LOCUS Exported 7475 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Cyan-fluorescent homoFRET/anisotropy-based biosensor for monitoring Protein Kinase A activity.. ACCESSION . VERSION . KEYWORDS pcDNA3-Cerulean3-Cerulean3-FLARE-AKAR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7475) 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 7475) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7475 /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(3028..3046) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3029..3046) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(3066..3083) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3072..3296 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3342..3770 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3429..3448) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3639..3660 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3779..3799) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3784..4113 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3964..4099 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4026..4045 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4180..4974 /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(4234..4253) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4844..4863 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5148..5269 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5185..5204) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5239..5258 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5318..5334) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5318..5334) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5331..5353) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5342..5358 /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(5366..5396) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5411..5432 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5549..5566) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5720..6308) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5800..5819) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6479..7339) /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 7102..7121 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7340..7444) /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 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 1081 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 1141 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 1201 ctgagctggg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc 1261 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 1321 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 1381 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 1441 aacgccatcc acggcaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc 1501 aacttcggcc tcaactgcaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 1561 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 1621 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 1681 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagcgcat gcataagttt 1741 tctcaagaac agatcggcga aaacattgtg tgcagggtca tttgtaccac gggtcaaatt 1801 cccatccgag atttgtcagc tgatatttca caagtgctta aggaaaaacg atccataaag 1861 aaagtttgga catttggtag aaacccagcc tgtgactatc atttaggaaa catttcaaga 1921 ctgtcaaata agcatttcca aatactacta ggagaagacg gtaacctttt attgaatgac 1981 atttccacta atgggacctg gttaaatggg caaaaagtcg agaagaacag caatcagtta 2041 ctgtctcaag gtgatgaaat aaccgttggt gtaggcgtgg aatcagatat tttatctctg 2101 gtcattttca taaacgacaa atttaagcag tgcctcgagc agaacaaagt tgatcgctct 2161 gcaggtaagc caggcagcgg cgagggcagc accaagggcc tgcgtcgcgc caccctggtg 2221 gacggcggca ccggcggcag cgagctcatg gtgagcaagg gcgaggagct gttcaccggg 2281 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 2341 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 2401 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgagctgggg cgtgcagtgc 2461 ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 2521 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 2581 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 2641 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acgccatcca cggcaacgtc 2701 tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcggcct caactgcaac 2761 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 2821 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccaagct gagcaaagac 2881 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 2941 ctcggcatgg acgagctgta caagtaagaa ttctgcagat atccatcaca ctggcggccg 3001 ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg 3061 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 3121 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 3181 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 3241 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc 3301 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc 3361 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 3421 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 3481 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 3541 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 3601 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 3661 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 3721 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg 3781 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 3841 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 3901 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 3961 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 4021 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga 4081 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt 4141 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 4201 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 4261 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 4321 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta 4381 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 4441 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 4501 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 4561 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 4621 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 4681 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc 4741 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 4801 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 4861 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 4921 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga 4981 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt 5041 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 5101 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg 5161 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 5221 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta 5281 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 5341 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 5401 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 5461 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 5521 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 5581 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 5641 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 5701 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 5761 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 5821 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 5881 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 5941 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 6001 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 6061 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 6121 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 6181 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 6241 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 6301 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 6361 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 6421 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 6481 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 6541 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 6601 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 6661 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 6721 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 6781 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 6841 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 6901 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 6961 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 7021 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 7081 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 7141 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 7201 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 7261 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 7321 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 7381 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 7441 cgcgcacatt tccccgaaaa gtgccacctg acgtc //