LOCUS       sequence-170101-        4463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Type II-C anti-CRISPR protein AcrIIC2Nme 
            from Neisseria meningitidis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4463)
  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 4463)
  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: pEJS436-pCSDest2-AcrIIC2Nme
FEATURES             Location/Qualifiers
     source          1..4463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        914..932
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     914..931
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     polyA_signal    1394..1528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1418..1437)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1472..1491
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1641..1661)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1641..1659)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1680..1696)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1680..1696)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1693..1715)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1704..1720
                     /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(1728..1734,1735..1752,1753..1758))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1773..1794
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1911..1928)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2082..2670)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2162..2181)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2841..3632,3633..3701))
                     /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     3464..3483
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3702..3806)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3832..4287)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3969..3990)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4181..4200
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(4358..4463,1..879)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 atatggtgga tctggacccc agccaattca atatggcgga cttggcacca tgccaattca
       61 atatggcgga cctggcactg tgccaactgg ggaggggtct acttggcacg gtgccaagtt
      121 tgaggagggg tcttggccct gtgccaagtc cgccatattg aattggcatg gtgccaataa
      181 tggcggccat attggctata tgccaggatc aatatatagg caatatccaa tatggcccta
      241 tgccaatatg gctattggcc aggttcaata ctatgtattg gccctatgcc atatagtatt
      301 ccatatatgg gttttcctat tgacgtagat agcccctccc aatgggcggt cccatatacc
      361 atatatgggg cttcctaata ccgcccatag ccactccccc attgacgtca atggtctcta
      421 tatatggtct ttcctattga cgtcatatgg gcggtcctat tgacgtatat ggcgcctccc
      481 ccattgacgt caattacggt aaatggcccg cctggctcaa tgcccattga cgtcaatagg
      541 accacccacc attgacgtca atgggatggc tcattgccca ttcatatccg ttctcacgcc
      601 ccctattgac gtcaatgacg gtaaatggcc cacttggcag tacatcaata tctattaata
      661 gtaacttggc aagtacatta ctattggaag tacgccaggg tacattggca gtactcccat
      721 tgacgtcaat ggcggtaaat ggcccgcgat ggctgccaag tacatcccca ttgacgtcaa
      781 tggggagggg caatgacgca aatgggcgtt ccattgacgt aaatgggcgg taggcgtgcc
      841 taatgggagg tctatataag caatgctcgt ttagggaacc gccattctgc ctggggacgt
      901 cggagcaagc ttgatttagg tgacactata gaatacaagc tacttgttct ttttgcagga
      961 tcccatcgat tcgaagccgc caccatgagc aaaaacaata ttttcaacaa gtatccaaca
     1021 attattcacg gcgaagcgcg gggggagaat gacgaatttg tggtgcatac gcgctacccg
     1081 cgattcttgg cgcggaaatc ttttgacgac aatttcacgg gcgaaatgcc tgcaaaacct
     1141 gttaacgggg aattgggaca aatcggcgaa ccgcgccgcc ttgcttatga ttcacggctt
     1201 ggtttgtggc tttctgactt catcatgttg gacaacaaca agccgaaaaa catggaggat
     1261 tggcttgggc aattaaaagc cgcctgcgat cgaatcgcgg cggatgattt gatgctgaat
     1321 gaagatgcgg cggatttgga gggctgggat gattgactcg agcctctaga actatagtga
     1381 gtcgtattac gtagatccag acatgataag atacattgat gagtttggac aaaccacaac
     1441 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     1501 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     1561 ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa tgcattgggc
     1621 ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     1681 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     1741 gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     1801 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     1861 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     1921 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     1981 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2041 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2101 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2161 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2221 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2281 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2341 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     2401 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2461 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2521 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2581 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2641 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2701 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     2761 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     2821 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     2881 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     2941 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3001 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3061 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3121 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3181 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3241 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3301 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3361 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3421 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3481 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3541 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3601 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3661 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     3721 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     3781 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     3841 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     3901 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     3961 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4021 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4081 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4141 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4201 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4261 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     4321 aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat tcaatatggc
     4381 gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat tcaatatggc
     4441 gtatatggac tcgtgccaat tca
//
