LOCUS Exported 6096 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Kinetochore localised FRB tagged with FLAG. ACCESSION . VERSION . KEYWORDS MIS12-FRB-FLAG 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6096 /organism="synthetic DNA construct" /mol_type="other DNA" 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 2033..2605 /label=IRES /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(3777..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(4890..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 //