LOCUS Exported 9209 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION a biosensor comprised of a fast-maturing pH-stable red fluorescent protein fused to a pH-sensitive green fluorescent protein variant. Mitochodrial localization.. ACCESSION . VERSION . KEYWORDS pAS1NB m Rosella I SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9209) AUTHORS Rosado CJ, Mijaljica D, Hatzinisiriou I, Prescott M, Devenish RJ TITLE Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast. JOURNAL Autophagy. 2008 Feb;4(2):205-13. Epub 2007 Nov 21. PUBMED 18094608 REFERENCE 2 (bases 1 to 9209) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9209 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 106..127 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 142..172 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 180..196 /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)." primer_bind 185..207 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 204..220 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 204..220 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" misc_feature 1825..1877 /label=MCS /note="multiple cloning site" regulatory 1887..1896 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1893..2567 /codon_start=1 /product="rapidly maturing tetrameric variant of DsRed fluorescent protein (Bevis and Glick, 2002)" /label=DsRed-Express /note="mammalian codon-optimized" /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGRFIYKVKFIGVNFPSDGPVMQKKTMGWEPSTERLYPRDGVLKGEIHK ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRHH LFL" primer_bind complement(2023..2041) /label=mCherry-R /note="mCherry, reverse primer" primer_bind complement(2073..2093) /label=DsRed1-N /note="DsRed1, reverse primer" primer_bind 2291..2310 /label=mCherry-F /note="mCherry, forward primer" primer_bind 2485..2508 /label=DsRed1-C /note="DsRed1, forward primer" CDS 2598..3305 /codon_start=1 /product="pH-sensitive mutant of green fluorescent protein (Ng et al., 2002)" /label=superecliptic pHluorin /note="excited strongly by blue light at neutral pH but minimally fluorescent at acidic pH" /translation="KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI CTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNY KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKANF KIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEFV TAAGITQGMDELYK" primer_bind complement(2622..2650) /label=GFP-R /note="GFP, reverse primer. Does NOT anneal to EGFP" primer_bind 3245..3265 /label=GFP-F /note="GFP, forward primer. Does NOT anneal to EGFP" terminator 3421..3694 /gene="S. cerevisae PGK1" /label=PGK1 terminator /note="transcription terminator for 3-phosphoglycerate kinase gene" misc_feature 3701..3757 /label=MCS /note="pUC18/19 multiple cloning site" primer_bind complement(3758..3775) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3758..3774) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3767..3789) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4211..5557 /label=2u ori /note="yeast 2u plasmid origin of replication" primer_bind complement(7217..7235) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7303..7407 /gene="bla" /label=AmpR promoter CDS 7408..8268 /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 complement(7626..7645) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8439..9027 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8928..8947 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 9181..9198 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttcg 241 aattctgcag tcgactctag agctgtagaa ttttctcatt ggtcacatta caacctgaaa 301 atactttatc tacaatcata ccattcttat aacatgtccc cttaatacta ggatcaggca 361 tgaacgcatc acagacaaaa tcttcttgac aaacgtcaca attgatccct ccccatccgt 421 tatcacaatg acaggtgtca ttttgtgctc ttatgggacg atccttatta ccgctttcat 481 ccggtgatag accgccacag aggggcagag agcaatcatc acctgcaaac ccttctatac 541 actcacatct accagtgtac gaattgcatt cagaaaactg tttgcattca aaaataggta 601 gcatacaatt aaaacatggc gggcacgtat cattgccctt atcttgtgca gttagacgcg 661 aatttttcga agaagtacct tcaaagaatg gggtctcatc ttgtttggaa gtaccactga 721 gcaggataat aatagaaatg ataatatact atagtagaga taacgtcgat gacttcccat 781 actgtaattg cttttagttg tgtattttta gtgtgcaagt ttctgtaaat cgattaattt 841 ttttttcttt cctcttttta ttaaccttaa tttttatttt agattcctga cttcaactca 901 agacgcacag atattataac atctgcacaa taggcatttg caagaattac tcgtgagtaa 961 ggaaagagtg aggaactatc gcatacctgc atttaaagat gccgatttgg gcgcgaatcc 1021 tttattttgg cttcaccctc atactattat cagggccaga aaaaggaagt gtttccctcc 1081 ttcttgaatt gatgttaccc tcataaagca cgtggcctct tatcgagaaa gaaattaccg 1141 tcgctcgtga tttgtttgca aaaagaacaa aactgaaaaa acccagacac gctcgacttc 1201 tctcttccta ttgattgcag cttccaattt cgtcacacaa caaggtccta gcgacggctc 1261 acaggttttg taacaagcaa tcgaaggttc tggaatggcg ggaaagggtt tactaccaca 1321 tgctatgatg cccactgtga tctccagagc aaagttcgtt cgatcgtact gttactctct 1381 ctctttcaaa cagaattgtc cgaatcgtgt gacaacaaca gcctgttctc acacactctt 1441 ttcttctaac caagggggtg gtttagttta gtagaacctc gtgaaactta catttacata 1501 tatataaact tgcataaatt ggtcaatgca agaaatacat atttggtctt ttctaattcg 1561 tagtttttca agttcttaga tgctttcttt ttctcttttt tacagatcat caaggaagta 1621 attatctact ttttctctag aagatccaaa caatgtcagc gatattatca acaactagca 1681 aaagtttctt atcaaggggc tccacaagac aatgtcaaaa tatgcaaaag gctctttttg 1741 cactattgaa tgctcgccac tatagtagcg cctccgaaca aacgttgaag gagagatttg 1801 ctgaaattac agatctaaac atgtctcgag ctcaagcttc gaattctgca gtcgacggta 1861 ccgcgggccc gggatcccca ctagtcgcca ccatggcctc ctccgaggac gtcatcaagg 1921 agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag ttcgagatcg 1981 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca 2041 agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca 2101 aggtgtacgt gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg 2161 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg 2221 actcctccct gcaggacggc cgcttcatct acaaggtgaa gttcatcggc gtgaacttcc 2281 cctccgacgg ccccgtaatg cagaagaaga ctatgggctg ggagccctcc accgagcgcc 2341 tgtacccccg cgacggcgtg ctgaagggcg agatccacaa ggccctgaag ctgaaggacg 2401 gcggccacta cctggtggag ttcaagtcca tctacatggc caagaagccc gtgcagctgc 2461 ccggctacta ctacgtggac tccaagctgg acatcacctc ccacaacgag gactacacca 2521 tcgtggagca gtacgagcgc accgagggcc gccaccacct gttcctgtct gatccctggt 2581 cttcttcggg agatctaaaa ggagaagaac ttttcactgg agttgtccca attcttgttg 2641 aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg 2701 caacatacgg aaaacttacc cttaaattta tttgcactac tggaaaacta cctgttcctt 2761 ggccaacact tgtcactact ttaacttatg gtgttcaatg cttttcaaga tacccagatc 2821 atatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaaagaa 2881 ctatattttt caaagatgac gggaactaca agacacgtgc tgaagtcaag tttgaaggtg 2941 atacccttgt taatagaatc gagttaaaag gtattgattt taaagaagat ggaaacattc 3001 ttggacacaa attggaatac aactataacg atcaccaggt gtacatcatg gcagacaaac 3061 aaaagaatgg aatcaaagct aacttcaaaa ttagacacaa cattgaagat ggaggcgttc 3121 aactagcaga ccattatcaa caaaatactc caattggcga tgggcccgtc cttttaccag 3181 acaaccatta cctgtttaca acttctactc tttcgaaaga tcccaacgaa aagagagacc 3241 acatggtcct tcttgagttt gtaacagctg ctgggattac acaaggcatg gatgaactat 3301 acaaatctga tcactgagcg gccgcggatc tcccatgtct ctactggtgg tggtgcttct 3361 ttggaattat tggaaggtaa ggaattgcca ggtgttgctt tcttatccga aaagaaataa 3421 attgaattga attgaaatcg atagatcaat ttttttcttt tctctttccc catcctttac 3481 gctaaaataa tagtttattt tattttttga atatttttta tttatatacg tatatataga 3541 ctattattat cttttaatag attattaaga tttttattaa aaaaaaattc gtccctcttt 3601 ttaatgcctt ttatgcagtt tttttttccc attcgatatt tctatgttcg ggtttcagcg 3661 tattttaagt ttaataactc gaaaattctg cgtttcgaaa aagcttgcat gcctgcaggt 3721 cgactctaga gaatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc 3781 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 3841 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 3901 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 3961 accgcatata tcggatcgta cttgttaccc atcattgaat tttgaacatc cgaacctggg 4021 agttttccct gaaacagata gtatatttga acctgtataa taatatatag tctagcgctt 4081 tacggaagac aatgtatgta tttcggttcc tggagaaact attgcatcta ttgcataggt 4141 aatcttgcac gtcgcatccc cggttcattt tctgcgtttc catcttgcac ttcaatagca 4201 tatctttgtt aacgaagcat ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc 4261 gctaattttt caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgaa 4321 agcgctattt taccaacgaa gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg 4381 agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac 4441 gcgagagcgc tattttacca acaaagaatc tatacttctt ttttgttcta caaaaatgca 4501 tcccgagagc gctatttttc taacaaagca tcttagatta ctttttttct cctttgtgcg 4561 ctctataatg cagtctcttg ataacttttt gcactgtagg tccgttaagg ttagaagaag 4621 gctactttgg tgtctatttt ctcttccata aaaaaagcct gactccactt cccgcgttta 4681 ctgattacta gcgaagctgc gggtgcattt tttcaagata aaggcatccc cgattatatt 4741 ctataccgat gtggattgcg catactttgt gaacagaaag tgatagcgtt gatgattctt 4801 cattggtcag aaaattatga acggtttctt ctattttgtc tctatatact acgtatagga 4861 aatgtttaca ttttcgtatt gttttcgatt cactctatga atagttctta ctacaatttt 4921 tttgtctaaa gagtaatact agagataaac ataaaaaatg tagaggtcga gtttagatgc 4981 aagttcaagg agcgaaaggt ggatgggtag gttatatagg gatatagcac agagatatat 5041 agcaaagaga tacttttgag caatgtttgt ggaagcggta ttcgcaatat tttagtagct 5101 cgttacagtc cggtgcgttt ttggtttttt gaaagtgcgt cttcagagcg cttttggttt 5161 tcaaaagcgc tctgaagttc ctatactttc tagctagaga ataggaactt cggaatagga 5221 acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac 5281 agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga 5341 agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg 5401 atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc 5461 ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct 5521 catccttcaa tgctatcatt tcctttgata ttggatcgat ccgatgataa gctgtcaaac 5581 atgagaatta attctaccct atgaacatat tccattttgt aatttcgtgt cgtttctatt 5641 atgaatttca tttataaagt ttatgtacaa atatcataaa aaaagagaat ctttttaagc 5701 aaggattttc ttaacttctt cggcgacagc atcaccgact tcggtggtac tgttggaacc 5761 acctaaatca ccagttctga tacctgcatc caaaaccttt ttaactgcat cttcaatggc 5821 cttaccttct tcaggcaagt tcaatgacaa tttcaacatc attgcagcag acaagatagt 5881 ggcgataggg tcaaccttat tctttggcaa atctggagca gaaccgtggc atggttcgta 5941 caaaccaaat gcggtgttct tgtctggcaa agaggccaag gacgcagatg gcaacaaacc 6001 caaggaacct gggataacgg aggcttcatc ggagatgata tcaccaaaca tgttgctggt 6061 gattataata ccatttaggt gggttgggtt cttaactagg atcatggcgg cagaatcaat 6121 caattgatgt tgaaccttca atgtaggaaa ttcgttcttg atggtttcct ccacagtttt 6181 tctccataat cttgaagagg ccaaaacatt agctttatcc aaggaccaaa taggcaatgg 6241 tggctcatgt tgtagggcca tgaaagcggc cattcttgtg attctttgca cttctggaac 6301 ggtgtattgt tcactatccc aagcgacacc atcaccatcg tcttcctttc tcttaccaaa 6361 gtaaatacct cccactaatt ctctgacaac aacgaagtca gtacctttag caaattgtgg 6421 cttgattgga gataagtcta aaagagagtc ggatgcaaag ttacatggtc ttaagttggc 6481 gtacaattga agttctttac ggatttttag taaaccttgt tcaggtctaa cactacctgt 6541 accccattta ggaccaccca cagcacctaa caaaacggca tcaaccttct tggaggcttc 6601 cagcgcctca tctggaagtg ggacacctgt agcatcgata gcagcaccac caattaaatg 6661 attttcgaaa tcgaacttga cattggaacg aacatcagaa atagctttaa gaaccttaat 6721 ggcttcggct gtgatcttga ccaacgtggt cacctggcaa aacgacgatc ttcttagggg 6781 cagacatagg ggcagacatt agaatgtata tccttgaaat atatatatat attgctgaaa 6841 tgtaaaaggt aagaaaagtt agaataagac gattgctaac cacctattgg aaaaaacaat 6901 aggtccttaa ataatattgt caacttcaag tattgtgatg caagcattta gtcatgaacg 6961 cttctctatt ctatatgaaa agccggttcc ggcctctcac ctttcctttt tctcccaatt 7021 tttcagttga aaaaggtata tgcgcggcga cctctgaaat taacaaaaaa tttccagtca 7081 tcgaatttgt ctgtgcgata gcgcccctgt gtgttctcgt tatgttgagg aaaaaaataa 7141 tggttgctaa gagattcgaa ctcttgcatc ttacgatacc tgagtattcc cacagttaat 7201 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata 7261 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 7321 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 7381 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 7441 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 7501 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 7561 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 7621 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 7681 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 7741 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 7801 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 7861 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 7921 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 7981 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 8041 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 8101 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 8161 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 8221 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 8281 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 8341 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 8401 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 8461 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 8521 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 8581 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 8641 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 8701 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 8761 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 8821 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 8881 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 8941 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 9001 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 9061 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 9121 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 9181 agcgagtcag tgagcgagga agcggaaga //