LOCUS       sequence-446865-        3376 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing 
            Lifeact-mScarlet-I (far red f-actin reporter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3376)
  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 3376)
  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: pDONR221-Lifeact- mScarlet-HA (JDW 1247)
FEATURES             Location/Qualifiers
     source          1..3376
                     /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..1455
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(893..911)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1161..1180
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1456..1482
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1501..1600)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1617..1636)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1618..1636)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1641..1657)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1641..1657)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1759..1778)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1770..2579
                     /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(1837..1856)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2726..3314
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3215..3234
                     /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 acctgatcaa gaagttcgag
      721 agcatcagca aggaggaggg ggatccaccg gtcgccacca tggtgagcaa gggcgaggca
      781 gtgatcaagg agttcatgcg gttcaaggtg cacatggagg gctccatgaa cggccacgag
      841 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
      901 aaggtgacca agggtggccc cctgcccttc tcctgggaca tcctgtcccc tcagttcatg
      961 tacggctcca gggccttcac caagcacccc gccgacatcc ccgactacta taagcagtcc
     1021 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgccgtgacc
     1081 gtgacccagg acacctccct ggaggacggc accctgatct acaaggtgaa gctccgcggc
     1141 accaacttcc ctcctgacgg ccccgtaatg cagaagaaga caatgggctg ggaagcgtcc
     1201 accgagcggt tgtaccccga ggacggcgtg ctgaagggcg acattaagat ggccctgcgc
     1261 ctgaaggacg gcggccgcta cctggcggac ttcaagacca cctacaaggc caagaagccc
     1321 gtgcagatgc ccggcgccta caacgtcgac cgcaagttgg acatcacctc ccacaacgag
     1381 gactacaccg tggtggaaca gtacgaacgc tccgagggcc gccactccac cggcggcatg
     1441 gacgagctgt acaagtatcc atatgacgtt ccagattacg cttaataaac gcgtactagt
     1501 gcccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca atttgttgca
     1561 acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct gatatcccct
     1621 atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa
     1681 atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg
     1741 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc
     1801 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     1861 tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt
     1921 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     1981 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     2041 atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat
     2101 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     2161 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     2221 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     2281 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     2341 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     2401 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     2461 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     2521 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     2581 cagaattggt taattggttg taacactggc agagcattac gctgacttga cgggacggcg
     2641 caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc
     2701 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2761 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2821 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2881 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2941 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3001 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3061 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3121 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3181 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3241 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3301 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3361 cttttgctca catgtt
//
