LOCUS       sequence-495337-        6517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of AcrIIA4-cpGR2 hybrid in mammalian cells after Bubeck 
            et al Nat Methods 2018; enables chemogenetic control of SpyCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6517)
  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 6517)
  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_AcrIIA4_cpGR2(GGS5)-Delta-N64_Q65_E66
FEATURES             Location/Qualifiers
     source          1..6517
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        134..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        514..717
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     668..688
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     762..781
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        762..780
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             806..826
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2007..2024)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2013..2237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2283..2711
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2370..2389)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2580..2601
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2720..2740)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2725..3054
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2905..3040
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2967..2986
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3121..3915
                     /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(3175..3194)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3785..3804
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4089..4210
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4126..4145)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4180..4199
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4259..4275)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4259..4275)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4272..4294)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4283..4299
                     /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(4307..4313,4314..4331,4332..4337))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4352..4373
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4490..4507)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4661..5249)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4741..4760)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5420..6211,6212..6280))
                     /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     6043..6062
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6281..6385)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6460..6479)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
       61 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
      121 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      541 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      601 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      661 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      721 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
      781 gagacccaag ctggctagca tggccccgaa gaaaaagcga aaggtgggcg gaagcggagg
      841 ttccatgaac attaacgacc tgattcggga aataaagaac aaggactaca cggtcaagct
      901 gtcaggcacc gactccaact ctataacaca gctcattata agggttaata atgatggcaa
      961 cgaatatgtc atctctgaat ccgagaatga atcaatcgtg gagaaattca tcagtgcatt
     1021 taaaaacggc tggggtccgg ggaacagcag ccagaactgg cagaggttct accagctgac
     1081 caagctgctg gacagcatgc acgagatggt gggcggcctg ctccagttct gcttctacac
     1141 cttcgtgaac aagagcctga gcgtggagtt ccccgagatg ctggccgaga tcatcagcaa
     1201 ccagctgcct aagtttaatg ccggcagcgt gaagcccctg ctgttccacc agaagggggg
     1261 cggaagcggc ggatccggcg gatctggcgg atcgggcggg agtggactga tcagcctgct
     1321 ggaggtgatc gagcccgagg tgctgtacag cggctacgac agcaccctgc ccgacaccag
     1381 caccaggctg atgagcaccc tgaacaggct gggcggcagg caggtggtga gcgccgtgaa
     1441 gtgggccaag gccctgcccg gcttcaggaa cctgcacctg gacgaccaga tgaccctgct
     1501 gcagtacagc tggatgagcc tgatggcctt cagcctgggc tggaggagct acaagcagag
     1561 caacggcaac atgctgtgct tcgcccccga cctggtgatc aacgaggaga ggatgcagct
     1621 gccctacatg tacgaccagt gccagcagat gctgaagatc agcagcgagt tcgtgaggct
     1681 gcaggtgagc tacgacgagt acctgtgcat gaaggtgctg ctgctgctga gcaccgtgcc
     1741 caaggacggc ctgaagagcc aggccgtgtt cgacgagatc aggatgacct acatcaagga
     1801 gctgggcaag gccatcgtga agagggaggg cggtccgggg tacgaagatg aggaagagtt
     1861 ctacaatgat atgcaaacta tcacactgaa atctgagctg aactagctcg agcggccgcc
     1921 actgtgctgg atatctgcag aattccacca cactggacta gtggatccga gctcggtacc
     1981 aagcttaagt ttaaaccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2041 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2101 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2161 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2221 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2281 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2341 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2401 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2461 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2521 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2581 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2641 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2701 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2761 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2821 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2881 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2941 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3001 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3061 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     3121 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3181 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3241 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3301 caggacgagg cagcgcggct atcgtgactg gccacgacgg gcgttccttg cgcagctgtg
     3361 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3421 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3481 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3541 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3601 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3661 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3721 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3781 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3841 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3901 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     3961 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4021 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4081 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4141 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4201 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4261 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atgcgagccg
     4321 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4381 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4441 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4501 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4561 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4621 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4681 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4741 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4801 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4861 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4921 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4981 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5041 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5101 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5161 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5221 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5281 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5341 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5401 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5461 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5521 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5581 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5641 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5701 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5761 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5821 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5881 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5941 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6001 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6061 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6121 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6181 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6241 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6301 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6361 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     6421 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     6481 atagttaagc cagtatctgc tccctgcttg tgtgttg
//
