LOCUS       sequence-302038-        2483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutisite Gateway mediated vector construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2483)
  AUTHORS   Yoshimatsu S, Sone T, Nakajima M, Sato T, Okochi R, Ishikawa M, 
            Nakamura M, Sasaki E, Shiozawa S, Okano H
  TITLE     A versatile toolbox for knock-in gene targeting based on the 
            Multisite Gateway technology.
  JOURNAL   PLoS One. 2019 Aug 27;14(8):e0221164. doi: 
            10.1371/journal.pone.0221164. eCollection 2019.
  PUBMED    31454364
REFERENCE   2  (bases 1 to 2483)
  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: "PLoS One.";
            date: "2019-08-27"; volume: "14(8):e0221164. doi"; pages: " 
            10.1371/journal.pone.0221164. eCollection 2019"
COMMENT     Sequence Label: pENTR-L1-U6-BsmBI-R5
FEATURES             Location/Qualifiers
     source          1..2483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(253..272)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             264..1073
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(331..350)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1216..1804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1705..1724
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1931..2030
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2061..2309
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2061..2081
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2232..2251
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    2310..2329
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        2351..2426
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    2427..2446
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    join(2476..2483,1..116)
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 tatacaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
       61 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatgaatc
      121 aactacttag atggtattag tgacctgtac tgcagctctg gcccgtgtct caaaatctct
      181 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
      241 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat
      301 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
      361 aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga
      421 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
      481 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
      541 ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg
      601 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
      661 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tcttgctcag gcgcaatcac
      721 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
      781 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
      841 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
      901 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
      961 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     1021 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat
     1081 tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcgcaagct
     1141 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1201 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1261 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1321 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     1381 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1441 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1501 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1561 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1621 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1681 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1741 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1801 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1861 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ctagcatgga
     1921 tctcgggccc caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat
     1981 tgatgagcaa tgctttttta taatgccaac tttgtacaaa aaagcagtcg actggatccg
     2041 gtaccaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac
     2101 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
     2161 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2221 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2281 tatatatctt gtggaaagga cgaaacaccg ttcactgccg tataggcagt gagacgatta
     2341 atgcgtctca gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     2401 ttgaaaaagt ggcaccgagt cggtgcgttc actgccgtat aggcagtttt tttaagcttg
     2461 ggccgctcga ggtaccaact ttg
//
