LOCUS       sequence-484483-        5103 bp    DNA     circular SYN 25-AUG-2025
DEFINITION  Gateway middle entry clone containing zebrafish FUCCI reporter; For 
            visualizing cell cycle state..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5103)
  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 5103)
  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-zFUCCI (JDW 1464)
FEATURES             Location/Qualifiers
     source          1..5103
                     /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"
     protein_bind    complement(3228..3327)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3344..3363)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3345..3363)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3368..3384)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3368..3384)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3486..3505)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3497..4306
                     /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(3564..3583)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4453..5041
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4942..4961
                     /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 agcaggctac tgtcaagctt cagtcaggat ccggtatgga 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 caaagagtaa accggtacct gagtcatcga tcagactacc cagctttctt
     3241 gtacaaagtt ggcattataa gaaagcattg cttatcaatt tgttgcaacg aacaggtcac
     3301 tatcagtcaa aataaaatca ttatttgcca tccagctgat atcccctata gtgagtcgta
     3361 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     3421 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     3481 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     3541 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     3601 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     3661 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     3721 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     3781 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     3841 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     3901 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     3961 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     4021 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     4081 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     4141 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     4201 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     4261 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     4321 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc
     4381 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     4441 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4501 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4561 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4621 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4681 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4741 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4801 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4861 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4921 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4981 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5041 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5101 gtt
//
