LOCUS       sequence-261428-        8358 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8358)
  AUTHORS   Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, 
            Kinzler KW, Vogelstein B
  TITLE     Mutations of mitotic checkpoint genes in human cancers.
  JOURNAL   Nature. 1998 Mar 19. 392(6673):300-3.
  PUBMED    9521327
REFERENCE   2  (bases 1 to 8358)
  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: "Nature. 
            1998 Mar 19. 392(6673):300-3."
COMMENT     Sequence Label: pBI-GFP-Bub1-wt
FEATURES             Location/Qualifiers
     source          1..8358
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    41..59
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    83..101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    167..185
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    209..227
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    251..269
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      410..419
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3704..3838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3728..3747)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3782..3801
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4201..4219)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4287..4391
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4392..4460,4461..5252)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4610..4629)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5423..6011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5912..5931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6165..6182
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6562..6581
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    6580..6635
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     6681..6700
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     intron          6832..7404
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             complement(join(7436..8149,8150..8152,8153..8155))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7481..7502)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     7830..7849
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8089..8110
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      8152..8161
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(join(8328..8358,1..8))
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 cgcctacctc gacccgggta ccgagctcga ctttcacttt tctctatcac tgatagggag
       61 tggtaaactc gactttcact tttctctatc actgataggg agtggtaaac tcgactttca
      121 cttttctcta tcactgatag ggagtggtaa actcgacttt cacttttctc tatcactgat
      181 agggagtggt aaactcgact ttcacttttc tctatcactg atagggagtg gtaaactcga
      241 ctttcacttt tctctatcac tgatagggag tggtaaactc gacctatata agcagagctc
      301 gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa
      361 gacaccggga ccgatccagc ctccgcggcc ccgaattcct gcagcggccg ccaccatgga
      421 caccccggaa aatgtccttc agatgcttga agcccacatg cagagctaca agggcaatga
      481 ccctcttggt gaatgggaaa gatacataca gtgggtagaa gagaattttc ctgagaataa
      541 agaatacttg ataactttac tagaacattt aatgaaggaa tttttagata agaagaaata
      601 ccacaatgac ccaagattca tcagttattg tttaaaattt gctgagtaca acagtgacct
      661 ccatcaattt tttgagtttc tgtacaacca tgggattgga accctgtcat cccctctgta
      721 cattgcctgg gcggggcatc tggaagccca aggagagctg cagcatgcca gtgctgtcct
      781 tcagagagga attcaaaacc aggctgaacc cagagagttc ctgcaacaac aatacaggtt
      841 atttcagaca cgcctcactg aaacccattt gccagctcaa gctagaacct cagaacctct
      901 gcataatgtt caggttttaa atcaaatgat aacatcaaaa tcaaatccag gaaataacat
      961 ggcctgcatt tctaagaatc agggttcaga gctttctgga gtgatatctt cagcttgtga
     1021 taaagagtca aatatggaac gaagagtgat cacgatttct aaatcagaat attctgtgca
     1081 ctcatctttg gcatccaaag ttgatgttga gcaggttgtt atgtattgca aggagaagct
     1141 tattcgtggg gaatcagaat tttcctttga agaattgaga gcccagaaat acaatcaacg
     1201 gagaaagcat gagcaatggg taaatgaaga cagacattat atgaaaagga aagaagcaaa
     1261 tgcttttgaa gaacagctat taaaacagaa aatggatgaa cttcataaga agttgcatca
     1321 ggtggtggag acatcccatg aggatctgcc cgcttcccag gaaaggtccg aggttaatcc
     1381 agcacgtatg gggccaagtg taggctccca gcaggaactg agagcgccat gtcttccagt
     1441 aacctatcag cagacaccag tgaacatgga aaagaaccca agagaggcac ctcctgttgt
     1501 tcctcctttg gcaaatgcta tttctgcagc tttggtgtcc ccagccacca gccagagcat
     1561 tgctcctcct gttcctttga aagcccagac agtaacagac tccatgtttg cagtggccag
     1621 caaagatgct ggatgtgtga ataagagtac tcatgaattc aagccacaga gtggagcaga
     1681 gatcaaagaa gggtgtgaaa cacataaggt tgccaacaca agttcttttc acacaactcc
     1741 aaacacatca ctgggaatgg ttcaggcaac gccatccaaa gtgcagccat cacccaccgt
     1801 gcacacaaaa gaagcattag gtttcatcat gaatatgttt caggctccta cacttcctga
     1861 tatttctgat ggcaaagatg aatggcaatc tctagatcaa aatgaagatg catttgaagc
     1921 ccagtttcaa aaaaatgtaa ggtcatctgg ggcttgggga gtcaataaga tcatctcttc
     1981 tttgtcatct gcttttcatg tgtttgaaga tggaaacaaa gaaaattatg gattaccaca
     2041 gcctaaaaat aaacccacag gagccaggac ctttggagaa cgctctgtca gcagacttcc
     2101 ttcaaaacca aaggaggaag tgcctcatgc tgaagagttt ttggatgact caactgtatg
     2161 gggtattcgc tgcaacaaaa ccctggcacc cagtcctaag agcccaggag acttcacatc
     2221 tgctgcacaa cttgcgtcta caccattcca caagcttcca gtggagtcag tgcacatttt
     2281 agaagataaa gaaaatgtgg tagcaaaaca gtgtacccag gcgactttgg attcttgtga
     2341 ggaaaacatg gtggtgcctt caagggatgg aaaattcagt ccaattcaag agaaaagccc
     2401 aaaacaggcc ttgtcgtctc acatgtattc agcatcctta cttcgtctga gccagcctgc
     2461 tgcaggtggg gtacttacct gtgaggcaga gttgggcgtt gaggcttgca gactcacaga
     2521 cactgatgct gccattgcag aagatccacc agatgctatt gctgggctcc aagcagaatg
     2581 gatgcagatg agttcacttg ggactgttga tgctccaaac ttcattgttg ggaacccatg
     2641 ggatgataag ctgattttca aacttttatc tgggctttct aaaccagtga gttcctatcc
     2701 aaatactttt gaatggcaat gtaaacttcc agccatcaag cccaagactg aatttcaatt
     2761 gggttctaag ctggtctatg tccatcacct tcttggagaa ggagcctttg cccaggtgta
     2821 cgaagctacc cagggagatc tgaatgatgc taaaaataaa cagaaatttg ttttaaaggt
     2881 ccaaaagcct gccaacccct gggaattcta cattgggacc cagttgatgg aaagactaaa
     2941 gccatctatg cagcacatgt ttatgaagtt ctattctgcc cacttattcc agaatggcag
     3001 tgtattagta ggagagctct acagctatgg aacattatta aatgccatta acctctataa
     3061 aaatacccct gaaaaagtga tgcctcaagg tcttgtcatc tcttttgcta tgagaatgct
     3121 ttacatgatt gagcaagtgc atgactgtga aatcattcat ggagacatta aaccagacaa
     3181 tttcatactt ggaaacggat ttttggaaca ggatgatgaa gatgatttat ctgctggctt
     3241 ggcactgatt gacctgggtc agagtataga tatgaaactt tttccaaaag gaactatatt
     3301 cacagcaaag tgtgaaacat ctggttttca gtgtgttgag atgctcagca acaaaccatg
     3361 gaactaccag atcgattact ttggggttgc tgcaacagta tattgcatgc tctttggcac
     3421 ttacatgaaa gtgaaaaatg aaggaggaga gtgtaagcct gaaggtcttt ttagaaggct
     3481 tcctcatttg gatatgtgga atgaattttt tcatgttatg ttgaatattc cagattgtca
     3541 tcatcttcca tctttggatt tgttaaggca aaagctgaag aaagtatttc aacaacacta
     3601 tactaacaag attagggccc tacgtaatag gctaattgta ctgctcttag aatgtaagcg
     3661 ttcacgaaaa taaaatttgg atatagtcga cctagttcta gaggatccag acatgataag
     3721 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     3781 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     3841 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     3901 aagcaagtaa aacctctaca aatgtggtat ggctgattat gatcctgcaa gcctcgtcgt
     3961 cctggccgga ccacgctatc tgtgcaaggt ccccggcccc ggacgcgcgc tccatgagca
     4021 gagcgcccgc cgccgaggcg aagactcggg cggcgccctg cccgtcccac caggtcaaca
     4081 ggcggtaacc ggcctcttca tcgggaatgc gcgcgacctt cagcatcgcc ggcatgtccc
     4141 cctggcggac gggaagtatc cagctcgacc aagctggccg cgtcgagtga agacgaaagg
     4201 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     4261 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4321 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4381 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4441 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4501 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4561 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4621 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4681 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4741 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4801 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4861 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4921 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4981 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5041 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5101 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5161 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5221 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5281 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5341 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5401 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5461 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5521 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5581 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5641 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5701 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5761 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5821 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5881 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5941 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6001 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6061 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6121 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6181 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     6241 aatgcagtcg agggatctcc ataagagaag agggacagct atgactggga gtagtcagga
     6301 gaggaggaaa aatctggcta gtaaaacatg taaggaaaat tttagggatg ttaaagaaaa
     6361 aaataacaca aaacaaaata taaaaaaaat ctaacctcaa gtcaaggctt ttctatggaa
     6421 taaggaatgg acagcagggg gctgtttcat atactgatga cctctttata gccaaccttt
     6481 gttcatggca gccagcatat gggcatatgt tgccaaactc taaaccaaat actcattctg
     6541 atgttttaaa tgatttgccc tcccatatgt ccttccgagt gagagacaca aaaaattcca
     6601 acacactatt gcaatgaaaa taaatttcct ttattagcca gaagtcagat gctcaagggg
     6661 cttcatgatg tccccataat ttttggcaga gggaaaaaga tctcagtggt atttgtgagc
     6721 cagggcattg gccacaccag ccaccacctt ctgataggca gcctgcacct gaggagtgaa
     6781 ttctttgcca aaatgatgag acagcacaac aaccagcacg ttgcccagga gctgtaggaa
     6841 agagaagaag gcatgaacat ggttagcaga ggggcccggt ttggactcag agtattttat
     6901 cctcatctca aacagtgtat atcattgtaa ccataaagag aaaggcagga tgatgaccag
     6961 ggtgtagttg tttctaccaa taagaatatt tccacgccag ccagaattta tatgcagaaa
     7021 tattctacct tatcatttaa ttataacaat tgttctctaa aactgtgctg aagtacaata
     7081 taatataccc tgattgcctt gaaaaaaaag tgattagaga aagtacttac aatctgacaa
     7141 ataaacaaaa gtgaatttaa aaattcgtta caaatgcaag ctaaagttta acgaaaaagt
     7201 tacagaaaat gaaaagaaaa taagaggaga caatggttgt caacagagta gaaagtgaaa
     7261 gaaacaaaat tatcatgagg gtccatggtg atacaaggga catcttccca ttctaaacaa
     7321 caccctgaaa actttgcccc ctccatataa catgaatttt acaatagcga aaaagaaaga
     7381 acaatcaagg gtccccaaac tcaccctgaa gttctcagct ctagagatgg ccgctttact
     7441 tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca
     7501 tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc aggtagtggt
     7561 tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga
     7621 gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct
     7681 tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc
     7741 ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg
     7801 tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga
     7861 tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca
     7921 tgtggtcggg gtagcggctg aagcactgca cgccgtaggt gaaggtggtc acgagggtgg
     7981 gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg
     8041 tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca
     8101 gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc accatggtgg
     8161 cggcaagcta tcgtcgaggc cgcgctagca cgcgtcagct gactagagga tccccgggta
     8221 ccgagctcga attcggggcc gcggaggctg gatcggtccc ggtgtcttct atggaggtca
     8281 aaacagcgtg gatggcgtct ccaggcgatc tgacggttca ctaaacgagc tctgcttata
     8341 taggcctccc accgtaca
//
