LOCUS       sequence-385666-        3425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kanamycin resistance donor for N7HELIX containing 14bp of spacing 
            between the I-AniI site and LE/RE using ShCAST flanking sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3425)
  AUTHORS   Tou CJ, Orr B, Kleinstiver BP
  TITLE     Precise cut-and-paste DNA insertion using engineered type V-K 
            CRISPR-associated transposases.
  JOURNAL   Nat Biotechnol. 2023 Jan 2. doi: 10.1038/s41587-022-01574-x.
  PUBMED    36593413
REFERENCE   2  (bases 1 to 3425)
  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 
            Biotechnol. 2023 Jan 2. doi: 10.1038/s41587-022-01574-x."
COMMENT     Sequence Label: pDonor14-N7HELIX-KanR
FEATURES             Location/Qualifiers
     source          1..3425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            259..367
                     /note="incP origin of transfer"
     protein_bind    complement(1084..1131)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             1518..2312
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1572..1591)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2182..2201
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(2420..2467)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     terminator      complement(2515..2601)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(2637..2731)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      join(3277..3425,1..208)
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
ORIGIN
        1 cttaaaacct tagaggctat ttaagttgct gatttatatt aattttattg ttcaaacatg
       61 agagcttagt acgtgaaaca tgagagctta gtacgttagc catgagagct tagtacgtta
      121 gccatgaggg tttagttcgt taaacatgag agcttagtac gttaaacttg agagcttagt
      181 acgtgaaaca tgagagctta gtacgtacta tcaacaggtt gaactgccca tgttctttcc
      241 tgcgttatca gagcttatcg gccagcctcg cagagcagga ttcccgttga gcaccgccag
      301 gtgcgaataa gggacagtga agaaggaaca cccgctcgcg ggtgggccta cttcacctat
      361 cctgcccggc tgacgccgtt ggatacacca aggaaagtct acacgaaccc tttggcaaaa
      421 tcctgtatat cgtgcgaaaa aggatggata taccgaaaaa atcgctataa tgaccccgaa
      481 gcagggttat gcagcggaaa gtagtacttt agacatctct ttacagagaa acctcctcac
      541 acaaaaggcg tagtgtcgaa taactaatta tttgtcgttg ttaacagatt gctgtcgcta
      601 ttaacaaatt aatgtcactg ttaacaaatt agtgtcgtat aatgctaatt gcgaaacgtt
      661 aacaaattaa tgtcgtctaa ccaatttgat aaagtgtttg cagacatcta ttgtacagga
      721 aatatagcta aatctttatt tgatgacttc cctgataata ttcataaata tgcttacaag
      781 tcggatgcac ctttcaaccc tctgttaaat attttctgac gctctttcaa ctcatcccta
      841 gctgggatag ttgttgaaac ttagagtcac ccagtttggc attagatact atcttttttc
      901 aacctacccc taaccaggat ggtcgttgaa acctggatat gctcaataca agggtacaac
      961 cacggtagcg cctttcaacc cacctctagc cgggatggtt gttgaaactt cgagtcctgg
     1021 atagcctggg gggatccact agtgagctca tgcatgatct cgaattagct tcaaaagcgc
     1081 tctgaagttc ctatactttc tagagaatag gaacttcgga ataggaactt caagatcccc
     1141 tgattccctt tgtcaacagc aatggataat tcgatttaac aaatgcatgg cgcaagggct
     1201 gctaaaggaa gcggaacacg tagaaagcca gtccgcagaa acggtgctga ccccggatga
     1261 atgtcagcta ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag
     1321 cttgcagtgg gcttacatgg cgatagctag actgggcggt tttatggaca gcaagcgaac
     1381 cggaattgcc agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga
     1441 tggctttctt gccgccaagg atctgatggc gcaggggatc aagatctgat caagagacag
     1501 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     1561 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     1621 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     1681 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     1741 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     1801 gcgaagtgcc ggggcaggat ctcctgtcat cccaccttgc tcctgccgag aaagtatcca
     1861 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     1921 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     1981 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     2041 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     2101 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     2161 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     2221 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     2281 ccttctatcg ccttcttgac gagttcttct gaattgaaaa aggaagagta tgaggatcca
     2341 acatttccaa tcactagtga attatctaga attattccat tgagtaagtt tttaagcaca
     2401 tcagcttcaa aagcgctctg aagttcctat actttctaga gaataggaac ttcggaatag
     2461 gtacttcaag atccccaatt cgagatcgtc cgggccgcaa gctcctagcg gcggatttgt
     2521 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga
     2581 ctgagccttt cgttttattt gatgcctcaa gctagagagt cattacccta gcttggattc
     2641 tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct
     2701 gaggtcatta ctggatctat caacaggagt ccaagctcag ctaattaaga ggaaaacttg
     2761 attcatactc aaaacagtaa tcacaatctc gctattgtgc gagaacatcc aaacttccta
     2821 aagcagttga cccctcaatg gacgcggcaa cttttcggta taaggatgta ttatttagtg
     2881 caaatgtact aaataaaatt ataataccac tattcaagct aaaaagcgac agctaatttg
     2941 ttatgaaact agaaaatttt agaaaacgta aaattttaaa agacgacgtt tattttgtta
     3001 ttatttaaat caacgacaag taaagtgtta aataaactac taacccatta cataataaaa
     3061 aacgttgtaa acactcatgt agcaacattt ttgatagttt tatatttgac gacattattt
     3121 tgttaagacg acaaataatt agttattcaa cacgtagaga cgtagctgag gaggtttctc
     3181 tgtaaaaatg ctacctctcg cgatatggtg cactctcagt acaatcttgt agcaccgcct
     3241 acatacctcg ctctgctaat cctgttacca gccggttgtc agccgttaag tgttcctgtg
     3301 tcactcaaaa ttgctttgag aggctctaag ggcttctcag tgcgttacat ccctggcttg
     3361 ttgtccacaa ccgttaaacc ttaaaagctt taaaagcctt atatattctt ttttttctta
     3421 taaaa
//
