LOCUS Exported 8458 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION ish1 mCherry insertion cassette with G418 resistance gene. ACCESSION . VERSION . KEYWORDS pMGF169 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8458) AUTHORS Gu M, LaJoie D, Chen OS, von Appen A, Ladinsky MS, Redd MJ, Nikolova L, Bjorkman PJ, Sundquist WI, Ullman KS, Frost A TITLE LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells. JOURNAL Proc Natl Acad Sci U S A. 2017 Mar 14;114(11):E2166-E2175. doi: 10.1073/pnas.1613916114. Epub 2017 Feb 27. PUBMED 28242692 REFERENCE 2 (bases 1 to 8458) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8458 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 129..159 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 167..183 /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)." primer_bind 191..207 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 191..207 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 203..730 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR QPSEAVVWLCRS" primer_bind complement(223..240) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(223..239) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(232..254) /label=M13/pUC Forward /note="In lacZ gene" promoter 832..860 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 868..884 /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)." CDS 907..1560 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK" primer_bind 1518..1540 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 1567..1590 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 2185..2895 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(2330..2348) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2598..2617 /label=mCherry-F /note="mCherry, forward primer" terminator 2916..3103 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind complement(2971..2995) /label=pHybLex-R /note="ADH terminator, reverse primer" gene 3178..4534 /label=kanMX /note="yeast selectable marker conferring kanamycin resistance (Wach et al., 1994)" promoter 3178..3521 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 3522..4331 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin" /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(3589..3608) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" terminator 4337..4534 /label=TEF terminator /note="Ashbya gossypii TEF terminator" primer_bind complement(5157..5179) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5324..5342) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5410..5514 /gene="bla" /label=AmpR promoter CDS 5515..6375 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5733..5752) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6546..7134 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7035..7054 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7288..7305 /label=L4440 /note="L4440 vector, forward primer" promoter 7378..7455 /gene="lacI" /label=lacI promoter CDS 7456..80 /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind complement(7475..7494) /label=LacI-R /note="LacI, reverse primer" ORIGIN 1 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 61 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca 121 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 181 caatttcaca caggaaacag ctatgaccat gattacggat tcactggccg tcgttttaca 241 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 301 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 361 cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag 421 ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat 481 gcacggttac gatgcgccca tctacaccaa cgtgacctat cccattacgg tcaatccgcc 541 gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag 601 ctggctacag gaaggccaga cgcgaattat ttttgatggc gttggaatta gcttatcgac 661 tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg 721 caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg 781 ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt 841 aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac acaggaaaca 901 gtattcatgt cccctatact aggttattgg aaaattaagg gccttgtgca acccactcga 961 cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt 1021 gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct tccttattat 1081 attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat agctgacaag 1141 cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg 1201 gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt tgaaactctc 1261 aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt 1321 cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct 1381 cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt 1441 tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat 1501 atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa 1561 tcggatctgg aagttctgtt ccaggggccc ctggctgttc ctaattgggc tgcttatttt 1621 gatactttga gctccaaaga gctttctgcc tggattaagg aatacaaaaa acattaccat 1681 ggaaaacttc acccatccaa agatcgtgaa catcttttcc aaaatgcctg taatatttat 1741 cgcgctattg ctgaaaaggc tgacaaatct gttcttcaat cgattcaatc aaagtttcct 1801 aaggcaactg ttcccggtta tgactcttgg tctaatgaag atctaaagtc tgcactcaag 1861 gaatatggtg acagtattgg taaggtgttt aatcgtaaag atgctattga acgcttaaag 1921 cgtcatgata ttttattcta tggtcctgtt gttgccgaca aggctaaatc aggagtctct 1981 ggcttccttc gacgtgttgc ttcttttatg ggatttggca ctcgcgacgt agctgaaatt 2041 aagctaggag aaaacattca aaaggtaaaa gatactgcct ctaaagcctc atctgctgta 2101 tcttcagcag gcgactatgt cgaaactaat gtaaagaaag cccaaagggt tttgggagga 2161 acgggcggta gtggaggcac tggtatggtg agcaagggcg aggaggataa catggccatc 2221 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc 2281 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag 2341 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac 2401 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc 2461 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg 2521 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc 2581 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc 2641 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg 2701 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg 2761 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac 2821 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac 2881 gagctgtaca agtgaggcgc gccacttcta aataagcgaa tttcttatga tttatgattt 2941 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg 3001 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc 3061 aggtatagta tgaggtcgct cttattgacc acacctctac cggcagatcc gctagggata 3121 acagggtaat atagatctgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac 3181 atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt 3241 gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat 3301 tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac ctgcgagcag 3361 ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat 3421 ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg 3481 ctaggataca gttctcacat cacatccgaa cataaacaac catgggtaag gaaaagactc 3541 acgtttcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg 3601 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg 3661 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga 3721 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc 3781 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggcaaaaca gcattccagg 3841 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc 3901 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc 3961 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg 4021 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt ttgccattct 4081 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg 4141 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc 4201 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc 4261 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg 4321 agtttttcta atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt 4381 atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt 4441 ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg 4501 cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca 4561 tccataaatt gttcatgaac gttcttaatg tctatttata ttatttaaaa agaaggagat 4621 tacgtctaaa acgagttctc gtttaatata tgacttttaa tgaaatatct atgttcatat 4681 tcgctactta accttccttg gcaagtaatc catcctattg ttaacattaa tataatccat 4741 caagatactt ttttattaat ttaagttttt cagaattaga tgttggtgca cctcctccaa 4801 atctagagct gtggaaagaa ttggtaaaat cttatcgcga tgttcgtgat tctctaaaaa 4861 ttgcagtaaa acatttctag catattcttt gttaatcaat tctttagaaa tattgcttat 4921 aacaggtggc tcgaatgaac ctgctgaatc acttgaagct ttgggcaata tttcactgct 4981 cataacactg gacagctcgc tctcgtgaga tttgacactt tccttttgaa ccttttgctt 5041 ttcaatgagt tcttttattt catcccttga ctccactaac tcgtttccaa caccgagcgg 5101 ccgcatcgtg actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc 5161 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 5221 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag 5281 tcacgtagcg atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta 5341 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 5401 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 5461 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 5521 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 5581 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 5641 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 5701 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt 5761 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 5821 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 5881 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 5941 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 6001 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca 6061 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 6121 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 6181 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 6241 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 6301 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 6361 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 6421 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 6481 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 6541 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 6601 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 6661 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 6721 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6781 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6841 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6901 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6961 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 7021 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 7081 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 7141 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 7201 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 7261 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 7321 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac 7381 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa 7441 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc 7501 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg 7561 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa 7621 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg 7681 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg 7741 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc 7801 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct 7861 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc 7921 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc 7981 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt 8041 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa 8101 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag 8161 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc 8221 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat 8281 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg 8341 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc 8401 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca cgctagcgcc caatacgc //