LOCUS Exported 7719 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Fluorescent biosensor for super-resolution imaging of PKA activity; targeted to plasma membrane.. ACCESSION . VERSION . KEYWORDS FLINC-AKAR1-CAAX SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7719) AUTHORS Mo GC, Ross B, Hertel F, Manna P, Yang X, Greenwald E, Booth C, Plummer AM, Tenner B, Chen Z, Wang Y, Kennedy EJ, Cole PA, Fleming KG, Palmer A, Jimenez R, Xiao J, Dedecker P, Zhang J TITLE Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution. JOURNAL Nat Methods. 2017 Apr;14(4):427-434. doi: 10.1038/nmeth.4221. Epub 2017 Mar 13. PUBMED 28288122 REFERENCE 2 (bases 1 to 7719) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7719 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 901..910 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 922..939 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 943..975 /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 979..1002 /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /label=Xpress(TM) tag /translation="DLYDDDDK" CDS 1009..1680 /codon_start=1 /product="monomeric photoswitchable Dronpa-Green1 fluorescent protein" /label=Dronpa-Green1 /translation="MSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVK EGGPLPFAYDILTTVFCYGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNAT NDITLDGDCYIYEIRFDGVNFPANGPVMQKRTVKWEPSTEKLYVRDGVLKGDVNMALSL EGGGHYRCDFKTTYKAKKVVQLPDYHFVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQ AK" promoter complement(3271..3289) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3272..3289) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(3309..3326) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3315..3539 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3585..4013 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3672..3691) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3882..3903 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4022..4042) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4027..4357 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4208..4343 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4270..4289 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4424..5218 /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(4478..4497) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5088..5107 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5392..5513 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5429..5448) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5483..5502 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5562..5578) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5562..5578) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5575..5597) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5586..5602 /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(5610..5640) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5655..5676 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5793..5810) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5964..6552) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6044..6063) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6723..7583) /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 7346..7365 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7584..7688) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc 901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt 961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat gagtgtgatt 1021 aaaccagaca tgaagatcaa gctgcgtatg gaaggcgctg taaatggaca cccgttcgcg 1081 attgaaggag ttggccttgg gaagcctttc gagggaaaac agagtatgga ccttaaagtc 1141 aaagaaggcg gacctctgcc tttcgcctat gacatcttga caactgtgtt ctgttacggc 1201 aacagggtat tcgccaaata cccagaaaat atagtagact atttcaagca gtcgtttcct 1261 gagggctact cttgggaacg aagcatgaat tacgaagacg ggggcatttg taacgcgaca 1321 aacgacataa ccctggatgg tgactgttat atctatgaaa ttcgatttga tggtgtgaac 1381 tttcctgcca atggtccagt tatgcagaag aggactgtga aatgggagcc atccactgag 1441 aaattgtatg tgcgtgatgg agtgctgaag ggtgatgtta acatggctct gtcgcttgaa 1501 ggaggtggcc attaccgatg tgacttcaaa actacttata aagctaagaa ggttgtccag 1561 ttgccagact atcactttgt ggaccaccac attgagatta aaagccacga caaagattac 1621 agtaatgtta atctgcatga gcatgccgaa gcgcattctg agctgccgag gcaggccaag 1681 agatctatgc ataagttttc tcaagaacag atcggcgaaa acattgtgtg cagggtcatt 1741 tgtaccacgg gtcaaattcc catccgagat ttgtcagctg atatttcaca agtgcttaag 1801 gaaaaacgat ccataaagaa agtttggaca tttggtagaa acccagcctg tgactatcat 1861 ttaggaaaca tttcaagact gtcaaataag catttccaaa tactactagg agaagacggt 1921 aaccttttat tgaatgacat ttccactaat gggacctggt taaatgggca aaaagtcgag 1981 aagaacagca atcagttact gtcccaaggt gatgaaataa ccgttggtgt aggcgtggaa 2041 tcagatattt tatctctggt cattttcata aacgacaaat ttaagcagtg cctggagcag 2101 aacaaagttg atcgcggtac cagtgctggt ggtagtgctg gtggtagtgc tggtggtagt 2161 gctggtggta gtgctggtgg ttccggcagt gctggtggta gtgctggtgg tagtaccagt 2221 gctggtggta gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc tggtggttcc 2281 ggcagtgctg gtggtagtgc tggtggtagt accagtgctg gtggtagtgc tggtggtagt 2341 gctggtggta gtgctggtgg tagtgctggt ggttccggca gtgctggtgg tagtgctggt 2401 ggtagtacca gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc tggtggtagt 2461 gctggtggtt ccggattgag gcgcgcgacg ctggttgacg gcggcagcga gctcatgagc 2521 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt gaacaaccac 2581 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga 2641 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc taccagcttc 2701 atgtacggca gcagaacctt catcaaccac acccagggca tccccgattt ctttaagcag 2761 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg 2821 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga 2881 ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc 2941 aacaccgaga tgctgtaccc cgctgacggc ggcctggaag gcagaaccga catggccctg 3001 aagctcgtgg gcgggggcca cctgatctgc aacttcaaga ccacatacag atccaagaaa 3061 cccgctaaga acctcaagat gcccggcgtc tactatgtgg accacagact ggaaagaatc 3121 aaggaggccg acaaagagac ctacgtcgag cagcacgagg tggctgtggc cagatactgc 3181 gacctcccta gcaaactggg gcacaaggaa ttcaagaaga agaagaagag caagaccaag 3241 tgcgtgatca tgtaatctag agggccctat tctatagtgt cacctaaatg ctagagctcg 3301 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt 3361 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 3421 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 3481 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 3541 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg 3601 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 3661 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 3721 ccgtcaagct ctaaatcggg gcatcccttt agggttccga tttagtgctt tacggcacct 3781 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 3841 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 3901 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttggggat 3961 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg 4021 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccaggcag gcagaagtat 4081 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 4141 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 4201 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 4261 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta 4321 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc 4381 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg 4441 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 4501 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 4561 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg 4621 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 4681 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 4741 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 4801 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 4861 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 4921 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt 4981 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 5041 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 5101 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 5161 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc 5221 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc 5281 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 5341 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt 5401 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 5461 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 5521 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg 5581 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 5641 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 5701 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 5761 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 5821 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 5881 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 5941 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 6001 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 6061 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 6121 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc 6181 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 6241 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 6301 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 6361 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 6421 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 6481 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 6541 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 6601 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 6661 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 6721 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 6781 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 6841 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 6901 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 6961 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 7021 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 7081 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 7141 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 7201 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 7261 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 7321 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 7381 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 7441 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 7501 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 7561 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 7621 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 7681 gttccgcgca catttccccg aaaagtgcca cctgacgtc //