LOCUS       sequence-359723-        8906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing E. coli codon optimized His-MBP-TEV-PlmCasX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8906)
  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 8906)
  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: pJJGL002
FEATURES             Location/Qualifiers
     source          1..8906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(61..66,67..83,84..89))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(116..134)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        202..306
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(307..375,376..1167)
                     /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(525..544)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1338..1926
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1827..1846
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2080..2097
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2112..2251
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2177..2196)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2337..2359
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2353..2544)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4002..4021
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4105..4124
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4105..4123
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4212..4234
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4254..4283
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4293..5393
                     /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     5367..5390
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5448..5468
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(8540..8558)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8554..8601
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc
       61 attaaagctt atcgatgata agctgtcaaa catgagaatt cttgaagacg aaagggcctc
      121 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
      181 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
      241 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataaca ttgaaaaagg
      301 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
      361 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
      421 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
      481 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      541 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
      601 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
      661 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
      721 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
      781 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
      841 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
      901 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
      961 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     1021 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     1081 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     1141 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     1201 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1261 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1321 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1381 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1441 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     1501 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1561 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1621 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1681 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1741 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1801 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     1861 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1921 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     1981 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2041 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2101 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2161 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     2221 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     2281 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     2341 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     2401 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     2461 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     2521 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     2581 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     2641 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     2701 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     2761 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     2821 ccgagtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     2881 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     2941 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     3001 gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat
     3061 ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa
     3121 gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc
     3181 attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg
     3241 tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa
     3301 atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat
     3361 ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg
     3421 ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc
     3481 gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc
     3541 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     3601 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     3661 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     3721 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     3781 ttgaaggctc tcaagggcat cggtcgacgc tctcccttat gcgactcctg cattaggaag
     3841 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     3901 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     3961 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     4021 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     4081 atcgagatct cgatcccgcg aaattaatac gactcactat agggagacca caacggtttc
     4141 cctctagtgc cggctccgga gagctcttta attaagcggc cgccctgcag gactcgagtt
     4201 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgaaatct tctcaccatc
     4261 accatcacca tcaccatcac catggttctt ctatgaaaat cgaagaaggt aaactggtaa
     4321 tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga
     4381 aagataccgg aattaaagtc accgttgagc atccggataa actggaagag aaattcccac
     4441 aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg
     4501 gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc
     4561 tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg
     4621 ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct
     4681 gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt
     4741 tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt
     4801 tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga
     4861 aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg
     4921 attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc
     4981 cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga
     5041 ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg
     5101 ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag
     5161 gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg
     5221 aagagttggc gaaagatcca cgtattgccg ccactatgga aaacgcccag aaaggtgaaa
     5281 tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca
     5341 acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actaattcga
     5401 gctcgaacaa caacaacaat aacaataaca acaacctcgg gatcgaggaa aacctgtact
     5461 tccaatccaa tgcaatgcag gaaataaaaa ggataaataa aatacgaagg agattggtaa
     5521 aggatagcaa cacgaaaaaa gccggcaaaa ccggccctat gaaaaccttg ctcgttcggg
     5581 ttatgacacc tgacctgaga gaaaggttag agaatcttcg caaaaagccg gaaaacattc
     5641 ctcagcccat ttcaaatact tcacgtgcaa atttaaataa actcctcact gactatacgg
     5701 aaatgaagaa agcaatcctg catgtttatt gggaagagtt ccaaaaagac cctgtcggat
     5761 tgatgagcag ggttgcacaa ccagcgccca agaatattga tcagagaaaa ttgattccgg
     5821 tgaaggacgg aaatgagaga ctaacaagtt ctggatttgc ctgttctcag tgctgtcaac
     5881 ccctctatgt ttataagctt gaacaagtga atgacaaggg taagccccat acaaattact
     5941 ttggccgttg taatgtctcc gagcatgaac gtttgatatt gctctcgccg cataaaccgg
     6001 aggcaaatga cgagctagta acgtattcgt tggggaagtt cggtcaaagg gcattggact
     6061 tttattcaat ccacgtaaca agagaatcga accatcctgt aaagccgcta gaacagatcg
     6121 gtggcaatag ctgcgcaagt ggtcccgttg gtaaggcttt atctgatgcc tgtatgggag
     6181 cagtagccag tttccttaca aagtaccagg acatcatcct cgaacaccaa aaggttataa
     6241 aaaaaaacga aaagagattg gcaaatctaa aggatatagc aagtgcaaac gggcttgcat
     6301 ttcctaaaat cactcttcca ccgcaaccgc atacaaaaga agggattgaa gcttataaca
     6361 atgttgttgc tcagatagtg atctgggtaa acctgaatct ttggcagaaa ctcaaaattg
     6421 gcagggatga ggcaaagccc ttacagcggc ttaagggttt tccgtccttc cctcttgttg
     6481 aacgccaggc gaatgaggtt gattggtggg atatggtctg taatgtcaaa aagttgatta
     6541 acgaaaagaa agaggacggg aaggtcttct ggcaaaatct tgctggatat aaaaggcagg
     6601 aagccttgct tccatatctt tcgtctgaag aagaccgtaa aaaaggaaaa aagtttgcgc
     6661 gttatcagtt tggtgacctt ttgcttcacc ttgaaaagaa acacggtgaa gattggggca
     6721 aagtttatga tgaggcatgg gaaagaatag ataaaaaagt tgaaggtctg agtaagcaca
     6781 taaagttgga ggaagaaaga aggtctgaag atgctcaatc aaaggctgcc ctcactgatt
     6841 ggctcagggc aaaggcctct tttgttattg aagggctcaa agaagctgat aaggatgagt
     6901 tttgcaggtg tgagttaaag cttcaaaagt ggtatggaga tttgagagga aaaccatttg
     6961 ctatagaagc agagaacagc attttagata taagcggatt ttctaaacag tataattgtg
     7021 catttatatg gcagaaagac ggcgtaaaga agttaaatct ttatttaata ataaattact
     7081 tcaaaggtgg taagctacgc ttcaaaaaaa tcaagccaga agcttttgaa gcaaataggt
     7141 tttatacagt aattaataaa aaaagcggtg agattgtgcc tatggaggtc aacttcaatt
     7201 ttgatgaccc gaatttgata attctgcctt tggcctttgg aaaaaggcag gggagggagt
     7261 ttatctggaa cgacctattg agccttgaga cgggttcatt gaaactcgcc aatggcaggg
     7321 ttattgaaaa aacgctctat aacagaagga cgagacagga tgaaccagca ctttttgttg
     7381 ccctgacatt tgaaagaaga gaggtgcttg actcatcgaa tataaaaccg atgaatctga
     7441 taggaataga ccggggagaa aatatcccgg cagtcatagc attaacagac ccggaaggat
     7501 gccccttgtc aagattcaaa gattcattgg gcaatccaac gcatattttg cgaataggag
     7561 aaagttataa ggaaaaacaa cggactattc aggctgctaa agaagttgaa caaaggcggg
     7621 caggcggata ttcgagaaaa tatgcatcaa aggcgaagaa tctggcggac gatatggtaa
     7681 gaaatacagc tcgtgacctc ttatattatg ctgttactca agatgcaatg ctcatttttg
     7741 aaaatctttc ccgcggtttt ggtagacaag gcaagaggac ttttatggcg gaaaggcagt
     7801 acacgaggat ggaagactgg ctgactgcaa agcttgccta tgaaggtctg ccatcaaaaa
     7861 cctatctttc aaagactctg gcacagtata cctcaaagac atgttctaat tgtggtttta
     7921 caatcacaag tgcagattat gacagggtgc tcgaaaagct caagaagacg gctactggat
     7981 ggatgactac aatcaatgga aaagagttaa aagttgaagg acagataaca tactataacc
     8041 ggtataaaag gcagaatgtg gtaaaagacc tctctgtaga gctggataga ctttcggaag
     8101 agtcggtaaa taatgatatt tctagttgga caaaaggccg cagtggtgaa gctttatctc
     8161 tgctaaaaaa gagatttagt cacaggccgg tgcaggaaaa gtttgtttgc ctgaactgtg
     8221 gttttgaaac ccatgcagac gaacaagcag cactgaatat tgcaaggtcg tggctctttc
     8281 tccgttctca agaatataag aagtatcaaa ccaataaaac gaccggaaat actgacaaaa
     8341 gggcatttgt tgaaacatgg caatcctttt acagaaagaa gctcaaagaa gtatggaaac
     8401 cagccgtctg ataataacat tggaagtgga taacggatcc gcgatcgcgg cgcgccacct
     8461 ggtggccggc cggtaccacg cgtgcgcgct gatccggctg ctaacaaagc ccgaaaggaa
     8521 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     8581 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc acaggacggg
     8641 tgtggtcgcc atgatcgcgt agtcgatagt ggctccaagt agcgaagcga gcaggactgg
     8701 gcggcggcca aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa attgcatcaa
     8761 cgcatatagc gctagcagca cgccatagtg actggcgatg ctgtcggaat ggacgatatc
     8821 ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac
     8881 ggtgccgagg atgacgatga gcgcat
//
