LOCUS       sequence-218013-        4973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for CASANOVA (AcrIIA4-LOV2 hybrid) expression in 
            mammalian cells; enables optogenetic control of SpyCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4973)
  AUTHORS   Bubeck F, Hoffmann MD, Harteveld Z, Aschenbrenner S, Bietz A, 
            Waldhauer MC, Borner K, Fakhiri J, Schmelas C, Dietz L, Grimm D, 
            Correia BE, Eils R, Niopek D
  TITLE     Engineered anti-CRISPR proteins for optogenetic control of 
            CRISPR-Cas9.
  JOURNAL   Nat Methods. 2018 Oct 30. pii: 10.1038/s41592-018-0178-9. doi: 
            10.1038/s41592-018-0178-9.
  PUBMED    30377362
REFERENCE   2  (bases 1 to 4973)
  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: "Nat 
            Methods. 2018 Oct 30. pii: 10.1038/s41592-018-0178-9. doi: 
            10.1038/s41592-018-0178-9."
COMMENT     Sequence Label: pAAV-CMV-CASANOVA
FEATURES             Location/Qualifiers
     source          1..4973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        849..1228
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1229..1432
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1383..1403
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        1477..1495
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1521..1541
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2344..2361)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2350..2574
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2785..2805)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2785..2803)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2824..2840)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2824..2840)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2837..2859)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2848..2864
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2872..2878,2879..2896,2897..2902))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2917..2938
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3226..3814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3306..3325)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3985..4776,4777..4845))
                     /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     4608..4627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4846..4950)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctctc
      661 tagaatgcag gggggggggg gggggggggc cactccctct ctgcgcgctc gctcgctcac
      721 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
      781 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct agatctgata
      841 tcgtcgacga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      901 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      961 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     1021 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     1081 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     1141 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     1201 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     1261 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     1321 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     1381 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct
     1441 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
     1501 ccaagctggc tagcatggcc ccgaagaaaa agcgaaaggt gggcggaagc ggaggttcca
     1561 tgaacattaa cgacctgatt cgggaaataa agaacaagga ctacacggtc aagctgtcag
     1621 gcaccgactc caactctata acacagctca ttataagggt taataatgat ggcaacgaat
     1681 atgtcatctc tgaatccgag aatgaatcaa tcgtggagaa attcatcagt gcatttaaaa
     1741 acggctggct tgctacaact ttggagcgga ttgagaagaa tttcgtcata actgatccca
     1801 ggttgcctga taatcctata atattcgcct ccgatagttt tcttcaactt acggagtact
     1861 ccagagagga gatactggga aggaattgca gattcctcca aggtccagaa actgatagag
     1921 ctactgtgag gaagatacgg gatgccattg acaatcaaac tgaagtgaca gtgcaactta
     1981 tcaattacac caagtcaggg aaaaaattct ggaacctgtt ccatttgcaa cccatgaggg
     2041 accaaaaggg cgatgtgcaa tatttcatag gcgttcaact tgatggtact gaacacgtca
     2101 gagatgcggc ggagcgagaa ggagtgatgc tgattaagaa aacagcagag aatatcgacg
     2161 aagcggcgaa agagttgtac gaagatgagg aagagttcta caatgatatg caaactatca
     2221 cactgaaatc tgagctgaac tagctcgagc ggccgccact gtgctggata tctgcagaat
     2281 tccaccacac tggactagtg gatccgagct cggtaccaag cttaagttta aaccgctgat
     2341 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2401 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2461 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2521 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggggcgcg
     2581 ccgatatcag atctggcaaa cctagatgat ggaggtaccc actccctcta tgcgcgctcg
     2641 ctcactcact cggccctgcc ggccagaggc cggcagtctg gagacctttg gtctccaggg
     2701 ccgagtgagt gagcgagcgc gcatagaggg agtgggtagg acgcgtcctg caggatgcat
     2761 actagtggta cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     2821 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2881 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2941 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3001 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3061 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3121 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3181 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3241 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3301 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3361 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3421 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3481 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3541 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3601 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3661 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3721 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3781 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3841 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3901 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3961 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4021 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4081 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4141 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4201 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4261 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4321 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4381 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4441 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4501 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4561 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4621 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4681 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4741 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4801 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4861 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4921 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac
//
