LOCUS       sequence-305030-        4748 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 4748)
  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 4748)
  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-PuroTK-L2
FEATURES             Location/Qualifiers
     source          1..4748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(43..129)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    182..281
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        288..787
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(323..346)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     720..739
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             811..1407
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(811..830)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1307..1327
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1414..2409
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     protein_bind    complement(2775..2874)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2986..3005)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2997..3806
                     /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(3064..3083)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3949..4537
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4438..4457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4672..4699)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag
       61 agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg
      121 ttttatttga tgcctggcag ttccctactc tcgcgttaac gctagcatgg atctcgggcc
      181 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca
      241 atgctttttt ataatgccaa ctttgtacaa aaaagcagaa ttctaccggg taggggaggc
      301 gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta
      361 cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc
      421 gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc
      481 cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct
      541 cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca
      601 atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg
      661 cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc
      721 attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg
      781 cctttcgacc gatcatcaag cttgatcctc atgaccgagt acaagcccac ggtgcgcctc
      841 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
      901 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
      961 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     1021 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     1081 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     1141 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     1201 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     1261 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     1321 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     1381 tggtgcatga cccgcaagcc cggtgccgga tccatgccca cgctactgcg ggtttatata
     1441 gacggtcccc acgggatggg gaaaaccacc accacgcaac tgctggtggc cctgggttcg
     1501 cgcgacgata tcgtctacgt acccgagccg atgacttact ggcgggtgct gggggcttcc
     1561 gagacaatcg cgaacatcta caccacacaa caccgcctcg accagggtga gatatcggcc
     1621 ggggacgcgg cggtggtaat gacaagcgcc cagataacaa tgggcatgcc ttatgccgtg
     1681 accgacgccg ttctggctcc tcatatcggg ggggaggctg ggagctcaca tgccccgccc
     1741 ccggccctca ccctcatctt cgaccgccat cccatcgccg ccctcctgtg ctacccggcc
     1801 gcgcggtacc ttatgggcag catgaccccc caggccgtgc tggcgttcgt ggccctcatc
     1861 ccgccgacct tgcccggcac caacatcgtg cttggggccc ttccggagga cagacacatc
     1921 gaccgcctgg ccaaacgcca gcgccccggc gagcggctgg acctggctat gctggctgcg
     1981 attcgccgcg tttacgggct acttgccaat acggtgcggt atctgcagtg cggcgggtcg
     2041 tggcgggagg actggggaca gctttcgggg acggccgtgc cgccccaggg tgccgagccc
     2101 cagagcaacg cgggcccacg accccatatc ggggacacgt tatttaccct gtttcgggcc
     2161 cccgagttgc tggcccccaa cggcgacctg tataacgtgt ttgcctgggc cttggacgtc
     2221 ttggccaaac gcctccgttc catgcacgtc tttatcctgg attacgacca atcgcccgcc
     2281 ggctgccggg acgccctgct gcaacttacc tccgggatgg tccagaccca cgtcaccacc
     2341 cccggctcca taccgacgat atgcgacctg gcgcgcacgt ttgcccggga gatgggggag
     2401 gctaactgag ctctagctta tcgactcgcc gatagtggaa accgacgccc cagcactcgt
     2461 ccgagggcaa aggaatagtc gagaaattga tgatctatta aacaataaag atgtccacat
     2521 ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct acattttgaa
     2581 tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg cctgctcttt
     2641 actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat cataatttaa
     2701 acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta tagatcctct
     2761 agagtcgagg gctgcagcag ctttcttgta caaagttggc attataagaa agcattgctt
     2821 atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc
     2881 agctgcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
     2941 tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     3001 gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat
     3061 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt
     3121 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     3181 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     3241 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggaa
     3301 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     3361 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     3421 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     3481 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     3541 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     3601 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     3661 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     3721 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     3781 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga
     3841 gcattacgct gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt
     3901 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3961 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4021 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4081 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4141 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4201 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4261 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4321 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4381 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4441 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4501 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4561 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4621 ttctgtggat aaccgtatta ccgctagcca ggaagagttt gtagaaacgc aaaaaggcca
     4681 tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg gcgtcctgcc
     4741 cgccaccc
//
