LOCUS       sequence-304880-        3344 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 3344)
  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 3344)
  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-WPRE-pA-L4
FEATURES             Location/Qualifiers
     source          1..3344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..49
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     41..58
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     42..58
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(129..253)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    276..864
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(329..349)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(747..758)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    883..1017
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(920..939)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     974..993
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1028..1123)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1236..1255)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1247..2056
                     /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(1314..1333)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2199..2787
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2688..2707
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2941..2958
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3117..3144)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3236..3322)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cgcgttaacg ctagcatgga tgttttccca gtcacgacgt tgtaaaacga cggccagtct
       61 taagctcggg ccctgcagct ctagagctcg aattctacag gtcactaata ccatctaagt
      121 agttggttca tagtgactgc atatgttgtg ttttacagta ttatgtagtc tgttttttat
      181 gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcaactttct
      241 tgtacaaagt ggtctagaga attcggtcat atgataatca acctctggat tacaaaattt
      301 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
      361 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
      421 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
      481 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
      541 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
      601 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
      661 tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc
      721 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
      781 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
      841 tctccctttg ggccgcctcc ccgcatcggt aaattcagat ctaacttgtt tattgcagct
      901 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
      961 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcctc
     1021 gagttaacaa cttttctata caaagttggc attataagaa agcattgctt atcaatttgt
     1081 tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgcagct
     1141 ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     1201 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     1261 acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     1321 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc
     1381 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     1441 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     1501 tatccgtact cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt
     1561 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     1621 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     1681 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     1741 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     1801 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     1861 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     1921 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     1981 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     2041 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct
     2101 gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2161 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2221 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2281 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2341 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2401 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2461 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2521 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2581 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2641 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2701 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2761 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2821 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2881 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2941 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     3001 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     3061 gagcgcaacg caattaatac gcgtaccgct agccaggaag agtttgtaga aacgcaaaaa
     3121 ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc
     3181 ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc ggcggatttg
     3241 tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtcttccg
     3301 actgagcctt tcgttttatt tgatgcctgg cagttcccta ctct
//
