LOCUS       sequence-170085-        7263 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses Type II-C anti-CRISPR protein, AcrIIC3Nme from Neisseria 
            meningitidis, and mTagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7263)
  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 7263)
  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..7263
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(25..41)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(25..41)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(38..60)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    49..65
                     /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(73..79,80..97,98..103))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    118..139
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(256..273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(427..1015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(507..526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1186..1977,1978..2046))
                     /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     1809..1828
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2047..2151)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2177..2632)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2314..2335)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2526..2545
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2703..3687
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        3722..3740
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3722..3739
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     polyA_signal    4184..4318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        4615..4994
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4995..5206
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5203..5227
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          5367..5499
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     5482..5501
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        5544..5562
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5568..6278
                     /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(6332..6906,6907..6909,6910..6918)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6499..6516)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     6726..6745
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    6989..7110
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(7245..7263)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
       61 cacaattcca cacaacatac gagccgggag cataaagtgt aaagcctggg gtgcctaatg
      121 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
      181 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
      241 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
      301 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      361 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
      421 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      481 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
      541 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
      601 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
      661 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
      721 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
      781 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
      841 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
      901 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
      961 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     1021 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     1081 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     1141 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     1201 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     1261 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     1321 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     1381 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     1441 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     1501 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     1561 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     1621 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     1681 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     1741 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     1801 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     1861 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     1921 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     1981 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     2041 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     2101 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     2161 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     2221 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     2281 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     2341 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     2401 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     2461 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2521 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     2581 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     2641 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     2701 cagtcgaccg ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc
     2761 tggacctgtg ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc
     2821 tggaccccag ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggacc
     2881 tggcactgtg ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc
     2941 ttggccctgt gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat
     3001 tggctatatg ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc
     3061 tattggccag gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt
     3121 tttcctattg acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct
     3181 tcctaatacc gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt
     3241 cctattgacg tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca
     3301 attacggtaa atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat
     3361 tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt
     3421 caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa
     3481 gtacattact attggaagta cgccagggta cattggcagt actcccattg acgtcaatgg
     3541 cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca
     3601 atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc
     3661 tatataagca atgctcgttt agggaaccgc cattctgcct ggggacgtcg gagcaagctt
     3721 gatttaggtg acactataga atacaagcta cttgttcttt ttgcaggatc ccatcgattc
     3781 gaagccgcca ccatgttcaa acgcgctatt atcttcactt ctttcaacgg ctttgaaaaa
     3841 gtttctcgaa ctgaaaaacg ccgccttgcc aaaatcatca atgctcgagt ttccatcatc
     3901 gacgaatact tgagagccaa agacaccaac gcatcgcttg acggtcagta ccgcgctttc
     3961 ttgttcaacg acgaatcgcc cgcaatgacc gaatttctgg caaaacttaa agcctttgcc
     4021 gaaagttgca ccggaatcag catcgacgca tgggaaattg aagaaagcga atacgtccgc
     4081 ctgccggtgg aacgcaggga tttcttagcg gcagccaacg gcaaagagat ttttaaaatt
     4141 taatgactcg agcctctaga actatagtga gtcgtattac gtagatccag acatgataag
     4201 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     4261 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     4321 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     4381 attcgcggcc gcggcgccaa tgcattgggc ccggtacctc acmksgsycg msrsrycwwy
     4441 rmwwtkggcc cggtacctca catggctcga cagatcttca atattggcca ttagccatat
     4501 tattcattgg ttatatagca taaatcaata ttggctattg gccattgcat acgttgtatc
     4561 tatatcataa tatgtacatt tatattggct catgtccaat atgaccgcca tgttggcatt
     4621 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     4681 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     4741 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     4801 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     4861 atcatatgcc aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     4921 atgcccagta catgacctta cgggactttc ctacttggca gtacatctac gtattagtca
     4981 tcgctattac catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg
     5041 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     5101 aaaatcaacg ggactttcca aaatgtcgta acaactgcga tcgcccgccc cgttgacgca
     5161 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
     5221 tcagatcact agaagcttta ttgcggtagt ttatcacagt taaattgcta acgcagtcag
     5281 tgcttctgac acaacagtct cgaacttaag ctgcagtgac tctcttaagg tagccttgca
     5341 gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag
     5401 accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta
     5461 ttggtcttac tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt
     5521 acagctctta aggctagagt acttaatacg actcactata ggctagaatg gtgtctaagg
     5581 gcgaagagct gattaaggag aacatgcaca tgaagctgta catggagggc accgtggaca
     5641 accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca
     5701 tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacta
     5761 gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc gacttcttca
     5821 agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg
     5881 tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga
     5941 tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg
     6001 aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga aacgacatgg
     6061 ccctgaagct cgtgggcggg agccatctga tcgcaaacgc caagaccaca tatagatcca
     6121 agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac agactggaaa
     6181 gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca gtggccagat
     6241 actgcgacct ccctagcaaa ctggggcaca agcttaattg aacgcgtcga gcatgcatct
     6301 agggcggcca attccgcccc tctccccccc ccccctctcc ctcccccccc cctaacgtta
     6361 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca
     6421 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca
     6481 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg
     6541 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc
     6601 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
     6661 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag
     6721 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
     6781 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt
     6841 taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat
     6901 gataagcttg ccacaacccg ggatcctcta gagtcgaccc gggcggccgc ttccctttag
     6961 tgagggttaa tgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     7021 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     7081 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     7141 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     7201 aaaatccgat aaggatcgat ccgggcggta cccagctttt gttcccttta gtgagggtta
     7261 att
//
