LOCUS       sequence-447313-        3948 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing a Rox flanked 
            3xNLS-mCherry-V5-2xStop cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3948)
  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 3948)
  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-rox-nls-mCherry-V5-2xStop-rox (JDW 1167)
FEATURES             Location/Qualifiers
     source          1..3948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    185..284
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    327..358
                     /label=rox
                     /bound_moiety="Dre recombinase"
     regulatory      386..395
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             401..421
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             425..445
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             449..469
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     892..911
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1086..1109
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1187..1228
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    1284..1405
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1482..1603
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    1681..1712
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(1741..1840)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1858..1874)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1858..1874)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1871..1893)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1882..1898
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1906..1912,1913..1930,1931..1936))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1951..1972
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2089..2106)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2260..2848)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2340..2359)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3026..3835)
                     /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     3749..3768
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3827..3846
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accacaaata atgattttat tttgactgat agtgacctgt tcgttgcaac acattgatga
      241 gcaatgcttt tttataatgc caactttgta caaaaaagca ggctttaatt aaccgtctta
      301 agcctgtaac aaccggtaca gttcgataac tttaaataat gccaattatt taaagttaca
      361 agcttggtac cctaggttcc cgcgggccac catggccggc ccaaaaaaga agagaaaggt
      421 agatccaaaa aagaagagaa aggtagatcc aaaaaagaag agaaaggtag gatccggcat
      481 ggtgagcaag ggcgaggagg acaacatggc catcatcaag gagttcatgc gcttcaaggt
      541 gcacatggag ggctccgtga acggccacga gttcgagata gaaggagaag gagaaggacg
      601 gccatacgaa ggtacgcaaa cagctaaact caaggtgacc aagggcggcc ccctgccctt
      661 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
      721 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
      781 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tccaggacgg
      841 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
      901 gcagaagaag acgatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
      961 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgccga
     1021 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     1081 catcaagctg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg
     1141 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagggta agcctatccc
     1201 taaccctctc ctcggtctcg attctacgta gtaagagcta agcggccgca cttgcaggct
     1261 gcctatatcg ataccagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1321 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1381 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     1441 tcagggggag gtgtgggagg tttttttcta gacagacatg ataagataca ttgatgagtt
     1501 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     1561 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     1621 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaagctt ctgcagctag
     1681 taactttaaa taatgccaat tatttaaagt tactcgaggc gatcgccgat atcaacgcgt
     1741 acccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca atttgttgca
     1801 acgaacaggt cactatcagt caaaataaaa tcattatttg agcttggtgt aatcatggtc
     1861 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     1921 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     1981 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2041 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2101 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2161 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2221 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2281 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2341 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2401 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2461 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2521 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2581 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2641 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2701 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2761 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2821 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2881 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2941 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaagcccaa tctgaataat
     3001 gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca
     3061 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     3121 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     3181 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     3241 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     3301 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     3361 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     3421 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     3481 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt tttccgggga
     3541 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     3601 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     3661 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaagcgat
     3721 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     3781 catccatgtt ggaatttaat cgcggcctcg acgtttcccg ttgaatatgg ctcataacac
     3841 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat
     3901 cttgtgcaat gtaacatcag agattttgag acacgggcca gagctgca
//
