LOCUS Exported 7405 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expression of HEC1 M2 mutant tagged with GFP. ACCESSION . VERSION . KEYWORDS pcDNA5-puro-eGFP-HEC1-M2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7405) AUTHORS Hiruma Y, Sacristan C, Pachis ST, Adamopoulos A, Kuijt T, Ubbink M, von Castelmur E, Perrakis A, Kops GJ TITLE CELL DIVISION CYCLE. Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling. JOURNAL Science. 2015 Jun 12;348(6240):1264-7. doi: 10.1126/science.aaa4055. Epub 2015 Jun 11. PUBMED 26068855 REFERENCE 2 (bases 1 to 7405) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7405 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 820..838 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 841..859 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 869..893 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 1074..1083 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1080..1796 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1080..1796 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1125..1146) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1386..1405) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1733..1754 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(3783..3800) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3789..4013 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(4146..4165) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 4297..4344 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." CDS 4352..4948 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind 4845..4865 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5078..5199 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5115..5134) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5169..5188 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5248..5264) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5248..5264) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5261..5283) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5272..5288 /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(5296..5326) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5341..5362 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5479..5496) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5650..6238) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5730..5749) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6409..7269) /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 7032..7051 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7270..7374) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc 841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga 901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga 961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga 1021 attctgcaga tatccagcac agtggcggcc gctcgagtct agcgctaccg gtcgccacca 1081 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 1141 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 1201 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 1261 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 1321 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 1381 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 1441 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 1501 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 1561 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 1621 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 1681 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 1741 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg 1801 gactcagatc catgaagcgc agttcagttt ccagcggtgg tgctggccgc ctctccatgc 1861 aggagttaag atcccaggat gtaaataaac aaggcctcta tacccctcaa accaaagaga 1921 aaccaacctt tggaaagttg agtataaaca aaccgacatc tgaaagaaaa gtctcgctat 1981 ttggcaaaag aactagtgga catggatccc ggaatagtca acttggtata ttttccagtt 2041 ctgagaaaat cgaggacccg agaccacttg aagacgaagc attcattcag cagtgtattc 2101 gacaactctg tgagtttctt acagaaaatg gttatgcaca taatgtgtcc atgaaatctc 2161 tacaagctcc ctctgttaaa gacttcctga agatcttcac atttctttat ggcttcctgt 2221 gcccctcata cgaacttcct gacacaaagt ttgaagaaga ggttccaaga atctttaaag 2281 accttgggta tccttttgca ctatccaaaa gctccatgta cacagtgggg gctcctcata 2341 catggcctca cattgtggca gccttagttt ggctaataga ctgcatcaag atacatactg 2401 ccatgaaaga aagctcacct ttatttgatg atgggcagcc ttggggagaa gaaactgaag 2461 atggaattat gcataataag ttgtttttgg actacaccat aaaatgctat gagagtttta 2521 tgagtggtgc cgacagcttt gatgagatga atgcagagct gcagtcaaaa ctgaaggatt 2581 tatttaatgt ggatgctttt aagctggaat cattagaagc aaaaaacaga gcattgaatg 2641 aacagattgc aagattggaa caagaaagag aaaaagaacc gaatcgtcta gagtcgttga 2701 gaaaactgaa ggcttcctta caaggagatg ttcaaaagta tcaggcatac atgagcaatt 2761 tggagtctca ttcagccatt cttgaccaga aattaaatgg tctcaatgag gaaattgcta 2821 gagtagaact agaatgtgaa acaataaaac aggagaacac tcgactacag aatatcattg 2881 acaaccagaa gtactcagtt gcagacattg agcgaataaa tcatgaaaga aatgaattgc 2941 agcagactat taataaatta accaaggacc tggaagctga acaacagaag ttgtggaatg 3001 aggagttaaa atatgccaga ggcaaagaag cgattgaaac acaattagca gagtatcaca 3061 aattggctag aaaattaaaa cttattccta aaggtgctga gaattccaaa ggttatgact 3121 ttgaaattaa gtttaatccc gaggctggtg ccaactgcct tgtcaaatac agggctcaag 3181 tttatgtacc tcttaaggaa ctcctgaatg aaactgaaga agaaattaat aaagccctaa 3241 ataaaaaaat gggtttggag gatactttag aacaattgaa tgcaatgata acagaaagca 3301 agagaagtgt gagaactctg aaagaagaag ttcaaaaatt ggatgatctt taccaacaaa 3361 aaattaagga agcagaggaa gaggatgaaa aatgtgccag tgagcttgag tccttggaga 3421 aacacaagca cctgctagaa agtactgtta accaggggct cagtgaagct atgaatgaat 3481 tagatgctgt tcagcgggaa taccaactag ttgtgcaaac cacgactgaa gaaagacgaa 3541 aagtgggaaa taacttgcaa cgtctgttag agatggttgc tacacatgtt gggtctgtag 3601 agaaacatct tgaggagcag attgctaaag ttgatagaga atatgaagaa tgcatgtcag 3661 aagatctctc ggaaaatatt aaagagatta gagataagta tgagaagaaa gctactctaa 3721 ttaagtcttc tgaagaatga ctcgaggacg gtaccgcggg cccgtttaaa cccgctgatc 3781 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 3841 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 3901 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 3961 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga 4021 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt 4081 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 4141 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 4201 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 4261 caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta 4321 ttctctagaa agtataggaa cttccttggc caccgagtac aagcccacgg tgcgcctcgc 4381 cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg ccgactaccc 4441 cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg agctgcaaga 4501 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc 4561 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat 4621 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg 4681 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc 4741 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc 4801 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta 4861 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg 4921 gtgcatgacc cgcaagcccg gtgcctgaca cgtactacga gatttcgatt ccaccgccgc 4981 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 5041 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa 5101 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 5161 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac 5221 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 5281 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 5341 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 5401 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 5461 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 5521 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 5581 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 5641 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 5701 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 5761 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 5821 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 5881 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 5941 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 6001 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 6061 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 6121 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 6181 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 6241 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 6301 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 6361 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 6421 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 6481 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 6541 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 6601 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 6661 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 6721 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 6781 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 6841 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 6901 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 6961 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 7021 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 7081 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 7141 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 7201 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 7261 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 7321 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 7381 tccccgaaaa gtgccacctg acgtc //