LOCUS       sequence-495449-        7151 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  Gateway middle entry clone containing mammalian FastFUCCI reporter 
            followed by H2A tdiRFP reporter; For visualizing cell cycle state. 
            tdiRFP reporter labels all cells regardless of cell cycle state.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7151)
  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 7151)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 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-FastFucci-P2A-H2A-tdiRFP (JDW 1399)
FEATURES             Location/Qualifiers
     source          1..7151
                     /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             696..1346
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAH"
     CDS             1401..1673
                     /codon_start=1
                     /product="degron consisting of residues 30-120 of human 
                     Cdt1 (Zielke and Edgar, 2015)"
                     /label=Cdt1 (30-120)
                     /translation="PSPARPALRAPASATSGSRKRARPPAAPGRDQARPPARRRLRLSV
                     DEVSSPSTPEAPDIPACPSPGQKIKKSTPAAGQPPHLTSAQDQDTI"
     CDS             1680..1733
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1740..2417
                     /codon_start=1
                     /product="humanized monomeric Azami-Green fluorescent 
                     protein"
                     /label=hmAzami-Green
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             1740..2417
                     /codon_start=1
                     /product="monomeric Azami-Green fluorescent protein 
                     (Karasawa et al., 2003)"
                     /label=mAzami-Green
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             2448..2777
                     /codon_start=1
                     /product="degron consisting of residues 1-110 of human 
                     Geminin (Zielke and Edgar, 2015)"
                     /label=Gem1 (1-110)
                     /translation="MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELS
                     AGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAE
                     KRRKAL"
     CDS             2793..2849
                     /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(5276..5375)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5392..5411)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5393..5411)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5416..5432)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5416..5432)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5534..5553)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5545..6354
                     /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(5612..5631)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6501..7089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6990..7009
                     /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 ttagattaat taagaattgg gatccatggt gtccgtgatc aagcccgaga
      721 tgaagatgcg gtactacatg gacggcagcg tgaacggcca cgagttcacc atcgagggcg
      781 agggcaccgg cagaccttac gagggccacc aggaaatgac cctgagagtc accatggccg
      841 agggcggacc catgcccttc gccttcgacc tggtgtccca cgtgttctgc tacggccacc
      901 gggtgttcac caagtacccc gaggaaatcc ccgactactt caagcaggcc ttccccgagg
      961 gcctgagctg ggagcggagc ctggaattcg aggatggcgg cagcgcctct gtgtctgccc
     1021 acatcagcct gcggggcaac accttctacc acaagagcaa gttcaccggc gtgaacttcc
     1081 ccgccgacgg ccccatcatg cagaaccaga gcgtggactg ggagcccagc accgagaaga
     1141 tcaccgccag cgacggcgtg ctgaagggcg acgtgaccat gtacctgaag ctggaaggcg
     1201 gcggaaacca caagtgccag atgaaaacca cctacaaggc cgccaaagaa atcctggaaa
     1261 tgcccggcga ccactacatc ggccaccggc tggtgcggaa aaccgagggc aacatcaccg
     1321 agcaggtgga ggatgccgtg gcccactaca gcatgctgcc tagcctcggc ggctacccta
     1381 gccattggag gcccctggaa ccctctcctg ccagacctgc cctgagagcc cctgcctccg
     1441 ccacctccgg cagcagaaag agagccagac ctcctgccgc ccctggcaga gatcaggcca
     1501 ggccccctgc ccgcagacgg ctccggctgt ccgtggatga ggtgtccagc cctagcaccc
     1561 ccgaggcccc cgatatccct gcctgtccca gccctggcca gaaaatcaag aagagcacac
     1621 ctgccgccgg acagcctcct cacctgacca gcgcccagga ccaggacacc atcgtcgacg
     1681 agggcagggg cagcctgctg acctgcggcg acgtggagga gaaccccggc cccagatcca
     1741 tggtgtccgt gatcaagccc gagatgaaga tcaagctgtg catgcgggga accgtgaacg
     1801 gccacaactt cgtgatcgag ggcgagggca agggaaaccc ctacgagggc acccagatcc
     1861 tggacctgaa cgtgaccgaa ggcgcccctc tgcccttcgc ctacgacatc ctgaccaccg
     1921 tgttccagta cggcaaccgg gccttcacca agtaccccgc cgacatccag gactacttca
     1981 agcagacctt ccccgagggc taccactggg agcggagcat gacctacgag gaccagggca
     2041 tctgcaccgc cacctccaac atcagcatga ggggcgactg ctttttctac gacatcagat
     2101 tcgacggcac caacttcccc cccaacggcc ccgtgatgca gaaaaagacc ctgaagtggg
     2161 agcccagcac cgagaagatg tacgtggagg acggcgtgct gaagggcgac gtgaacatgc
     2221 ggctgctgct ggaaggcggc ggacactaca gatgcgactt caagaccacc tacaaggcca
     2281 agaaagaagt gcggctgccc gacgcccaca agatcgacca ccggatcgag atcctgaagc
     2341 acgacaagga ctacaacaaa gtgaagctgt acgagaacgc cgtggccagg tacagcatgc
     2401 tgcccagcca ggccaagggc taccccagcc actggcggcc tctggaaatg aaccccagca
     2461 tgaagcagaa gcaggaagag atcaaagaga acatcaagaa cagcagcgtg cccaggcgga
     2521 ccctgaagat gatccagccc agcgccagcg gatctctggt gggccgggag aatgagctgt
     2581 ctgccggcct gagcaagcgg aagcaccgga acgaccacct gaccagcacc acctctagcc
     2641 caggcgtgat cgtgcccgag agcagcgaga acaagaacct gggcggcgtg acccaagaaa
     2701 gcttcgacct gatgatcaaa gaaaacccca gcagccagta ctggaaagag gtggccgaga
     2761 agcggcggaa ggccctggga tctggcagcg gcgccacgaa cttctctctg ttaaagcaag
     2821 caggcgacgt ggaagaaaac cccggtcccg gtaccatggc aggtggaaaa gcaggtaaag
     2881 acagtggaaa agccaagacg aaagccgtct ccaggtctca gagagctggt ctgcagttcc
     2941 ctgtaggtcg tatccacagg cacctgaaga cccgaaccac cagccatggc cgtgtcggag
     3001 ctacagctgc ggtttacagt gctgctatcc tggagtatct caccgctgag gtgctggagt
     3061 tggctggaaa cgcttctaag gatctgaagg tgaagcgaat cactcccaga catttacagc
     3121 tggccatccg cggagacgag gagctggact cgcttatcaa ggccaccatt gctggaggag
     3181 gagtcattcc tcacatccac aagtctctga ttggtaagaa gggccagcag aaaaccgcag
     3241 atccaccggt cgacaccatg gccgaaggga gcgtggcaag acagcccgat ctgctgacat
     3301 gcgacgacga accaatccac attccaggag ctatccagcc tcatggactg ctgctggctc
     3361 tggccgctga catgactatc gtggcaggct ctgataacct gccagagctg accgggctgg
     3421 ctatcggagc actgattggc cggtccgcag ccgacgtgtt cgattctgag actcacaata
     3481 gactgaccat cgccctggct gaacctgggg ctgcagtggg agccccaatt accgtcggct
     3541 tcacaatgcg aaaggacgct ggctttatcg ggtcctggca ccgccatgat cagctgattt
     3601 ttctggagct ggaaccacct cagagagacg tggcagagcc acaggctttc tttcggagaa
     3661 caaacagcgc catcaggcgc ctgcaggccg ctgagactct ggaatccgct tgcgcagccg
     3721 ctgcacagga agtgaggaag atcaccgggt tcgatcgcgt catgatctac agattcgcct
     3781 cagactttag cggagaagtg atcgcagaag atcgctgtgc cgaggtcgaa agtaaactgg
     3841 gcctgcatta cccagcctca accgtgcctg cacaggcacg acggctgtat acaatcaacc
     3901 ccgtgcggat cattcccgac attaattaca gaccagtgcc cgtcacacct gacctgaacc
     3961 cagtgactgg gaggcccatt gatctgagct tcgccattct gcgcagcgtg tcccctgtcc
     4021 acctggagtt tatgaggaat atcggaatgc atggcaccat gtctatcagt attctgagag
     4081 gcgaaaggct gtgggggctg atcgtgtgcc accatcggac accttactat gtggacctgg
     4141 acggcaggca ggcttgtgaa ctggtggctc aggtgctggc ttggcagatt ggcgtgatga
     4201 aggaggggca tggcaccggc agcaccggca gcggcagcag cggccgctcc ggcaccgcct
     4261 cctccgagga caacaacatg gccgccgaag ggagcgtggc aagacagccc gatctgctga
     4321 catgcgacga cgaaccaatc cacattccag gagctatcca gcctcatgga ctgctgctgg
     4381 ctctggccgc tgacatgact atcgtggcag gctctgataa cctgccagag ctgaccgggc
     4441 tggctatcgg agcactgatt ggccggtccg cagccgacgt gttcgattct gagactcaca
     4501 atagactgac catcgccctg gctgaacctg gggctgcagt gggagcccca attaccgtcg
     4561 gcttcacaat gcgaaaggac gctggcttta tcgggtcctg gcaccgccat gatcagctga
     4621 tttttctgga gctggaacca cctcagagag acgtggcaga gccacaggct ttctttcgga
     4681 gaacaaacag cgccatcagg cgcctgcagg ccgctgagac tctggaatcc gcttgcgcag
     4741 ccgctgcaca ggaagtgagg aagatcaccg ggttcgatcg cgtcatgatc tacagattcg
     4801 cctcagactt tagcggagaa gtgatcgcag aagatcgctg tgccgaggtc gaaagtaaac
     4861 tgggcctgca ttacccagcc tcaaccgtgc ctgcacaggc acgacggctg tatacaatca
     4921 accccgtgcg gatcattccc gacattaatt acagaccagt gcccgtcaca cctgacctga
     4981 acccagtgac tgggaggccc attgatctga gcttcgccat tctgcgcagc gtgtcccctg
     5041 tccacctgga gtttatgagg aatatcggaa tgcatggcac catgtctatc agtattctga
     5101 gaggcgaaag gctgtggggg ctgatcgtgt gccaccatcg gacaccttac tatgtggacc
     5161 tggacggcag gcaggcttgt gaactggtgg ctcaggtgct ggcttggcag attggcgtga
     5221 tgaaggagga ataactcgag taatgataaa ctagtgttta aactacgtaa ctaggaccca
     5281 gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa
     5341 caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgatat cccctatagt
     5401 gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc
     5461 tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac
     5521 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga
     5581 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     5641 caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg
     5701 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     5761 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     5821 tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct
     5881 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     5941 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     6001 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     6061 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
     6121 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     6181 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     6241 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     6301 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa
     6361 ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc
     6421 tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta
     6481 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6541 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6601 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6661 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6721 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6781 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6841 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6901 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6961 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7021 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7081 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7141 gctcacatgt t
//
