LOCUS       sequence-495335-        6640 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of AcrIIA5-cpGR2 hybrid in mammalian cells; insertion 
            before D41; enables chemogenetic control of SauCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6640)
  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 6640)
  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_cpGR2(GGS5)-D41
FEATURES             Location/Qualifiers
     source          1..6640
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     244..263
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(482..586)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(661..680)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        852..1231
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1232..1435
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1386..1406
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1480..1499
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1480..1498
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2848..2865)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2854..3078
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3124..3552
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3211..3230)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3421..3442
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3561..3581)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3566..3895
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3746..3881
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3808..3827
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3962..4756
                     /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(4016..4035)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4626..4645
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4930..5051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4967..4986)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5021..5040
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5100..5116)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5100..5116)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5113..5135)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5124..5140
                     /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(5148..5154,5155..5172,5173..5178))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5193..5214
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5331..5348)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5502..6090)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5582..5601)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6261..6640,1..412,413..481))
                     /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"
ORIGIN
        1 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
       61 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
      121 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
      181 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
      241 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
      301 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
      361 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
      421 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
      481 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
      541 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
      601 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
      661 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
      721 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
      781 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
      841 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      901 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      961 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     1021 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     1081 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     1141 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     1201 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     1261 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     1321 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     1381 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     1441 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     1501 gacccaagct ggctagcatg gcatacggta aaagccgata taactcttac cggaaacgaa
     1561 attttagtat cagtgacaac cagagaagag aatacgccaa aaaaatgaag gaattggagc
     1621 aagcgtttga gaatttgggt ccggggaaca gcagccagaa ctggcagagg ttctaccagc
     1681 tgaccaagct gctggacagc atgcacgaga tggtgggcgg cctgctccag ttctgcttct
     1741 acaccttcgt gaacaagagc ctgagcgtgg agttccccga gatgctggcc gagatcatca
     1801 gcaaccagct gcctaagttt aatgccggca gcgtgaagcc cctgctgttc caccagaagg
     1861 ggggcggaag cggcggatcc ggcggatctg gcggatcggg cgggagtgga ctgatcagcc
     1921 tgctggaggt gatcgagccc gaggtgctgt acagcggcta cgacagcacc ctgcccgaca
     1981 ccagcaccag gctgatgagc accctgaaca ggctgggcgg caggcaggtg gtgagcgccg
     2041 tgaagtgggc caaggccctg cccggcttca ggaacctgca cctggacgac cagatgaccc
     2101 tgctgcagta cagctggatg agcctgatgg ccttcagcct gggctggagg agctacaagc
     2161 agagcaacgg caacatgctg tgcttcgccc ccgacctggt gatcaacgag gagaggatgc
     2221 agctgcccta catgtacgac cagtgccagc agatgctgaa gatcagcagc gagttcgtga
     2281 ggctgcaggt gagctacgac gagtacctgt gcatgaaggt gctgctgctg ctgagcaccg
     2341 tgcccaagga cggcctgaag agccaggccg tgttcgacga gatcaggatg acctacatca
     2401 aggagctggg caaggccatc gtgaagaggg agggcggtcc gggggacggg tggtacctct
     2461 catccatgaa ggatagcgcc tacaaagact tcggaaagta cgagataaga ctgtcaaacc
     2521 acagtgcaga caataggtat cacgaccttg aaaatggtcg ccttatcgtt aatgtaaagg
     2581 cttcaaaact taatttcgtt gatattatag agaacaagct gggcaaaatt attgagaaga
     2641 tcgatacgct tgatttggat aagtacagat tcattaacgc tactaaactg gaaagggata
     2701 taaaatgtta ctataagggg tataaaacaa aaaaggatgt tatctgactc gagcggccgc
     2761 cactgtgctg gatatctgca gaattccacc acactggact agtggatccg agctcggtac
     2821 caagcttaag tttaaaccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2881 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2941 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3001 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3061 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3121 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3181 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3241 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3301 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3361 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3421 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3481 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3541 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3601 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3661 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3721 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3781 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3841 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3901 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     3961 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4021 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4081 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4141 gcaggacgag gcagcgcggc tatcgtgact ggccacgacg ggcgttcctt gcgcagctgt
     4201 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4261 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4321 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4381 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4441 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4501 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4561 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4621 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4681 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4741 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4801 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4861 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4921 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4981 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5041 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5101 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catgcgagcc
     5161 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5221 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5281 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5341 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5401 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5461 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5521 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5581 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5641 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5701 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5761 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5821 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5881 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5941 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6001 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6061 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6121 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6181 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6241 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6301 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6361 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6421 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6481 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6541 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6601 tcgtttggta tggcttcatt cagctccggt tcccaacgat
//
