LOCUS       sequence-302052-        5282 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 5282)
  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 5282)
  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-tdTomato-PBL-R1
FEATURES             Location/Qualifiers
     source          1..5282
                     /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             1444..2868
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYE
                     GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMN
                     FEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVL
                     KGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYER
                     SEGRHHLFLYGMDELYK"
     repeat_region   2878..2940
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    3112..3235
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3296..3313)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3302..3509
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3524..3543)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3525..3543)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3548..3564)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3548..3564)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3666..3685)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3677..4486
                     /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(3744..3763)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4633..5221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5122..5141
                     /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 ggggsggggg 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 ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag
     1501 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     1561 ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac
     1621 atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc
     1681 cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     1741 gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc
     1801 tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaaaaag
     1861 accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc
     1921 gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc
     1981 atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg
     2041 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc
     2101 cgccaccacc tgttcctggg gcatggcacc ggcagcaccg gcagcggcag ctccggcacc
     2161 gcctcctccg aggacaacaa catggccgtc atcaaagagt tcatgcgctt caaggtgcgc
     2221 atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     2281 tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc
     2341 tgggacatcc tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc
     2401 gacatccccg attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     2461 aacttcgagg acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg
     2521 ctgatctaca aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag
     2581 aaaaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg
     2641 aagggcgaga tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc
     2701 aagaccatct acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc
     2761 aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc
     2821 gagggccgcc accacctgtt cctgtacggc atggacgagc tgtacaagga attttaaccc
     2881 tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg
     2941 tgttttatcg gtctgtatat cgaggtcccg tgaggcgttt gaatagatat taagttttat
     3001 tatatttaca cttacatact aataataaat tcaacaaaca attcttttac gtgactttta
     3061 agatttaact caaacaaaaa ctcaaaattt cttctataaa gtaacaaaac tcaagtttgt
     3121 acaaaaaagt tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt
     3181 tgcataaaaa acagactaca taatactgta aaacacaaca tatgcagtca ctatgaacca
     3241 actacttaga tggtattagt gacctgtaga attctaacta gagctcgctg atcagcctcg
     3301 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     3361 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     3421 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     3481 tgggaagaga atagcaggca tgctggggag cggccgcgat atcccctata gtgagtcgta
     3541 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     3601 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     3661 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     3721 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     3781 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     3841 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     3901 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     3961 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     4021 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     4081 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     4141 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     4201 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     4261 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     4321 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     4381 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     4441 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     4501 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc
     4561 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     4621 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4681 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4741 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4801 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4861 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4921 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4981 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5041 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5101 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5161 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5221 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5281 gt
//
