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" terminator 51..97 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" protein_bind complement(161..194) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." 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 1149..1166 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind complement(5182..5215) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." 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 //