LOCUS       sequence-221374-        6096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kinetochore localised FRB tagged with FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6096)
  AUTHORS   Kuijt TE, Omerzu M, Saurin AT, Kops GJ
  TITLE     Conditional targeting of MAD1 to kinetochores is sufficient to 
            reactivate the spindle assembly checkpoint in metaphase.
  JOURNAL   Chromosoma. 2014 Oct;123(5):471-80. doi: 10.1007/s00412-014-0458-9. 
            Epub 2014 Apr 4.
  PUBMED    24695965
REFERENCE   2  (bases 1 to 6096)
  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"; doi: 
            "10.1007/s00412-014-0458-9"; journalName: "Chromosoma"; date: 
            "2014-10"; volume: "123"; issue: "5"; pages: "471-80"
COMMENT     Sequence Label: MIS12-FRB-FLAG
FEATURES             Location/Qualifiers
     source          1..6096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        25..404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        405..608
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     559..579
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     653..672
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        653..671
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1356..1634
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             1644..1667
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     intron          1731..1960
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1943..1962
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2033..2606,2607..2609,2610..2618)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2199..2216)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2426..2445
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2638..3237
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2638..2657)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3134..3154
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3373..3390)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3379..3603
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3650..3669
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3729..3745)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3729..3745)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3742..3764)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3753..3769
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3777..3783,3784..3801,3802..3807))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3822..3843
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3960..3977)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4131..4719)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4211..4230)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4890..5681,5682..5750))
                     /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     5513..5532
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5751..5855)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5930..5949)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
       61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      361 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      421 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      481 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      541 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      601 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
      661 ctcactatag ggagacccaa gcttggcgcg ccaccatgtc tgtggatcca atgacctacg
      721 aggcccagtt ctttggcttc acgccacaaa cgtgcatgct tcggatctac attgcatttc
      781 aagactacct atttgaagtg atgcaggccg ttgaacaggt tattctgaag aagctggatg
      841 gcatcccaga ctgtgacatt agcccagtgc agattcgcaa atgcacagag aagtttcttt
      901 gcttcatgaa aggacatttt gataaccttt ttagcaaaat ggagcaactg tttttgcagc
      961 tgattttacg tattccctca aacatcttgc ttcctgaaga taaatgtaag gagacacctt
     1021 atagtgagga agattttcag catctccaga aagaaattga acagttacag gagaagtaca
     1081 agactgaatt atgtactaag caggcccttc ttgcagaatt agaagagcaa aaaattgttc
     1141 aggccaaact caaacagacg ttgactttct ttgatgagct tcataatgtt ggcagagatc
     1201 atgggactag tgattttagg gagagtttag tatccctggt tcagaactcc agaaaactac
     1261 agaacattag agacaatgtg gaaaaggaat cgaaacgact gaaaatatcg ctagcaagat
     1321 ctcgagctca agcttcgaat tcaatggctt ctagaatcct ctggcatgag atgtggcatg
     1381 aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg
     1441 aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg aaggaaacat
     1501 cctttaatca ggcctatggt cgagatttaa tggaggccca agagtggtgc aggaagtaca
     1561 tgaaatcagg gaatgtcaag gacctcctcc aagcctggga cctctattat catgtgttcc
     1621 gacgaatctc aaagactagt atcgattaca aggatgacga tgacaagggc atagataact
     1681 gatccagtgt gctggaatta attcgctgtc tgcgagggcc agctgttggg gtgagtactc
     1741 cctctcaaaa gcgggcatga cttctgcgct aagattgtca gtttccaaaa acgaggagga
     1801 tttgatattc acctggcccg cggtgatgcc tttgagggtg gccgcgtcca tctggtcaga
     1861 aaagacaatc tttttgttgt caagcttgag gtgtggcagg cttgagatct ggccatacac
     1921 ttgagtgaca atgacatcca ctttgccttt ctctccacag gtgtccactc ccaggtccaa
     1981 ctgcaggtcg agcatgcatc tagggcggcc aattccgccc ctctcccccc cccccctctc
     2041 cctccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt
     2101 ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg
     2161 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg
     2221 tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc
     2281 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca
     2341 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag
     2401 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa
     2461 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt
     2521 tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt
     2581 tttcctttga aaaacacgat gataagcttg ccacaaccca caaggagacg accttccatg
     2641 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc
     2701 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc
     2761 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     2821 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     2881 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     2941 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     3001 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     3061 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     3121 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     3181 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc
     3241 ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgg ctccgaccga
     3301 agccacccgg ggcggccccg ccgaccccgc acccgccccc gaggcccacc gactctagag
     3361 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3421 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3481 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3541 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3601 tggcttctga ggcggaaaga accagctggg gctcgagtgc attctagttg tggtttgtcc
     3661 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     3721 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3781 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3841 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3901 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3961 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4021 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4081 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4141 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4201 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4261 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4321 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4381 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4441 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4501 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4561 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4621 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4681 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4741 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4801 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4861 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4921 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4981 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5041 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5101 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5161 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5221 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5281 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5341 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5401 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5461 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5521 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5581 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5641 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5701 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5761 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5821 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5881 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     5941 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     6001 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     6061 atctgcttag ggttaggcgt tttgcgctgc ttcgcg
//
