LOCUS       sequence-351051-        7827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AsCas12f1-based E. coli genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2021 Sep 2. pii: 10.1038/s41589-021-00868-6. doi: 
            10.1038/s41589-021-00868-6."
COMMENT     Sequence Label: p15a-AsCas12f-blaZ
FEATURES             Location/Qualifiers
     source          1..7827
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             join(1721..1789,1790..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
//
