LOCUS       sequence-446937-        3745 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing a 3x nuclear 
            localization signal followed by a multiple cloning site and a 
            flexible linker fused to Cre .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3745)
  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 3745)
  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-(G2SG2)2-Cre (JDW 1149)
FEATURES             Location/Qualifiers
     source          1..3745
                     /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             695..715
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             719..739
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             743..763
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             827..1858
                     /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(1870..1969)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1986..2005)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1987..2005)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2010..2026)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2010..2026)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2128..2147)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2139..2948
                     /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(2206..2225)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3095..3683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3584..3603
                     /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 agcaggctct cgagttattc tagacgccac catgccaaaa aagaagagaa aggtaggtcc
      721 aaaaaagaag agaaaggtag gtccaaaaaa gaagagaaag gtaggtggtg ctggtggtag
      781 atctgagctc ttctgcagtc gacgcgggcc cggtggtgct ggtggtatgg ccaatttact
      841 gaccgtacac caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa
      901 gaacctgatg gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct
      961 tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc
     1021 cgcagaacct gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt
     1081 aaaaactatc cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc
     1141 acgaccaagt gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt
     1201 tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt
     1261 tcgttcactc atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg
     1321 gattgcttat aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat
     1381 ctcacgtact gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag
     1441 caccgcaggt gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat
     1501 ttccgtctct ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa
     1561 tggtgttgcc gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt
     1621 tgaagcaact catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc
     1681 ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc
     1741 aataccggag atcatgcaag ctggtggctg gaccaatgta aatattgtca tgaactatat
     1801 ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattagta
     1861 gtagactagt cccagctttc ttgtacaaag ttggcattat aagaaagcat tgcttatcaa
     1921 tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc catccagctg
     1981 atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg
     2041 tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa
     2101 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     2161 cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
     2221 gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc
     2281 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
     2341 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
     2401 ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat
     2461 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     2521 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg
     2581 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
     2641 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac
     2701 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     2761 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     2821 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     2881 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     2941 ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac
     3001 gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag
     3061 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3121 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3181 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3241 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3301 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3361 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3421 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3481 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3541 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3601 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3661 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3721 tttgctggcc ttttgctcac atgtt
//
