LOCUS       sequence-495346-        5459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of AcrIIA5-cpGR2 hybrid in mammalian cells; insertion 
            before N77; enables chemogenetic control of SauCas9; contains ITRs 
            for AAV packaging with Rep2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5459)
  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 5459)
  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: AAV_CMV-AcrIIA5_cpGR2(GGS5)-N77
FEATURES             Location/Qualifiers
     source          1..5459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..34
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     18..34
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     53..73
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        55..73
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        266..645
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        646..849
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     800..820
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        894..912
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2262..2279)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2268..2492
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2516..2645
                     /label=AAV2 ITR
     promoter        complement(2700..2718)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2728..2745)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2728..2744)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2737..2759)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2885..3340)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3022..3043)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3234..3253
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3367..3471
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3472..3540,3541..4332)
                     /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     complement(3690..3709)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4503..5091
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4992..5011
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5379..5400
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5415..5420,5421..5438,5439..5445)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(5453..5459,1..10)
                     /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)."
     primer_bind     join(5458..5459,1..21)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
       61 ccctcactaa agggaacaaa agctgggtac cactagtatg catcctgcag gacgcgtcct
      121 acccactccc tctatgcgcg ctcgctcact cactcggccc tggagaccaa aggtctccag
      181 ggccgagtga gtgagcgagc gcgcatagag ggagtgggta cctccatcat ctggtttccc
      241 agatctcggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
      301 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      361 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      421 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      481 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      541 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      601 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      661 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      721 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      781 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      841 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
      901 actcactata gggagaccca agctggctag catggcatac ggtaaaagcc gatataactc
      961 ttaccggaaa cgaaatttta gtatcagtga caaccagaga agagaatacg ccaaaaaaat
     1021 gaaggaattg gagcaagcgt ttgagaattt ggacgggtgg tacctctcat ccatgaagga
     1081 tagcgcctac aaagacttcg gaaagtacga gataagactg tcaaaccaca gtgcagacaa
     1141 taggtatcac gaccttgaag gtccggggaa cagcagccag aactggcaga ggttctacca
     1201 gctgaccaag ctgctggaca gcatgcacga gatggtgggc ggcctgctcc agttctgctt
     1261 ctacaccttc gtgaacaaga gcctgagcgt ggagttcccc gagatgctgg ccgagatcat
     1321 cagcaaccag ctgcctaagt ttaatgccgg cagcgtgaag cccctgctgt tccaccagaa
     1381 ggggggcgga agcggcggat ccggcggatc tggcggatcg ggcgggagtg gactgatcag
     1441 cctgctggag gtgatcgagc ccgaggtgct gtacagcggc tacgacagca ccctgcccga
     1501 caccagcacc aggctgatga gcaccctgaa caggctgggc ggcaggcagg tggtgagcgc
     1561 cgtgaagtgg gccaaggccc tgcccggctt caggaacctg cacctggacg accagatgac
     1621 cctgctgcag tacagctgga tgagcctgat ggccttcagc ctgggctgga ggagctacaa
     1681 gcagagcaac ggcaacatgc tgtgcttcgc ccccgacctg gtgatcaacg aggagaggat
     1741 gcagctgccc tacatgtacg accagtgcca gcagatgctg aagatcagca gcgagttcgt
     1801 gaggctgcag gtgagctacg acgagtacct gtgcatgaag gtgctgctgc tgctgagcac
     1861 cgtgcccaag gacggcctga agagccaggc cgtgttcgac gagatcagga tgacctacat
     1921 caaggagctg ggcaaggcca tcgtgaagag ggagggcggt ccggggaatg gtcgccttat
     1981 cgttaatgta aaggcttcaa aacttaattt cgttgatatt atagagaaca agctgggcaa
     2041 aattattgag aagatcgata cgcttgattt ggataagtac agattcatta acgctactaa
     2101 actggaaagg gatataaaat gttactataa ggggtataaa acaaaaaagg atgttatctg
     2161 actcgagcgg ccgccactgt gctggatatc tgcagaattc caccacactg gactagtgga
     2221 tccgagctcg gtaccaagct taagtttaaa ccgctgatca gcctcgactg tgccttctag
     2281 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2341 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2401 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2461 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagag atctgaggaa
     2521 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     2581 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     2641 cgcagagagg gagtggcccc cccccccccc cccctgcatt ctagagagct ccaattcgcc
     2701 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     2761 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     2821 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2881 atggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     2941 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     3001 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     3061 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     3121 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     3181 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     3241 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3301 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     3361 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3421 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3481 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3541 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3601 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3661 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     3721 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3781 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3841 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3901 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3961 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4021 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4081 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4141 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4201 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4261 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4321 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4381 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4441 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4501 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4561 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4621 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4681 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4741 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4801 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4861 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4921 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4981 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5041 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5101 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5161 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5221 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     5281 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     5341 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     5401 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgag
//
