LOCUS       sequence-272422-        6221 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CNC3G (CASANOVA-C3 with glycine linkers), a blue light 
            dependent NmeCas9 inhibitory protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6221)
  AUTHORS   Hoffmann MD, Mathony J, Upmeier Zu Belzen J, Harteveld Z, 
            Aschenbrenner S, Stengl C, Grimm D, Correia BE, Eils R, Niopek D
  TITLE     Optogenetic control of Neisseria meningitidis Cas9 genome editing 
            using an engineered, light-switchable anti-CRISPR protein.
  JOURNAL   Nucleic Acids Res. 2021 Mar 18;49(5):e29. doi: 10.1093/nar/gkaa1198.
  PUBMED    33330940
REFERENCE   2  (bases 1 to 6221)
  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: "Nucleic 
            Acids Res."; date: "2021-03-18"; volume: "49(5):e29. doi"; pages: " 
            10.1093/nar/gkaa1198"
COMMENT     Sequence Label: CNC3G (CASANOVA-C3 with glycine linkers)
FEATURES             Location/Qualifiers
     source          1..6221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        129..332
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     283..303
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      415..424
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1326..1343)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1332..1556
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1602..2030
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1689..1708)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1899..1920
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2039..2059)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2044..2373
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2224..2359
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2286..2305
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2440..3234
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2494..2513)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3104..3123
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3408..3529
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3445..3464)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3499..3518
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3578..3594)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3578..3594)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3591..3613)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3602..3618
                     /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(3626..3632,3633..3650,3651..3656))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3671..3692
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3809..3826)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3980..4568)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4060..4079)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4739..5530,5531..5599))
                     /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     5362..5381
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5600..5704)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5779..5798)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(5970..6221,1..128)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
       61 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      121 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      181 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      241 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      301 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt
      361 actggcttat cgaaattaat acgactcact atagggagac ccaagctggc tagcgccacc
      421 atggctttca agagagctat tatatttacg tcctttaatg gttttgagaa ggtctctcga
      481 acagagaagc gacgccttgc caagataatt aacgcgaggg tctcaattat tgatgagtat
      541 ctcagagcca aggatacaaa cgcgagcctg gatggccaat accgagcctt tctgtttgga
      601 cttgctacaa ctttggagcg gattgagaag aatttcgtca taactgatcc caggttgcct
      661 gataatccta taatattcgc ctccgatagt tttcttcaac ttacggagta ctccagagag
      721 gagatactgg gaaggaattg cagattcctc caaggtccag aaactgatag agctactgtg
      781 aggaagatac gggatgccat tgacaatcaa actgaagtga cagtgcaact tatcaattac
      841 accaagtcag ggaaaaaatt ctggaacctg ttccatttgc aacccatgag ggaccaaaag
      901 ggcgatgtgc aatatttcat aggcgttcaa cttgatggta ctgaacacgt cagagatgcg
      961 gcggagcgag aaggagtgat gctgattaag aaaacagcag agaatatcga cgaagcggcg
     1021 aaagagttgg gaaacgacga aagccccgcg atgacagagt tcctcgcaaa actgaaagca
     1081 ttcgcggaga gctgcacggg aatatcaata gatgcgtggg agattgaaga gagcgagtat
     1141 gttcgactgc ccgttgaacg ccgggatttc ctggccgctg cgaatggcaa ggaaatcttc
     1201 aagatataac tcgagcggcc gccactgtgc tggatatctg cagaattaga gacgccgttg
     1261 atgttgaagt ttatataccg ttgatgttga agtcgtctct agcttaagtt taaaccgctg
     1321 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     1381 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     1441 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     1501 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     1561 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     1621 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1681 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1741 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1801 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1861 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1921 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1981 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     2041 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2101 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2161 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2221 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2281 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     2341 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     2401 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     2461 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     2521 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     2581 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     2641 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     2701 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     2761 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     2821 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     2881 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     2941 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     3001 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3061 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3121 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     3181 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     3241 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3301 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3361 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     3421 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3481 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3541 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3601 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3661 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3721 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3781 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3841 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3901 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3961 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4021 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4081 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4141 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4201 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4261 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4321 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4381 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4441 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4501 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4561 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4621 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4681 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4741 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4801 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4861 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4921 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4981 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5041 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5101 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5161 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5221 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5281 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5341 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5401 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5461 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5521 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5581 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5641 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5701 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     5761 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     5821 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     5881 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     5941 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     6001 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     6061 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     6121 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     6181 acggtaaact gcccacttgg cagtacatca agtgtatcat a
//
