LOCUS       sequence-447070-        3392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone containing a Myristoylated 
            mTagBP2 with a c-terminal Flag tag (cell membrane BFP marker).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3392)
  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 3392)
  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-myr-mTagBFP2-Flag (JDW 1183)
FEATURES             Location/Qualifiers
     source          1..3392
                     /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      675..684
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             705..1403
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    1404..1469
                     /label=MCS
                     /note="multiple cloning site"
     CDS             1482..1505
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(1517..1616)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1633..1652)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1634..1652)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1657..1673)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1657..1673)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1775..1794)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1786..2595
                     /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(1853..1872)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2742..3330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3231..3250
                     /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 agcaggcttt aaacgccacc atgggttgct gtttctccaa gaccatgagc gagctgatta
      721 aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat cacttcaagt
      781 gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg
      841 tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc ctctacggca
      901 gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag tccttccctg
      961 agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc
     1021 aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact
     1081 tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga
     1141 cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg aagctcgtgg
     1201 gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa cccgctaaga
     1261 acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc aaggaggcca
     1321 acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc gacctcccta
     1381 gcaaactggg acacaagctt aattccggac tcagatctcg agctcaagct tcgaattctg
     1441 cagtcgacgg taccgcgggc ccgggatcca ccggatctag agactacaaa gacgatgacg
     1501 acaagtagcc atctagaccc agctttcttg tacaaagttg gcattataag aaagcattgc
     1561 ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat
     1621 ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc
     1681 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     1741 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     1801 cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     1861 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc
     1921 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     1981 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     2041 atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc
     2101 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     2161 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     2221 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     2281 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     2341 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     2401 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     2461 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     2521 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     2581 gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg
     2641 acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc
     2701 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2761 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2821 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2881 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2941 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3001 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3061 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3121 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3181 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3241 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3301 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3361 ctggcctttt gctggccttt tgctcacatg tt
//
