LOCUS Exported 5418 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Homology arms and linker-mEGFP sequence for N-terminus tagging of human MYH10. ACCESSION . VERSION . KEYWORDS AICSDP-24:MYH10-mEGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5418) AUTHORS Roberts B, Haupt A, Tucker A, Grancharova T, Arakaki J, Fuqua MA, Nelson A, Hookway C, Ludmann SA, Mueller IA, Yang R, Horwitz R, Rafelski SM, Gunawardane RN TITLE Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. JOURNAL Mol Biol Cell. 2017 Oct 15;28(21):2854-2874. doi: 10.1091/mbc.E17-03-0209. Epub 2017 Aug 16. PUBMED 28814507 REFERENCE 2 (bases 1 to 5418) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5418 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 364..386 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 378..395 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 379..395 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 1413..2129 /codon_start=1 /product="enhanced GFP with monomerizing A206K mutation (Zacharias et al., 2002)" /label=mEGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1458..1479) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1719..1738) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2066..2087 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(3197..3213) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3197..3213) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3210..3232) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3221..3237 /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(3245..3275) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3290..3311 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3428..3445) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3599..4187) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3679..3698) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4358..5218) /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 4981..5000 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5219..5323) /gene="bla" /label=AmpR promoter primer_bind 5391..5409 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggataagtg 421 atggagcttc ctctgctagc cctttgtgag ccaatggtaa atgggtgcta aataaaacaa 481 ctaggtcttg agatacatta attgtaaatg tcacagaacc agtactttcc tcaatgtggc 541 taagatagtt gatggttcct ttttcttctg cactggtcag accatatctg ggctatgatg 601 tttgcttctg ggccacacac tttagaggga agacaagcag catgttggag tctgtttagg 661 cgagagatcc ggcaggggaa ggagtcttgg tgaatgaggg gtggaggagc tgcaggatgg 721 gaatagaggc ctgaactgct accatgacat attcaaaagg ctgccgtgtg aagccaagtt 781 atgcttgtct tttgtggtcc cagttgatca cattaagacc tcatggggcc atagaaaagc 841 tagagggaga ctgatttggg ttattcataa gaagaacttt aagtctgtta tctgagggta 901 gaatgagagg catgttttag attctttaga ttctttactc ttctgacaat catgtgtttt 961 tgtagctgtt tccttgtggt catattaatt ctggtaccac ttcatgaacc ttttattacc 1021 catctttgtt ttcttttttt ttttcccttc ttaactccct gtttaatttg tggtgagggt 1081 gaaagaggag ataaagaaaa aaaagggtca acttgtaact ttgccttttc ttttcttttc 1141 ttttcttttt tttgccctca gtaactgagg gcaaacccat cagacaacca gagccataat 1201 ttgtggtcac cgctgaaatt taccttggaa actcggttag tatggctgtg aagaggtata 1261 ccccagctcc ttaacacaga gttaatgctt aatctaaggt tttaagtttc ttagaaaaga 1321 aaaacgtgta cattcttttg tttcttaaac atctaatttt gccctcctcc tcttctctta 1381 gaggcaattg cttttggatc gttccattta caatggtgag caagggcgag gagctgttca 1441 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 1501 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 1561 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc 1621 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 1681 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 1741 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 1801 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 1861 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 1921 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 1981 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca 2041 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 2101 tcactctcgg catggacgag ctgtacaagt acagcgatct ggagctgaag ctgcggatcc 2161 ctgcacaaag gacagggctg gaggatccag agaggtacct gttcgtcgat cgtgctgtca 2221 tctacaaccc tgccactcaa gctgattgga cagctaaaaa gctagtgtgg attccatcag 2281 aacgccatgg ttttgaggca gctagtatca aagaagaacg gggagatgaa gttatggtgg 2341 agttggcaga gaatggaaag aaagcaatgg tcaacaaaga tgatattcag aagatgaacc 2401 cacctaagtt ttccaaggtg gaggatatgg cagaattgac atgcttgaat gaagcttccg 2461 ttttacataa tctgaaggat cgctactatt caggactaat ctatgtaagt atttcttcca 2521 aataatcatg tgaagtggta gctaggaatt aatgtaaatt atacatcttg tcataatcaa 2581 atgagaatgt ggaataccca aactctctgt ttaacatttc tatttctctt taagatagaa 2641 agatttgttg cttgcttacc catgtcttgc ttttctttga atcttaacac attaagttta 2701 aataatacag gctgcaatta catataataa aatggcattt gaagactttt gtagtggtct 2761 tctggagcat aataaggtgg gagagagcat gtaacaggaa gaccagaagg tttaataagg 2821 taaagagagt tgcattaatt ggacgcagac agcaaaacgg tcaaaaatca agtgcatacc 2881 caagagtaaa gtggaggggc tgtaagctga gaaatttctg tggacagcat gaacagcttc 2941 actggatgta gtagggaagt aggaaagatg aatgctgagg tttttaagag gaaacaatta 3001 gggtaagatt gaggctggct ggggtcgtcc tgtggttagc agctgacatg aatgttggag 3061 tcaccgactt tgtcactgac catgtagaag aagttattga aaatcataag ggataatgta 3121 gagagggata atgtagagag gaaaaatgta agccagatac taatcccaat ggcgcgccga 3181 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 3241 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 3301 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 3361 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 3421 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 3481 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 3541 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 3601 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 3661 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 3721 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 3781 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 3841 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 3901 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 3961 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 4021 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 4081 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 4141 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 4201 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 4261 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 4321 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 4381 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 4441 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 4501 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 4561 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 4621 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 4681 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 4741 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 4801 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 4861 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 4921 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 4981 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 5041 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 5101 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 5161 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 5221 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 5281 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 5341 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta 5401 tcacgaggcc ctttcgtc //