LOCUS Exported 6643 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses biosensor for cAMP in mammalian cells. ACCESSION . VERSION . KEYWORDS Flamindo2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6643) AUTHORS Odaka H, Arai S, Inoue T, Kitaguchi T TITLE Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging. JOURNAL PLoS One. 2014 Jun 24;9(6):e100252. doi: 10.1371/journal.pone.0100252. eCollection 2014. PUBMED 24959857 REFERENCE 2 (bases 1 to 6643) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6643 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 629..648 /label=T7 /note="T7 promoter, forward primer" promoter 629..647 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(795..816) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1056..1075) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" CDS 1719..1970 /codon_start=1 /product="C-terminal fragment of mVenus for use in bimolecular fluorescence complementation (BiFC) (Kodama and Hu, 2010)" /label=VC155 /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK" primer_bind 1907..1928 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(2000..2017) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2006..2230 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2276..2704 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2363..2382) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2573..2594 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2713..2733) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2718..3047 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2898..3033 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2960..2979 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3114..3908 /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(3168..3187) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3778..3797 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4082..4203 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4119..4138) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4173..4192 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4252..4268) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4252..4268) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4265..4287) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4276..4292 /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(4300..4330) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4345..4366 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4483..4500) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4654..5242) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4734..4753) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5413..6273) /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 6036..6055 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6274..6378) /gene="bla" /label=AmpR promoter primer_bind complement(6453..6472) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg 661 gctagcgttt aaacgggccc tctagactcg agcggccgcc actgtgctgg atatctgcag 721 aattccacca cactggacta gtggatccca tggtgagcaa gggcgaggag ctgttcaccg 781 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt 841 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca 901 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgatgt 961 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg 1021 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg 1081 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact 1141 tcaaggagga cggcaacatc ctggggcaca agctggagta caacctccgc ggggcactga 1201 agaaagagga gcttgttgag gctatggccc tcctgtccca gcgagggcct gatgccctac 1261 tcaccgtggc gctccggaag cccccaggtc agcgtacgga tgaagaactg gacctgatct 1321 ttgaagagct gctgcatatc aaggcggtgg cacacctttc taactcggtg aagcgggaac 1381 tagctgctgt tctgctcttt gaaccacaca gcaaggcagg aactgtgttg ttcagccagg 1441 gggacaaggg tacctcgtgg tacattatct ggaagggatc tgtcaatgtg gtgacccatg 1501 gcaaggggct ggtgaccacg ttgcacgagg gagatgactt tggacagctg gctctggtga 1561 acgacgcacc tcgggcagcc accatcatcc ttcgagaaaa taactgtcac tttctgcgtg 1621 tggacaagca ggacttcaac cgcatcatca aggatgtgga agcaaaaacc atgagactgg 1681 aagaagaatt caacagccac aacgtctata tcatggccga caagcagaag aacggcatca 1741 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact 1801 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga 1861 gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg 1921 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taaaagctta 1981 agtttaaacc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc 2041 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa 2101 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg 2161 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg 2221 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg 2281 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 2341 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 2401 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 2461 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg 2521 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 2581 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg 2641 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 2701 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag 2761 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag 2821 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc 2881 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc 2941 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat 3001 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag 3061 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg 3121 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 3181 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 3241 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 3301 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 3361 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 3421 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 3481 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 3541 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 3601 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 3661 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 3721 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 3781 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 3841 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 3901 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc 3961 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg 4021 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc 4081 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 4141 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc 4201 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct 4261 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 4321 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 4381 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 4441 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 4501 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 4561 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 4621 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 4681 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 4741 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 4801 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 4861 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 4921 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 4981 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 5041 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 5101 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 5161 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 5221 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 5281 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 5341 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 5401 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 5461 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 5521 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 5581 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 5641 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 5701 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 5761 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 5821 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 5881 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 5941 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 6001 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 6061 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 6121 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 6181 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 6241 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 6301 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 6361 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg 6421 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 6481 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt 6541 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg 6601 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt //