LOCUS       sequence-446913-        4301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing loxP flanked 
            3xNLS-mCherry-V5-3xSTOP cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4301)
  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 4301)
  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-lox-3xNLS-mCherry-V5-SV40pA-3xstop-lox (JD...
FEATURES             Location/Qualifiers
     source          1..4301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    33..132
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(133..166)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      210..219
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             222..242
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             246..266
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             270..290
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             312..1019
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKGFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(457..475)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     725..744
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1020..1061
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    1088..1209
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1329..1345)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1329..1345)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     polyA_signal    1346..1457
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    1549..1683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1769..1789)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1769..1787)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1808..1824)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1821..1843)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1832..1848
                     /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)."
     protein_bind    complement(1856..1889)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1890..1989)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2007..2025)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2030..2046)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2148..2167)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2159..2968
                     /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(2226..2245)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3115..3703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3604..3623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3857..3874
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4033..4060)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4152..4238)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(4287..4301,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4301,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag
       61 tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtaca
      121 aaaaagcagg ctataacttc gtatagcata cattatacga agttatcgga gcaagcttca
      181 tatggtcgac gatatcatcg atgctgcagg ccaccatggg tccaaaaaag aagagaaagg
      241 taggtccaaa aaagaagaga aaggtaggtc caaaaaagaa gagaaaggta ggtggtgctg
      301 gtggtagatc tatggtgagc aagggcgagg aggataacat ggccatcatc aaggggttca
      361 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
      421 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
      481 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
      541 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
      601 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
      661 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
      721 acggccccgt aatgcagaag aagactatgg gctgggaggc ctcctccgag cggatgtacc
      781 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
      841 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
      901 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
      961 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg
     1021 gtaagcctat ccctaaccct ctcctcggtc tcgattctac gggtacctaa taattttaaa
     1081 aaacgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     1141 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     1201 tgtatcttaa cgcgtctcga gggatcataa tcagccatac cacatttgta gaggttttac
     1261 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     1321 ttgttgttga tccactagtt ctagactgtg ccttctagtt gccagccatc tgttgtttgc
     1381 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1441 aatgaggaaa ttgcatcatc gagggggggc ccggtaacca ccgcggtggc ggcgatctcg
     1501 cggccgcaag taaagcggcc ctagaactat agtgagtcgt attacgtaga tccagacatg
     1561 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     1621 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     1681 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     1741 ttttgggccc ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     1801 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacaataac
     1861 ttcgtatagc atacattata cgaagttata cccagctttc ttgtacaaag ttggcattat
     1921 aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat
     1981 cattatttgc catccagctg atatccccta tagtgagtcg tattacatgg tcatagctgt
     2041 ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa
     2101 tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     2161 gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt
     2221 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt
     2281 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     2341 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     2401 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     2461 aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     2521 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     2581 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     2641 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     2701 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     2761 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     2821 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     2881 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     2941 tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca
     3001 gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag
     3061 ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3121 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3181 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3241 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3301 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3361 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3421 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3481 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3541 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3601 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3661 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3721 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3781 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3841 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     3901 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     3961 ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt accgctagcc aggaagagtt
     4021 tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg atgcctggca
     4081 gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc
     4141 gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga
     4201 aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt tccctactct
     4261 cgcgttaacg ctagcatgga tgttttccca gtcacgacgt t
//
