LOCUS       sequence-231532-        4541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a FLAG/NLS tagged type II-C anti-CRISPR protein from S. 
            muelleri in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4541)
  AUTHORS   Lee J, Mir A, Edraki A, Garcia B, Amrani N, Lou HE, Gainetdinov I, 
            Pawluk A, Ibraheim R, Gao XD, Liu P, Davidson AR, Maxwell KL, 
            Sontheimer EJ
  TITLE     Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and 
            AcrIIC5 Anti-CRISPR Proteins.
  JOURNAL   MBio. 2018 Dec 4;9(6). pii: mBio.02321-18. doi: 
            10.1128/mBio.02321-18.
  PUBMED    30514786
REFERENCE   2  (bases 1 to 4541)
  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: "MBio."; 
            date: "2018-12-4"; volume: "9(6). pii: mBio.02321-18. doi"; pages: "
            10.1128/mBio.02321-18"
COMMENT     Sequence Label: pEJS583-pCSDest2-AcrIIC5Smu-FLAG-NLS
FEATURES             Location/Qualifiers
     source          1..4541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        63..1047
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1082..1100
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1082..1099
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1549..1572
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1579..1599
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1640..1774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1664..1683)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1718..1737
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1887..1907)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1887..1905)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1926..1942)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1926..1942)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1939..1961)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1950..1966
                     /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(1974..1980,1981..1998,1999..2004))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2019..2040
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2157..2174)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2328..2916)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2408..2427)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3087..3878,3879..3947))
                     /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     3710..3729
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3948..4052)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4078..4533)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4215..4236)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4427..4446
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
       61 cagtcgaccg ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc
      121 tggacctgtg ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc
      181 tggaccccag ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggacc
      241 tggcactgtg ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc
      301 ttggccctgt gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat
      361 tggctatatg ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc
      421 tattggccag gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt
      481 tttcctattg acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct
      541 tcctaatacc gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt
      601 cctattgacg tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca
      661 attacggtaa atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat
      721 tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt
      781 caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa
      841 gtacattact attggaagta cgccagggta cattggcagt actcccattg acgtcaatgg
      901 cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca
      961 atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc
     1021 tatataagca atgctcgttt agggaaccgc cattctgcct ggggacgtcg gagcaagctt
     1081 gatttaggtg acactataga atacaagcta cttgttcttt ttgcaggatc ccatcgattc
     1141 gaagccgcca ccatgaacaa cagcatcaag ttccacgtga gctacgacgg taccgcgcgt
     1201 gcgctgttta acaccaagga gcaggcggaa aaatactgcc tggttgagga aattaacgat
     1261 gagatgaacg gctataagcg taaaagctgg gaggaaaagc tgcgtgagga aaactgcgcg
     1321 agcgtgcagg actgggttga gaagaactac accagcagct atagcgacct gttcaacatc
     1381 tgcgagattg aagtgagcag cgcgggtcaa ctggttaaga tcgacaacac cgaggtggac
     1441 gatttcgttg aaaactgcta tggctttacc ctggaggacg atctggagga attcaacaag
     1501 gcgaaacagt acctgcaaaa attttatgcg gagtgcgaaa acggcgggga ttataaggat
     1561 gatgatgata agggctcccc taagaagaag agaaaggtgt gactcgagcc tctagaacta
     1621 tagtgagtcg tattacgtag atccagacat gataagatac attgatgagt ttggacaaac
     1681 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     1741 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     1801 gtttcaggtt cagggggagg tgtgggaggt tttttaattc gcggccgcgg cgccaatgca
     1861 ttgggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     1921 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     1981 cgagccggga gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     2041 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     2101 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     2161 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2221 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2281 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2341 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2401 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2461 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2521 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2581 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2641 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2701 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2761 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2821 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2881 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2941 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3001 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3061 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3121 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3181 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3241 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3301 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3361 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3421 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3481 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3541 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3601 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3661 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3721 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3781 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3841 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3901 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3961 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4021 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     4081 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     4141 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     4201 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     4261 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     4321 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     4381 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     4441 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     4501 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc g
//
