LOCUS       sequence-307533-        9149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses codon-optimized FnCpf1 .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9149)
  AUTHORS   Yan MY, Yan HQ, Ren GX, Zhao JP, Guo XP, Sun YC
  TITLE     CRISPR-Cas12a-Assisted Recombineering in Bacteria.
  JOURNAL   Appl Environ Microbiol. 2017 Aug 17;83(17). pii: AEM.00947-17. doi: 
            10.1128/AEM.00947-17. Print 2017 Sep 1.
  PUBMED    28646112
REFERENCE   2  (bases 1 to 9149)
  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: "Appl 
            Environ Microbiol."; date: "2017-08-17"; volume: "83(17). pii: 
            AEM.00947-17. doi"; pages: " 10.1128/AEM.00947-17. Print 2017 Sep 1"
COMMENT     Sequence Label: pMV261-Cas12a
FEATURES             Location/Qualifiers
     source          1..9149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(51..97)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    161..194
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(211..834)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     742..761
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    5182..5215
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(7305..7893)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7385..7404)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(8224..9039)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     8947..8966
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     9031..9050
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 cccccccccc cgacgtcagg tggctagctg atcaccgcgg ccatgatggc ataaaacgaa
       61 aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtc gatcgtacgc
      121 tagttaacta cgtcgacatc gataagcttg gcgtgaattc ataacttcgt ataatgtatg
      181 ctatacgaag ttattaattg cccatggtca ttaagaccca ctttcacatt taagttgttt
      241 ttctaatccg cagatgatca attcaaggcc gaataagaag gctggctctg caccttggtg
      301 atcaaataat tcgatagctt gtcgtaataa tggcggcata ctatcagtag taggtgtttc
      361 cctttcttct ttagcgactt gatgctcttg atcttccaat acgcaaccta aggtaaaatg
      421 ccccacagcg ctgagtgcat ataacgcgtt ctctagtgaa aaaccttgtt ggcataaaaa
      481 ggctaattga ttttcgagag tttcatactg tttttctgta ggccgtgtac ctaaatgtac
      541 ttttgctcca tcgcgatgac ttagtaaagc acatctaaaa cttttagcgt tattacgcaa
      601 aaaatcttgc cagctttccc cttctaaagg gcaaaagtga gtatggtgcc tatctaacat
      661 ctcaatggct aaggcgtcga gcagggctcg cttatttttt acatgccaat acaatgtagg
      721 ctgctctaca ccaagtttct gggcgagttt acgggttgtt aaaccttcga ttccgacctc
      781 attaagcagc tctaatgcgc tgttaatcac tttactttta tctaatctag acattgcgaa
      841 gtgattcctc cgatccgtgg caggagggtc gcctgcgtcg gcaactcgcc caggctacac
      901 gagagccctc gactgcgaaa atgccgacca ggcaccaccg cactccaggc ttaacccggt
      961 gcgacgatcc aatattaata ttggatcgtc ggcaccgtca cggccgtggg aggcggcacg
     1021 atccgcgacg tgatgatcgg ccgcatcccc acggtgctgc gcagtgagct ctacgccatc
     1081 ccggcgttga tctgtgcgtt cgcacgcaca ggcccggtgt gagaagggtc tctgacgagc
     1141 gggagaactc cctatcagtg atagagtttg tcctccctat cagtgataga taggctctgg
     1201 gagtacccgt gtgtacgacc agcacggcat acatcatttc gacgccgaga gattcgccgc
     1261 ccgaaatgag cacgatccat gtcgatctac caagagttcg tgaataagta tagcctgagc
     1321 aagaccttgc ggttcgagtt gatcccgcag ggcaaaacct tggaaaacat taaggcgcgc
     1381 gggctgatcc tggatgatga gaagcgggcc aaggactata aaaaggccaa gcagattatc
     1441 gataaatatc atcagttctt catcgaggaa atcctgtcga gcgtgtgcat cagcgaagac
     1501 ttgttgcaga actatagcga cgtgtacttc aaattgaaaa agtcggacga cgacaatctg
     1561 caaaaagact tcaagtcggc caaagacacc atcaagaaac agatttcgga gtacatcaag
     1621 gactcggaaa agttcaaaaa cctgttcaac cagaacttga tcgacgcgaa aaagggccag
     1681 gaaagcgatt tgatcttgtg gctgaagcag tcgaaggata atggcattga gttgttcaag
     1741 gccaactcgg acattacgga cattgacgag gccctggaaa tcattaaatc gtttaagggc
     1801 tggaccacgt acttcaaggg cttccatgag aaccggaaaa acgtgtattc gtcgaacgac
     1861 attcccacgt cgatcatcta ccggatcgtc gatgacaact tgcccaagtt cttggaaaat
     1921 aaagcgaaat acgagagctt gaaggacaaa gcgccggagg ccattaacta tgagcagatc
     1981 aagaaggacc tggccgaaga gctgaccttt gacattgact acaagaccag cgaggtcaat
     2041 cagcgggtct tctcgttgga tgaggtcttt gaaatcgcca atttcaacaa ctatttgaac
     2101 cagagcggca tcaccaaatt taataccatt atcggcggga agttcgtcaa cggcgagaac
     2161 acgaagcgga aggggatcaa cgaatacatc aatctgtact cgcagcagat caacgacaag
     2221 accctgaaaa agtacaagat gagcgtgctg ttcaagcaaa ttctgagcga caccgagagc
     2281 aagtcgttcg tcattgacaa gctggaggat gacagcgatg tggtcaccac gatgcagtcg
     2341 ttttacgagc aaatcgcggc cttcaagacc gtggaggaaa agagcatcaa ggaaaccctg
     2401 agcctgctgt tcgatgatct gaaggcgcag aagttggatc tgtcgaaaat ctacttcaag
     2461 aacgataagt cgctgaccga cctgagccaa caggtcttcg atgattacag cgtcatcggg
     2521 accgccgtcc tggagtacat cacccagcag attgcgccca aaaacctgga caatccctcg
     2581 aaaaaagagc aggagctgat tgccaagaag accgagaagg cgaagtactt gtcgctggaa
     2641 accatcaaac tggcgctgga ggagttcaac aagcaccgcg acattgataa acaatgccgg
     2701 ttcgaggaga ttctggccaa ttttgccgcc attcccatga tcttcgatga gatcgcgcag
     2761 aataaagaca acctggcgca aatctcgatc aaatatcaga atcagggcaa aaaagacctg
     2821 ttacaagcct cggccgaaga cgacgtgaaa gccatcaagg acctgttgga tcagacgaac
     2881 aacttgttgc acaagctgaa aattttccac atcagccaga gcgaggacaa ggccaacatc
     2941 ttggacaaag acgagcactt ctacttggtg ttcgaagagt gctacttcga attggccaac
     3001 atcgtccccc tgtacaataa gattcgcaat tatattacgc agaagcccta tagcgacgag
     3061 aaattcaagt tgaattttga aaattcgacc ctggcgaatg gctgggacaa gaacaaagag
     3121 cccgacaaca ccgccatctt gttcatcaag gatgataaat attatctggg ggtgatgaac
     3181 aaaaagaata acaagatctt tgatgacaag gccatcaagg agaataaggg cgaggggtac
     3241 aagaaaatcg tctacaaact gttgcccggc gccaacaaga tgttgccgaa ggtcttcttc
     3301 agcgcgaaga gcattaaatt ctataacccc agcgaggaca tcctgcgcat ccggaatcat
     3361 tcgacccaca cgaagaatgg cagcccgcaa aagggctacg agaagttcga gtttaacatc
     3421 gaggattgtc ggaaattcat cgacttctat aaacagtcga tcagcaagca ccccgagtgg
     3481 aaggatttcg gcttccgctt cagcgatacc caacgctaca acagcatcga cgagttttat
     3541 cgggaagtgg aaaaccaggg gtacaagttg accttcgaga acatttcgga gtcgtacatt
     3601 gactcggtcg tcaaccaagg gaaactgtac ctgttccaaa tctacaataa ggacttcagc
     3661 gcgtactcga aagggcggcc caacttgcac accctgtatt ggaaggcctt gtttgacgag
     3721 cggaacttac aagacgtcgt ctataaactg aacggggagg cggagctgtt ttaccgcaag
     3781 cagtcgatcc cgaaaaagat tacccacccg gcgaaggagg cgatcgccaa caaaaacaag
     3841 gacaacccga agaaagagtc ggtcttcgaa tacgatctga tcaaagacaa gcgctttacc
     3901 gaggacaagt tctttttcca ttgtccgatc accatcaatt ttaagagcag cggcgcgaac
     3961 aagttcaacg acgagatcaa cctgctgttg aaagaaaagg ccaatgatgt gcacatcctg
     4021 tcgatcgacc ggggggaacg gcacctggcc tactacaccc tggtggacgg caagggcaac
     4081 atcatcaagc aggatacctt caatatcatt gggaacgacc ggatgaaaac gaactaccac
     4141 gacaagctgg cggccatcga aaaggatcgc gacagcgccc gcaaggactg gaagaagatt
     4201 aacaatatca aggagatgaa ggaggggtac ttgagccagg tcgtgcacga gatcgccaag
     4261 ctggtcattg agtacaacgc gattgtggtc tttgaagacc tgaactttgg cttcaaacgg
     4321 gggcggttca aggtcgagaa gcaggtgtat cagaagctgg aaaaaatgtt gatcgagaag
     4381 ctgaactatc tggtgttcaa agacaacgag tttgacaaaa cggggggcgt cctgcgggcc
     4441 taccagttga ccgccccctt cgaaaccttc aagaaaatgg gcaagcagac gggcatcatc
     4501 tactacgtcc cggcgggctt tacgagcaag atctgtccgg tgaccggctt cgtcaaccag
     4561 ctgtatccga agtatgagag cgtgtcgaaa agccaggagt ttttctcgaa gttcgataaa
     4621 atctgttata acctggataa ggggtacttc gaattttcgt tcgattacaa aaacttcggg
     4681 gacaaggcgg ccaaggggaa atggacgatc gccagcttcg gctcgcgcct gattaacttt
     4741 cgcaactcgg acaagaacca taactgggac acccgggaag tgtacccgac caaagaattg
     4801 gaaaaactgt tgaaggacta tagcatcgag tacgggcacg gggagtgcat caaagccgcc
     4861 atctgcgggg aaagcgacaa gaagtttttt gcgaagctga ccagcgtgct gaatacgatc
     4921 ttacaaatgc gtaattctaa gaccggcacc gagctggact acctgatctc gccggtggcg
     4981 gatgtcaacg gcaacttctt cgacagccgc caggccccga agaacatgcc gcaggacgcg
     5041 gatgcgaatg gcgcgtacca catcgggctg aaagggctga tgctgctggg gcggatcaag
     5101 aacaaccagg aggggaagaa gctgaatctg gtgattaaga acgaggaata tttcgagttc
     5161 gtccaaaatc gcaacaattg aataacttcg tataatgtat gctatacgaa gttattaatt
     5221 ctgcagagtc ctactagtat ttgatatcgg taccgcggcc gcgccacgga tgccaccaca
     5281 ggcactacaa cggagttcgc cacgtacatc accacaacca ccgattctgg cggtgagctc
     5341 cccgatattc agcggaaatg gcttggtatc gaccaagatt cgtagaaccc cgtctcgtct
     5401 ggctggtatt caaaacgggc gcaacgaaac acgcaacgag acaggcatgg cccaaaccag
     5461 aaaactagcg tctaccagga cttttacctg tccgacccgt tgcaacggaa ccccccacgg
     5521 aacccccgcg acacccgctc cccaattgcg ttagaacagc ggtggattgt cggcttcgtt
     5581 gtgggccttt tgagccgctt cctgttctgc cgcacgctct ttcctcgccc gatagccgag
     5641 tcgcttaacg gtgtccagat gcagcccgaa atgtttggcc gtttgcggcc aagagtggcc
     5701 ctcgtcgtcg tgataggcgc ggatgcgttc gcggcgtgca gcctgctcgg cgagccactc
     5761 gctgcgttcc tgcgccacga gccggacgac gtggcgttcg gatagtccgg tgattcgagc
     5821 gccttcggcg gcggtcacgc gccgcttttt gcggacagtc ggctgccggt tgtagccgtc
     5881 gctgtagccg tcgctcatag caatgcctcc atggctgacg cggactttgc gcgccgcgca
     5941 actgtgctcg ccgccgtgcg cgctgctgcg cccttccgcg agatggccga ctggcgcgca
     6001 ctgagtgtgg cctcgtagac cacgatcccg tccgcccaaa tgcgcgactt ggttgtgatc
     6061 caacgccaaa tgctgttggc gatggcgcgg acctcgctgt ccggtagcgg tccgggacac
     6121 acgtcgttgc acggaaattc ggcgtttcgc gcgtggcact cggcatagat cgcgcggccg
     6181 agtccgtcca cgttccgggt cggcaggtag atccgcatga gggcgggacg ataggcccac
     6241 aacctgacgg aatcgaacag tgcgcaattc cgccctagcg gcgtcggagc cgctttgtac
     6301 gtggtctgct gacgccagcg cggcggtggc atgttcgcgc cgagctcggc ctcgatgtgg
     6361 ctgagtgtgt agagatctga gtggagccat tccgtttccc aggcgatgtg gccggggttt
     6421 ttggtcatga ggcctgagta actgcggtcg ccatcgacgg cgcgccgaag gccttcggcg
     6481 cacgccgcca tgtatgcgag cggcttacgc cgcgcgtatt cggtgcgtgg aacaggggcg
     6541 ttgagtgccc acactgcgtg tgcgtggccg ttggcgcgat tgcccacgat cgcgttgggc
     6601 agcggatggg acccccgggc gctgagcgct cggagcgctg cgtctggatg gtctacgtcc
     6661 acgaccagca ggtttgccag cgctgttggg ttcgcctcga tgtaccggcg gcctagggcc
     6721 gacgcgcggc tttggcggta gatcccctcg agcagatcgt cgcttgccag cggccagtac
     6781 ggcagccaga gctgctcaaa ttcgtcggcg acgtggctca cgcttggtag tagaccacga
     6841 ttaatcaccg gtgtatggtc cgacacgagc tccaagtcag atatttcgct gaggggccac
     6901 cccacaactg cacactcccc cgctctcccg tcgagccctg gtggtggaac accagcgaca
     6961 gccgagcacc cccaaccacc tgtaccaacc aggaggaaca catgcgtcgt ttcgaggacg
     7021 tttccgggcc gctgagagcc gctgtggcgg ccgtacacgc cgccttagac ccgttagacc
     7081 ccctgccgcc tgaatgcgcg ggtacgagcc acacagcgcc cgaacttacg gagctggtgg
     7141 gctcacgcgt tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     7201 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     7261 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     7321 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     7381 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     7441 ctgccgctta ccggatacct gtccgccttt ctcccctcgg gaagcgtggc gctttctcat
     7501 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     7561 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     7621 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     7681 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     7741 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     7801 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7861 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7921 tctgacgctc atggaactag tgaggtctgc ctcgtgaaga aggtgttgct gactcatacc
     7981 aggcctgaat cgccccatca tccagccaga aagtgaggga gccacggttg gtgagagctt
     8041 tgttgtaggt ggaccagttg gtgattttga acttttgctt tgccacggaa cggtctgcgt
     8101 tgtcgggaag atgcgtgatc tgatccttca actcagcaaa agttcgattt attcaacaaa
     8161 gccgccgtcc cgtcaagtca gcgtaatgct ctgccagtgt tacaaccaat taaccaattc
     8221 tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     8281 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     8341 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     8401 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     8461 gactgaatcc ggtgagaatg gcaaaagatt atgcatttct ttccagactt gttcaacagg
     8521 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     8581 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     8641 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     8701 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     8761 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     8821 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     8881 aaacaactct ggcgcatggg gcttcccata caagcgatag attgtcgcac ctgattgccc
     8941 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     9001 cggcctcgag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     9061 tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca
     9121 tcagagattt tgagacacaa cgtggcttt
//
