LOCUS       sequence-232930-        8490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a 6X-His tagged type II-C Cas9 from S. muelleri in 
            bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8490)
  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 8490)
  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: pEJS1027-pMCSG7-SmuCas9
FEATURES             Location/Qualifiers
     source          1..8490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             66..83
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(136..154)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      150..197
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(321..340)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        507..610
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(611..679,680..1471)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(829..848)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1642..2230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2131..2150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2384..2401
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2416..2558
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2484..2503)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2644..2666
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2660..2851)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3660..4742)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4704..4723
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4743..4820)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5026..5045
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5133..5152
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5133..5151
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5152..5176
                     /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)."
     RBS             5191..5213
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5224..5241
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5266..5286
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
ORIGIN
        1 aacattggaa gtggataacg gatccgaatt cgagctccgt cgacaagctt gcggccgcac
       61 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
      121 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      181 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
      241 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      301 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      361 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      421 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacattaac gcttacaatt
      481 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
      541 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatttgaaa
      601 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
      661 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
      721 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
      781 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
      841 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
      901 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
      961 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     1021 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     1081 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     1141 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     1201 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     1261 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     1321 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     1381 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     1441 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     1501 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     1561 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1621 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1681 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1741 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     1801 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1861 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1921 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1981 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2041 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2101 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2161 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2221 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     2281 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     2341 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     2401 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2461 ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     2521 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     2581 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     2641 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     2701 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     2761 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     2821 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     2881 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     2941 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     3001 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     3061 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     3121 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     3181 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     3241 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     3301 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     3361 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     3421 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     3481 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     3541 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     3601 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     3661 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3721 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     3781 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     3841 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     3901 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     3961 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     4021 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     4081 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     4141 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     4201 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     4261 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     4321 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     4381 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     4441 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     4501 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     4561 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     4621 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     4681 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     4741 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     4801 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     4861 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     4921 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     4981 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     5041 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     5101 gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     5161 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
     5221 atgcaccatc atcatcatca ttcttctggt gtagatctgg gtaccgagaa cctgtacttc
     5281 caatccaatg ccatgatgat ggaaaaattt cactatgtat tgggtttgga tttgggtatc
     5341 gcctctgtgg ggtgggctgc cattgaaatt gacaaggaaa ccgaaacatc aatcggttta
     5401 ttggattgcg gtgtcagaac atttgaacgt gcagaagtac ccaaaacagg cgattctctt
     5461 gccaaagctc gccgtgaagc cagaagtact cgccgtttaa ttcgcagacg ttcgcatcgc
     5521 ttattacgtt taaaacgttt attgaaacgt gaaattttca ggcagcctga aacgtttaaa
     5581 gacttaccaa tcaatgcttg gcaattgcgt gttaaaggct tggatagtcg gttgaatgaa
     5641 tatgaatggg cggccgtttt attgcatttg gtgaagcatc gcggttattt atcgcaacgc
     5701 aaaagcgaaa tgagcgaaac agacagcaaa tctgaaatgg gcagattact ggcaggtgtg
     5761 gcggaaaatc accaactttt acaacaagaa caatatcgta caccagccga attagcactc
     5821 aaaaaatttg tgaaacattt tcgcaataaa ggtggcgatt atgcacacac tttcaaccgt
     5881 ttggatttgc aagccgaatt gcatttattg tttcaaaaac aacgtgaatt aggcaatcca
     5941 ttcacttcac cagaattgga acggcaagtt gatgatttgt tgatgacgca gcgcagtgct
     6001 ttacaaggtg atgcgatttt gaaaatgttg ggtcattgtg ggtttgaacc tgaacaattc
     6061 aaagcagcga aaaacacatt cagtgccgaa cgttttattt ggttgacaaa actcaataat
     6121 cttcgcattc aagaccaagg caaagaacgt gcgttaactg ccgatgagcg taccaaattg
     6181 ttggacgagc cttataaaaa aagtaaattg acttacgcac aagttcgcaa attattaagc
     6241 ttgcctcaaa ctgctatttt taaaggtttg cgttatgatt tggaacatga caaaaaagca
     6301 gaaaacagta cgttgatgga aatgaaatcc tatcacaaca tccgccaaac attggaaaaa
     6361 tcaggtttga aaacagaatg gcaaagtatt gccacgcagc ctgaaatttt agatgcaatt
     6421 ggcacggcgt tttccattta taaaaccgat gaagatattt cgcatgaatt aaaaacgtgc
     6481 aggctgcctg aaaacgtatt gaatgaatta ctgaaaaaca tcaattttga tggattcatt
     6541 caattatcgt tgactgcatt acgcaaaatt ttgcccttga tggaacaagg ctaccgttat
     6601 gatgaagcgt gtacccaaat ttacggtaat catcattcag gcagcttgca acaagaatca
     6661 aagcaatttt tgccacatat tccgattgat gatgtccgaa atcctgtggt gttccgtact
     6721 ttgacccaag caagaaaagt ggtgaatgcg attattcgtc ggtatggttc gccagctcgt
     6781 gtgcatattg agatggcgcg tgaattgggt aagtctaaat cagaccgtga ccgaattgaa
     6841 aaacaacaac aaaaaaataa aaaagaacgt gaaaacgcag tcgccaaatt caaagaagat
     6901 ttccctgatt ttgtgggcga acccagaggg aaagatattt tgaaaatgcg tttgtatgaa
     6961 caacaacacg gcaaatgttt gtattcgggt catgatattg atattaatcg attgaatgaa
     7021 aaaggttatg ttgaaattga ccatgccctg ccattttcac ggacttggga tgatagtcaa
     7081 aataataaag ttttggtact tggcagcgaa aaccaaaata aacgcaatca aacgcctgat
     7141 gaatatttgg acggtgcaaa caatagccaa cgttggcttg aatttcaagc gcgtgtacaa
     7201 acttgtcatt tttcttacgg taaaaaacaa cgcattcaat tagccaaatt agacgatgaa
     7261 accgaaaaag gatttttaga acgcaatcta aatgatacgc ggtatattgc tcgttttatg
     7321 tgtcaatttg tccaagaaaa tttatatttg acaggtaaag gaaaacgtct tgtttttgca
     7381 tcaaacggcg gaatgaccgc aacattgaga aatttatggg gtttgagaaa agtccgtgaa
     7441 gacaacgacc gccatcatgc tcttgatgcg attgtggtgg cgtgttccac tgcttctatg
     7501 caacaaaaaa taaccaaagc atttcaacgc catgaaagca ttgaatatgt ggataccgaa
     7561 acgggcgaag taaaatttcg tattccacag ccttgggatt ttttccgtca ggaagtgatg
     7621 attcgtgtgt tcagcgacca accgtgtgaa gatttggtag aaaaattgtc ggctcgtccc
     7681 gaagctttgc atgacaacgt aacgccttta tttgtctcgc gtgcaccaaa tcgcaaaatg
     7741 tcggggcaag ggcatttgga aaccatcaaa tctgcaaaaa ggctgtctga agaaaacagt
     7801 atggttaaaa aaccattaac cacattgaaa ttaaaagata ttccagaaat cgtaggctac
     7861 ccgagtcgtg aacctcaatt gtatgccgca ttgaaaacac gtttagaaac gcatgatgat
     7921 gacccaatta aagcctttgc caaacccttt tacaaaccca ataaaaatgg tgaattgggg
     7981 gcgttggttc gatcggtgcg tgtgaaaggt gtacaaaata cgggtgtaat ggttcatgat
     8041 ggcaaaggca ttgccgataa tgccacaatg gttcgtgttg atgtctatac caaagcgggc
     8101 aaaaattacc ttgttcctgt gtatgtttgg caggtggctc aaggaatttt gccaaatcgg
     8161 gcggttactt ctggcaaaag tgaagcagat tgggatttaa ttgatgaaag ttttgaattt
     8221 aaattttcgc tgtctcgtgg ggatttagtg gaaatgatta gcaataaagg aagaattttt
     8281 ggttattaca atgggttaga tcgtgcaaat ggaagtattg ggattcgtga acatgatttg
     8341 gaaaagtcca aaggaaaaga tggtgttcat cgtgttggcg tgaaaaccgc caccgcattc
     8401 aacaaatacc acgttgaccc acttggtaaa gaaattcatc ggtgttcatc tgaaccacgc
     8461 cccacattaa aaatcaaatc caagaaataa
//
