LOCUS       sequence-273821-        5217 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 5217)
  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 5217)
  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..5217
                     /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"
     CDS             join(1445..1447,1448..2158)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1445..1447,1448..2158)
                     /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(1487..1508)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1748..1767)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2095..2116
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2159..2212
                     /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             2300..2812
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     repeat_region   2813..2875
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    3047..3170
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3231..3248)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3237..3444
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3459..3478)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3460..3478)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3483..3499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3483..3499)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3601..3620)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3612..4421
                     /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(3679..3698)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4568..5156
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5057..5076
                     /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 tcctgttagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1501 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1561 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1621 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1681 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1741 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1801 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1861 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1921 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1981 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2041 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2101 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga
     2161 gggcagggga agtcttctaa catgcgggga cgtggaggaa aatcccggcc ccatgaactc
     2221 cttctccaca agcgccttcg gtccagttgc cttctccctg ggcctgctcc tggtgttgcc
     2281 tgctgccttc cctgccccag tcttcacact cgaagatttc gttggggact ggcgacagac
     2341 agccggctac aacctggacc aagtccttga acagggaggt gtgtccagtt tgtttcagaa
     2401 tctcggggtg tccgtaactc cgatccaaag gattgtcctg agcggtgaaa atgggctgaa
     2461 gatcgacatc catgtcatca tcccgtatga aggtctgagc ggcgaccaaa tgggccagat
     2521 cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat cactttaagg tgatcctgca
     2581 ctatggcaca ctggtaatcg acggggttac gccgaacatg atcgactatt tcggacggcc
     2641 gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact gtaacaggga ccctgtggaa
     2701 cggcaacaaa attatcgacg agcgcctgat caaccccgac ggctccctgc tgttccgagt
     2761 aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc attctggctt aaccctagaa
     2821 agataatcat attgtgacgt acgttaaaga taatcatgcg taaaattgac gcatgtgttt
     2881 tatcggtctg tatatcgagg tcccgtgagg cgtttgaata gatattaagt tttattatat
     2941 ttacacttac atactaataa taaattcaac aaacaattct tttacgtgac ttttaagatt
     3001 taactcaaac aaaaactcaa aatttcttct ataaagtaac aaaactcaag tttgtacaaa
     3061 aaagttgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
     3121 aaaaaacaga ctacataata ctgtaaaaca caacatatgc agtcactatg aatcaactac
     3181 ttagatggta ttagtgacct gtagaattct aactagagct cgctgatcag cctcgactgt
     3241 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3301 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3361 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3421 agagaatagc aggcatgctg gggagcggcc gcgatatccc ctatagtgag tcgtattaca
     3481 tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg
     3541 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata
     3601 caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca
     3661 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     3721 caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     3781 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     3841 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     3901 ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     3961 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     4021 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     4081 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     4141 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     4201 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     4261 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     4321 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     4381 ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt
     4441 tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat
     4501 cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4561 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4621 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4681 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4741 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4801 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4861 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4921 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4981 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5041 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5101 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5161 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
