LOCUS       sequence-446886-        4367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible 3' entry clone containing an 
            IRES-FLAG-NLS-mTagBFP2-pA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4367)
  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 4367)
  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: p3E-IRES-mTagBFP2-pA (JDW 1361)
FEATURES             Location/Qualifiers
     source          1..4367
                     /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    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    join(749..1323,1324..1326,1327..1335)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(916..933)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1143..1162
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1339..1404
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1423..1443
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1447..1467
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1471..1491
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1513..2223
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     polyA_signal    2280..2414
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2304..2323)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2358..2377
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2476..2571)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2608..2627)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2609..2627)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2632..2648)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2632..2648)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2750..2769)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2761..3570
                     /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(2828..2847)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3717..4305
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4206..4225
                     /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 tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtggccc cctctccctc ccccccccct aacgttactg
      781 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
      841 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
      901 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
      961 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc
     1021 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac
     1081 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca
     1141 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt
     1201 gtatgggatc tgatctgggg cctcggtaca catgctttac atgtgtttag tcgaggttaa
     1261 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat
     1321 aatatggcca caaccatgga ctacaaagac catgacggtg attataaaga tcatgacatc
     1381 gactacaagg atgacgatga caaaggatcc accggtgaat tcccgaagaa gaaaagaaaa
     1441 gtaggaccca agaagaaacg caaagtcgga ccgaagaaga aacgaaaagt cagatctggc
     1501 ggtgctggtg gtatggtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag
     1561 ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc
     1621 aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc
     1681 ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac
     1741 acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga
     1801 gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac
     1861 ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg
     1921 atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc
     1981 ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca
     2041 aacgccaaga ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc
     2101 tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag
     2161 cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctc
     2221 aatgctagcg gctgatatct ataatgaggc gcgccgtcga cacgcgttaa tgatacgtag
     2281 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     2341 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     2401 gcaataaaca agttaacaac atctagacat tcattttatg tttcaggttc agggggaggt
     2461 gtgggaggtt ttttccaact ttattataca aagttggcat tataaaaaag cattgcttat
     2521 caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt tggagctcca
     2581 tggtagcgtt aacgcggccg cgatatcccc tatagtgagt cgtattacat ggtcatagct
     2641 gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa
     2701 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     2761 atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat
     2821 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc
     2881 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     2941 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     3001 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     3061 ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     3121 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     3181 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     3241 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     3301 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     3361 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     3421 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     3481 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     3541 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg
     3601 cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg
     3661 agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3721 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3781 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3841 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     3901 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3961 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4021 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4081 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4141 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4201 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4261 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4321 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtt
//
