LOCUS       sequence-338133-        8299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses catalytically inactive Campylobacter jejuni Cas9 
            (CjedCas9) in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8299)
  AUTHORS   Saha C, Mohanraju P, Stubbs A, Dugar G, Hoogstrate Y, Kremers GJ, 
            van Cappellen WA, Horst-Kreft D, Laffeber C, Lebbink JHG, Bruens S, 
            Gaskin D, Beerens D, Klunder M, Joosten R, Demmers JAA, van Gent D, 
            Mouton JW, van der Spek PJ, van der Oost J, van Baarlen P, Louwen R
  TITLE     Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes
            severe damage to DNA.
  JOURNAL   Sci Adv. 2020 Jun 17;6(25):eaaz4849. doi: 10.1126/sciadv.aaz4849. 
            eCollection 2020 Jun.
  PUBMED    32596446
REFERENCE   2  (bases 1 to 8299)
  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: "Sci Adv."; 
            date: "2020-06-17"; volume: "6(25):eaaz4849. doi"; pages: " 
            10.1126/sciadv.aaz4849. eCollection 2020 Jun"
COMMENT     Sequence Label: pCjedCas9
FEATURES             Location/Qualifiers
     source          1..8299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     964..983
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1003..1080)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1286..1305
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1389..1408
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1389..1407
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1408..1432
                     /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             1447..1469
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1488..1505
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1506..1526
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4551..4568
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4621..4639)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4635..4682
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4719..5174
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4806..4825)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5016..5037
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5267..6082)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5267..6082)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5990..6009
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6074..6093
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6204..6792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6693..6712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6946..6963
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6978..7117
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7043..7062)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7203..7225
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7219..7410)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(8219..8299,1..1002))
                     /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"
ORIGIN
        1 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
       61 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
      121 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
      181 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
      241 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgataccctc
      301 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
      361 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
      421 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
      481 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
      541 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
      601 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
      661 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
      721 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
      781 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
      841 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
      901 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
      961 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     1021 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     1081 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     1141 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     1201 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     1261 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     1321 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     1381 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     1441 aataattttg tttaacttta agaaggagat ataccatggg ttcttctcac catcaccatc
     1501 accatgaaaa cctgtacttc caatccaatg cagcaagaat tttggcattt gcgataggaa
     1561 taagctctat tggctgggca tttagtgaaa atgatgagtt aaaagattgt ggagttagga
     1621 tttttacaaa ggttgaaaat cccaaaacag gagaatcctt agccttgcca agacgccttg
     1681 caagaagtgc tagaaaacgc cttgctagac gcaaagcaag gctaaatcac ttaaaacatt
     1741 taatcgcaaa tgaatttaag ttaaattacg aagattatca aagctttgat gaaagtttag
     1801 ccaaggctta taaaggatct ttaattagcc cttatgagtt aagatttaga gctttaaacg
     1861 agcttttaag taagcaagat tttgcaagag tgattttaca tattgcaaaa aggcgtggct
     1921 atgatgatat taaaaatagt gatgataaag aaaaaggtgc gattttaaaa gctatcaagc
     1981 aaaatgaaga aaaactagca aattatcaaa gtgtgggcga gtatctttat aaagagtatt
     2041 ttcaaaaatt caaagaaaat tccaaagaat ttacaaatgt tagaaataaa aaagaaagtt
     2101 atgaacgctg catagcgcaa agttttttaa aagatgaatt aaagcttatt tttaaaaaac
     2161 aaagagaatt tggcttttct ttttcaaaaa aatttgaaga agaggtgctt agtgtagcat
     2221 tttataaaag ggctttaaaa gatttttctc atttggtggg aaattgtagt ttttttaccg
     2281 atgaaaaaag agcgccaaaa aattcgccct tagcttttat gtttgtagca cttacaagaa
     2341 ttatcaatct tttaaacaat cttaaaaata ctgaaggaat tttatataca aaagatgatt
     2401 taaacgccct tttaaatgaa gttttaaaaa acggcactct aacttataaa caaacaaaaa
     2461 agcttttggg cttaagcgat gattatgaat ttaaaggaga aaaaggaact tattttattg
     2521 agtttaaaaa atacaaagaa tttataaaag ccctaggtga gcataattta agccaagatg
     2581 atttaaatga aatagcaaaa gatattactc ttataaaaga tgagattaaa cttaaaaagg
     2641 ctttagcaaa atatgattta aatcaaaatc aaattgatag tttaagcaaa ttggaattta
     2701 aagaccattt aaatataagt tttaaagcct taaaacttgt cactccttta atgcttgaag
     2761 gtaaaaaata cgatgaagct tgcaatgagc ttaatttaaa agtagcaatc aatgaagata
     2821 aaaaagactt tttacccgca tttaatgaaa cttattacaa agatgaagta actaatcctg
     2881 tggttttaag ggcgattaaa gaatatagaa aggtcttaaa tgccttactt aaaaaatacg
     2941 gtaaagtgca taaaataaac attgaattag caagagaagt aggtaaaaat cacagccaaa
     3001 gagctaaaat agaaaaagag caaaacgaaa actacaaggc caaaaaagac gccgaacttg
     3061 aatgtgaaaa actaggctta aaaataaata gcaaaaatat tttaaaatta agattattta
     3121 aagaacaaaa agaattttgt gcttatagtg gagaaaagat taaaattagt gatttgcaag
     3181 atgaaaaaat gcttgaaatt gatgcgattt acccttattc tagaagtttt gatgattctt
     3241 atatgaataa agttttagtt tttaccaagc aaaatcaaga aaaacttaat caaaccccct
     3301 ttgaagcctt tggcaatgat agtgcaaaat ggcagaaaat tgaagtttta gcaaaaaatt
     3361 tgcctactaa aaagcaaaaa agaattttag ataaaaatta caaagacaaa gagcaaaaaa
     3421 actttaaaga tagaaatcta aacgatactc gctacatagc aaggcttgtt ttaaactaca
     3481 ctaaagatta tttagatttt ttacctttaa gcgatgatga aaatacaaaa ttaaacgata
     3541 cacaaaaagg ctcaaaagtg catgtagaag caaaaagcgg tatgcttaca tcggctttaa
     3601 gacacacttg gggctttagc gctaaggata gaaataacca tcttcatcac gccatcgatg
     3661 cagtaattat tgcttatgca aacaactcta tcgtaaaggc tttttcagat tttaaaaaag
     3721 agcaagaaag caacagtgca gaactttatg caaaaaaaat tagtgaatta gattataaaa
     3781 acaaaaggaa gttttttgag ccttttagtg gttttaggca aaaggtttta gataaaattg
     3841 atgaaatttt tgtctcaaaa cctgagagaa aaaagccaag tggagcttta catgaagaaa
     3901 catttagaaa agaagaagaa ttctatcaat catacggcgg aaaagaagga gttttaaaag
     3961 ccctagagct tggtaaaatc cgcaaagtaa atggtaaaat agttaaaaat ggcgatatgt
     4021 ttagagtgga tatttttaaa cacaaaaaga caaataaatt ttatgctgtg ccaatttata
     4081 ctatggattt tgctttaaaa gtattaccaa ataaagctgt agcacgaagt aaaaaaggtg
     4141 agataaaaga ctggatttta atggatgaaa attatgagtt ttgcttttct ttatataaag
     4201 atagtttgat tttaattcaa actaaagata tgcaagagcc agaatttgtt tattataatg
     4261 cttttacaag ctctactgtt agtttgattg tttcaaaaca cgataataaa tttgaaacgc
     4321 taagcaaaaa tcaaaaaata ttgtttaaaa atgcaaatga aaaagaagta atcgcaaaaa
     4381 gcataggaat tcaaaatcta aaagtatttg aaaaatacat agtttcagca cttggggaag
     4441 ttacaaaggc tgagtttaga caaagagaag attttaaaaa ataataacat tggaagtgga
     4501 taacggatcc gaattcgagc gccgtcgaca agcttgcggc cgcactcgag caccaccacc
     4561 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     4621 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     4681 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     4741 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4801 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4861 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4921 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4981 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5041 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5101 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaactagt
     5161 aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5221 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     5281 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     5341 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     5401 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     5461 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     5521 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     5581 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     5641 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     5701 ggaacactgc cagcgcatca acaatgtttt cacctgaatc aggatattct tctaatacct
     5761 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     5821 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     5881 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     5941 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     6001 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     6061 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     6121 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6181 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6241 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6301 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     6361 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6421 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6481 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6541 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6601 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6661 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6721 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6781 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6841 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6901 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6961 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     7021 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     7081 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     7141 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     7201 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     7261 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     7321 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     7381 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     7441 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     7501 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     7561 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     7621 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     7681 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     7741 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     7801 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     7861 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     7921 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     7981 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     8041 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     8101 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     8161 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     8221 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     8281 cgcggggaga ggcggtttg
//
