LOCUS Exported 7828 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Inducible expression of eGFP-tagged HEC1 in FLPin Trex cell lines. Due to insertion of a STOP-codon after the eGFP-tag, this plasmid only expressed eGFP. . ACCESSION . VERSION . KEYWORDS pcDNA5-hygro-eGFP-STOP-HEC1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7828) AUTHORS Etemad B, Kuijt TE, Kops GJ TITLE Kinetochore-microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint. JOURNAL Nat Commun. 2015 Dec 1;6:8987. doi: 10.1038/ncomms9987. PUBMED 26621779 REFERENCE 2 (bases 1 to 7828) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7828 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 62..441 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 442..645 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 596..616 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 647..665 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 668..686 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 696..720 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 901..910 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 907..1623 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 907..1623 /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(952..973) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1213..1232) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1560..1581 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(3607..3624) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3613..3837 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3970..3989) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 4121..4168 /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 4176..5198 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE " polyA_signal 5328..5449 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5365..5384) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5419..5438 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5498..5514) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5498..5514) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5511..5533) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5522..5538 /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(5546..5576) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5591..5612 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5729..5746) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5900..6488) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5980..5999) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6659..7519) /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 7282..7301 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7520..7624) /gene="bla" /label=AmpR promoter primer_bind complement(7699..7718) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt 61 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 121 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 181 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 241 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 301 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 361 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 421 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 481 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 541 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 601 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctccc tatcagtgat 661 agagatctcc ctatcagtga tagagatcgt cgacgagctc gtttagtgaa ccgtcagatc 721 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc 781 ctccggactc tagcgtttaa acttaagctt ggtaccgagc tcggatccac tagtccagtg 841 tggtggaatt ctgcagatat ccagcacagt ggcggccgct cgagtctagc gctaccggtc 901 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 961 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 1021 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 1081 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac 1141 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 1201 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 1261 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 1321 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 1381 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 1441 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 1501 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 1561 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 1621 aagtccggac tcagatccta gaagcgcagt tcagtttcca gcggtggtgc tggccgcctc 1681 tccatgcagg agttaagatc ccaggatgta aataaacaag gcctctatac ccctcaaacc 1741 aaagagaaac caacctttgg aaagttgagt ataaacaaac cgacatctga aagaaaagtc 1801 tcgctatttg gcaaaagaac tagtggacat ggatcacgga atagtcaact tggtatattt 1861 tccagttctg agaaaatcaa ggacccgaga ccacttaacg acaaagcatt cattcagcag 1921 tgtattcgac aactctgtga gtttcttaca gaaaatggtt atgcacataa tgtgtccatg 1981 aaatctctac aagctccctc tgttaaagac ttcctgaaga tcttcacatt tctttatggc 2041 ttcctgtgcc cctcatacga acttcctgac acaaagtttg aagaagaggt tccaagaatc 2101 tttaaagacc ttgggtatcc ttttgcacta tccaaaagct ccatgtacac agtgggggct 2161 cctcatacat ggcctcacat tgtggcagcc ttagtttggc taatagactg catcaagata 2221 catactgcca tgaaagaaag ctcaccttta tttgatgatg ggcagccttg gggagaagaa 2281 actgaagatg gaattatgca taataagttg tttttggact acaccataaa atgctatgag 2341 agttttatga gtggtgccga cagctttgat gagatgaatg cagagctgca gtcaaaactg 2401 aaggatttat ttaatgtgga tgcttttaag ctggaatcat tagaagcaaa aaacagagca 2461 ttgaatgaac agattgcaag attggaacaa gaaagagaaa aagaaccgaa tcgtctagag 2521 tcgttgagaa aactgaaggc ttccttacaa ggagatgttc aaaagtatca ggcatacatg 2581 agcaatttgg agtctcattc agccattctt gaccagaaat taaatggtct caatgaggaa 2641 attgctagag tagaactaga atgtgaaaca ataaaacagg agaacactcg actacagaat 2701 atcattgaca accagaagta ctcagttgca gacattgagc gaataaatca tgaaagaaat 2761 gaattgcagc agactattaa taaattaacc aaggacctgg aagctgaaca acagaagttg 2821 tggaatgagg agttaaaata tgccagaggc aaagaagcga ttgaaacaca attagcagag 2881 tatcacaaat tggctagaaa attaaaactt attcctaaag gtgctgagaa ttccaaaggt 2941 tatgactttg aaattaagtt taatcccgag gctggtgcca actgccttgt caaatacagg 3001 gctcaagttt atgtacctct taaggaactc ctgaatgaaa ctgaagaaga aattaataaa 3061 gccctaaata aaaaaatggg tttggaggat actttagaac aattgaatgc aatgataaca 3121 gaaagcaaga gaagtgtgag aactctgaaa gaagaagttc aaaagctgga tgatctttac 3181 caacaaaaaa ttaaggaagc agaggaagag gatgaaaaat gtgccagtga gcttgagtcc 3241 ttggagaaac acaagcacct gctagaaagt actgttaacc aggggctcag tgaagctatg 3301 aatgaattag atgctgttca gcgggaatac caactagttg tgcaaaccac gactgaagaa 3361 agacgaaaag tgggaaataa cttgcaacgt ctgttagaga tggttgctac acatgttggg 3421 tctgtagaga aacatcttga ggagcagatt gctaaagttg atagagaata tgaagaatgc 3481 atgtcagaag atctctcgga aaatattaaa gagattagag ataagtatga gaagaaagct 3541 actctaatta agtcttctga agaatgactc gacggtaccg cgggcccgtt taaacccgct 3601 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 3661 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 3721 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 3781 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 3841 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg 3901 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 3961 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 4021 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 4081 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt 4141 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt 4201 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg 4261 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg 4321 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg 4381 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt 4441 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg 4501 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga 4561 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt 4621 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg 4681 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg 4741 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc 4801 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg 4861 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg 4921 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg 4981 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc 5041 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg 5101 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa 5161 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt 5221 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 5281 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg 5341 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 5401 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta 5461 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 5521 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 5581 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 5641 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 5701 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 5761 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 5821 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 5881 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 5941 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 6001 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 6061 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 6121 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 6181 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 6241 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 6301 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 6361 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 6421 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 6481 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 6541 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 6601 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 6661 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 6721 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 6781 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 6841 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 6901 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 6961 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 7021 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 7081 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 7141 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 7201 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 7261 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 7321 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 7381 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 7441 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 7501 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 7561 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 7621 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc 7681 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct 7741 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag 7801 gcaaggcttg accgacaatt gcatgaag //