LOCUS       sequence-485815-        3481 bp    DNA     circular SYN 11-SEP-2025
DEFINITION  Gateway middle entry clone containing Lifeact-mRuby2-3xMyc; Red 
            F-actin reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3481)
  AUTHORS   Gillespie W, Zhang Y, Ruiz OE, Cerda J, Ortiz-Guzman J, Turner WD, 
            Largoza G, Sherman M, 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 with diverse applications in vertebrate model 
            systems.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267.
  PUBMED    39026881
REFERENCE   2  (bases 1 to 3481)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267."
COMMENT     Sequence Label: pME-Lifeact-mRuby2-3xMyc (JDW 1246)
FEATURES             Location/Qualifiers
     source          1..3481
                     /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      682..691
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             688..738
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     regulatory      754..763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             760..1470
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             1480..1509
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1516..1545
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1552..1581
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     protein_bind    complement(1606..1705)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1722..1741)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1723..1741)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1746..1762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1746..1762)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1864..1883)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1875..2684
                     /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(1942..1961)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2831..3419
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3320..3339
                     /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 agcaggcaac cggtggatcc agccaccatg ggcgtggccg acttgatcaa gaagttcgag
      721 tccatctcca aggaggaggg ggatccaccg gtcgccacca tggtgtctaa gggcgaagag
      781 ctgatcaagg aaaatatgcg tatgaaggtg gtcatggaag gttcggtcaa cggccaccaa
      841 ttcaaatgca caggtgaagg agaaggcaat ccgtacatgg gaactcaaac catgaggatc
      901 aaagtcatcg agggaggacc cctgccattt gcctttgaca ttcttgccac gtcgttcatg
      961 tatggcagcc gtacttttat caagtacccg aaaggcattc ctgatttctt taaacagtcc
     1021 tttcctgagg gttttacttg ggaaagagtt acgagatacg aagatggtgg agtcgtcacc
     1081 gtcatgcagg acaccagcct tgaggatggc tgtctcgttt accacgtcca agtcagaggg
     1141 gtaaactttc cctccaatgg tcccgtgatg cagaagaaga ccaagggttg ggagcctaat
     1201 acagagatga tgtatccagc agatggtggt ctgaggggat acactcatat ggcactgaaa
     1261 gttgatggtg gtggccatct gtcttgctct ttcgtaacaa cttacaggtc aaaaaagacc
     1321 gtcgggaaca tcaagatgcc cggtatccat gccgttgatc accgcctgga aaggttagag
     1381 gaaagtgaca atgaaatgtt cgtagtacaa cgcgaacacg cagttgccaa gttcgccggg
     1441 cttggtggtg ggatggacga gctgtacaag agatccttag agcagaaact cattagcgag
     1501 gaggacctga acagcgaaca gaaactcatt tccgaagagg atctcaactc cgagcagaag
     1561 ctgatcagcg aggaggacct gagatcctag taaacgcgta ctagtgccca gctttcttgt
     1621 acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta
     1681 tcagtcaaaa taaaatcatt atttgccatc cagctgatat cccctatagt gagtcgtatt
     1741 acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     1801 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     1861 atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca
     1921 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     1981 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     2041 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     2101 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     2161 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     2221 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     2281 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     2341 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     2401 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     2461 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     2521 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     2581 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     2641 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     2701 ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa
     2761 aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca
     2821 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2881 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2941 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     3001 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3061 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3121 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3181 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3241 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3301 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3361 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3421 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3481 t
//
