LOCUS       sequence-456039-        4310 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing a 
            3x-NLS-mTagBFP2-FLAG for labeling the nucleus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4310)
  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 4310)
  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 nls BFP FLAG WPRE bGH pA (JDW 484)
FEATURES             Location/Qualifiers
     source          1..4310
                     /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      677..686
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             710..730
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             734..754
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             758..778
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             794..1492
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    1493..1558
                     /label=MCS
                     /note="multiple cloning site"
     CDS             1571..1594
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1664..1684)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2082..2093)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2202..2218)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2203..2219)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2220..2237)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2226..2337
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2435..2534)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2551..2570)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2552..2570)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2575..2591)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2575..2591)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2693..2712)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2704..3513
                     /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(2771..2790)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3660..4248
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4149..4168
                     /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 agcaggctgc gatcgcgcca ccatggccac aaccatggtg cacgtggatc caaaaaagaa
      721 gagaaaggta gatccaaaaa agaagagaaa ggtagatcca aaaaagaaga gaaaggtaca
      781 cgtgtccacg cgtatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga
      841 gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga
      901 gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga
      961 catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat
     1021 ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata
     1081 cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
     1141 ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa
     1201 aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg
     1261 cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac
     1321 cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga
     1381 ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt
     1441 ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta attccggact
     1501 cagatctcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc cgggatccac
     1561 cggatctaga gactacaaag acgatgacga caagtagagg gagctcatct agtcaacctc
     1621 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     1681 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     1741 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     1801 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     1861 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     1921 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     1981 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     2041 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     2101 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     2161 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgac
     2221 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2281 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2341 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2401 ggattgggaa gacaatagca ggcatggcga tcgcacccag ctttcttgta caaagttggc
     2461 attataagaa agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat
     2521 aaaatcatta tttgccatcc agctgatatc ccctatagtg agtcgtatta catggtcata
     2581 gctgtttcct ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat
     2641 aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     2701 gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct
     2761 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     2821 cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     2881 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     2941 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     3001 cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     3061 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     3121 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     3181 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     3241 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     3301 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     3361 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     3421 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     3481 cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac
     3541 tggcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa atcccttaac
     3601 gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3661 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3721 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3781 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3841 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3901 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3961 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4021 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4081 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4141 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4201 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4261 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
//
