LOCUS       sequence-495343-        6739 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 and GGS2 
            linkers; enables chemogenetic control of MbCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6739)
  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 6739)
  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_(GGS)2-(GGS)2
FEATURES             Location/Qualifiers
     source          1..6739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        120..499
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        500..703
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     654..674
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     748..767
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        748..766
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2215..2232)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2221..2445
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2491..2919
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2578..2597)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2788..2809
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2928..2948)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2933..3262
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3113..3248
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3175..3194
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3329..4123
                     /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(3383..3402)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3993..4012
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4297..4418
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4334..4353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4388..4407
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4467..4483)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4467..4483)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4480..4502)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4491..4507
                     /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(4515..4521,4522..4539,4540..4545))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4560..4581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4698..4715)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4869..5457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4949..4968)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5628..6419,6420..6488))
                     /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     6251..6270
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6489..6593)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6668..6687)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
       61 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      121 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      181 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      241 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      301 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      361 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      421 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      481 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      541 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      601 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      661 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
      721 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
      781 ctagcatgta tgaagctaaa gagcgctatg ccaagaagaa gatgcaggag aatactaaga
      841 ttgacacctt gacagatgaa cagcatgatg ctcttgcaca gctttgcgct ttcagacata
      901 agttccatag caacaaggac agtctgttcc tgtctgagtc tgctttcagc ggggaattct
      961 ccttcgaaat gcagtcagac gaaaatagca aactgaggga ggtgggattg ccaacaatag
     1021 aatggagctt ctatgacaac tcccacattc ccgacgactc tttccgagag tggtttaact
     1081 ttgctaatta cagcgggggc agcggagggt ccaacagcag ccagaactgg cagaggttct
     1141 accagctgac caagctgctg gacagcatgc acgagatggt gggcggcctg ctccagttct
     1201 gcttctacac cttcgtgaac aagagcctga gcgtggagtt ccccgagatg ctggccgaga
     1261 tcatcagcaa ccagctgcct aagtttaatg ccggcagcgt gaagcccctg ctgttccacc
     1321 agaagggggg cggaagcggc ggatccggcg gatctggcgg atcgggcggg agtggactga
     1381 tcagcctgct ggaggtgatc gagcccgagg tgctgtacag cggctacgac agcaccctgc
     1441 ccgacaccag caccaggctg atgagcaccc tgaacaggct gggcggcagg caggtggtga
     1501 gcgccgtgaa gtgggccaag gccctgcccg gcttcaggaa cctgcacctg gacgaccaga
     1561 tgaccctgct gcagtacagc tggatgagcc tgatggcctt cagcctgggc tggaggagct
     1621 acaagcagag caacggcaac atgctgtgct tcgcccccga cctggtgatc aacgaggaga
     1681 ggatgcagct gccctacatg tacgaccagt gccagcagat gctgaagatc agcagcgagt
     1741 tcgtgaggct gcaggtgagc tacgacgagt acctgtgcat gaaggtgctg ctgctgctga
     1801 gcaccgtgcc caaggacggc ctgaagagcc aggccgtgtt cgacgagatc aggatgacct
     1861 acatcaagga gctgggcaag gccatcgtga agagggaggg cggcggatcc gggggaagcg
     1921 aaaccatcca agagcaggga ctggagctcg atctggacga cgatgaaacg tacgaactcg
     1981 tatacgacga attgtatact gaggccatgg gagaatatga agagctaaac caagacattg
     2041 agaagtatct gaggcgtatt gacgaagagc acggcactca gtattgtccg acagggtttg
     2101 cgaggcttcg gtgactcgag cggccgccac tgtgctggat atctgcagaa ttccaccaca
     2161 ctggactagt ggatccgagc tcggtaccaa gcttaagttt aaaccgctga tcagcctcga
     2221 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2281 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     2341 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     2401 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     2461 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     2521 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2581 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2641 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2701 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     2761 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2821 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2881 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     2941 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3001 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3061 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3121 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3181 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     3241 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     3301 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     3361 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     3421 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     3481 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtgactggc
     3541 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     3601 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     3661 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     3721 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     3781 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     3841 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     3901 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     3961 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     4021 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4081 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     4141 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     4201 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4261 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4321 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4381 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4441 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4501 ctcacaattc cacacaacat gcgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4561 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4621 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4681 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4741 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4801 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4861 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4921 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4981 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5041 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5101 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5161 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5221 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5281 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5341 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5401 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5461 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5521 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5581 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5641 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5701 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5761 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5821 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5881 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5941 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6001 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6061 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6121 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6181 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6241 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6301 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6361 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6421 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6481 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6541 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6601 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     6661 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     6721 tgttggaggt cgctgagta
//
