LOCUS       sequence-447031-        3818 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone containing a V5-tagged 
            mScarlet fused to Giantin (For visualizing the Golgi).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3818)
  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 3818)
  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-mScarlet3-Giantin (JDW 1356)
FEATURES             Location/Qualifiers
     source          1..3818
                     /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      681..690
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             690..731
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             762..1448
                     /codon_start=1
                     /product="bright cysteine-free monomeric red fluorescent 
                     protein with fast and complete maturation (Gadella et al., 
                     2023)"
                     /label=mScarlet3
                     /translation="MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LRVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGA
                     VSVAQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKM
                     ALRLKDGGRYLADFKTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHS
                     TGGSGGS"
     primer_bind     1160..1179
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1943..2042)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2059..2078)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2060..2078)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2083..2099)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2083..2099)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2201..2220)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2212..3021
                     /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(2279..2298)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3168..3756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3657..3676
                     /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 gccaccatgg gtaagcctat ccctaaccct ctcctcggtc
      721 tcgattctac gggtaccgga tcaggaggat caggagctag catggatagc accgaggcag
      781 tgatcaagga gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac ggccacgagt
      841 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
      901 gggtgaccaa gggtggcccc ctgcccttct cctgggacat cctgtcccct cagttcatgt
      961 acggctccag ggccttcacg aagcaccccg ccgacatccc cgactactgg aagcagtcct
     1021 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gccgtgtccg
     1081 tggcccagga cacctccctg gaggacggca ccctgatcta caaggtgaag ctccgcggca
     1141 ccaacttccc tcctgacggc cccgtaatgc agaagaagac aatgggctgg gaagcatcca
     1201 ccgagcggtt gtaccccgag gacgtcgtgc tgaagggcga cattaagatg gccctgcgcc
     1261 tgaaggacgg cggccgctac ctggcggact tcaagaccac ctacagggcc aagaagcccg
     1321 tgcagatgcc cggcgccttc aacatcgacc gcaagttgga catcacatcc cacaacgagg
     1381 actacaccgt ggtggaacag tacgaacgct ccgtggcccg ccactccacc ggcggctccg
     1441 gtggctccga attcgatggt ggaggcggtt caggcggagg tggctctggc ggtggcggat
     1501 cgccatcaag cttttctgaa gctcagcagc agctatgcaa caccagacag gaagtgaatg
     1561 aattaaggaa gctgctggaa gaagaacgag accaaagagt ggctgctgag aatgctctct
     1621 ctgtggccga ggagcagatc agacggttag agcacagtga atgggactct tcccggactc
     1681 ctatcattgg ctcctgtggc actcaggagc aggcactgtt aatagatctt acaagcaaca
     1741 gttgtcgaag gacccggagt ggcgttggat ggaagcgagt cctgcgttca ctctgtcatt
     1801 cacggacccg agtgccactt ctagcagcca tctactttct aatgattcat gtcctgctca
     1861 ttctgtgttt tacgggccat ctatgatatc tataatgagg cgcgccgtcg acgttaacac
     1921 gcgttaatga tacgtatcta gaacccagct ttcttgtaca aagttggcat tataagaaag
     1981 cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt
     2041 tgccatccag ctgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg
     2101 cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
     2161 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     2221 attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg
     2281 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg
     2341 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     2401 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     2461 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca
     2521 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     2581 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     2641 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     2701 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     2761 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     2821 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     2881 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     2941 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     3001 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt
     3061 acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg
     3121 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3181 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3241 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3301 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3361 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3421 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3481 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3541 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3601 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3661 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3721 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3781 cggttcctgg ccttttgctg gccttttgct cacatgtt
//
