LOCUS Exported 5438 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Produces equimolar levels of two fluorescent proteins. It can be used to measure practical brightness and as a negative control for FRET. ACCESSION . VERSION . KEYWORDS mKO2-2A-mTurquoise2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5438) AUTHORS Mastop M, Bindels DS, Shaner NC, Postma M, Gadella TWJ Jr, Goedhart J TITLE Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2. JOURNAL Sci Rep. 2017 Sep 20;7(1):11999. doi: 10.1038/s41598-017-12212-x. PUBMED 28931898 REFERENCE 2 (bases 1 to 5438) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5438 /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" regulatory 607..616 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 634..1281 /codon_start=1 /product="monomeric Kusabira-Orange 2 fluorescent protein" /label=mKO2 /translation="SVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTM AEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSASV SAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYLK LEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS" CDS 1309..1362 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" regulatory 1378..1387 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1384..2103 /codon_start=1 /product="enhanced monomeric variant of CFP (Goedhart et al., 2012)" /label=mTurquoise2 /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1429..1450) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1690..1709) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2037..2058 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2226..2347 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2263..2282) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2317..2336 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2354..2809) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2491..2512) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2703..2722 /label=F1ori-R /note="F1 origin, reverse primer" promoter 2836..2940 /gene="bla" /label=AmpR promoter promoter 2942..3299 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2965..2985) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3150..3285 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3212..3231 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3334..4128 /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(3388..3407) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3998..4017 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4316..4335) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4360..4407 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 4736..5324 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5225..5244 /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 gctagcgcta 601 ccggtcgcca ccatggtgag caagggcgag gagagtgtga ttaaaccaga gatgaagatg 661 aggtactaca tggacggctc cgtcaatggg catgagttca caattgaagg tgaaggcaca 721 ggcagacctt acgagggaca tcaagagatg acactacgcg tcacaatggc cgagggcggg 781 ccaatgcctt tcgcgtttga cttagtgtca cacgtgttct gttacggcca cagagtattt 841 actaaatatc cagaagagat accagactat ttcaaacaag catttcctga aggcctgtca 901 tgggaaaggt cgttggagtt cgaagatggt gggtccgctt cagtcagtgc gcatataagc 961 cttagaggaa acaccttcta ccacaaatcc aaatttactg gggttaactt tcctgccgat 1021 ggtcctatca tgcaaaacca aagtgttgat tgggagccat caaccgagaa aattactgcc 1081 agcgacggag ttctgaaggg tgatgttacg atgtacctaa aacttgaagg aggcggcaat 1141 cacaaatgcc aaatgaagac tacttacaag gcggcaaaag agattcttga aatgccagga 1201 gaccattaca tcggccatcg cctcgtcagg aaaaccgaag gcaacattac tgagcaggta 1261 gaagatgcag tagctcattc cactggcgac gagctgtaca agtccggaga gggcagagga 1321 agtcttctaa catgcggtga cgtggaggag aatcccggcc ctggggatcc accggtcgcc 1381 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 1441 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 1501 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 1561 accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg 1621 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 1681 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 1741 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 1801 cacaagctgg agtacaacta ctttagcgac aacgtctata tcaccgccga caagcagaag 1861 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc 1921 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 1981 cactacctga gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg 2041 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 2101 taaagcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg 2161 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg 2221 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 2281 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 2341 tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt 2401 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa 2461 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag 2521 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt 2581 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 2641 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 2701 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 2761 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact 2821 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 2881 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 2941 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag 3001 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 3061 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 3121 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 3181 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg 3241 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag 3301 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca 3361 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc 3421 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc 3481 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg 3541 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg 3601 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct 3661 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct 3721 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa 3781 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa 3841 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc 3901 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt 3961 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct 4021 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc 4081 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg 4141 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg 4201 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc 4261 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa 4321 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata 4381 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact 4441 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc 4501 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag 4561 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt 4621 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 4681 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 4741 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 4801 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 4861 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 4921 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 4981 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 5041 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 5101 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 5161 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 5221 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 5281 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 5341 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 5401 cccctgattc tgtggataac cgtattaccg ccatgcat //