctgcactgg
     4921 ctcaggcacc agcacccgtg cctgtgctgg ctcctggacc tccacaggct gtggctccac
     4981 cagcccctaa acctacacag gccggcgagg gcacactgtc tgaagctctg ctgcagctgc
     5041 agttcgacga cgaggatctg ggagccctgc tgggaaacag caccgatcct gccgtgttca
     5101 ccgacctggc cagcgtggac aacagcgagt tccagcagct gctgaaccag ggcatccctg
     5161 tggcccctca caccaccgag cccatgctga tggaataccc cgaggccatc acccggctcg
     5221 tgacaggcgc tcagaggcct cctgatccag ctcctgcccc tctgggagca ccaggcctgc
     5281 ctaatggact gctgtctggc gacgaggact tcagctctat cgccgatatg gatttctcag
     5341 ccttgctgtc aggcggtggt agtggtggga gcggtagtga cctttcccat ccgcccccaa
     5401 ggggccatct ggatgagctg acaaccacac ttgagtccat gaccgaggat ctgaacctgg
     5461 actcacccct gaccccggaa ttgaacgaga ttctggatac cttcctgaac gacgagtgcc
     5521 tcttgcatgc catgcatatc agcacaggac tgtccatctt cgacacatct ctgttttagg
     5581 aattcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgcaattg
     5641 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     5701 ctgaggccgg gcgaccaaag gttgcccgac gcccgggctt tgcccgggcg gcctcagtga
     5761 gcgagcgagc gcgcagctgc ctgcaggaca tgtgagcaaa aggccagcaa aaggccagga
     5821 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5881 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5941 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6001 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6061 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6121 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6181 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6241 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6301 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6361 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6421 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6481 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6541 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6601 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6661 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6721 ctggccccag tgctgcaatg ataccgcggc ttccacgctc accggctcca gatttatcag
     6781 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6841 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6901 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6961 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7021 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7081 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7141 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7201 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7261 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7321 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7381 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7441 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7501 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7561 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     7621 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     7681 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     7741 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
//
