LOCUS       sequence-447068-        3485 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone containing Lifeact-mCherry 
            (RFP F-actin reporter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3485)
  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 3485)
  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-Lifeact-mCherry-3xHA (JDW 1321)
FEATURES             Location/Qualifiers
     source          1..3485
                     /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      687..696
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             693..743
                     /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"
     CDS             756..1463
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(901..919)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1169..1188
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1363..1386
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1479..1568
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(1610..1709)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1726..1745)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1727..1745)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1750..1766)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1750..1766)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1868..1887)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1879..2688
                     /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(1946..1965)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2835..3423
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3324..3343
                     /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 agcaggctgg atccactagt gctagcgcca ccatgggtgt cgcagatttg atcaagaaat
      721 tcgaaagcat ctcaaaggaa gaaggaggag gatcgatggt gagcaagggc gaggaggaca
      781 acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg
      841 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
      901 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc
      961 agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga
     1021 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
     1081 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc
     1141 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg
     1201 aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga
     1261 ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc tacaaggcca
     1321 agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagctggac atcacctccc
     1381 acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg
     1441 gcggcatgga cgagctgtac aagggtggtg ctggtggtta cccatacgat gttcctgact
     1501 atgcgggcta tccctatgac gtcccggact atgcaggttc ctatccatat gacgttccag
     1561 attacgctta ataatgaggc gcgccacgcg tgaattcgat atcaagctta cccagctttc
     1621 ttgtacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc
     1681 actatcagtc aaaataaaat cattatttgc catccagctg atatccccta tagtgagtcg
     1741 tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt
     1801 tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac
     1861 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat
     1921 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     1981 ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     2041 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     2101 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     2161 ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca
     2221 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     2281 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     2341 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     2401 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     2461 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     2521 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     2581 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     2641 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt
     2701 aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga
     2761 ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag
     2821 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2881 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2941 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     3001 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3061 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3121 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3181 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3241 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3301 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3361 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3421 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3481 atgtt
//
