LOCUS       sequence-245795-        8927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for inactive casX (triple mutations)--Used
            for CasX protein purification .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8927)
  AUTHORS   Liu JJ, Orlova N, Oakes BL, Ma E, Spinner HB, Baney KLM, Chuck J, 
            Tan D, Knott GJ, Harrington LB, Al-Shayeb B, Wagner A, Brotzmann J, 
            Staahl BT, Taylor KL, Desmarais J, Nogales E, Doudna JA
  TITLE     CasX enzymes comprise a distinct family of RNA-guided genome 
            editors.
  JOURNAL   Nature. 2019 Feb 4. pii: 10.1038/s41586-019-0908-x. doi: 
            10.1038/s41586-019-0908-x.
  PUBMED    30718774
REFERENCE   2  (bases 1 to 8927)
  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: "Nature. 
            2019 Feb 4. pii: 10.1038/s41586-019-0908-x. doi: 
            10.1038/s41586-019-0908-x."
COMMENT     Sequence Label: pET 2C-T10-dCasX
FEATURES             Location/Qualifiers
     source          1..8927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             108..130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             150..179
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             189..1289
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1263..1286
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1344..1364
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(4457..4475)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4471..4518
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4884..4889,4890..4906,4907..4912))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     promoter        5025..5129
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5130..5198,5199..5990)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5348..5367)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6161..6749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6650..6669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6903..6920
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6935..7074
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7000..7019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7160..7182
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7176..7367)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     8825..8844
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagtgccggc tccggagagc
       61 tctttaatta agcggccgcc ctgcaggact cgagttctag aaataatttt gtttaacttt
      121 aagaaggaga tatacatatg aaatcttctc accatcacca tcaccatcac catcaccatg
      181 gttcttctat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
      241 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
      301 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
      361 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
      421 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
      481 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
      541 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
      601 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
      661 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
      721 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
      781 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
      841 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
      901 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
      961 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     1021 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     1081 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     1141 ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     1201 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     1261 tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac aacaataaca
     1321 ataacaacaa cctcgggatc gaggaaaacc tgtacttcca atccaatgca atggagaagc
     1381 gtattaacaa gattcgtaag aagttatcgg ctgataatgc cacaaaacct gtaagccgct
     1441 caggacctat gaaaacgctt ttagttcgtg ttatgacaga tgatctgaaa aaacgtcttg
     1501 agaagcgtcg taaaaagccg gaggtcatgc ctcaagtcat ttcgaataat gccgccaata
     1561 atttacgtat gcttcttgat gactatacaa agatgaaaga agcaatcctg caagtctact
     1621 ggcaagagtt caaagatgat catgtaggtt tgatgtgtaa gtttgcacag cccgctagta
     1681 aaaagatcga ccaaaacaag ttaaagccag agatggacga gaaagggaat cttaccactg
     1741 ccgggttcgc ttgctctcaa tgcggccaac cgctgttcgt atataagctg gagcaagttt
     1801 cagaaaaggg taaggcatac acaaattatt tcggacgttg caacgtcgct gagcacgaga
     1861 agcttattct tttagcccag ctgaaaccag agaaggactc tgatgaagcg gttacctact
     1921 ccttgggaaa gttcggacag cgcgctcttg acttttattc catccacgtg acaaaggaaa
     1981 gtacacaccc cgtcaaacca ctggcccaaa ttgcagggaa ccgctacgcg agcgggccag
     2041 tgggaaaagc attgtcagac gcttgtatgg gcacgattgc ttcctttctt agtaaatacc
     2101 aggatattat cattgaacat caaaaggtag ttaaggggaa ccagaagcgt ctggaaagct
     2161 tacgtgaact tgcagggaaa gagaaccttg agtacccatc ggtcacattg cctccgcagc
     2221 cgcacacaaa ggagggtgtc gatgcttata acgaggtgat tgcacgcgta cgcatgtggg
     2281 taaatttgaa tctgtggcag aagttgaaac tgtcgcgtga tgacgccaaa cctcttttac
     2341 gcctgaaggg cttcccctca tttccagtcg tagagcgccg tgaaaatgaa gtagactggt
     2401 ggaatactat caacgaagta aaaaagctga ttgacgcgaa gcgcgacatg ggacgcgtgt
     2461 tttggagcgg cgttactgct gaaaagcgca acaccatcct tgaaggatac aactatttac
     2521 ccaacgagaa cgatcataaa aagcgcgagg gcagtttaga gaatccgaag aagccggcca
     2581 aacgtcagtt tggcgacctt ttattatacc tggaaaagaa atatgcgggt gactggggca
     2641 aagtgttcga tgaagcctgg gagcgcattg acaagaaaat cgctggtctg acgtcgcaca
     2701 ttgagcgcga ggaagctcgt aacgcagaag acgcccaatc caaagcggtc ctgacagact
     2761 ggctgcgcgc taaggcgtcc tttgttcttg agcgtttaaa agagatggac gaaaaggaat
     2821 tctatgcctg tgagattcaa ttacaaaagt ggtatggtga cttacgcggg aacccctttg
     2881 ctgtagaggc tgagaatcgt gtagtggaca tttcaggttt cagcatcgga tcggatggac
     2941 attctatcca atatcgtaac cttcttgcat ggaaatattt ggagaatggt aagcgcgagt
     3001 tctatctgct tatgaactac gggaaaaaag gacgtattcg cttcaccgat ggtacagaca
     3061 ttaaaaaaag tggcaaatgg caagggttgt tatatggggg tggcaaagca aaggttatcg
     3121 atttaacgtt tgatcccgat gatgagcagc tgattatctt gcctttagct ttcggaaccc
     3181 gccagggccg tgaatttatc tggaacgact tgctttctct tgagacagga cttatcaaac
     3241 ttgcgaatgg acgcgtgatc gaaaagacta tttacaataa aaagatcggt cgcgatgagc
     3301 cggccctgtt tgtcgcctta acattcgagc gtcgcgaagt tgttgatccg tcaaatatca
     3361 agccggtaaa cttaatcggg gtcgcccgtg gtgaaaatat cccggcagta atcgctctta
     3421 cagacccaga aggctgcccc ttacccgagt tcaaggactc atccggcggt ccaacagaca
     3481 tccttcgcat tggcgaagga tataaagaga aacagcgtgc aatccaggca gcaaaagaag
     3541 tggaacaacg tcgcgccgga ggctacagcc gcaaatttgc gtcaaaatcg cgtaacttgg
     3601 cagatgatat ggtccgcaac agcgcccgtg atttgtttta ccacgcggtg acacatgacg
     3661 ctgttttagt ttttgccaac ttgtcacgtg ggttcggacg tcagggaaaa cgcaccttca
     3721 tgactgagcg ccaatacaca aaaatggaag actggttgac ggctaaactg gcttacgagg
     3781 ggcttactag taaaacgtat ttgtcgaaaa ctcttgctca gtacacctct aaaacttgtt
     3841 ctaactgcgg gttcacgatt accacagcgg actacgatgg catgctggta cgccttaaaa
     3901 agacgagcga cgggtgggca acgacattga acaataaaga acttaaggcc gagggacaaa
     3961 tcacctacta taatcgttat aagcgccaga cggtagaaaa agagttaagc gccgaacttg
     4021 accgcctgag cgaggagagt gggaacaacg acatttcaaa atggactaag gggcgccgtg
     4081 acgaggcttt gtttttgctg aagaaacgtt tctcccatcg tcccgtacaa gagcagttcg
     4141 tttgcttaga ttgtggccat gaagttcatg ctgccgaaca ggctgcttta aatatcgcgc
     4201 gttcctggtt gtttcttaat agtaactcaa ctgagttcaa atcatataag tcagggaaac
     4261 agccgtttgt gggagcatgg caggcttttt ataaacgccg cctgaaggag gtctggaagc
     4321 cgaatgcttg ataacattgg aagtggataa cggatccgcg atcgcggcgc gccacctggt
     4381 ggccggccgg taccacgcgt gcgcgctgat ccggctgcta acaaagcccg aaaggaagct
     4441 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     4501 gtcttgaggg gttttttgct gaaaggagga actatatccg gatatccaca ggacgggtgt
     4561 ggtcgccatg atcgcgtagt cgatagtggc tccaagtagc gaagcgagca ggactgggcg
     4621 gcggccaaag cggtcggaca gtgctccgag aacgggtgcg catagaaatt gcatcaacgc
     4681 atatagcgct agcagcacgc catagtgact ggcgatgctg tcggaatgga cgatatcccg
     4741 caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt
     4801 gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca
     4861 atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga
     4921 attcttgaag acgaaagggc ctcgtgatac acctattttt ataggttaat gtcatgataa
     4981 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5041 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5101 tgcttcaata acattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5161 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5221 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5281 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5341 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     5401 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5461 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5521 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5581 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5641 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     5701 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5761 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5821 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5881 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5941 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6001 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6061 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6121 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6181 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6241 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6301 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6361 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6421 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6481 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6541 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6601 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6661 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6721 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6781 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6841 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     6901 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     6961 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     7021 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     7081 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     7141 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     7201 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     7261 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     7321 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     7381 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     7441 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     7501 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     7561 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     7621 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     7681 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     7741 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     7801 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg
     7861 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt
     7921 tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg
     7981 ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
     8041 gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
     8101 atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta
     8161 ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc
     8221 tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa
     8281 tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg
     8341 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     8401 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     8461 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     8521 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     8581 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga cgctctccct
     8641 tatgtgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     8701 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     8761 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     8821 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     8881 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
