LOCUS       sequence-273819-        4806 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 4806)
  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 4806)
  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..4806
                     /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..1233
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     primer_bind     1244..1261
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1245..1261
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1278..1373
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      1376..1385
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1388..1444
                     /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   2402..2464
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    2636..2759
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2820..2837)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2826..3033
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3048..3067)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3049..3067)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3072..3088)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3072..3088)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3190..3209)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3201..4010
                     /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(3268..3287)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4157..4745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4646..4665
                     /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 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgtaaaa cgacggccag
     1261 tcttaagctc gggccccaaa taatgatttt atttcgactg atagtgacct gttcgttgca
     1321 acaaattgat gagcaatgct tttttataat gccaactttg tataataaag ttgttgccac
     1381 catgggcgcc acgaacttct ctctgttaaa gcaagcagga gacgtggaag aaaaccccgg
     1441 tcctgttatg gccgaaggga gcgtggcaag acagcccgat ctgctgacat gcgacgacga
     1501 accaatccac attccaggag ctatccagcc tcatggactg ctgctggctc tggccgctga
     1561 catgactatc gtggcaggct ctgataacct gccagagctg accgggctgg ctatcggagc
     1621 actgattggc cggtccgcag ccgacgtgtt cgattctgag actcacaata gactgaccat
     1681 cgccctggct gaacctgggg ctgcagtggg agccccaatt accgtcggct tcacaatgcg
     1741 aaaggacgct ggctttatcg ggtcctggca ccgccatgat cagctgattt ttctggagct
     1801 ggaaccacct cagagagacg tggcagagcc acaggctttc tttcggagaa caaacagcgc
     1861 catcaggcgg ctgcaggccg ctgagactct ggaatccgct tgcgcagccg ctgcacagga
     1921 agtgaggaag atcaccgggt tcgatcgcgt catgatctac agattcgcct cagactttag
     1981 cggagaagtg atcgcagaag atcgctgtgc cgaggtcgaa agtaaactgg gcctgcatta
     2041 cccagcctca accgtgcctg cacaggcacg acggctgtat acaatcaacc ccgtgcggat
     2101 cattcccgac attaattaca gaccagtgcc cgtcacacct gacctgaacc cagtgactgg
     2161 gaggcccatc gatctgagct tcgccattct gcgcagcgtg tcccctgtcc acctggagtt
     2221 tatgaggaat atcggaatgc atggcaccat gtctatcagt attctgagag gcgaaaggct
     2281 gtgggggctg atcgtgtgcc accatcggac accttactat gtggacctgg acggcaggca
     2341 ggcttgtgaa ctggtggctc aggtgctggc ttggcagatt ggcgtgatgg aagaacttta
     2401 accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg
     2461 catgtgtttt atcggtctgt atatcgaggt cccgtgaggc gtttgaatag atattaagtt
     2521 ttattatatt tacacttaca tactaataat aaattcaaca aacaattctt ttacgtgact
     2581 tttaagattt aactcaaaca aaaactcaaa atttcttcta taaagtaaca aaactcaagt
     2641 ttgtacaaaa aagttgaacg agaaacgtaa aatgatataa atatcaatat attaaattag
     2701 attttgcata aaaaacagac tacataatac tgtaaaacac aacatatgca gtcactatga
     2761 atcaactact tagatggtat tagtgacctg tagaattcta actagagctc gctgatcagc
     2821 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2881 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2941 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3001 ggattgggaa gagaatagca ggcatgctgg ggagcggccg cgatatcccc tatagtgagt
     3061 cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat
     3121 gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa
     3181 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa
     3241 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     3301 caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac
     3361 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     3421 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     3481 tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt
     3541 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     3601 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     3661 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     3721 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     3781 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     3841 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     3901 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     3961 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg
     4021 ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat
     4081 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     4141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     4321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4801 acatgt
//
