LOCUS       sequence-305025-        2738 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 2738)
  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 2738)
  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-R2-pA-L4
FEATURES             Location/Qualifiers
     source          1..2738
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(10..105)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(218..237)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             229..1038
                     /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(296..315)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1181..1769
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1670..1689
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1923..1940
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2099..2126)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2218..2304)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2353..2375
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2367..2384
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2368..2384
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2455..2579)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    2603..2737
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2640..2659)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2694..2713
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tcgagttaac aacttttcta tacaaagttg gcattataag aaagcattgc ttatcaattt
       61 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgcag
      121 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
      181 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
      241 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
      301 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
      361 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
      421 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
      481 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
      541 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
      601 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
      661 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
      721 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
      781 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
      841 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
      901 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
      961 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     1021 ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg
     1081 ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     1141 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     1201 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1261 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     1321 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     1381 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     1441 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     1501 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     1561 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     1621 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     1681 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     1741 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     1801 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     1861 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     1921 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     1981 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2041 gtgagcgcaa cgcaattaat acgcgtaccg ctagccagga agagtttgta gaaacgcaaa
     2101 aaggccatcc gtcaggatgg ccttctgctt agtttgatgc ctggcagttt atggcgggcg
     2161 tcctgcccgc caccctccgg gccgttgctt cacaacgttc aaatccgctc ccggcggatt
     2221 tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttc
     2281 cgactgagcc tttcgtttta tttgatgcct ggcagttccc tactctcgcg ttaacgctag
     2341 catggatgtt ttcccagtca cgacgttgta aaacgacggc cagtcttaag ctcgggccct
     2401 gcagctctag agctcgaatt ctacaggtca ctaataccat ctaagtagtt ggttcatagt
     2461 gactgcatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt
     2521 aatatattga tatttatatc attttacgtt tctcgttcaa ctttcttgta caaagtggtc
     2581 tagagaattc gtcgacagat ctaacttgtt tattgcagct tataatggtt acaaataaag
     2641 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     2701 gtccaaactc atcaatgtat cttatcatgt ctggatcc
//
