LOCUS       Exported                9149 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  expresses codon-optimized FnCpf1 .
ACCESSION   .
VERSION     .
KEYWORDS    pMV261-Cas12a
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 May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9149
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    complement(1..34)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(51..674)
                     /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     582..601
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    989..1006
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    complement(5022..5055)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     rep_origin      complement(7145..7733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7225..7244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(8064..8879)
                     /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     8787..8806
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8871..8890
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      9040..9086
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ataacttcgt ataatgtatg ctatacgaag ttattaattg cccatggtca ttaagaccca
       61 ctttcacatt taagttgttt ttctaatccg cagatgatca attcaaggcc gaataagaag
      121 gctggctctg caccttggtg atcaaataat tcgatagctt gtcgtaataa tggcggcata
      181 ctatcagtag taggtgtttc cctttcttct ttagcgactt gatgctcttg atcttccaat
      241 acgcaaccta aggtaaaatg ccccacagcg ctgagtgcat ataacgcgtt ctctagtgaa
      301 aaaccttgtt ggcataaaaa ggctaattga ttttcgagag tttcatactg tttttctgta
      361 ggccgtgtac ctaaatgtac ttttgctcca tcgcgatgac ttagtaaagc acatctaaaa
      421 cttttagcgt tattacgcaa aaaatcttgc cagctttccc cttctaaagg gcaaaagtga
      481 gtatggtgcc tatctaacat ctcaatggct aaggcgtcga gcagggctcg cttatttttt
      541 acatgccaat acaatgtagg ctgctctaca ccaagtttct gggcgagttt acgggttgtt
      601 aaaccttcga ttccgacctc attaagcagc tctaatgcgc tgttaatcac tttactttta
      661 tctaatctag acattgcgaa gtgattcctc cgatccgtgg caggagggtc gcctgcgtcg
      721 gcaactcgcc caggctacac gagagccctc gactgcgaaa atgccgacca ggcaccaccg
      781 cactccaggc ttaacccggt gcgacgatcc aatattaata ttggatcgtc ggcaccgtca
      841 cggccgtggg aggcggcacg atccgcgacg tgatgatcgg ccgcatcccc acggtgctgc
      901 gcagtgagct ctacgccatc ccggcgttga tctgtgcgtt cgcacgcaca ggcccggtgt
      961 gagaagggtc tctgacgagc gggagaactc cctatcagtg atagagtttg tcctccctat
     1021 cagtgataga taggctctgg gagtacccgt gtgtacgacc agcacggcat acatcatttc
     1081 gacgccgaga gattcgccgc ccgaaatgag cacgatccat gtcgatctac caagagttcg
     1141 tgaataagta tagcctgagc aagaccttgc ggttcgagtt gatcccgcag ggcaaaacct
     1201 tggaaaacat taaggcgcgc gggctgatcc tggatgatga gaagcgggcc aaggactata
     1261 aaaaggccaa gcagattatc gataaatatc atcagttctt catcgaggaa atcctgtcga
     1321 gcgtgtgcat cagcgaagac ttgttgcaga actatagcga cgtgtacttc aaattgaaaa
     1381 agtcggacga cgacaatctg caaaaagact tcaagtcggc caaagacacc atcaagaaac
     1441 agatttcgga gtacatcaag gactcggaaa agttcaaaaa cctgttcaac cagaacttga
     1501 tcgacgcgaa aaagggccag gaaagcgatt tgatcttgtg gctgaagcag tcgaaggata
     1561 atggcattga gttgttcaag gccaactcgg acattacgga cattgacgag gccctggaaa
     1621 tcattaaatc gtttaagggc tggaccacgt acttcaaggg cttccatgag aaccggaaaa
     1681 acgtgtattc gtcgaacgac attcccacgt cgatcatcta ccggatcgtc gatgacaact
     1741 tgcccaagtt cttggaaaat aaagcgaaat acgagagctt gaaggacaaa gcgccggagg
     1801 ccattaacta tgagcagatc aagaaggacc tggccgaaga gctgaccttt gacattgact
     1861 acaagaccag cgaggtcaat cagcgggtct tctcgttgga tgaggtcttt gaaatcgcca
     1921 atttcaacaa ctatttgaac cagagcggca tcaccaaatt taataccatt atcggcggga
     1981 agttcgtcaa cggcgagaac acgaagcgga aggggatcaa cgaatacatc aatctgtact
     2041 cgcagcagat caacgacaag accctgaaaa agtacaagat gagcgtgctg ttcaagcaaa
     2101 ttctgagcga caccgagagc aagtcgttcg tcattgacaa gctggaggat gacagcgatg
     2161 tggtcaccac gatgcagtcg ttttacgagc aaatcgcggc cttcaagacc gtggaggaaa
     2221 agagcatcaa ggaaaccctg agcctgctgt tcgatgatct gaaggcgcag aagttggatc
     2281 tgtcgaaaat ctacttcaag aacgataagt cgctgaccga cctgagccaa caggtcttcg
     2341 atgattacag cgtcatcggg accgccgtcc tggagtacat cacccagcag attgcgccca
     2401 aaaacctgga caatccctcg aaaaaagagc aggagctgat tgccaagaag accgagaagg
     2461 cgaagtactt gtcgctggaa accatcaaac tggcgctgga ggagttcaac aagcaccgcg
     2521 acattgataa acaatgccgg ttcgaggaga ttctggccaa ttttgccgcc attcccatga
     2581 tcttcgatga gatcgcgcag aataaagaca acctggcgca aatctcgatc aaatatcaga
     2641 atcagggcaa aaaagacctg ttacaagcct cggccgaaga cgacgtgaaa gccatcaagg
     2701 acctgttgga tcagacgaac aacttgttgc acaagctgaa aattttccac atcagccaga
     2761 gcgaggacaa ggccaacatc ttggacaaag acgagcactt ctacttggtg ttcgaagagt
     2821 gctacttcga attggccaac atcgtccccc tgtacaataa gattcgcaat tatattacgc
     2881 agaagcccta tagcgacgag aaattcaagt tgaattttga aaattcgacc ctggcgaatg
     2941 gctgggacaa gaacaaagag cccgacaaca ccgccatctt gttcatcaag gatgataaat
     3001 attatctggg ggtgatgaac aaaaagaata acaagatctt tgatgacaag gccatcaagg
     3061 agaataaggg cgaggggtac aagaaaatcg tctacaaact gttgcccggc gccaacaaga
     3121 tgttgccgaa ggtcttcttc agcgcgaaga gcattaaatt ctataacccc agcgaggaca
     3181 tcctgcgcat ccggaatcat tcgacccaca cgaagaatgg cagcccgcaa aagggctacg
     3241 agaagttcga gtttaacatc gaggattgtc ggaaattcat cgacttctat aaacagtcga
     3301 tcagcaagca ccccgagtgg aaggatttcg gcttccgctt cagcgatacc caacgctaca
     3361 acagcatcga cgagttttat cgggaagtgg aaaaccaggg gtacaagttg accttcgaga
     3421 acatttcgga gtcgtacatt gactcggtcg tcaaccaagg gaaactgtac ctgttccaaa
     3481 tctacaataa ggacttcagc gcgtactcga aagggcggcc caacttgcac accctgtatt
     3541 ggaaggcctt gtttgacgag cggaacttac aagacgtcgt ctataaactg aacggggagg
     3601 cggagctgtt ttaccgcaag cagtcgatcc cgaaaaagat tacccacccg gcgaaggagg
     3661 cgatcgccaa caaaaacaag gacaacccga agaaagagtc ggtcttcgaa tacgatctga
     3721 tcaaagacaa gcgctttacc gaggacaagt tctttttcca ttgtccgatc accatcaatt
     3781 ttaagagcag cggcgcgaac aagttcaacg acgagatcaa cctgctgttg aaagaaaagg
     3841 ccaatgatgt gcacatcctg tcgatcgacc ggggggaacg gcacctggcc tactacaccc
     3901 tggtggacgg caagggcaac atcatcaagc aggatacctt caatatcatt gggaacgacc
     3961 ggatgaaaac gaactaccac gacaagctgg cggccatcga aaaggatcgc gacagcgccc
     4021 gcaaggactg gaagaagatt aacaatatca aggagatgaa ggaggggtac ttgagccagg
     4081 tcgtgcacga gatcgccaag ctggtcattg agtacaacgc gattgtggtc tttgaagacc
     4141 tgaactttgg cttcaaacgg gggcggttca aggtcgagaa gcaggtgtat cagaagctgg
     4201 aaaaaatgtt gatcgagaag ctgaactatc tggtgttcaa agacaacgag tttgacaaaa
     4261 cggggggcgt cctgcgggcc taccagttga ccgccccctt cgaaaccttc aagaaaatgg
     4321 gcaagcagac gggcatcatc tactacgtcc cggcgggctt tacgagcaag atctgtccgg
     4381 tgaccggctt cgtcaaccag ctgtatccga agtatgagag cgtgtcgaaa agccaggagt
     4441 ttttctcgaa gttcgataaa atctgttata acctggataa ggggtacttc gaattttcgt
     4501 tcgattacaa aaacttcggg gacaaggcgg ccaaggggaa atggacgatc gccagcttcg
     4561 gctcgcgcct gattaacttt cgcaactcgg acaagaacca taactgggac acccgggaag
     4621 tgtacccgac caaagaattg gaaaaactgt tgaaggacta tagcatcgag tacgggcacg
     4681 gggagtgcat caaagccgcc atctgcgggg aaagcgacaa gaagtttttt gcgaagctga
     4741 ccagcgtgct gaatacgatc ttacaaatgc gtaattctaa gaccggcacc gagctggact
     4801 acctgatctc gccggtggcg gatgtcaacg gcaacttctt cgacagccgc caggccccga
     4861 agaacatgcc gcaggacgcg gatgcgaatg gcgcgtacca catcgggctg aaagggctga
     4921 tgctgctggg gcggatcaag aacaaccagg aggggaagaa gctgaatctg gtgattaaga
     4981 acgaggaata tttcgagttc gtccaaaatc gcaacaattg aataacttcg tataatgtat
     5041 gctatacgaa gttattaatt ctgcagagtc ctactagtat ttgatatcgg taccgcggcc
     5101 gcgccacgga tgccaccaca ggcactacaa cggagttcgc cacgtacatc accacaacca
     5161 ccgattctgg cggtgagctc cccgatattc agcggaaatg gcttggtatc gaccaagatt
     5221 cgtagaaccc cgtctcgtct ggctggtatt caaaacgggc gcaacgaaac acgcaacgag
     5281 acaggcatgg cccaaaccag aaaactagcg tctaccagga cttttacctg tccgacccgt
     5341 tgcaacggaa ccccccacgg aacccccgcg acacccgctc cccaattgcg ttagaacagc
     5401 ggtggattgt cggcttcgtt gtgggccttt tgagccgctt cctgttctgc cgcacgctct
     5461 ttcctcgccc gatagccgag tcgcttaacg gtgtccagat gcagcccgaa atgtttggcc
     5521 gtttgcggcc aagagtggcc ctcgtcgtcg tgataggcgc ggatgcgttc gcggcgtgca
     5581 gcctgctcgg cgagccactc gctgcgttcc tgcgccacga gccggacgac gtggcgttcg
     5641 gatagtccgg tgattcgagc gccttcggcg gcggtcacgc gccgcttttt gcggacagtc
     5701 ggctgccggt tgtagccgtc gctgtagccg tcgctcatag caatgcctcc atggctgacg
     5761 cggactttgc gcgccgcgca actgtgctcg ccgccgtgcg cgctgctgcg cccttccgcg
     5821 agatggccga ctggcgcgca ctgagtgtgg cctcgtagac cacgatcccg tccgcccaaa
     5881 tgcgcgactt ggttgtgatc caacgccaaa tgctgttggc gatggcgcgg acctcgctgt
     5941 ccggtagcgg tccgggacac acgtcgttgc acggaaattc ggcgtttcgc gcgtggcact
     6001 cggcatagat cgcgcggccg agtccgtcca cgttccgggt cggcaggtag atccgcatga
     6061 gggcgggacg ataggcccac aacctgacgg aatcgaacag tgcgcaattc cgccctagcg
     6121 gcgtcggagc cgctttgtac gtggtctgct gacgccagcg cggcggtggc atgttcgcgc
     6181 cgagctcggc ctcgatgtgg ctgagtgtgt agagatctga gtggagccat tccgtttccc
     6241 aggcgatgtg gccggggttt ttggtcatga ggcctgagta actgcggtcg ccatcgacgg
     6301 cgcgccgaag gccttcggcg cacgccgcca tgtatgcgag cggcttacgc cgcgcgtatt
     6361 cggtgcgtgg aacaggggcg ttgagtgccc acactgcgtg tgcgtggccg ttggcgcgat
     6421 tgcccacgat cgcgttgggc agcggatggg acccccgggc gctgagcgct cggagcgctg
     6481 cgtctggatg gtctacgtcc acgaccagca ggtttgccag cgctgttggg ttcgcctcga
     6541 tgtaccggcg gcctagggcc gacgcgcggc tttggcggta gatcccctcg agcagatcgt
     6601 cgcttgccag cggccagtac ggcagccaga gctgctcaaa ttcgtcggcg acgtggctca
     6661 cgcttggtag tagaccacga ttaatcaccg gtgtatggtc cgacacgagc tccaagtcag
     6721 atatttcgct gaggggccac cccacaactg cacactcccc cgctctcccg tcgagccctg
     6781 gtggtggaac accagcgaca gccgagcacc cccaaccacc tgtaccaacc aggaggaaca
     6841 catgcgtcgt ttcgaggacg tttccgggcc gctgagagcc gctgtggcgg ccgtacacgc
     6901 cgccttagac ccgttagacc ccctgccgcc tgaatgcgcg ggtacgagcc acacagcgcc
     6961 cgaacttacg gagctggtgg gctcacgcgt tgcgctcggt cgttcggctg cggcgagcgg
     7021 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     7081 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     7141 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     7201 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     7261 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccctcgg
     7321 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     7381 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     7441 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     7501 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     7561 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     7621 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     7681 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     7741 ctttgatctt ttctacgggg tctgacgctc atggaactag tgaggtctgc ctcgtgaaga
     7801 aggtgttgct gactcatacc aggcctgaat cgccccatca tccagccaga aagtgaggga
     7861 gccacggttg gtgagagctt tgttgtaggt ggaccagttg gtgattttga acttttgctt
     7921 tgccacggaa cggtctgcgt tgtcgggaag atgcgtgatc tgatccttca actcagcaaa
     7981 agttcgattt attcaacaaa gccgccgtcc cgtcaagtca gcgtaatgct ctgccagtgt
     8041 tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat
     8101 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     8161 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     8221 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     8281 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagatt atgcatttct
     8341 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     8401 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     8461 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     8521 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     8581 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     8641 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     8701 ctacctttgc catgtttcag aaacaactct ggcgcatggg gcttcccata caagcgatag
     8761 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     8821 tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat atggctcata
     8881 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     8941 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttc cccccccccc
     9001 gacgtcaggt ggctagctga tcaccgcggc catgatggca taaaacgaaa ggcccagtct
     9061 ttcgactgag cctttcgttt tatttgatgc ctggcagtcg atcgtacgct agttaactac
     9121 gtcgacatcg ataagcttgg cgtgaattc
//
