LOCUS Exported 7684 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Pyronic, FRET sensor that allow quantitative measurement of pyruvate transport, pyruvate production and mitochondrial pyruvate consumption in intact individual cells at high temporal resolution.. ACCESSION . VERSION . KEYWORDS Pyronic/pcDNA3.1(-) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7684) AUTHORS San_Martin A, Ceballo S, Baeza-Lehnert F, Lerchundi R, Valdebenito R, Contreras-Baeza Y, Alegria K, Barros LF TITLE Imaging Mitochondrial Flux in Single Cells with a FRET Sensor for Pyruvate. JOURNAL PLoS One. 2014 Jan 21;9(1):e85780. doi: 10.1371/journal.pone.0085780. eCollection 2014 Jan 21. PUBMED 24465702 REFERENCE 2 (bases 1 to 7684) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7684 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(42..61) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 233..612 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 613..816 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 767..787 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 861..880 /label=T7 /note="T7 promoter, forward primer" promoter 861..879 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 985..1692 /codon_start=1 /product="monomeric teal (cyan) fluorescent protein (Ai et al., 2006)" /label=mTFP1 /note="mammalian codon-optimized" /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG TNTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLK GDVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESA VARNSTDGMDELYK" protein_bind 1705..1729 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 2527..3246 /codon_start=1 /product="yellow fluorescent protein (YFP) with fast and efficient maturation (Nagai et al., 2002)" /label=Venus /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2572..2593) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2833..2852) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3180..3201 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(3273..3290) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3279..3503 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3549..3977 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3636..3655) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3846..3867 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3986..4006) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3991..4320 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4171..4306 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4233..4252 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4387..5181 /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(4441..4460) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5051..5070 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5355..5476 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5392..5411) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5446..5465 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5525..5541) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5525..5541) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5538..5560) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5549..5565 /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(5573..5603) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5618..5639 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5756..5773) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5927..6515) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6007..6026) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6686..7546) /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 7309..7328 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7547..7651) /gene="bla" /label=AmpR promoter ORIGIN 1 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc 61 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg 121 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt 181 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga 241 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg 301 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 361 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 421 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat 481 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 541 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 601 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac 661 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa 721 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt 781 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact 841 gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgt 901 ttaaacgggc cctctagact cgagcggccg ccactgtgct ggatatctgc agaattccac 961 cacactggac tagtggatcc gaacatggtg agcaagggcg aggagaccac aatgggcgta 1021 atcaagcccg acatgaagat caagctgaag atggagggca acgtgaatgg ccacgccttc 1081 gtgatcgagg gcgagggcga gggcaagccc tacgacggca ccaacaccat caacctggag 1141 gtgaaggagg gagcccccct gcccttctcc tacgacattc tgaccaccgc gttcgcctac 1201 ggcaacaggg ccttcaccaa gtaccccgac gacatcccca actacttcaa gcagtccttc 1261 cccgagggct actcttggga gcgcaccatg accttcgagg acaagggcat cgtgaaggtg 1321 aagtccgaca tctccatgga ggaggactcc ttcatctacg agatacacct caagggcgag 1381 aacttccccc ccaacggccc cgtgatgcag aagaagacca ccggctggga cgcctccacc 1441 gagaggatgt acgtgcgcga cggcgtgctg aagggcgacg tcaagcacaa gctgctgctg 1501 gagggcggcg gccaccaccg cgttgacttc aagaccatct acagggccaa gaaggcggtg 1561 aagctgcccg actatcactt tgtggaccac cgcatcgaga tcctgaacca cgacaaggac 1621 tacaacaagg tgaccgttta cgagagcgcc gtggcccgca actccaccga cggcatggac 1681 gagctgtaca agagatctgg taccacaagt ttgtacaaaa aagcaggctc cgaattcgcc 1741 cttggtacca tggcctacag caaaatccgc caaccaaaac tctccgatgt gattgagcag 1801 caactggagt ttttgatcct cgaaggcact ctccgcccgg gcgaaaaact cccaccggaa 1861 cgcgaactgg caaaacagtt tgacgtctcc cgtccctcct tgcgtgaggc gattcaacgt 1921 ctcgaagcga agggcttgtt gcttcgtcgc cagggtggcg gcacttttgt ccagagcagc 1981 ctatggcaaa gtttcagcga tccgctggtg gagctgctct ccgaccatcc tgagtcacag 2041 tatgacttgc tcgaaacacg acacgccctg gaaggtatcg ccgcttatta cgccgcgctg 2101 cgtagtaccg atgaagacaa ggaacgcatc cgtgaactcc accacgccat agagctggcg 2161 cagcagtctg gcgatctgga cgcggaatca aacgccgtac tccagtatca gattgccgtc 2221 accgaagcgg cccacaatgt ggttctgctt catctgctaa ggtgtatgga gccgatgttg 2281 gcccagaatg tccgccagaa cttcgaattg ctctattcgc gtcgcgagat gctgccgctg 2341 gtgagtagtc accgcacccg catatttgaa gcgattatgg ccggtaagcc ggaagaagcg 2401 cgcgaagcat cgcatcgcca tctggccttt atcgaagaaa ttttgctcga cagaagtcgt 2461 gaagagagcc gccgtgagcg ttctctgcgt cgtctggagc aacgaaagaa tcttaagaga 2521 tctaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 2581 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 2641 acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg 2701 cccaccctcg tgaccaccct gggctacggc ctgcagtgct tcgcccgcta ccccgaccac 2761 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 2821 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 2881 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 2941 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcaccgc cgacaagcag 3001 aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag 3061 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 3121 aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 3181 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 3241 aagtaaaagc ttaagtttaa accgctgatc agcctcgact gtgccttcta gttgccagcc 3301 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 3361 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 3421 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 3481 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg 3541 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 3601 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 3661 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 3721 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 3781 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 3841 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 3901 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 3961 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca 4021 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt 4081 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 4141 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc 4201 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct 4261 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa 4321 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg 4381 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 4441 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 4501 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 4561 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 4621 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 4681 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 4741 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 4801 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 4861 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg 4921 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 4981 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 5041 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 5101 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 5161 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc 5221 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg 5281 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt 5341 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 5401 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 5461 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat 5521 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 5581 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 5641 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 5701 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 5761 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 5821 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 5881 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 5941 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 6001 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 6061 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 6121 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 6181 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 6241 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 6301 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6361 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6421 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6481 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 6541 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 6601 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 6661 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 6721 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 6781 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 6841 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 6901 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 6961 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 7021 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 7081 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 7141 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 7201 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 7261 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 7321 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 7381 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 7441 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 7501 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 7561 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 7621 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 7681 tcga //