LOCUS Exported 7457 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Red-fluorescent homoFRET/anisotropy-based biosensor for monitoring Protein Kinase A activity.. ACCESSION . VERSION . KEYWORDS pcDNA3-mCherry-mCherry-FLARE-AKAR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7457) AUTHORS Ross BL, Tenner B, Markwardt ML, Zviman A, Shi G, Kerr JP, Snell NE, McFarland JJ, Mauban JR, Ward CW, Rizzo MA, Zhang J TITLE Single-color, ratiometric biosensors for detecting signaling activities in live cells. JOURNAL Elife. 2018 Jul 3;7. pii: 35458. doi: 10.7554/eLife.35458. PUBMED 29968564 REFERENCE 2 (bases 1 to 7457) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7457 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(55..74) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 246..625 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 626..829 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 780..800 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 874..893 /label=T7 /note="T7 promoter, forward primer" promoter 874..892 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 912..921 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 933..950 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 954..986 /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10" /label=T7 tag (gene 10 leader) /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS 990..1013 /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /label=Xpress(TM) tag /translation="DLYDDDDK" CDS 1020..1727 /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" CDS 2250..2960 /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" promoter complement(3021..3039) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3022..3039) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(3059..3076) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3065..3289 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3335..3763 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3422..3441) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3632..3653 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3772..3792) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3777..4106 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3957..4092 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4019..4038 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4173..4967 /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(4227..4246) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4837..4856 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5141..5262 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5178..5197) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5232..5251 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5311..5327) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5311..5327) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5324..5346) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5335..5351 /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(5359..5389) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5404..5425 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5542..5559) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5713..6301) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5793..5812) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6472..7332) /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 7095..7114 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7333..7437) /gene="bla" /label=AmpR promoter ORIGIN 1 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat 61 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc 121 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat 181 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac 241 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca 301 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 361 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 421 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 481 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 541 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 601 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 661 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 721 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 781 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac 841 tagagaaccc actgcttact ggcttatcga aattaatacg actcactata gggagaccca 901 agcttgcggc cgccaccatg gtgcggggtt ctcatcatca tcatcatcat ggtatggcta 961 gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatccca 1021 tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg 1081 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc 1141 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct 1201 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc 1261 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg 1321 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg 1381 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa 1441 tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg 1501 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg 1561 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca 1621 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac 1681 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagcgc atgcataagt 1741 tttctcaaga acagatcggc gaaaacattg tgtgcagggt catttgtacc acgggtcaaa 1801 ttcccatccg agatttgtca gctgatattt cacaagtgct taaggaaaaa cgatccataa 1861 agaaagtttg gacatttggt agaaacccag cctgtgacta tcatttagga aacatttcaa 1921 gactgtcaaa taagcatttc caaatactac taggagaaga cggtaacctt ttattgaatg 1981 acatttccac taatgggacc tggttaaatg ggcaaaaagt cgagaagaac agcaatcagt 2041 tactgtctca aggtgatgaa ataaccgttg gtgtaggcgt ggaatcagat attttatctc 2101 tggtcatttt cataaacgac aaatttaagc agtgcctcga gcagaacaaa gttgatcgct 2161 ctgcaggtaa gccaggcagc ggcgagggca gcaccaaggg cctgcgtcgc gccaccctgg 2221 tggacggcgg caccggcggc agcgagctca tggtgagcaa gggcgaggag gataacatgg 2281 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg 2341 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc 2401 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca 2461 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt 2521 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga 2581 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg 2641 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct 2701 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga 2761 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc 2821 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg 2881 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca 2941 tggacgagct gtacaagtaa gaattctgca gatatccatc acactggcgg ccgctcgagc 3001 atgcatctag agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc 3061 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 3121 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 3181 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 3241 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg 3301 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc 3361 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 3421 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 3481 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 3541 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 3601 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 3661 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 3721 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt 3781 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 3841 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta 3901 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc 3961 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta 4021 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct 4081 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat 4141 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag 4201 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg 4261 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca 4321 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc 4381 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg 4441 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg 4501 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta 4561 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag 4621 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac 4681 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg 4741 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg 4801 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg 4861 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg 4921 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg 4981 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc 5041 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 5101 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta 5161 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 5221 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc 5281 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 5341 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 5401 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 5461 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 5521 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 5581 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 5641 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 5701 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 5761 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 5821 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 5881 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 5941 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 6001 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 6061 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 6121 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 6181 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 6241 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 6301 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 6361 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 6421 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 6481 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 6541 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 6601 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 6661 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 6721 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 6781 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 6841 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 6901 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 6961 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 7021 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 7081 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 7141 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 7201 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 7261 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 7321 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 7381 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 7441 atttccccga aaagtgc //