LOCUS       sequence-305031-        4562 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 4562)
  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 4562)
  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..4562
                     /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..2148
                     /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(1586..1604)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1854..1873
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     repeat_region   2158..2220
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    2392..2515
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2576..2593)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2582..2789
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2804..2823)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2805..2823)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2828..2844)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2828..2844)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2946..2965)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2957..3766
                     /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(3024..3043)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3913..4501
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4402..4421
                     /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 ggataacatg gccatcatca aggagttcat gcgcttcaag
     1501 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1561 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     1621 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     1681 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     1741 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     1801 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     1861 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacgga
     1921 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     1981 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggagc ctacaacgtc
     2041 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     2101 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagga attttaaccc
     2161 tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg
     2221 tgttttatcg gtctgtatat cgaggtcccg tgaggcgttt gaatagatat taagttttat
     2281 tatatttaca cttacatact aataataaat tcaacaaaca attcttttac gtgactttta
     2341 agatttaact caaacaaaaa ctcaaaattt cttctataaa gtaacaaaac tcaagtttgt
     2401 acaaaaaagt tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt
     2461 tgcataaaaa acagactaca taatactgta aaacacaaca tatgcagtca ctatgaacca
     2521 actacttaga tggtattagt gacctgtaga attctaacta gagctcgctg atcagcctcg
     2581 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2641 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2701 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2761 tgggaagaga atagcaggca tgctggggag cggccgcgat atcccctata gtgagtcgta
     2821 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     2881 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     2941 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     3001 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     3061 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     3121 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     3181 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     3241 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     3301 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     3361 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     3421 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     3481 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     3541 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     3601 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     3661 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     3721 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     3781 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc
     3841 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     3901 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3961 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4021 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4081 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4141 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4201 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4261 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4321 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4381 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4441 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4501 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4561 gt
//
