LOCUS       sequence-446929-        4471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway-compatible middle entry clone containing a 
            3xNLS-mTagBFP2-Cre fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4471)
  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 4471)
  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-3xNLS-mTagBFP2-Cre (JDW 918)
FEATURES             Location/Qualifiers
     source          1..4471
                     /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             679..699
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             703..723
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             727..747
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             769..1467
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1477..1503
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1507..1533
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1564..2595
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     protein_bind    complement(2596..2695)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2712..2731)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2713..2731)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2736..2752)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2736..2752)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2854..2873)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2865..3674
                     /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(2932..2951)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3821..4409
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4310..4329
                     /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 agcaggctcg ccaccatgcc aaaaaagaag agaaaggtag gtccaaaaaa gaagagaaag
      721 gtaggtccaa aaaagaagag aaaggtaggt ggtgctggtg gtagatctat gagcgagctg
      781 attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc
      841 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
      901 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac
      961 ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc
     1021 cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct
     1081 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
     1141 aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc
     1201 gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc
     1261 gtgggcggga gccatctgat cgcaaacgcc aagaccacat atagatccaa gaaacccgct
     1321 aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag
     1381 gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc
     1441 cctagcaaac tggggcacaa gcttaatgga ggtggctacc catacgatgt tccagattac
     1501 gccatgtacc catacgatgt tccagattac gccggtcgac gcgggcccgg tggtgctggt
     1561 ggtatggcca atttactgac cgtacaccaa aatttgcctg cattaccggt cgatgcaacg
     1621 agtgatgagg ttcgcaagaa cctgatggac atgttcaggg atcgccaggc gttttctgag
     1681 catacctgga aaatgcttct gtccgtttgc cggtcgtggg cggcatggtg caagttgaat
     1741 aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg attatcttct atatcttcag
     1801 gcgcgcggtc tggcagtaaa aactatccag caacatttgg gccagctaaa catgcttcat
     1861 cgtcggtccg ggctgccacg accaagtgac agcaatgctg tttcactggt tatgcggcgg
     1921 atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac aggctctagc gttcgaacgc
     1981 actgatttcg accaggttcg ttcactcatg gaaaatagcg atcgctgcca ggatatacgt
     2041 aatctggcat ttctggggat tgcttataac accctgttac gtatagccga aattgccagg
     2101 atcagggtta aagatatctc acgtactgac ggtgggagaa tgttaatcca tattggcaga
     2161 acgaaaacgc tggttagcac cgcaggtgta gagaaggcac ttagcctggg ggtaactaaa
     2221 ctggtcgagc gatggatttc cgtctctggt gtagctgatg atccgaataa ctacctgttt
     2281 tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct atcaactcgc
     2341 gccctggaag ggatttttga agcaactcat cgattgattt acggcgctaa ggatgactct
     2401 ggtcagagat acctggcctg gtctggacac agtgcccgtg tcggagccgc gcgagatatg
     2461 gcccgcgctg gagtttcaat accggagatc atgcaagctg gtggctggac caatgtaaat
     2521 attgtcatga actatatccg taacctggat agtgaaacag gggcaatggt gcgcctgctg
     2581 gaagatggcg attagaccca gctttcttgt acaaagttgg cattataaga aagcattgct
     2641 tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttgccatc
     2701 cagctgatat cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct
     2761 ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa
     2821 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     2881 gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     2941 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg
     3001 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     3061 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     3121 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc
     3181 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     3241 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     3301 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     3361 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     3421 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     3481 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     3541 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     3601 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     3661 atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga
     3721 cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc
     3781 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3841 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3901 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3961 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4021 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4081 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4141 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4201 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4261 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4321 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4381 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4441 tggccttttg ctggcctttt gctcacatgt t
//
