LOCUS Exported 5994 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses nuclear-targeted non-polymerizing R62D mutant of human actin, with an mCherry expression reporter after a P2A protease cleavage site, on a CMV promoter. ACCESSION . VERSION . KEYWORDS pmCherry-C1 R62D actin-3XNLS P2A mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5994) AUTHORS Belin BJ, Lee T, Mullins RD TITLE DNA damage induces nuclear actin filament assembly by formin-2 and Spire-1/2 that promotes efficient DNA repair. JOURNAL Elife. 2015 Aug 19;4. doi: 10.7554/eLife.07735. PUBMED 26287480 REFERENCE 2 (bases 1 to 5994) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5994 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 61..364 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 365..568 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 519..539 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 1738..1758 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1762..1782 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1813..1869 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" regulatory 1879..1888 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1885..2592 /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(2030..2048) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2298..2317 /label=mCherry-F /note="mCherry, forward primer" misc_feature 2593..2658 /label=MCS /note="multiple cloning site" polyA_signal 2782..2903 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2819..2838) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2873..2892 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2910..3365) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3047..3068) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3259..3278 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3392..3496 /gene="bla" /label=AmpR promoter promoter 3498..3855 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3521..3541) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3706..3841 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3768..3787 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3890..4684 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3944..3963) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4554..4573 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4872..4891) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4916..4963 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5292..5880 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5781..5800 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgatg 601 gatgatgata tcgccgcgct cgtcgtcgac aacggctccg gcatgtgcaa ggccggcttc 661 gcgggcgacg atgccccccg ggccgtcttc ccctccatcg tggggcgccc caggcaccag 721 ggcgtgatgg tgggcatggg tcagaaggat tcctatgtgg gcgacgaggc ccagagcaag 781 gacggcatcc tcaccctgaa gtaccccatc gagcacggca tcgtcaccaa ctgggacgac 841 atggagaaaa tctggcacca caccttctac aatgagctgc gtgtggctcc cgaggagcac 901 cccgtgctgc tgaccgaggc ccccctgaac cccaaggcca accgcgagaa gatgacccag 961 atcatgtttg agaccttcaa caccccagcc atgtacgttg ctatccaggc tgtgctatcc 1021 ctgtacgcct ctggccgtac cactggcatc gtgatggact ccggtgacgg ggtcacccac 1081 actgtgccca tctacgaggg gtatgccctc ccccatgcca tcctgcgtct ggacctggct 1141 ggccgggacc tgactgacta cctcatgaag atcctcaccg agcgcggcta cagcttcacc 1201 accacggccg agcgggaaat cgtgcgtgac attaaggaga agctgtgcta cgtcgccctg 1261 gacttcgagc aagagatggc cacggctgct tccagctcct ccctggagaa gagctacgag 1321 ctgcctgacg gccaggtcat caccattggc aatgagcggt tccgctgccc tgaggcactc 1381 ttccagcctt ccttcctggg catggagtcc tgtggcatcc acgaaactac cttcaactcc 1441 atcatgaagt gtgacgtgga catccgcaaa gacctgtacg ccaacacagt gctgtctggc 1501 ggcaccacca tgtaccctgg cattgccgac aggatgcaga aggagatcac tgccctggca 1561 cccagcacaa tgaagatcaa gatcattgct cctcctgagc gcaagtactc cgtgtggatc 1621 ggcggctcca tcctggcctc gctgtccacc ttccagcaga tgtggatcag caagcaggag 1681 tatgacgagt ccggcccctc catcgtccac cgcaaatgct tcctaccggt aatggatcca 1741 aaaaagaaga gaaaggtaga tccaaaaaag aagagaaagg tagatccaaa aaagaagaga 1801 aaggcggccg cagccacgaa cttctctctg ttaaagcaag caggagacgt ggaagaaaac 1861 cccggtccta aaccggtcgc caccatggtg agcaagggcg aggaggataa catggccatc 1921 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc 1981 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag 2041 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac 2101 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc 2161 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg 2221 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc 2281 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc 2341 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg 2401 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg 2461 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac 2521 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac 2581 gagctgtaca agtccggact cagatctcga gctcaagctt cgaattctgc agtcgacggt 2641 accgcgggcc cgggatccac cggatctaga taactgatca taatcagcca taccacattt 2701 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa 2761 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc 2821 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 2881 tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt 2941 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat 3001 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa 3061 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg 3121 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa 3181 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc 3241 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag 3301 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg 3361 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 3421 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 3481 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg 3541 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 3601 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 3661 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 3721 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 3781 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 3841 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca 3901 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg 3961 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg 4021 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc 4081 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt 4141 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc 4201 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca 4261 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc 4321 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg 4381 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct 4441 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc 4501 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc 4561 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta 4621 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt 4681 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga 4741 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac 4801 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg 4861 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca 4921 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg 4981 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc 5041 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc 5101 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat 5161 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 5221 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 5281 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 5341 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 5401 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 5461 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 5521 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 5581 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 5641 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 5701 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 5761 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 5821 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 5881 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 5941 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat //