LOCUS       sequence-484474-        4696 bp    DNA     circular SYN 25-AUG-2025
DEFINITION  Gateway middle entry clone containing FastFUCCI reporter; For 
            visualizing cell cycle state.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4696)
  AUTHORS   Gillespie W, Zhang Y, Ruiz OE, Cerda J, Ortiz-Guzman J, Turner WD, 
            Largoza G, Sherman M, 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 with diverse applications in vertebrate model 
            systems.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267.
  PUBMED    39026881
REFERENCE   2  (bases 1 to 4696)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267."
COMMENT     Sequence Label: pME-FastFUCCI (JDW 1488)
FEATURES             Location/Qualifiers
     source          1..4696
                     /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             710..1360
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAH"
     CDS             1415..1687
                     /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             1694..1747
                     /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             1754..2431
                     /codon_start=1
                     /product="humanized monomeric Azami-Green fluorescent 
                     protein"
                     /label=hmAzami-Green
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             1754..2431
                     /codon_start=1
                     /product="monomeric Azami-Green fluorescent protein 
                     (Karasawa et al., 2003)"
                     /label=mAzami-Green
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             2462..2791
                     /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"
     protein_bind    complement(2821..2920)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2937..2956)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2938..2956)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2961..2977)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2961..2977)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3079..3098)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3090..3899
                     /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(3157..3176)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4046..4634
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4535..4554
                     /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 agcaggctgg tctgactggt acctcgtgat taattaagaa ttgggatcca tggtgtccgt
      721 gatcaagccc gagatgaaga tgcggtacta catggacggc agcgtgaacg gccacgagtt
      781 caccatcgag ggcgagggca ccggcagacc ttacgagggc caccaggaaa tgaccctgag
      841 agtcaccatg gccgagggcg gacccatgcc cttcgccttc gacctggtgt cccacgtgtt
      901 ctgctacggc caccgggtgt tcaccaagta ccccgaggaa atccccgact acttcaagca
      961 ggccttcccc gagggcctga gctgggagcg gagcctggaa ttcgaggatg gcggcagcgc
     1021 ctctgtgtct gcccacatca gcctgcgggg caacaccttc taccacaaga gcaagttcac
     1081 cggcgtgaac ttccccgccg acggccccat catgcagaac cagagcgtgg actgggagcc
     1141 cagcaccgag aagatcaccg ccagcgacgg cgtgctgaag ggcgacgtga ccatgtacct
     1201 gaagctggaa ggcggcggaa accacaagtg ccagatgaaa accacctaca aggccgccaa
     1261 agaaatcctg gaaatgcccg gcgaccacta catcggccac cggctggtgc ggaaaaccga
     1321 gggcaacatc accgagcagg tggaggatgc cgtggcccac tacagcatgc tgcctagcct
     1381 cggcggctac cctagccatt ggaggcccct ggaaccctct cctgccagac ctgccctgag
     1441 agcccctgcc tccgccacct ccggcagcag aaagagagcc agacctcctg ccgcccctgg
     1501 cagagatcag gccaggcccc ctgcccgcag acggctccgg ctgtccgtgg atgaggtgtc
     1561 cagccctagc acccccgagg cccccgatat ccctgcctgt cccagccctg gccagaaaat
     1621 caagaagagc acacctgccg ccggacagcc tcctcacctg accagcgccc aggaccagga
     1681 caccatcgtc gacgagggca ggggcagcct gctgacctgc ggcgacgtgg aggagaaccc
     1741 cggccccaga tccatggtgt ccgtgatcaa gcccgagatg aagatcaagc tgtgcatgcg
     1801 gggaaccgtg aacggccaca acttcgtgat cgagggcgag ggcaagggaa acccctacga
     1861 gggcacccag atcctggacc tgaacgtgac cgaaggcgcc cctctgccct tcgcctacga
     1921 catcctgacc accgtgttcc agtacggcaa ccgggccttc accaagtacc ccgccgacat
     1981 ccaggactac ttcaagcaga ccttccccga gggctaccac tgggagcgga gcatgaccta
     2041 cgaggaccag ggcatctgca ccgccacctc caacatcagc atgaggggcg actgcttttt
     2101 ctacgacatc agattcgacg gcaccaactt cccccccaac ggccccgtga tgcagaaaaa
     2161 gaccctgaag tgggagccca gcaccgagaa gatgtacgtg gaggacggcg tgctgaaggg
     2221 cgacgtgaac atgcggctgc tgctggaagg cggcggacac tacagatgcg acttcaagac
     2281 cacctacaag gccaagaaag aagtgcggct gcccgacgcc cacaagatcg accaccggat
     2341 cgagatcctg aagcacgaca aggactacaa caaagtgaag ctgtacgaga acgccgtggc
     2401 caggtacagc atgctgccca gccaggccaa gggctacccc agccactggc ggcctctgga
     2461 aatgaacccc agcatgaagc agaagcagga agagatcaaa gagaacatca agaacagcag
     2521 cgtgcccagg cggaccctga agatgatcca gcccagcgcc agcggatctc tggtgggccg
     2581 ggagaatgag ctgtctgccg gcctgagcaa gcggaagcac cggaacgacc acctgaccag
     2641 caccacctct agcccaggcg tgatcgtgcc cgagagcagc gagaacaaga acctgggcgg
     2701 cgtgacccaa gaaagcttcg acctgatgat caaagaaaac cccagcagcc agtactggaa
     2761 agaggtggcc gagaagcggc ggaaggccct gtagagatct gactgagcgg ccgccagtca
     2821 acccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca atttgttgca
     2881 acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct gatatcccct
     2941 atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa
     3001 atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg
     3061 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc
     3121 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     3181 tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt
     3241 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     3301 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     3361 atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat
     3421 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     3481 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     3541 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     3601 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     3661 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     3721 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     3781 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     3841 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     3901 cagaattggt taattggttg taacactggc agagcattac gctgacttga cgggacggcg
     3961 caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc
     4021 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4081 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4141 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     4201 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4261 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4321 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4381 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4441 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4501 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4561 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4621 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4681 cttttgctca catgtt
//
