LOCUS       sequence-273818-        4563 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 4563)
  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 4563)
  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-mCherry-PBL-R1
FEATURES             Location/Qualifiers
     source          1..4563
                     /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             1445..2149
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1587..1605)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1855..1874
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     repeat_region   2159..2221
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    2393..2516
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2577..2594)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2583..2790
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2805..2824)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2806..2824)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2829..2845)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2829..2845)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2947..2966)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2958..3767
                     /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(3025..3044)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3914..4502
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4403..4422
                     /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 aggataacat ggccatcatc aaggagttca tgcgcttcaa
     1501 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     1561 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     1621 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     1681 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     1741 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     1801 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     1861 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     1921 agccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     1981 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggag cctacaacgt
     2041 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     2101 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg aattttaacc
     2161 ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa attgacgcat
     2221 gtgttttatc ggtctgtata tcgaggtccc gtgaggcgtt tgaatagata ttaagtttta
     2281 ttatatttac acttacatac taataataaa ttcaacaaac aattctttta cgtgactttt
     2341 aagatttaac tcaaacaaaa actcaaaatt tcttctataa agtaacaaaa ctcaagtttg
     2401 tacaaaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
     2461 ttgcataaaa aacagactac ataatactgt aaaacacaac atatgcagtc actatgaatc
     2521 aactacttag atggtattag tgacctgtag aattctaact agagctcgct gatcagcctc
     2581 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2641 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2701 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2761 ttgggaagag aatagcaggc atgctgggga gcggccgcga tatcccctat agtgagtcgt
     2821 attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt
     2881 acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca
     2941 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt
     3001 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     3061 gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     3121 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     3181 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     3241 tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag
     3301 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     3361 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     3421 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     3481 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     3541 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     3601 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     3661 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     3721 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta
     3781 attggttgta acactggcag agcattacgc tgacttgacg ggacggcgca agctcatgac
     3841 caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga
     3901 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3961 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4021 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4081 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4141 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4201 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4261 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4321 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4381 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4441 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4501 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4561 tgt
//
