LOCUS       sequence-302055-        2858 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 2858)
  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 2858)
  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: pENTR2-L3-PBL-R1
FEATURES             Location/Qualifiers
     source          1..2858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    34..129
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   134..196
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    368..491
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(563..582)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(564..582)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(587..603)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(587..603)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(705..724)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             716..1525
                     /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(783..802)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1672..2260
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2161..2180
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2414..2431
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2590..2617)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2709..2795)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(2844..2858,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(2858,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag
       61 tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtata
      121 ataaagttgt taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc
      181 gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtcccgtgag gcgtttgaat
      241 agatattaag ttttattata tttacactta catactaata ataaattcaa caaacaattc
      301 ttttacgtga cttttaagat ttaactcaaa caaaaactca aaatttcttc tataaagtaa
      361 caaaactcaa gtttgtacaa aaaagttgaa cgagaaacgt aaaatgatat aaatatcaat
      421 atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatg
      481 cagtcactat gaaccaacta cttagatggt attagtgacc tgtagaattc gagctctaga
      541 gctgcagggc ggccgcgata tcccctatag tgagtcgtat tacatggtca tagctgtttc
      601 ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
      661 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
      721 ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata
      781 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta
      841 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
      901 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
      961 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa
     1021 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     1081 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     1141 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     1201 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     1261 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     1321 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     1381 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     1441 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     1501 tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag
     1561 cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagtta
     1621 cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     1681 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     1741 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     1801 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     1861 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     1921 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     1981 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2041 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2101 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2161 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2221 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2281 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2341 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     2401 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     2461 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     2521 gaaagcgggc agtgagcgca acgcaattaa tacgcgtacc gctagccagg aagagtttgt
     2581 agaaacgcaa aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt
     2641 tatggcgggc gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct
     2701 cccggcggat ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag
     2761 gcccagtctt ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc
     2821 gtcaacgcta gcatggatgt tttcccagtc acgacgtt
//
