LOCUS Exported 7651 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION a genetically-encoded sensor for lactate concentration, transport and metabolic flux. ACCESSION . VERSION . KEYWORDS Laconic/pcDNA3.1(-) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7651) AUTHORS San_Martin A, Ceballo S, Ruminot I, Lerchundi R, Frommer WB, Barros LF TITLE A Genetically Encoded FRET Lactate Sensor and Its Use To Detect the Warburg Effect in Single Cancer Cells. JOURNAL PLoS One. 2013;8(2):e57712. doi: 10.1371/journal.pone.0057712. Epub 2013 Feb 26. PUBMED 23469056 REFERENCE 2 (bases 1 to 7651) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7651 /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" CDS 987..1694 /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" CDS 2496..3215 /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(2541..2562) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2802..2821) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3149..3170 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(3242..3259) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3248..3472 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3518..3946 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3605..3624) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3815..3836 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3955..3975) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3960..4289 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4140..4275 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4202..4221 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4356..5150 /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(4410..4429) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5020..5039 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5324..5445 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5361..5380) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5415..5434 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5494..5510) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5494..5510) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5507..5529) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5518..5534 /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(5542..5572) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5587..5608 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5725..5742) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5896..6484) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5976..5995) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6655..7515) /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 7278..7297 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7516..7620) /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 ccgaacatgg tgagcaaggg cgaggagacc acaatgggcg 1021 taatcaagcc cgacatgaag atcaagctga agatggaggg caacgtgaat ggccacgcct 1081 tcgtgatcga gggcgagggc gagggcaagc cctacgacgg caccaacacc atcaacctgg 1141 aggtgaagga gggagccccc ctgcccttct cctacgacat tctgaccacc gcgttcgcct 1201 acggcaacag ggccttcacc aagtaccccg acgacatccc caactacttc aagcagtcct 1261 tccccgaggg ctactcttgg gagcgcacca tgaccttcga ggacaagggc atcgtgaagg 1321 tgaagtccga catctccatg gaggaggact ccttcatcta cgagatacac ctcaagggcg 1381 agaacttccc ccccaacggc cccgtgatgc agaagaagac caccggctgg gacgcctcca 1441 ccgagaggat gtacgtgcgc gacggcgtgc tgaagggcga cgtcaagcac aagctgctgc 1501 tggagggcgg cggccaccac cgcgttgact tcaagaccat ctacagggcc aagaaggcgg 1561 tgaagctgcc cgactatcac tttgtggacc accgcatcga gatcctgaac cacgacaagg 1621 actacaacaa ggtgaccgtt tacgagagcg ccgtggcccg caactccacc gacggcatgg 1681 acgagctgta caagagatct ggtaccatga ttgttttacc cagacgcctg tcagacgagg 1741 ttgccgatcg tgtgcgggcg ctgattgatg aaaaaaacct ggaagcgggc atgaagttgc 1801 ccgctgagcg ccaactggcg atgcaactcg gcgtatcacg taattcactg cgcgaggcgc 1861 tggcaaaact ggtgagtgaa ggcgtgctgc tcagtcgacg cggcggcggg acgtttattc 1921 gctggcgtca tgacacatgg tcggagcaaa acatcgtcca gccgctaaaa acactgatgg 1981 ccgatgatcc ggattacagt ttcgatattc tggaagcccg ctacgccatt gaagccagca 2041 ccgcatggca tgcggcaatg cgcgccacac ctggcgacaa agaaaagatt cagctttgct 2101 ttgaagcaac gctaagtgaa gacccggata tcgcctcaca agcggacgtt cgttttcatc 2161 tggcgattgc cgaagcctca cataacatcg tgctgctgca aaccatgcgc ggtttcttcg 2221 atgtcctgca atcctcagtg aagcatagcc gtcagcggat gtatctggtg ccaccggttt 2281 tttcacaact gaccgaacaa catcaggctg tcattgacgc catttttgcc ggtgatgctg 2341 acggggcgcg taaagcaatg atggcgcacc ttagttttgt tcacaccacc atgaaacgat 2401 tcgatgaaga tcaggctcgc cacgcacgga ttacccgcct gcccggtgag cataatgagc 2461 attcgaggga gaaaaacgca cttaagagat ctaccatggt gagcaagggc gaggagctgt 2521 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca 2581 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagctgatct 2641 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg ggctacggcc 2701 tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca 2761 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga 2821 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca 2881 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc 2941 acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc 3001 gccacaacat cgaggacggc ggcgtgcagc tcgccgacca ctaccagcag aacaccccca 3061 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag tccgccctga 3121 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg 3181 ggatcactct cggcatggac gagctgtaca agtaaaagct taagtttaaa ccgctgatca 3241 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 3301 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 3361 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 3421 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 3481 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta 3541 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 3601 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 3661 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 3721 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 3781 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 3841 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 3901 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 3961 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 4021 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 4081 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 4141 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 4201 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 4261 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg 4321 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 4381 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 4441 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 4501 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 4561 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 4621 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 4681 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 4741 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 4801 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 4861 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg 4921 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 4981 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 5041 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 5101 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 5161 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac 5221 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat 5281 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc 5341 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 5401 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc 5461 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 5521 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 5581 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 5641 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 5701 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 5761 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 5821 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 5881 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 5941 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 6001 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 6061 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 6121 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 6181 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 6241 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 6301 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 6361 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 6421 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 6481 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 6541 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 6601 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 6661 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 6721 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 6781 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 6841 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 6901 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 6961 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 7021 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 7081 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 7141 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 7201 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 7261 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 7321 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 7381 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 7441 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 7501 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 7561 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 7621 cacatttccc cgaaaagtgc cacctgacgt c //