LOCUS       sequence-167159-        7294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Type II-C anti-CRISPR protein, AcrIIC3Nme from Neisseria 
            meningitidis, and mTagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7294)
  AUTHORS   Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, 
            Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR
  TITLE     Naturally Occurring Off-Switches for CRISPR-Cas9.
  JOURNAL   Cell. 2016 Dec 15;167(7):1829-1838.e9. doi: 
            10.1016/j.cell.2016.11.017. Epub 2016 Dec 8.
  PUBMED    27984730
REFERENCE   2  (bases 1 to 7294)
  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"; doi: 
            "10.1016/j.cell.2016.11.017"; journalName: "Cell"; date: 
            "2016-12-15- 15"; volume: "167"; issue: "7"; pages: "1829-1838.e9"
COMMENT     Sequence Label: pEJS482-pCDest2-AcrIIC3Nme-mTagBFP2-IRES
FEATURES             Location/Qualifiers
     source          1..7294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     polyA_signal    497..631
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        928..1307
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1308..1519
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1516..1540
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1680..1812
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1795..1814
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1857..1875
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1881..2591
                     /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    join(2645..3219,3220..3222,3223..3231)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2812..2829)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3039..3058
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    3302..3423
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(3558..3576)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(3590..3608)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3629..3645)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3629..3645)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3642..3664)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3653..3669
                     /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(3677..3683,3684..3701,3702..3707))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3722..3743
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3860..3877)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4031..4619)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4111..4130)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4790..5581,5582..5650))
                     /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     5413..5432
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5651..5755)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5781..6236)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5918..5939)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6130..6149
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6310..7294
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaagccg ccaccatgtt caaacgcgct
      121 attatcttca cttctttcaa cggctttgaa aaagtttctc gaactgaaaa acgccgcctt
      181 gccaaaatca tcaatgctcg agtttccatc atcgacgaat acttgagagc caaagacacc
      241 aacgcatcgc ttgacggtca gtaccgcgct ttcttgttca acgacgaatc gcccgcaatg
      301 accgaatttc tggcaaaact taaagccttt gccgaaagtt gcaccggaat cagcatcgac
      361 gcatgggaaa ttgaagaaag cgaatacgtc cgcctgccgg tggaacgcag ggatttctta
      421 gcggcagcca acggcaaaga gatttttaaa atttaatgac tcgagcctct agaactatag
      481 tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac
      541 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
      601 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
      661 tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg gccgcggcgc caatgcattg
      721 ggcccggtac taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc tcacatggct
      781 cgacagatct tcaatattgg ccattagcca tattattcat tggttatata gcataaatca
      841 atattggcta ttggccattg catacgttgt atctatatca taatatgtac atttatattg
      901 gctcatgtcc aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat
      961 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     1021 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     1081 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     1141 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg
     1201 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact
     1261 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1321 ggcagtacac caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1381 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1441 gtaacaactg cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga
     1501 ggtctatata agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt
     1561 agtttatcac agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt
     1621 aagctgcagt gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg
     1681 taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac
     1741 agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
     1801 ttctctccac aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat
     1861 acgactcact ataggctaga atggtgtcta agggcgaaga gctgattaag gagaacatgc
     1921 acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg
     1981 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc
     2041 ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca
     2101 tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat
     2161 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc
     2221 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg
     2281 gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg
     2341 ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc
     2401 tgatcgcaaa cgccaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc
     2461 ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct
     2521 acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc
     2581 acaagcttaa ttgaacgcgt cgagcatgca tctagggcgg ccaattccgc ccctctcccc
     2641 cccccccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     2701 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     2761 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     2821 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     2881 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     2941 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     3001 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     3061 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     3121 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     3181 acggggacgt ggttttcctt tgaaaaacac gatgataagc ttgccacaac ccgggatcct
     3241 ctagagtcga cccgggcggc cgcttccctt tagtgagggt taatgcttcg agcagacatg
     3301 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     3361 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     3421 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat gtgggaggtt
     3481 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcc gataaggatc gatccgggcg
     3541 gtacccagct tttgttccct ttagtgaggg ttaattccag cttttgttcc ctttagtgag
     3601 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3661 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3721 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3781 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3841 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3901 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3961 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4021 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4081 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4141 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4201 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4261 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4321 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4381 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4441 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     4501 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4561 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4621 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4681 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4741 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4801 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4861 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4921 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4981 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5041 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5101 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5161 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5221 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5281 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5341 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5401 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5461 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5521 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5581 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5641 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5701 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5761 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5821 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5881 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5941 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     6001 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     6061 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     6121 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     6181 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     6241 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     6301 acgccagtcg accatagcca attcaatatg gcgtatatgg actcatgcca attcaatatg
     6361 gtggatctgg acctgtgcca attcaatatg gcgtatatgg actcgtgcca attcaatatg
     6421 gtggatctgg accccagcca attcaatatg gcggacttgg caccatgcca attcaatatg
     6481 gcggacttgg cactgtgcca actggggagg ggtctacttg gcacggtgcc aagtttgagg
     6541 aggggtcttg gccctgtgcc aagtccgcca tattgaattg gcatggtgcc aataatggcg
     6601 gccatattgg ctatatgcca ggatcaatat ataggcaata tccaatatgg ccctatgcca
     6661 atatggctat tggccaggtt caatactatg tattggccct atgccatata gtattccata
     6721 tatgggtttt cctattgacg tagatagccc ctcccaatgg gcggtcccat ataccatata
     6781 tggggcttcc taataccgcc catagccact cccccattga cgtcaatggt ctctatatat
     6841 ggtctttcct attgacgtca tatgggcggt cctattgacg tatatggcgc ctcccccatt
     6901 gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc attgacgtca ataggaccac
     6961 ccaccattga cgtcaatggg atggctcatt gcccattcat atccgttctc acgcccccta
     7021 ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat caatatctat taatagtaac
     7081 ttggcaagta cattactatt ggaaggacgc cagggtacat tggcagtact cccattgacg
     7141 tcaatggcgg taaatggccc gcgatggctg ccaagtacat ccccattgac gtcaatgggg
     7201 aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg ggcggtaggc gtgcctaatg
     7261 ggaggtctat ataagcaatg ctcgtttagg gaac
//
