LOCUS Exported 6640 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 6640) 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 6640) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6640 /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" primer_bind complement(1029..1050) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1290..1309) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" CDS 1953..2204 /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 2141..2162 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(2234..2251) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2240..2464 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2510..2938 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2597..2616) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2807..2828 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2947..2967) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2952..3281 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3132..3267 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3194..3213 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3348..4142 /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(3402..3421) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4012..4031 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4316..4437 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4353..4372) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4407..4426 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4486..4502) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4486..4502) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4499..4521) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4510..4526 /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(4534..4564) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4579..4600 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4717..4734) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4888..5473) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4968..4987) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5644..6504) /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 6267..6286 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6505..6609) /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 gtttaaacgg gccctctaga ctcgagcggc cgccactgtg ctggatatct gcagaattcc 961 accacactgg actagtggat cccatggtga gcaagggcga ggagctgttc accggggtgg 1021 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 1081 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 1141 agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg atgtgcttcg 1201 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 1261 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 1321 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 1381 aggacggcaa catcctgggg cacaagctgg agtacaacct ccgcggggca ctgaagaaag 1441 aggagcttgt tgaggctatg gccctcctgt cccagcgagg gcctgatgcc ctactcaccg 1501 tggcgctccg gaagccccca ggtcagcgta cggatgaaga actggacctg atctttgaag 1561 agctgctgca tatcaaggcg gtggcacacc tttctaactc ggtgaagcgg gaactagctg 1621 ctgttctgct ctttgaacca cacagcaagg caggaactgt gttgttcagc cagggggaca 1681 agggtacctc gtggtacatt atctggaagg gatctgtcaa tgtggtgacc catggcaagg 1741 ggctggtgac cacgttgcac gagggagatg actttggaca gctggctctg gtgaacgacg 1801 cacctcgggc agccaccatc atccttcgag aaaataactg tcactttctg cgtgtggaca 1861 agcaggactt caaccgcatc atcaaggatg tggaagcaaa aaccatgaga ctggaagaag 1921 aattcaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga 1981 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 2041 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc 2101 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 2161 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaaag cttaagttta 2221 aaccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 2281 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 2341 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 2401 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct 2461 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt 2521 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 2581 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 2641 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 2701 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 2761 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 2821 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg 2881 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa 2941 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 3001 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 3061 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 3121 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 3181 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga 3241 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta 3301 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag 3361 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg 3421 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc 3481 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag 3541 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca 3601 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat 3661 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata 3721 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac 3781 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc 3841 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg 3901 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg 3961 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta 4021 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg 4081 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct 4141 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga 4201 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc 4261 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt 4321 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 4381 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 4441 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc 4501 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 4561 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 4621 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 4681 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 4741 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 4801 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 4861 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 4921 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 4981 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 5041 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 5101 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 5161 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 5221 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 5281 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 5341 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 5401 caaacaaacc accgctggta gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa 5461 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 5521 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 5581 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 5641 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 5701 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 5761 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5821 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5881 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 5941 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 6001 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 6061 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 6121 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 6181 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 6241 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 6301 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 6361 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 6421 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 6481 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 6541 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 6601 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc //