LOCUS       sequence-486846-        7571 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway middle entry clone containing a zebrafish FUCCI reporter 
            followed by H2A tdiRFP reporter; For visualizing cell cycle state..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7571)
  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 7571)
  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-zFUCCI-P2A-H2A-tdiRFP (JDW 1380)
FEATURES             Location/Qualifiers
     source          1..7571
                     /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             699..764
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(777..779,780..782,783..1295)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(822..843)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1083..1102)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1433..1453
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1833..1889
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1890..2597
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2035..2053)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2303..2322
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3213..3269
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    complement(5696..5795)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5812..5831)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5813..5831)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5836..5852)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5836..5852)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5954..5973)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5965..6774
                     /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(6032..6051)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6921..7509
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7410..7429
                     /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 caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc ttagccggga tcccttcgat tggagatgga ctacaaagac catgacggtg
      721 attataaaga tcatgacatc gactacaagg atgacgatga caagctcaga tccaccatgg
      781 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      841 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
      901 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
      961 tgaccaccct gacctggggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc
     1021 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1081 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1141 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1201 tggagtacaa cgccatcagc gacaacgtct atatcaccgc cgacaagcag aagaacggca
     1261 tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1321 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1381 tgagcaccca gtccaagctg tcgaaagatc ccaacgaaaa gagagaccac atggtccttc
     1441 ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac aaaggatatc
     1501 catcacactg gcggccgctc gagatgagtt ccatcagaag accaaagaat gcagaaaacc
     1561 cgtctgaaaa catcaagaaa ttcctggtag cgccaacatc tggagcagga atggggagga
     1621 ggacgcttca ggtgcttcag ccctctgctg taaacaaaaa ccttggtcgt ataattgaga
     1681 atggtaaagc agtggccaaa aggaagatgt ggagcgccga gcaagttaag ggttcaaaga
     1741 gagtcaaagc agaggtggct gtcaaatcca caaatgcaga aaatgaaaac cagcctgagg
     1801 gggtcacaca agaggcctat gagggatctg gagccacgaa cttctctctg ttaaagcaag
     1861 caggagacgt ggaagaaaac cccggtccta tggtgagcaa gggcgaggag gataacatgg
     1921 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     1981 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     2041 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     2101 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     2161 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     2221 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     2281 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     2341 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
     2401 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     2461 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     2521 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     2581 tggacgagct gtacaaggga tatccatcac actggcggcc gctcgagatg gctcaagctc
     2641 gtgttactga ttattttgca cagagtaaaa aagctggtgt ttctcgatct ttacggtcta
     2701 agggacaaaa ggtgtccggg gatgtagtag aatcagcggt gatcaataaa ccgaggagtt
     2761 cgagcagagc gtccggttct tcacgcaaag cgacgcacag tgagatcacc acagcagagc
     2821 cgcagaaaca aactcagctg gagtttctca aagtcatcga cgaggcttta tcgacggaga
     2881 ctgcagatac cgtcgcggat tcgcgtgatg tgaagattga gggcttaacg gcgagtccca
     2941 ggacccctaa gcggagttca ccggagttcg acgtgtgttc ggtgttgttt ccgtccacag
     3001 ccgagctgca tagcagcgcc aagaagcggc agagactcaa cgcgggccac aactgcagga
     3061 gctcaccgga ggagcgcgct ggacagaaaa ctgcgaggaa aaaactctat ctactggcaa
     3121 gcgacgacaa agtaaagacc actgagccct tggccagcag cagccctcag gcacctcaac
     3181 aaactgcccg caaagagcga tctggcagcg gcgccacgaa cttctctctg ttaaagcaag
     3241 caggcgacgt ggaagaaaac cccggtcccg gtaccatggc aggtggaaaa gcaggtaaag
     3301 acagtggaaa agccaagacg aaagccgtct ccaggtctca gagagctggt ctgcagttcc
     3361 ctgtaggtcg tatccacagg cacctgaaga cccgaaccac cagccatggc cgtgtcggag
     3421 ctacagctgc ggtttacagt gctgctatcc tggagtatct caccgctgag gtgctggagt
     3481 tggctggaaa cgcttctaag gatctgaagg tgaagcgaat cactcccaga catttacagc
     3541 tggccatccg cggagacgag gagctggact cgcttatcaa ggccaccatt gctggaggag
     3601 gagtcattcc tcacatccac aagtctctga ttggtaagaa gggccagcag aaaaccgcag
     3661 atccaccggt cgacaccatg gccgaaggga gcgtggcaag acagcccgat ctgctgacat
     3721 gcgacgacga accaatccac attccaggag ctatccagcc tcatggactg ctgctggctc
     3781 tggccgctga catgactatc gtggcaggct ctgataacct gccagagctg accgggctgg
     3841 ctatcggagc actgattggc cggtccgcag ccgacgtgtt cgattctgag actcacaata
     3901 gactgaccat cgccctggct gaacctgggg ctgcagtggg agccccaatt accgtcggct
     3961 tcacaatgcg aaaggacgct ggctttatcg ggtcctggca ccgccatgat cagctgattt
     4021 ttctggagct ggaaccacct cagagagacg tggcagagcc acaggctttc tttcggagaa
     4081 caaacagcgc catcaggcgc ctgcaggccg ctgagactct ggaatccgct tgcgcagccg
     4141 ctgcacagga agtgaggaag atcaccgggt tcgatcgcgt catgatctac agattcgcct
     4201 cagactttag cggagaagtg atcgcagaag atcgctgtgc cgaggtcgaa agtaaactgg
     4261 gcctgcatta cccagcctca accgtgcctg cacaggcacg acggctgtat acaatcaacc
     4321 ccgtgcggat cattcccgac attaattaca gaccagtgcc cgtcacacct gacctgaacc
     4381 cagtgactgg gaggcccatt gatctgagct tcgccattct gcgcagcgtg tcccctgtcc
     4441 acctggagtt tatgaggaat atcggaatgc atggcaccat gtctatcagt attctgagag
     4501 gcgaaaggct gtgggggctg atcgtgtgcc accatcggac accttactat gtggacctgg
     4561 acggcaggca ggcttgtgaa ctggtggctc aggtgctggc ttggcagatt ggcgtgatga
     4621 aggaggggca tggcaccggc agcaccggca gcggcagcag cggccgctcc ggcaccgcct
     4681 cctccgagga caacaacatg gccgccgaag ggagcgtggc aagacagccc gatctgctga
     4741 catgcgacga cgaaccaatc cacattccag gagctatcca gcctcatgga ctgctgctgg
     4801 ctctggccgc tgacatgact atcgtggcag gctctgataa cctgccagag ctgaccgggc
     4861 tggctatcgg agcactgatt ggccggtccg cagccgacgt gttcgattct gagactcaca
     4921 atagactgac catcgccctg gctgaacctg gggctgcagt gggagcccca attaccgtcg
     4981 gcttcacaat gcgaaaggac gctggcttta tcgggtcctg gcaccgccat gatcagctga
     5041 tttttctgga gctggaacca cctcagagag acgtggcaga gccacaggct ttctttcgga
     5101 gaacaaacag cgccatcagg cgcctgcagg ccgctgagac tctggaatcc gcttgcgcag
     5161 ccgctgcaca ggaagtgagg aagatcaccg ggttcgatcg cgtcatgatc tacagattcg
     5221 cctcagactt tagcggagaa gtgatcgcag aagatcgctg tgccgaggtc gaaagtaaac
     5281 tgggcctgca ttacccagcc tcaaccgtgc ctgcacaggc acgacggctg tatacaatca
     5341 accccgtgcg gatcattccc gacattaatt acagaccagt gcccgtcaca cctgacctga
     5401 acccagtgac tgggaggccc attgatctga gcttcgccat tctgcgcagc gtgtcccctg
     5461 tccacctgga gtttatgagg aatatcggaa tgcatggcac catgtctatc agtattctga
     5521 gaggcgaaag gctgtggggg ctgatcgtgt gccaccatcg gacaccttac tatgtggacc
     5581 tggacggcag gcaggcttgt gaactggtgg ctcaggtgct ggcttggcag attggcgtga
     5641 tgaaggagga ataactcgag taatgataaa ctagtgttta aactacgtaa ctaggaccca
     5701 gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa
     5761 caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgatat cccctatagt
     5821 gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc
     5881 tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac
     5941 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga
     6001 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     6061 caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg
     6121 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     6181 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     6241 tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct
     6301 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     6361 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     6421 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     6481 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
     6541 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     6601 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     6661 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     6721 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa
     6781 ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc
     6841 tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta
     6901 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6961 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     7021 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     7081 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     7141 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     7201 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     7261 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7321 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7381 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7441 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7501 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7561 gctcacatgt t
//
