LOCUS       sequence-224728-        6139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  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 6139)
  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 6139)
  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: CMV-CASANOVA
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..52)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        224..603
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        604..807
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     758..778
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     852..871
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        852..870
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             896..916
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1719..1736)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1725..1949
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1995..2423
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2082..2101)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2292..2313
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2432..2452)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2437..2766
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2617..2752
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2679..2698
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2833..3627
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLS*LATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2887..2906)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3497..3516
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3801..3922
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3838..3857)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3892..3911
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3971..3987)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3971..3987)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3984..4006)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3995..4011
                     /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(4019..4025,4026..4043,4044..4049))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4064..4085
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4202..4219)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4373..4961)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4453..4472)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5132..5923,5924..5992))
                     /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     5755..5774
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5993..6097)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
       61 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
      121 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
      181 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
      241 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      301 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      361 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      421 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      481 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      541 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      601 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      661 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      721 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      781 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
      841 cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagca tggccccgaa
      901 gaaaaagcga aaggtgggcg gaagcggagg ttccatgaac attaacgacc tgattcggga
      961 aataaagaac aaggactaca cggtcaagct gtcaggcacc gactccaact ctataacaca
     1021 gctcattata agggttaata atgatggcaa cgaatatgtc atctctgaat ccgagaatga
     1081 atcaatcgtg gagaaattca tcagtgcatt taaaaacggc tggcttgcta caactttgga
     1141 gcggattgag aagaatttcg tcataactga tcccaggttg cctgataatc ctataatatt
     1201 cgcctccgat agttttcttc aacttacgga gtactccaga gaggagatac tgggaaggaa
     1261 ttgcagattc ctccaaggtc cagaaactga tagagctact gtgaggaaga tacgggatgc
     1321 cattgacaat caaactgaag tgacagtgca acttatcaat tacaccaagt cagggaaaaa
     1381 attctggaac ctgttccatt tgcaacccat gagggaccaa aagggcgatg tgcaatattt
     1441 cataggcgtt caacttgatg gtactgaaca cgtcagagat gcggcggagc gagaaggagt
     1501 gatgctgatt aagaaaacag cagagaatat cgacgaagcg gcgaaagagt tgtacgaaga
     1561 tgaggaagag ttctacaatg atatgcaaac tatcacactg aaatctgagc tgaactagct
     1621 cgagcggccg ccactgtgct ggatatctgc agaattccac cacactggac tagtggatcc
     1681 gagctcggta ccaagcttaa gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg
     1741 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     1801 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     1861 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     1921 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     1981 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2041 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2101 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2161 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2221 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2281 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2341 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2401 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     2461 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2521 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2581 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2641 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2701 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     2761 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     2821 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     2881 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     2941 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3001 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtgac tggccacgac gggcgttcct
     3061 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3121 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3181 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3241 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3301 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3361 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3421 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     3481 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3541 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3601 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     3661 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3721 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     3781 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     3841 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     3901 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     3961 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4021 acatgcgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4081 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4141 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4201 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4261 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4321 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4381 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4441 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4501 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4561 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4621 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4681 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4741 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4801 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4861 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4921 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4981 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5041 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5101 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5161 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5221 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5281 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5341 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5401 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5461 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5521 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5581 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5641 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5701 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5761 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5821 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5881 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5941 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6001 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6061 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6121 ctgacgtcga cggatcggg
//
