LOCUS Exported 5964 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Double fluorescent stalling reporter K0. ACCESSION . VERSION . KEYWORDS pmGFP-P2A-K0-P2A-RFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5964) AUTHORS Juszkiewicz S, Hegde RS TITLE Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination. JOURNAL Mol Cell. 2017 Feb 16;65(4):743-750.e4. doi: 10.1016/j.molcel.2016.11.039. Epub 2017 Jan 5. PUBMED 28065601 REFERENCE 2 (bases 1 to 5964) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5964 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..304 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 305..508 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 459..479 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 547..556 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 553..1269 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK EFVTAAGITLGMDELYK" primer_bind complement(598..619) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(859..878) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" CDS 1285..1341 /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" CDS 1348..1413 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1789..1845 /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" CDS 1852..2559 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(1994..2012) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2262..2281 /label=mCherry-F /note="mCherry, forward primer" polyA_signal 2692..2813 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2729..2748) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2783..2802 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2820..3275) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2957..2978) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3169..3188 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3302..3406 /gene="bla" /label=AmpR promoter promoter 3408..3765 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3431..3451) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3616..3751 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3678..3697 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3800..4594 /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(3854..3873) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4464..4483 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4782..4801) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4826..4873 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5202..5790 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5691..5710 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 541 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 601 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 661 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 721 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 781 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 841 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 901 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 961 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 1021 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 1081 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 1141 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 1201 gatcacatgg tccttaagga gttcgtgacc gccgccggga tcactctcgg catggacgag 1261 ctgtacaagt ccggaggcag cggcgccacc aacttttccc tgctcaagca ggccggcgac 1321 gtggaagaga atcccggccc cggtactgac tacaaagacc atgacggtga ttataaagat 1381 catgacatcg attacaagga tgacgatgac aaggccggat ctcctggtcc gacccccagt 1441 ggcactaacg tgggatcctc actctctgac gaggacttca aggctgtttt tggcatgacc 1501 cgctctgcct ttgccaactt gcccttgtgg aaacagcaga acctcaagaa ggagaaagga 1561 ctcttcggat cagggcgctc tcccagcaaa gcagtggccg cccgggcggc gggatccact 1621 gtccggcaga ggaaaaatgc cagctgtggg acaaggagtg caggccgcac aacctcggca 1681 ggcaccgggg ggatgtggcg attctacaca gaagattcac ctgggctcaa agttggccct 1741 aggtcgaccg ccatggcgac ccccggggga tccggtaccg gaagcggagc tactaacttc 1801 agcctgctga agcaggctgg agacgtggag gagaaccctg gacctctcga ggtgagcaag 1861 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag 1921 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag 1981 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac 2041 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc 2101 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc 2161 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc 2221 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag 2281 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc 2341 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc 2401 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg 2461 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc 2521 cgccactcca ccggcggcat ggacgagctg tacaagtaaa ctggatccac cggatctaga 2581 taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc 2641 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt 2701 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 2761 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta 2821 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt 2881 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat 2941 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa 3001 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta 3061 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc 3121 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc 3181 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac 3241 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt 3301 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 3361 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag 3421 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc 3481 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc 3541 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg 3601 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 3661 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc 3721 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac 3781 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc 3841 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc 3901 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc 3961 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg 4021 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt 4081 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc 4141 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga 4201 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga 4261 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct 4321 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc 4381 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt 4441 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg 4501 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat 4561 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc 4621 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa 4681 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat 4741 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca 4801 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt 4861 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca 4921 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca 4981 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc 5041 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 5101 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 5161 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 5221 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 5281 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 5341 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 5401 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 5461 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 5521 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 5581 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 5641 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 5701 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 5761 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 5821 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 5881 gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt 5941 tcatagccca tatatggagt tccg //