LOCUS       sequence-335049-        9251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of Cas12i1 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_083 Cas12i1
FEATURES             Location/Qualifiers
     source          1..9251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(2..7,8..24,25..30))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(57..75)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        143..247
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(248..316,317..1108)
                     /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(466..485)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1279..1867
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1768..1787
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2021..2038
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2053..2192
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2118..2137)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2278..2300
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2294..2485)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3943..3962
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4046..4065
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4046..4064
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4153..4175
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4195..4224
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4234..5334
                     /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     5308..5331
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5389..5409
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(8826..8844)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8840..8887
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct
       61 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
      121 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
      181 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataac attgaaaaag
      241 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
      301 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
      361 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
      421 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
      481 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
      541 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
      601 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
      661 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
      721 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
      781 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
      841 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
      901 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
      961 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     1021 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     1081 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     1141 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     1201 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1261 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1321 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1381 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     1441 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1501 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1561 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1621 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1681 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1741 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1801 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1861 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     1921 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     1981 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     2041 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2101 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     2161 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     2221 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     2281 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     2341 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     2401 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     2461 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     2521 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     2581 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     2641 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     2701 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     2761 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     2821 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     2881 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     2941 tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga
     3001 tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca
     3061 agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc
     3121 cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag
     3181 gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata
     3241 aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga
     3301 tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc
     3361 gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg
     3421 cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc
     3481 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     3541 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     3601 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     3661 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     3721 gttgaaggct ctcaagggca tcggtcgacg ctctccctta tgcgactcct gcattaggaa
     3781 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     3841 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     3901 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     3961 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     4021 gatcgagatc tcgatcccgc gaaattaata cgactcacta tagggagacc acaacggttt
     4081 ccctctagtg ccggctccgg agagctcttt aattaagcgg ccgccctgca ggactcgagt
     4141 tctagaaata attttgttta actttaagaa ggagatatac atatgaaatc ttctcaccat
     4201 caccatcacc atcaccatca ccatggttct tctatgaaaa tcgaagaagg taaactggta
     4261 atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag
     4321 aaagataccg gaattaaagt caccgttgag catccggata aactggaaga gaaattccca
     4381 caggttgcgg caactggcga tggccctgac attatcttct gggcacacga ccgctttggt
     4441 ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag
     4501 ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc
     4561 gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc
     4621 tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg
     4681 ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
     4741 ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg
     4801 aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc
     4861 gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc
     4921 ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg
     4981 accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc
     5041 gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa
     5101 ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag
     5161 gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca gaaaggtgaa
     5221 atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc
     5281 aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactaattcg
     5341 agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgagga aaacctgtac
     5401 ttccaatcca atgcaatgag caacaaagaa aagaacgcga gcgaaacccg taaggcgtac
     5461 accaccaaaa tgatcccgcg tagccacgac cgtatgaagc tgctgggcaa ctttatggac
     5521 tatctgatgg atggcacccc gattttcttt gaactgtgga accagttcgg tggcggtatc
     5581 gaccgtgata tcattagcgg caccgcgaac aaagataaga ttagcgacga tctgctgctg
     5641 gcggttaact ggtttaaagt gatgccgatc aacagcaaac cgcaaggcgt gagcccgagc
     5701 aacctggcga acctgttcca gcaatacagc ggtagcgaac cggacatcca ggcgcaagag
     5761 tatttcgcga gcaactttga caccgaaaag caccagtgga aagatatgcg tgttgagtac
     5821 gaacgtctgc tggcggagct gcaactgagc cgtagcgaca tgcaccatga tctgaagctg
     5881 atgtacaaag aaaagtgcat tggcctgagc ctgagcaccg cgcactatat caccagcgtg
     5941 atgtttggca ccggtgcgaa gaacaaccgt cagaccaaac atcaattcta tagcaaagtg
     6001 attcagctgc tggaggaaag cacccagatc aacagcgttg agcaactggc gagcatcatt
     6061 ctgaaagcgg gtgactgcga tagctaccgt aaactgcgta tccgttgcag ccgtaagggt
     6121 gcgaccccga gcattctgaa aatcgtgcaa gactatgaac tgggcaccaa ccacgacgat
     6181 gaggtgaacg ttccgagcct gattgcgaac ctgaaagaaa agctgggccg ttttgagtac
     6241 gaatgcgagt ggaagtgcat ggagaaaatc aaggcgttcc tggcgagcaa agttggcccg
     6301 tactatctgg gtagctatag cgcgatgctg gagaacgcgc tgagcccgat taagggtatg
     6361 accaccaaga actgcaaatt cgtgctgaaa cagattgacg cgaagaacga tatcaaatac
     6421 gaaaacgagc cgtttggcaa gatcgttgaa ggtttctttg acagcccgta tttcgagagc
     6481 gataccaacg tgaaatgggt tctgcacccg caccatattg gcgaaagcaa catcaagacc
     6541 ctgtgggagg acctgaacgc gattcatagc aaatacgagg aagacatcgc gagcctgagc
     6601 gaagataaga aagagaagcg tatcaaggtt taccagggcg atgtgtgcca aaccatcaac
     6661 acctattgcg aggaagtggg caaggaagcg aaaaccccgc tggttcagct gctgcgttac
     6721 ctgtatagcc gtaaagacga tattgcggtg gacaagatca ttgatggtat caccttcctg
     6781 agcaagaaac acaaggtgga gaaacagaag attaacccgg ttatccaaaa atacccgagc
     6841 ttcaactttg gcaacaacag caagctgctg ggtaaaatca ttagcccgaa agacaagctg
     6901 aaacataacc tgaagtgcaa ccgtaaccaa gtggataact acatctggat tgaaatcaag
     6961 gttctgaaca ccaagaccat gcgttgggag aaacaccatt atgcgctgag cagcacccgt
     7021 tttctggagg aagtttacta tccggcgacc agcgaaaacc cgccggatgc gctggcggcg
     7081 cgtttccgta ccaaaaccaa cggttatgag ggcaagccgg cgctgagcgc ggaacagatt
     7141 gagcaaatcc gtagcgcgcc ggttggcctg cgtaaagtga agaaacgtca gatgcgtctg
     7201 gaagcggcgc gtcagcaaaa cctgctgccg cgttatacct ggggcaagga ttttaacatt
     7261 aacatctgca aacgtggtaa caacttcgaa gttaccctgg cgaccaaggt gaagaaaaag
     7321 aaagagaaga actacaaagt ggttctgggt tatgacgcga acattgtgcg taaaaacacc
     7381 tatgcggcga ttgaggcgca tgcgaacggt gatggtgtga ttgactataa cgatctgccg
     7441 gttaagccga tcgaaagcgg ctttgtgacc gttgagagcc aggttcgtga caaaagctac
     7501 gatcaactga gctataacgg tgtgaagctg ctgtactgca aaccgcatgt tgaaagccgt
     7561 cgtagcttcc tggagaaata tcgtaacggc accatgaagg acaaccgtgg taacaacatt
     7621 cagatcgact tcatgaagga ttttgaagcg atcgcggacg atgaaaccag cctgtactat
     7681 tttaacatga agtactgcaa actgctgcag agcagcattc gtaaccacag cagccaagcg
     7741 aaagaatatc gtgaggaaat cttcgagctg ctgcgtgatg gcaagctgag cgtgctgaaa
     7801 ctgagcagcc tgagcaacct gagcttcgtg atgtttaagg ttgcgaaaag cctgattggc
     7861 acctactttg gtcatctgct gaagaaaccg aagaacagca aaagcgacgt taaggctccg
     7921 ccgatcaccg acgaagataa gcagaaagcg gacccggaga tgttcgcgct gcgtctggcg
     7981 ctggaggaaa aacgtctgaa caaggttaaa agcaagaaag aagtgattgc gaacaagatc
     8041 gttgcgaaag cgctggagct gcgtgacaaa tacggcccgg tgctgattaa gggcgagaac
     8101 atcagcgata ccaccaagaa aggtaagaaa agcagcacca acagcttcct gatggactgg
     8161 ctggcgcgtg gcgtggcgaa caaggttaaa gaaatggtga tgatgcacca gggtctggag
     8221 ttcgttgagg tgaacccgaa ctttaccagc caccaagatc cgttcgttca taagaacccg
     8281 gagaacacct ttcgtgcgcg ttacagccgt tgcaccccga gcgaactgac cgagaagaac
     8341 cgtaaagaaa tcctgagctt cctgagcgac aagccgagca aacgtccgac caacgcgtac
     8401 tataacgagg gtgcgatggc gtttctggcg acctatggcc tgaagaaaaa cgacgtgctg
     8461 ggtgttagcc tggaaaagtt caaacagatt atggcgaaca tcctgcatca gcgtagcgag
     8521 gatcaactgc tgtttccgag ccgtggcggt atgttctacc tggcgaccta taagctggac
     8581 gcggatgcga ccagcgttaa ctggaacggc aaacagtttt gggtgtgcaa cgcggacctg
     8641 gttgcggcgt acaacgtggg tctggttgac atccaaaaag atttcaagaa aaagtaataa
     8701 taacattgga agtggataac ggatccgcga tcgcggcgcg ccacctggtg gccggccggt
     8761 accacgcgtg cgcgctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     8821 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     8881 ttttttgctg aaaggaggaa ctatatccgg atatccacag gacgggtgtg gtcgccatga
     8941 tcgcgtagtc gatagtggct ccaagtagcg aagcgagcag gactgggcgg cggccaaagc
     9001 ggtcggacag tgctccgaga acgggtgcgc atagaaattg catcaacgca tatagcgcta
     9061 gcagcacgcc atagtgactg gcgatgctgt cggaatggac gatatcccgc aagaggcccg
     9121 gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga
     9181 cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg
     9241 ataaactacc g
//
