LOCUS       sequence-191797-        8602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses tripartite VP64-p65-RTA activator fused to C term. of dead
            Sa Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8602)
  TITLE     Church Lab CRISPR plasmids 2017
REFERENCE   2  (bases 1 to 8602)
  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"
COMMENT     Sequence Label: pAAV-dSa-VPR
FEATURES             Location/Qualifiers
     source          1..8602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   21..161
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   32..161
                     /label=AAV2 ITR
     enhancer        181..560
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        561..764
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     715..735
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      783..792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             795..815
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3996..4043
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4074..4223
                     /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             4248..4268
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4698..5054
                     /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"
     CDS             5073..5642
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     primer_bind     complement(5670..5687)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5676..5883
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5896..6025
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(6087..6675)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6167..6186)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6846..7637,7638..7706))
                     /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     7469..7488
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7707..7811)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7879..7897
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7935..7957)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8057..8076
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(8093..8548)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8230..8251)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8442..8461
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aaaataccgc atcaggcgcc cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc
       61 ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
      121 gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccgcc tcgaggcgtt
      181 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      241 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      301 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      361 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      421 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      481 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      541 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      601 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      661 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      721 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactaccg
      781 gtgccaccat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagcca
      841 agcggaacta catcctgggc ctggccatcg gcatcaccag cgtgggctac ggcatcatcg
      901 actacgagac acgggacgtg atcgatgccg gcgtgcggct gttcaaagag gccaacgtgg
      961 aaaacaacga gggcaggcgg agcaagagag gcgccagaag gctgaagcgg cggaggcggc
     1021 atagaatcca gagagtgaag aagctgctgt tcgactacaa cctgctgacc gaccacagcg
     1081 agctgagcgg catcaacccc tacgaggcca gagtgaaggg cctgagccag aagctgagcg
     1141 aggaagagtt ctctgccgcc ctgctgcacc tggccaagag aagaggcgtg cacaacgtga
     1201 acgaggtgga agaggacacc ggcaacgagc tgtccaccaa agagcagatc agccggaaca
     1261 gcaaggccct ggaagagaaa tacgtggccg aactgcagct ggaacggctg aagaaagacg
     1321 gcgaagtgcg gggcagcatc aacagattca agaccagcga ctacgtgaaa gaagccaaac
     1381 agctgctgaa ggtgcagaag gcctaccacc agctggacca gagcttcatc gacacctaca
     1441 tcgacctgct ggaaacccgg cggacctact atgagggacc tggcgagggc agccccttcg
     1501 gctggaagga catcaaagaa tggtacgaga tgctgatggg ccactgcacc tacttccccg
     1561 aggaactgcg gagcgtgaag tacgcctaca acgccgacct gtacaacgcc ctgaacgacc
     1621 tgaacaatct cgtgatcacc agggacgaga acgagaagct ggaatattac gagaagttcc
     1681 agatcatcga gaacgtgttc aagcagaaga agaagcccac cctgaagcag atcgccaaag
     1741 aaatcctcgt gaacgaagag gatattaagg gctacagagt gaccagcacc ggcaagcccg
     1801 agttcaccaa cctgaaggtg taccacgaca tcaaggacat taccgcccgg aaagagatta
     1861 ttgagaacgc cgagctgctg gatcagattg ccaagatcct gaccatctac cagagcagcg
     1921 aggacatcca ggaagaactg accaatctga actccgagct gacccaggaa gagatcgagc
     1981 agatctctaa tctgaagggc tataccggca cccacaacct gagcctgaag gccatcaacc
     2041 tgatcctgga cgagctgtgg cacaccaacg acaaccagat cgctatcttc aaccggctga
     2101 agctggtgcc caagaaggtg gacctgtccc agcagaaaga gatccccacc accctggtgg
     2161 acgacttcat cctgagcccc gtcgtgaaga gaagcttcat ccagagcatc aaagtgatca
     2221 acgccatcat caagaagtac ggcctgccca acgacatcat tatcgagctg gcccgcgaga
     2281 agaactccaa ggacgcccag aaaatgatca acgagatgca gaagcggaac cggcagacca
     2341 acgagcggat cgaggaaatc atccggacca ccggcaaaga gaacgccaag tacctgatcg
     2401 agaagatcaa gctgcacgac atgcaggaag gcaagtgcct gtacagcctg gaagccatcc
     2461 ctctggaaga tctgctgaac aaccccttca actatgaggt ggaccacatc atccccagaa
     2521 gcgtgtcctt cgacaacagc ttcaacaaca aggtgctcgt gaagcaggaa gaagccagca
     2581 agaagggcaa ccggacccca ttccagtacc tgagcagcag cgacagcaag atcagctacg
     2641 aaaccttcaa gaagcacatc ctgaatctgg ccaagggcaa gggcagaatc agcaagacca
     2701 agaaagagta tctgctggaa gaacgggaca tcaacaggtt ctccgtgcag aaagacttca
     2761 tcaaccggaa cctggtggat accagatacg ccaccagagg cctgatgaac ctgctgcgga
     2821 gctacttcag agtgaacaac ctggacgtga aagtgaagtc catcaatggc ggcttcacca
     2881 gctttctgcg gcggaagtgg aagtttaaga aagagcggaa caaggggtac aagcaccacg
     2941 ccgaggacgc cctgatcatt gccaacgccg atttcatctt caaagagtgg aagaaactgg
     3001 acaaggccaa aaaagtgatg gaaaaccaga tgttcgagga aaagcaggcc gagagcatgc
     3061 ccgagatcga aaccgagcag gagtacaaag agatcttcat caccccccac cagatcaagc
     3121 acattaagga cttcaaggac tacaagtaca gccaccgggt ggacaagaag cctaatagag
     3181 agctgattaa cgacaccctg tactccaccc ggaaggacga caagggcaac accctgatcg
     3241 tgaacaatct gaacggcctg tacgacaagg acaatgacaa gctgaaaaag ctgatcaaca
     3301 agagccccga aaagctgctg atgtaccacc acgaccccca gacctaccag aaactgaagc
     3361 tgattatgga acagtacggc gacgagaaga atcccctgta caagtactac gaggaaaccg
     3421 ggaactacct gaccaagtac tccaaaaagg acaacggccc cgtgatcaag aagattaagt
     3481 attacggcaa caaactgaac gcccatctgg acatcaccga cgactacccc aacagcagaa
     3541 acaaggtcgt gaagctgtcc ctgaagccct acagattcga cgtgtacctg gacaatggcg
     3601 tgtacaagtt cgtgaccgtg aagaatctgg atgtgatcaa aaaagaaaac tactacgaag
     3661 tgaatagcaa gtgctatgag gaagctaaga agctgaagaa gatcagcaac caggccgagt
     3721 ttatcgcctc cttctacaac aacgatctga tcaagatcaa cggcgagctg tatagagtga
     3781 tcggcgtgaa caacgacctg ctgaaccgga tcgaagtgaa catgatcgac atcacctacc
     3841 gcgagtacct ggaaaacatg aacgacaaga ggccccccag gatcattaag acaatcgcct
     3901 ccaagaccca gagcattaag aagtacagca cagacattct gggcaacctg tatgaagtga
     3961 aatctaagaa gcaccctcag atcatcaaaa agggcaaaag gccggcggcc acgaaaaagg
     4021 ccggccaggc aaaaaagaaa aagggatccg aggccagcgg ttccggacgg gctgacgcat
     4081 tggacgattt tgatctggat atgctgggaa gtgacgccct cgatgatttt gaccttgaca
     4141 tgcttggttc ggatgccctt gatgactttg acctcgacat gctcggcagt gacgcccttg
     4201 atgatttcga cctggacatg ctgattaact ctagaagttc cggatctccg aaaaagaaac
     4261 gcaaagttgg tagccagtac ctgcccgaca ccgacgaccg gcaccggatc gaggaaaagc
     4321 ggaagcggac ctacgagaca ttcaagagca tcatgaagaa gtcccccttc agcggcccca
     4381 ccgaccctag acctccacct agaagaatcg ccgtgcccag cagatccagc gccagcgtgc
     4441 caaaacctgc cccccagcct taccccttca ccagcagcct gagcaccatc aactacgacg
     4501 agttccctac catggtgttc cccagcggcc agatctctca ggcctctgct ctggctccag
     4561 cccctcctca ggtgctgcct caggctcctg ctcctgcacc agctccagcc atggtgtctg
     4621 cactggctca ggcaccagca cccgtgcctg tgctggctcc tggacctcca caggctgtgg
     4681 ctccaccagc ccctaaacct acacaggccg gcgagggcac actgtctgaa gctctgctgc
     4741 agctgcagtt cgacgacgag gatctgggag ccctgctggg aaacagcacc gatcctgccg
     4801 tgttcaccga cctggccagc gtggacaaca gcgagttcca gcagctgctg aaccagggca
     4861 tccctgtggc ccctcacacc accgagccca tgctgatgga ataccccgag gccatcaccc
     4921 ggctcgtgac aggcgctcag aggcctcctg atccagctcc tgcccctctg ggagcaccag
     4981 gcctgcctaa tggactgctg tctggcgacg aggacttcag ctctatcgcc gatatggatt
     5041 tctcagcctt gctgggctct ggcagcggca gccgggattc cagggaaggg atgtttttgc
     5101 cgaagcctga ggccggctcc gctattagtg acgtgtttga gggccgcgag gtgtgccagc
     5161 caaaacgaat ccggccattt catcctccag gaagtccatg ggccaaccgc ccactccccg
     5221 ccagcctcgc accaacacca accggtccag tacatgagcc agtcgggtca ctgaccccgg
     5281 caccagtccc tcagccactg gatccagcgc ccgcagtgac tcccgaggcc agtcacctgt
     5341 tggaggatcc cgatgaagag acgagccagg ctgtcaaagc ccttcgggag atggccgata
     5401 ctgtgattcc ccagaaggaa gaggctgcaa tctgtggcca aatggacctt tcccatccgc
     5461 ccccaagggg ccatctggat gagctgacaa ccacacttga gtccatgacc gaggatctga
     5521 acctggactc acccctgacc ccggaattga acgagattct ggataccttc ctgaacgacg
     5581 agtgcctctt gcatgccatg catatcagca caggactgtc catcttcgac acatctctgt
     5641 tttgagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     5701 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     5761 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     5821 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg
     5881 ggagctagag gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     5941 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcggc ctcagtgagc
     6001 gagcgagcgc gcagctgcct gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac
     6061 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6121 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6181 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6241 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6301 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6361 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6421 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6481 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6541 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6601 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6661 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6721 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6781 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6841 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6901 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6961 ggccccagtg ctgcaatgat accgcggctt ccacgctcac cggctccaga tttatcagca
     7021 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     7081 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     7141 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     7201 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     7261 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7321 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7381 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7441 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7501 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7561 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7621 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7681 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7741 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7801 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     7861 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     7921 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     7981 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     8041 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca taaaattgta
     8101 aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     8161 caatagaccg aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagagttg
     8221 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     8281 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt
     8341 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     8401 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     8461 gcgggcgcta aggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     8521 gcgcttaatg cgccgctaca gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa
     8581 taccgcacag atgcgtaagg ag
//
