LOCUS       sequence-335050-        9251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of Cas12i1 D647A (RuvC-inhibiting mutation)
            with an N-terminal His-MBP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9251)
  AUTHORS   Cofsky JC, Karandur D, Huang CJ, Witte IP, Kuriyan J, Doudna JA
  TITLE     CRISPR-Cas12a exploits R-loop asymmetry to form double-strand 
            breaks.
  JOURNAL   Elife. 2020 Jun 10;9. pii: 55143. doi: 10.7554/eLife.55143.
  PUBMED    32519675
REFERENCE   2  (bases 1 to 9251)
  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: "Elife."; 
            date: "2020-06-10"; volume: "9. pii: 55143. doi"; pages: " 
            10.7554/eLife.55143"
COMMENT     Sequence Label: pJCC_084 Cas12i1 D647A
FEATURES             Location/Qualifiers
     source          1..9251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(55..73)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        141..245
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(246..314,315..1106)
                     /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(464..483)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1277..1865
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1766..1785
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2019..2036
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2051..2190
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2116..2135)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2276..2298
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2292..2483)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3941..3960
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4044..4063
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4044..4062
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4151..4173
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4193..4222
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4232..5332
                     /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     5306..5329
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5387..5407
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(8824..8842)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8838..8885
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(9251,1..5,6..22,23..28))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttaaagctta tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg
       61 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
      121 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
      181 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataacat tgaaaaagga
      241 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      301 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      361 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      421 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      481 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      541 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      601 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      661 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      721 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      781 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      841 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      901 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      961 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     1021 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     1081 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1141 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1201 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1261 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1321 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1381 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1441 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1501 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1561 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1621 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1681 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1741 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1801 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1861 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1921 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1981 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2041 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2101 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     2161 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     2221 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     2281 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     2341 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     2401 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     2461 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     2521 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     2581 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     2641 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     2701 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     2761 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     2821 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     2881 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     2941 cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg
     3001 gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag
     3061 aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca
     3121 ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt
     3181 atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa
     3241 tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc
     3301 cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg
     3361 gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg
     3421 tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct
     3481 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     3541 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     3601 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     3661 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3721 tgaaggctct caagggcatc ggtcgacgct ctcccttatg cgactcctgc attaggaagc
     3781 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     3841 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     3901 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     3961 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     4021 tcgagatctc gatcccgcga aattaatacg actcactata gggagaccac aacggtttcc
     4081 ctctagtgcc ggctccggag agctctttaa ttaagcggcc gccctgcagg actcgagttc
     4141 tagaaataat tttgtttaac tttaagaagg agatatacat atgaaatctt ctcaccatca
     4201 ccatcaccat caccatcacc atggttcttc tatgaaaatc gaagaaggta aactggtaat
     4261 ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa
     4321 agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca
     4381 ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
     4441 ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct
     4501 gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc
     4561 tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg
     4621 ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt
     4681 caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt
     4741 caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
     4801 agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga
     4861 ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc
     4921 gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac
     4981 cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc
     5041 cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg
     5101 tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
     5161 agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga aaggtgaaat
     5221 catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa
     5281 cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag
     5341 ctcgaacaac aacaacaata acaataacaa caacctcggg atcgaggaaa acctgtactt
     5401 ccaatccaat gcaatgagca acaaagaaaa gaacgcgagc gaaacccgta aggcgtacac
     5461 caccaaaatg atcccgcgta gccacgaccg tatgaagctg ctgggcaact ttatggacta
     5521 tctgatggat ggcaccccga ttttctttga actgtggaac cagttcggtg gcggtatcga
     5581 ccgtgatatc attagcggca ccgcgaacaa agataagatt agcgacgatc tgctgctggc
     5641 ggttaactgg tttaaagtga tgccgatcaa cagcaaaccg caaggcgtga gcccgagcaa
     5701 cctggcgaac ctgttccagc aatacagcgg tagcgaaccg gacatccagg cgcaagagta
     5761 tttcgcgagc aactttgaca ccgaaaagca ccagtggaaa gatatgcgtg ttgagtacga
     5821 acgtctgctg gcggagctgc aactgagccg tagcgacatg caccatgatc tgaagctgat
     5881 gtacaaagaa aagtgcattg gcctgagcct gagcaccgcg cactatatca ccagcgtgat
     5941 gtttggcacc ggtgcgaaga acaaccgtca gaccaaacat caattctata gcaaagtgat
     6001 tcagctgctg gaggaaagca cccagatcaa cagcgttgag caactggcga gcatcattct
     6061 gaaagcgggt gactgcgata gctaccgtaa actgcgtatc cgttgcagcc gtaagggtgc
     6121 gaccccgagc attctgaaaa tcgtgcaaga ctatgaactg ggcaccaacc acgacgatga
     6181 ggtgaacgtt ccgagcctga ttgcgaacct gaaagaaaag ctgggccgtt ttgagtacga
     6241 atgcgagtgg aagtgcatgg agaaaatcaa ggcgttcctg gcgagcaaag ttggcccgta
     6301 ctatctgggt agctatagcg cgatgctgga gaacgcgctg agcccgatta agggtatgac
     6361 caccaagaac tgcaaattcg tgctgaaaca gattgacgcg aagaacgata tcaaatacga
     6421 aaacgagccg tttggcaaga tcgttgaagg tttctttgac agcccgtatt tcgagagcga
     6481 taccaacgtg aaatgggttc tgcacccgca ccatattggc gaaagcaaca tcaagaccct
     6541 gtgggaggac ctgaacgcga ttcatagcaa atacgaggaa gacatcgcga gcctgagcga
     6601 agataagaaa gagaagcgta tcaaggttta ccagggcgat gtgtgccaaa ccatcaacac
     6661 ctattgcgag gaagtgggca aggaagcgaa aaccccgctg gttcagctgc tgcgttacct
     6721 gtatagccgt aaagacgata ttgcggtgga caagatcatt gatggtatca ccttcctgag
     6781 caagaaacac aaggtggaga aacagaagat taacccggtt atccaaaaat acccgagctt
     6841 caactttggc aacaacagca agctgctggg taaaatcatt agcccgaaag acaagctgaa
     6901 acataacctg aagtgcaacc gtaaccaagt ggataactac atctggattg aaatcaaggt
     6961 tctgaacacc aagaccatgc gttgggagaa acaccattat gcgctgagca gcacccgttt
     7021 tctggaggaa gtttactatc cggcgaccag cgaaaacccg ccggatgcgc tggcggcgcg
     7081 tttccgtacc aaaaccaacg gttatgaggg caagccggcg ctgagcgcgg aacagattga
     7141 gcaaatccgt agcgcgccgg ttggcctgcg taaagtgaag aaacgtcaga tgcgtctgga
     7201 agcggcgcgt cagcaaaacc tgctgccgcg ttatacctgg ggcaaggatt ttaacattaa
     7261 catctgcaaa cgtggtaaca acttcgaagt taccctggcg accaaggtga agaaaaagaa
     7321 agagaagaac tacaaagtgg ttctgggtta tgccgcgaac attgtgcgta aaaacaccta
     7381 tgcggcgatt gaggcgcatg cgaacggtga tggtgtgatt gactataacg atctgccggt
     7441 taagccgatc gaaagcggct ttgtgaccgt tgagagccag gttcgtgaca aaagctacga
     7501 tcaactgagc tataacggtg tgaagctgct gtactgcaaa ccgcatgttg aaagccgtcg
     7561 tagcttcctg gagaaatatc gtaacggcac catgaaggac aaccgtggta acaacattca
     7621 gatcgacttc atgaaggatt ttgaagcgat cgcggacgat gaaaccagcc tgtactattt
     7681 taacatgaag tactgcaaac tgctgcagag cagcattcgt aaccacagca gccaagcgaa
     7741 agaatatcgt gaggaaatct tcgagctgct gcgtgatggc aagctgagcg tgctgaaact
     7801 gagcagcctg agcaacctga gcttcgtgat gtttaaggtt gcgaaaagcc tgattggcac
     7861 ctactttggt catctgctga agaaaccgaa gaacagcaaa agcgacgtta aggctccgcc
     7921 gatcaccgac gaagataagc agaaagcgga cccggagatg ttcgcgctgc gtctggcgct
     7981 ggaggaaaaa cgtctgaaca aggttaaaag caagaaagaa gtgattgcga acaagatcgt
     8041 tgcgaaagcg ctggagctgc gtgacaaata cggcccggtg ctgattaagg gcgagaacat
     8101 cagcgatacc accaagaaag gtaagaaaag cagcaccaac agcttcctga tggactggct
     8161 ggcgcgtggc gtggcgaaca aggttaaaga aatggtgatg atgcaccagg gtctggagtt
     8221 cgttgaggtg aacccgaact ttaccagcca ccaagatccg ttcgttcata agaacccgga
     8281 gaacaccttt cgtgcgcgtt acagccgttg caccccgagc gaactgaccg agaagaaccg
     8341 taaagaaatc ctgagcttcc tgagcgacaa gccgagcaaa cgtccgacca acgcgtacta
     8401 taacgagggt gcgatggcgt ttctggcgac ctatggcctg aagaaaaacg acgtgctggg
     8461 tgttagcctg gaaaagttca aacagattat ggcgaacatc ctgcatcagc gtagcgagga
     8521 tcaactgctg tttccgagcc gtggcggtat gttctacctg gcgacctata agctggacgc
     8581 ggatgcgacc agcgttaact ggaacggcaa acagttttgg gtgtgcaacg cggacctggt
     8641 tgcggcgtac aacgtgggtc tggttgacat ccaaaaagat ttcaagaaaa agtaataata
     8701 acattggaag tggataacgg atccgcgatc gcggcgcgcc acctggtggc cggccggtac
     8761 cacgcgtgcg cgctgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     8821 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     8881 ttttgctgaa aggaggaact atatccggat atccacagga cgggtgtggt cgccatgatc
     8941 gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga ctgggcggcg gccaaagcgg
     9001 tcggacagtg ctccgagaac gggtgcgcat agaaattgca tcaacgcata tagcgctagc
     9061 agcacgccat agtgactggc gatgctgtcg gaatggacga tatcccgcaa gaggcccggc
     9121 agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg
     9181 atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat
     9241 aaactaccgc a
//
