LOCUS       sequence-307603-        4546 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 4546)
  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 4546)
  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-TKBsd-L2
FEATURES             Location/Qualifiers
     source          1..4546
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             6..404
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(793..892)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1004..1023)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1015..1824
                     /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(1082..1101)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1967..2555
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2456..2475
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2690..2717)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2809..2895)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    2948..3047
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3054..3553
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3089..3112)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3486..3505
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
ORIGIN
        1 gatcgatggc caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
       61 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg
      121 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
      181 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
      241 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
      301 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
      361 ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaagggaga tgggggaggc
      421 taactgagct ctagcttatc gactcgccga tagtggaaac cgacgcccca gcactcgtcc
      481 gagggcaaag gaatagtcga gaaattgatg atctattaaa caataaagat gtccacatgg
      541 aagtttttcc tgtcatactt tgttaagaag ggtgagaaca gagtacctac attttgaatg
      601 gaaggattgg agctacgggg gtgggggtgg ggtgggatta gataaatgcc tgctctttac
      661 tgaaggctct ttactattgc tttatgataa tgtttcatag ttggatatca taatttaaac
      721 aagcaaaacc aaattaaggg ccagctcatt cctcccactc atgatctata gatcctctag
      781 agtcgagggc tgcagcagct ttcttgtaca aagttggcat tataagaaag cattgcttat
      841 caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag
      901 ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
      961 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     1021 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     1081 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     1141 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     1201 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     1261 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     1321 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     1381 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     1441 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     1501 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     1561 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     1621 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     1681 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     1741 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     1801 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     1861 attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagtttt
     1921 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     1981 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2041 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2101 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2161 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2221 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2281 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2341 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2401 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2461 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2521 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2581 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2641 ctgtggataa ccgtattacc gctagccagg aagagtttgt agaaacgcaa aaaggccatc
     2701 cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc gtcctgcccg
     2761 ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat ttgtcctact
     2821 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt ccgactgagc
     2881 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc gttaacgcta gcatggatct
     2941 cgggccccaa ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga
     3001 tgagcaatgc ttttttataa tgccaacttt gtacaaaaaa gcagaattct accgggtagg
     3061 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     3121 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     3181 ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     3241 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     3301 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     3361 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     3421 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     3481 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     3541 tccgggcctt tcgaccgatc atcaagctga tccatgccca cgctactgcg ggtttatata
     3601 gacggtcccc acgggatggg gaaaaccacc accacgcaac tgctggtggc cctgggttcg
     3661 cgcgacgata tcgtctacgt acccgagccg atgacttact ggcgggtgct gggggcttcc
     3721 gagacaatcg cgaacatcta caccacacaa caccgcctcg accagggtga gatatcggcc
     3781 ggggacgcgg cggtggtaat gacaagcgcc cagataacaa tgggcatgcc ttatgccgtg
     3841 accgacgccg ttctggctcc tcatatcggg ggggaggctg ggagctcaca tgccccgccc
     3901 ccggccctca ccctcatctt cgaccgccat cccatcgccg ccctcctgtg ctacccggcc
     3961 gcgcggtacc ttatgggcag catgaccccc caggccgtgc tggcgttcgt ggccctcatc
     4021 ccgccgacct tgcccggcac caacatcgtg cttggggccc ttccggagga cagacacatc
     4081 gaccgcctgg ccaaacgcca gcgccccggc gagcggctgg acctggctat gctggctgcg
     4141 attcgccgcg tttacgggct acttgccaat acggtgcggt atctgcagtg cggcgggtcg
     4201 tggcgggagg actggggaca gctttcgggg acggccgtgc cgccccaggg tgccgagccc
     4261 cagagcaacg cgggcccacg accccatatc ggggacacgt tatttaccct gtttcgggcc
     4321 cccgagttgc tggcccccaa cggcgacctg tataacgtgt ttgcctgggc cttggacgtc
     4381 ttggccaaac gcctccgttc catgcacgtc tttatcctgg attacgacca atcgcccgcc
     4441 ggctgccggg acgccctgct gcaacttacc tccgggatgg tccagaccca cgtcaccacc
     4501 cccggctcca taccgacgat atgcgacctg gcgcgcacgt ttgccc
//
