LOCUS       sequence-244076-        6416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Cyclin B1 with R42A mutation in destruction 
            box to make cyclin B1 non-destructable.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6416)
  AUTHORS   Clute P, Pines J
  TITLE     Temporal and spatial control of cyclin B1 destruction in metaphase.
  JOURNAL   Nat Cell Biol. 1999 Jun;1(2):82-7.
  PUBMED    10559878
REFERENCE   2  (bases 1 to 6416)
  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: "Nat Cell 
            Biol."; date: "1999-06"; volume: "1(2)"; pages: "82-7"
COMMENT     Sequence Label: (R42A) Cyclin B1-GFP (62)
FEATURES             Location/Qualifiers
     source          1..6416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        118..497
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        498..701
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     652..672
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     746..765
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        746..764
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             784..813
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2131..2844
                     /codon_start=1
                     /gene="mgfp5"
                     /product="GFP with folding enhancement mutations"
                     /label=mgfp5
                     /note="suitable for expression in plants due to removal of 
                     a cryptic intron"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKTRHNIEDGGVQLADHYQQNTPIGDDPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2158..2186)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2781..2801
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     intron          2951..3016
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             3146..3166
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3438..3572
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3475..3494)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3529..3548
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4030..4164
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4229..4246)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4400..4988)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4480..4499)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5159..5950,5951..6019))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5782..5801
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6020..6124)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6192..6210
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6248..6270)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6370..6389
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg
       61 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gcgcgttgac
      121 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      181 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      241 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      301 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      361 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      421 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      481 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      541 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      601 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      661 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
      721 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagcttatc
      781 atggagcaaa aacttatatc tgaggaggac ctttccatgg cgctccgagc caccaggaac
      841 tcgaaaatta atgctgaaaa taaggcgaag atcaacatgg caggcgcaaa gcgcgttcct
      901 acggcccctg ctgcaacctc caagcccgga ctgaggccag caacagctct tggggacatt
      961 ggtaacaaag tcagtgaaca actgcaggcc aaaatgccta tgaagaagga agcaaaacct
     1021 tcagctactg gaaaagtcat tgataaaaaa ctaccaaaac ctcttgaaaa ggtacctatg
     1081 ctggtgccag tgccagtgtc tgagccagtg ccagagccag aacctgagcc agaacctgag
     1141 cctgttaaag aagaaaaact ttcgcctgag cctattttgg ttgatactgc ctctccaagc
     1201 ccaatggaaa catctggatg tgcccctgca gaagaagacc tgtgtcaggc tttctctgat
     1261 gtaattcttg cagtaaatga tgtggatgca gaagatggag ctgatccaaa cctttgtagt
     1321 gaatatgtga aagatattta tgcttatctg agacaacttg aggaagagca agcagtcaga
     1381 ccaaaatacc tactgggtcg ggaagtcact ggaaacatga gagccatcct aattgactgg
     1441 ctagtacagg ttcaaatgaa attcaggttg ttgcaggaga ccatgtacat gactgtctcc
     1501 attattgatc ggttcatgca gaataattgt gtgcccaaga agatgctgca gctggttggt
     1561 gtcactgcca tgtttattgc aagcaaatat gaagaaatgt accctccaga aattggtgac
     1621 tttgcttttg tgactgacaa cacttatact aagcaccaaa tcagacagat ggaaatgaag
     1681 attctaagag ctttaaactt tggtctgggt cggcctctac ctttgcactt ccttcggaga
     1741 gcatctaaga ttggagaggt tgatgtcgag caacatactt tggccaaata cctgatggaa
     1801 ctaactatgt tggactatga catggtgcac tttcctcctt ctcaaattgc agcaggagct
     1861 ttttgcttag cactgaaaat tctggataat ggtgaatgga caccaactct acaacattac
     1921 ctgtcatata ctgaagaatc tcttcttcca gttatgcagc acctggctaa gaatgtagtc
     1981 atggtaaatc aaggacttac aaagcacatg actgtcaaga acaagtatgc cacatcgaag
     2041 catgctaaga tcagcactct accacagctg aattctgcac tagttcaaga tttagccaag
     2101 gctgtggcaa aggtggcagg agcgcaattc agtaaaggag aagaactttt cactggagtt
     2161 gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtcagtgga
     2221 gagggtgaag gtgatgcaac atacggaaaa cttaccctta aatttatttg cactactgga
     2281 aaactacctg ttccatggcc aacacttgtc actactctca cttatggtgt tcaatgcttt
     2341 tcaagatatc cagatcatat gaagcggcac gacttcttca agagcgccat gcctgaggga
     2401 tacgtgcagg agaggaccat cttcttcaag gacgacggga actacaagac acgtgctgaa
     2461 gtcaagtttg agggagacac cctcgtcaac aggatcgagc ttaagggaat cgatttcaag
     2521 gaggacggaa acatcctcgg ccacaagttg gaatacaact acaactccca caacgtatac
     2581 atcatggccg acaagcaaaa gaacggcatc aaagccaact tcaagacccg ccacaacatc
     2641 gaagacggcg gcgtgcaact cgctgatcat tatcaacaaa atactccaat tggcgatgac
     2701 cctgtccttt taccagacaa ccattacctg tccacacaat ctgccctttc gaaagatccc
     2761 aacgaaaaga gagaccacat ggtccttctt gagtttgtaa cggctgctgg gattacacat
     2821 ggcatggatg aactatacaa ataggatcct ggccagctag ctaggtagct agaggatctt
     2881 tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta
     2941 aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat
     3001 tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc
     3061 ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac
     3121 tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga
     3181 ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc
     3241 ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga
     3301 aaaatatttg atgtatagtg ccttgactag agatcataat cagccatacc acatttgtag
     3361 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     3421 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3481 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3541 aactcatcaa tgtatcttat catgtctgga tcagcttcag aagatggcgg agggcctcca
     3601 acacagtaat tttcctcccg actcttaaaa tagaaaatgt caagtcagtt aagcaggaag
     3661 tgactaactg accgcagctg gccgtgcgac atcctctttt aattagttgc taggcaactg
     3721 ccctccagag ggcagtgtgg ttttgcaaga ggaagcaaaa agcctctcca cccaggccta
     3781 gaatgtttcc acccaatcat tactatgaca acagctgttt tttttagtat taagcagagg
     3841 ccgggggccc ctggcctccg cttactctgg agaaaaagaa gagaggcatt gtagaggctt
     3901 ccagaggcaa cttgtcaaaa caggactgct tctatttctg tcacactgtc tggccctgtc
     3961 acaaggtcca gcacctccat acccccttta ataagcagtt tgggaacggg tgcgggtctt
     4021 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4081 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4141 tagtgaggag gcttttttgg aggctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4201 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4261 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4321 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4381 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4441 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4501 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4561 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4621 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4681 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4741 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4801 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4861 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4921 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4981 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5041 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5101 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5161 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5221 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5281 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5341 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5401 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5461 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5521 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5581 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5641 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5701 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5761 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5821 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5881 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5941 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6001 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6061 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6121 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6181 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     6241 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     6301 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     6361 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaa
//
