LOCUS       sequence-215342-        6837 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 6837)
  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 6837)
  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..6837
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        43..422
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        423..626
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     577..597
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     671..690
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        671..689
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1374..1652
                     /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             1662..1685
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1692..2405
                     /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(2415..2433)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2416..2433)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     intron          2504..2733
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2716..2735
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2791..3365,3366..3368,3369..3377)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2958..2975)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3185..3204
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3397..3996
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     primer_bind     complement(3397..3416)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3893..3913
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4132..4149)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4138..4362
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4409..4428
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4488..4504)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4488..4504)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4501..4523)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4512..4528
                     /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(4536..4542,4543..4560,4561..4566))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4581..4602
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4719..4736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4890..5478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4970..4989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5649..6440,6441..6509))
                     /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     6272..6291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6510..6614)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6689..6708)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
       61 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      121 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      181 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      241 gggtggacta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      301 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      361 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      421 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      481 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      541 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      601 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc
      661 ttatcgaaat taatacgact cactataggg agacccaagc ttggcgcgcc accatgtctg
      721 tggatccaat gacctacgag gcccagttct ttggcttcac gccacaaacg tgcatgcttc
      781 ggatctacat tgcatttcaa gactacctat ttgaagtgat gcaggccgtt gaacaggtta
      841 ttctgaagaa gctggatggc atcccagact gtgacattag cccagtgcag attcgcaaat
      901 gcacagagaa gtttctttgc ttcatgaaag gacattttga taaccttttt agcaaaatgg
      961 agcaactgtt tttgcagctg attttacgta ttccctcaaa catcttgctt cctgaagata
     1021 aatgtaagga gacaccttat agtgaggaag attttcagca tctccagaaa gaaattgaac
     1081 agttacagga gaagtacaag actgaattat gtactaagca ggcccttctt gcagaattag
     1141 aagagcaaaa aattgttcag gccaaactca aacagacgtt gactttcttt gatgagcttc
     1201 ataatgttgg cagagatcat gggactagtg attttaggga gagtttagta tccctggttc
     1261 agaactccag aaaactacag aacattagag acaatgtgga aaaggaatcg aaacgactga
     1321 aaatatcgct agcaagatct cgagctcaag cttcgaattc aatggcttct agaatcctct
     1381 ggcatgagat gtggcatgaa ggcctggaag aggcatctcg tttgtacttt ggggaaagga
     1441 acgtgaaagg catgtttgag gtgctggagc ccttgcatgc tatgatggaa cggggccccc
     1501 agactctgaa ggaaacatcc tttaatcagg cctatggtcg agatttaatg gaggcccaag
     1561 agtggtgcag gaagtacatg aaatcaggga atgtcaagga cctcctccaa gcctgggacc
     1621 tctattatca tgtgttccga cgaatctcaa agactagtat cgattacaag gatgacgatg
     1681 acaagggatc catggtgtct aagggcgaag agctgattaa ggagaacatg cacatgaagc
     1741 tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca
     1801 agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct
     1861 tcgccttcga catcctggct accagcttca tgtacggcag cagaaccttc atcaaccaca
     1921 cccagggcat ccccgacttc tttaagcagt ccttccctga gggcttcaca tgggagagag
     1981 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     2041 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga
     2101 tgcagaagaa aacactcggc tgggaggcca acaccgagat gctgtacccc gctgacggcg
     2161 gcctggaagg cagaagcgac atggccctga agctcgtggg cgggggccac ctgatctgca
     2221 acttcaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct
     2281 actatgtgga ccacagactg gaaagaatca aggaggccga caaagagacc tacgtcgagc
     2341 agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg cacaaactta
     2401 attgagggcc ctattctata gtgtcaccta aatgcgcggc cgcatagata actgatccag
     2461 tgtgctggaa ttaattcgct gtctgcgagg gccagctgtt ggggtgagta ctccctctca
     2521 aaagcgggca tgacttctgc gctaagattg tcagtttcca aaaacgagga ggatttgata
     2581 ttcacctggc ccgcggtgat gcctttgagg gtggccgcgt ccatctggtc agaaaagaca
     2641 atctttttgt tgtcaagctt gaggtgtggc aggcttgaga tctggccata cacttgagtg
     2701 acaatgacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcagg
     2761 tcgagcatgc atctagggcg gccaattccg cccctctccc tccccccccc ctaacgttac
     2821 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgtgat tttccaccat
     2881 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     2941 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     3001 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     3061 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     3121 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     3181 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     3241 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     3301 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     3361 ataagcttgc cacaacccac aaggagacga ccttccatga ccgagtacaa gcccacggtg
     3421 cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc
     3481 gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag
     3541 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     3601 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     3661 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     3721 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     3781 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     3841 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     3901 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgagtgccc gaaggaccgc
     3961 gcgacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc
     4021 ccgaccgaaa ggagcgcacg accccatggc tccgaccgaa gccgacccgg gcggccccgc
     4081 cgaccccgca cccgcccccg aggcccaccg actctagagc tcgctgatca gcctcgactg
     4141 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     4201 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     4261 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     4321 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     4381 ccagctgggg ctcgagtgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4441 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     4501 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4561 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4621 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4681 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4741 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4801 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4861 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4921 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4981 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5041 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     5101 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5161 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5221 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5281 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     5341 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5401 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5461 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5521 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5581 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5641 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5701 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5761 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5821 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5881 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5941 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6001 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6061 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6121 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6181 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6241 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6301 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6361 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6421 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6481 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6541 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6601 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     6661 ctcccgatcc cctatggtcg actctcagta caatctgctc tgatgccgca tagttaagcc
     6721 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     6781 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggc
//
