LOCUS       sequence-486787-        3587 bp    DNA     circular SYN 19-SEP-2025
DEFINITION  Gateway middle entry clone containing an EGFP fused to a microRNA 
            multiple cloning site. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3587)
  AUTHORS   Gillespie W, Zhang Y, Ruiz OE, Cerda J, Ortiz-Guzman J, Turner WD, 
            Largoza G, Sherman M, 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 with diverse applications in vertebrate model 
            systems.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267.
  PUBMED    39026881
REFERENCE   2  (bases 1 to 3587)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 19, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267."
COMMENT     Sequence Label: pME-EGFP-miR-MCS (JDW 1231)
FEATURES             Location/Qualifiers
     source          1..3587
                     /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      700..709
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(706..708,709..711,712..1170)
                     /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(751..772)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1012..1031)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1616..1637
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1712..1811)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1828..1847)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1829..1847)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1852..1868)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1852..1868)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1970..1989)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1981..2790
                     /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(2048..2067)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2937..3525
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3426..3445
                     /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 agcaggcagc tagcgctacc ggactcagat ccaccggtcg ccaccatggt gagcaagggc
      721 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
      781 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
      841 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
      901 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
      961 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1021 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1081 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1141 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgagt
     1201 ggatcgggta ccgaattcca tgatcttttt tttttttttt tttcccatga tcatcccggg
     1261 gatcctgcag atgcagtcag atttctgaac tgtctcagca aatggtaagt aacttagagc
     1321 tagtaattaa taacctgtcc tttgatttct gattcagcca agaatggcca tatttgggaa
     1381 aggcagatct ggagagtaac cacgttttca tgcagtctag actgttaacg cagtgtttct
     1441 ctaactctca ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1501 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1561 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1621 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1681 aaagcggccg cgactctaga cgcgtactag tgcccagctt tcttgtacaa agttggcatt
     1741 ataagaaagc attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa
     1801 atcattattt gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagct
     1861 gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa
     1921 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     1981 atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat
     2041 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc
     2101 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     2161 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     2221 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     2281 ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     2341 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     2401 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     2461 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     2521 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     2581 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     2641 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     2701 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     2761 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg
     2821 cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg
     2881 agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2941 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3001 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3061 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     3121 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3181 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3241 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3301 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3361 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3421 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3481 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3541 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtt
//
