LOCUS Exported 7742 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION ddRFP-based single-color biosensor for monitoring Protein Kinase A activity.. ACCESSION . VERSION . KEYWORDS pcDNA3-RAB-AKARev SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7742) AUTHORS Mehta S, Zhang Y, Roth RH, Zhang JF, Mo A, Tenner B, Huganir RL, Zhang J TITLE Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities. JOURNAL Nat Cell Biol. 2018 Sep 24. pii: 10.1038/s41556-018-0200-6. doi: 10.1038/s41556-018-0200-6. PUBMED 30250062 REFERENCE 2 (bases 1 to 7742) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7742 /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" promoter complement(3295..3313) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3296..3313) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(3333..3350) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3339..3563 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3609..4037 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3696..3715) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3906..3927 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4046..4066) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4051..4380 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4231..4366 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4293..4312 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4447..5241 /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(4501..4520) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5111..5130 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5415..5536 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5452..5471) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5506..5525 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5585..5601) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5585..5601) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5598..5620) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5609..5625 /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(5633..5663) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5678..5699 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5816..5833) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5987..6575) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6067..6086) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6746..7606) /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 7369..7388 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7607..7711) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 ggtgagcaag 1021 agcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc gcttggaggg ctccatgaac 1081 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc 1141 gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc 1201 cagatcatgt acggctccaa ggcgtacgtg aagcaccccg ccgacgtccc cgattacatg 1261 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgcacttcga ggacggcggt 1321 ctggtgaccg tgactcagga cacctccctg caggacggca cgctgatcta caaggtgaag 1381 atgcgcggca ccaacttccc ccccgacggc cccgtaatgc agaagaagac cttgggctgg 1441 gattatgcca ccgagcgcct gtaccccgaa gacggcgtgc tgaagggcga gcttctgggg 1501 cgcctgaagc tgaaggacgg cggcctcaac ctggtggagt tcaagaccat ctacatggcc 1561 aagaagcccg tgcaactgcc cggctactac ttcgtggaca ccaagctggg catcacctcc 1621 cacaacgagg actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg 1681 gggcatggca ccggcagcag atctatgcat aagttttctc aagaacagat cggcgaaaac 1741 attgtgtgca gggtcatttg taccacgggt caaattccca tccgagattt gtcagctgat 1801 atttcacaag tgcttaagga aaaacgatcc ataaagaaag tttggacatt tggtagaaac 1861 ccagcctgtg actatcattt aggaaacatt tcaagactgt caaataagca tttccaaata 1921 ctactaggag aagacggtaa ccttttattg aatgacattt ccactaatgg gacctggtta 1981 aatgggcaaa aagtcgagaa gaacagcaat cagttactgt cccaaggtga tgaaataacc 2041 gttggtgtag gcgtggaatc agatatttta tctctggtca ttttcataaa cgacaaattt 2101 aagcagtgcc tggagcagaa caaagttgat cgcggtacca gtgctggtgg tagtgctggt 2161 ggtagtgctg gtggtagtgc tggtggtagt gctggtggtt ccggcagtgc tggtggtagt 2221 gctggtggta gtaccagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt 2281 ggtagtgctg gtggttccgg cagtgctggt ggtagtgctg gtggtagtac cagtgctggt 2341 ggtagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg ttccggcagt 2401 gctggtggta gtgctggtgg tagtaccagt gctggtggta gtgctggtgg tagtgctggt 2461 ggtagtgctg gtggtagtgc tggtggttcc ggattgaggc gcgcgacgct ggttgacggc 2521 ggcagcgagc tcgcctcctc cgaggacaac aacatggcca ccatcaaaga gttcatgcgc 2581 ttcaaggtgc gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc 2641 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc 2701 ctgcccttcg cctgggacat cctgtccccc cagttcatgt acggctccga ggcgtacgtg 2761 aggcaccccg ccgacatccc cgattacaag aagctgccct tccccgaggg cttcaagtgg 2821 gagcgcgtga tgaacttcga agacggcggt ctggtgaccg ttacccagga ctcctccctg 2881 caggacggca cgctgatctg caaggtgaag atgcgcggca ccaacttccc ccccgacggc 2941 cccgtaatgc agaagaagac catgggctgg gaggcctcca ccgagatgct gtaccccgaa 3001 gacggcgtgc tgaagggcca tagctatcag gccctgaagc tgaaggacgg cggccactac 3061 ctggtggagt tcgagaccat ctacatggcc aagaagcccg tgcaactgcc cggcgattac 3121 tgtgtggaca ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag 3181 tacgggcgct ccgagggccg ccaccgtctg ggcatggacg agcggtacaa gtaagaattc 3241 tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc ctattctata 3301 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 3361 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 3421 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 3481 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 3541 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg 3601 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 3661 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 3721 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 3781 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 3841 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 3901 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 3961 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 4021 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca 4081 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt 4141 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 4201 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc 4261 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct 4321 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa 4381 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg 4441 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 4501 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 4561 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 4621 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 4681 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 4741 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 4801 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 4861 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 4921 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg 4981 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 5041 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 5101 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 5161 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 5221 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc 5281 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg 5341 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt 5401 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 5461 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 5521 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat 5581 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 5641 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 5701 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 5761 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 5821 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 5881 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 5941 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 6001 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 6061 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 6121 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 6181 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 6241 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 6301 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 6361 aagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6421 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6481 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6541 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 6601 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 6661 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 6721 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 6781 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 6841 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 6901 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 6961 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 7021 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 7081 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 7141 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 7201 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 7261 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 7321 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 7381 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 7441 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 7501 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 7561 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 7621 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 7681 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 7741 tc //