LOCUS       sequence-359724-        8927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing E. coli codon optimized His-MBP-TEV-DpbCasX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8927)
  AUTHORS   Tsuchida CA, Zhang S, Doost MS, Zhao Y, Wang J, O'Brien E, Fang H, 
            Li CP, Li D, Hai ZY, Chuck J, Brotzmann J, Vartoumian A, Burstein D,
            Chen XW, Nogales E, Doudna JA, Liu JG
  TITLE     Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome 
            editing activity.
  JOURNAL   Mol Cell. 2022 Mar 17;82(6):1199-1209.e6. doi: 
            10.1016/j.molcel.2022.02.002. Epub 2022 Feb 25.
  PUBMED    35219382
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"; doi: 
            "10.1016/j.molcel.2022.02.002"; journalName: "Mol Cell"; date: 
            "2022-03-17- 17"; volume: "82"; issue: "6"; pages: "1199-1209.e6"
COMMENT     Sequence Label: pJJGL001
FEATURES             Location/Qualifiers
     source          1..8927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1578..1597
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1681..1700
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1681..1699
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1788..1810
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1830..1859
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1869..2969
                     /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     2943..2966
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3024..3044
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6137..6155)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6151..6198
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6564..6569,6570..6586,6587..6592))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6619..6637)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6705..6809
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6810..6878,6879..7670)
                     /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(7028..7047)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7841..8429
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8330..8349
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8583..8600
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8615..8754
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8680..8699)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8840..8862
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(8856..8927,1..120))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
       61 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
      121 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
      181 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
      241 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
      301 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
      361 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
      421 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
      481 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
      541 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
      601 gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg
      661 ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg
      721 agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc
      781 catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat
      841 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
      901 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
      961 tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg
     1021 agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag
     1081 cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     1141 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     1201 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     1261 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     1321 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     1381 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     1441 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     1501 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1561 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1621 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     1681 taatacgact cactataggg agaccacaac ggtttccctc tagtgccggc tccggagagc
     1741 tctttaatta agcggccgcc ctgcaggact cgagttctag aaataatttt gtttaacttt
     1801 aagaaggaga tatacatatg aaatcttctc accatcacca tcaccatcac catcaccatg
     1861 gttcttctat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
     1921 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
     1981 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
     2041 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
     2101 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
     2161 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
     2221 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
     2281 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
     2341 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
     2401 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
     2461 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
     2521 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
     2581 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
     2641 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     2701 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     2761 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     2821 ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     2881 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     2941 tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac aacaataaca
     3001 ataacaacaa cctcgggatc gaggaaaacc tgtacttcca atccaatgca atggagaagc
     3061 gtattaacaa gattcgtaag aagttatcgg ctgataatgc cacaaaacct gtaagccgct
     3121 caggacctat gaaaacgctt ttagttcgtg ttatgacaga tgatctgaaa aaacgtcttg
     3181 agaagcgtcg taaaaagccg gaggtcatgc ctcaagtcat ttcgaataat gccgccaata
     3241 atttacgtat gcttcttgat gactatacaa agatgaaaga agcaatcctg caagtctact
     3301 ggcaagagtt caaagatgat catgtaggtt tgatgtgtaa gtttgcacag cccgctagta
     3361 aaaagatcga ccaaaacaag ttaaagccag agatggacga gaaagggaat cttaccactg
     3421 ccgggttcgc ttgctctcaa tgcggccaac cgctgttcgt atataagctg gagcaagttt
     3481 cagaaaaggg taaggcatac acaaattatt tcggacgttg caacgtcgct gagcacgaga
     3541 agcttattct tttagcccag ctgaaaccag agaaggactc tgatgaagcg gttacctact
     3601 ccttgggaaa gttcggacag cgcgctcttg acttttattc catccacgtg acaaaggaaa
     3661 gtacacaccc cgtcaaacca ctggcccaaa ttgcagggaa ccgctacgcg agcgggccag
     3721 tgggaaaagc attgtcagac gcttgtatgg gcacgattgc ttcctttctt agtaaatacc
     3781 aggatattat cattgaacat caaaaggtag ttaaggggaa ccagaagcgt ctggaaagct
     3841 tacgtgaact tgcagggaaa gagaaccttg agtacccatc ggtcacattg cctccgcagc
     3901 cgcacacaaa ggagggtgtc gatgcttata acgaggtgat tgcacgcgta cgcatgtggg
     3961 taaatttgaa tctgtggcag aagttgaaac tgtcgcgtga tgacgccaaa cctcttttac
     4021 gcctgaaggg cttcccctca tttccagtcg tagagcgccg tgaaaatgaa gtagactggt
     4081 ggaatactat caacgaagta aaaaagctga ttgacgcgaa gcgcgacatg ggacgcgtgt
     4141 tttggagcgg cgttactgct gaaaagcgca acaccatcct tgaaggatac aactatttac
     4201 ccaacgagaa cgatcataaa aagcgcgagg gcagtttaga gaatccgaag aagccggcca
     4261 aacgtcagtt tggcgacctt ttattatacc tggaaaagaa atatgcgggt gactggggca
     4321 aagtgttcga tgaagcctgg gagcgcattg acaagaaaat cgctggtctg acgtcgcaca
     4381 ttgagcgcga ggaagctcgt aacgcagaag acgcccaatc caaagcggtc ctgacagact
     4441 ggctgcgcgc taaggcgtcc tttgttcttg agcgtttaaa agagatggac gaaaaggaat
     4501 tctatgcctg tgagattcaa ttacaaaagt ggtatggtga cttacgcggg aacccctttg
     4561 ctgtagaggc tgagaatcgt gtagtggaca tttcaggttt cagcatcgga tcggatggac
     4621 attctatcca atatcgtaac cttcttgcat ggaaatattt ggagaatggt aagcgcgagt
     4681 tctatctgct tatgaactac gggaaaaaag gacgtattcg cttcaccgat ggtacagaca
     4741 ttaaaaaaag tggcaaatgg caagggttgt tatatggggg tggcaaagca aaggttatcg
     4801 atttaacgtt tgatcccgat gatgagcagc tgattatctt gcctttagct ttcggaaccc
     4861 gccagggccg tgaatttatc tggaacgact tgctttctct tgagacagga cttatcaaac
     4921 ttgcgaatgg acgcgtgatc gaaaagacta tttacaataa aaagatcggt cgcgatgagc
     4981 cggccctgtt tgtcgcctta acattcgagc gtcgcgaagt tgttgatccg tcaaatatca
     5041 agccggtaaa cttaatcggg gtcgatcgtg gtgaaaatat cccggcagta atcgctctta
     5101 cagacccaga aggctgcccc ttacccgagt tcaaggactc atccggcggt ccaacagaca
     5161 tccttcgcat tggcgaagga tataaagaga aacagcgtgc aatccaggca gcaaaagaag
     5221 tggaacaacg tcgcgccgga ggctacagcc gcaaatttgc gtcaaaatcg cgtaacttgg
     5281 cagatgatat ggtccgcaac agcgcccgtg atttgtttta ccacgcggtg acacatgacg
     5341 ctgttttagt ttttgagaac ttgtcacgtg ggttcggacg tcagggaaaa cgcaccttca
     5401 tgactgagcg ccaatacaca aaaatggaag actggttgac ggctaaactg gcttacgagg
     5461 ggcttactag taaaacgtat ttgtcgaaaa ctcttgctca gtacacctct aaaacttgtt
     5521 ctaactgcgg gttcacgatt accacagcgg actacgatgg catgctggta cgccttaaaa
     5581 agacgagcga cgggtgggca acgacattga acaataaaga acttaaggcc gagggacaaa
     5641 tcacctacta taatcgttat aagcgccaga cggtagaaaa agagttaagc gccgaacttg
     5701 accgcctgag cgaggagagt gggaacaacg acatttcaaa atggactaag gggcgccgtg
     5761 acgaggcttt gtttttgctg aagaaacgtt tctcccatcg tcccgtacaa gagcagttcg
     5821 tttgcttaga ttgtggccat gaagttcatg ctgatgaaca ggctgcttta aatatcgcgc
     5881 gttcctggtt gtttcttaat agtaactcaa ctgagttcaa atcatataag tcagggaaac
     5941 agccgtttgt gggagcatgg caggcttttt ataaacgccg cctgaaggag gtctggaagc
     6001 cgaatgcttg ataacattgg aagtggataa cggatccgcg atcgcggcgc gccacctggt
     6061 ggccggccgg taccacgcgt gcgcgctgat ccggctgcta acaaagcccg aaaggaagct
     6121 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     6181 gtcttgaggg gttttttgct gaaaggagga actatatccg gatatccaca ggacgggtgt
     6241 ggtcgccatg atcgcgtagt cgatagtggc tccaagtagc gaagcgagca ggactgggcg
     6301 gcggccaaag cggtcggaca gtgctccgag aacgggtgcg catagaaatt gcatcaacgc
     6361 atatagcgct agcagcacgc catagtgact ggcgatgctg tcggaatgga cgatatcccg
     6421 caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt
     6481 gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca
     6541 atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga
     6601 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     6661 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     6721 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     6781 tgcttcaata acattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     6841 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     6901 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     6961 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     7021 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     7081 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     7141 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     7201 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     7261 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     7321 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     7381 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     7441 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     7501 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     7561 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     7621 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     7681 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     7741 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     7801 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     7861 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     7921 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     7981 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     8041 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     8101 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     8161 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     8221 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     8281 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     8341 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     8401 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     8461 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     8521 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     8581 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     8641 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     8701 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     8761 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     8821 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     8881 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaag
//
