LOCUS       sequence-305028-        4953 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 4953)
  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 4953)
  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-PGK-TKNeo-L2
FEATURES             Location/Qualifiers
     source          1..4953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             6..809
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(69..88)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     679..698
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(1200..1299)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1411..1430)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1422..2231
                     /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(1489..1508)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2374..2962
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2863..2882
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3097..3124)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3216..3302)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    3355..3454
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3461..3960
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3496..3519)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3893..3912
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
ORIGIN
        1 gatcgatggg atcggccatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
       61 tggagaggct attcggctat gactgggcac aacagacgat cggctgctct gatgccgccg
      121 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
      181 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
      241 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
      301 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
      361 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
      421 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
      481 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
      541 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
      601 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
      661 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
      721 gggctgaccg cttcctcgtg ctttacggta tcgccgcccc cgattcgcag cgcatcgcct
      781 tctatcgcct tcttgacgag ttcttctgag cgggagatgg gggaggctaa ctgagctcta
      841 gcttatcgac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa
      901 tagtcgagaa attgatgatc tattaaacaa taaagatgtc cacatggaag tttttcctgt
      961 catactttgt taagaagggt gagaacagag tacctacatt ttgaatggaa ggattggagc
     1021 tacgggggtg ggggtggggt gggattagat aaatgcctgc tctttactga aggctcttta
     1081 ctattgcttt atgataatgt ttcatagttg gatatcataa tttaaacaag caaaaccaaa
     1141 ttaagggcca gctcattcct cccactcatg atctatagat cctctagagt cgagggctgc
     1201 agcagctttc ttgtacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa
     1261 cgaacaggtc actatcagtc aaaataaaat cattatttgc catccagctg cagctctggc
     1321 ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     1381 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     1441 aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     1501 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg
     1561 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     1621 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     1681 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg
     1741 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     1801 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     1861 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     1921 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     1981 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     2041 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     2101 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     2161 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     2221 agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt
     2281 gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2341 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2401 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2461 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2521 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2581 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2641 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2701 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2761 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2821 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2881 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2941 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3001 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3061 tattaccgct agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc
     3121 ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc
     3181 cgttgcttca caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc
     3241 accgacaaac aacagataaa acgaaaggcc cagtcttccg actgagcctt tcgttttatt
     3301 tgatgcctgg cagttcccta ctctcgcgtt aacgctagca tggatctcgg gccccaaata
     3361 atgattttat tttgactgat agtgacctgt tcgttgcaac aaattgatga gcaatgcttt
     3421 tttataatgc caactttgta caaaaaagca gaattctacc gggtagggga ggcgcttttc
     3481 ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
     3541 ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt
     3601 ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg
     3661 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
     3721 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
     3781 ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
     3841 aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
     3901 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg
     3961 accgatcatc aagctgatcc atgcccacgc tactgcgggt ttatatagac ggtccccacg
     4021 ggatggggaa aaccaccacc acgcaactgc tggtggccct gggttcgcgc gacgatatcg
     4081 tctacgtacc cgagccgatg acttactggc gggtgctggg ggcttccgag acaatcgcga
     4141 acatctacac cacacaacac cgcctcgacc agggtgagat atcggccggg gacgcggcgg
     4201 tggtaatgac aagcgcccag ataacaatgg gcatgcctta tgccgtgacc gacgccgttc
     4261 tggctcctca tatcgggggg gaggctggga gctcacatgc cccgcccccg gccctcaccc
     4321 tcatcttcga ccgccatccc atcgccgccc tcctgtgcta cccggccgcg cggtacctta
     4381 tgggcagcat gaccccccag gccgtgctgg cgttcgtggc cctcatcccg ccgaccttgc
     4441 ccggcaccaa catcgtgctt ggggcccttc cggaggacag acacatcgac cgcctggcca
     4501 aacgccagcg ccccggcgag cggctggacc tggctatgct ggctgcgatt cgccgcgttt
     4561 acgggctact tgccaatacg gtgcggtatc tgcagtgcgg cgggtcgtgg cgggaggact
     4621 ggggacagct ttcggggacg gccgtgccgc cccagggtgc cgagccccag agcaacgcgg
     4681 gcccacgacc ccatatcggg gacacgttat ttaccctgtt tcgggccccc gagttgctgg
     4741 cccccaacgg cgacctgtat aacgtgtttg cctgggcctt ggacgtcttg gccaaacgcc
     4801 tccgttccat gcacgtcttt atcctggatt acgaccaatc gcccgccggc tgccgggacg
     4861 ccctgctgca acttacctcc gggatggtcc agacccacgt caccaccccc ggctccatac
     4921 cgacgatatg cgacctggcg cgcacgtttg ccc
//
