LOCUS       sequence-446936-        4415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing a NLS-EGFP-Cre 
            fusion .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4415)
  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 4415)
  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 EGFP-Cre (JDW 1329)
FEATURES             Location/Qualifiers
     source          1..4415
                     /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             692..712
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(728..730,731..733,734..1444)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(728..730,731..733,734..1444)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1034..1053)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1381..1402
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1499..2530
                     /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(2540..2639)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2656..2675)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2657..2675)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2680..2696)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2680..2696)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2798..2817)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2809..3618
                     /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(2876..2895)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3765..4353
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4254..4273
                     /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 agcaggctac tagtttaatt aagccaccat gccaaaaaag aagagaaagg taggtggtgc
      721 tggtggtatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
      781 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
      841 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
      901 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
      961 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     1021 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1081 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1141 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     1201 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     1261 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1321 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     1381 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     1441 caagtccggt acagctctcg acggagaaag ctcaggctct ggctcagagt ctgactccat
     1501 ggccaattta ctgaccgtac accaaaattt gcctgcatta ccggtcgatg caacgagtga
     1561 tgaggttcgc aagaacctga tggacatgtt cagggatcgc caggcgtttt ctgagcatac
     1621 ctggaaaatg cttctgtccg tttgccggtc gtgggcggca tggtgcaagt tgaataaccg
     1681 gaaatggttt cccgcagaac ctgaagatgt tcgcgattat cttctatatc ttcaggcgcg
     1741 cggtctggca gtaaaaacta tccagcaaca tttgggccag ctaaacatgc ttcatcgtcg
     1801 gtccgggctg ccacgaccaa gtgacagcaa tgctgtttca ctggttatgc ggcggatccg
     1861 aaaagaaaac gttgatgccg gtgaacgtgc aaaacaggct ctagcgttcg aacgcactga
     1921 tttcgaccag gttcgttcac tcatggaaaa tagcgatcgc tgccaggata tacgtaatct
     1981 ggcatttctg gggattgctt ataacaccct gttacgtata gccgaaattg ccaggatcag
     2041 ggttaaagat atctcacgta ctgacggtgg gagaatgtta atccatattg gcagaacgaa
     2101 aacgctggtt agcaccgcag gtgtagagaa ggcacttagc ctgggggtaa ctaaactggt
     2161 cgagcgatgg atttccgtct ctggtgtagc tgatgatccg aataactacc tgttttgccg
     2221 ggtcagaaaa aatggtgttg ccgcgccatc tgccaccagc cagctatcaa ctcgcgccct
     2281 ggaagggatt tttgaagcaa ctcatcgatt gatttacggc gctaaggatg actctggtca
     2341 gagatacctg gcctggtctg gacacagtgc ccgtgtcgga gccgcgcgag atatggcccg
     2401 cgctggagtt tcaataccgg agatcatgca agctggtggc tggaccaatg taaatattgt
     2461 catgaactat atccgtaacc tggatagtga aacaggggca atggtgcgcc tgctggaaga
     2521 tggcgattag tagtaggcta gccagctttc ttgtacaaag ttggcattat aagaaagcat
     2581 tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc
     2641 catccagctg atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag
     2701 ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     2761 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     2821 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     2881 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
     2941 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     3001 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     3061 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
     3121 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     3181 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     3241 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     3301 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     3361 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     3421 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     3481 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     3541 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     3601 ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg
     3661 ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg
     3721 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3781 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3841 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3901 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3961 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4021 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4081 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4141 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4201 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4261 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4321 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4381 ttcctggcct tttgctggcc ttttgctcac atgtt
//
