LOCUS       sequence-305027-        5216 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 5216)
  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 5216)
  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-secNanoLuc-PBL-R1
FEATURES             Location/Qualifiers
     source          1..5216
                     /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"
     CDS             2158..2211
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2299..2811
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     repeat_region   2812..2874
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    3046..3169
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3230..3247)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3236..3443
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3458..3477)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3459..3477)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3482..3498)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3482..3498)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3600..3619)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3611..4420
                     /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(3678..3697)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4567..5155
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5056..5075
                     /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 gggggggggg ggggsggggg gsggggsggg gsgrggcgga
      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 gtacaaggag
     2161 ggcaggggaa gtcttctaac atgcggggac gtggaggaaa atcccggccc catgaactcc
     2221 ttctccacaa gcgccttcgg tccagttgcc ttctccctgg gcctgctcct ggtgttgcct
     2281 gctgccttcc ctgccccagt cttcacactc gaagatttcg ttggggactg gcgacagaca
     2341 gccggctaca acctggacca agtccttgaa cagggaggtg tgtccagttt gtttcagaat
     2401 ctcggggtgt ccgtaactcc gatccaaagg attgtcctga gcggtgaaaa tgggctgaag
     2461 atcgacatcc atgtcatcat cccgtatgaa ggtctgagcg gcgaccaaat gggccagatc
     2521 gaaaaaattt ttaaggtggt gtaccctgtg gatgatcatc actttaaggt gatcctgcac
     2581 tatggcacac tggtaatcga cggggttacg ccgaacatga tcgactattt cggacggccg
     2641 tatgaaggca tcgccgtgtt cgacggcaaa aagatcactg taacagggac cctgtggaac
     2701 ggcaacaaaa ttatcgacga gcgcctgatc aaccccgacg gctccctgct gttccgagta
     2761 accatcaacg gagtgaccgg ctggcggctg tgcgaacgca ttctggctta accctagaaa
     2821 gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg catgtgtttt
     2881 atcggtctgt atatcgaggt cccgtgaggc gtttgaatag atattaagtt ttattatatt
     2941 tacacttaca tactaataat aaattcaaca aacaattctt ttacgtgact tttaagattt
     3001 aactcaaaca aaaactcaaa atttcttcta taaagtaaca aaactcaagt ttgtacaaaa
     3061 aagttgaacg agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata
     3121 aaaaacagac tacataatac tgtaaaacac aacatatgca gtcactatga accaactact
     3181 tagatggtat tagtgacctg tagaattcta actagagctc gctgatcagc ctcgactgtg
     3241 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     3301 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     3361 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     3421 gagaatagca ggcatgctgg ggagcggccg cgatatcccc tatagtgagt cgtattacat
     3481 ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc
     3541 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
     3601 aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat
     3661 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     3721 aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     3781 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     3841 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     3901 tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     3961 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     4021 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     4081 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     4141 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     4201 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     4261 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     4321 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     4381 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt
     4441 gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc
     4501 ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4561 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4621 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4681 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4741 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4801 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4861 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4921 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4981 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5041 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5101 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5161 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
