LOCUS Exported 5503 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Genetically encoded calcium sensor (GECI) that reports with lifetime and intensity changes. ACCESSION . VERSION . KEYWORDS FHL-cpmTq2-Calcium-lifetime-sensor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5503) AUTHORS Goedhart, J, Gadella, D. TITLE Goedhart/Gadella plasmids JOURNAL unpublished REFERENCE 2 (bases 1 to 5503) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5503 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(202..221) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 246..293 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 622..1210 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1111..1130 /label=pBR322ori-F /note="pBR322 origin, forward primer" enhancer 1358..1661 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1663..1862 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1816..1836 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 2068..2090 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 2152..2171 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" promoter 2213..2331 /label=rhaB promoter /note="promoter of the E. coli rhaBAD operon, conferring tight induction with L-rhamnose and repression with D-glucose in the presence of RhaR and RhaS (Giacalone et al., 2006)" RBS 2402..2413 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 2568..2585 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 2589..2621 /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 2625..2648 /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /label=Xpress(TM) tag /translation="DLYDDDDK" CDS 2676..2732 /codon_start=1 /product="calmodulin-binding peptide from avian smooth muscle myosin light chain kinase" /label=calmodulin-binding peptide /translation="RRKWNKAGHAVRAIGRLSS" CDS 2757..3005 /codon_start=1 /product="C-terminal fragment of mVenus for use in bimolecular fluorescence complementation (BiFC) (Kodama and Hu, 2010)" /label=VC155 /translation="DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH YLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELY" primer_bind complement(3069..3090) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3330..3349) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" CDS 3480..3923 /codon_start=1 /product="calcium-binding protein" /label=calmodulin /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA ELQDMINEVDADGDGTFDFPEFLTMMARKMNDTDSEEEIREAFRVFDKDGNGYIGAAEL RHVMTDLGEKLTDEEVDEMIRVADIDGDGQVNYEEFVQMMTAK" primer_bind complement(4016..4035) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 4081..4136 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(4135..4154) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" terminator 4224..4271 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter 4297..4401 /gene="bla" /label=AmpR promoter primer_bind complement(4426..4446) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4470..4680 /label=SV40 promoter /note="SV40 early promoter" promoter 4492..4688 /label=SV40 promoter /note="SV40 early promoter" rep_origin 4539..4674 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4601..4620 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4723..14 /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 GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4777..4796) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5387..5406 /label=Neo-F /note="Neomycin resistance gene, forward primer" ORIGIN 1 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct 61 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt 121 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc 181 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc 241 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg 301 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca 361 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg 421 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact 481 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 541 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 601 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 661 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 721 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 781 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 841 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 901 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 961 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 1021 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 1081 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 1141 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 1201 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 1261 tgctcacatg ttctttcctg cgttatcccc tgaggccggc ctattaatag taatcaatta 1321 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 1381 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 1441 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 1501 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca 1561 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta 1621 cttggcagta catctacgta ttagtcatcg ctattaccat gctgatgcgg ttttggcagt 1681 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 1741 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata 1801 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1861 gacgtcgttt agtgaaccgt cagatcacta gatgctttat tgcggtagtt tatcacagtt 1921 aaattgctaa cgccagtctc gaacttaacg tgcagaagtt ggtcgtgagg cactgggcag 1981 gtaagtatcg ggccctttgt gcggggggag cggctcgggg ctgtccgcgg ggggacggct 2041 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta 2101 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc 2161 tggttattgt gctgtctcat cattttggca aagaattgga tcggaccgaa atcaccacaa 2221 ttcagcaaat tgtgaacatc atcacgttca tctttccctg gttgccaaag gcccattttc 2281 ctgtcagtaa cgagaaggtc gcgaattcag gcgcttttta gactggtcgt agggagacca 2341 caacggtttc cctctagaaa taattttgtt taactataag aaggagatat accatgggtt 2401 taaagaggag aaaggtcatg aacaataacg atctctttca ggcatcacgt cggcgttttc 2461 tggcacaact cggcagctta accgacgccg ggacgctggg gccgtcattg ttaacgccgc 2521 gacgtgcgac tgcggcgcaa gcggcgactg acgcttctag aggttctcat catcatcatc 2581 atcatggtat ggctagcatg actggaggac agcaaatggg tcgggatctg tacgacgatg 2641 atgataagga tctcgccaca atggtcgact cttcacgtcg taagtggaat aaggcaggtc 2701 acgcagtcag agctataggt cggctgagct cacccgtggt tgtctatatc accgccgaca 2761 agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag gacggcggcg 2821 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc 2881 ccgacaacca ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg 2941 atcacatggt cctactggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc 3001 tgtaccaggg tggcagcggt ggcatggtga gcaagggcga ggagctgttc accggggtgg 3061 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 3121 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 3181 agctgcccgt gccctggccc accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg 3241 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 3301 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 3361 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 3421 aggacggcaa catcctgggg cacaagctgg agtacaacta ctatagcgac aacacgcgtg 3481 accaactgac tgaagagcag atcgcagaat ttaaagaggc tttctcccta tttgacaagg 3541 acggggatgg gacgataaca accaaggagc tggggacggt gatgcggtct ctggggcaga 3601 accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac ggtgacggca 3661 cattcgactt ccctgagttc ctgacgatga tggcaagaaa aatgaatgac acagacagtg 3721 aagaggaaat tagagaagcg ttccgcgtgt ttgataagga cggcaatggc tacatcggcg 3781 cagcagagct tcgccacgtg atgacagacc ttggagagaa gttaacagat gaggaggttg 3841 atgaaatgat cagggtagca gacatcgatg gggatggtca ggtaaactac gaagagtttg 3901 tacaaatgat gacagcgaag tagaagcttg tgattaacct caggtgcagg ctgcctatca 3961 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atcgatcttt 4021 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct 4081 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg 4141 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt 4201 tggcaacata tgcccatatg taactagcat aaccccttgg ggcctctaaa cgggtcttga 4261 ggggtttttt gctgaaagca tgctcgggga aatgtgcgcg gaacccctat ttgtttattt 4321 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 4381 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt 4441 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 4501 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 4561 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 4621 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 4681 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga 4741 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 4801 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 4861 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg 4921 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 4981 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 5041 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 5101 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 5161 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 5221 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg 5281 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 5341 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 5401 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 5461 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttg //