LOCUS       sequence-221370-        6835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kinetochore localised FRB tagged with FLAG and tagRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6835)
  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 6835)
  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-tagRFP
FEATURES             Location/Qualifiers
     source          1..6835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        158..537
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        538..741
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     692..712
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     786..805
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        786..804
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1489..1767
                     /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             1777..1800
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1807..2520
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     promoter        complement(2530..2548)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2531..2548)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     intron          2619..2848
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2831..2850
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2906..3477
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3071..3088)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3298..3317
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3510..4109
                     /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(3510..3529)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4006..4026
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4245..4262)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4251..4475
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4522..4541
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4601..4617)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4601..4617)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4614..4636)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4625..4641
                     /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(4649..4655,4656..4673,4674..4679))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4694..4715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4832..4849)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5003..5591)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5083..5102)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5762..6553,6554..6622))
                     /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     6385..6404
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6623..6727)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6802..6821)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
       61 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
      121 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat
      181 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      241 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      301 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      361 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      421 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      481 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      541 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      601 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      661 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      721 gaggtctata taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc
      781 gaaattaata cgactcacta tagggagacc caagcttggc gcgccaccat gtctgtggat
      841 ccaatgacct acgaggccca gttctttggc ttcacgccac aaacgtgcat gcttcggatc
      901 tacattgcat ttcaagacta cctatttgaa gtgatgcagg ccgttgaaca ggttattctg
      961 aagaagctgg atggcatccc agactgtgac attagcccag tgcagattcg caaatgcaca
     1021 gagaagtttc tttgcttcat gaaaggacat tttgataacc tttttagcaa aatggagcaa
     1081 ctgtttttgc agctgatttt acgtattccc tcaaacatct tgcttcctga agataaatgt
     1141 aaggagacac cttatagtga ggaagatttt cagcatctcc agaaagaaat tgaacagtta
     1201 caggagaagt acaagactga attatgtact aagcaggccc ttcttgcaga attagaagag
     1261 caaaaaattg ttcaggccaa actcaaacag acgttgactt tctttgatga gcttcataat
     1321 gttggcagag atcatgggac tagtgatttt agggagagtt tagtatccct ggttcagaac
     1381 tccagaaaac tacagaacat tagagacaat gtggaaaagg aatcgaaacg actgaaaata
     1441 tcgctagcaa gatctcgagc tcaagcttcg aattcaatgg cttctagaat cctctggcat
     1501 gagatgtggc atgaaggcct ggaagaggca tctcgtttgt actttgggga aaggaacgtg
     1561 aaaggcatgt ttgaggtgct ggagcccttg catgctatga tggaacgggg cccccagact
     1621 ctgaaggaaa catcctttaa tcaggcctat ggtcgagatt taatggaggc ccaagagtgg
     1681 tgcaggaagt acatgaaatc agggaatgtc aaggacctcc tccaagcctg ggacctctat
     1741 tatcatgtgt tccgacgaat ctcaaagact agtatcgatt acaaggatga cgatgacaag
     1801 ggatccatgg tgtctaaggg cgaagagctg attaaggaga acatgcacat gaagctgtac
     1861 atggagggca ccgtgaacaa ccaccacttc aagtgcacat ccgagggcga aggcaagccc
     1921 tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc
     1981 ttcgacatcc tggctaccag cttcatgtac ggcagcagaa ccttcatcaa ccacacccag
     2041 ggcatccccg acttctttaa gcagtccttc cctgagggct tcacatggga gagagtcacc
     2101 acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc
     2161 ctcatctaca acgtcaagat cagaggggtg aacttcccat ccaacggccc tgtgatgcag
     2221 aagaaaacac tcggctggga ggccaacacc gagatgctgt accccgctga cggcggcctg
     2281 gaaggcagaa gcgacatggc cctgaagctc gtgggcgggg gccacctgat ctgcaacttc
     2341 aagaccacat acagatccaa gaaacccgct aagaacctca agatgcccgg cgtctactat
     2401 gtggaccaca gactggaaag aatcaaggag gccgacaaag agacctacgt cgagcagcac
     2461 gaggtggctg tggccagata ctgcgacctc cctagcaaac tggggcacaa acttaattga
     2521 gggccctatt ctatagtgtc acctaaatgc gcggccgcat agataactga tccagtgtgc
     2581 tggaattaat tcgctgtctg cgagggccag ctgttggggt gagtactccc tctcaaaagc
     2641 gggcatgact tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt tgatattcac
     2701 ctggcccgcg gtgatgcctt tgagggtggc cgcgtccatc tggtcagaaa agacaatctt
     2761 tttgttgtca agcttgaggt gtggcaggct tgagatctgg ccatacactt gagtgacaat
     2821 gacatccact ttgcctttct ctccacaggt gtccactccc aggtccaact gcaggtcgag
     2881 catgcatcta gggcggccaa ttccgcccct ctcccccccc cccctaacgt tactggccga
     2941 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     3001 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     3061 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     3121 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     3181 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     3241 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     3301 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     3361 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa
     3421 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataagct
     3481 tgccacaacc cacaaggaga cgaccttcca tgaccgagta caagcccacg gtgcgcctcg
     3541 ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc gccgactacc
     3601 ccgccacgcg ccacaccgtc gacccggacc gccacatcga gcgggtcacc gagctgcaag
     3661 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     3721 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     3781 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     3841 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     3901 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     3961 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     4021 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     4081 ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg
     4141 aaaggagcgc acgaccccat ggctccgacc gaagccaccc ggggcggccc cgccgacccc
     4201 gcacccgccc ccgaggccca ccgactctag agctcgctga tcagcctcga ctgtgccttc
     4261 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     4321 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     4381 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     4441 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     4501 gggctcgagt gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4561 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4621 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4681 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4741 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4801 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4861 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4921 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4981 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5041 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5101 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5161 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5221 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5281 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5341 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5401 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5461 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5521 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5581 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5641 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5701 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5761 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5821 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5881 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5941 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6001 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6061 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6121 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6181 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6241 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6301 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6361 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6421 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6481 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6541 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6601 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6661 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6721 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     6781 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccag
//
