LOCUS       sequence-338280-        8662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of FnCas12a D917A (RuvC-inactivating 
            mutation) with a C-terminal sorting motif and His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8662)
  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 8662)
  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_058 FnCas12a D917A
FEATURES             Location/Qualifiers
     source          1..8662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..23)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        91..195
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(196..264,265..1056)
                     /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(414..433)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1227..1815
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1716..1735
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1969..1986
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2001..2140
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2066..2085)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2226..2248
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2242..2433)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3891..3910
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3994..4013
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3994..4012
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4101..4123
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             8058..8075
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(8185..8203)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8199..8246
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(8612..8617,8618..8634,8635..8640))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
       61 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
      121 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataacat
      181 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
      241 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
      301 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
      361 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
      421 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
      481 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
      541 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
      601 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
      661 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
      721 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
      781 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
      841 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
      901 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
      961 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     1021 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     1081 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     1141 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt agttccactg agcgtcagac
     1201 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     1261 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     1321 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     1381 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1441 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1501 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1561 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1621 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1681 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1741 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1801 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     1861 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     1921 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     1981 agcgaggaag cggaacagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     2041 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     2101 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     2161 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     2221 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     2281 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     2341 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     2401 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     2461 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     2521 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     2581 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     2641 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     2701 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     2761 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     2821 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     2881 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct
     2941 gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt
     3001 tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg
     3061 ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg
     3121 cagacaaggt atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag
     3181 gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc
     3241 gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc
     3301 tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc
     3361 cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg
     3421 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     3481 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     3541 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     3601 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     3661 ctgactgggt tgaaggctct caagggcatc ggtcgacgct ctcccttatg cgactcctgc
     3721 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     3781 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     3841 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     3901 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     3961 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata gggagaccac
     4021 aacggtttcc ctctagtgcc ggctccggag agctctttaa ttaagcggcc gccctgcagg
     4081 actcgagttc tagaaataat tttgtttaac tttaagaagg agatatacat atgtcaattt
     4141 atcaagaatt tgttaataaa tatagtttaa gtaaaactct aagatttgag ttaatcccac
     4201 agggtaaaac acttgaaaac ataaaagcaa gaggtttgat tttagatgat gagaaaagag
     4261 ctaaagacta caaaaaggct aaacaaataa ttgataaata tcatcagttt tttatagagg
     4321 agatattaag ttcggtttgt attagcgaag atttattaca aaactattct gatgtttatt
     4381 ttaaacttaa aaagagtgat gatgataatc tacaaaaaga ttttaaaagt gcaaaagata
     4441 cgataaagaa acaaatatct gaatatataa aggactcaga gaaatttaag aatttgttta
     4501 atcaaaacct tatcgatgct aaaaaagggc aagagtcaga tttaattcta tggctaaagc
     4561 aatctaagga taatggtata gaactattta aagccaatag tgatatcaca gatatagatg
     4621 aggcgttaga aataatcaaa tcttttaaag gttggacaac ttattttaag ggttttcatg
     4681 aaaatagaaa aaatgtttat agtagcaatg atattcctac atctattatt tataggatag
     4741 tagatgataa tttgcctaaa tttctagaaa ataaagctaa gtatgagagt ttaaaagaca
     4801 aagctccaga agctataaac tatgaacaaa ttaaaaaaga tttggcagaa gagctaacct
     4861 ttgatattga ctacaaaaca tctgaagtta atcaaagagt tttttcactt gatgaagttt
     4921 ttgagatagc aaactttaat aattatctaa atcaaagtgg tattactaaa tttaatacta
     4981 ttattggtgg taaatttgta aatggtgaaa atacaaagag aaaaggtata aatgaatata
     5041 taaatctata ctcacagcaa ataaatgata aaacactcaa aaaatataaa atgagtgttt
     5101 tatttaagca aattttaagt gatacagaat ctaaatcttt tgtaattgat aagttagaag
     5161 atgatagtga tgtagttaca acgatgcaaa gtttttatga gcaaatagca gcttttaaaa
     5221 cagtagaaga aaaatctatt aaagaaacac tatctttatt atttgatgat ttaaaagctc
     5281 aaaaacttga tttgagtaaa atttatttta aaaatgataa atctcttact gatctatcac
     5341 aacaagtttt tgatgattat agtgttattg gtacagcggt actagaatat ataactcaac
     5401 aaatagcacc taaaaatctt gataacccta gtaagaaaga gcaagaatta atagccaaaa
     5461 aaactgaaaa agcaaaatac ttatctctag aaactataaa gcttgcctta gaagaattta
     5521 ataagcatag agatatagat aaacagtgta ggtttgaaga aatacttgca aactttgcgg
     5581 ctattccgat gatatttgat gaaatagctc aaaacaaaga caatttggca cagatatcta
     5641 tcaaatatca aaatcaaggt aaaaaagacc tacttcaagc tagtgcggaa gatgatgtta
     5701 aagctatcaa ggatctttta gatcaaacta ataatctctt acataaacta aaaatatttc
     5761 atattagtca gtcagaagat aaggcaaata ttttagacaa ggatgagcat ttttatctag
     5821 tatttgagga gtgctacttt gagctagcga atatagtgcc tctttataac aaaattagaa
     5881 actatataac tcaaaagcca tatagtgatg agaaatttaa gctcaatttt gagaactcga
     5941 ctttggctaa tggttgggat aaaaataaag agcctgacaa tacggcaatt ttatttatca
     6001 aagatgataa atattatctg ggtgtgatga ataagaaaaa taacaaaata tttgatgata
     6061 aagctatcaa agaaaataaa ggcgagggtt ataaaaaaat tgtttataaa cttttacctg
     6121 gcgcaaataa aatgttacct aaggttttct tttctgctaa atctataaaa ttttataatc
     6181 ctagtgaaga tatacttaga ataagaaatc attccacaca tacaaaaaat ggtagtcctc
     6241 aaaaaggata tgaaaaattt gagtttaata ttgaagattg ccgaaaattt atagattttt
     6301 ataaacagtc tataagtaag catccggagt ggaaagattt tggatttaga ttttctgata
     6361 ctcaaagata taattctata gatgaatttt atagagaagt tgaaaatcaa ggctacaaac
     6421 taacttttga aaatatatca gagagctata ttgatagcgt agttaatcag ggtaaattgt
     6481 acctattcca aatctataat aaagattttt cagcttatag caaagggcga ccaaatctac
     6541 atactttata ttggaaagcg ctgtttgatg agagaaatct tcaagatgtg gtttataagc
     6601 taaatggtga ggcagagctt ttttatcgta aacaatcaat acctaaaaaa atcactcacc
     6661 cagctaaaga ggcaatagct aataaaaaca aagataatcc taaaaaagag agtgtttttg
     6721 aatatgattt aatcaaagat aaacgcttta ctgaagataa gtttttcttt cactgtccta
     6781 ttacaatcaa ttttaaatct agtggagcta ataagtttaa tgatgaaatc aatttattgc
     6841 taaaagaaaa agcaaatgat gttcatatat taagtatagc tagaggtgaa agacatttag
     6901 cttactatac tttggtagat ggtaaaggca atatcatcaa acaagatact ttcaacatca
     6961 ttggtaatga tagaatgaaa acaaactacc atgataagct tgctgcaata gagaaagata
     7021 gggattcagc taggaaagac tggaaaaaga taaataacat caaagagatg aaagagggct
     7081 atctatctca ggtagttcat gaaatagcta agctagttat agagtataat gctattgtgg
     7141 tttttgagga tttaaatttt ggatttaaaa gagggcgttt caaggtagag aagcaggtct
     7201 atcaaaagtt agaaaaaatg ctaattgaga aactaaacta tctagttttc aaagataatg
     7261 agtttgataa aactggggga gtgcttagag cttatcagct aacagcacct tttgagactt
     7321 ttaaaaagat gggtaaacaa acaggtatta tctactatgt accagctggt tttacttcaa
     7381 aaatttgtcc tgtaactggt tttgtaaatc agttatatcc taagtatgaa agtgtcagca
     7441 aatctcaaga gttctttagt aagtttgaca agatttgtta taaccttgat aagggctatt
     7501 ttgagtttag ttttgattat aaaaactttg gtgacaaggc tgccaaaggc aagtggacta
     7561 tagctagctt tgggagtaga ttgattaact ttagaaattc agataaaaat cataattggg
     7621 atactcgaga agtttatcca actaaagagt tggagaaatt gctaaaagat tattctatcg
     7681 aatatgggca tggcgaatgt atcaaagcag ctatttgcgg tgagagcgac aaaaagtttt
     7741 ttgctaagct aactagtgtc ctaaatacta tcttacaaat gcgtaactca aaaacaggta
     7801 ctgagttaga ttatctaatt tcaccagtag cagatgtaaa tggcaatttc tttgattcgc
     7861 gacaggcgcc aaaaaatatg cctcaagatg ctgatgccaa tggtgcttat catattgggc
     7921 taaaaggtct gatgctacta ggtaggatca aaaataatca agagggcaaa aaactcaatt
     7981 tggttatcaa aaatgaagag tattttgagt tcgtgcagaa taggaataac ggttcttctc
     8041 tgccagaaac gggaggccat caccatcatc accactaacg gatccgcgat cgtggcgcgc
     8101 cacctggtgg ccggccggta ccacgcgtgc gcgctgatcc ggctgctaac aaagcccgaa
     8161 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     8221 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tatccacagg
     8281 acgggtgtgg tcgccatgat cgcgtagtcg atagtggctc caagtagcga agcgagcagg
     8341 actgggcggc ggccaaagcg gtcggacagt gctccgagaa cgggtgcgca tagaaattgc
     8401 atcaacgcat atagcgctag cagcacgcca tagtgactgg cgatgctgtc ggaatggacg
     8461 atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg
     8521 gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg
     8581 cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa
     8641 acatgagaat tcttgaagac ga
//
