LOCUS       sequence-495344-        6721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of AcrVA1-cpGR2 hybrid in mammalian cells; insertion 
            before S103 with deletion of surrounding amino acids ELS; enables 
            chemogenetic control of MbCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6721)
  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 6721)
  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: AcrVA1_cpGR2(GGS5)-S103-Delta-ELS
FEATURES             Location/Qualifiers
     source          1..6721
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        143..522
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        523..726
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     677..697
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     771..790
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        771..789
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2220..2237)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2226..2450
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2496..2924
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2583..2602)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2793..2814
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2933..2953)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2938..3267
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3118..3253
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3180..3199
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3334..4128
                     /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(3388..3407)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3998..4017
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4302..4423
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4339..4358)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4393..4412
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4472..4488)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4472..4488)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4485..4507)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4496..4512
                     /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(4520..4526,4527..4544,4545..4550))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4565..4586
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4703..4720)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4874..5462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4954..4973)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5633..6424,6425..6493))
                     /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     6256..6275
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6494..6598)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6673..6692)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
       61 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
      121 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
      181 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      241 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      301 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      361 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      421 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      481 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      541 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      601 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      661 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      721 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
      781 cactataggg agacccaagc tggctagcat gtatgaagct aaagagcgct atgccaagaa
      841 gaagatgcag gagaatacta agattgacac cttgacagat gaacagcatg atgctcttgc
      901 acagctttgc gctttcagac ataagttcca tagcaacaag gacagtctgt tcctgtctga
      961 gtctgctttc agcggggaat tctccttcga aatgcagtca gacgaaaata gcaaactgag
     1021 ggaggtggga ttgccaacaa tagaatggag cttctatgac aactcccaca ttcccgacga
     1081 ctctttccga gagtggttta actttgctaa ttacagcggt ccggggaaca gcagccagaa
     1141 ctggcagagg ttctaccagc tgaccaagct gctggacagc atgcacgaga tggtgggcgg
     1201 cctgctccag ttctgcttct acaccttcgt gaacaagagc ctgagcgtgg agttccccga
     1261 gatgctggcc gagatcatca gcaaccagct gcctaagttt aatgccggca gcgtgaagcc
     1321 cctgctgttc caccagaagg ggggcggaag cggcggatcc ggcggatctg gcggatcggg
     1381 cgggagtgga ctgatcagcc tgctggaggt gatcgagccc gaggtgctgt acagcggcta
     1441 cgacagcacc ctgcccgaca ccagcaccag gctgatgagc accctgaaca ggctgggcgg
     1501 caggcaggtg gtgagcgccg tgaagtgggc caaggccctg cccggcttca ggaacctgca
     1561 cctggacgac cagatgaccc tgctgcagta cagctggatg agcctgatgg ccttcagcct
     1621 gggctggagg agctacaagc agagcaacgg caacatgctg tgcttcgccc ccgacctggt
     1681 gatcaacgag gagaggatgc agctgcccta catgtacgac cagtgccagc agatgctgaa
     1741 gatcagcagc gagttcgtga ggctgcaggt gagctacgac gagtacctgt gcatgaaggt
     1801 gctgctgctg ctgagcaccg tgcccaagga cggcctgaag agccaggccg tgttcgacga
     1861 gatcaggatg acctacatca aggagctggg caaggccatc gtgaagaggg agggcggtcc
     1921 gggggaaacc atccaagagc agggactgga gctcgatctg gacgacgatg aaacgtacga
     1981 actcgtatac gacgaattgt atactgaggc catgggagaa tatgaagagc taaaccaaga
     2041 cattgagaag tatctgaggc gtattgacga agagcacggc actcagtatt gtccgacagg
     2101 gtttgcgagg cttcggtgac tcgagcggcc gccactgtgc tggatatctg cagaattcca
     2161 ccacactgga ctagtggatc cgagctcggt accaagctta agtttaaacc gctgatcagc
     2221 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2281 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2341 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2401 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2461 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2521 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2581 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2641 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2701 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2761 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2821 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2881 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2941 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3001 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3061 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3121 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3181 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3241 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3301 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3361 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3421 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3481 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtga
     3541 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3601 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3661 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3721 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3781 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3841 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3901 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3961 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4021 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4081 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4141 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4201 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4261 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4321 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4381 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4441 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4501 atccgctcac aattccacac aacatgcgag ccggaagcat aaagtgtaaa gcctggggtg
     4561 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4621 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4681 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4741 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4801 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4861 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4921 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4981 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5041 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5101 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5161 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5221 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5281 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5341 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5401 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5461 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5521 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5581 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5641 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5701 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5761 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5821 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5881 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5941 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6001 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6061 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6121 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6181 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6241 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6301 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6361 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6421 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6481 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6541 tcatgagcgg atacatattt gaatttattt agaaaaataa acaaataggg gttccgcgca
     6601 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     6661 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     6721 t
//
