LOCUS       sequence-446938-        2931 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing a 3x FLAG tagged 
            human Ubiquitin for monitoring ubiquitylation status.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2931)
  AUTHORS   Gillespie WB 3rd, Zhang Y, Ruiz OE, Cerda J 3rd, Ortiz-Guzman J, 
            Sherman M, Turner WD, Largoza G, Mosser LE, Fujimoto E, Chien CB, 
            Kwan KM, Arenkiel BR, Devine WP, Wythe JD
  TITLE     MultiSite Assembly of Gateway Induced Clones (MAGIC): a flexible 
            cloning toolbox for use in vertebrate model systems.
  JOURNAL   Development. 2025 Nov 15;152(22):dev204308. doi: 10.1242/dev.204308.
            Epub 2025 Nov 13.
  PUBMED    41230856
REFERENCE   2  (bases 1 to 2931)
  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: 
            "Development."; date: "2025-11-15"; volume: "152(22):dev204308. 
            doi"; pages: " 10.1242/dev.204308. Epub 2025 Nov 13"
COMMENT     Sequence Label: pME-3x-FLAG-Ubiquitin (JDW 1098)
FEATURES             Location/Qualifiers
     source          1..2931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      684..693
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             693..758
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             765..989
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="QIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFA
                     GKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     protein_bind    complement(1056..1155)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1172..1191)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1173..1191)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1196..1212)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1196..1212)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1314..1333)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1325..2134
                     /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(1392..1411)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2281..2869
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2770..2789
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctac tagtgctagc gccgccacca tggactacaa agaccatgac ggtgattata
      721 aagatcatga catcgattac aaggatgacg atgacaagct tgcacagatt ttcgtgaaaa
      781 cccttacggg gaagaccatc acccttgagg ttgaaccctc ggatacgata gaaaatgtaa
      841 aggccaagat ccaggataag gaaggaattc ctcctgatca gcagagactg atctttgctg
      901 gcaagcagct ggaagatgga cgtactttgt ctgactacaa tattcaaaag gagtctactc
      961 ttcatcttgt gttgagactt cgtggtggtt gaaagcttgc ggccgcgaat tcatcgatag
     1021 atctgatatc ggtaccagtc gactctagag gatccaccca gctttcttgt acaaagttgg
     1081 cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa
     1141 taaaatcatt atttgccatc cagctgatat cccctatagt gagtcgtatt acatggtcat
     1201 agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga
     1261 taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     1321 tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc
     1381 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     1441 tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     1501 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     1561 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     1621 ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     1681 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     1741 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     1801 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     1861 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     1921 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     1981 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     2041 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     2101 gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca
     2161 ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa
     2221 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2281 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2341 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2401 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2461 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2521 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2581 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2641 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2701 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2761 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2821 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2881 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt t
//
