LOCUS Exported 7045 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Express PKA-SPARK, an activity reporter of protein kinase A in mammalian cells.. ACCESSION . VERSION . KEYWORDS pcDNA3-PKA-SPARK SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7045) AUTHORS Zhang Q, Huang H, Lu Q, Wu R, Chung CI, Zhang SQ, Torra J, Schepis A, Coughlin SR, Kornberg TB, Shu X TITLE Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter. JOURNAL Mol Cell. 2018 Jan 3. pii: S1097-2765(17)30936-X. doi: 10.1016/j.molcel.2017.12.008. PUBMED 29307513 REFERENCE 2 (bases 1 to 7045) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7045 /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 997..1713 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1042..1063) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1303..1322) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1650..1671 /label=EGFP-C /note="EGFP, forward primer" CDS 1909..1962 /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" primer_bind complement(2636..2653) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2642..2866 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2912..3340 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2999..3018) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3209..3230 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3349..3369) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3354..3683 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3534..3669 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3596..3615 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3750..4544 /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(3804..3823) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4414..4433 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4718..4839 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4755..4774) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4809..4828 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4888..4904) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4888..4904) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4901..4923) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4912..4928 /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(4936..4966) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4981..5002 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5119..5136) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5290..5878) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5370..5389) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6049..6909) /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 6672..6691 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6910..7014) /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 gctggctagc 901 gccaccatgg gcggtggagg gagcctgcgt cgcgccaccc tggtggacgg cggactcgag 961 tccggactca gatctggcag cggtggaggc gatatcatgg tgagcaaggg cgaggagctg 1021 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 1081 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 1141 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 1201 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 1261 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 1321 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 1381 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 1441 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggccaa cttcaagatc 1501 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 1561 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 1621 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 1681 gggatcactc tcggcatgga cgagctgtac aagggatccg gcagtgctgg tggtagtgct 1741 ggtggtagtg ctggtggtag tgctggtggc agtgctggtg gtagtgctgg tggtagtgct 1801 ggtggctcta gaggagaaat cgcaaagagt ctgaaagaaa ttgcaaaatc cttgaaggaa 1861 atagcgtggt cactgaaaga aattgccaag tccctcaaag ggggtaccga gggcagagga 1921 agtctgctaa catgcggtga cgtcgaggag aatcctggcc caaagcttgc caccatgcat 1981 aagttttctc aagaacagat cggcgaaaac attgtgtgca gggtcatttg taccacgggt 2041 caaattccca tccgagattt gtcagctgat atttcacaag tgcttaagga aaaacgatcc 2101 ataaagaaag tttggacatt tggtagaaac ccagcctgtg actatcattt aggaaacatt 2161 tcaagactgt caaataagca tttccaaata ctactaggag aagacggtaa ccttttattg 2221 aatgacattt ccactaatgg gacctggtta aatgggcaaa aagtcgagaa gaacagcaat 2281 cagttactgt ctcaaggtga tgaaataacc gttggtgtag gcgtggaatc agatatttta 2341 tctctggtca ttttcataaa cgacaaattt aagcagtgcc tcgaacagaa caaagttgat 2401 cgcggatccg gcagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggc 2461 agtgctggtg gtagtgctgg tggtagtgct ggtggctcta gaaccttgag agaaatcgag 2521 gaattgttga ggaaaatcat tgaagacagc gtgcgcagtg tggccgagct ggaggacatt 2581 gagaagtggc tgaagaaaat ctaagaattc gggcccgttt aaacccgctg atcagcctcg 2641 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 2701 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 2761 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 2821 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa 2881 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg 2941 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 3001 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 3061 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 3121 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 3181 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 3241 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 3301 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc 3361 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 3421 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 3481 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 3541 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 3601 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 3661 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 3721 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt 3781 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct 3841 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga 3901 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg 3961 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact 4021 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg 4081 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct 4141 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg 4201 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt 4261 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg 4321 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc 4381 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag 4441 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt 4501 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt 4561 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc 4621 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 4681 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa 4741 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 4801 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac 4861 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 4921 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 4981 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 5041 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 5101 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 5161 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 5221 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 5281 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 5341 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 5401 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 5461 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 5521 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 5581 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 5641 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 5701 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 5761 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 5821 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 5881 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 5941 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 6001 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 6061 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 6121 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 6181 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 6241 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 6301 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 6361 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 6421 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 6481 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 6541 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 6601 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 6661 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 6721 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 6781 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 6841 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 6901 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 6961 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 7021 tccccgaaaa gtgccacctg acgtc //