LOCUS       sequence-359722-        8942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing E. coli codon optimized 
            His-MBP-TEV-DpbCasX+Region3 insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8942)
  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 8942)
  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: pJJGL003
FEATURES             Location/Qualifiers
     source          1..8942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     500..519
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     603..622
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        603..621
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             710..732
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             752..781
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             791..1891
                     /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     1865..1888
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1946..1966
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5074..5092)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5088..5135
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5501..5506,5507..5523,5524..5529))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5556..5574)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5642..5746
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5747..5815,5816..6607)
                     /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(5965..5984)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6778..7366
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7267..7286
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7520..7537
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7552..7691
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7617..7636)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7777..7799
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7793..7984)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
       61 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
      121 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
      181 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
      241 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
      301 gtcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
      361 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
      421 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
      481 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
      541 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
      601 attaatacga ctcactatag ggagaccaca acggtttccc tctagtgccg gctccggaga
      661 gctctttaat taagcggccg ccctgcagga ctcgagttct agaaataatt ttgtttaact
      721 ttaagaagga gatatacata tgaaatcttc tcaccatcac catcaccatc accatcacca
      781 tggttcttct atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg
      841 ctataacggt ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac
      901 cgttgagcat ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg
      961 ccctgacatt atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt
     1021 ggctgaaatc accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc
     1081 cgtacgttac aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat
     1141 ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga
     1201 taaagaactg aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt
     1261 cacctggccg ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta
     1321 cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt
     1381 tgacctgatt aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc
     1441 ctttaataaa ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga
     1501 caccagcaaa gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa
     1561 accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc
     1621 aaaagagttc ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga
     1681 caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg
     1741 tattgccgcc actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat
     1801 gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac
     1861 tgtcgatgaa gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa
     1921 caataacaac aacctcggga tcgaggaaaa cctgtacttc caatccaatg caatggagaa
     1981 gcgtattaac aagattcgta agaagttatc ggctgataat gccacaaaac ctgtaagccg
     2041 ctcaggacct atgaaaacgc ttttagttcg tgttatgaca gatgatctga aaaaacgtct
     2101 tgagaagcgt cgtaaaaagc cggaggtcat gcctcaagtc atttcgaata atgccgccaa
     2161 taatttacgt atgcttcttg atgactatac aaagatgaaa gaagcaatcc tgcaagtcta
     2221 ctggcaagag ttcaaagatg atcatgtagg tttgatgtgt aagtttgcac agcccgctag
     2281 taaaaagatc gaccaaaaca agttaaagcc agagatggac gagaaaggga atcttaccac
     2341 tgccgggttc gcttgctctc aatgcggcca accgctgttc gtatataagc tggagcaagt
     2401 ttcagaaaag ggtaaggcat acacaaatta tttcggacgt tgcaacgtcg ctgagcacga
     2461 gaagcttatt cttttagccc agctgaaacc agagaaggac tctgatgaag cggttaccta
     2521 ctccttggga aagttcggac agcgcgctct tgacttttat tccatccacg tgacaaagga
     2581 aagtacacac cccgtcaaac cactggccca aattgcaggg aaccgctacg cgagcgggcc
     2641 agtgggaaaa gcattgtcag acgcttgtat gggcacgatt gcttcctttc ttagtaaata
     2701 ccaggatatt atcattgaac atcaaaaggt agttaagggg aaccagaagc gtctggaaag
     2761 cttacgtgaa cttgcaggga aagagaacct tgagtaccca tcggtcacat tgcctccgca
     2821 gccgcacaca aaggagggtg tcgatgctta taacgaggtg attgcacgcg tacgcatgtg
     2881 ggtaaatttg aatctgtggc agaagttgaa actgtcgcgt gatgacgcca aacctctttt
     2941 acgcctgaag ggcttcccct catttccagt cgtagagcgc cgtgaaaatg aagtagactg
     3001 gtggaatact atcaacgaag taaaaaagct gattgacgcg aagcgcgaca tgggacgcgt
     3061 gttttggagc ggcgttactg ctgaaaagcg caacaccatc cttgaaggat acaactattt
     3121 acccaacgag aacgatcata aaaagcgcga gggcagttta gagaatccga agaagccggc
     3181 caaacgtcag tttggcgacc ttttattata cctggaaaag aaatatgcgg gtgactgggg
     3241 caaagtgttc gatgaagcct gggagcgcat tgacaagaaa atcgctggtc tgacgtcgca
     3301 cattgagcgc gaggaagctc gtaacgcaga agacgcccaa tccaaagcgg tcctgacaga
     3361 ctggctgcgc gctaaggcgt cctttgttct tgagcgttta aaagagatgg acgaaaagga
     3421 attctatgcc tgtgagattc aattacaaaa gtggtatggt gacttacgcg ggaacccctt
     3481 tgctgtagag gctgagaatc gtgtagtgga catttcaggt ttcagcatcg gatcggatgg
     3541 acattctatc caatatcgta accttcttgc atggaaatat ttggagaatg gtaagcgcga
     3601 gttctatctg cttatgaact acgggaaaaa aggacgtatt cgcttcaccg atggtacaga
     3661 cattaaaaaa agtggcaaat ggcaagggtt gttatatggg ggtggcaaag caaaggttat
     3721 cgatttaacg tttgatcccg atgatgagca gctgattatc ttgcctttag ctttcggaac
     3781 ccgccagggc cgtgaattta tctggaacga cttgctttct cttgagacag gacttatcaa
     3841 acttgcgaat ggacgcgtga tcgaaaagac tatttacaat aaaaagatcg gtcgcgatga
     3901 gccggccctg tttgtcgcct taacattcga gcgtcgcgaa gttgttgatc cgtcaaatat
     3961 caagccggta aacttaatcg gggtcgatcg tggtgaaaat atcccggcag taatcgctct
     4021 tacagaccca gaaggctgcc ccttacccga gttcaaggac tcatccggcg gtccaacaga
     4081 catccttcgc attggcgaag gatataaaga gaaacagcgt gcaatccagg cagcaaaaga
     4141 agtggaacaa cgtcgcgccg gaggctacag ccgcaaattt gcgtcaaaat cgcgtaactt
     4201 ggcagatgat atggtccgca acagcgcccg tgatttgttt taccacgcgg tgacacatga
     4261 cgctgtttta gtttttgaga acttgtcacg tgggttcgga cgtcagggaa aacgcacctt
     4321 catgactgag cgccaataca caaaaatgga agactggttg acggctaaac tggcttacga
     4381 ggggcttact agtaaaacgt atttgtcgaa aactcttgct cagtacacct ctaaaacttg
     4441 ttctaactgc gggttcacga ttaccacagc ggactacgat ggcatgctgg tacgccttaa
     4501 aaagacgagc gacgggtggg caacgacatt gaacaataaa gaacttaagg ccgagggaca
     4561 aatcacctac tataatcgtt ataagcgcca gacggtagaa aaagagttaa gcgccgaact
     4621 tgaccgcctg agcgaggaga gtgggaacaa cgacatttca aaatggacta aggggcgccg
     4681 tgacgaggct ttgtttttgc tgaagaaacg tttctcccat cgtcccgtac aagagcagtt
     4741 cgtttgctta gattgtggcc atgaagttca tgctgatgaa caggctgctt taaatatcgc
     4801 gcgttcctgg ttgtttcttc gttctcaaga atataagaag tatcaaacca ataaaacgac
     4861 cggaaatact gacaaaaggg catttgttga aacatggcaa tccttttata aacgccgcct
     4921 gaaggaggtc tggaagccga atgcttgata acattggaag tggataacgg atccgcgatc
     4981 gcggcgcgcc acctggtggc cggccggtac cacgcgtgcg cgctgatccg gctgctaaca
     5041 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     5101 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     5161 atccacagga cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa
     5221 gcgagcagga ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat
     5281 agaaattgca tcaacgcata tagcgctagc agcacgccat agtgactggc gatgctgtcg
     5341 gaatggacga tatcccgcaa gaggcccggc agtaccggca taaccaagcc tatgcctaca
     5401 gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt catacacggt
     5461 gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt atcgatgata
     5521 agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     5581 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5641 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5701 acaataaccc tgataaatgc ttcaataaca ttgaaaaagg aagagtatga gtattcaaca
     5761 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5821 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5881 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5941 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     6001 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6061 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6121 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6181 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6241 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     6301 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6361 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6421 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6481 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6541 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6601 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6661 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6721 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6781 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6841 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6901 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     6961 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     7021 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     7081 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7141 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7201 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7261 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7321 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7381 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     7441 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     7501 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     7561 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     7621 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     7681 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     7741 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     7801 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     7861 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     7921 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     7981 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     8041 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     8101 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     8161 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     8221 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     8281 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     8341 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     8401 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggccaggacc
     8461 caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg atggatatgt
     8521 tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg gctccaattc
     8581 ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg
     8641 gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg cgcctacaat
     8701 ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga cgatcagcgg
     8761 tccaatgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct gtccctgatg
     8821 gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga tgccgccgga
     8881 agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac
     8941 gt
//
