LOCUS       sequence-495334-        6262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of AcrIIA5-AsLOV2 hybrid in mammalian cells; insertion 
            before N77; enables optogenetic control of SauCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6262)
  AUTHORS   Brenker L, Aschenbrenner S, Bubeck F, Staykov K, Gebhardt C, Wolf B,
            Jendrusch M, Kroll AS, Becker J, Ambiel I, Fackler OT, Grimm D, 
            Mathony J, Niopek D
  TITLE     A versatile anti-CRISPR platform for opto- and chemogenetic control 
            of CRISPR-Cas9 and Cas12 across a wide range of orthologs.
  JOURNAL   Nucleic Acids Res. 2025 Jul 19;53(14):gkaf752. doi: 
            10.1093/nar/gkaf752.
  PUBMED    40794863
REFERENCE   2  (bases 1 to 6262)
  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: "Nucleic 
            Acids Res."; date: "2025-07-19"; volume: "53(14):gkaf752. doi"; 
            pages: " 10.1093/nar/gkaf752"
COMMENT     Sequence Label: CMV_AcrIIA5_AsLOV2-N77
FEATURES             Location/Qualifiers
     source          1..6262
                     /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"
     primer_bind     complement(1842..1859)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1848..2072
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2118..2546
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2205..2224)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2415..2436
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2555..2575)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2560..2889
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2740..2875
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2802..2821
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2956..3750
                     /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(3010..3029)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3620..3639
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3924..4045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3961..3980)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4015..4034
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4094..4110)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4094..4110)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4107..4129)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4118..4134
                     /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(4142..4148,4149..4166,4167..4172))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4187..4208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4325..4342)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4496..5084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4576..4595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5255..6046,6047..6115))
                     /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     5878..5897
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6116..6220)
                     /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 tggcatacgg
      901 taaaagccga tataactctt accggaaacg aaattttagt atcagtgaca accagagaag
      961 agaatacgcc aaaaaaatga aggaattgga gcaagcgttt gagaatttgg acgggtggta
     1021 cctctcatcc atgaaggata gcgcctacaa agacttcgga aagtacgaga taagactgtc
     1081 aaaccacagt gcagacaata ggtatcacga ccttgaactt gctacaactt tggagcggat
     1141 tgagaagaat ttcgtcataa ctgatcccag gttgcctgat aatcctataa tattcgcctc
     1201 cgatagtttt cttcaactta cggagtactc cagagaggag atactgggaa ggaattgcag
     1261 attcctccaa ggtccagaaa ctgatagagc tactgtgagg aagatacggg atgccattga
     1321 caatcaaact gaagtgacag tgcaacttat caattacacc aagtcaggga aaaaattctg
     1381 gaacctgttc catttgcaac ccatgaggga ccaaaagggc gatgtgcaat atttcatagg
     1441 cgttcaactt gatggtactg aacacgtcag agatgcggcg gagcgagaag gagtgatgct
     1501 gattaagaaa acagcagaga atatcgacga agcggcgaaa gagctgaatg gtcgccttat
     1561 cgttaatgta aaggcttcaa aacttaattt cgttgatatt atagagaaca agctgggcaa
     1621 aattattgag aagatcgata cgcttgattt ggataagtac agattcatta acgctactaa
     1681 actggaaagg gatataaaat gttactataa ggggtataaa acaaaaaagg atgttatctg
     1741 actcgagcgg ccgccactgt gctggatatc tgcagaattc caccacactg gactagtgga
     1801 tccgagctcg gtaccaagct taagtttaaa ccgctgatca gcctcgactg tgccttctag
     1861 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     1921 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     1981 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2041 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2101 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2161 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2221 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2281 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2341 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2401 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2461 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2521 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     2581 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2641 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2701 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2761 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2821 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2881 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     2941 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3001 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3061 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3121 gccctgaatg aactgcagga cgaggcagcg cggctatcgt gactggccac gacgggcgtt
     3181 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3241 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3301 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3361 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3421 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3481 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3541 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3601 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3661 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3721 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     3781 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     3841 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     3901 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     3961 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4021 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4081 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4141 acaacatgcg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4201 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4261 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4321 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4381 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4441 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4501 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4561 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4621 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4681 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4741 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4801 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4861 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4921 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4981 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5041 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5101 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5161 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5221 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5281 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5341 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5401 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5461 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5521 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5581 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5641 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5701 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5761 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5821 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5881 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5941 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6001 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6061 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6121 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6181 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6241 cacctgacgt cgacggatcg gg
//
