LOCUS       sequence-447099-        3401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone containing V5 tagged 
            m-Scarlet-I (Cytosolic far red fluorescent reporter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3401)
  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 3401)
  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 V5 mScarlet-I (JDW 1323)
FEATURES             Location/Qualifiers
     source          1..3401
                     /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             696..737
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             753..1448
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(886..904)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1154..1173
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1526..1625)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1642..1661)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1643..1661)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1666..1682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1666..1682)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1784..1803)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1795..2604
                     /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(1862..1881)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2751..3339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3240..3259
                     /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 ccatgggtaa gcctatccct aaccctctcc
      721 tcggtctcga ttctacgggt ggtgctggtg gtatggtgag caagggcgag gcagtgatca
      781 aggagttcat gcggttcaag gtgcacatgg agggctccat gaacggccac gagttcgaga
      841 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
      901 ccaagggtgg ccccctgccc ttctcctggg acatcctgtc ccctcagttc atgtacggct
      961 ccagggcctt catcaagcac cccgccgaca tccccgacta ctataagcag tccttccccg
     1021 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc
     1081 aggacacctc cctggaggac ggcaccctga tctacaaggt gaagctccgc ggcaccaact
     1141 tccctcctga cggccccgta atgcagaaga agacaatggg ctgggaagca tccaccgagc
     1201 ggttgtaccc cgaggacggc gtgctgaagg gcgacattaa gatggccctg cgcctgaagg
     1261 acggcggccg ctacctggcg gacttcaaga ccacctacaa ggccaagaag cccgtgcaga
     1321 tgcccggcgc ctacaacgtc gaccgcaagt tggacatcac ctcccacaac gaggactaca
     1381 ccgtggtgga acagtacgaa cgctccgagg gccgccactc caccggcggc atggacgagc
     1441 tgtacaagta ctcctccgga ctcagatctg gaggctccgg aggctaataa tgaggcgcgc
     1501 cacgcgtgaa ttcgatatca agcttaccca gctttcttgt acaaagttgg cattataaga
     1561 aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt
     1621 atttgccatc cagctgatat cccctatagt gagtcgtatt acatggtcat agctgtttcc
     1681 tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
     1741 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     1801 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     1861 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     1921 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     1981 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     2041 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     2101 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     2161 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     2221 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     2281 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     2341 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     2401 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     2461 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     2521 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     2581 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     2641 attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac
     2701 gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2761 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2821 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2881 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     2941 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3001 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3061 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3121 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3181 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3241 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3301 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3361 ttacggttcc tggccttttg ctggcctttt gctcacatgt t
//
