LOCUS       sequence-485805-        4198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway middle entry clone containing an EGFP fused to the murine 
            microRNA 500. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4198)
  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 4198)
  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-EGFP-mm-miR-500 (JDW 8)
FEATURES             Location/Qualifiers
     source          1..4198
                     /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"
     CDS             join(689..691,692..694,695..1153)
                     /codon_start=1
                     /product="improved N-terminal fragment of mVenus for use in
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN155(I152L)
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA"
     primer_bind     complement(734..755)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(995..1014)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             complement(1674..1685)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2231..2252
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2323..2422)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2439..2458)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2440..2458)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2463..2479)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2463..2479)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2581..2600)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2592..3401
                     /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(2659..2678)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3548..4136
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4037..4056
                     /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 agcaggctcc gcggccgccc ccttcaccat ggtgagcaag ggcgaggagc tgttcaccgg
      721 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      781 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      841 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
      901 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
      961 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     1021 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     1081 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     1141 ctatatcatg gccgacaagc agaagaacgg catcaaggtg agtggatcgg gtaccgaatt
     1201 ccatgatcag acccgcctaa ggctagcggg gcccatgatc atcccgggga tccaccatgc
     1261 tgagcaggtg gtagtcccca agatagacta gctctcaccc tcaaaagaga tgcgtaggga
     1321 gtggcatctc catgttttca gttgatggtg agtcacaaaa tgggactttt taatctacag
     1381 aaacacacaa agtgtaagac ttctcatttc tgctttcata ttggtgtata gtacagtgct
     1441 tcccgccccc cccatctccc ccccccgcct ccccccctgc acattgagta ctcaaggccc
     1501 tggtccttct cctctgctcc ccctctctaa tccttgctat ctgggtgctt agtgctatct
     1561 caatgcaatg cacctgggca agggttcaga gaaggtgagc tcatctgcat caagaaggac
     1621 cagagaccct gtgtgtcagc ctacccctcc ctccttcttt ccccctcccc cttccttccc
     1681 tctattgagt aaaatagttg tttgagtgag tcatgtatac cagggaaccc cctaacagat
     1741 tatttaagag atttcaggta ctctagttgg cttctttcta cagctaactt ataaaatact
     1801 caggtacctg gagtgtgtag gctttctctg tattctgtcc tgtatggtta tacaggatga
     1861 ggtcaggagt tcaaacctgc tgcagatgca gtcagatttc tgaactgtct cagcaaatgg
     1921 taagtaactt agagctagta attaataacc tgtcctttga tttctgattc agccaagaat
     1981 ggccatattt gggaaaggca gatctggaga gtaaccacgt tttcatgcag tctagactgt
     2041 taacgcagtg tttctctaac tctcaggtga acttcaagat ccgccacaac atcgaggacg
     2101 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2161 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     2221 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2281 acgagctgta caagtaaagc ggccgcaagg gtgggcgcgc cgacccagct ttcttgtaca
     2341 aagttggcat tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca
     2401 gtcaaaataa aatcattatt tgccatccag ctgatatccc ctatagtgag tcgtattaca
     2461 tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg
     2521 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata
     2581 caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca
     2641 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     2701 caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     2761 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     2821 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     2881 ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     2941 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     3001 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     3061 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     3121 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     3181 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     3241 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     3301 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     3361 ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt
     3421 tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat
     3481 cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3541 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3601 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3661 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     3721 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3781 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3841 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3901 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3961 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4021 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4081 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4141 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtt
//
