LOCUS       sequence-405078-        8037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  p15a-based plasmid containing a rpsL driven AcCas12n cassette and 
            the araBAD-driven lambda-red recombinase cassette. Apramycin 
            resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8037)
  AUTHORS   Chen W, Ma J, Wu Z, Wang Z, Zhang H, Fu W, Pan D, Shi J, Ji Q
  TITLE     Cas12n nucleases, early evolutionary intermediates of type V CRISPR,
            comprise a distinct family of miniature genome editors.
  JOURNAL   Mol Cell. 2023 Jun 27:S1097-2765(23)00463-X. doi: 
            10.1016/j.molcel.2023.06.014.
  PUBMED    37402371
REFERENCE   2  (bases 1 to 8037)
  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: "Mol Cell. 
            2023 Jun 27:S1097-2765(23)00463-X. doi: 
            10.1016/j.molcel.2023.06.014."
COMMENT     Sequence Label: p15a_AcCas12n
FEATURES             Location/Qualifiers
     source          1..8037
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(380..397)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(514..1059)
                     /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(594..613)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1177..1279
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1346..1365)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1735..2535
                     /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(2765..3643)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3670..3954
                     /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     3877..3896
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             4013..4429
                     /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             4435..5220
                     /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             5217..5897
                     /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 gtggtaaata cccatatgga cagttttccc tttgatatgt aacggtgaac agttgttcta
       61 cttttgtttg ttagtcttga tgcttcactg atagatacaa gagcgtcggg tgatgctgcc
      121 aacttactga tttagtgtat gatggtgttt ttgaggtgct ccagtggctt ctgtttctat
      181 cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt aacggcaaag gcaccgccgg
      241 acatcagcgc tagcggagtg tatactggct tactatgttg gcactgatga gggtgtcagt
      301 gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat
      361 acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc
      421 gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta
      481 acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa
      541 gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata
      601 ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac
      661 cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg
      721 ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct
      781 gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac
      841 tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa
      901 ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc
      961 aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt
     1021 tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag
     1081 ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
     1141 cccatacgat ataagttgtg acgtaaaatg agacgttgat cggcacgtaa gaggttccaa
     1201 ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat cgagattttc
     1261 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     1321 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     1381 ccagaccgtt cagctgggtc atccaccgga tcaattcccc tgctcgcgca ggctgggtgc
     1441 caagctctcg ggtaacatca aggcccgatc cttggagccc ttgccctccc gcacgatgat
     1501 cgtgccgtga tcgaaatcca gatccttgac ccgcagttgc aaaccctcac tgatccggct
     1561 cacggtaact gatgccgtat ttgcagtacc agcgtacggc ccacagaatg atgtcacgct
     1621 gaaaatgccg gcctttgaat gggttcatgt gcagctccat cagcaaaagg ggatgataag
     1681 tttatcacca ccgactattt gcaacagtgc cgttgatcgt gctatgatcg actgatgtca
     1741 tcagcggtgg agtgcaatgt cgtgcaatac gaatggcgaa aagccgagct catcggtcag
     1801 cttctcaacc ttggggttac ccccggcggt gtgctgctgg tccacagctc cttccgtagc
     1861 gtccggcccc tcgaagatgg gccacttgga ctgatcgagg ccctgcgtgc tgcgctgggt
     1921 ccgggaggga cgctcgtcat gccctcgtgg tcaggtctgg acgacgagcc gttcgatcct
     1981 gccacgtcgc ccgttacacc ggaccttgga gttgtctctg acacattctg gcgcctgcca
     2041 aatgtaaagc gcagcgccca tccatttgcc tttgcggcag cggggccaca ggcagagcag
     2101 atcatctctg atccattgcc cctgccacct cactcgcctg caagcccggt cgcccgtgtc
     2161 catgaactcg atgggcaggt acttctcctc ggcgtgggac acgatgccaa cacgacgctg
     2221 catcttgccg agttgatggc aaaggttccc tatggggtgc cgagacactg caccattctt
     2281 caggatggca agttggtacg cgtcgattat ctcgagaatg accactgctg tgagcgcttt
     2341 gccttggcgg acaggtggct caaggagaag agccttcaga aggaaggtcc agtcggtcat
     2401 gcctttgctc ggttgatccg ctcccgcgac attgtggcga cagccctggg tcaactgggc
     2461 cgagatccgt tgatcttcct gcatccgcca gaggcgggat gcgaagaatg cgatgccgct
     2521 cgccagtcga ttggctgagc tcatgagcgg agaacgagat gacgttggag gggcaaggtc
     2581 gcgctgattg ctggggcaac acgtgaaagg cgagatcacc aaggtagtcg gcaaataatg
     2641 tctaacggta cccggggatc ctctagagtc gacctgcata atgtgcctgt caaatggacg
     2701 aagcagggat tctgcaaacc ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta
     2761 ccaattatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
     2821 cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
     2881 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
     2941 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
     3001 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa
     3061 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
     3121 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
     3181 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
     3241 caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg
     3301 caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
     3361 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
     3421 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
     3481 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
     3541 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
     3601 acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccgc
     3661 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
     3721 actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
     3781 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
     3841 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
     3901 gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt
     3961 ttttttgggc tagcgaaaag atgtttcgtg aagccgtcga cgcttataaa aaatggatat
     4021 taatactgaa actgagatca agcaaaagca ttcactaacc ccctttcctg ttttcctaat
     4081 cagcccggca tttcgcgggc gatattttca cagctatttc aggagttcag ccatgaacgc
     4141 ttattacatt caggatcgtc ttgaggctca gagctgggcg cgtcactacc agcagctcgc
     4201 ccgtgaagag aaagaggcag aactggcaga cgacatggaa aaaggcctgc cccagcacct
     4261 gtttgaatcg ctatgcatcg atcatttgca acgccacggg gccagcaaaa aatccattac
     4321 ccgtgcgttt gatgacgatg ttgagtttca ggagcgcatg gcagaacaca tccggtacat
     4381 ggttgaaacc attgctcacc accaggttga tattgattca gaggtataaa acgaatgagt
     4441 actgcactcg caacgctggc tgggaagctg gctgaacgtg tcggcatgga ttctgtcgac
     4501 ccacaggaac tgatcaccac tcttcgccag acggcattta aaggtgatgc cagcgatgcg
     4561 cagttcatcg cattactgat cgttgccaac cagtacggcc ttaatccgtg gacgaaagaa
     4621 atttacgcct ttcctgataa gcagaatggc atcgttccgg tggtgggcgt tgatggctgg
     4681 tcccgcatca tcaatgaaaa ccagcagttt gatggcatgg actttgagca ggacaatgaa
     4741 tcctgtacat gccggattta ccgcaaggac cgtaatcatc cgatctgcgt taccgaatgg
     4801 atggatgaat gccgccgcga accattcaaa actcgcgaag gcagagaaat cacggggccg
     4861 tggcagtcgc atcccaaacg gatgttacgt cataaagcca tgattcagtg tgcccgtctg
     4921 gccttcggat ttgctggtat ctatgacaag gatgaagccg agcgcattgt cgaaaatact
     4981 gcatacactg cagaacgtca gccggaacgc gacatcactc cggttaacga tgaaaccatg
     5041 caggagatta acactctgct gatcgccctg gataaaacat gggatgacga cttattgccg
     5101 ctctgttccc agatatttcg ccgcgacatt cgtgcatcgt cagaactgac acaggccgaa
     5161 gcagtaaaag ctcttggatt cctgaaacag aaagccgcag agcagaaggt ggcagcatga
     5221 caccggacat tatcctgcag cgtaccggga tcgatgtgag agctgtcgaa cagggggaag
     5281 atgcgtggca caaattacgg ctcggcgtca tcaccgcttc agaagttcac aacgtgatag
     5341 caaaaccccg ctccggaaag aagtggcctg acatgaaaat gtcctacttc cacaccctgc
     5401 ttgctgaggt ttgcaccggt gtggctccgg aagttaacgc taaagcactg gcctggggaa
     5461 aacagtacga gaacgacgcc agaaccctgt ttgaattcac ttccggcgtg aatgttactg
     5521 aatccccgat catctatcgc gacgaaagta tgcgtaccgc ctgctctccc gatggtttat
     5581 gcagtgacgg caacggcctt gaactgaaat gcccgtttac ctcccgggat ttcatgaagt
     5641 tccggctcgg tggtttcgag gccataaagt cagcttacat ggcccaggtg cagtacagca
     5701 tgtgggtgac gcgaaaaaat gcctggtact ttgccaacta tgacccgcgt atgaagcgtg
     5761 aaggcctgca ttatgtcgtg attgagcggg atgaaaagta catggcgagt tttgacgaga
     5821 tcgtgccgga gttcatcgaa aaaatggacg aggcactggc tgaaattggt tttgtatttg
     5881 gggagcaatg gcgatgacgc atcctcacga taataagctt cctgctgaac atcaaaggca
     5941 agaaaacatc tgttgtcaaa gacagcatcc ttgaacaagg acaattaaca gttaacaaat
     6001 aaaaacgcaa aagaaaatgc cgatattgac taccggaagc agtgtgaccg tgtgcttctc
     6061 aaatgcctga ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg
     6121 ttcaatttca tgttctagtt gctttgtttt actggtttca cctgttctat taggtgttac
     6181 atgctgttca tctgttacat tgtcgatctg ttcatggtga acagctttaa atgcaccaaa
     6241 aactcgtaaa agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatatagc
     6301 tatactgatt tcgtcagact cacagtcaaa catgccggtc agttggcctg gtgatggcgg
     6361 gatcgttgta tatttcttga caccttttcg gcaccgccct aaaattctgc gtcctcataa
     6421 tatatgaggc gatttattac gtgtttacga agcaaaagct aaaaccagga gctatttaat
     6481 ggtgcagacg gagatcctga aggcgtttcg gtttgcgttg gatccgacgt ctgtgcaggt
     6541 tgctgccctt tcccggcatg ctggtgcggc caggtgggcg ttcaaccatg ctctcgcggc
     6601 caaggtcggc gcgcatgagc ggtggcgggc ggaggtagcc aagcttgtcg gggacggtgt
     6661 tcctgaggag caggctcgta ggcaggtgcg cgtcccggtt ccgatgaagc cggcgatcca
     6721 gaaggcgttg aacgcggtca agggtgactc tcgcaagggc ttggatggtg cgtgcccgtg
     6781 gtggcatgag gtcaacactt acgcgttcca gtccgctttc atcgacgcgg accaggcgtg
     6841 gaagaactgg ttggactcgt tgtcggggaa gcgggtcggc cgccgggtgg ggtacccgcg
     6901 gttcaagaag aaggggcggg cccgcgactc gtttcgcttg caccacgacg tgaagaagcc
     6961 aagcatccgg ctggccgggt accggcggtt gcggctgccg aggatcgggg aagtccggct
     7021 tcatgactcg gggaagcgtc tggcacggct gatcgaccga ggtgacgcgg tggtgcagtc
     7081 ggtgacggtg tcgcggggtg gccatcgctg gtatgcgtct gtgctgtgca aggtcaccgt
     7141 gcaggtaccg gatcggccgt cacggcgcca gcgggaacga ggcgctgttg gggtggacct
     7201 tggcgttaag gtcctcgcgg ctttgtccaa gccactggtg gtcgacgacc cgtcgagtgc
     7261 cctggtgcgg aatccgcagc atctgcggca ggccgagcgg cgtctactga aggctcagcg
     7321 ggcgttggcg cgtacacaaa aaggttcagc ccgacgcgag aaggccaagc ggcgagttgg
     7381 tcgcgcgcat cacgaggttg ctgtgcgccg gcatgctgcg ctccatcaga tcaccaagcg
     7441 cctgaccacc ggcttcgcgg tcgtcgcgct tgaggatctg aacgtggccg gtatgacaag
     7501 gtcagcgcgg ggaacagtgg cagcgccggg aaagaacgtg cggcagaagg caggcctgaa
     7561 ccgcgtcatc ctggactccg ctccagccga gttgcgtcgc caggtgaact acaaggccac
     7621 ctggtatgga tctgagctgg cggtcgcaga ccgctggttc ccctcgagta agacctgttc
     7681 gggttgcgga tggcaaaacc cacacctgaa gctgtccgac agggtgtttc gttgcacaga
     7741 ctgcgggctg gtgatggacc gggacatgaa cgcggcccgg aacatcgaac gccacgcagt
     7801 gctgatcgat cgaaacgtcg cctgcgatag gcgggagacg ctaaacgccc gtggagcatc
     7861 cataagacca accacctctc ggggcggtag gcacgacgca tcgaagcggg aaggctccgg
     7921 cgctcggcct gagtcacctc agcagagtga tctgctgacg ctcccaacct tgaataacga
     7981 aacggcaacg cctccatagc ggtgcaggtc aataaggtgg ctggttggcg tactgtt
//
