LOCUS       sequence-304867-        4805 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 4805)
  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 4805)
  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-SfoI-iRFP713-PBL-R1
FEATURES             Location/Qualifiers
     source          1..4805
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     primer_bind     1243..1260
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1244..1260
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1277..1372
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      1375..1384
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1387..1443
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     repeat_region   2401..2463
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    2635..2758
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2819..2836)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2825..3032
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3047..3066)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3048..3066)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3071..3087)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3071..3087)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3189..3208)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3200..4009
                     /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(3267..3286)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4156..4744
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4645..4664
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccrggs ggggsggggs ggggsggggg gsggggsggg gsgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgtaaaac gacggccagt
     1261 cttaagctcg ggccccaaat aatgatttta tttcgactga tagtgacctg ttcgttgcaa
     1321 caaattgatg agcaatgctt ttttataatg ccaactttgt ataataaagt tgttgccacc
     1381 atgggcgcca cgaacttctc tctgttaaag caagcaggag acgtggaaga aaaccccggt
     1441 cctgttatgg ccgaagggag cgtggcaaga cagcccgatc tgctgacatg cgacgacgaa
     1501 ccaatccaca ttccaggagc tatccagcct catggactgc tgctggctct ggccgctgac
     1561 atgactatcg tggcaggctc tgataacctg ccagagctga ccgggctggc tatcggagca
     1621 ctgattggcc ggtccgcagc cgacgtgttc gattctgaga ctcacaatag actgaccatc
     1681 gccctggctg aacctggggc tgcagtggga gccccaatta ccgtcggctt cacaatgcga
     1741 aaggacgctg gctttatcgg gtcctggcac cgccatgatc agctgatttt tctggagctg
     1801 gaaccacctc agagagacgt ggcagagcca caggctttct ttcggagaac aaacagcgcc
     1861 atcaggcggc tgcaggccgc tgagactctg gaatccgctt gcgcagccgc tgcacaggaa
     1921 gtgaggaaga tcaccgggtt cgatcgcgtc atgatctaca gattcgcctc agactttagc
     1981 ggagaagtga tcgcagaaga tcgctgtgcc gaggtcgaaa gtaaactggg cctgcattac
     2041 ccagcctcaa ccgtgcctgc acaggcacga cggctgtata caatcaaccc cgtgcggatc
     2101 attcccgaca ttaattacag accagtgccc gtcacacctg acctgaaccc agtgactggg
     2161 aggcccatcg atctgagctt cgccattctg cgcagcgtgt cccctgtcca cctggagttt
     2221 atgaggaata tcggaatgca tggcaccatg tctatcagta ttctgagagg cgaaaggctg
     2281 tgggggctga tcgtgtgcca ccatcggaca ccttactatg tggacctgga cggcaggcag
     2341 gcttgtgaac tggtggctca ggtgctggct tggcagattg gcgtgatgga agaactttaa
     2401 ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc
     2461 atgtgtttta tcggtctgta tatcgaggtc ccgtgaggcg tttgaataga tattaagttt
     2521 tattatattt acacttacat actaataata aattcaacaa acaattcttt tacgtgactt
     2581 ttaagattta actcaaacaa aaactcaaaa tttcttctat aaagtaacaa aactcaagtt
     2641 tgtacaaaaa agttgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga
     2701 ttttgcataa aaaacagact acataatact gtaaaacaca acatatgcag tcactatgaa
     2761 ccaactactt agatggtatt agtgacctgt agaattctaa ctagagctcg ctgatcagcc
     2821 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2881 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2941 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3001 gattgggaag agaatagcag gcatgctggg gagcggccgc gatatcccct atagtgagtc
     3061 gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg
     3121 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     3181 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa
     3241 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     3301 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     3361 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     3421 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     3481 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc
     3541 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     3601 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     3661 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     3721 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     3781 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     3841 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     3901 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     3961 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     4021 taattggttg taacactggc agagcattac gctgacttga cgggacggcg caagctcatg
     4081 accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa
     4141 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4201 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     4261 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     4321 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     4381 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4441 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     4501 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4561 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4621 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4681 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4741 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4801 catgt
//
