LOCUS       sequence-304482-        2326 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 2326)
  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 2326)
  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-R4-H1-BbsI-L2
FEATURES             Location/Qualifiers
     source          1..2326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(71..194)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     357..376
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        464..539
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(555..654)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(766..785)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             777..1586
                     /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
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(844..863)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1676..2264
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2165..2184
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgtataca cagccagtct
       61 gcaggtcgac catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt
      121 atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc gttcaacttt
      181 tctatacaaa gttgatagct tgatatcaat actctgaatt cgaacgctga cgtcatcaac
      241 ccgctccaag gaatcgcggg cccagtgtca ctaggcggga acacccagcg cgcgtgcgcc
      301 ctggcaggaa gatggctgtg agggacaggg gagtggcgcc ctgcaatatt tgcatgtcgc
      361 tatgtgttct gggaaatcac cataaacgtg aaatgtcttt ggatttggga atcttataag
      421 ttctgtatga ggaccacaga tcaccgggtc ttcgagaaga cctgttttag agctagaaat
      481 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
      541 tttttgatat ctagacccag ctttcttgta caaagttggc attataagaa agcattgctt
      601 atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc
      661 agctgcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
      721 tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
      781 gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat
      841 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt
      901 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
      961 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     1021 tcaagcattt tatccgtact cctggtgatg catggttact caccactgcg atccccggaa
     1081 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     1141 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     1201 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     1261 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     1321 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     1381 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     1441 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     1501 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     1561 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cattattcag
     1621 attgggcccc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1681 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1741 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1801 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     1861 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1921 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1981 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2041 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2101 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2161 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2221 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2281 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt
//
