LOCUS       sequence-358230-        2905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDonor for ShoHELIX containing 14bp of spacing between the I-AniI 
            site and LE/RE using ShCAST flanking sequence and encoding a 
            kanamycin resistance gene as cargo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2905)
  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 2905)
  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-ShoHELIX-KanR (BO4)
FEATURES             Location/Qualifiers
     source          1..2905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      118..474
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            525..633
                     /note="incP origin of transfer"
     protein_bind    complement(1077..1124)
                     /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             1511..2305
                     /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(1565..1584)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2175..2194
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      complement(2460..2546)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(2582..2674)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 acgtagctga ggaggtttct ctgtaaaaat gctacctctc gcgatatggt gcactctcag
       61 tacaatcttg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agccggttgt
      121 cagccgttaa gtgttcctgt gtcactcaaa attgctttga gaggctctaa gggcttctca
      181 gtgcgttaca tccctggctt gttgtccaca accgttaaac cttaaaagct ttaaaagcct
      241 tatatattct tttttttctt ataaaactta aaaccttaga ggctatttaa gttgctgatt
      301 tatattaatt ttattgttca aacatgagag cttagtacgt gaaacatgag agcttagtac
      361 gttagccatg agagcttagt acgttagcca tgagggttta gttcgttaaa catgagagct
      421 tagtacgtta aacttgagag cttagtacgt gaaacatgag agcttagtac gtactatcaa
      481 caggttgaac tgcccatgtt ctttcctgcg ttatcagagc ttatcggcca gcctcgcaga
      541 gcaggattcc cgttgagcac cgccaggtgc gaataaggga cagtgaagaa ggaacacccg
      601 ctcgcgggtg ggcctacttc acctatcctg cccggctgac gccgttggat acaccaagga
      661 aagtctacac gaaccctttg gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc
      721 gaaaaaatcg ctataatgac cccgaagcag ggttatgcag cggaaagtag tactttagac
      781 atctctttac agagaaacct cctcacacaa aaggcgtagt gtacattcgc aaattaaatg
      841 tcgtaattcg caaatttgtg tcgtttttcg caaattaatg tcgtttagaa tagtttgtct
      901 catcaattca attataggaa cttttcgcaa attaatgtcg tcctgtttct ccatttagtg
      961 tcgattaaca aattaatgtc gctgttaacg aattaatgtc gtcgaattag ttccaactaa
     1021 cgggggatcc actagtgagc tcatgcatga tctcgaatta gcttcaaaag cgctctgaag
     1081 ttcctatact ttctagagaa taggaacttc ggaataggaa cttcaagatc ccctgattcc
     1141 ctttgtcaac agcaatggat aattcgattt aacaaatgca tggcgcaagg gctgctaaag
     1201 gaagcggaac acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag
     1261 ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag
     1321 tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt
     1381 gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt
     1441 cttgccgcca aggatctgat ggcgcagggg atcaagatct gatcaagaga caggatgagg
     1501 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     1561 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     1621 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     1681 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     1741 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     1801 gccggggcag gatctcctgt catcccacct tgctcctgcc gagaaagtat ccatcatggc
     1861 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     1921 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     1981 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     2041 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     2101 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     2161 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     2221 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     2281 tcgccttctt gacgagttct tctgaattga aaaaggaaga gtatgaggat ccaacatttc
     2341 caatcactag tgaattatct agaattattc cattgagtaa gtttttaagc acatcagctt
     2401 caaaagcgct ctaagatccc caattcgaga tcgtccgggc cgcaagctcc tagcggcgga
     2461 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
     2521 ttcgactgag cctttcgttt tatttgatgc ctcaagctag agagtcatta ccctagcttg
     2581 gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaaaatc cagatggagt
     2641 tctgaggtca ttactggatc tatcaacagg agtccaagct cagctaatta aggacatcta
     2701 atttgcgaaa caggcaaatc ttaataaacg acatttaatt tgcgaaaata ggatttgcga
     2761 catctaattt gcgaaacagg caaattactc agttttatgg ataaatagct tgtaagtcct
     2821 acgcaataaa gatctcagct attagaagta attgcgacac taatttgcga attgcgacat
     2881 ataatttgcg aatgtacacg tagag
//
