LOCUS Exported 6824 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of RFP-TEVprotease. ACCESSION . VERSION . KEYWORDS pcDNA_RFP-TEVprotease SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6824) AUTHORS Schuster BS, Reed EH, Parthasarathy R, Jahnke CN, Caldwell RM, Bermudez JG, Ramage H, Good MC, Hammer DA TITLE Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. JOURNAL Nat Commun. 2018 Jul 30;9(1):2985. doi: 10.1038/s41467-018-05403-1. PUBMED 30061688 REFERENCE 2 (bases 1 to 6824) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6824 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 124..503 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 504..707 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 658..678 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 752..771 /label=T7 /note="T7 promoter, forward primer" promoter 752..770 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 824..1531 /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(969..987) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1237..1256 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(2304..2321) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2310..2534 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2580..3008 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2667..2686) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2877..2898 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3017..3037) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3022..3351 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3202..3337 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3264..3283 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3418..4212 /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 GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3472..3491) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4082..4101 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4386..4507 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4423..4442) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4477..4496 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4556..4572) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4556..4572) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4569..4591) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4580..4596 /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(4604..4634) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4649..4670 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4787..4804) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4958..5546) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5038..5057) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5717..6577) /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 6340..6359 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6578..6682) /gene="bla" /label=AmpR promoter primer_bind complement(6757..6776) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 61 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 121 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 181 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 241 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 301 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 361 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 421 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 481 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 541 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 601 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 661 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 721 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 781 ctggctagcg tttaaactta agcttggtac cgagctcgga tccatggtga gcaagggcga 841 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc 901 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac 961 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct 1021 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga 1081 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga 1141 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa 1201 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat 1261 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat 1321 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta 1381 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat 1441 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca 1501 ctccaccggc ggcatggacg agctgtacaa gggtggtggg ggaagcggag gaggaggttc 1561 gggtgaaagc ttgtttaaag ggccgcgcga ttataacccg attagtagca cgatttgtca 1621 tttgacaaat gaatccgatg gacatacaac gagcctttat gggatcggtt tcggaccatt 1681 catcatcaca aacaagcact tatttcgccg taacaatggc acattggttg ttcaaagtct 1741 tcacggagtg tttaaagtca agaacactac cacccttcag caacacttga tcgatgggcg 1801 cgacatgatt attattcgta tgccgaaaga tttccctccc ttcccgcaga agcttaaatt 1861 tcgtgaaccc caacgtgaag aacgcatttg tttggtgaca acgaactttc aaaccaaatc 1921 tatgtctagc atggtgtccg acacttcttg cacatttccg agtggagacg gtattttttg 1981 gaaacattgg attcaaacaa aagacggaca atgtggatct cctctggtaa gcacacgtga 2041 cggatttatc gtcgggattc attcggcatc gaactttact aatacgaata attactttac 2101 gtcagttccg aaaaatttta tggagttact tactaatcag gaggcgcaac aatgggtcag 2161 tgggtggcgc ttaaatgccg acagcgtgtt gtggggtgga cataaagtgt ttatggtaaa 2221 accggaagag ccattccagc ccgtgaagga ggcgacgcag ttgatgaact aatctagagg 2281 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt 2341 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta 2401 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 2461 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc 2521 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca 2581 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 2641 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 2701 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 2761 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 2821 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 2881 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata 2941 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 3001 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca 3061 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 3121 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 3181 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 3241 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag 3301 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg 3361 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg 3421 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 3481 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 3541 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 3601 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 3661 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 3721 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 3781 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 3841 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 3901 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 3961 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 4021 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 4081 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 4141 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 4201 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 4261 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 4321 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 4381 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 4441 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 4501 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat 4561 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 4621 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 4681 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4741 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 4801 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4861 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4921 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4981 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 5041 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 5101 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 5161 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 5221 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 5281 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 5341 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 5401 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 5461 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 5521 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 5581 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 5641 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 5701 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 5761 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 5821 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 5881 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 5941 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 6001 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 6061 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 6121 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 6181 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 6241 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 6301 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 6361 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 6421 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 6481 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 6541 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 6601 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 6661 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat 6721 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata 6781 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctg //