LOCUS       sequence-335044-        8662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial expression of FnCas12a 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_039 FnCas12a
FEATURES             Location/Qualifiers
     source          1..8662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(63..68,69..85,86..91))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(118..136)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        204..308
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(309..377,378..1169)
                     /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(527..546)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1340..1928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1829..1848
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2082..2099
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2114..2253
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2179..2198)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2339..2361
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2355..2546)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4004..4023
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4107..4126
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4107..4125
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4214..4236
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             8171..8188
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(8298..8316)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8312..8359
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc
       61 gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga cgaaagggcc
      121 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
      181 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
      241 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa cattgaaaaa
      301 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      361 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      421 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      481 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      541 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      601 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      661 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      721 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      781 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      841 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      901 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      961 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     1021 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     1081 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     1141 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1201 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1261 atctcatgac caaaatccct taacgtgagt tttagttcca ctgagcgtca gaccccgtag
     1321 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1381 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1441 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     1501 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1561 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1621 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1681 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1741 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1801 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1861 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1921 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1981 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     2041 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     2101 aagcggaaca gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2161 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     2221 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     2281 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     2341 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     2401 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     2461 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     2521 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     2581 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     2641 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     2701 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     2761 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     2821 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     2881 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     2941 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     3001 atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag
     3061 atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc
     3121 aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt
     3181 ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa
     3241 ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat
     3301 aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg
     3361 atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg
     3421 cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc
     3481 gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg
     3541 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     3601 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     3661 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     3721 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     3781 ggttgaaggc tctcaagggc atcggtcgac gctctccctt atgcgactcc tgcattagga
     3841 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     3901 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     3961 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     4021 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     4081 ggatcgagat ctcgatcccg cgaaattaat acgactcact atagggagac cacaacggtt
     4141 tccctctagt gccggctccg gagagctctt taattaagcg gccgccctgc aggactcgag
     4201 ttctagaaat aattttgttt aactttaaga aggagatata catatgtcaa tttatcaaga
     4261 atttgttaat aaatatagtt taagtaaaac tctaagattt gagttaatcc cacagggtaa
     4321 aacacttgaa aacataaaag caagaggttt gattttagat gatgagaaaa gagctaaaga
     4381 ctacaaaaag gctaaacaaa taattgataa atatcatcag ttttttatag aggagatatt
     4441 aagttcggtt tgtattagcg aagatttatt acaaaactat tctgatgttt attttaaact
     4501 taaaaagagt gatgatgata atctacaaaa agattttaaa agtgcaaaag atacgataaa
     4561 gaaacaaata tctgaatata taaaggactc agagaaattt aagaatttgt ttaatcaaaa
     4621 ccttatcgat gctaaaaaag ggcaagagtc agatttaatt ctatggctaa agcaatctaa
     4681 ggataatggt atagaactat ttaaagccaa tagtgatatc acagatatag atgaggcgtt
     4741 agaaataatc aaatctttta aaggttggac aacttatttt aagggttttc atgaaaatag
     4801 aaaaaatgtt tatagtagca atgatattcc tacatctatt atttatagga tagtagatga
     4861 taatttgcct aaatttctag aaaataaagc taagtatgag agtttaaaag acaaagctcc
     4921 agaagctata aactatgaac aaattaaaaa agatttggca gaagagctaa cctttgatat
     4981 tgactacaaa acatctgaag ttaatcaaag agttttttca cttgatgaag tttttgagat
     5041 agcaaacttt aataattatc taaatcaaag tggtattact aaatttaata ctattattgg
     5101 tggtaaattt gtaaatggtg aaaatacaaa gagaaaaggt ataaatgaat atataaatct
     5161 atactcacag caaataaatg ataaaacact caaaaaatat aaaatgagtg ttttatttaa
     5221 gcaaatttta agtgatacag aatctaaatc ttttgtaatt gataagttag aagatgatag
     5281 tgatgtagtt acaacgatgc aaagttttta tgagcaaata gcagctttta aaacagtaga
     5341 agaaaaatct attaaagaaa cactatcttt attatttgat gatttaaaag ctcaaaaact
     5401 tgatttgagt aaaatttatt ttaaaaatga taaatctctt actgatctat cacaacaagt
     5461 ttttgatgat tatagtgtta ttggtacagc ggtactagaa tatataactc aacaaatagc
     5521 acctaaaaat cttgataacc ctagtaagaa agagcaagaa ttaatagcca aaaaaactga
     5581 aaaagcaaaa tacttatctc tagaaactat aaagcttgcc ttagaagaat ttaataagca
     5641 tagagatata gataaacagt gtaggtttga agaaatactt gcaaactttg cggctattcc
     5701 gatgatattt gatgaaatag ctcaaaacaa agacaatttg gcacagatat ctatcaaata
     5761 tcaaaatcaa ggtaaaaaag acctacttca agctagtgcg gaagatgatg ttaaagctat
     5821 caaggatctt ttagatcaaa ctaataatct cttacataaa ctaaaaatat ttcatattag
     5881 tcagtcagaa gataaggcaa atattttaga caaggatgag catttttatc tagtatttga
     5941 ggagtgctac tttgagctag cgaatatagt gcctctttat aacaaaatta gaaactatat
     6001 aactcaaaag ccatatagtg atgagaaatt taagctcaat tttgagaact cgactttggc
     6061 taatggttgg gataaaaata aagagcctga caatacggca attttattta tcaaagatga
     6121 taaatattat ctgggtgtga tgaataagaa aaataacaaa atatttgatg ataaagctat
     6181 caaagaaaat aaaggcgagg gttataaaaa aattgtttat aaacttttac ctggcgcaaa
     6241 taaaatgtta cctaaggttt tcttttctgc taaatctata aaattttata atcctagtga
     6301 agatatactt agaataagaa atcattccac acatacaaaa aatggtagtc ctcaaaaagg
     6361 atatgaaaaa tttgagttta atattgaaga ttgccgaaaa tttatagatt tttataaaca
     6421 gtctataagt aagcatccgg agtggaaaga ttttggattt agattttctg atactcaaag
     6481 atataattct atagatgaat tttatagaga agttgaaaat caaggctaca aactaacttt
     6541 tgaaaatata tcagagagct atattgatag cgtagttaat cagggtaaat tgtacctatt
     6601 ccaaatctat aataaagatt tttcagctta tagcaaaggg cgaccaaatc tacatacttt
     6661 atattggaaa gcgctgtttg atgagagaaa tcttcaagat gtggtttata agctaaatgg
     6721 tgaggcagag cttttttatc gtaaacaatc aatacctaaa aaaatcactc acccagctaa
     6781 agaggcaata gctaataaaa acaaagataa tcctaaaaaa gagagtgttt ttgaatatga
     6841 tttaatcaaa gataaacgct ttactgaaga taagtttttc tttcactgtc ctattacaat
     6901 caattttaaa tctagtggag ctaataagtt taatgatgaa atcaatttat tgctaaaaga
     6961 aaaagcaaat gatgttcata tattaagtat agatagaggt gaaagacatt tagcttacta
     7021 tactttggta gatggtaaag gcaatatcat caaacaagat actttcaaca tcattggtaa
     7081 tgatagaatg aaaacaaact accatgataa gcttgctgca atagagaaag atagggattc
     7141 agctaggaaa gactggaaaa agataaataa catcaaagag atgaaagagg gctatctatc
     7201 tcaggtagtt catgaaatag ctaagctagt tatagagtat aatgctattg tggtttttga
     7261 ggatttaaat tttggattta aaagagggcg tttcaaggta gagaagcagg tctatcaaaa
     7321 gttagaaaaa atgctaattg agaaactaaa ctatctagtt ttcaaagata atgagtttga
     7381 taaaactggg ggagtgctta gagcttatca gctaacagca ccttttgaga cttttaaaaa
     7441 gatgggtaaa caaacaggta ttatctacta tgtaccagct ggttttactt caaaaatttg
     7501 tcctgtaact ggttttgtaa atcagttata tcctaagtat gaaagtgtca gcaaatctca
     7561 agagttcttt agtaagtttg acaagatttg ttataacctt gataagggct attttgagtt
     7621 tagttttgat tataaaaact ttggtgacaa ggctgccaaa ggcaagtgga ctatagctag
     7681 ctttgggagt agattgatta actttagaaa ttcagataaa aatcataatt gggatactcg
     7741 agaagtttat ccaactaaag agttggagaa attgctaaaa gattattcta tcgaatatgg
     7801 gcatggcgaa tgtatcaaag cagctatttg cggtgagagc gacaaaaagt tttttgctaa
     7861 gctaactagt gtcctaaata ctatcttaca aatgcgtaac tcaaaaacag gtactgagtt
     7921 agattatcta atttcaccag tagcagatgt aaatggcaat ttctttgatt cgcgacaggc
     7981 gccaaaaaat atgcctcaag atgctgatgc caatggtgct tatcatattg ggctaaaagg
     8041 tctgatgcta ctaggtagga tcaaaaataa tcaagagggc aaaaaactca atttggttat
     8101 caaaaatgaa gagtattttg agttcgtgca gaataggaat aacggttctt ctctgccaga
     8161 aacgggaggc catcaccatc atcaccacta acggatccgc gatcgtggcg cgccacctgg
     8221 tggccggccg gtaccacgcg tgcgcgctga tccggctgct aacaaagccc gaaaggaagc
     8281 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     8341 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatccac aggacgggtg
     8401 tggtcgccat gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc aggactgggc
     8461 ggcggccaaa gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg
     8521 catatagcgc tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc
     8581 gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg
     8641 tgccgaggat gacgatgagc gc
//
