LOCUS       sequence-225650-        7240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses type II-C anti-CRISPR protein from S. muelleri and a BFP 
            from IRES in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7240)
  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 7240)
  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: pEJS593-pCSDest2-AcrIIC5Smu-BFPv2-IRES
FEATURES             Location/Qualifiers
     source          1..7240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..1014
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1049..1067
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1049..1066
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     polyA_signal    1550..1684
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        1916..2295
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2296..2507
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2504..2528
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          2668..2800
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2783..2802
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        2845..2863
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2869..3579
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    3723..4185
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3779..3796)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4006..4025
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    4269..4390
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4553..4571)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4592..4608)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4592..4608)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4605..4627)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4616..4632
                     /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(4640..4646,4647..4664,4665..4670))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4685..4706
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4823..4840)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4994..5582)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5074..5093)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5753..6544,6545..6613))
                     /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     6376..6395
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6614..6718)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6744..7199)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6881..6902)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7093..7112
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcggtgcggg cctcttcgct attacgccag tcgaccgcca attcaatatg gcgtatatgg
       61 actcatgcca attcaatatg gtggatctgg acctgtgcca attcaatatg gcgtatatgg
      121 actcgtgcca attcaatatg gtggatctgg accccagcca attcaatatg gcggacttgg
      181 caccatgcca attcaatatg gcggacctgg cactgtgcca actggggagg ggtctacttg
      241 gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc aagtccgcca tattgaattg
      301 gcatggtgcc aataatggcg gccatattgg ctatatgcca ggatcaatat ataggcaata
      361 tccaatatgg ccctatgcca atatggctat tggccaggtt caatactatg tattggccct
      421 atgccatata gtattccata tatgggtttt cctattgacg tagatagccc ctcccaatgg
      481 gcggtcccat ataccatata tggggcttcc taataccgcc catagccact cccccattga
      541 cgtcaatggt ctctatatat ggtctttcct attgacgtca tatgggcggt cctattgacg
      601 tatatggcgc ctcccccatt gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc
      661 attgacgtca ataggaccac ccaccattga cgtcaatggg atggctcatt gcccattcat
      721 atccgttctc acgcccccta ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat
      781 caatatctat taatagtaac ttggcaagta cattactatt ggaagtacgc cagggtacat
      841 tggcagtact cccattgacg tcaatggcgg taaatggccc gcgatggctg ccaagtacat
      901 ccccattgac gtcaatgggg aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg
      961 ggcggtaggc gtgcctaatg ggaggtctat ataagcaatg ctcgtttagg gaaccgccat
     1021 tctgcctggg gacgtcggag caagcttgat ttaggtgaca ctatagaata caagctactt
     1081 gttctttttg caggatccca tcgattcgaa gccgccacca tgaacaacag catcaagttc
     1141 cacgtgagct acgacggtac cgcgcgtgcg ctgtttaaca ccaaggagca ggcggaaaaa
     1201 tactgcctgg ttgaggaaat taacgatgag atgaacggct ataagcgtaa aagctgggag
     1261 gaaaagctgc gtgaggaaaa ctgcgcgagc gtgcaggact gggttgagaa gaactacacc
     1321 agcagctata gcgacctgtt caacatctgc gagattgaag tgagcagcgc gggtcaactg
     1381 gttaagatcg acaacaccga ggtggacgat ttcgttgaaa actgctatgg ctttaccctg
     1441 gaggacgatc tggaggaatt caacaaggcg aaacagtacc tgcaaaaatt ttatgcggag
     1501 tgcgaaaact gactcgagcc tctagaacta tagtgagtcg tattacgtag atccagacat
     1561 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     1621 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     1681 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     1741 tttttaattc gcggccgctc acatggctcg acagatcttc aatattggcc attagccata
     1801 ttattcattg gttatatagc ataaatcaat attggctatt ggccattgca tacgttgtat
     1861 ctatatcata atatgtacat ttatattggc tcatgtccaa tatgaccgcc atgttggcat
     1921 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     1981 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     2041 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     2101 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     2161 tatcatatgc caagtccgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     2221 tatgcccagt acatgacctt acgggacttt cctacttggc agtacatcta cgtattagtc
     2281 atcgctatta ccatggtgat gcggttttgg cagtacacca atgggcgtgg atagcggttt
     2341 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
     2401 caaaatcaac gggactttcc aaaatgtcgt aacaactgcg atcgcccgcc ccgttgacgc
     2461 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc
     2521 gtcagatcac tagaagcttt attgcggtag tttatcacag ttaaattgct aacgcagtca
     2581 gtgcttctga cacaacagtc tcgaacttaa gctgcagtga ctctcttaag gtagccttgc
     2641 agaagttggt cgtgaggcac tgggcaggta agtatcaagg ttacaagaca ggtttaagga
     2701 gaccaataga aactgggctt gtcgagacag agaagactct tgcgtttctg ataggcacct
     2761 attggtctta ctgacatcca ctttgccttt ctctccacag gtgtccactc ccagttcaat
     2821 tacagctctt aaggctagag tacttaatac gactcactat aggctagaat ggtgtctaag
     2881 ggcgaagagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac
     2941 aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
     3001 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact
     3061 agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc
     3121 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     3181 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
     3241 atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg
     3301 gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg
     3361 gccctgaagc tcgtgggcgg gagccatctg atcgcaaacg ccaagaccac atatagatcc
     3421 aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa
     3481 agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga
     3541 tactgcgacc tccctagcaa actggggcac aagcttaatt gaacgcgtcg agcatgcatc
     3601 tagggcggcc aattccgccc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa
     3661 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat
     3721 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
     3781 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct
     3841 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc
     3901 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa
     3961 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc
     4021 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
     4081 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc
     4141 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataagcttg ccacaacccg
     4201 ggatcctcta gagtcgaccc gggcggccgc ttccctttag tgagggttaa tgcttcgagc
     4261 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     4321 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     4381 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg
     4441 ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatccgat aaggatcgat
     4501 ccgggcgcgg ccgcggcgcc aatgcattgg gcccggtacc cagcttttgt tccctttagt
     4561 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4621 atccgctcac aattccacac aacatacgag ccgggagcat aaagtgtaaa gcctggggtg
     4681 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4741 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4801 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4861 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4921 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4981 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5041 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5101 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5161 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5221 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5281 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5341 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5401 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5461 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5521 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5581 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5641 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5701 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5761 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5821 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5881 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5941 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6001 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6061 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6121 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6181 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6241 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6301 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6361 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6421 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6481 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6541 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6601 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6661 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6721 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     6781 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     6841 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     6901 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     6961 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     7021 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     7081 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     7141 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     7201 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
//
