LOCUS       sequence-225083-        7116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses type II-C anti-CRISPR protein from H. parainfluenzae and a
            BFP from IRES in mammalian cells .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7116)
  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 7116)
  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: pEJS592-pCSDest2-AcrIIC4Hpa-BFPv2-IRES
FEATURES             Location/Qualifiers
     source          1..7116
                     /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    1424..1558
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        1790..2169
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2170..2381
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2378..2402
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          2542..2674
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2657..2676
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        2719..2737
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2743..3453
                     /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    3599..4061
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3655..3672)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3882..3901
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    4145..4266
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4429..4447)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4468..4484)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4468..4484)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4481..4503)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4492..4508
                     /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(4516..4522,4523..4540,4541..4546))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4561..4582
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4699..4716)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4870..5458)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4950..4969)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5629..6420,6421..6489))
                     /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     6252..6271
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6490..6594)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6620..7075)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6757..6778)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6969..6988
                     /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 tgaagatcac cagcagcaac
     1141 ttcgcgacca ttgcgaccag cgagaacttt gcgaagctga gcgtgctgcc gaaaaaccac
     1201 cgtgagccga tcaagggtct gttcaaaagc gcggttgaac agtttagcag cgcgcgtgac
     1261 ttctttaaga acgagaacta cagcaaagag ctggcggaaa agttcaacaa agaagcggtg
     1321 aacgaggcgg ttgaaaagct gcaaaaagcg atcgatctgg cggaaaaaca gggcattcaa
     1381 ttttgactcg agcctctaga actatagtga gtcgtattac gtagatccag acatgataag
     1441 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     1501 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     1561 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     1621 attcgcggcc gctcacatgg ctcgacagat cttcaatatt ggccattagc catattattc
     1681 attggttata tagcataaat caatattggc tattggccat tgcatacgtt gtatctatat
     1741 cataatatgt acatttatat tggctcatgt ccaatatgac cgccatgttg gcattgatta
     1801 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     1861 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     1921 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     1981 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     2041 atgccaagtc cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     2101 cagtacatga ccttacggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     2161 attaccatgg tgatgcggtt ttggcagtac accaatgggc gtggatagcg gtttgactca
     2221 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     2281 caacgggact ttccaaaatg tcgtaacaac tgcgatcgcc cgccccgttg acgcaaatgg
     2341 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
     2401 tcactagaag ctttattgcg gtagtttatc acagttaaat tgctaacgca gtcagtgctt
     2461 ctgacacaac agtctcgaac ttaagctgca gtgactctct taaggtagcc ttgcagaagt
     2521 tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta aggagaccaa
     2581 tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc acctattggt
     2641 cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccagtt caattacagc
     2701 tcttaaggct agagtactta atacgactca ctataggcta gaatggtgtc taagggcgaa
     2761 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     2821 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     2881 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     2941 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     3001 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     3061 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     3121 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     3181 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     3241 aagctcgtgg gcgggagcca tctgatcgca aacgccaaga ccacatatag atccaagaaa
     3301 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     3361 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     3421 gacctcccta gcaaactggg gcacaagctt aattgaacgc gtcgagcatg catctagggc
     3481 ggccaattcc gcccctctcc cccsccccta acgttactgg ccgaagccgc ttggaataag
     3541 gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga
     3601 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg
     3661 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt
     3721 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca
     3781 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc
     3841 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat
     3901 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc
     3961 ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga
     4021 accacgggga cgtggttttc ctttgaaaaa cacgatgata agcttgccac aacccgggat
     4081 cctctagagt cgacccgggc ggccgcttcc ctttagtgag ggttaatgct tcgagcagac
     4141 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     4201 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     4261 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag
     4321 gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg atcgatccgg
     4381 gcgcggccgc ggcgccaatg cattgggccc ggtacccagc ttttgttccc tttagtgagg
     4441 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4501 gctcacaatt ccacacaaca tacgagccgg gagcataaag tgtaaagcct ggggtgccta
     4561 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4621 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4681 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4741 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4801 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4861 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4921 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4981 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5041 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5101 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5161 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5221 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5281 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5341 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5401 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5461 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5521 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5581 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5641 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5701 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5761 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5821 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5881 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5941 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6001 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6061 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6121 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6181 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6241 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6301 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6361 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6421 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6481 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6541 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6601 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     6661 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     6721 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     6781 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     6841 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     6901 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     6961 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     7021 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     7081 tcgccattca ggctgcgcaa ctgttgggaa gggcga
//
