LOCUS       sequence-194575-        7590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSa Cas9 fused to gold standard VP64 activator domain and 
            Sa sgRNA for Afp promoter, vector allows for activation of mouse 
            Afp, can be packaged and delivered as AAV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7590)
  TITLE     Church Lab CRISPR plasmids 2017
REFERENCE   2  (bases 1 to 7590)
  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-CMV-dSa VP64 Afp
FEATURES             Location/Qualifiers
     source          1..7590
                     /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        842..1090
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     842..862
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1013..1032
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1112..1187
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        1212..1591
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1592..1795
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1746..1766
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1814..1823
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1826..1846
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5027..5074
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5105..5254
                     /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             5279..5299
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5330..5347)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5336..5543
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5556..5685
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(5747..6335)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5827..5846)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6506..7297,7298..7366))
                     /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     7129..7148
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7367..7471)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7539..7557
                     /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 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
      901 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
      961 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     1021 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     1081 acgaaacacc gctaacttca acgtaaggaa agttttagta ctctggaaac agaatctact
     1141 aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttttggccgc
     1201 ctcgaggcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     1261 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     1321 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     1381 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     1441 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     1501 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     1561 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     1621 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     1681 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     1741 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     1801 gctaactacc ggtgccacca tggccccaaa gaagaagcgg aaggtcggta tccacggagt
     1861 cccagcagcc aagcggaact acatcctggg cctggccatc ggcatcacca gcgtgggcta
     1921 cggcatcatc gactacgaga cacgggacgt gatcgatgcc ggcgtgcggc tgttcaaaga
     1981 ggccaacgtg gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa ggctgaagcg
     2041 gcggaggcgg catagaatcc agagagtgaa gaagctgctg ttcgactaca acctgctgac
     2101 cgaccacagc gagctgagcg gcatcaaccc ctacgaggcc agagtgaagg gcctgagcca
     2161 gaagctgagc gaggaagagt tctctgccgc cctgctgcac ctggccaaga gaagaggcgt
     2221 gcacaacgtg aacgaggtgg aagaggacac cggcaacgag ctgtccacca aagagcagat
     2281 cagccggaac agcaaggccc tggaagagaa atacgtggcc gaactgcagc tggaacggct
     2341 gaagaaagac ggcgaagtgc ggggcagcat caacagattc aagaccagcg actacgtgaa
     2401 agaagccaaa cagctgctga aggtgcagaa ggcctaccac cagctggacc agagcttcat
     2461 cgacacctac atcgacctgc tggaaacccg gcggacctac tatgagggac ctggcgaggg
     2521 cagccccttc ggctggaagg acatcaaaga atggtacgag atgctgatgg gccactgcac
     2581 ctacttcccc gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc tgtacaacgc
     2641 cctgaacgac ctgaacaatc tcgtgatcac cagggacgag aacgagaagc tggaatatta
     2701 cgagaagttc cagatcatcg agaacgtgtt caagcagaag aagaagccca ccctgaagca
     2761 gatcgccaaa gaaatcctcg tgaacgaaga ggatattaag ggctacagag tgaccagcac
     2821 cggcaagccc gagttcacca acctgaaggt gtaccacgac atcaaggaca ttaccgcccg
     2881 gaaagagatt attgagaacg ccgagctgct ggatcagatt gccaagatcc tgaccatcta
     2941 ccagagcagc gaggacatcc aggaagaact gaccaatctg aactccgagc tgacccagga
     3001 agagatcgag cagatctcta atctgaaggg ctataccggc acccacaacc tgagcctgaa
     3061 ggccatcaac ctgatcctgg acgagctgtg gcacaccaac gacaaccaga tcgctatctt
     3121 caaccggctg aagctggtgc ccaagaaggt ggacctgtcc cagcagaaag agatccccac
     3181 caccctggtg gacgacttca tcctgagccc cgtcgtgaag agaagcttca tccagagcat
     3241 caaagtgatc aacgccatca tcaagaagta cggcctgccc aacgacatca ttatcgagct
     3301 ggcccgcgag aagaactcca aggacgccca gaaaatgatc aacgagatgc agaagcggaa
     3361 ccggcagacc aacgagcgga tcgaggaaat catccggacc accggcaaag agaacgccaa
     3421 gtacctgatc gagaagatca agctgcacga catgcaggaa ggcaagtgcc tgtacagcct
     3481 ggaagccatc cctctggaag atctgctgaa caaccccttc aactatgagg tggaccacat
     3541 catccccaga agcgtgtcct tcgacaacag cttcaacaac aaggtgctcg tgaagcagga
     3601 agaagccagc aagaagggca accggacccc attccagtac ctgagcagca gcgacagcaa
     3661 gatcagctac gaaaccttca agaagcacat cctgaatctg gccaagggca agggcagaat
     3721 cagcaagacc aagaaagagt atctgctgga agaacgggac atcaacaggt tctccgtgca
     3781 gaaagacttc atcaaccgga acctggtgga taccagatac gccaccagag gcctgatgaa
     3841 cctgctgcgg agctacttca gagtgaacaa cctggacgtg aaagtgaagt ccatcaatgg
     3901 cggcttcacc agctttctgc ggcggaagtg gaagtttaag aaagagcgga acaaggggta
     3961 caagcaccac gccgaggacg ccctgatcat tgccaacgcc gatttcatct tcaaagagtg
     4021 gaagaaactg gacaaggcca aaaaagtgat ggaaaaccag atgttcgagg aaaagcaggc
     4081 cgagagcatg cccgagatcg aaaccgagca ggagtacaaa gagatcttca tcacccccca
     4141 ccagatcaag cacattaagg acttcaagga ctacaagtac agccaccggg tggacaagaa
     4201 gcctaataga gagctgatta acgacaccct gtactccacc cggaaggacg acaagggcaa
     4261 caccctgatc gtgaacaatc tgaacggcct gtacgacaag gacaatgaca agctgaaaaa
     4321 gctgatcaac aagagccccg aaaagctgct gatgtaccac cacgaccccc agacctacca
     4381 gaaactgaag ctgattatgg aacagtacgg cgacgagaag aatcccctgt acaagtacta
     4441 cgaggaaacc gggaactacc tgaccaagta ctccaaaaag gacaacggcc ccgtgatcaa
     4501 gaagattaag tattacggca acaaactgaa cgcccatctg gacatcaccg acgactaccc
     4561 caacagcaga aacaaggtcg tgaagctgtc cctgaagccc tacagattcg acgtgtacct
     4621 ggacaatggc gtgtacaagt tcgtgaccgt gaagaatctg gatgtgatca aaaaagaaaa
     4681 ctactacgaa gtgaatagca agtgctatga ggaagctaag aagctgaaga agatcagcaa
     4741 ccaggccgag tttatcgcct ccttctacaa caacgatctg atcaagatca acggcgagct
     4801 gtatagagtg atcggcgtga acaacgacct gctgaaccgg atcgaagtga acatgatcga
     4861 catcacctac cgcgagtacc tggaaaacat gaacgacaag aggcccccca ggatcattaa
     4921 gacaatcgcc tccaagaccc agagcattaa gaagtacagc acagacattc tgggcaacct
     4981 gtatgaagtg aaatctaaga agcaccctca gatcatcaaa aagggcaaaa ggccggcggc
     5041 cacgaaaaag gccggccagg caaaaaagaa aaagggatcg gaggccagcg gttccggacg
     5101 ggctgacgca ttggacgatt ttgatctgga tatgctggga agtgacgccc tcgatgattt
     5161 tgaccttgac atgcttggtt cggatgccct tgatgacttt gacctcgaca tgctcggcag
     5221 tgacgccctt gatgatttcg acctggacat gctgattaac tctagaagtt ccggatctcc
     5281 gaaaaagaaa cgcaaagttg gttaagaatt cctagagctc gctgatcagc ctcgactgtg
     5341 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     5401 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     5461 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     5521 gagaatagca ggcatgctgg ggagctagag gccgcaggaa cccctagtga tggagttggc
     5581 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     5641 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggacatg tgagcaaaag
     5701 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5761 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5821 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5881 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5941 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6001 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6061 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6121 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6181 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6241 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6301 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6361 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6421 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6481 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6541 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6601 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcggctt ccacgctcac
     6661 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6721 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6781 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6841 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6901 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6961 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7021 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7081 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7141 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7201 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7261 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7321 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7381 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7441 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7501 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     7561 ttcgtctcgc gcgtttcggt gatgacggtg
//
