LOCUS       Exported                7767 bp ds-DNA     circular SYN 10-SEP-2021
DEFINITION  AsCas12f1-based K. pneumoniae genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    p15a-AsCas12f-apmR
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7767)
  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 7767)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 10, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7767
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(400..417)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(534..1079)
                     /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(614..633)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1197..1299
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1366..1385)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1755..2555
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     CDS             complement(2785..3663)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3690..3974
                     /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     3897..3916
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             4033..4449
                     /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             4455..5240
                     /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             5237..5917
                     /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 tggctggttg gcgtactgtt gtggtaaata cccatatgga cagttttccc tttgatatgt
       61 aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg atagatacaa
      121 gagcgtcggg tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct
      181 ccagtggctt ctgtttctat cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt
      241 aacggcaaaa gcaccgccgg acatcagcgc tagcggagtg tatactggct tactatgttg
      301 gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt
      361 gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg
      421 ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga
      481 agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat
      541 aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac
      601 ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct
      661 gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc
      721 acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc
      781 ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga
      841 aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct
      901 tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc
      961 caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct
     1021 gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag
     1081 aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa
     1141 atgtagcacc tgaagtcagc cccatacgat ataagttgtg acgtaaaatg agacgttgat
     1201 cggcacgtaa gaggttccaa ctttcaccat aatgaaataa gatcactacc gggcgtattt
     1261 tttgagttat cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga
     1321 tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca
     1381 gttgctcaat gtacctataa ccagaccgtt cagctgggtc atccaccgga tcaattcccc
     1441 tgctcgcgca ggctgggtgc caagctctcg ggtaacatca aggcccgatc cttggagccc
     1501 ttgccctccc gcacgatgat cgtgccgtga tcgaaatcca gatccttgac ccgcagttgc
     1561 aaaccctcac tgatccggct cacggtaact gatgccgtat ttgcagtacc agcgtacggc
     1621 ccacagaatg atgtcacgct gaaaatgccg gcctttgaat gggttcatgt gcagctccat
     1681 cagcaaaagg ggatgataag tttatcacca ccgactattt gcaacagtgc cgttgatcgt
     1741 gctatgatcg actgatgtca tcagcggtgg agtgcaatgt cgtgcaatac gaatggcgaa
     1801 aagccgagct catcggtcag cttctcaacc ttggggttac ccccggcggt gtgctgctgg
     1861 tccacagctc cttccgtagc gtccggcccc tcgaagatgg gccacttgga ctgatcgagg
     1921 ccctgcgtgc tgcgctgggt ccgggaggga cgctcgtcat gccctcgtgg tcaggtctgg
     1981 acgacgagcc gttcgatcct gccacgtcgc ccgttacacc ggaccttgga gttgtctctg
     2041 acacattctg gcgcctgcca aatgtaaagc gcagcgccca tccatttgcc tttgcggcag
     2101 cggggccaca ggcagagcag atcatctctg atccattgcc cctgccacct cactcgcctg
     2161 caagcccggt cgcccgtgtc catgaactcg atgggcaggt acttctcctc ggcgtgggac
     2221 acgatgccaa cacgacgctg catcttgccg agttgatggc aaaggttccc tatggggtgc
     2281 cgagacactg caccattctt caggatggca agttggtacg cgtcgattat ctcgagaatg
     2341 accactgctg tgagcgcttt gccttggcgg acaggtggct caaggagaag agccttcaga
     2401 aggaaggtcc agtcggtcat gcctttgctc ggttgatccg ctcccgcgac attgtggcga
     2461 cagccctggg tcaactgggc cgagatccgt tgatcttcct gcatccgcca gaggcgggat
     2521 gcgaagaatg cgatgccgct cgccagtcga ttggctgagc tcatgagcgg agaacgagat
     2581 gacgttggag gggcaaggtc gcgctgattg ctggggcaac acgtgaaagg cgagatcacc
     2641 aaggtagtcg gcaaataatg tctaacggta cccggggatc ctctagagtc gacctgcata
     2701 atgtgcctgt caaatggacg aagcagggat tctgcaaacc ctatgctact ccgtcaagcc
     2761 gtcaattgtc tgattcgtta ccaattatga caacttgacg gctacatcat tcactttttc
     2821 ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga
     2881 gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt
     2941 ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgct
     3001 aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctg
     3061 tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc
     3121 ctcgcgtacc cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg
     3181 cagtaacaat tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg
     3241 cccggcgtta atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg
     3301 gcgaaagaac cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc
     3361 gcgcggacga aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgta
     3421 gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg
     3481 tccctgattt ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat
     3541 tcccagcggt cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc
     3601 cgccaccaga tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc
     3661 catacttttc atactcccgc cattcagaga agaaaccaat tgtccatatt gcatcagaca
     3721 ttgccgtcac tgcgtctttt actggctctt ctcgctaacc aaaccggtaa ccccgcttat
     3781 taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt
     3841 ctataatcac ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgc
     3901 catagcattt ttatccataa gattagcgga tcctacctga cgctttttat cgcaactctc
     3961 tactgtttct ccatacccgt ttttttgggc tagcgaaaag atgtttcgtg aagccgtcga
     4021 cgcttataaa aaatggatat taatactgaa actgagatca agcaaaagca ttcactaacc
     4081 ccctttcctg ttttcctaat cagcccggca tttcgcgggc gatattttca cagctatttc
     4141 aggagttcag ccatgaacgc ttattacatt caggatcgtc ttgaggctca gagctgggcg
     4201 cgtcactacc agcagctcgc ccgtgaagag aaagaggcag aactggcaga cgacatggaa
     4261 aaaggcctgc cccagcacct gtttgaatcg ctatgcatcg atcatttgca acgccacggg
     4321 gccagcaaaa aatccattac ccgtgcgttt gatgacgatg ttgagtttca ggagcgcatg
     4381 gcagaacaca tccggtacat ggttgaaacc attgctcacc accaggttga tattgattca
     4441 gaggtataaa acgaatgagt actgcactcg caacgctggc tgggaagctg gctgaacgtg
     4501 tcggcatgga ttctgtcgac ccacaggaac tgatcaccac tcttcgccag acggcattta
     4561 aaggtgatgc cagcgatgcg cagttcatcg cattactgat cgttgccaac cagtacggcc
     4621 ttaatccgtg gacgaaagaa atttacgcct ttcctgataa gcagaatggc atcgttccgg
     4681 tggtgggcgt tgatggctgg tcccgcatca tcaatgaaaa ccagcagttt gatggcatgg
     4741 actttgagca ggacaatgaa tcctgtacat gccggattta ccgcaaggac cgtaatcatc
     4801 cgatctgcgt taccgaatgg atggatgaat gccgccgcga accattcaaa actcgcgaag
     4861 gcagagaaat cacggggccg tggcagtcgc atcccaaacg gatgttacgt cataaagcca
     4921 tgattcagtg tgcccgtctg gccttcggat ttgctggtat ctatgacaag gatgaagccg
     4981 agcgcattgt cgaaaatact gcatacactg cagaacgtca gccggaacgc gacatcactc
     5041 cggttaacga tgaaaccatg caggagatta acactctgct gatcgccctg gataaaacat
     5101 gggatgacga cttattgccg ctctgttccc agatatttcg ccgcgacatt cgtgcatcgt
     5161 cagaactgac acaggccgaa gcagtaaaag ctcttggatt cctgaaacag aaagccgcag
     5221 agcagaaggt ggcagcatga caccggacat tatcctgcag cgtaccggga tcgatgtgag
     5281 agctgtcgaa cagggggaag atgcgtggca caaattacgg ctcggcgtca tcaccgcttc
     5341 agaagttcac aacgtgatag caaaaccccg ctccggaaag aagtggcctg acatgaaaat
     5401 gtcctacttc cacaccctgc ttgctgaggt ttgcaccggt gtggctccgg aagttaacgc
     5461 taaagcactg gcctggggaa aacagtacga gaacgacgcc agaaccctgt ttgaattcac
     5521 ttccggcgtg aatgttactg aatccccgat catctatcgc gacgaaagta tgcgtaccgc
     5581 ctgctctccc gatggtttat gcagtgacgg caacggcctt gaactgaaat gcccgtttac
     5641 ctcccgggat ttcatgaagt tccggctcgg tggtttcgag gccataaagt cagcttacat
     5701 ggcccaggtg cagtacagca tgtgggtgac gcgaaaaaat gcctggtact ttgccaacta
     5761 tgacccgcgt atgaagcgtg aaggcctgca ttatgtcgtg attgagcggg atgagaagta
     5821 catggcgagt tttgacgaga tcgtgccgga gttcatcgaa aaaatggacg aggcactggc
     5881 tgaaattggt tttgtatttg gggagcaatg gcgatgacgc atcctcacga taataagctt
     5941 cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa gacagcatcc ttgaacaagg
     6001 acaattaaca gttaacaaat aaaaacgcaa aagaaaatgc cgatattgac taccggaagc
     6061 agtgtgaccg tgtgcttctc aaatgcctga ttcaggctgt ctatgtgtga ctgttgagct
     6121 gtaacaagtt gtctcaggtg ttcaatttca tgttctagtt gctttgtttt actggtttca
     6181 cctgttctat taggtgttac atgctgttca tctgttacat tgtcgatctg ttcatggtga
     6241 acagctttaa atgcaccaaa aactcgtaaa agctctgatg tatctatctt ttttacaccg
     6301 ttttcatctg tgcatatagc tatactgatt tcgtcagact cacagtcaaa catgccggtc
     6361 agttggcctg gtgatggcgg gatcgttgta tatttcttga caccttttcg gcaccgccct
     6421 aaaattctgc gtcctcataa tatatgaggc gatttattac gtgtttacga agcaaaagct
     6481 aaaaccagga gctatttaat gatcaaagtt taccgttatg aaatcgttaa accgctggac
     6541 ctggattgga aagaattcgg caccatcctg cgtcagctgc agcaggaaac ccgcttcgcg
     6601 ctgaacaaag caacccagct ggcgtgggaa tggatgggtt tcagctctga ttacaaagac
     6661 aaccatggcg aatacccgaa aagcaaagac atcctgggct acaccaacgt tcacggttac
     6721 gcataccaca ccatcaaaac caaagcttat cgtctgaaca gcggcaacct gagccagacc
     6781 atcaaacgtg cgaccgatcg cttcaaagcg taccagaaag aaatcctgcg tggtgacatg
     6841 tccatcccgt cttacaaacg tgatatcccg ctggatctga tcaaagaaaa catcagcgtt
     6901 aaccgtatga accacggtga ttatatcgct tccctgtccc tgctgtctaa cccggcgaaa
     6961 caggaaatga acgttaaacg taaaatctcc gtgattatca tcgttcgtgg cgcgggtaaa
     7021 accatcatgg atcgcatcct gagcggcgaa taccaggtga gcgcgagcca gatcatccac
     7081 gacgaccgta aaaacaaatg gtacctgaac atctcttatg atttcgaacc gcagacccgt
     7141 gttctggatc tgaacaaaat catgggcatc gatctgggtg ttgcggtggc ggtgtacatg
     7201 gcgttccagc acaccccggc gcgctacaaa ctggaaggcg gtgaaatcga aaacttccgt
     7261 cgtcaggtgg agtctcgtcg catctccatg ctgcgccagg gtaaatacgc gggtggtgct
     7321 cgtggcggtc acggccgtga taaacgtatc aaaccgatcg aacagctgcg tgacaaaatc
     7381 gctaacttcc gcgataccac taaccaccgt tactctcgtt acatcgttga tatggctatc
     7441 aaagaaggtt gcggtaccat tcagatggaa gatctgacca acatccgtga tatcggctct
     7501 cgtttcctgc agaactggac ctactacgat ctgcagcaga aaatcatcta caaagcggaa
     7561 gaagcgggca tcaaagttat caaaattgat ccgcagtaca ccagccagcg ttgcagcgaa
     7621 tgcggcaaca tcgattccgg taaccgtatc ggtcaggcga tcttcaaatg ccgtgcttgc
     7681 ggctacgaag cgaacgcgga ctacaacgca gctcgtaaca tcgcgatccc gaacatcgat
     7741 aaaatcatcg cggaaagcat caaataa
//
