LOCUS Exported 6126 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION GFP with robust performance in protein fusions and useful as FRET acceptor for mRuby2 in a FRET pair that offers bright fluorescence, dynamic range, and photostability while limiting emissions overlap. ACCESSION . VERSION . KEYWORDS pcDNA3-Clover SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6126) AUTHORS Lam AJ, St-Pierre F, Gong Y, Marshall JD, Cranfill PJ, Baird MA, McKeown MR, Wiedenmann J, Davidson MW, Schnitzer MJ, Tsien RY, Lin MZ TITLE Improving FRET dynamic range with bright green and red fluorescent proteins. JOURNAL Nat Methods. 2012 Sep 9. doi: 10.1038/nmeth.2171. PUBMED 22961245 REFERENCE 2 (bases 1 to 6126) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6126 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 177..556 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 557..760 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 711..731 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 805..824 /label=T7 /note="T7 promoter, forward primer" promoter 805..823 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 889..1605 /codon_start=1 /product="bright green-yellow fluorescent protein derived from GFP (Lam et al., 2012)" /label=Clover /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL EFVTAAGITHGMDELYK" primer_bind complement(934..955) /label=EGFP-N /note="EGFP, reverse primer" primer_bind 1542..1563 /label=EGFP-C /note="EGFP, forward primer" promoter complement(1621..1639) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(1622..1639) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(1659..1676) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1665..1889 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 1935..2363 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2022..2041) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2232..2253 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2372..2392) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2377..2706 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2557..2692 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2619..2638 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 2773..3567 /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(2827..2846) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3437..3456 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 3741..3862 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3778..3797) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3832..3851 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3911..3927) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3911..3927) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3924..3946) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3935..3951 /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(3959..3989) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4004..4025 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4142..4159) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4313..4901) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4393..4412) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5072..5932) /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 5695..5714 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5933..6037) /gene="bla" /label=AmpR promoter primer_bind complement(6112..5) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 61 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct 121 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca 181 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 241 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 301 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 361 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 421 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 481 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 541 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 601 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 661 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 721 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc 781 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagcttggta 841 ccgagctcgg atccactagt aacggccgcc agtgtgctgg aattcggcat ggtgagcaag 901 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 961 ggccacaagt tcagcgtccg cggcgagggc gagggcgatg ccaccaacgg caagctgacc 1021 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 1081 ttcggctacg gcgtggcctg cttcagccgc taccccgacc acatgaagca gcacgacttc 1141 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatctcttt caaggacgac 1201 ggtacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 1261 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 1321 aacttcaaca gccacaacgt ctatatcacg gccgacaagc agaagaacgg catcaaggct 1381 aacttcaaga tccgccacaa cgttgaggac ggcagcgtgc agctcgccga ccactaccag 1441 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagccat 1501 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 1561 gtgaccgccg ccgggattac acatggcatg gacgagctgt acaagtctag agggccctat 1621 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg 1681 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 1741 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 1801 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 1861 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 1921 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 1981 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 2041 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 2101 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 2161 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 2221 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 2281 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 2341 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag 2401 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 2461 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 2521 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 2581 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 2641 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 2701 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga 2761 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg 2821 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg 2881 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc 2941 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct 3001 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa 3061 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg 3121 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa 3181 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat 3241 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg 3301 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc 3361 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac 3421 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg 3481 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc 3541 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag 3601 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg 3661 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc 3721 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca 3781 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 3841 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg 3901 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 3961 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 4021 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 4081 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 4141 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 4201 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 4261 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 4321 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 4381 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 4441 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 4501 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 4561 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 4621 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 4681 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 4741 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 4801 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 4861 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 4921 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 4981 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 5041 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 5101 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 5161 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 5221 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 5281 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 5341 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 5401 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 5461 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 5521 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 5581 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 5641 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 5701 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 5761 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 5821 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 5881 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 5941 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 6001 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 6061 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg 6121 ctctga //