LOCUS Exported 7827 bp ds-DNA circular SYN 23-DEC-2021 DEFINITION AsCas12f1-based E. coli genome editing. ACCESSION . VERSION . KEYWORDS p15a-AsCas12f-blaZ SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7827) AUTHORS Wu Z, Zhang Y, Yu H, Pan D, Wang Y, Wang Y, Li F, Liu C, Nan H, Chen W, Ji Q TITLE Programmed genome editing by a miniature CRISPR-Cas12f nuclease. JOURNAL Nat Chem Biol. 2021 Sep 2. pii: 10.1038/s41589-021-00868-6. doi: 10.1038/s41589-021-00868-6. PUBMED 34475565 REFERENCE 2 (bases 1 to 7827) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 23, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7827 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(366..383) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(500..1045) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(580..599) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 1163..1265 /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" primer_bind complement(1332..1351) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 1721..2581 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1939..1958) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" CDS complement(2811..3689) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" promoter 3716..4000 /gene="araBAD" /label=araBAD promoter /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)" primer_bind 3923..3942 /label=pBAD Forward /note="For vectors with E. coli araBAD promoter, forward primer" CDS 4059..4475 /codon_start=1 /gene="gam" /product="inhibitor of the host RecBCD nuclease in the lambda Red system" /label=Gam /note="protects the double-stranded DNA substrate for recombination" /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV" CDS 4481..5266 /codon_start=1 /gene="bet" /product="single-stranded DNA binding recombinase in the lambda Red system" /label=Beta /note="mediates pairing of a single-stranded DNA with a complementary target" /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA" CDS 5263..5943 /codon_start=1 /gene="exo" /product="5' to 3' double-stranded DNA exonuclease in the lambda Red system" /label=Exo /note="creates 3' single-stranded DNA tails" /translation="MTPDIILQRTGIDVRAVEQGEDAWHKLRLGVITASEVHNVIAKPR SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG EQWR" ORIGIN 1 tatggacagt tttccctttg atatgtaacg gtgaacagtt gttctacttt tgtttgttag 61 tcttgatgct tcactgatag atacaagagc gtcgggtgat gctgccaact tactgattta 121 gtgtatgatg gtgtttttga ggtgctccag tggcttctgt ttctatcagc tgtccctcct 181 gttcagctac tgacggggtg gtgcgtaacg gcaaaagcac cgccggacat cagcgctagc 241 ggagtgtata ctggcttact atgttggcac tgatgagggt gtcagtgaag tgcttcatgt 301 ggcaggagaa aaaaggctgc accggtgcgt cagcagaata tgtgatacag gatatattcc 361 gcttcctcgc tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct 421 tacgaacggg gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga 481 gggccgcggc aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct 541 gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc 601 ctggcggctc cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc 661 tgttatggcc gcgtttgtct cattccacgc ctgacactca gttccgggta ggcagttcgc 721 tccaagctgg actgtatgca cgaacccccc gttcagtccg accgctgcgc cttatccggt 781 aactatcgtc ttgagtccaa cccggaaaga catgcaaaag caccactggc agcagccact 841 ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag 901 gacaagtttt ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc 961 tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat 1021 tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt aatcagataa aatatttcta 1081 gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca tacgatataa 1141 gttgtgacgt aaaatgagac gttgatcggc acgtaagagg ttccaacttt caccataatg 1201 aaataagatc actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag 1261 ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta 1321 aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc 1381 tgggtcatcc accggatcaa ttcccctgct cgcgcaggct gggtgccaag ctctcgggta 1441 acatcaaggc ccgatccttg gagcccttgc cctcccgcac gatgatcgtg ccgtgatcga 1501 aatccagatc cttgacccgc agttgcaaac cctcactgat ccggctcacg gtaactgatg 1561 ccgtatttgc agtaccagcg tacggcccac agaatgatgt cacgctgaaa atgccggcct 1621 ttgaatgggt tcatgtgcag ctccatcagc aaaaggggat gataagttta tcaccaccga 1681 ctatttgcaa cagtgccgtt gatcgtgcta tgatcgactg atgagtattc aacatttccg 1741 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 1801 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 1861 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 1921 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga 1981 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 2041 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 2101 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 2161 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 2221 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 2281 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 2341 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 2401 gtttattgct gataaatctg gagccggtga gcgtgggtcg cgcggtatca ttgcagcact 2461 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 2521 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 2581 atgagctcat gagcggagaa cgagatgacg ttggaggggc aaggtcgcgc tgattgctgg 2641 ggcaacacgt gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta acggtacccg 2701 gggatcctct agagtcgacc tgcataatgt gcctgtcaaa tggacgaagc agggattctg 2761 caaaccctat gctactccgt caagccgtca attgtctgat tcgttaccaa ttatgacaac 2821 ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct cgggctggcc 2881 ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc aacattgcga 2941 ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg gctgatacgt 3001 tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag atgtgacaga 3061 cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt gctgtctgcc 3121 aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg tggatggagc 3181 gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt tatcgccagc 3241 agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa caggtcgctg 3301 aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa tattgacggc 3361 cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg gtgataccat 3421 tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa cagcaaaata 3481 tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg accgcgaatg 3541 gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa atcgagataa 3601 ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc 3661 agcaggggat cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa 3721 accaattgtc catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg 3781 ctaaccaaac cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc 3841 atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat 3901 ttgcacggcg tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct 3961 acctgacgct ttttatcgca actctctact gtttctccat acccgttttt ttgggctagc 4021 gaaaagatgt ttcgtgaagc cgtcgacgct tataaaaaat ggatattaat actgaaactg 4081 agatcaagca aaagcattca ctaaccccct ttcctgtttt cctaatcagc ccggcatttc 4141 gcgggcgata ttttcacagc tatttcagga gttcagccat gaacgcttat tacattcagg 4201 atcgtcttga ggctcagagc tgggcgcgtc actaccagca gctcgcccgt gaagagaaag 4261 aggcagaact ggcagacgac atggaaaaag gcctgcccca gcacctgttt gaatcgctat 4321 gcatcgatca tttgcaacgc cacggggcca gcaaaaaatc cattacccgt gcgtttgatg 4381 acgatgttga gtttcaggag cgcatggcag aacacatccg gtacatggtt gaaaccattg 4441 ctcaccacca ggttgatatt gattcagagg tataaaacga atgagtactg cactcgcaac 4501 gctggctggg aagctggctg aacgtgtcgg catggattct gtcgacccac aggaactgat 4561 caccactctt cgccagacgg catttaaagg tgatgccagc gatgcgcagt tcatcgcatt 4621 actgatcgtt gccaaccagt acggccttaa tccgtggacg aaagaaattt acgcctttcc 4681 tgataagcag aatggcatcg ttccggtggt gggcgttgat ggctggtccc gcatcatcaa 4741 tgaaaaccag cagtttgatg gcatggactt tgagcaggac aatgaatcct gtacatgccg 4801 gatttaccgc aaggaccgta atcatccgat ctgcgttacc gaatggatgg atgaatgccg 4861 ccgcgaacca ttcaaaactc gcgaaggcag agaaatcacg gggccgtggc agtcgcatcc 4921 caaacggatg ttacgtcata aagccatgat tcagtgtgcc cgtctggcct tcggatttgc 4981 tggtatctat gacaaggatg aagccgagcg cattgtcgaa aatactgcat acactgcaga 5041 acgtcagccg gaacgcgaca tcactccggt taacgatgaa accatgcagg agattaacac 5101 tctgctgatc gccctggata aaacatggga tgacgactta ttgccgctct gttcccagat 5161 atttcgccgc gacattcgtg catcgtcaga actgacacag gccgaagcag taaaagctct 5221 tggattcctg aaacagaaag ccgcagagca gaaggtggca gcatgacacc ggacattatc 5281 ctgcagcgta ccgggatcga tgtgagagct gtcgaacagg gggaagatgc gtggcacaaa 5341 ttacggctcg gcgtcatcac cgcttcagaa gttcacaacg tgatagcaaa accccgctcc 5401 ggaaagaagt ggcctgacat gaaaatgtcc tacttccaca ccctgcttgc tgaggtttgc 5461 accggtgtgg ctccggaagt taacgctaaa gcactggcct ggggaaaaca gtacgagaac 5521 gacgccagaa ccctgtttga attcacttcc ggcgtgaatg ttactgaatc cccgatcatc 5581 tatcgcgacg aaagtatgcg taccgcctgc tctcccgatg gtttatgcag tgacggcaac 5641 ggccttgaac tgaaatgccc gtttacctcc cgggatttca tgaagttccg gctcggtggt 5701 ttcgaggcca taaagtcagc ttacatggcc caggtgcagt acagcatgtg ggtgacgcga 5761 aaaaatgcct ggtactttgc caactatgac ccgcgtatga agcgtgaagg cctgcattat 5821 gtcgtgattg agcgggatga gaagtacatg gcgagttttg acgagatcgt gccggagttc 5881 atcgaaaaaa tggacgaggc actggctgaa attggttttg tatttgggga gcaatggcga 5941 tgacgcatcc tcacgataat aagcttcctg ctgaacatca aaggcaagaa aacatctgtt 6001 gtcaaagaca gcatccttga acaaggacaa ttaacagtta acaaataaaa acgcaaaaga 6061 aaatgccgat attgactacc ggaagcagtg tgaccgtgtg cttctcaaat gcctgattca 6121 ggctgtctat gtgtgactgt tgagctgtaa caagttgtct caggtgttca atttcatgtt 6181 ctagttgctt tgttttactg gtttcacctg ttctattagg tgttacatgc tgttcatctg 6241 ttacattgtc gatctgttca tggtgaacag ctttaaatgc accaaaaact cgtaaaagct 6301 ctgatgtatc tatctttttt acaccgtttt catctgtgca tatagctata ctgatttcgt 6361 cagactcaca gtcaaacatg ccggtcagtt ggcctggtga tggcgggatc gttgtatatt 6421 tcttgacacc ttttcggcac cgccctaaaa ttctgcgtcc tcataatata tgaggcgatt 6481 tattacgtgt ttacgaagca aaagctaaaa ccaggagcta tttaatgatc aaagtttacc 6541 gttatgaaat cgttaaaccg ctggacctgg attggaaaga attcggcacc atcctgcgtc 6601 agctgcagca ggaaacccgc ttcgcgctga acaaagcaac ccagctggcg tgggaatgga 6661 tgggtttcag ctctgattac aaagacaacc atggcgaata cccgaaaagc aaagacatcc 6721 tgggctacac caacgttcac ggttacgcat accacaccat caaaaccaaa gcttatcgtc 6781 tgaacagcgg caacctgagc cagaccatca aacgtgcgac cgatcgcttc aaagcgtacc 6841 agaaagaaat cctgcgtggt gacatgtcca tcccgtctta caaacgtgat atcccgctgg 6901 atctgatcaa agaaaacatc agcgttaacc gtatgaacca cggtgattat atcgcttccc 6961 tgtccctgct gtctaacccg gcgaaacagg aaatgaacgt taaacgtaaa atctccgtga 7021 ttatcatcgt tcgtggcgcg ggtaaaacca tcatggatcg catcctgagc ggcgaatacc 7081 aggtgagcgc gagccagatc atccacgacg accgtaaaaa caaatggtac ctgaacatct 7141 cttatgattt cgaaccgcag acccgtgttc tggatctgaa caaaatcatg ggcatcgatc 7201 tgggtgttgc ggtggcggtg tacatggcgt tccagcacac cccggcgcgc tacaaactgg 7261 aaggcggtga aatcgaaaac ttccgtcgtc aggtggagtc tcgtcgcatc tccatgctgc 7321 gccagggtaa atacgcgggt ggtgctcgtg gcggtcacgg ccgtgataaa cgtatcaaac 7381 cgatcgaaca gctgcgtgac aaaatcgcta acttccgcga taccactaac caccgttact 7441 ctcgttacat cgttgatatg gctatcaaag aaggttgcgg taccattcag atggaagatc 7501 tgaccaacat ccgtgatatc ggctctcgtt tcctgcagaa ctggacctac tacgatctgc 7561 agcagaaaat catctacaaa gcggaagaag cgggcatcaa agttatcaaa attgatccgc 7621 agtacaccag ccagcgttgc agcgaatgcg gcaacatcga ttccggtaac cgtatcggtc 7681 aggcgatctt caaatgccgt gcttgcggct acgaagcgaa cgcggactac aacgcagctc 7741 gtaacatcgc gatcccgaac atcgataaaa tcatcgcgga aagcatcaaa taatggctgg 7801 ttggcgtact gttgtggtaa ataccca //