LOCUS       sequence-232748-        4560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway cloning vector for the cell cycle indicator PIP-FUCCI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4560)
  AUTHORS   Grant GD, Kedziora KM, Limas JC, Cook JG, Purvis JE
  TITLE     Accurate delineation of cell cycle phase transitions in living cells
            with PIP-FUCCI.
  JOURNAL   Cell Cycle. 2018;17(21-22):2496-2516. doi: 
            10.1080/15384101.2018.1547001.
  PUBMED    30421640
REFERENCE   2  (bases 1 to 4560)
  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: "Cell 
            Cycle."; date: "2018"; volume: "17(21-22):2496-2516. doi"; pages: " 
            10.1080/15384101.2018.1547001"
COMMENT     Sequence Label: pENTR-PIP-FUCCI
FEATURES             Location/Qualifiers
     source          1..4560
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             162..182
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             192..218
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             222..248
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(255..257,258..260,261..968)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(300..321)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(561..580)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     908..929
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             984..1040
                     /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             1047..1754
                     /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(1192..1210)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1460..1479
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1761..2090
                     /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(2107..2206)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2223..2242)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2224..2242)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2247..2263)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2247..2263)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2365..2384)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2376..3185
                     /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(2443..2462)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3332..3920
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3821..3840
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4074..4091
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4250..4277)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4369..4455)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4504..4526
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4518..4535
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4519..4535
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    join(4551..4560,1..90)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa tgctttttta
       61 taatgccaac tttgtacaaa aaagcaggct tcgaattcat ggagcagcgc cgcgtcaccg
      121 acttcttcgc gcgccgccgc cccgggcccc ggacacgtgg cccaaaaaag aagagaaagg
      181 taggagctca atacccatac gatgttcctg actatgcggg ctatccctat gacgtcccgg
      241 actatgcaac tagtatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      301 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      361 gcgatgccac ctacggcaag ctgaccctga agctgatctg caccaccggc aagctgcccg
      421 tgccctggcc caccctcgtg accaccctgg gctacggcct gcagtgcttc gcccgctacc
      481 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      541 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      601 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      661 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcaccgccg
      721 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggcg
      781 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
      841 tgcccgacaa ccactacctg agctaccagt ccgccctgag caaagacccc aacgagaagc
      901 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
      961 agctgtacct cgagggaagc ggagctacta acttcagcct gctgaagcag gctggagacg
     1021 tggaggagaa ccctggacct caattgatgg tgagcaaggg cgaggaggat aacatggcca
     1081 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
     1141 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     1201 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
     1261 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
     1321 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     1381 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     1441 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
     1501 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
     1561 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg
     1621 tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg
     1681 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
     1741 acgagctgta caaggtcgag atgaatccca gtatgaagca gaaacaagaa gaaatcaaag
     1801 agaatataaa gaatagttct gtcccaagaa gaactctgaa gatgattcag ccttctgcat
     1861 ctggatctct tgttggaaga gaaaatgagc tgtccgcagg cttgtccaaa aggaaacatc
     1921 ggaatgacca cttaacatct acaacttcca gccctggggt tattgtccca gaatctagtg
     1981 aaaataaaaa tcttggagga gtcacccagg agtcatttga tcttatgatt aaagaaaatc
     2041 catcctctca gtattggaag gaagtggcag aaaaacggag aaaggcgctg taacaattgg
     2101 gatcctaccc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt
     2161 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata
     2221 tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt
     2281 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac
     2341 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     2401 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     2461 ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag
     2521 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     2581 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     2641 ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag
     2701 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     2761 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     2821 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     2881 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     2941 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     3001 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     3061 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     3121 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     3181 tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg
     3241 acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt
     3301 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3361 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3421 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     3481 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3541 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3601 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3661 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3721 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3781 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3841 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3901 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3961 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4021 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4081 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     4141 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     4201 acgcaattaa tacgcgtacc gctagccagg aagagtttgt agaaacgcaa aaaggccatc
     4261 cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc gtcctgcccg
     4321 ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat ttgtcctact
     4381 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt ccgactgagc
     4441 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc gttaacgcta gcatggatgt
     4501 tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc caaataatga
//
