LOCUS       sequence-301958-        4571 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 4571)
  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 4571)
  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-EGFP-PBL-R1
FEATURES             Location/Qualifiers
     source          1..4571
                     /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"
     CDS             join(1444..1446,1447..2157)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1444..1446,1447..2157)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1486..1507)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1747..1766)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2094..2115
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   2167..2229
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    2401..2524
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2585..2602)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2591..2798
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2813..2832)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2814..2832)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2837..2853)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2837..2853)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2955..2974)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2966..3775
                     /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(3033..3052)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3922..4510
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4411..4430
                     /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 ggggsgrggg 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 cctgttagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1501 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1561 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1621 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1681 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1741 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1801 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1861 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1921 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1981 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2041 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2101 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggaa
     2161 ttttaaccct agaaagataa tcatattgtg acgtacgtta aagataatca tgcgtaaaat
     2221 tgacgcatgt gttttatcgg tctgtatatc gaggtcccgt gaggcgtttg aatagatatt
     2281 aagttttatt atatttacac ttacatacta ataataaatt caacaaacaa ttcttttacg
     2341 tgacttttaa gatttaactc aaacaaaaac tcaaaatttc ttctataaag taacaaaact
     2401 caagtttgta caaaaaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa
     2461 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atgcagtcac
     2521 tatgaaccaa ctacttagat ggtattagtg acctgtagaa ttctaactag agctcgctga
     2581 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2641 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2701 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2761 ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcgata tcccctatag
     2821 tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct
     2881 ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
     2941 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     3001 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     3061 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     3121 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     3181 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     3241 atggttactc accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc
     3301 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     3361 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     3421 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     3481 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     3541 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     3601 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     3661 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     3721 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     3781 attggttaat tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag
     3841 ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt
     3901 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3961 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4021 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4081 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4141 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4201 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4261 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4321 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4381 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4441 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4501 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4561 tgctcacatg t
//
